© 2014. Published by The Company of Biologists Ltd.
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The Role of the Plexin-A2 Receptor in Semaphorin-3a and Semaphorin-3b Signal
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Transduction
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By: Adi D. [Sabag]1#, Tatyana [Smolkin]1#, Yelena [Mumblat]1, Marius [Ueffing]2,3, Ofra
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[Kessler]1, Christian Johannes [Gloeckner]2,3, and Gera [Neufeld]1*
Journal of Cell Science
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From the 1Cancer Research and vascular Biology Center, The Bruce Rappaport Faculty
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of Medicine, Technion, Israel Institute of Technology, Haifa, Israel, 2Eberhard Karls
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University Tübingen, Institute for Ophthalmic Research, Medical Proteome Center,
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Tübingen, Germany and 3Helmholtz Zentrum München – German Research Center for
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Environmental Health, Research Unit Protein Science, Neuherberg, Germany.
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*To whom correspondence should be addressed: E-mail: [email protected]
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Tel: +972-4-8295430, Fax: +972-4-8523672
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#
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Keywords: cell guidance/neuropilins/plexins/receptor complexes/semaphorins
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Running title: Plexin-A2 in semaphorin signaling
These authors contributed equally to the manuscript
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JCS Advance Online Article. Posted on 21 October 2014
Accepted manuscript Journal of Cell Science
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ABSTRACT
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Class-3 semaphorins are anti-angiogenic and anti-tumorigenic guidance factors that bind
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to neuropilins which in turn associate with class-A plexins to transduce semaphorin
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signals. To study the role of the plexin-A2 receptor in semaphorin signaling, we silenced
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its expression in endothelial cells and in glioblastoma cells. The silencing did not affect
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sema3A signaling which depended on neuropilin-1, plexin-A1 and plexin-A4, but
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abolished completely sema3B signaling which required in addition plexin-A4 and one of
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the two neuropilins. Interestingly, over-expression of plexin-A2 in plexin-A1 or plexin-
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A4 silenced cells restored responses to both semaphorins although it nullified their ability
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to differentiate between them, suggesting that when over-expressed plexin-A2 is
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functionally interchangeable with other class-A plexins. In-contrast, although plexin-A4
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over-expression restored sema3A signaling in plexin-A1 silenced cells, it failed to restore
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sema3B signaling in plexin-A2 silenced cells. It follows that the identity of plexins in
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functional semaphorin receptors can be flexible depending on their expression level. Our
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results suggest that changes in the expression of plexins induced by microenvironmental
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cues can trigger differential responses of different populations of migrating cells to
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encountered gradients of semaphorins.
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INTRODUCTION
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During embryonic development, cells migrate extensively to distant locations. Similar
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migrations, although much less extensive, take pace in adult animals and in diseases such
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as in the various forms of cancer (Trepat et al., 2012). Migrating cells are guided to their
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correct destination by a variety of guidance cues which include gradients of secreted
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repulsive and attractive guidance factors. Essential to the understanding of the
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mechanisms by which cells interpret guidance cues is the characterization of the receptors
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that interact with these guidance cues and of mechanisms that modulate the activity and
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expression of such receptors since the activity of these receptors determines which signal
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transducing cascades will be activated downstream.
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The semaphorins were identified as repulsive axon guidance factors (Kolodkin et al.,
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1993) but have also emerged as important regulators of angiogenesis, immune responses
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and tumor progression (Tamagnone, 2012; Kumanogoh and Kikutani, 2013; Sakurai et
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al., 2011). Class-3 semaphorins are a subfamily of secreted semaphorins containing seven
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members of which six, with the exception of sema3E (Gu et al., 2005), bind to one of the
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two neuropilin receptors. Neuropilins contain short intracellular domains that limit their
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signal transducing ability, and associate with members of the class-A plexin receptor
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subfamily or with plexin-D1 to transduce class-3 semaphorin signals. These plexins also
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function in addition as direct binding and signal transducing receptors for some
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membrane anchored members of the class-4, class-5 and class-6 semaphorin subfamilies
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(reviewed in (Neufeld et al., 2012; Gu and Giraudo, 2013)).
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Sema3A is an anti-angiogenic semaphorin that binds to neuropilin-1 (np1) but not to
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neuropilin-2 (np2) (Kolodkin et al., 1997; Chen et al., 1997; Acevedo et al., 2008).
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Sema3B is a tumor suppressor of lung cancer (Tomizawa et al., 2001) that binds to both 3
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neuropilins and functions in addition as an inhibitor of angiogenesis (Varshavsky et al.,
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2008). The roles of specific class-A plexins in sema3B signal transduction have not been
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well characterized. Plexin-A1 was found to be required, along with the adhesion receptor
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NrCAM for the transduction of sema3B signals guiding commissural projections at the
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vertebrate ventral midline during the development of the nervous system (Nawabi et al.,
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2010), but the possible roles of additional plexins in sema3B signaling have not been
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investigated. In contrast, sema3A signal transduction had been studied more extensively.
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Studies conducted using knockout mice lacking either functional plexin-A1, plexin-A3 or
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plexin-A4 receptors found evidence suggesting that these three receptors all play a role in
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sema3A signal transduction (Yaron et al., 2005; Suto et al., 2005; Schwarz et al., 2008;
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Moretti et al., 2007; Bouvree et al., 2012; Katayama et al., 2013). Interestingly, another
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study suggested that plexin-A3 transduces exclusively pro-apoptotic signals of sema3A
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but not signals affecting cytoskeletal organization (Ben-Zvi et al., 2008). Additional
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studies in which plexin-A2 was ectopically expressed at higher expression levels in cells
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suggested that plexin-A2 can also transduce sema3A signals (Janssen et al., 2012;
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Takahashi and Strittmatter, 2001). Taken together these observations seem to suggest that
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all four class-A plexins can transduce sema3A signals, which in turn seems to suggest
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that for sema3A signaling the class-A plexins may be interchangeable. By extension
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these studies suggest that the class-A plexins may not display high specificity for any of
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the class-3 semaphorins. However, it should be noted that in most studies using gene
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targeted mouse models it was unclear if the observed loss of responsiveness to sema3A
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was due to a direct effect on sema3A signaling, or if the targeting affected the expression
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of a secondary modulator. Likewise, much of the information obtained from cell culture
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experiments was derived from experiments in which specific plexins were over-
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expressed, and thus may not be relevant physiologically (Takahashi and Strittmatter,
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2001; Janssen et al., 2012).
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In all these studies it was assumed that successful sema3A signaling requires np1 and a
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single plexin. However, we have recently observed that in endothelial cells and in
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U87MG glioblastoma cells sema3A signaling affecting cytoskeletal organization requires
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the simultaneous presence of plexin-A1 and plexin-A4 which form spontaneous hetero-
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complexes. Inhibition of the expression of any one of these plexins but not of plexin-A3
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was sufficient to completely inhibit the cytoskeletal collapse induced by sema3A
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suggesting that physiologically relevant functional sema3A receptors consist of a
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complex containing np1, plexin-A1 and plexin-A4 (Kigel et al., 2011). These
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observations suggest that specific combinations of plexins and neuropilins determine the
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specificity of receptor complexes to specific semaphorins and that the plexins are not
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interchangeable.
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. To gain a better understanding of the roles of the class-A plexins in class-3 semaphorin
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signal transduction, and to clarify the possible role of plexin-A2 in sema3A signaling, we
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studied in more detail the contribution of plexin-A2 as well as additional class-A plexins
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to sema3A and sema3B signaling. We provide evidence suggesting that in endothelial
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cells and glioblastoma cells plexin-A4 is a required component of both sema3A and
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sema3B receptor complexes and inhibition of its expression nullifies both sema3A and
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sema3B signaling. The specificity for sema3A or sema3B is determined by the presence
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of plexin-A1 in sema3A receptors and plexin-A2 in sema3B receptors and silencing each
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abrogates signaling by the appropriate semaphorin. Interestingly, when plexin-A2 was
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over-expressed in cells silenced for plexin-A1 or plexin-A4 expression it restored both
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sema3A and sema3B signaling, suggesting that the requirement for specific plexins may
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be abolished if the expression levels of some of the plexins exceed certain threshold 5
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levels and further suggesting that when highly expressed the different plexins are
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functionally interchangeable. This comes at a price as such cells lost their ability to
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distinguish between sema3A and sema3B. However, this may not always be true since
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plexin-A4 over-expression failed to restore sema3B signaling in plexin-A2 silenced cells
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Accepted manuscript Journal of Cell Science
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RESULTS
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Plexin-A2 is not required for sema3A signal transduction but is required for the
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transduction of sema3B and sema6A signals in U87MG glioblastoma cells and in
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endothelial cells: U87MG glioblastoma multiforme cells and human umbilical vein
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derived endothelial cells (HUVEC) respond to stimulation by semaphorins such as
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sema3A with localized disassembly of their actin cytoskeleton that is associated with loss
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of focal contacts followed by cell contraction, and with the appearance of "spikes" that
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remain attached to the substrate around the contracted cell (Fig. 1C & 1E, arrows). In
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both cell types the number of mRNA molecules/cell encoding np2, type-A and type-D
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plexins is pretty similar with some variations while the number/cell of mRNA molecules
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encoding np1 is 2-5 fold higher (Fig. S1A). We have previously observed that np1,
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plexin-A1 and plexin-A4, but not plexin-A3, are required for the transduction of sema3A
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induced signals resulting in cytoskeletal collapse in HUVEC and in U87MG cells.
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Inhibition of the expression of any of these genes using specific shRNA species that do
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not cross-inhibit the expression of the other plexins, completely inhibited signal
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transduction by sema3A (Kigel et al., 2011). These observations suggested that the
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sema3A signaling complex contains these three receptors, an assumption that was
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supported by co-immunoprecipitation experiments. Because plexin-A2 was also reported
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to transduce sema3A signals and to participate in the formation of functional sema3A
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receptors (Takahashi and Strittmatter, 2001; Janssen et al., 2012), we sought to determine
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if plexin-A2 also participates in the transduction of sema3A signals in these cells. To
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examine this possibility we inhibited the expression of plexin-A2 in HUVEC and in
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U87MG cells using two specific shRNA species which do not inhibit the expression of
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the other class-A plexins or the expression of neuropilins (Fig. 1A and Fig. S1B-D).
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However, even though the shRNA silenced the expression of plexin-A2 effectively in 7
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HUVEC and in U87MG cells, the silencing did not inhibit sema3A induced cell
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contraction (Figs. 1C, 1D, S1E) as does plexin-A1 or plexin-A4 silencing (Kigel et al.,
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2011).
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To gain a better understanding of the role of plexin-A2 in the transduction of class-3
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semaphorin signals, we determined if plexin-A2 is required for the transduction of the
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signals of other class-3 semaphorins. Silencing plexin-A2 expression had no effect on
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sema3F induced contraction of U87MG cells (Figs. 1C & 1D) while in agreement with
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prior reports, silencing plexin-A3 inhibited sema3F signaling completely (Fig. 2B)
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(Yaron et al., 2005; Schwarz et al., 2008). However, the collapse of the actin
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cytoskeleton, the disassembly of vinculin containing focal contacts, and the subsequent
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cell contraction induced by sema3B were strongly inhibited in the plexin-A2 silenced
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cells (Figs. 1C-1E). The silencing also inhibited completely sema6A induced contraction
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of these cells (Figs. 1C & 1D), a surprising result considering that these cells also express
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plexin-A4, a plexin that is known to function as a signal transducing sema6A receptor
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(Suto et al., 2007; Okada et al., 2007; Kigel et al., 2011), indicating that the concentration
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of plexin-A4 in these cells, which seems rather low (Fig. S1A), is probably not sufficient
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to enable sema6A signaling on its own. The contractile response of the U87MG cells to
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sema3B and sema6A was rescued following re-expression of recombinant plexin-A2 in
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the silenced cells. These observations attest to the specificity of the silencing and indicate
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that plexin-A2 is essential the transduction of sema3B and sema6A signals in these cells
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(Figs. 1B-D). Likewise, silencing the expression of plexin-A2 in HUVEC inhibited
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completely sema3B and sema6A induced cytoskeletal collapse (Figs. S2A-S2C).
