J Mol Neurosci DOI 10.1007/s12031-014-0388-2
Transcriptional Down-regulation of Epidermal Growth Factor (EGF) Receptors by Nerve Growth Factor (NGF) in PC12 Cells Gadi Cohen & Keren Ettinger & Shimon Lecht & Peter I. Lelkes & Philip Lazarovici
Received: 8 July 2014 / Accepted: 22 July 2014 # Springer Science+Business Media New York 2014
Abstract Nerve growth factor (NGF) treatment causes a profound down-regulation of epidermal growth factor (EGF) receptors (EGFR) during the neuronal differentiation of PC12 cells. This process was characterized by a progressive decrease in EGFR level, as measured by 125I-EGF binding and Scatchard analysis, tyrosine phosphorylation, Western blotting, and bio-imaging using EGF-labeled with a near-infrared probe. Differentiation of the cells with NGF for 5–7 days produces a 95 % reduction in the amount of 35S-methioninelabeled EGFR. This down-regulation does not occur in PC12nnr5 cells, which lack the TrkA NGF receptor but is reconstituted in these cells upon their stable transfection with TrkA. The process of NGF-induced EGFR down-regulation was inhibited by K252a, a TrkA antagonist and by anti-TrkA antibodies but not by Thx-B, a blocker of the interaction of NGF with p75NTR receptors. NGF-induced (heterologous) down-regulation, but not EGF-induced (homologous) downregulation of EGFR, was blocked in Ras-deficient PC12 cells. NGF treatment for 5–7 days of PC12 cells, grown in suspension or in 3D collagen gels, induces down-regulation of EGFR independent of neurite outgrowth. The messenger RNA (mRNA) for EGFR decreased in a comparable fashion. This process was correlated temporally with a decrease in the transcription of the EGFR gene. Treatment with NGF also
Mini Review for the special issue: From Molecular Biology to Neurobiology in memory of Uriel Littauer. G. Cohen : K. Ettinger : S. Lecht : P. Lazarovici (*) Faculty of Medicine, School of Pharmacy Institute for Drug Research, The Hebrew University of Jerusalem, Jerusalem 91120, Israel e-mail: [email protected] S. Lecht : P. I. Lelkes : P. Lazarovici Department of Bioengineering and Temple Institute for Regenerative Medicine and Engineering, Temple University, Philadelphia, PA 19122, USA
increased the cellular content of GCF2, a putative inhibitory transcription factor of the EGFR gene. The temporal increase in GCF2, like the decrease in the EGFR mRNA, was not seen in TrkA deficient PC12 cells nor in cells expressing dominantnegative Ras. The results suggest that NGF-induced downregulation of the EGFR is under transcriptional control, is TrkA and Ras-dependent, may involve transcriptional repression by GCF2, and independent of mechanisms that lead to NGF-induced neurite outgrowth in PC12cells. This heterologous down-regulation of EGFR would appear to be an efficient mean of desensitizing the neuron to proliferative stimuli, thereby representing a safety latch for initiating and sustaining NGF-induced neuronal differentiation. Keywords NGF . EGFR . Down-regulation . TrkA . Ras . PC12 cells . Proliferation . Differentiation . Neurite outgrowth . 2D . 3D . Suspension
Introduction Neuropeptides, neurotrophins, cytokines, chemokines, and different growth factors play a key role in the mechanism of action of growth factors regulating the development of the nervous system (Sieber-Blum 1998; Johnson 2001; Wang and Zoghbi 2001; Bernd 2008). However, the interactions and cross talk between these diverse factors are still poorly understood (Hall and Ekanayake 1991; Sieber-Blum 1998; Fargali et al. 2012; Williamson and Bilbo 2013). Physiological neuronal development requires a proper delicate balance between cell proliferation, differentiation, and apoptosis (de la Rosa and de Pablo 2000; Liu and Greene 2001; Becker and Bonni 2005). Neurogenesis, neuronal differentiation, neuroprotection, and repair are usually controlled by the neurotrophin family of growth factors, which includes nerve growth factor (NGF) (Sofroniew et al. 2001).
