Fractalkine promotes chemotaxis of bone marrow-derived mesenchymal stem cells towards ischemic brain lesions through Jak2 signaling and cytoskeletal reorganization Yuan Zhang1, Jian Zheng2, Zhujuan Zhou2, Huadong Zhou3, Yanjiang Wang3, Zili Gong2 and Jie Zhu3 1 Department of Orthopedics, Xinqiao Hospital, Third Military Medical University, Chongqing, China 2 Department of Neurology, Xinqiao Hospital, Third Military Medical University, Chongqing, China 3 Department of Neurology, Daping Hospital, Third Military Medical University, Chongqing, China

Keywords bone marrow-derived mesenchymal stem cells; chemotaxis; cytoskeleton; fractalkine; Jak2 Correspondence J. Zhu, Department of Neurology, Daping Hospital, Third Military Medical University, Chongqing 40042, China Fax: +86 23 68757852 Tel: +86 23 68757853 E-mail: [email protected] (Received 18 September 2014, revised 1 December 2014, accepted 27 December 2014) doi:10.1111/febs.13187

The fractalkine (FKN)–CX3CR1 (FKN receptor) axis reportedly plays an important role in the progression of many neural pathologies. However, its role in the recruitment of bone marrow-derived progenitor cells for neurogenesis remains elusive. The chemokine-based mechanism underlying the migration of bone marrow-derived mesenchymal stem cells (BMSCs) was investigated in a double-chamber transmigration model with recombinant FKN and endogenous FKN extract, and the results confirmed the involvement of FKN in migration. This chemotactic response was CX3CR1dependent and FKN-sensitive. Western blotting, immunoprecipitation and transmigration assays revealed that the Janus kinase (Jak)2–signal transducer and activator of transcription (Stat)5a–extracellular signal-related kinase (ERK)1/2 pathway was activated by FKN. Confocal laser scanning microscopy was used to demonstrate cytoskeletal reorganization caused by remodeling of the surface receptor integrin a5b1, intracellular phosphorylation of Fak and Pax, and upregulation of intercellular adhesion molecule-1 during BMSC migration. Moreover, significant inhibition of signaling and migration was detected after treatment of cells with Jak2-interfering RNA or the antagonist AG490. In addition, the results of a fluorescence immunohistochemical analysis of an in vivo chemotactic model, developed via transplantation of BMSCs into transient middle cerebral artery-occluded rats, were consistent with the in vitro results. These findings suggest that FKN activates Jak2–Stat5a–ERK1/2 signaling through CX3CR1, thereby triggering integrin-dependent machinery reorganization to allow chemotactic migration of BMSCs towards an ischemic cerebral lesion.

Introduction Stroke is a devastating disorder that is secondary to cardiac ischemia as a major cause of mortality and

disability worldwide [1]. Cerebral infarctions are generally considered to be incurable, owing to irreversible

Abbreviations a-FKN, membrane-anchored fractalkine; BMSC, bone marrow-derived mesenchymal stem cell; CLSM, confocal laser scanning microscopy; DMEM, Dulbecco’s modified Eagle’s medium; ERK, extracellular signal-related kinase; Fak, focal adhesion kinase; FKN, fractalkine; H-R, hypoxia–reoxygenation; ICAM-1, intercellular adhesion molecule-1; I-R, ischemia–reperfusion; Jak, Janus kinase; MAPK, mitogen-activated protein kinase; MCA, middle cerebral artery; MCAO, middle cerebral artery occlusion; Pax, paxillin; PTEN, phosphatase and tensin homolog deleted on chromosome 10; qPCR, quantitative real-time PCR; r-FKN, recombinant fractalkine; SDF-1a, stromal cell-derived factor-1a; s-FKN, soluble fractalkine; shRNA, small hairpin RNA; Stat, signal transducer and activator of transcription; TTC, triphenyl tetrazolium chloride.