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In order to verify these results by a different method we also expressed in the U87MG
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cells a truncated form of the plexin-A2 receptor lacking the intracellular domain of
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plexin-A2 which was tagged at its C-terminal with a FLAG tag (A2ExTm) (Fig. S3A). It 8
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was previously observed that a similarly truncated form of plexin-A1 functions as a
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dominant negative inhibitor of sema3A (Serini et al., 2003). The truncated plexin-A2
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formed complexes with full length plexin-A2 (Fig. S3B) as well as with plexin-A4 (Fig.
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S3C). Over-expression of A2ExTm in U87MG cells inhibited sema3B and sema6A
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induced contraction of the U87MG cells independently suggesting that plexin-A2 is
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indeed required for sema3B and sema6A signal transduction in these cells (Figs. S3D &
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S3E).
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Sema3A transduces signals exclusively using np1 while sema3B utilizes both
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neuropilins for signal transduction: HUVEC and U87MG cells express the two
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neuropilins as well as the four class-A plexins as well as plexins belonging to additional
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plexin subclasses (Shraga-Heled et al., 2007; Kigel et al., 2011; Basile et al., 2005;
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Artigiani et al., 2004; Gu et al., 2005). The primary binding receptors of class-3
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semaphorins are the neuropilins which cannot transduce signals on their own and
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associate with class-A plexins or plexin-D1 to transduce semaphorin signals. We have
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previously observed that sema3B can utilize both neuropilins for signal transduction.
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However, in HUVEC sema3B transduces signals using primarily np1 and np2 only plays
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a secondary role (Varshavsky et al., 2008). In contrast, in U87MG cells only inhibition of
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the expression of both neuropilins completely abrogated efficiently sema3B signal
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transduction while inhibition of either np1 or np2 expression alone was not sufficient to
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inhibit sema3B signaling (Figs. S4A & S4B). In contrast, silencing np1 expression alone
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completely inhibited sema3A induced cell contraction while silencing np2 alone was
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sufficient to abrogate sema3F induced cell contraction (Figs. S4A & S4B). Thus, plexin-
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A2 seems to associate with both neuropilins to transduce sema3B signals in U87MG
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cells.
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Plexin-A2 and plexin-A4 are required simultaneously for the transduction of sema3B
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signals in HUVEC and in U87MG cells: Our previous work on sema3A demonstrated
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that more than one plexin is required, in addition to np1, for the transduction of sema3A
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signals (Kigel et al., 2011). To find out if any additional plexins are required besides
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plexin-A2 for sema3B induced cell contraction, we silenced the expression of additional
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class-A plexins in HUVEC and in U87MG cells (Fig. 2A) using shRNA species that do
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not affect the expression of neuropilins or the expression of other class-A plexins (Kigel
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et al., 2011). Silencing plexin-A1 or plexin-A3 did not inhibit sema3B or sema6A
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induced contraction of U87MG cells, suggesting that they do not play an essential role in
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sema3B or sema6A induced signal transduction (Figs. 2B & 2C). This lack of an effect
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was not due to insufficient silencing because silencing plexin-A1 expression inhibited
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completely the response to sema3A and silencing plexin-A3 inhibited completely cell
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contraction induced by sema3F (Figs. 2B-2E). As before, silencing plexin-A2 aborted
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completely responses to sema3B and sema6A but had no effect on sema3A induced cell
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contraction. However, cells in which we silenced the expression of plexin-A4 also failed
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to contract in response to sema3B (Figs. 2B & 2C) (Kigel et al., 2011). Identical results
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were observed when the expression of plexin-A4 was silenced in HUVEC (Figs. S2B &
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S2C). These results suggest that plexin-A4 is an essential shared component of the
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sema3A and sema3B receptor complexes and that in its absence sema3A and sema3B
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signaling are inhibited despite the presence of plexin-A1 and plexin-A2. These
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observations were also confirmed by observing the response of cultured HUVEC to
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gradients of semaphorin produced by the seeding of a low concentration of semaphorin
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producing HEK293 cells on top of monolayers of HUVEC. While cells expressing
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sema3A, sema3B or sema3E repulsed HUVEC infected with lentiviruses inducing
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expression of a control non-targeting shRNA, HUVEC in which the expression of plexin10
Accepted manuscript Journal of Cell Science
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A4 was silenced were only repulsed by sema3E while HUVEC in which plexin-A2 was
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silenced were repulsed by cells expressing sema3A but not by cells expressing sema3B
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(Fig. 2F). We hypothesize that plexin-A4 facilitates the association of plexin-A2 with
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neuropilins, and that silencing plexin-A4 inhibits responses to sema3B because plexin-
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A2 is no longer able to associate with neuropilins. Silencing plexin-A4 did not seem to
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inhibit the association of over-expressed plexin-A2 with np1 in co-immunoprecipitation
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experiments (Fig. S4C). However, when plexin-A2 is over-expressed it may be able to
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associate with neuropilins independently of plexin-A4, and we have not been able to
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perform a similar experiment in cells that express only endogenous receptors.
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In contrast to the very strong inhibitory effect that the silencing of plexin-A4 had on
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sema3B induced cell contraction, the silencing of plexin-A4 expression only produced a
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modest ~20% reduction in the percentage of cells that contracted in response to sema6A.
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This is a much weaker effect compared with the effect produced by plexin-A2 silencing,
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which results in almost complete inhibition of sema6A induced cell contraction (Figs. 2B
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& 2C). Taken together, these results suggest that the main signal transducing receptor of
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sema6A in these cells is plexin-A2 and that plexin-A4 functions as a less effective
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sema6A receptor in these cells.
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Sema3B induces the association of np1 with plexin-A2 but not the association of
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plexin-A2 with plexin-A4: The experiments described above suggested that the
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functional sema3B receptor in HUVEC and in U87MG cells consists of a complex
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containing np1 or np2, plexin-A2 and plexin-A4. In order to map in more detail the
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associations formed by the plexin-A2 receptor in response to sema3B and sema6A we
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expressed recombinant plexin-A2 tagged with a C-terminal Strep/FLAG epitope tag
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(Gloeckner et al., 2007) in U87MG cells. We then labeled the cells metabolically with
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arginine and lysine containing heavy or light nitrogen isotopes following established 11
Accepted manuscript Journal of Cell Science
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stable isotope labeling by amino acids in cell culture (SILAC) protocols (Walther and
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Mann, 2010; Pimienta et al., 2009). Cells that incorporated amino-acids labeled with the
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heavy isotopes were then stimulated with conditioned medium derived from heavy-
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isotope labeled HEK293 cells secreting recombinant sema3B or sema6A. Cells
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containing amino-acids labeled with the light isotopes served as controls. In order to
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exclude false-positive hits labels were switched in a second experiment. The cells were
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then solubilized using NP-40, and following centrifugation to remove insoluble debris,
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cell lysates from the control and stimulated cells were independently affinity purified on
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an anti-FLAG affinity resin. The eluants from columns loaded with conditioned medium
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from control and sema3B stimulated cells were mixed, and analyzed by mass
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spectroscopy. These experiments revealed that sema3B enhances strongly the association
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of np1 with plexin-A2 (Fig. 3A & 3B). As expected, sema6A did not promote the
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association of plexin-A2 with neuropilins (Fig. 3A). Surprisingly, we could not detect in
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the SILAC experiments a similar sema3B induced association of np2 with plexin-A2
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(Fig. 3A). This may be due to a weaker association between these receptors since when
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eluants recovered from the anti-FLAG affinity column were examined by western blot
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analysis, we found that np2 co-purified with plexin-A2 only in cell extracts purified from
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sema3B stimulated cells but not in control cells (Fig. 3C). These observations suggest
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that sema3B is nevertheless able to induce the association of np2 with plexin-A2
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although this association seems to be less stable than the sema3B induced association of
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plexin-A2 with np1.
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We have shown previously that plexin-A4 forms spontaneous complexes with plexin-A1
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(Kigel et al., 2011). We find that plexin-A4 is also able to form complexes with plexin-
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A2 as revealed by experiments in which plexin-A4 was co-immunoprecipitated with
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plexin-A2 from cell lysates prepared from PAE cells in which we expressed the two 12
Accepted manuscript Journal of Cell Science
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receptors (Fig. 3D). However, the formation of plexin-A2/plexin-A4 complexes was not
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enhanced by sema3B or by sema6A (Fig. 3A).
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Over-expression of plexin-A2 in cells silenced for plexin-A4 expression restores
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responsiveness to sema3B but not vice-versa: Silencing plexin-A2 or plexin-A4
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completely inhibits the sema3B induced collapse of the cytoskeleton of U87MG cells and
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HUVEC. A likely explanation is that each plexin provides a unique property essential for
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the function of the sema3B receptor complex that the other plexin cannot contribute.
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Alternatively, it is possible that the identity of the plexins that participate in the formation
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of the sema3B signaling complex is not important so long as the total concentration of the
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plexins exceeds a certain threshold required to transduce sema3B signals. To differentiate
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between these two possibilities we expressed in U87MG cells a plexin-A2 shRNA under
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the control of doxycycline. In these cells the expression of plexin-A2 is inhibited
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following stimulation with doxycycline (Fig. 4A). Following exposure to doxycycline
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these cells lost their contractile response to sema3B and sema6A (Figs. 4B & 4C). When
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recombinant plexin-A4 was ectopically over-expressed in the silenced cells (Fig. 4A) it
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restored the response to sema6A but not the response to sema3B (Figs. 4B & 4C). It
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should be noted that over expression of plexin A4 did not restore plexin A2 expression
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(Fig. 4A). This experiment therefore suggests that plexin-A2 and plexin-A4 are not
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interchangeable with respect to sema3B signaling but can be interchanged to restore
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sema6A signal transduction. We have also conducted the reciprocal experiment, in which
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we have ectopically over-expressed plexin-A2 under the control of doxycycline in cells
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that have been silenced for plexin-A4 expression (Fig. 5A). Surprisingly, ectopic over-
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expression of plexin-A2 in the plexin-A4 silenced cells fully restored responsiveness to
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sema3B (Figs. 5B & 5C). Thus, although at elevated expression levels plexin-A2 is able
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to compensate for missing plexin-A4 activity and thus restore sema3B signal transduction 13
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in the plexin-A4 silenced cells, the opposite is not true as plexin-A4 cannot replace
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plexin-A2 in plexin-A2 silenced cells to restore sema3B signaling.
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We also determined if over-expression of np1 can restore responsiveness to sema3B
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following the silencing of plexin-A2 expression. For that experiment we used cells in
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which ectopic over-expression of np1 is under the control of the tet-repressor (Fig. 6A &
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6B). When np1 was not over-expressed, silencing plexin-A2 inhibited, as expected,
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sema3B and sema6A induced signaling but not sema3A induced signal transduction.
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When neuropilin-1 was ectopically over-expressed by addition of doxycycline, the
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response to sema3B was restored but not the response to sema6A (Fig. 6C-6D),
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suggesting that up-regulation of np1 enables a higher percentage of the remaining plexin-
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A2 in the silenced cell to associate with np1 thus enabling signal transduction.