J Mol Neurosci
NGF is a 13-kD protein that influences the growth and survival of sympathetic neurons and chromaffin cells, affects cholinergic neuronal pathways in the brain frontal cortex, stimulates proliferation and differentiation of neuronal stem cells (Ip et al. 1994; Bath and Lee 2010), and contributes to the maintenance of the differentiated neuronal phenotype in vitro and in vivo (Manni et al. 2013). Embryonic and adult nervous tissues alike also contain a variety of mitogens, including insulin-like growth factors-I and -II (Devaskar 1991), platelet-derived growth factor (Yeh et al. 1991), basic fibroblast growth factor (Abe and Saito 2001), and epidermal growth factor (Plata-Salaman 1991). In their absence, neuronal cells are growth-arrested and turn into post-mitotic neurons. Epidermal growth factor (EGF) and its receptors are widely expressed in both the central and peripheral nervous systems, including in dopaminergic neurons in the basal ganglia (Piao et al. 2005; Abe et al. 2009) and play an important role in the proliferation, migration, and survival of numerous cell populations in the embryonic brain (Kornblum et al. 1997). Mice lacking epidermal growth factor receptors (EGFRs) exhibit brain defects including progressive neurodegeneration in the frontal cortex, olfactory bulb, and thalamus, as characterized by massive apoptosis (Sibilia et al. 1998). These observations suggest that in EGFR levels, signaling and regulation may play an important role during the development of the nervous system. The rat pheochromocytoma cell line (PC12) is an extensively used model to study neuronal differentiation (Fujita et al. 1989; Ravni et al. 2006). Stimulation of PC12 cells with NGF induces a neuronal differentiation program: The cells exit the cell cycle, develop an elaborate network of branching neurites (neuritogenesis), and become electrically excitable (Greene and Tischler 1976). Acquisition of a sympathetic neuronal phenotype entails a complex cascade of transcription-dependent and -independent biological processes, which are tightly coordinated topographically and temporally (Greene and Angelastro 2005; van Dijkmans et al. 2008; Mullenbrock et al. 2011). The initial signaling steps triggered by NGF stimulation are well characterized (Kaplan and Miller 2000). After NGF binding to its receptors p75NTR and the tropomyosin-related kinase A (TrkA) receptor, TrkA receptors dimerize and subsequently autophosphorylate followed by recruitment of complex of signaling molecules initiating intracellular signaling cascades involving, among others, PI3KAkt, PLCγ-IP3, and Ras-mitogen activated protein kinase (MAPK). NGF-induced neuronal differentiation requires activation of the TrkA receptor and proceeds downstream through the Ras/MAPK pathway (Vaudry et al. 2002). Once activated and translocated to the nucleus, phosphorylated transcription factors such as Elk-1, CREB, and many others start the transcriptional reprogramming of the cell by turningon differentiation-related and turning-off proliferation-related
genes (van Dijkmans et al. 2008). A critical regulatory prerequisite step for NGF-induced differentiation of PC12 cells is the arrest of the cell cycle, as measured by cessation of DNA synthesis and cell division (Greene and Tischler 1976; Gunning et al. 1981; Ignatius et al. 1985; Buchkovich and Ziff 1994). NGF decreases the growth rate of PC12 cells and, in the short term, causes synchronized PC12 cells to accumulate in the G1 phase of the cell cycle with a decrease in DNA synthesis. Acute exposure to NGF arrests the PC12 cell population in G1 with an increased number in the G2/M phase (Rudkin et al. 1989). Chronic treatment of PC12 cells with NGF promotes terminal differentiation, in which the terminal phenotype resembles that of sympathetic neurons, as inferred from the cessation of cell division, increased adherence to extracellular matrix proteins, neurite extension, and shift from dopamine to norepinephrine synthesis. Although the mechanism by which NGF arrests PC12 cells in the G1 phase of the cell cycle is not yet fully understood, there seems to be a direct correlation between the differential regulation of specific cell cycle regulatory components and NGF-induced neuronal differentiation (Hughes et al. 2000). Furthermore, NGF seems to mediate down-regulation of EGFR during the differentiation of PC12 cells therefore making the cells refractory to EGF mitogen signaling (Lazarovici et al. 1987, 1997a). The molecular mechanism responsible for this process is addressed in the present review, hopefully helping to understand in more detail the role of down-regulation of EGFR in neuronal differentiation.