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neuronal necrosis and multiple neurochemical cascades that are triggered by ischemia–reperfusion (I-R) [2,3]. Fortunately, current chemokine-based approaches provide hope that lost neurons can be replenished and that communication between neural networks can be restored. Chemokines form a superfamily of glycoproteins that induce directional chemotaxis of cells via corresponding receptors. Fractalkine (FKN, also known as CX3CL1), one of the few chemokines that is constitutively expressed in the central nervous system [4], interacts with its specific seven-transmembrane, G-protein-coupled receptor CX3CR1. FXN participates in multiple neuropathologies involving inflammation, degeneration, angiogenesis, and tumorigenesis, by improving the migration and adhesion of CX3CR1positive cells [5–7]. Endogenous and exogenous nonneural cells have the potential to migrate and accumulate in ischemic regions through an incompletely known mechanism. These cells also provide support for the formation of neurons and associated cells, and maintain tissue homeostasis during neurogenesis [8]. For example, one type of macrophage that characteristically expresses chondroitin sulfate proteoglycan can be induced to attach to the vascular wall and migrate towards ischemic brain lesions via monocyte chemoattractant protein-1 and its receptor CCR2 [9]. Pluripotent cells, primarily bone marrow-derived mesenchymal stem cells (BMSCs), show therapeutic superiority, because they specifically localize to injury sites and show multilineage differentiation. Some in vivo experimental data suggest that BMSCs, engrafted either systemically or locally, migrate towards the ischemic hemisphere and improve functional outcomes via a stromal cell-derived factor-1a (SDF-1a)–CXCR4 axis-dependent mechanism [10,11]. Another FKN–CX3CR1 axis has also been reported to be involved in chemotaxis towards ischemic injury, but most studies have focused on its role in recruiting proinflammatory cells, such as leukocytes, monocytes, and lymphocytes, and its contribution to neuroinflammation [3,9,12]. For example, neurons and astrocytes cause microglia to exert neuroprotective or neurotoxic effects through an interaction between FKN and CX3CR1 [13]. Up to now, there have been few studies on the role that stem cells play in chemotactic migration, especially stem cells employed in transplantation therapy. In a previous study, we verified that the FKN– CX3CR1 axis participates in the directional migration of systemically grafted BMSCs to an ischemic brain lesion. We also showed histopathological evidence of the codistribution of BMSCs with endogenous neurons 892

and astrocytes in ischemic boundary zones showing high expression of FKN. In addition, RNA interference targeting CX3CR1 significantly inhibited cell migration [14]. However, the molecular mechanisms underlying these effects remain unclear. Given the controversial role of FKN in the diversified outcomes of stroke, this issue is of high importance. We hypothesized that the FKN–CX3CR1 axis shares certain G-protein-coupled receptor signaling pathways and reassembles the adhesion machinery to modulate the migration of BMSCs. Therefore, we further investigated the role of the FKN–CX3CR1 axis and its effects on downstream molecules and cytoskeletal components by using both in vitro and in vivo models.

Results I–R increases the expression of FKN and CX3CR1 A rat middle cerebral artery (MCA) occlusion (MCAO) model was employed to replicate a local cerebral infarction that mimicked pathological I-R at a predetermined time and to illustrate the characteristics of FKN expression in ischemic brains ex vivo. After occlusion for 2 h, the spatial distribution of the ischemic core and peri-ischemic region could be grossly recognized via triphenyl tetrazolium chloride (TTC) staining, and differential production of FKN within the ischemic core could be differentiated by microscopic immunohistology (Fig. 1A,B). A previous study reported that cerebral FKN was primarily produced by neurons and hypoxic astrocytes in the cortex [9]; however, our histopathological results indicated that FKN was notably upregulated in the peri-ischemic region 12 h after MCAO. At 24 h, FKN peaked at a value of 2.7-fold the level of FKN at MCAO onset (P < 0.01), and this augmentation was maintained for 72 h (Fig. 1C). The corresponding contralateral region showed compensatory elevation of FKN 12 h after IR, and this increase was sustained for 72 h (P < 0.05). Previous studies showed that FKN functioned in both the membrane-anchored form (a-FKN, for adhesion) and the released soluble form (s-FKN, chemoattraction), and the expression of these two forms might occur in different patterns [7]. Hence, in this study, ELISA-based quantification of these two forms was employed to discriminate their composition within an endogenous cerebral filtrate. The results revealed that the level of s-FKN was markedly increased in the entire central ischemic block, accounting for nearly 80% of the total FKN content 48 h after I-R onset. However, a mild increase in the level of a-FKN was also noted (Fig. 1D). FEBS Journal 282 (2015) 891–903 ª 2015 FEBS