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Silencing plexin-A4 in the U87MG cells affected only minimally the response to sema6A
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(Fig. 2C). Doxycycline induced expression of plexin-A2 in cells silenced for plexin-A4
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expression resulted in enhanced contraction in response to sema6A (Figs. 5B & 5C)
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indicating that for sema6A signal transduction plexin-A2 and plexin-A4 are fully
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interchangeable.
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Over-expression of plexin-A2 in cells silenced for plexin-A1 or plexin-A4 expression
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restores responsiveness to sema3A: Silencing plexin-A1 or plexin-A4 in U87MG cells or
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in HUVEC inhibits sema3A induced cell contraction while silencing plexin-A2 has no
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effect (Fig. 2B). However, conditional ectopic over-expression of plexin-A2 in cells
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silenced for plexin-A4 or plexin-A1 expression completely restored their responsiveness
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to sema3A (Figs. 5D, 5E, 7A & 7B). Similar results were obtained when recombinant
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plexin-A2 was expressed in porcine aortic endothelial (PAE) cells expressing either np1
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or np2. In the absence of plexin-A2 PAE cells expressing np1 contract weakly in 14
Accepted manuscript Journal of Cell Science
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response to either sema3A or sema3B while PAE cells expressing np2 do not contract at
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all in response to these semaphorins (Fig. S2D). When recombinant plexin-A2 was
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expressed in these cells in addition to np1 the cells contracted strongly in response to
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both sema3A and sema3B while cells co-expressing np2 and plexin-A2 only contracted
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in response to sema3B (Fig. S2D). These results suggest again that when plexin-A2 is
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highly expressed it can transduce sema3A and sema3B signals independently of plexin-
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A4. These results are also in agreement with previous reports which showed that at high
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plexin-A2 expression levels sema3A can transduce signals via receptors containing
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complexes of np1 and plexin-A2 (Janssen et al., 2012). Indeed, we found that sema3A
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was able to induce the association of plexin-A2 with np1 in cells over-expressing plexin-
322
A2 while sema3F was not able to promote such an association (Fig. 5F). These
323
observations imply that the ability to distinguish between sema3A and sema3B can be
324
lost if the concentration of plexin-A2 in the cell membrane exceeds a certain threshold.
325
These observations also predict that over-expression of A2ExTm in the U87MG cells
326
should also inhibit sema3A activity. Indeed, A2ExTm over-expression in U87MG cells
327
also inhibited sema3A signaling in addition to inhibition of sema3B and sema6A
328
signaling (Figs. S3D & S3E). Finally, we have ectopically over-expressed plexin-A4 in
329
cells silenced for plexin-A1 expression. To our surprise we found that although plexin-
330
A4 was not able to restore sema3B signaling due to loss of plexin-A2 expression, it was
331
able to restore responsiveness to sema3A in cells silenced for plexin-A1 expression (Figs.
332
7C & 7D).
333
15
334
DISCUSSION
335
Cells employ cell surface receptors in order to sense changes in their environment. These
336
receptors include receptors for guidance factors such as semaphorins. When cells
337
transverse gradients of semaphorins in the course of their migrations, the activation of
338
semaphorin receptors triggers local changes in the actin cytoskeleton that are translated
339
into changes in the direction of their movement. The characterization of the semaphorin
Accepted manuscript
340
receptors and of the molecular mechanisms which ensure that cells interpret correctly
341
guidance instructions delivered by semaphorins is thus important for the understanding of
342
the mechanisms that regulate cell migrations in-vivo.
343
Research conducted in the last two decades revealed that functional receptors are
344
frequently complexes that contain a ligand binding receptor as well as additional
Journal of Cell Science
345
membrane anchored proteins that can affect the specificity of binding and can modulate
346
signal transduction. In endothelial cells and in U87MG gliobalstoma cells sema3A
347
induces the collapse of the actin cytoskeleton and consequent cell contraction. Our results
348
and the results of a previous study (Kigel et al., 2011) indicate that the sema3A induced
349
collapse of the actin cytoskeleton in these cells is completely inhibited if the expression
350
of either np1, plexin-A1 or plexin-A4, but not plexin-A3 or plexin-A2, are silenced.
351
These results suggest that the minimal functional sema3A receptor in these cells consists
352
of a complex containing these three receptors. While silencing plexin-A2 expression did
353
not affect sema3A signaling, it completely inhibited sema3B signaling in these cells,
354
indicating that the endogenous plexin-A2 levels of expression in these cells are
355
sufficiently high to enable signal transduction, and further suggesting that plexin-A2 is an
356
essential component of sema3B receptors but not of sema3A receptors.
16
Accepted manuscript Journal of Cell Science
357
Further examination of the composition of the functional sema3B receptor revealed that
358
unlike sema3A which signals exclusively using the np1 receptor, sema3B utilizes both
359
np1 and np2 for signal transduction. These observations suggested that signal
360
transduction by semaphorins that utilize np2 as their exclusive or primary binding
361
receptor may require the presence of plexin-A2. However, cell contraction induced by the
362
specific np2 ligand sema3F was not inhibited following plexin-A2 silencing and was
363
inhibited only following the silencing of plexin-A3. We conclude from these experiments
364
that when plexin-A2 is expressed at its physiological level of expression in endothelial
365
cells and in U87MG cells it functions as an essential component of sema3B receptors, but
366
not of functional sema3A or sema3F receptors. This was supported by observations that
367
revealed that upon stimulation with sema3B both np1 and np2 form stable complexes
368
with plexin-A2. Taken together, these experiments suggest that plexin-A1 and plexin-A2
369
and not the neuropilins, confer specificity to sema3A and sema3B within sema3A and
370
sema3B receptor complexes.
371
Interestingly, sema3B induced signaling was also completely inhibited by the silencing of
372
plexin-A4 expression, suggesting that plexin-A4 is an essential shared component of both
373
sema3A and sema3B receptors. In agreement with these results, we have observed that
374
plexin-A4 forms spontaneous complexes with plexin-A2 as well as with plexin-A1 (Kigel
375
et al., 2011) suggesting that hetero-complexes of plexin-A1 and plexin-A2 with plexin-
376
A4 pre-exist in HUVEC and U87MG cells. We postulate that stimulation with sema3B
377
recruits such pre-formed complexes to np1 or np2. It is possible that when plexin-A4 is in
378
complex with either plexin-A1 or plexin-A2 it is able to increase the affinity of these two
379
plexins to neuropilins, and that may be of crucial importance when plexin-A1 or plexin-
380
A2 are expressed at their physiological levels of expression.
17
Accepted manuscript Journal of Cell Science
381
These observations have profound implications for our understanding of the mechanisms
382
by which responses to guidance factors such as semaphorins are interpreted by migrating
383
cells, and highlight the advantages of multi-component receptors. They suggest that local
384
factors encountered in the microenvironments traversed by the migrating cells may
385
modulate the expression of essential constituents of semaphorin receptor complexes and
386
as a result change the way by which the migrating cells interpret signals of encountered
387
gradients of semaphorins. For example, a migrating cell that encounters two intersecting
388
gradients of sema3A and sema3B may also encounter local factors that affect the
389
expression of specific plexins. Such changes may render cells blind to a specific
390
semaphorin or to several semaphorins and as a result change the direction of migration
391
(Fig. 8A). In cases in which two populations of migrating cells that are destined to
392
migrate to different targets encounter several intersecting gradients of guidance factors
393
this mechanism may enable each cell population to interpret differently semaphorin
394
signals. Thus, multi-component receptor complexes provide plasticity and flexibility that
395
is not available in simple single component receptors.
396
Previous studies provided evidence suggesting that all the four class-A plexins including
397
plexin-A2 may be able to transduce sema3A signals (Takahashi and Strittmatter, 2001;
398
Bouvree et al., 2012; Janssen et al., 2012; Katayama et al., 2013; Yaron et al., 2005).
399
These results seemingly contradict our findings which suggest that functional receptor
400
complexes for sema3A and sema3B contain specific essential plexins. We hypothesized
401
that these apparent discrepancies may perhaps be the result of abnormally high plexin
402
expression levels since many of these studies were conducted using ectopically over-
403
expressed plexins. Indeed, when we over-expressed plexin-A2 in U87MG cells in which
404
plexin-A4 or plexin-A1 expression was silenced, the over-expressed plexin-A2 was able
405
to restore both sema3A and sema3B signaling, and the cells lost their ability to 18
Accepted manuscript Journal of Cell Science
406
differentiate between sema3A and sema3B. These observations suggested that at higher
407
levels of expression all the class-A plexins may be interchangeable. However, further
408
experiments revealed that this assumption may not be general since plexin-A4 was unable
409
to restore sema3B signaling in cells silenced for plexin-A2 expression even when it was
410
strongly over-expressed (Fig. 8B).
411
We hypothesize that the association of plexin-A4 with plexin-A2 increases the affinity of
412
plexin-A2 to neuropilins and thus enables the formation of the functional signaling
413
receptor complexes. This may also explain why over-expression of plexin-A2 can
414
substitute for plexin-A4 but not vice versa. When plexin-A2 is over-expressed it can form
415
complexes with neuropilins without plexin-A4. We hypothesize that the reason plexin-A4
416
cannot replace plexin-A2 even when highly expressed is because it does not play a real
417
functional role in sema3B signal transduction and its only role is to facilitate the
418
recruitment of plexin-A2 to neuropilins when the concentration of plexin-A2 is below a
419
threshold at which it cannot associate with neuropilins on its own.
420
It remains to be determined if similar combinations of plexins regulate in a similar
421
manner signal transduction induced by other class-3 semaphorins, and if signal
422
transduction by these semaphorins is gated similarly by changes in the expression levels
423
of specific plexins. Semaphorins have the potential to be used as therapeutic agents for
424
the treatment of diseases such as cancer (Tamagnone, 2012). Our observations suggest
425
that changes in the expression levels of plexins in the tumor microenvironment may
426
change the sensitivity of tumor cells and endothelial cells found in the tumor
427
microenvironment to specific semaphorins, indicating that successful future use of
428
semaphorins as therapeutics may require the monitoring of the expression levels of
429
plexins in the tumors.
430 19
Accepted manuscript Journal of Cell Science
431
MATERIALS AND METHODS
432
Antibodies and Reagents: The following antibodies were used: Goat anti mouse IgG
433
peroxidase conjugate: Sigma (A4416), Goat anti rabbit IgG peroxidase conjugate: Sigma
434
(A6154), Bovine anti goat IgG eproxidase conjugate: Santa-Cruz (sc-2350), Mouse anti-
435
actin clone AC-74: Sigma (A5316), Mouse anti-FLAG: Sigma (F3165), Mouse anti-flag
436
agarose beads: Sigma (A2220), Goat anti-human NP1: Santa Cruz (sc-7239), Mouse anti-
437
human NP2: Santa Cruz (sc-13117), Cy3 conjugated donkey anti-mouse antibody:
438
Jackson Immunoresearch laboratories (705-165-147), Cy2 conjugated anti-rabbit
439
antibody: Jackson Immunoresearch laboratories, Mouse anti-V5: Invitrogen (R960-25),
440
Rabbit anti-plexin-A1: Cell signaling (3813s), Rabbit anti Plexin-A2: Santa cruz (sc-
441
25640), Rabbit anti plexin-A2: abcam (ab39357), Rabbit anti plexin-A4: Sigma
442
(R30914), Rabbit anti sema6A: abcam (ab72369), Anti HA: Boeringer Manheim 1583-
443
816, Mouse anti c-myc: Santa Cruz (s.c. 789), Mouse anti-vinculin: Chemicon (3574).
444
Kits: The RNA reverse PCR kit- 5-Prime was from PerfectPure (Gaithersburg, MD), The
445
Verso cDNA kit was from Thermo scientific, Fugene-6 was purchased from Roche Ltd
446
(Switzerland), and the Block-it Inducible H1 Lentiviral RNAi System from Invitrogen.