The Process of NGF-induced Heterologous Down-regulation of EGFR in PC12 Cells The capacity of NGF-differentiated PC12 cells to interact with 125 I-EGF (Fig. 1a, b) was correlated to the NGF-induced neuronal differentiated as inferred from changes in cell morphology (Fig. 1a-inset). Treatment of the cells with 50 ng/ml NGF significantly reduced the binding capacity of the cells for EGF after a latency period of 1–2 days. After 5–7 days of NGF treatment, a near-total disappearance of EGF-binding capacity was observed (Fig. 1a). The decrease in EGF binding capacity correlated well with the NGF-induced morphological changes, e.g. cell hypertrophy, neurite outgrowth formation, and elongation (Fig. 1a-inset), properties typical of terminally differentiated neurons (Fujita et al. 1989). Reduction in the binding of 125I-EGF to NGF-differentiated PC12 cells may be due to either reduction in the affinity or in the number of EGFR. To distinguish between these two possibilities, binding experiments were performed with both undifferentiated and NGF-differentiated PC12 cultures and the data was evaluated by Scatchard analysis (Fig. 1b). While the slope of the curves (affinity) did not change, a 50 and 95 % reduction in the number of binding sites was measured after 3 and 6 days of
J Mol Neurosci Fig. 1 Time course of the NGFmediated heterologous downregulation of 125I-EGF binding to PC12 cells. a Cells were grown in control medium (white circles) or in medium with 50 ng/ml NGF (black circles). Specific binding of 125I-EGF (0.2×106 cpm/l05 cells) was measured by incubation for 45 min at 37 °C at the indicated time points, in the presence or absence of excess unlabeled EGF (100 ng/ml). The cells were exposed to NGF at 0 time, and fresh NGF was added every 2 days. Each binding point represents the mean±SD of two experiments performed in triplicate. The upper insets show control PC12 cells and typical neurite outgrowth induced by 50 ng/ml NGF after 3 (center) and 6 (right) days of treatment. The lower inset shows the homologous down-regulation of EGFR by EGF. PC12 cells were incubated at 37 °C for the indicated time period with EGF (50 ng/ml) in DMEM. Monolayers were washed to remove the unbound factor, and the extent of receptor downregulation was determined by measuring cell associated radioactivity. b Scatchard plots of 125 I-EGF binding to cells grown in control medium (white circles) or in medium with 50 ng/ml NGF for 3 days (black circles) or 6 days (triangle)
NGF treatment, respectively. These findings suggest that NGF-induced neuronal differentiation of the cells causes a reduction in the number of functional EGFR (Lazarovici et al. 1987). To obtain further proof that a reduction in the number of EGFR is the cause of the reduced binding capacity of PC12 cells differentiated with NGF, EGF was cross-linked to its receptors with succinimidyl suberate. Plasma membranes
isolated by sucrose gradient from control undifferentiated PC12 cell cultures and from differentiated cells were isolated, carefully normalized for protein, exposed to 125I-EGF, crosslinked, and analyzed by electrophoresis, and autoradiography (Fig. 2a). Cross-linking of 125I-EGF to membranes from control PC12 cells resulted in the labeling of a major component of 170 kD and a minor component of 150 kD, as repeatedly documented that the 150-kD species is the Ca2+-activated
J Mol Neurosci
protein product of the 170-kD EGFR (Seger et al. 1988). Analysis of the cross-linked EGFR bands in PC12 cells treated for 6 days with NGF indicate complete disappearance of the labeling of these bands. This decrease in labeling is consistent with a lower number of EGFR in NGF-differentiated PC12 cells. Another indication for an NGF-induced decrease in the number of EGFR would be a decrease in EGFstimulated tyrosine kinase activity in differentiated PC12 cells. As seen in a typical experiment (Fig. 2b), plasma membranes from PC12 cells treated for 6 days with NGF show a 95 % reduction in the autophosphorylation intensity of the 170-kD band, when compared with membranes from control undifferentiated cells. A possible mechanism by which NGF might decrease EGFR number is by decreased synthesis. The biosynthesis of the EGFR was studied by labeling control and NGF-differentiated PC12 cells with 35S-methionine for 18 h (Lazarovici et al. 1987). The radioactive cell membranes were solubilized with detergent, and the EGFR was enriched by immunoprecipitation with anti-EGFR antibody (Fig. 2c). The levels of 35S-methionine-labeled EGFR in the membranes of NGF-differentiated cells decreased by more than 90 % by comparison to membranes isolated from control, untreated cells (Fig. 2c). This result is in accordance with the