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Fig. 1. Expression profiles of FKN in ischemic brain tissue and its receptor CX3CR1 on BMSCs. (A) The gross appearance of TTC-stained brain tissue shows the morphological distribution of the ischemic core and the peri-ischemic region following occlusion of the MCA for 2 h. (B) The morphological difference of FKN expression within the ischemic core was determined through anti-FKN immunohistochemistry (9 100). And the striatum and cortex were labeled as dark and light brown areas, respectively. (C) The spatiotemporal expression of FKN in different groups was observed at various reperfusion time points after MCAO for 2 h. The sham control is the group in which the rats underwent the same surgical operation but without insertion of the monofilament nylon suture. *P < 0.05 as compared with the sham control; #P < 0.05 as compared with corresponding regions of the MCAO sample collected at 6 h; §P < 0.05 as compared with the contralateral region at the same time points after MCAO. (D) Two constitutive forms of FKN, s-FKN and a-FKN, were quantified via ELISA at 450 nm. *P < 0.05 as compared with the sham control. (E) Gene and protein expression of CX3CR1 in BMSCs subjected to different treatments, determined via qPCR, western blotting, and anti-CX3CR1 immunohistochemistry (9 200). *P < 0.05 and #P < 0.05 as compared with normoxia and CX3CR1 shRNA + r-FKN-treated samples, respectively. Con-shRNA, control shRNA; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

The CX3CR1 expression profile in BMSCs cultured in vitro was investigated to test the response of the BMSCs to physical treatment [hypoxia–reoxygenation (H-R)] and biochemical stimulation [chemokine FEBS Journal 282 (2015) 891–903 ª 2015 FEBS

domain of recombinant FKN (r-FKN)]. We observed markedly increased CX3CR1 expression following exposure of BMSCs toH-R for 2 h or incubation of BMSCs with r-FKN at 100 ng
mL 1, as demonstrated

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by the results of quantitative real-time PCR (qPCR), western blotting of cell lysates, and whole-cell immunohistochemistry. When BMSCs were pretreated with CX3CR1 small hairpin RNA (shRNA), the upregulation of CX3CR1 caused by r-FKN stimulation was significantly inhibited at the mRNA level (by ~ 60%) (Fig. 1E).

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The FKN–CX3CR1 axis contributes greatly to BMSC migration We investigated whether the FKN–CX3CR1 axis modulated chemotactic cell migration by using agonist and antagonist strategies. The migration of cells in a transwell assay was quantified by the use of fluorescence values from the bottom of the transwell chamber. The results revealed a dose-dependent increase in the number of BMSCs following r-FKN stimulation at an initial concentration of 10 ng
mL 1 for 30 min, and the maximal effect was observed at 100 ng
mL 1 (P < 0.01). We also transfected CX3CR1 shRNA into cultured BMSCs, and exposed these cells to r-FKN. We found that the blockade of surface CX3CR1 significantly inhibited the migration of BMSCs into the bottom chamber, by as much as 78% (Fig. 2A). Verifying that the ability of exogenous FKN to stimulate BMSC migration is also present in the ischemic brain, the filtrate from the ischemic region of the brain markedly promoted the migration of BMSCs after I-R for 24 h (Fig. 2B), and CX3CR1 knockdown by shRNA also decreased the number of migrated cells (~ 40%, P < 0.05). Notably, the blockade efficiency of CX3CR1 shRNA did not fully replicate the results of the in vitro assay, suggesting that other chemokines or factors are present in the filtrate, possibly including macrophage inflammatory protein-1a, SDF-1a, monocyte chemoattractant protein-1, and CXCL10 [11,15].

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FKN–CX3CR1 interaction activates the Janus kinase (Jak)2–signal transducer and activator of transcription (Stat)5a–extracellular signal-related kinase (ERK)1/2 signaling pathway

Fig. 2. Quantitative migration of BMSCs towards exogenous and endogenous FKN gradients in vitro. (A) BMSCs were exposed to intermittent gradients of r-FKN, and fluorescence-based quantitation of the migration of BMSCs in transwell chamber assay was performed. The effect of CX3CR1 shRNA on cell migration, with or without r-FKN treatment at 100 ng
mL 1, was also determined. *P < 0.05 and #P < 0.05 as compared with non-FKN treated samples and CX3CR1 shRNA + r-FKN-treated samples, respectively. (B) Quantitative analysis of migrated BMSCs was performed in cells exposed to the cerebral filtrates from MCAOtreated brains collected at consecutive reperfusion time points. The effect of CX3CR1 shRNA on cell migration, with or without filtrate collected after reperfusion for 48 h, was also examined. §P < 0.05, †P < 0.05 and ‡P < 0.05 as compared with non-filtrate-treated samples, CX3CR1 shRNA  filtrate-treated samples, and ConshRNA  filtrate-treated samples, respectively. Con-shRNA, control shRNA.