447
Plasmids: The NSPI-CMV-MCS-myc-His lentiviral expression vector was given to us by
448
Dr. Gal Akiri, Mount Sinai School of Medicine, NY, The PcDNA3-Sema6A plasmid and
449
the Plexin-A2/myc cDNAs were provided by Dr. Oded Behar, Hebrew University,
450
Jerusalem. The ∆NRF (pCMV dR 8.74) and pMD2-VSV-G vectors for lentiviruse
451
production were obtained from Dr. Tal Kafri, University of North Carolina at Chapel
452
Hill. The pDonor221, pLenti6/V5-DEST, pLenti6.3/TO/V5-DEST, pENTR/H1/TO, and
453
the pLenti4/Block-iT-DEST plasmids were purchased from Invitrogen. The pENTR1A-
454
GFP-N2, pEF-1α/pENTRA, pLenti-CMV-GFP-DEST, pLenti-CMV-Puro-DEST and 20
Accepted manuscript Journal of Cell Science
455
pLenti-CMV-Neo-DEST plasmids were from Addgene. The pLKO-Tet-On plasmid was
456
kindly provided by Dr. Ayoub Nabieh, Faculty of Biology, Technion. The ShRNA
457
containing lentiviral vectors were purchased from Sigma Aldrich.
458
Cell lines: HUVEC were isolated and cultured as previously described (Gospodarowicz
459
et al., 1978). Porcine aortic endothelial (PAE) cells and HEK293 cells were cultured as
460
previously described (Gluzman-Poltorak et al., 2000; Kigel et al., 2011). U87MG cells
461
were purchased from the American Type Culture Collection (ATCC) and were
462
maintained in MEM-Eagle Earl's medium supplemented with 10% fetal bovine serum
463
(FBS), 2 mM Glutamine, 50µg/ml gentamicine, 2.5 µg/ml fungizone, 1mM Sodium
464
Pyruvate, and non-essential amino acids (Biological Industries, Beth Haemek, Israel). All
465
shRNA-expressing stable cell lines were grown in this medium which was supplemented
466
with 10% Tet-approved FBS (Biological Industries). Expression of the shRNA was
467
induced by addition of 1 µg/ml doxycycline (Sigma) dissolved in deionized water for 72
468
hours.
469
Expression and production of semaphorins: Class-3 semaphorin cDNAs were sub-
470
cloned into the NSPI-CMV-myc-his lentiviral expression vector as previously described
471
(Kigel et al., 2008; Varshavsky et al., 2008). An expression plasmid containing the
472
extracellular domain of the sema6A cDNA was kindly provided by Dr. Behar of the
473
Hebrew University. The production of lentiviruses and the generation of conditioned
474
media containing various semaphorins were done as previously described (Kigel et al.,
475
2008). For repulsion assays, HEK293 cells expressing various semaphorins were also
476
infected with a lentiviruses generated from the PGK-eGFP lentiviral vector (Chrenek et
477
al., 2011).
21
Accepted manuscript Journal of Cell Science
478
Quantitative real time PCR (qRT-PCR): QRT-PCR was performed using the StepOne
479
Plus Real Time PCR System with TaqMen Gene Expression Master Mix, according to
480
the instructions of the manufacturer (Applied biosystems). The normalizing gene was
481
RPLPO. Data was analyzed by the StepOne Software (Applied biosystems) using the
482
relative Quantitation-Comparative CT method. The following assays were used: NP1-
483
Hs00826128, NP2- Hs00187290, PlexinA1- Hs00413698, PlexinA2- Hs00300697,
484
PlexinA3- Hs00250178, PlexinA4- Hs00297356, PlexinD1- Hs00892410, RPLPO-
485
Hs99999902.
486
Quantitation of the number of mRNA molecules/cell by real time reverse PCR: The
487
number of mRNA molecules/cell of the plexins and neuropilins expressed in non-
488
stimulated HUVEC and U87MG cells was quantified using the TaqMen gene expression
489
assay. Plasmids containing the cDNA of each plexin or neuropilin were used to build a
490
standard curve of cycle threshold (Ct) vs. plasmid concentration. RNA from HUVEC and
491
U87MG cells was extracted and converted to cDNA. Real time PCR was performed in
492
triplicate as described above. The amount of each mRNA (ng/well) was calculated from
493
the standard curve and converted to moles/well taking into consideration the dilutions
494
made in the process to derive the number of mRNA molecules/cell for each plexin and
495
neuropilin in each HUVEC and U87MG cells.
496
Inhibition of gene expression with shRNA expressing lentiviruses: Lentiviral vectors
497
directing expression of various shRNAs were purchased from Sigma Aldrich
498
The following shRNA sequences were used: PlexinA1-CCGGGCACTTCTTCACGTCC
499
AAGATCTCGAGATCTTGGACGTGAAGAAGTGCTTTTTG, Plexin A2- First
500
shRNA: CCGGCGGCAATTTCATCATTGACAACTCGAGTTGTCAATGATGAAAT
501
TGCCGTTTTTG, Second shRNA: CCGGCGCCCAGATGAGTTTGGATTCTCGAGA 22
Accepted manuscript Journal of Cell Science
502
ATCCAAACTCATCTGGGCGTTTTTG, PlexinA3- CCGGGCTGTATTTCTATGTCA
503
CCAACTCGAGTTGGTGACATAGAAATACAGCTTTTTG, PlexinA4- CCGGGCAG
504
ATAAATGACCGCATTAACTCGAGTTAATGCGGTCATTTATCTGCTTTTTG.
505
The production of the lentiviruses, infection of cells and the selection of shRNA
506
expression cells in HEK293 cells were performed as previously described (Varshavsky et
507
al., 2008).
508
Inducible expression of shRNA species: To generate inducible shRNA-expressing
509
plasmids, double-stranded oligonucleotides encoding the desired shRNAs were generated
510
and cloned into the single-vector inducible shRNA expression vectors pLKO-Tet-On (gift
511
of Dr. Ayoub Nabieh) as described (Wiederschain et al., 2009).
512
Down-regulation of neuropilin expression using small interfering RNA (siRNA):
513
Inhibition of np1 and np2 expressions in U87MG cells using transfection of siRNAs was
514
performed as previously described (Varshavsky et al., 2008).
515
Expression of recombinant plexins: The full length cDNAs of plexin-A2 and plexin-A4
516
were cloned into the gateway pDonor221 plasmid and then transferred by recombination
517
into the pLenti6/V5-DEST or pLenti6.3/TO/V5-Dest lentiviral expression vector in frame
518
with a c-terminal V5 tag according to the instructions of the manufacturer (Invitrogen). A
519
deletion mutant of plexin-A2 containing the extracellular and transmembrane domains
520
(amino-acids 1-1323) of the protein fused to a c-terminal FLAG tag was constructed
521
according to the same procedures. Production of lentiviruses using these plasmids and
522
stable infection of target cells were performed essentially as previously described
523
(Brekhman and Neufeld, 2009). The sema6A expression vector was transfected into
524
target cells using FuGENE6 transfection reagent (Roche) as previously described
525
(Guttmann-Raviv et al., 2007). 23
Accepted manuscript Journal of Cell Science
526
Cell contraction and cell repulsion assays: Contraction of HUVEC and U87MG cells in
527
response to semaphorins as well as repulsion assays of endothelial cells were performed
528
as described (Varshavsky et al., 2008; Guttmann-Raviv et al., 2007).
529
Co-immunoprecipitation: Co-immunoprecipitation assays were performed as previously
530
described (Shraga-Heled et al., 2007).
531
Immunofluorescence: U87MG cells were plated on glass coverslips coated with PBS-
532
gelatin. Detection of vinculin and actin was done as previously described (Kigel et al.,
533
2011).
534
Stable isotope labeling by amino acids in cell culture (SILAC) and Affinity purification
535
of plexin-A2 tagged with a Strep/FLAG epitope tag: U87MG cells expressing plexin-A2
536
fused in-frame with a Strep/FLAG epitope tag (Gloeckner et al., 2007) as well as
537
HEK293 cells expressing recombinant sema3B (Varshavsky et al., 2008) or a secreted
538
soluble extracellular domain of sema6A (Kigel et al., 2011) were metabolically labeled
539
with lysine and arginine containing light (K0/R0) (Sigma), control cells) or heavy carbon,
540
nitrogen or hydrogen isotopes (K8/R10) (Cambridge Isotope Laboratories) for 10
541
passages in order to achieve complete labeling of cell proteins with these amino-acids
542
according to standard SILAC protocols (Pimienta et al., 2009). Preparation of the ligands:
543
cells labeled with amino-acids containing heavy isotopes were then stimulated with
544
conditioned medium containing saturating concentrations of recombinant sema3B or
545
sema6A for 5 min. at 37oC (five 20 cm diameter confluent plates for each treatment).
546
Control cells were incubated with conditioned medium from HEK293 cells labeled with
547
light isotopes. For reverse experiments, labels were switched. Both the control cells and
548
the cells stimulated with sema3B or sema6A were then washed briefly with ice cold TBS,
549
scraped, lysed quickly with lysis buffer (TBS supplemented with a protease inhibitor 24
Accepted manuscript Journal of Cell Science
550
cocktail (Roche) and 0.5% Np-40). Equal amounts of protein from each treatment were
551
loaded on an anti-FLAG affinity column (150ul bed volume). The column was washed
552
briefly with 1.5 ml of TBS, and bound proteins eluted using a buffer containing 100mM
553
Glycine (pH=2.5) and quick frozen in liquid nitrogen.
554
Proteomic analysis of proteins co purified with plexin-A2: Mass spectrometric analysis
555
was performed as described earlier (Gloeckner et al., 2010). Briefly, light isotopes and
556
heavy isotope labeled eluants from the anti-FLAG columns were mixed in a 1:1 ratio
557
based on protein content, proteolytically cleaved by trypsin and subsequently analyzed by
558
LC-MSMS using a nano-flow HPLC system (Dionex Ultimate 3000 RSLC, Thermo-
559
Fisher) coupled to an Orbitrap Velos (Thermo-Fisher) tandem mass spectrometer. To
560
exclude false-positive hits caused by labeling artifacts, reverse labeling was performed
561
were heavy/light labels were switched. In order to identify proteins that are induced to
562
bind to plexin-A2 following the stimulation, quantification of the resulting MS-data was
563
performed by the MaxQuant Software ver. 1.3.0.2 (Cox et al., 2009). Downstream
564
analysis and graphical representation was performed using in-house R-scripts. Only those
565
hits have been considered which were identified by at least 1 peptide in both, forward and
566
reverse experiments.
567
Statistical analysis: All experiments were repeated at least twice with similar results
568
unless otherwise stated. The one tailed unpaired with Welch's correction student's T-test
569
was used in most experiments. In cell proliferation experiments in which we normalized
570
the data across several experiments we used the paired student's T-test taking care that the
571
differences between the controls do not vary by more then 50%. Cell proliferation
572
experiments were performed in triplicates. Variations between replicates within single
573
experiments did not exceed 10%. Unless otherwise stated error bars represent the
574
standard error of the mean. Statistical significance is presented in the following manner: 25
575
*p
1
The Role of the Plexin-A2 Receptor in Semaphorin-3a and Semaphorin-3b Signal
2
Transduction
3
By: Adi D. [Sabag]1#, Tatyana [Smolkin]1#, Yelena [Mumblat]1, Marius [Ueffing]2,3, Ofra
4
[Kessler]1, Christian Johannes [Gloeckner]2,3, and Gera [Neufeld]1*
Journal of Cell Science
Accepted manuscript
5 6
From the 1Cancer Research and vascular Biology Center, The Bruce Rappaport Faculty
7
of Medicine, Technion, Israel Institute of Technology, Haifa, Israel, 2Eberhard Karls
8
University Tübingen, Institute for Ophthalmic Research, Medical Proteome Center,
9
Tübingen, Germany and 3Helmholtz Zentrum München – German Research Center for
10
Environmental Health, Research Unit Protein Science, Neuherberg, Germany.