disappearance of autophosphorylated receptor (Fig. 2b). We conclude that the decrease in EGF-binding capacity of the NGFdifferentiated PC12 cells (Fig. 1) is due to a reduction in the number of cell surface (plasma membrane) receptors, which, in turn, is caused by decreased receptor biosynthesis (Fig. 2). Thus, the NGF-induced loss of EGFR from PC12 cells fulfills the criteria for “heterologous down-regulation” (Sibley and Lefkowitz 1985). The process of NGF-induced heterologous down-regulation of EGFR can be visualized by a novel bio-imaging method that uses EGF labeled with a near-infrared dye (EGF-NIR). The EGF molecule can be conjugated with IRDye800CW at the amino-terminal of the protein due to a free amino group available (Cohen et al. 2012). After reaction with the IRDye800CW, the EGF-NIR was purified by gradient HPLC
chromatography (Fig. 3a), yielding a homogenous single peak as confirmed by SDS-PAGE electrophoresis and NIR visualization (Fig. 3a-inset). EGF-NIR specifically and selectively bound to EGFR-expressing A431 cells (Fig. 3b). The intensity of the EGF-NIR signal reflected EGFR levels (Cohen et al. 2012, 2013; Cohen and Lazarovici 2013a). Using NGFdifferentiated PC12 cultures, we demonstrated a significant 85 % reduction in EGF-NIR specific binding to the differentiated cells (Fig. 3c), indicative of a significant heterologous down-regulation of EGFR measured by optical imaging (Cohen and Lazarovici 2013b). In view of the above observation that the NGF-induced heterologous down-regulation of EGFR is temporally correlated with NGF-induced neurite outgrowth, we cultured in the presence or absence of 50 ng/ml NGF, enhanced green fluorescence protein (GFP) expressing PC12 cells for 7 days either in conventional two-dimensional (2D) cultures, PC12 cells embedded in a three-dimensional collagen hydrogel (3D cultures) (Arien-Zakay et al. 2009) and PC12 cells grown as suspension cultures (Lazarovici et al. 1997a). Under 3D conditions using collagen hydrogels as scaffolds, GFP-PC12 cells proliferated, formed aggregates, and respond to NGF by exuberant outgrowth of neurites of different lengths and complexity, very similar to wild-type PC12 cells (Arien-Zakay et al. 2009). In 2D and 3D cultures, the 50 ng/ml NGF-treated PC12 cells extend neurites, and in suspension, they aggregate but do not grow neurites (Fig. 4). Following 7 days of culture under the various conditions mentioned above, the cells were harvested and analyzed by Western blotting using an anti-EGFR antibody to measure EGFR down-regulation and an antineurofilament-M antibody (NF-M) to measure the increased expression of this cytoskeleton protein as marker for the neuronal differentiation of the cells. As seen in Fig. 4, NGFinduced heterologous down-regulation of EGFR under all three different growth conditions occurred independently of neurite outgrowth, which was well developed under 2D and 3D conditions but absent in cells growing in suspension culture. PC12 cells grown in suspension in a spinner flask
Fig. 2 Cross linking of 125I-EGF (a), tyrosine autophosphorylation (b) and 35S-methionine incorporation (c) into EGFR in control PC12 cells and cells after 6 days of differentiation with 50 ng/ml NGF. a Membranes of PC12 cells and A431 cells were incubated for 60 min with 5 nM 125IEGF on ice in the presence of 0.5 μM disuccinimidyl suberate. b Membranes of PC12 cells were incubated for 15 min at 40 °C in a kinase reaction buffer and thereafter incubated for 2 min at 4 °C with 2 μCi of
γ-32P-ATP 50 μM unlabeled ATP. c PC12 cells were incubated for 18 h in methionine-free DMEM containing 60 μCi/ml L-35S-methionine, and plasma membranes were prepared. In all experiments, the membranes were solubilized with RIPA buffer, and EGRR were immune-precipitated and analyzed by 7.5 % SDS-PAGE. The autoradiograms of the fixed dried gels are presented. A431 carcinoma cells were used as control. EGFR is represented by the band of 170 kD
J Mol Neurosci
aggregated into macroscopic organoids as evident by morphological and histological examinations (Fig. 4), as previously documented using different types of bioreactors (Manley and Lelkes 2006). In contrast, under all conditions, NGF increased the level of the NF-M (Fig. 4) and neuronal tubulin and microtubule-associated proteins (data not shown), indicating NGF-triggered cytoskeleton differentiation under all growth conditions. These findings suggest that NGF-induced heterologous down-regulation of EGFR is a molecular event accompanying neuronal differentiation and that this effect is independent of PC12 cell adhesion and the organization of the extracellular matrix (2D vs 3D) and can occur in the absence of neurite outgrowth (Lazarovici et al. 1997a).