Jak–Stat signaling was analyzed to explore the participation of specific downstream molecules in BMSCs treated with FKN. The phosphorylation of Jak2 was first examined after chemokine stimulation (Fig. 3A). Phosphorylation at Tyr317 of Jak2 showed a rapid increase, with a transient peak from 1 min to 5 min during r-FKN treatment (phosphorylation at Tyr221 and Tyr1007 was not investigated). Additionally, Stat5 phosphorylation at Tyr694 was detected at 5 min, peaked at 15 min, and lasted for

30 min. The total and phosphorylated levels of Jak1, Jak3, Stat1, Stat2, Stat3 and Stat6 remained unchanged under our experimental conditions (data not shown). To further determine which Stat5 isoform was phosphorylated, we employed an antibody against Stat5a or an antibody against Stat5b to immunoprecipitate protein extracts derived from cells incubated with r-FKN for 15 min or 30 min, and then blotted the

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Fig. 3. Activation of signal transduction triggered by FKN stimulation. (A) Western blotting analyses of Jak2, Stat5, Akt, ERK1/2 and PTEN were conducted after treatment of BMSCs with r-FKN (100 ng
mL 1) at consecutive time points. Immunoprecipitated PTEN from lysates of r-FKN-induced BMSCs was employed to determine the phosphatase activity of PTEN, by quantifying the phosphoinositide product dephosphorylated from phosphatidylinositol 3,4,5-triphosphate substrate, via a competitive ELISA.To further investigate which isoform of Stat5 was phosphorylated during BMSC migration, BMSCs were incubated with r-FKN at 100 ng
mL 1 for 15 min (left two lanes) or 30 min (right two lanes), and the protein extract was subjected to immunoprecipitation with antibodies against Stat5a and Stat5b and then blotting with antibody against pStat5. (B) BMSCs were pretreated with CX3CR1 shRNA and control shRNA, or Jak2 shRNA, control shRNA, and AG490, and the inhibition of the phosphorylation of Jak2 (at 5 min), Stat5 (at 10 min) and ERK1/2 (at 15 min) was assessed following incubation with r-FKN (100 ng
mL 1). (C) BMSCs were incubated with Jak2 shRNA or Jak2 inhibitor AG490 (100 lM) overnight, and then stimulated with r-FKN for 15 min. Western blotting analysis suggested that the phosphorylation of Stat5 and ERK1/2 was significantly inhibited. (D) The effects of an AG490 gradient and Jak2 shRNA on BMSC migration were determined in transwell chamber assays. Cells were treated with AG490 (100 lM) or Jak2 shRNA overnight, and then stimulated with r-FKN at 100 ng
mL 1 to trigger migration. Knockdown efficacy of Jak2 was shown by screening protein levels after virus infection. *P < 0.05 and #P < 0.05 as compared with FKNtreated samples and control shRNA + r-FKN-treated samples, respectively. Con-shRNA, control shRNA; GAPDH, glyceraldehyde-3phosphate dehydrogenase; IB, immunoblotting; IP, immunoprecipitation; WB, western blotting.

immunoprecipitated proteins with antibody against phospho-Stat5. The results demonstrated that Stat5a was predominantly phosphorylated following r-FKN stimulation. FEBS Journal 282 (2015) 891–903 ª 2015 FEBS

The expression of two other common participants in cellular migration, ERK1/2 and Akt, was also analyzed following Jak–Stat activation. The results demonstrated that ERK1/2 phosphorylation began at

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5 min and persisted for > 30 min. Akt remained unphosphorylated throughout the stimulation process. Quantification by western blotting and an ELISAbased phosphatase activity assay for phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a key regulator of the phosphoinositide 3-kinase–Akt pathway, indicated that neither protein expression nor phosphatase activity showed a significant change after r-FKN stimulation for 60 min; this result suggests that PTEN may not be involved in the continued dephosphorylation of Akt (Fig. 3A). To further elucidate the signaling of the CX3CR1– Jak–Stat and ERK pathways in our model, we examined the effects of CX3CR1 shRNA on downstream signaling, and found that the phosphorylation levels of Jak2, Stat5 and ERK1/2 were prominently blocked by CX3CR1 shRNA, although there was a minimal effect on total protein expression (Fig. 3B). Furthermore, AG490 (a Jak2 inhibitor) and Jak2 shRNA were employed to interfere with signal transduction mediated by Jak2. The levels of phosphorylated Stat5 and ERK1/2 were significantly decreased to different extents following overnight treatment with 10 lM AG490 or incubation with Jak2 shRNA for 24 h (Fig. 3C). Therefore, we re-examined the migration of BMSCs in response to r-FKN following treatment with AG490 and Jak2 shRNA (Fig. 3D). AG490 treatment (10 lM)