11
12
*To whom correspondence should be addressed: E-mail: [email protected]
13
Tel: +972-4-8295430, Fax: +972-4-8523672
14
#
15
Keywords: cell guidance/neuropilins/plexins/receptor complexes/semaphorins
16
Running title: Plexin-A2 in semaphorin signaling
These authors contributed equally to the manuscript
17
1
JCS Advance Online Article. Posted on 21 October 2014
Accepted manuscript Journal of Cell Science
18
ABSTRACT
19
Class-3 semaphorins are anti-angiogenic and anti-tumorigenic guidance factors that bind
20
to neuropilins which in turn associate with class-A plexins to transduce semaphorin
21
signals. To study the role of the plexin-A2 receptor in semaphorin signaling, we silenced
22
its expression in endothelial cells and in glioblastoma cells. The silencing did not affect
23
sema3A signaling which depended on neuropilin-1, plexin-A1 and plexin-A4, but
24
abolished completely sema3B signaling which required in addition plexin-A4 and one of
25
the two neuropilins. Interestingly, over-expression of plexin-A2 in plexin-A1 or plexin-
26
A4 silenced cells restored responses to both semaphorins although it nullified their ability
27
to differentiate between them, suggesting that when over-expressed plexin-A2 is
28
functionally interchangeable with other class-A plexins. In-contrast, although plexin-A4
29
over-expression restored sema3A signaling in plexin-A1 silenced cells, it failed to restore
30
sema3B signaling in plexin-A2 silenced cells. It follows that the identity of plexins in
31
functional semaphorin receptors can be flexible depending on their expression level. Our
32
results suggest that changes in the expression of plexins induced by microenvironmental
33
cues can trigger differential responses of different populations of migrating cells to
34
encountered gradients of semaphorins.
35
2
36
INTRODUCTION
37
During embryonic development, cells migrate extensively to distant locations. Similar
38
migrations, although much less extensive, take pace in adult animals and in diseases such
39
as in the various forms of cancer (Trepat et al., 2012). Migrating cells are guided to their
40
correct destination by a variety of guidance cues which include gradients of secreted
41
repulsive and attractive guidance factors. Essential to the understanding of the
Accepted manuscript
42
mechanisms by which cells interpret guidance cues is the characterization of the receptors
43
that interact with these guidance cues and of mechanisms that modulate the activity and
44
expression of such receptors since the activity of these receptors determines which signal
45
transducing cascades will be activated downstream.
46
The semaphorins were identified as repulsive axon guidance factors (Kolodkin et al.,
Journal of Cell Science
47
1993) but have also emerged as important regulators of angiogenesis, immune responses
48
and tumor progression (Tamagnone, 2012; Kumanogoh and Kikutani, 2013; Sakurai et
49
al., 2011). Class-3 semaphorins are a subfamily of secreted semaphorins containing seven
50
members of which six, with the exception of sema3E (Gu et al., 2005), bind to one of the
51
two neuropilin receptors. Neuropilins contain short intracellular domains that limit their
52
signal transducing ability, and associate with members of the class-A plexin receptor
53
subfamily or with plexin-D1 to transduce class-3 semaphorin signals. These plexins also
54
function in addition as direct binding and signal transducing receptors for some
55
membrane anchored members of the class-4, class-5 and class-6 semaphorin subfamilies
56
(reviewed in (Neufeld et al., 2012; Gu and Giraudo, 2013)).
57
Sema3A is an anti-angiogenic semaphorin that binds to neuropilin-1 (np1) but not to
58
neuropilin-2 (np2) (Kolodkin et al., 1997; Chen et al., 1997; Acevedo et al., 2008).
59
Sema3B is a tumor suppressor of lung cancer (Tomizawa et al., 2001) that binds to both 3
Accepted manuscript Journal of Cell Science
60
neuropilins and functions in addition as an inhibitor of angiogenesis (Varshavsky et al.,
61
2008). The roles of specific class-A plexins in sema3B signal transduction have not been
62
well characterized. Plexin-A1 was found to be required, along with the adhesion receptor
63
NrCAM for the transduction of sema3B signals guiding commissural projections at the
64
vertebrate ventral midline during the development of the nervous system (Nawabi et al.,
65
2010), but the possible roles of additional plexins in sema3B signaling have not been
66
investigated. In contrast, sema3A signal transduction had been studied more extensively.
67
Studies conducted using knockout mice lacking either functional plexin-A1, plexin-A3 or
68
plexin-A4 receptors found evidence suggesting that these three receptors all play a role in
69
sema3A signal transduction (Yaron et al., 2005; Suto et al., 2005; Schwarz et al., 2008;
70
Moretti et al., 2007; Bouvree et al., 2012; Katayama et al., 2013). Interestingly, another
71
study suggested that plexin-A3 transduces exclusively pro-apoptotic signals of sema3A
72
but not signals affecting cytoskeletal organization (Ben-Zvi et al., 2008). Additional
73
studies in which plexin-A2 was ectopically expressed at higher expression levels in cells
74
suggested that plexin-A2 can also transduce sema3A signals (Janssen et al., 2012;
75
Takahashi and Strittmatter, 2001). Taken together these observations seem to suggest that
76
all four class-A plexins can transduce sema3A signals, which in turn seems to suggest
77
that for sema3A signaling the class-A plexins may be interchangeable. By extension
78
these studies suggest that the class-A plexins may not display high specificity for any of
79
the class-3 semaphorins. However, it should be noted that in most studies using gene
80
targeted mouse models it was unclear if the observed loss of responsiveness to sema3A
81
was due to a direct effect on sema3A signaling, or if the targeting affected the expression
82
of a secondary modulator. Likewise, much of the information obtained from cell culture
83
experiments was derived from experiments in which specific plexins were over-
4
Accepted manuscript Journal of Cell Science
84
expressed, and thus may not be relevant physiologically (Takahashi and Strittmatter,
85
2001; Janssen et al., 2012).
86
In all these studies it was assumed that successful sema3A signaling requires np1 and a
87
single plexin. However, we have recently observed that in endothelial cells and in
88
U87MG glioblastoma cells sema3A signaling affecting cytoskeletal organization requires
89
the simultaneous presence of plexin-A1 and plexin-A4 which form spontaneous hetero-
90
complexes. Inhibition of the expression of any one of these plexins but not of plexin-A3
91
was sufficient to completely inhibit the cytoskeletal collapse induced by sema3A
92
suggesting that physiologically relevant functional sema3A receptors consist of a
93
complex containing np1, plexin-A1 and plexin-A4 (Kigel et al., 2011). These
94
observations suggest that specific combinations of plexins and neuropilins determine the
95
specificity of receptor complexes to specific semaphorins and that the plexins are not
96
interchangeable.
97
. To gain a better understanding of the roles of the class-A plexins in class-3 semaphorin
98
signal transduction, and to clarify the possible role of plexin-A2 in sema3A signaling, we
99
studied in more detail the contribution of plexin-A2 as well as additional class-A plexins
100
to sema3A and sema3B signaling. We provide evidence suggesting that in endothelial
101
cells and glioblastoma cells plexin-A4 is a required component of both sema3A and
102
sema3B receptor complexes and inhibition of its expression nullifies both sema3A and
103
sema3B signaling. The specificity for sema3A or sema3B is determined by the presence
104
of plexin-A1 in sema3A receptors and plexin-A2 in sema3B receptors and silencing each
105
abrogates signaling by the appropriate semaphorin. Interestingly, when plexin-A2 was
106
over-expressed in cells silenced for plexin-A1 or plexin-A4 expression it restored both
107
sema3A and sema3B signaling, suggesting that the requirement for specific plexins may
108
be abolished if the expression levels of some of the plexins exceed certain threshold 5
109
levels and further suggesting that when highly expressed the different plexins are
110
functionally interchangeable. This comes at a price as such cells lost their ability to
111
distinguish between sema3A and sema3B. However, this may not always be true since
112
plexin-A4 over-expression failed to restore sema3B signaling in plexin-A2 silenced cells
Journal of Cell Science
Accepted manuscript
113
6
Accepted manuscript Journal of Cell Science
114
RESULTS
115
Plexin-A2 is not required for sema3A signal transduction but is required for the
116
transduction of sema3B and sema6A signals in U87MG glioblastoma cells and in
117
endothelial cells: U87MG glioblastoma multiforme cells and human umbilical vein
118
derived endothelial cells (HUVEC) respond to stimulation by semaphorins such as
119
sema3A with localized disassembly of their actin cytoskeleton that is associated with loss
120
of focal contacts followed by cell contraction, and with the appearance of "spikes" that
121
remain attached to the substrate around the contracted cell (Fig. 1C & 1E, arrows). In
122
both cell types the number of mRNA molecules/cell encoding np2, type-A and type-D
123
plexins is pretty similar with some variations while the number/cell of mRNA molecules
124
encoding np1 is 2-5 fold higher (Fig. S1A). We have previously observed that np1,
125
plexin-A1 and plexin-A4, but not plexin-A3, are required for the transduction of sema3A
126
induced signals resulting in cytoskeletal collapse in HUVEC and in U87MG cells.
127
Inhibition of the expression of any of these genes using specific shRNA species that do
128
not cross-inhibit the expression of the other plexins, completely inhibited signal
129
transduction by sema3A (Kigel et al., 2011). These observations suggested that the
130
sema3A signaling complex contains these three receptors, an assumption that was
131
supported by co-immunoprecipitation experiments. Because plexin-A2 was also reported
132
to transduce sema3A signals and to participate in the formation of functional sema3A
133
receptors (Takahashi and Strittmatter, 2001; Janssen et al., 2012), we sought to determine
134
if plexin-A2 also participates in the transduction of sema3A signals in these cells. To
135
examine this possibility we inhibited the expression of plexin-A2 in HUVEC and in
136
U87MG cells using two specific shRNA species which do not inhibit the expression of
137
the other class-A plexins or the expression of neuropilins (Fig. 1A and Fig. S1B-D).
138
However, even though the shRNA silenced the expression of plexin-A2 effectively in 7
Accepted manuscript Journal of Cell Science
139
HUVEC and in U87MG cells, the silencing did not inhibit sema3A induced cell
140
contraction (Figs. 1C, 1D, S1E) as does plexin-A1 or plexin-A4 silencing (Kigel et al.,
141
2011).
142
To gain a better understanding of the role of plexin-A2 in the transduction of class-3
143
semaphorin signals, we determined if plexin-A2 is required for the transduction of the
144
signals of other class-3 semaphorins. Silencing plexin-A2 expression had no effect on
145
sema3F induced contraction of U87MG cells (Figs. 1C & 1D) while in agreement with
146
prior reports, silencing plexin-A3 inhibited sema3F signaling completely (Fig. 2B)
147
(Yaron et al., 2005; Schwarz et al., 2008). However, the collapse of the actin
148
cytoskeleton, the disassembly of vinculin containing focal contacts, and the subsequent
149
cell contraction induced by sema3B were strongly inhibited in the plexin-A2 silenced
150
cells (Figs. 1C-1E). The silencing also inhibited completely sema6A induced contraction
151
of these cells (Figs. 1C & 1D), a surprising result considering that these cells also express
152
plexin-A4, a plexin that is known to function as a signal transducing sema6A receptor
153
(Suto et al., 2007; Okada et al., 2007; Kigel et al., 2011), indicating that the concentration
154
of plexin-A4 in these cells, which seems rather low (Fig. S1A), is probably not sufficient
155
to enable sema6A signaling on its own. The contractile response of the U87MG cells to
156
sema3B and sema6A was rescued following re-expression of recombinant plexin-A2 in
157
the silenced cells. These observations attest to the specificity of the silencing and indicate
158
that plexin-A2 is essential the transduction of sema3B and sema6A signals in these cells
159
(Figs. 1B-D). Likewise, silencing the expression of plexin-A2 in HUVEC inhibited
160
completely sema3B and sema6A induced cytoskeletal collapse (Figs. S2A-S2C).