NGF-induced Heterologous Down-regulation of EGFR is TrkA and Ras Dependent
Fig. 3 Preparation and isolation of EGF-NIR probe and its use for visualization of NGF-induced heterologous down-regulation of the EGFR in PC12 cells. a HPLC separation of EGF-NIR. Solid line represents absorbance at 226 nm and dotted line indicates the acetonitrile gradient. Inset shows 12 % SDS-PAGE analysis of 10 μg of EGF-NIR scanned with Odyssey near-infrared scanner and of unmodified EGF stained with Coomassie blue. b The specificity and selectivity of EGF-NIR probe binding measured by NIR imaging. A431 carcinoma cells were incubated for 15 min at 37 °C with 7 ng/ml EGF-NIR in the presence (EGF/EGF-NIR) or absence (EGF-NIR) of 100 ng/ml EGF. Competition experiments with 500 ng/ml Neuroregulin 1 (NRG1/EGF-NIR) were also conducted. In control experiments (NIR-Dye), the cultures were incubated with 7 ng/ml NIR-Dye to evaluate non-specific labeling of the cells by comparison to untreated cultures (untreated). The NIR intensity at 800 nm was estimated under identical conditions for all cultures, and the mean±SD (n=9) is presented. Upper insets, NIR scans; lower insets, phase-contrast photomicrographs of the cultures, *p< 0.05 versus NIR-Dye; **p
Transcriptional Down-regulation of Epidermal Growth Factor (EGF) Receptors by Nerve Growth Factor (NGF) in PC12 Cells Gadi Cohen & Keren Ettinger & Shimon Lecht & Peter I. Lelkes & Philip Lazarovici
Received: 8 July 2014 / Accepted: 22 July 2014 # Springer Science+Business Media New York 2014
Abstract Nerve growth factor (NGF) treatment causes a profound down-regulation of epidermal growth factor (EGF) receptors (EGFR) during the neuronal differentiation of PC12 cells. This process was characterized by a progressive decrease in EGFR level, as measured by 125I-EGF binding and Scatchard analysis, tyrosine phosphorylation, Western blotting, and bio-imaging using EGF-labeled with a near-infrared probe. Differentiation of the cells with NGF for 5–7 days produces a 95 % reduction in the amount of 35S-methioninelabeled EGFR. This down-regulation does not occur in PC12nnr5 cells, which lack the TrkA NGF receptor but is reconstituted in these cells upon their stable transfection with TrkA. The process of NGF-induced EGFR down-regulation was inhibited by K252a, a TrkA antagonist and by anti-TrkA antibodies but not by Thx-B, a blocker of the interaction of NGF with p75NTR receptors. NGF-induced (heterologous) down-regulation, but not EGF-induced (homologous) downregulation of EGFR, was blocked in Ras-deficient PC12 cells. NGF treatment for 5–7 days of PC12 cells, grown in suspension or in 3D collagen gels, induces down-regulation of EGFR independent of neurite outgrowth. The messenger RNA (mRNA) for EGFR decreased in a comparable fashion. This process was correlated temporally with a decrease in the transcription of the EGFR gene. Treatment with NGF also
Mini Review for the special issue: From Molecular Biology to Neurobiology in memory of Uriel Littauer. G. Cohen : K. Ettinger : S. Lecht : P. Lazarovici (*) Faculty of Medicine, School of Pharmacy Institute for Drug Research, The Hebrew University of Jerusalem, Jerusalem 91120, Israel e-mail: [email protected] S. Lecht : P. I. Lelkes : P. Lazarovici Department of Bioengineering and Temple Institute for Regenerative Medicine and Engineering, Temple University, Philadelphia, PA 19122, USA
increased the cellular content of GCF2, a putative inhibitory transcription factor of the EGFR gene. The temporal increase in GCF2, like the decrease in the EGFR mRNA, was not seen in TrkA deficient PC12 cells nor in cells expressing dominantnegative Ras. The results suggest that NGF-induced downregulation of the EGFR is under transcriptional control, is TrkA and Ras-dependent, may involve transcriptional repression by GCF2, and independent of mechanisms that lead to NGF-induced neurite outgrowth in PC12cells. This heterologous down-regulation of EGFR would appear to be an efficient mean of desensitizing the neuron to proliferative stimuli, thereby representing a safety latch for initiating and sustaining NGF-induced neuronal differentiation. Keywords NGF . EGFR . Down-regulation . TrkA . Ras . PC12 cells . Proliferation . Differentiation . Neurite outgrowth . 2D . 3D . Suspension
Introduction Neuropeptides, neurotrophins, cytokines, chemokines, and different growth factors play a key role in the mechanism of action of growth factors regulating the development of the nervous system (Sieber-Blum 1998; Johnson 2001; Wang and Zoghbi 2001; Bernd 2008). However, the interactions and cross talk between these diverse factors are still poorly understood (Hall and Ekanayake 1991; Sieber-Blum 1998; Fargali et al. 2012; Williamson and Bilbo 2013). Physiological neuronal development requires a proper delicate balance between cell proliferation, differentiation, and apoptosis (de la Rosa and de Pablo 2000; Liu and Greene 2001; Becker and Bonni 2005). Neurogenesis, neuronal differentiation, neuroprotection, and repair are usually controlled by the neurotrophin family of growth factors, which includes nerve growth factor (NGF) (Sofroniew et al. 2001).