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significantly reduced BMSC migration, and 100 lM AG490 completely abolished the FKN-induced increase in cellular migration. A similar result regarding BMSC migration was obtained following Jak2 shRNA treatment, but the amount of migration was smaller than with AG490 treatment (~ 50%, P < 0.05). These data suggest that Jak2 signal transduction is required for BMSC migration in response to FKN. FKN–CX3CR1 triggers cytoskeletal reorganization via Jak2–Stat5a transduction Cell transmigration is dominated by complicated migration machinery between the transmembrane integrin receptor and the cell substratum interface. Therefore, we focused on the differential activation of components of this machinery, including integrin, focal adhesion kinase (Fak), paxillin (Pax), and intercellular adhesion molecule-1 (ICAM-1). The effects of r-FKN and Jak2 inhibitors on integrin remodeling were determined with confocal laser scanning microscopy (CLSM), and different integrin clustering statuses were examined on the cell surface. In particular, remodeling of integrin a5b1 was prominently activated after r-FKN stimulation, as demonstrated by strong spot/fiber-like fluorescence, but AG490 and Jak2 shRNA notably inhibited this remodeling (Fig. 4A).

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Fig. 4. Cytoskeletal reorganization induced by the FKN–CX3CR1 interaction. (A) BMSCs were incubated with DMEM or blocked with Jak2 shRNA/AG490 (100 lM) overnight, and this was followed by r-FKN treatment at 100 ng
mL 1 for 30 min.Integrin a5b1 remodeling on the cell surface was subsequently determined via CLSM at 550 nm. Integrin a5b1 and cell nuclei were labeled with rhodamine (in bright red) and Hoechst-33 342 (in blue), respectively (scale bar: 50 lm). (B) Western blotting analysis was performed to detect the phosphorylation of Fak and Pax, and ICAM-1 protein expression, in BMSCs upon exposure to r-FKN (100 ng
mL 1) at consecutive time points. (C) Inhibition of the phosphorylation of Fak and Pax was observed when BMSCs were treated with Jak2 shRNA/AG490 (100 lM) following subsequent r-FKN stimulation. Knockdown efficacy of Jak2 was shown by screening protein levels after virus infection. Con-shRNA, control shRNA; GAPDH, glyceraldehyde-3phosphate dehydrogenase.

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Western blotting results showed that stimulation with 100 ng
mL 1 r-FKN for 30 min significantly increased the expression of Fak phosphorylated at Tyr397, and this dynamic increase lasted for 60 min. Consequently, Pax phosphorylation at Tyr118 was significantly activated 30 min after r-FKN treatment. ICAM-1 protein expression increased at an early stage, 5 min after r-FKN incubation (Fig. 4B). However, Jak2 inhibitors (AG490 or shRNA) diminished the increase in both Fak and Pax phosphorylation in response to FKN (Fig. 4C), which suggests that their activation was dependent on Jak2 but also relied on two independent pathways downstream of Fak and Pax. These data indicate that Jak2–Stat5a signaling mediates the migration of BMSCs in response to FKN by reorganizing the cytoskeletal system. Jak2 mediates the chemotactic migration of transplanted BMSCs towards ischemic brain lesions An in vivo BMSC transplantation experiment was conducted in a rat MCAO model to verify the in vitro results. Frozen coronal brain slices were processed for immunofluorescence analysis with CLSM on the first day and third day after transplantation, and the migration of transplanted BMSCs in ischemic brains was investigated. The results indicated that GFPlabeled BMSCs were distributed throughout the ischemia-damaged brain in the ipsilateral hemisphere of recipient rats, including the cortex and striatum. However, the majority of the cells were located in the ischemic boundary region, where homogeneous FKN immunoreactivity was strongly increased, as observed through double immunolabeling with GFP and Cy3. The BMSCs that migrated towards the ischemic region 3 days after cell transplantation were distinctly more abundant than the BMSCs that migrated on the first day. In particular, the BMSCs transduced with the Jak2 shRNA construct were significantly less abundant in the corresponding ischemic area than in the control shRNA transplantation groups on the first and third days after transplantation (Fig. 5A). According to a well-established method [14], the number of GFP-positive BMSCs distributed in the peri-ischemic region was assessed by counting the average number of cells in five equally spaced sections in each brain block. The results showed that the numbers of directionally migrated cells at 24 h and 72 h after cell transplantation were 16.4-fold and 28.3-fold higher than in the MCAO sham group. In particular, the BMSCs that migrated in the first 24 h accounted for 57.8% of the total number of cells that accumulated FEBS Journal 282 (2015) 891–903 ª 2015 FEBS