161
In order to verify these results by a different method we also expressed in the U87MG
162
cells a truncated form of the plexin-A2 receptor lacking the intracellular domain of
163
plexin-A2 which was tagged at its C-terminal with a FLAG tag (A2ExTm) (Fig. S3A). It 8
Accepted manuscript Journal of Cell Science
164
was previously observed that a similarly truncated form of plexin-A1 functions as a
165
dominant negative inhibitor of sema3A (Serini et al., 2003). The truncated plexin-A2
166
formed complexes with full length plexin-A2 (Fig. S3B) as well as with plexin-A4 (Fig.
167
S3C). Over-expression of A2ExTm in U87MG cells inhibited sema3B and sema6A
168
induced contraction of the U87MG cells independently suggesting that plexin-A2 is
169
indeed required for sema3B and sema6A signal transduction in these cells (Figs. S3D &
170
S3E).
171
Sema3A transduces signals exclusively using np1 while sema3B utilizes both
172
neuropilins for signal transduction: HUVEC and U87MG cells express the two
173
neuropilins as well as the four class-A plexins as well as plexins belonging to additional
174
plexin subclasses (Shraga-Heled et al., 2007; Kigel et al., 2011; Basile et al., 2005;
175
Artigiani et al., 2004; Gu et al., 2005). The primary binding receptors of class-3
176
semaphorins are the neuropilins which cannot transduce signals on their own and
177
associate with class-A plexins or plexin-D1 to transduce semaphorin signals. We have
178
previously observed that sema3B can utilize both neuropilins for signal transduction.
179
However, in HUVEC sema3B transduces signals using primarily np1 and np2 only plays
180
a secondary role (Varshavsky et al., 2008). In contrast, in U87MG cells only inhibition of
181
the expression of both neuropilins completely abrogated efficiently sema3B signal
182
transduction while inhibition of either np1 or np2 expression alone was not sufficient to
183
inhibit sema3B signaling (Figs. S4A & S4B). In contrast, silencing np1 expression alone
184
completely inhibited sema3A induced cell contraction while silencing np2 alone was
185
sufficient to abrogate sema3F induced cell contraction (Figs. S4A & S4B). Thus, plexin-
186
A2 seems to associate with both neuropilins to transduce sema3B signals in U87MG
187
cells.
9
Accepted manuscript Journal of Cell Science
188
Plexin-A2 and plexin-A4 are required simultaneously for the transduction of sema3B
189
signals in HUVEC and in U87MG cells: Our previous work on sema3A demonstrated
190
that more than one plexin is required, in addition to np1, for the transduction of sema3A
191
signals (Kigel et al., 2011). To find out if any additional plexins are required besides
192
plexin-A2 for sema3B induced cell contraction, we silenced the expression of additional
193
class-A plexins in HUVEC and in U87MG cells (Fig. 2A) using shRNA species that do
194
not affect the expression of neuropilins or the expression of other class-A plexins (Kigel
195
et al., 2011). Silencing plexin-A1 or plexin-A3 did not inhibit sema3B or sema6A
196
induced contraction of U87MG cells, suggesting that they do not play an essential role in
197
sema3B or sema6A induced signal transduction (Figs. 2B & 2C). This lack of an effect
198
was not due to insufficient silencing because silencing plexin-A1 expression inhibited
199
completely the response to sema3A and silencing plexin-A3 inhibited completely cell
200
contraction induced by sema3F (Figs. 2B-2E). As before, silencing plexin-A2 aborted
201
completely responses to sema3B and sema6A but had no effect on sema3A induced cell
202
contraction. However, cells in which we silenced the expression of plexin-A4 also failed
203
to contract in response to sema3B (Figs. 2B & 2C) (Kigel et al., 2011). Identical results
204
were observed when the expression of plexin-A4 was silenced in HUVEC (Figs. S2B &
205
S2C). These results suggest that plexin-A4 is an essential shared component of the
206
sema3A and sema3B receptor complexes and that in its absence sema3A and sema3B
207
signaling are inhibited despite the presence of plexin-A1 and plexin-A2. These
208
observations were also confirmed by observing the response of cultured HUVEC to
209
gradients of semaphorin produced by the seeding of a low concentration of semaphorin
210
producing HEK293 cells on top of monolayers of HUVEC. While cells expressing
211
sema3A, sema3B or sema3E repulsed HUVEC infected with lentiviruses inducing
212
expression of a control non-targeting shRNA, HUVEC in which the expression of plexin10
Accepted manuscript Journal of Cell Science
213
A4 was silenced were only repulsed by sema3E while HUVEC in which plexin-A2 was
214
silenced were repulsed by cells expressing sema3A but not by cells expressing sema3B
215
(Fig. 2F). We hypothesize that plexin-A4 facilitates the association of plexin-A2 with
216
neuropilins, and that silencing plexin-A4 inhibits responses to sema3B because plexin-
217
A2 is no longer able to associate with neuropilins. Silencing plexin-A4 did not seem to
218
inhibit the association of over-expressed plexin-A2 with np1 in co-immunoprecipitation
219
experiments (Fig. S4C). However, when plexin-A2 is over-expressed it may be able to
220
associate with neuropilins independently of plexin-A4, and we have not been able to
221
perform a similar experiment in cells that express only endogenous receptors.
222
In contrast to the very strong inhibitory effect that the silencing of plexin-A4 had on
223
sema3B induced cell contraction, the silencing of plexin-A4 expression only produced a
224
modest ~20% reduction in the percentage of cells that contracted in response to sema6A.
225
This is a much weaker effect compared with the effect produced by plexin-A2 silencing,
226
which results in almost complete inhibition of sema6A induced cell contraction (Figs. 2B
227
& 2C). Taken together, these results suggest that the main signal transducing receptor of
228
sema6A in these cells is plexin-A2 and that plexin-A4 functions as a less effective
229
sema6A receptor in these cells.
230
Sema3B induces the association of np1 with plexin-A2 but not the association of
231
plexin-A2 with plexin-A4: The experiments described above suggested that the
232
functional sema3B receptor in HUVEC and in U87MG cells consists of a complex
233
containing np1 or np2, plexin-A2 and plexin-A4. In order to map in more detail the
234
associations formed by the plexin-A2 receptor in response to sema3B and sema6A we
235
expressed recombinant plexin-A2 tagged with a C-terminal Strep/FLAG epitope tag
236
(Gloeckner et al., 2007) in U87MG cells. We then labeled the cells metabolically with
237
arginine and lysine containing heavy or light nitrogen isotopes following established 11
Accepted manuscript Journal of Cell Science
238
stable isotope labeling by amino acids in cell culture (SILAC) protocols (Walther and
239
Mann, 2010; Pimienta et al., 2009). Cells that incorporated amino-acids labeled with the
240
heavy isotopes were then stimulated with conditioned medium derived from heavy-
241
isotope labeled HEK293 cells secreting recombinant sema3B or sema6A. Cells
242
containing amino-acids labeled with the light isotopes served as controls. In order to
243
exclude false-positive hits labels were switched in a second experiment. The cells were
244
then solubilized using NP-40, and following centrifugation to remove insoluble debris,
245
cell lysates from the control and stimulated cells were independently affinity purified on
246
an anti-FLAG affinity resin. The eluants from columns loaded with conditioned medium
247
from control and sema3B stimulated cells were mixed, and analyzed by mass
248
spectroscopy. These experiments revealed that sema3B enhances strongly the association
249
of np1 with plexin-A2 (Fig. 3A & 3B). As expected, sema6A did not promote the
250
association of plexin-A2 with neuropilins (Fig. 3A). Surprisingly, we could not detect in
251
the SILAC experiments a similar sema3B induced association of np2 with plexin-A2
252
(Fig. 3A). This may be due to a weaker association between these receptors since when
253
eluants recovered from the anti-FLAG affinity column were examined by western blot
254
analysis, we found that np2 co-purified with plexin-A2 only in cell extracts purified from
255
sema3B stimulated cells but not in control cells (Fig. 3C). These observations suggest
256
that sema3B is nevertheless able to induce the association of np2 with plexin-A2
257
although this association seems to be less stable than the sema3B induced association of
258
plexin-A2 with np1.
259
We have shown previously that plexin-A4 forms spontaneous complexes with plexin-A1
260
(Kigel et al., 2011). We find that plexin-A4 is also able to form complexes with plexin-
261
A2 as revealed by experiments in which plexin-A4 was co-immunoprecipitated with
262
plexin-A2 from cell lysates prepared from PAE cells in which we expressed the two 12
Accepted manuscript Journal of Cell Science
263
receptors (Fig. 3D). However, the formation of plexin-A2/plexin-A4 complexes was not
264
enhanced by sema3B or by sema6A (Fig. 3A).
265
Over-expression of plexin-A2 in cells silenced for plexin-A4 expression restores
266
responsiveness to sema3B but not vice-versa: Silencing plexin-A2 or plexin-A4
267
completely inhibits the sema3B induced collapse of the cytoskeleton of U87MG cells and
268
HUVEC. A likely explanation is that each plexin provides a unique property essential for
269
the function of the sema3B receptor complex that the other plexin cannot contribute.
270
Alternatively, it is possible that the identity of the plexins that participate in the formation
271
of the sema3B signaling complex is not important so long as the total concentration of the
272
plexins exceeds a certain threshold required to transduce sema3B signals. To differentiate
273
between these two possibilities we expressed in U87MG cells a plexin-A2 shRNA under
274
the control of doxycycline. In these cells the expression of plexin-A2 is inhibited
275
following stimulation with doxycycline (Fig. 4A). Following exposure to doxycycline
276
these cells lost their contractile response to sema3B and sema6A (Figs. 4B & 4C). When
277
recombinant plexin-A4 was ectopically over-expressed in the silenced cells (Fig. 4A) it
278
restored the response to sema6A but not the response to sema3B (Figs. 4B & 4C). It
279
should be noted that over expression of plexin A4 did not restore plexin A2 expression
280
(Fig. 4A). This experiment therefore suggests that plexin-A2 and plexin-A4 are not
281
interchangeable with respect to sema3B signaling but can be interchanged to restore
282
sema6A signal transduction. We have also conducted the reciprocal experiment, in which
283
we have ectopically over-expressed plexin-A2 under the control of doxycycline in cells
284
that have been silenced for plexin-A4 expression (Fig. 5A). Surprisingly, ectopic over-
285
expression of plexin-A2 in the plexin-A4 silenced cells fully restored responsiveness to
286
sema3B (Figs. 5B & 5C). Thus, although at elevated expression levels plexin-A2 is able
287
to compensate for missing plexin-A4 activity and thus restore sema3B signal transduction 13
288
in the plexin-A4 silenced cells, the opposite is not true as plexin-A4 cannot replace
289
plexin-A2 in plexin-A2 silenced cells to restore sema3B signaling.
290
We also determined if over-expression of np1 can restore responsiveness to sema3B
291
following the silencing of plexin-A2 expression. For that experiment we used cells in
292
which ectopic over-expression of np1 is under the control of the tet-repressor (Fig. 6A &
293
6B). When np1 was not over-expressed, silencing plexin-A2 inhibited, as expected,
294
sema3B and sema6A induced signaling but not sema3A induced signal transduction.