J Mol Neurosci
NGF is a 13-kD protein that influences the growth and survival of sympathetic neurons and chromaffin cells, affects cholinergic neuronal pathways in the brain frontal cortex, stimulates proliferation and differentiation of neuronal stem cells (Ip et al. 1994; Bath and Lee 2010), and contributes to the maintenance of the differentiated neuronal phenotype in vitro and in vivo (Manni et al. 2013). Embryonic and adult nervous tissues alike also contain a variety of mitogens, including insulin-like growth factors-I and -II (Devaskar 1991), platelet-derived growth factor (Yeh et al. 1991), basic fibroblast growth factor (Abe and Saito 2001), and epidermal growth factor (Plata-Salaman 1991). In their absence, neuronal cells are growth-arrested and turn into post-mitotic neurons. Epidermal growth factor (EGF) and its receptors are widely expressed in both the central and peripheral nervous systems, including in dopaminergic neurons in the basal ganglia (Piao et al. 2005; Abe et al. 2009) and play an important role in the proliferation, migration, and survival of numerous cell populations in the embryonic brain (Kornblum et al. 1997). Mice lacking epidermal growth factor receptors (EGFRs) exhibit brain defects including progressive neurodegeneration in the frontal cortex, olfactory bulb, and thalamus, as characterized by massive apoptosis (Sibilia et al. 1998). These observations suggest that in EGFR levels, signaling and regulation may play an important role during the development of the nervous system. The rat pheochromocytoma cell line (PC12) is an extensively used model to study neuronal differentiation (Fujita et al. 1989; Ravni et al. 2006). Stimulation of PC12 cells with NGF induces a neuronal differentiation program: The cells exit the cell cycle, develop an elaborate network of branching neurites (neuritogenesis), and become electrically excitable (Greene and Tischler 1976). Acquisition of a sympathetic neuronal phenotype entails a complex cascade of transcription-dependent and -independent biological processes, which are tightly coordinated topographically and temporally (Greene and Angelastro 2005; van Dijkmans et al. 2008; Mullenbrock et al. 2011). The initial signaling steps triggered by NGF stimulation are well characterized (Kaplan and Miller 2000). After NGF binding to its receptors p75NTR and the tropomyosin-related kinase A (TrkA) receptor, TrkA receptors dimerize and subsequently autophosphorylate followed by recruitment of complex of signaling molecules initiating intracellular signaling cascades involving, among others, PI3KAkt, PLCγ-IP3, and Ras-mitogen activated protein kinase (MAPK). NGF-induced neuronal differentiation requires activation of the TrkA receptor and proceeds downstream through the Ras/MAPK pathway (Vaudry et al. 2002). Once activated and translocated to the nucleus, phosphorylated transcription factors such as Elk-1, CREB, and many others start the transcriptional reprogramming of the cell by turningon differentiation-related and turning-off proliferation-related
genes (van Dijkmans et al. 2008). A critical regulatory prerequisite step for NGF-induced differentiation of PC12 cells is the arrest of the cell cycle, as measured by cessation of DNA synthesis and cell division (Greene and Tischler 1976; Gunning et al. 1981; Ignatius et al. 1985; Buchkovich and Ziff 1994). NGF decreases the growth rate of PC12 cells and, in the short term, causes synchronized PC12 cells to accumulate in the G1 phase of the cell cycle with a decrease in DNA synthesis. Acute exposure to NGF arrests the PC12 cell population in G1 with an increased number in the G2/M phase (Rudkin et al. 1989). Chronic treatment of PC12 cells with NGF promotes terminal differentiation, in which the terminal phenotype resembles that of sympathetic neurons, as inferred from the cessation of cell division, increased adherence to extracellular matrix proteins, neurite extension, and shift from dopamine to norepinephrine synthesis. Although the mechanism by which NGF arrests PC12 cells in the G1 phase of the cell cycle is not yet fully understood, there seems to be a direct correlation between the differential regulation of specific cell cycle regulatory components and NGF-induced neuronal differentiation (Hughes et al. 2000). Furthermore, NGF seems to mediate down-regulation of EGFR during the differentiation of PC12 cells therefore making the cells refractory to EGF mitogen signaling (Lazarovici et al. 1987, 1997a). The molecular mechanism responsible for this process is addressed in the present review, hopefully helping to understand in more detail the role of down-regulation of EGFR in neuronal differentiation.