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throughout the observation period. The chemotactic capability of the BMSCs was significantly impaired by Jak2 shRNA at both 24 h and 72 h (Fig. 5B).

Discussion In this study, the molecular mechanism by which transplanted multipotent cells restore infarcted cerebral microenvironments was studied by examining the specific role and molecular mechanism of FKN and its receptor, CX3CR1, in the migration of transplanted BMSCs towards ischemic brain lesions. We obtained definitive evidence that: (a) the increased expression of endogenous FKN in an ischemic brain shows defined spatiotemporal characteristics – the action of CX3CR1 on BMSCs is hypoxia-responsive and FKN-sensitive, and initiates intracellular biochemical changes and extrinsic mobility; (b) CX3CR1 triggers signal transduction via Jak2–Stat5a and the downstream activation of the ERK1/2 pathway, and these pathways are required for the chemotactic migration of BMSCs in response to FKN; and (c) the interaction between FKN and CX3CR1 triggers substantial cytoskeletal reorganization, including integrin a5b1 remodeling, Fak/Pax phosphorylation, and ICAM-1 upregulation, and this reorganization drives the chemotactic migration of BMSCs towards ischemic brain lesions. In this study, a well-developed MCAO model was first employed to determine the expression pattern of FKN in the ischemic brain from two aspects. Spatiotemporally, endogenous FKN is rapidly accumulated in both the ipsilateral lesion and contralateral brain, beginning 12 h after the onset of I-R. Consistent with a previous report, our preliminary result also suggested that the ischemic core contributed little to the total pattern of FKN in the ischemic brain, owing to progressive necrosis and intracerebral cyst formation [16]. We believe that the major contribution to the increase in FKN mRNA comes from the ipsilateral peri-ischemic region, followed by the contralateral brain. The expression profile of FKN was also validated in our transwell chamber migration assay with r-FKN at different concentrations and an endogenous FKN extract at predetermined times following MCAO. The majority of the increase in FKN is attributable to s-FKN, which is released from cell surfaces through proteolytic cleavage and behaves as a chemotactic factor. The level of a-FKN, which acts as an adhesion molecule, was also increased slightly in our H-R samples, which may have occurred to enhance local trafficking and crosstalk between neural cells, such as neurons, astrocytes, and microglia [17], and improve the neuroregenerative response.

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There is accumulating evidence that FKN promotes the activation of neural and non-neural cells and their migration to lesions through interactions with CX3CR1. Increased numbers of leukocytes, macrophages, natural killer cells and T lymphocytes play critical roles in the clearance of necrotic tissue, the regulation of inflammation, and the promotion of functional recovery [3,9,12]. One study explored the mechanism of FKN/CX3CR1-mediated microglial activation, and found that hypoxia induced microglial proliferation and cytokine secretion through p38 mitogen-activated protein kinase (MAPK)/protein kinase C [18]. Another study demonstrated monocyte chemotaxis in response to FKN, and revealed that activation of the cytoplasmic protein tyrosine kinase Syk and the formation of cell protrusions reorganized the F-actin cytoskeleton [19]. Furthermore, Cambien et al. identified phosphoinositide 3-kinase and members of the MAPK family, including ERK1/2, p38 MAPK, and c-Jun N-terminal kinase 1, as signaling components leading to cell adhesion to fibronectin in response to soluble FKN, by using monocytic cell 898

Fig. 5. Fluorescence immunohistochemical analysis of BMSCs that migrated towards the ischemic region in vivo. (A) The colocalization of BMSCs (GFP-labeled) and endogenous FKN (Cy3labeled) in the peri-ischemic area was determined through double-labeling under CLSM at 500 nm, 1 or 3 days after cells had been transplanted into MCAO rats (scale bar: 50 lm). (B) The number of GFP-positive cells in the ischemic brain was evaluated according to the average number of cells in five equally spaced sections in each brain block, and the difference in the numbers of migrated of BMSCs between different groups are summarized in the chart. *P < 0.01 as compared with the BMSC-transplanted MCAO sham control; #P < 0.05 as compared with control shRNA treatment at the time points of 24 h and 72 h after cell transplantation; §P < 0.05 as compared with control shRNA treatment at 24 h after cell transplantation. ConshRNA, control shRNA.