Accepted manuscript
295
When neuropilin-1 was ectopically over-expressed by addition of doxycycline, the
296
response to sema3B was restored but not the response to sema6A (Fig. 6C-6D),
297
suggesting that up-regulation of np1 enables a higher percentage of the remaining plexin-
298
A2 in the silenced cell to associate with np1 thus enabling signal transduction.
299
Silencing plexin-A4 in the U87MG cells affected only minimally the response to sema6A
Journal of Cell Science
300
(Fig. 2C). Doxycycline induced expression of plexin-A2 in cells silenced for plexin-A4
301
expression resulted in enhanced contraction in response to sema6A (Figs. 5B & 5C)
302
indicating that for sema6A signal transduction plexin-A2 and plexin-A4 are fully
303
interchangeable.
304
Over-expression of plexin-A2 in cells silenced for plexin-A1 or plexin-A4 expression
305
restores responsiveness to sema3A: Silencing plexin-A1 or plexin-A4 in U87MG cells or
306
in HUVEC inhibits sema3A induced cell contraction while silencing plexin-A2 has no
307
effect (Fig. 2B). However, conditional ectopic over-expression of plexin-A2 in cells
308
silenced for plexin-A4 or plexin-A1 expression completely restored their responsiveness
309
to sema3A (Figs. 5D, 5E, 7A & 7B). Similar results were obtained when recombinant
310
plexin-A2 was expressed in porcine aortic endothelial (PAE) cells expressing either np1
311
or np2. In the absence of plexin-A2 PAE cells expressing np1 contract weakly in 14
Accepted manuscript Journal of Cell Science
312
response to either sema3A or sema3B while PAE cells expressing np2 do not contract at
313
all in response to these semaphorins (Fig. S2D). When recombinant plexin-A2 was
314
expressed in these cells in addition to np1 the cells contracted strongly in response to
315
both sema3A and sema3B while cells co-expressing np2 and plexin-A2 only contracted
316
in response to sema3B (Fig. S2D). These results suggest again that when plexin-A2 is
317
highly expressed it can transduce sema3A and sema3B signals independently of plexin-
318
A4. These results are also in agreement with previous reports which showed that at high
319
plexin-A2 expression levels sema3A can transduce signals via receptors containing
320
complexes of np1 and plexin-A2 (Janssen et al., 2012). Indeed, we found that sema3A
321
was able to induce the association of plexin-A2 with np1 in cells over-expressing plexin-
322
A2 while sema3F was not able to promote such an association (Fig. 5F). These
323
observations imply that the ability to distinguish between sema3A and sema3B can be
324
lost if the concentration of plexin-A2 in the cell membrane exceeds a certain threshold.
325
These observations also predict that over-expression of A2ExTm in the U87MG cells
326
should also inhibit sema3A activity. Indeed, A2ExTm over-expression in U87MG cells
327
also inhibited sema3A signaling in addition to inhibition of sema3B and sema6A
328
signaling (Figs. S3D & S3E). Finally, we have ectopically over-expressed plexin-A4 in
329
cells silenced for plexin-A1 expression. To our surprise we found that although plexin-
330
A4 was not able to restore sema3B signaling due to loss of plexin-A2 expression, it was
331
able to restore responsiveness to sema3A in cells silenced for plexin-A1 expression (Figs.
332
7C & 7D).
333
15
334
DISCUSSION
335
Cells employ cell surface receptors in order to sense changes in their environment. These
336
receptors include receptors for guidance factors such as semaphorins. When cells
337
transverse gradients of semaphorins in the course of their migrations, the activation of
338
semaphorin receptors triggers local changes in the actin cytoskeleton that are translated
339
into changes in the direction of their movement. The characterization of the semaphorin
Accepted manuscript
340
receptors and of the molecular mechanisms which ensure that cells interpret correctly
341
guidance instructions delivered by semaphorins is thus important for the understanding of
342
the mechanisms that regulate cell migrations in-vivo.
343
Research conducted in the last two decades revealed that functional receptors are
344
frequently complexes that contain a ligand binding receptor as well as additional
Journal of Cell Science
345
membrane anchored proteins that can affect the specificity of binding and can modulate
346
signal transduction. In endothelial cells and in U87MG gliobalstoma cells sema3A
347
induces the collapse of the actin cytoskeleton and consequent cell contraction. Our results
348
and the results of a previous study (Kigel et al., 2011) indicate that the sema3A induced
349
collapse of the actin cytoskeleton in these cells is completely inhibited if the expression
350
of either np1, plexin-A1 or plexin-A4, but not plexin-A3 or plexin-A2, are silenced.
351
These results suggest that the minimal functional sema3A receptor in these cells consists
352
of a complex containing these three receptors. While silencing plexin-A2 expression did
353
not affect sema3A signaling, it completely inhibited sema3B signaling in these cells,
354
indicating that the endogenous plexin-A2 levels of expression in these cells are
355
sufficiently high to enable signal transduction, and further suggesting that plexin-A2 is an
356
essential component of sema3B receptors but not of sema3A receptors.
16
Accepted manuscript Journal of Cell Science
357
Further examination of the composition of the functional sema3B receptor revealed that
358
unlike sema3A which signals exclusively using the np1 receptor, sema3B utilizes both
359
np1 and np2 for signal transduction. These observations suggested that signal
360
transduction by semaphorins that utilize np2 as their exclusive or primary binding
361
receptor may require the presence of plexin-A2. However, cell contraction induced by the
362
specific np2 ligand sema3F was not inhibited following plexin-A2 silencing and was
363
inhibited only following the silencing of plexin-A3. We conclude from these experiments
364
that when plexin-A2 is expressed at its physiological level of expression in endothelial
365
cells and in U87MG cells it functions as an essential component of sema3B receptors, but
366
not of functional sema3A or sema3F receptors. This was supported by observations that
367
revealed that upon stimulation with sema3B both np1 and np2 form stable complexes
368
with plexin-A2. Taken together, these experiments suggest that plexin-A1 and plexin-A2
369
and not the neuropilins, confer specificity to sema3A and sema3B within sema3A and
370
sema3B receptor complexes.
371
Interestingly, sema3B induced signaling was also completely inhibited by the silencing of
372
plexin-A4 expression, suggesting that plexin-A4 is an essential shared component of both
373
sema3A and sema3B receptors. In agreement with these results, we have observed that
374
plexin-A4 forms spontaneous complexes with plexin-A2 as well as with plexin-A1 (Kigel
375
et al., 2011) suggesting that hetero-complexes of plexin-A1 and plexin-A2 with plexin-
376
A4 pre-exist in HUVEC and U87MG cells. We postulate that stimulation with sema3B
377
recruits such pre-formed complexes to np1 or np2. It is possible that when plexin-A4 is in
378
complex with either plexin-A1 or plexin-A2 it is able to increase the affinity of these two
379
plexins to neuropilins, and that may be of crucial importance when plexin-A1 or plexin-
380
A2 are expressed at their physiological levels of expression.
17
Accepted manuscript Journal of Cell Science
381
These observations have profound implications for our understanding of the mechanisms
382
by which responses to guidance factors such as semaphorins are interpreted by migrating
383
cells, and highlight the advantages of multi-component receptors. They suggest that local
384
factors encountered in the microenvironments traversed by the migrating cells may
385
modulate the expression of essential constituents of semaphorin receptor complexes and
386
as a result change the way by which the migrating cells interpret signals of encountered
387
gradients of semaphorins. For example, a migrating cell that encounters two intersecting
388
gradients of sema3A and sema3B may also encounter local factors that affect the
389
expression of specific plexins. Such changes may render cells blind to a specific
390
semaphorin or to several semaphorins and as a result change the direction of migration
391
(Fig. 8A). In cases in which two populations of migrating cells that are destined to
392
migrate to different targets encounter several intersecting gradients of guidance factors
393
this mechanism may enable each cell population to interpret differently semaphorin
394
signals. Thus, multi-component receptor complexes provide plasticity and flexibility that
395
is not available in simple single component receptors.
396
Previous studies provided evidence suggesting that all the four class-A plexins including
397
plexin-A2 may be able to transduce sema3A signals (Takahashi and Strittmatter, 2001;
398
Bouvree et al., 2012; Janssen et al., 2012; Katayama et al., 2013; Yaron et al., 2005).
399
These results seemingly contradict our findings which suggest that functional receptor
400
complexes for sema3A and sema3B contain specific essential plexins. We hypothesized
401
that these apparent discrepancies may perhaps be the result of abnormally high plexin
402
expression levels since many of these studies were conducted using ectopically over-
403
expressed plexins. Indeed, when we over-expressed plexin-A2 in U87MG cells in which
404
plexin-A4 or plexin-A1 expression was silenced, the over-expressed plexin-A2 was able
405
to restore both sema3A and sema3B signaling, and the cells lost their ability to 18
Accepted manuscript Journal of Cell Science
406
differentiate between sema3A and sema3B. These observations suggested that at higher
407
levels of expression all the class-A plexins may be interchangeable. However, further
408
experiments revealed that this assumption may not be general since plexin-A4 was unable
409
to restore sema3B signaling in cells silenced for plexin-A2 expression even when it was
410
strongly over-expressed (Fig. 8B).
411
We hypothesize that the association of plexin-A4 with plexin-A2 increases the affinity of
412
plexin-A2 to neuropilins and thus enables the formation of the functional signaling
413
receptor complexes. This may also explain why over-expression of plexin-A2 can
414
substitute for plexin-A4 but not vice versa. When plexin-A2 is over-expressed it can form
415
complexes with neuropilins without plexin-A4. We hypothesize that the reason plexin-A4
416
cannot replace plexin-A2 even when highly expressed is because it does not play a real
417
functional role in sema3B signal transduction and its only role is to facilitate the
418
recruitment of plexin-A2 to neuropilins when the concentration of plexin-A2 is below a
419
threshold at which it cannot associate with neuropilins on its own.
420
It remains to be determined if similar combinations of plexins regulate in a similar
421
manner signal transduction induced by other class-3 semaphorins, and if signal
422
transduction by these semaphorins is gated similarly by changes in the expression levels
423
of specific plexins. Semaphorins have the potential to be used as therapeutic agents for
424
the treatment of diseases such as cancer (Tamagnone, 2012). Our observations suggest
425
that changes in the expression levels of plexins in the tumor microenvironment may
426
change the sensitivity of tumor cells and endothelial cells found in the tumor
427
microenvironment to specific semaphorins, indicating that successful future use of
428
semaphorins as therapeutics may require the monitoring of the expression levels of
429
plexins in the tumors.
430 19
Accepted manuscript Journal of Cell Science
431
MATERIALS AND METHODS
432
Antibodies and Reagents: The following antibodies were used: Goat anti mouse IgG
433
peroxidase conjugate: Sigma (A4416), Goat anti rabbit IgG peroxidase conjugate: Sigma
434
(A6154), Bovine anti goat IgG eproxidase conjugate: Santa-Cruz (sc-2350), Mouse anti-
435
actin clone AC-74: Sigma (A5316), Mouse anti-FLAG: Sigma (F3165), Mouse anti-flag
436
agarose beads: Sigma (A2220), Goat anti-human NP1: Santa Cruz (sc-7239), Mouse anti-
437
human NP2: Santa Cruz (sc-13117), Cy3 conjugated donkey anti-mouse antibody:
438
Jackson Immunoresearch laboratories (705-165-147), Cy2 conjugated anti-rabbit
439
antibody: Jackson Immunoresearch laboratories, Mouse anti-V5: Invitrogen (R960-25),
440
Rabbit anti-plexin-A1: Cell signaling (3813s), Rabbit anti Plexin-A2: Santa cruz (sc-
441
25640), Rabbit anti plexin-A2: abcam (ab39357), Rabbit anti plexin-A4: Sigma
442
(R30914), Rabbit anti sema6A: abcam (ab72369), Anti HA: Boeringer Manheim 1583-
443
816, Mouse anti c-myc: Santa Cruz (s.c. 789), Mouse anti-vinculin: Chemicon (3574).