The Process of NGF-induced Heterologous Down-regulation of EGFR in PC12 Cells The capacity of NGF-differentiated PC12 cells to interact with 125 I-EGF (Fig. 1a, b) was correlated to the NGF-induced neuronal differentiated as inferred from changes in cell morphology (Fig. 1a-inset). Treatment of the cells with 50 ng/ml NGF significantly reduced the binding capacity of the cells for EGF after a latency period of 1–2 days. After 5–7 days of NGF treatment, a near-total disappearance of EGF-binding capacity was observed (Fig. 1a). The decrease in EGF binding capacity correlated well with the NGF-induced morphological changes, e.g. cell hypertrophy, neurite outgrowth formation, and elongation (Fig. 1a-inset), properties typical of terminally differentiated neurons (Fujita et al. 1989). Reduction in the binding of 125I-EGF to NGF-differentiated PC12 cells may be due to either reduction in the affinity or in the number of EGFR. To distinguish between these two possibilities, binding experiments were performed with both undifferentiated and NGF-differentiated PC12 cultures and the data was evaluated by Scatchard analysis (Fig. 1b). While the slope of the curves (affinity) did not change, a 50 and 95 % reduction in the number of binding sites was measured after 3 and 6 days of
J Mol Neurosci Fig. 1 Time course of the NGFmediated heterologous downregulation of 125I-EGF binding to PC12 cells. a Cells were grown in control medium (white circles) or in medium with 50 ng/ml NGF (black circles). Specific binding of 125I-EGF (0.2×106 cpm/l05 cells) was measured by incubation for 45 min at 37 °C at the indicated time points, in the presence or absence of excess unlabeled EGF (100 ng/ml). The cells were exposed to NGF at 0 time, and fresh NGF was added every 2 days. Each binding point represents the mean±SD of two experiments performed in triplicate. The upper insets show control PC12 cells and typical neurite outgrowth induced by 50 ng/ml NGF after 3 (center) and 6 (right) days of treatment. The lower inset shows the homologous down-regulation of EGFR by EGF. PC12 cells were incubated at 37 °C for the indicated time period with EGF (50 ng/ml) in DMEM. Monolayers were washed to remove the unbound factor, and the extent of receptor downregulation was determined by measuring cell associated radioactivity. b Scatchard plots of 125 I-EGF binding to cells grown in control medium (white circles) or in medium with 50 ng/ml NGF for 3 days (black circles) or 6 days (triangle)
NGF treatment, respectively. These findings suggest that NGF-induced neuronal differentiation of the cells causes a reduction in the number of functional EGFR (Lazarovici et al. 1987). To obtain further proof that a reduction in the number of EGFR is the cause of the reduced binding capacity of PC12 cells differentiated with NGF, EGF was cross-linked to its receptors with succinimidyl suberate. Plasma membranes
isolated by sucrose gradient from control undifferentiated PC12 cell cultures and from differentiated cells were isolated, carefully normalized for protein, exposed to 125I-EGF, crosslinked, and analyzed by electrophoresis, and autoradiography (Fig. 2a). Cross-linking of 125I-EGF to membranes from control PC12 cells resulted in the labeling of a major component of 170 kD and a minor component of 150 kD, as repeatedly documented that the 150-kD species is the Ca2+-activated
J Mol Neurosci
protein product of the 170-kD EGFR (Seger et al. 1988). Analysis of the cross-linked EGFR bands in PC12 cells treated for 6 days with NGF indicate complete disappearance of the labeling of these bands. This decrease in labeling is consistent with a lower number of EGFR in NGF-differentiated PC12 cells. Another indication for an NGF-induced decrease in the number of EGFR would be a decrease in EGFstimulated tyrosine kinase activity in differentiated PC12 cells. As seen in a typical experiment (Fig. 2b), plasma membranes from PC12 cells treated for 6 days with NGF show a 95 % reduction in the autophosphorylation intensity of the 170-kD band, when compared with membranes from control undifferentiated cells. A possible mechanism by which NGF might decrease EGFR number is by decreased synthesis. The biosynthesis of the EGFR was studied by labeling control and NGF-differentiated PC12 cells with 35S-methionine for 18 h (Lazarovici et al. 1987). The radioactive cell membranes were solubilized with detergent, and the EGFR was enriched by immunoprecipitation with anti-EGFR antibody (Fig. 2c). The levels of 35S-methionine-labeled EGFR in the membranes of NGF-differentiated cells decreased by more than 90 % by comparison to membranes isolated from control, untreated cells (Fig. 2c). This result is in accordance with the disappearance of autophosphorylated receptor (Fig. 2b). We conclude that the decrease in EGF-binding capacity of the NGFdifferentiated PC12 cells (Fig. 1) is due to a reduction in the number of cell surface (plasma membrane) receptors, which, in turn, is caused by decreased receptor biosynthesis (Fig. 2). Thus, the NGF-induced loss of EGFR from PC12 cells fulfills the criteria for “heterologous down-regulation” (Sibley and Lefkowitz 1985). The process of NGF-induced heterologous down-regulation of EGFR can be visualized by a novel bio-imaging method that uses EGF labeled with a near-infrared dye (EGF-NIR). The EGF molecule can be conjugated with IRDye800CW at the amino-terminal of the protein due to a free amino group available (Cohen et al. 2012). After reaction with the IRDye800CW, the EGF-NIR was purified by gradient HPLC