lines [20]. During the acute inflammation phase of atherosclerosis, soluble FKN is shed from the endothelium following I-R, and acts via CX3CR1 to promote neutrophil adhesion through activation of the Jak–Stat pathway [21]. This signaling, in addition to MAPK/ERK kinase/ERK1/2 phosphorylation, has also been shown to be involved in the SDF-1a/ CXCR4-mediated homing of BMSCs to the breast cancer milieu [22]. These studies inspired our current hypothesis. On the basis of our preliminary data concerning differences in mRNA expression levels among Jak isoforms, we conducted systemic immunoblotting, and found that Jak2– Stat5a signaling was involved in BMSC migration. In our supplementary studies, the expression patterns of the Jak and Stat isoforms were shown to be consistent with their involvement in BMSC migration. Additionally, two participants in common mechanisms of cell migration, ERK1/2 and Akt, were examined, and the results confirmed the role of ERK1/2 in BMSC migration. Cell migration relies on forces generated by dynamic machinery between integrins and the cell FEBS Journal 282 (2015) 891–903 ª 2015 FEBS

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substratum interface [23]. This study evaluated cytoskeletal reorganization by determining the expression of integrin, Fak, Pax, and ICAM-1. Integrins are heterodimeric transmembrane proteins involved in cell–cell and cell–extracellular matrix interactions. The formation of integrin clusters is a critical step in the transduction of extracellular stimuli and the triggering of intracellular structural or biochemical changes [24]. Previous studies suggested that specific integrins are expressed on neural cells, and significant alterations in cellular adhesion receptors and their ligands occur under conditions of cerebral ischemia. For example, the a6-subunit and b1-subunit of integrin are specifically responsible for the recruitment of microglia to disease-affected brain regions and the enhanced adhesion of neurons in situ [25]. We determined the activation of integrin a5b1, and we found that remodeling of a5b1 was a prominent trait of BMSCs following FKN treatment. Fak and Pax, which colocalize with the integrin-enriched focal adhesion contact site, showed significantly increased phosphorylation after FKN stimulation. Therefore, activated integrin, Fak and Pax form a complex that promotes the focal contact structure responsible for cell migration. Furthermore, we examined the effects of CX3CR1 shRNA, Jak2 shRNA and AG490 (a Jak2 antagonist) on molecular and cytoskeletal activation. The antagonist effect of AG490 has been well documented in previous studies [20,21], and the efficacy of suppression by shRNA was confirmed through real-time PCR and western blotting assays in vitro. We found that the phosphorylation levels of Jak2, Stat5 and ERK1/2 and the protein expression of ICAM-1 were markedly decreased to different extents by CX3CR1 shRNA, AG490 and Jak2 shRNA treatment. The morphological and quantitative results also suggest that Jak2 shRNA-transfected BMSCs have an impaired ability to migrate into FKN-rich regions both in vitro and in vivo. The neuroprotective or neurotoxic role of FKN has been debatable until now. Previous studies showed that the administration of exogenous FKN to rodents with experimentally induced stroke [26] and plasma FKN to stroke patients [27] was positively associated with a smaller infarction size and fewer neurological deficits. Conversely, genetically FKN-deficient and CX3CR-deficient mice (FKN / and CX3CR1 / ) are less susceptible to ischemic insult during an early stage [28,29]. This work elucidates some of the mechanisms underlying grafted cell chemotaxis, and provides insights into the role of FKN from a non-inflammatory perspective. Additional studies might be required FEBS Journal 282 (2015) 891–903 ª 2015 FEBS

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to broaden our knowledge of the exact mechanism that drives BMSC chemostaxis towards ischemic brain lesions, by determining the full influence of phosphorylation sites (e.g. Tyr221 and Tyr1007 of Jak2, Thr202 of ERK1/2, and Tyr925 of FAK) on signal transduction. In conclusion, we provide experimental evidence that the FKN–CX3CR1 chemokine axis triggers the activation of Jak2–Stat5–ERK1/2 signaling and the cytoskeletal reorganization mediated by integrin a5b1, and thereby promotes the directional migration of BMSCs towards an ischemic cerebral lesion. Therefore, elevated or prolonged expression of certain chemokines by genetic engineering of BMSCs may be one of the critical targets for novel therapeutic approaches for stroke therapy.