444
Kits: The RNA reverse PCR kit- 5-Prime was from PerfectPure (Gaithersburg, MD), The
445
Verso cDNA kit was from Thermo scientific, Fugene-6 was purchased from Roche Ltd
446
(Switzerland), and the Block-it Inducible H1 Lentiviral RNAi System from Invitrogen.
447
Plasmids: The NSPI-CMV-MCS-myc-His lentiviral expression vector was given to us by
448
Dr. Gal Akiri, Mount Sinai School of Medicine, NY, The PcDNA3-Sema6A plasmid and
449
the Plexin-A2/myc cDNAs were provided by Dr. Oded Behar, Hebrew University,
450
Jerusalem. The ∆NRF (pCMV dR 8.74) and pMD2-VSV-G vectors for lentiviruse
451
production were obtained from Dr. Tal Kafri, University of North Carolina at Chapel
452
Hill. The pDonor221, pLenti6/V5-DEST, pLenti6.3/TO/V5-DEST, pENTR/H1/TO, and
453
the pLenti4/Block-iT-DEST plasmids were purchased from Invitrogen. The pENTR1A-
454
GFP-N2, pEF-1α/pENTRA, pLenti-CMV-GFP-DEST, pLenti-CMV-Puro-DEST and 20
Accepted manuscript Journal of Cell Science
455
pLenti-CMV-Neo-DEST plasmids were from Addgene. The pLKO-Tet-On plasmid was
456
kindly provided by Dr. Ayoub Nabieh, Faculty of Biology, Technion. The ShRNA
457
containing lentiviral vectors were purchased from Sigma Aldrich.
458
Cell lines: HUVEC were isolated and cultured as previously described (Gospodarowicz
459
et al., 1978). Porcine aortic endothelial (PAE) cells and HEK293 cells were cultured as
460
previously described (Gluzman-Poltorak et al., 2000; Kigel et al., 2011). U87MG cells
461
were purchased from the American Type Culture Collection (ATCC) and were
462
maintained in MEM-Eagle Earl's medium supplemented with 10% fetal bovine serum
463
(FBS), 2 mM Glutamine, 50µg/ml gentamicine, 2.5 µg/ml fungizone, 1mM Sodium
464
Pyruvate, and non-essential amino acids (Biological Industries, Beth Haemek, Israel). All
465
shRNA-expressing stable cell lines were grown in this medium which was supplemented
466
with 10% Tet-approved FBS (Biological Industries). Expression of the shRNA was
467
induced by addition of 1 µg/ml doxycycline (Sigma) dissolved in deionized water for 72
468
hours.
469
Expression and production of semaphorins: Class-3 semaphorin cDNAs were sub-
470
cloned into the NSPI-CMV-myc-his lentiviral expression vector as previously described
471
(Kigel et al., 2008; Varshavsky et al., 2008). An expression plasmid containing the
472
extracellular domain of the sema6A cDNA was kindly provided by Dr. Behar of the
473
Hebrew University. The production of lentiviruses and the generation of conditioned
474
media containing various semaphorins were done as previously described (Kigel et al.,
475
2008). For repulsion assays, HEK293 cells expressing various semaphorins were also
476
infected with a lentiviruses generated from the PGK-eGFP lentiviral vector (Chrenek et
477
al., 2011).
21
Accepted manuscript Journal of Cell Science
478
Quantitative real time PCR (qRT-PCR): QRT-PCR was performed using the StepOne
479
Plus Real Time PCR System with TaqMen Gene Expression Master Mix, according to
480
the instructions of the manufacturer (Applied biosystems). The normalizing gene was
481
RPLPO. Data was analyzed by the StepOne Software (Applied biosystems) using the
482
relative Quantitation-Comparative CT method. The following assays were used: NP1-
483
Hs00826128, NP2- Hs00187290, PlexinA1- Hs00413698, PlexinA2- Hs00300697,
484
PlexinA3- Hs00250178, PlexinA4- Hs00297356, PlexinD1- Hs00892410, RPLPO-
485
Hs99999902.
486
Quantitation of the number of mRNA molecules/cell by real time reverse PCR: The
487
number of mRNA molecules/cell of the plexins and neuropilins expressed in non-
488
stimulated HUVEC and U87MG cells was quantified using the TaqMen gene expression
489
assay. Plasmids containing the cDNA of each plexin or neuropilin were used to build a
490
standard curve of cycle threshold (Ct) vs. plasmid concentration. RNA from HUVEC and
491
U87MG cells was extracted and converted to cDNA. Real time PCR was performed in
492
triplicate as described above. The amount of each mRNA (ng/well) was calculated from
493
the standard curve and converted to moles/well taking into consideration the dilutions
494
made in the process to derive the number of mRNA molecules/cell for each plexin and
495
neuropilin in each HUVEC and U87MG cells.
496
Inhibition of gene expression with shRNA expressing lentiviruses: Lentiviral vectors
497
directing expression of various shRNAs were purchased from Sigma Aldrich
498
The following shRNA sequences were used: PlexinA1-CCGGGCACTTCTTCACGTCC
499
AAGATCTCGAGATCTTGGACGTGAAGAAGTGCTTTTTG, Plexin A2- First
500
shRNA: CCGGCGGCAATTTCATCATTGACAACTCGAGTTGTCAATGATGAAAT
501
TGCCGTTTTTG, Second shRNA: CCGGCGCCCAGATGAGTTTGGATTCTCGAGA 22
Accepted manuscript Journal of Cell Science
502
ATCCAAACTCATCTGGGCGTTTTTG, PlexinA3- CCGGGCTGTATTTCTATGTCA
503
CCAACTCGAGTTGGTGACATAGAAATACAGCTTTTTG, PlexinA4- CCGGGCAG
504
ATAAATGACCGCATTAACTCGAGTTAATGCGGTCATTTATCTGCTTTTTG.
505
The production of the lentiviruses, infection of cells and the selection of shRNA
506
expression cells in HEK293 cells were performed as previously described (Varshavsky et
507
al., 2008).
508
Inducible expression of shRNA species: To generate inducible shRNA-expressing
509
plasmids, double-stranded oligonucleotides encoding the desired shRNAs were generated
510
and cloned into the single-vector inducible shRNA expression vectors pLKO-Tet-On (gift
511
of Dr. Ayoub Nabieh) as described (Wiederschain et al., 2009).
512
Down-regulation of neuropilin expression using small interfering RNA (siRNA):
513
Inhibition of np1 and np2 expressions in U87MG cells using transfection of siRNAs was
514
performed as previously described (Varshavsky et al., 2008).
515
Expression of recombinant plexins: The full length cDNAs of plexin-A2 and plexin-A4
516
were cloned into the gateway pDonor221 plasmid and then transferred by recombination
517
into the pLenti6/V5-DEST or pLenti6.3/TO/V5-Dest lentiviral expression vector in frame
518
with a c-terminal V5 tag according to the instructions of the manufacturer (Invitrogen). A
519
deletion mutant of plexin-A2 containing the extracellular and transmembrane domains
520
(amino-acids 1-1323) of the protein fused to a c-terminal FLAG tag was constructed
521
according to the same procedures. Production of lentiviruses using these plasmids and
522
stable infection of target cells were performed essentially as previously described
523
(Brekhman and Neufeld, 2009). The sema6A expression vector was transfected into
524
target cells using FuGENE6 transfection reagent (Roche) as previously described
525
(Guttmann-Raviv et al., 2007). 23
Accepted manuscript Journal of Cell Science
526
Cell contraction and cell repulsion assays: Contraction of HUVEC and U87MG cells in
527
response to semaphorins as well as repulsion assays of endothelial cells were performed
528
as described (Varshavsky et al., 2008; Guttmann-Raviv et al., 2007).
529
Co-immunoprecipitation: Co-immunoprecipitation assays were performed as previously
530
described (Shraga-Heled et al., 2007).
531
Immunofluorescence: U87MG cells were plated on glass coverslips coated with PBS-
532
gelatin. Detection of vinculin and actin was done as previously described (Kigel et al.,
533
2011).
534
Stable isotope labeling by amino acids in cell culture (SILAC) and Affinity purification
535
of plexin-A2 tagged with a Strep/FLAG epitope tag: U87MG cells expressing plexin-A2
536
fused in-frame with a Strep/FLAG epitope tag (Gloeckner et al., 2007) as well as
537
HEK293 cells expressing recombinant sema3B (Varshavsky et al., 2008) or a secreted
538
soluble extracellular domain of sema6A (Kigel et al., 2011) were metabolically labeled
539
with lysine and arginine containing light (K0/R0) (Sigma), control cells) or heavy carbon,
540
nitrogen or hydrogen isotopes (K8/R10) (Cambridge Isotope Laboratories) for 10
541
passages in order to achieve complete labeling of cell proteins with these amino-acids
542
according to standard SILAC protocols (Pimienta et al., 2009). Preparation of the ligands:
543
cells labeled with amino-acids containing heavy isotopes were then stimulated with
544
conditioned medium containing saturating concentrations of recombinant sema3B or
545
sema6A for 5 min. at 37oC (five 20 cm diameter confluent plates for each treatment).
546
Control cells were incubated with conditioned medium from HEK293 cells labeled with
547
light isotopes. For reverse experiments, labels were switched. Both the control cells and
548
the cells stimulated with sema3B or sema6A were then washed briefly with ice cold TBS,
549
scraped, lysed quickly with lysis buffer (TBS supplemented with a protease inhibitor 24
Accepted manuscript Journal of Cell Science
550
cocktail (Roche) and 0.5% Np-40). Equal amounts of protein from each treatment were
551
loaded on an anti-FLAG affinity column (150ul bed volume). The column was washed
552
briefly with 1.5 ml of TBS, and bound proteins eluted using a buffer containing 100mM
553
Glycine (pH=2.5) and quick frozen in liquid nitrogen.
554
Proteomic analysis of proteins co purified with plexin-A2: Mass spectrometric analysis
555
was performed as described earlier (Gloeckner et al., 2010). Briefly, light isotopes and
556
heavy isotope labeled eluants from the anti-FLAG columns were mixed in a 1:1 ratio
557
based on protein content, proteolytically cleaved by trypsin and subsequently analyzed by
558
LC-MSMS using a nano-flow HPLC system (Dionex Ultimate 3000 RSLC, Thermo-
559
Fisher) coupled to an Orbitrap Velos (Thermo-Fisher) tandem mass spectrometer. To
560
exclude false-positive hits caused by labeling artifacts, reverse labeling was performed
561
were heavy/light labels were switched. In order to identify proteins that are induced to
562
bind to plexin-A2 following the stimulation, quantification of the resulting MS-data was
563
performed by the MaxQuant Software ver. 1.3.0.2 (Cox et al., 2009). Downstream
564
analysis and graphical representation was performed using in-house R-scripts. Only those
565
hits have been considered which were identified by at least 1 peptide in both, forward and
566
reverse experiments.
567
Statistical analysis: All experiments were repeated at least twice with similar results
568
unless otherwise stated. The one tailed unpaired with Welch's correction student's T-test
569
was used in most experiments. In cell proliferation experiments in which we normalized
570
the data across several experiments we used the paired student's T-test taking care that the
571
differences between the controls do not vary by more then 50%. Cell proliferation
572
experiments were performed in triplicates. Variations between replicates within single
573
experiments did not exceed 10%. Unless otherwise stated error bars represent the
574
standard error of the mean. Statistical significance is presented in the following manner: 25
575
*p