chromatography (Fig. 3a), yielding a homogenous single peak as confirmed by SDS-PAGE electrophoresis and NIR visualization (Fig. 3a-inset). EGF-NIR specifically and selectively bound to EGFR-expressing A431 cells (Fig. 3b). The intensity of the EGF-NIR signal reflected EGFR levels (Cohen et al. 2012, 2013; Cohen and Lazarovici 2013a). Using NGFdifferentiated PC12 cultures, we demonstrated a significant 85 % reduction in EGF-NIR specific binding to the differentiated cells (Fig. 3c), indicative of a significant heterologous down-regulation of EGFR measured by optical imaging (Cohen and Lazarovici 2013b). In view of the above observation that the NGF-induced heterologous down-regulation of EGFR is temporally correlated with NGF-induced neurite outgrowth, we cultured in the presence or absence of 50 ng/ml NGF, enhanced green fluorescence protein (GFP) expressing PC12 cells for 7 days either in conventional two-dimensional (2D) cultures, PC12 cells embedded in a three-dimensional collagen hydrogel (3D cultures) (Arien-Zakay et al. 2009) and PC12 cells grown as suspension cultures (Lazarovici et al. 1997a). Under 3D conditions using collagen hydrogels as scaffolds, GFP-PC12 cells proliferated, formed aggregates, and respond to NGF by exuberant outgrowth of neurites of different lengths and complexity, very similar to wild-type PC12 cells (Arien-Zakay et al. 2009). In 2D and 3D cultures, the 50 ng/ml NGF-treated PC12 cells extend neurites, and in suspension, they aggregate but do not grow neurites (Fig. 4). Following 7 days of culture under the various conditions mentioned above, the cells were harvested and analyzed by Western blotting using an anti-EGFR antibody to measure EGFR down-regulation and an antineurofilament-M antibody (NF-M) to measure the increased expression of this cytoskeleton protein as marker for the neuronal differentiation of the cells. As seen in Fig. 4, NGFinduced heterologous down-regulation of EGFR under all three different growth conditions occurred independently of neurite outgrowth, which was well developed under 2D and 3D conditions but absent in cells growing in suspension culture. PC12 cells grown in suspension in a spinner flask
Fig. 2 Cross linking of 125I-EGF (a), tyrosine autophosphorylation (b) and 35S-methionine incorporation (c) into EGFR in control PC12 cells and cells after 6 days of differentiation with 50 ng/ml NGF. a Membranes of PC12 cells and A431 cells were incubated for 60 min with 5 nM 125IEGF on ice in the presence of 0.5 μM disuccinimidyl suberate. b Membranes of PC12 cells were incubated for 15 min at 40 °C in a kinase reaction buffer and thereafter incubated for 2 min at 4 °C with 2 μCi of
γ-32P-ATP 50 μM unlabeled ATP. c PC12 cells were incubated for 18 h in methionine-free DMEM containing 60 μCi/ml L-35S-methionine, and plasma membranes were prepared. In all experiments, the membranes were solubilized with RIPA buffer, and EGRR were immune-precipitated and analyzed by 7.5 % SDS-PAGE. The autoradiograms of the fixed dried gels are presented. A431 carcinoma cells were used as control. EGFR is represented by the band of 170 kD
J Mol Neurosci
aggregated into macroscopic organoids as evident by morphological and histological examinations (Fig. 4), as previously documented using different types of bioreactors (Manley and Lelkes 2006). In contrast, under all conditions, NGF increased the level of the NF-M (Fig. 4) and neuronal tubulin and microtubule-associated proteins (data not shown), indicating NGF-triggered cytoskeleton differentiation under all growth conditions. These findings suggest that NGF-induced heterologous down-regulation of EGFR is a molecular event accompanying neuronal differentiation and that this effect is independent of PC12 cell adhesion and the organization of the extracellular matrix (2D vs 3D) and can occur in the absence of neurite outgrowth (Lazarovici et al. 1997a).
NGF-induced Heterologous Down-regulation of EGFR is TrkA and Ras Dependent
Fig. 3 Preparation and isolation of EGF-NIR probe and its use for visualization of NGF-induced heterologous down-regulation of the EGFR in PC12 cells. a HPLC separation of EGF-NIR. Solid line represents absorbance at 226 nm and dotted line indicates the acetonitrile gradient. Inset shows 12 % SDS-PAGE analysis of 10 μg of EGF-NIR scanned with Odyssey near-infrared scanner and of unmodified EGF stained with Coomassie blue. b The specificity and selectivity of EGF-NIR probe binding measured by NIR imaging. A431 carcinoma cells were incubated for 15 min at 37 °C with 7 ng/ml EGF-NIR in the presence (EGF/EGF-NIR) or absence (EGF-NIR) of 100 ng/ml EGF. Competition experiments with 500 ng/ml Neuroregulin 1 (NRG1/EGF-NIR) were also conducted. In control experiments (NIR-Dye), the cultures were incubated with 7 ng/ml NIR-Dye to evaluate non-specific labeling of the cells by comparison to untreated cultures (untreated). The NIR intensity at 800 nm was estimated under identical conditions for all cultures, and the mean±SD (n=9) is presented. Upper insets, NIR scans; lower insets, phase-contrast photomicrographs of the cultures, *p< 0.05 versus NIR-Dye; **p