Experimental procedures Ethical statement The Research Council and Ethics Committee of the Third Military Medical University (Chongqing, China) approved the study protocol. Animals were housed and treated in accordance with the Guide for the Care and Use of Laboratory Animals published by the United States National Institutes of Health.

Materials and reagents r-FKN (chemokine domain) was obtained from R&D Systems (Minneapolis, MN, USA). The following antibodies were used in this study: antibodies aganist Jak, Stat, Akt, ERK, Fak, and Pax and their phosphorylated products were obtained from Cell Signaling (Boston, MA, USA); antibodies against ICAM-1 and PTEN were obtained from Abcam (Cambridge, UK); and antibodies against integrin, GFP and FKN were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). AG490 (a Jak2 inhibitor) was obtained from Calbiochem (Billerica, MA, USA). Dulbecco’s modified Eagle’s medium (DMEM) and FBS were obtained from Gibco (Boston, MA, USA). The protease inhibitor PMSF, the RNA extraction reagent Tripure, a Percoll gradient and Hoechst-33342 were obtained from Sigma (St Louis, MO, USA). Protein extraction RIPA buffer was obtained from Cell Signaling. The PTEN activity ELISA kit was obtained from Echelon Biosciences (Salt Lake City, UT, USA).

Cell isolation, culture, and treatment BMSCs were isolated and cultured in accordance with a previously published report [30]. Briefly, bone marrow was harvested from the femurs of Wistar rats (Laboratory Ani-

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mal Center, The Third Military Medical University, China) and isolated by use of a Percoll gradient. The upper cell-containing phase was initially plated in DMEM-F12 supplemented with 10% FBS at 37 °C in a humidified atmosphere containing 5% CO2. Cell passages 3–5 were used in subsequent experiments. Hypoxia was induced by exposing BMSCs to a mixture of 5% CO2, 94% N2 and 1% O2 gas in an airtight chamber in the presence of DMEM-F12 containing 10% FBS. The chamber containing the cell cultures was incubated for 2 h, and subsequently reoxygenated for up to 120 min.

Focal cerebral infarction model, endogenous FKN extraction, and quantification A transient MCAO model was employed to replicate ischemic brain lesions in adult Wistar rats, as described in a previous study by our group [14]. Briefly, the MCA was occluded for 120 min by use of the intraluminal filament technique to obstruct the origin of the right MCA. A 4-0 nylon monofilament suture with a 0.25-mm-diameter silicone tip was inserted into the internal carotid artery through the external carotid artery. Reperfusion was performed until 6–72 h after the occlusion was abolished through the removal of the nylon filament and tip. TTC staining and Longa’s behavioral scale were adopted to verify the occlusive efficacy. TTC was also used to distinguish ischemic and normal brain tissues and core and peri-ischemic regions. To prepare tissue extracts from the ischemic brains, the infarcted hemisphere, including the ischemic core and periischemic region, was collected in blocks and mechanically ground in a homogenizer with PBS (200 lL for 5 g of tissue). The samples were then subjected to filtration with a 0.45-lm vacuum filter (Corning, USA) to collect the extract. Endogenous FKN within the filtrate was measured with an ELISA kit (R&D Systems, USA), according to the manufacturer’s protocol.

Transwell chamber migration assay BMSCs (1 9 105 per well) were seeded on transwell filters (8-lm pores; Corning, USA) and grown to confluence. The bottom of the chamber was then filled with 600 lL per well of DMEM/0.2% BSA with or without the indicated concentrations of r-FKN at 10, 50, 100 or 200 ng
mL 1, or with brain filtrates extracted at different reperfusion time points starting from the onset of reperfusion to 72 h after MCAO. The top chamber was removed after incubation for 6 h at 37 °C, whereas the bottom chamber was subjected to centrifugation at 50 g for 5 min (Beckman Allegra X-22R centrifuge, USA). The adherent cells in the bottom chamber were quickly labeled with Hoechst-33342 (5 lg
mL 1, 5 min, 4 °C). Fluorescence values were read at 450 nm in a Thermo system (Varioskam Flash, USA) to

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calculate cell density. For inhibition assays, BMSCs were either incubated with AG490 (a Jak2 inhibitor) at 10, 50 or 100 lM for 12 h, or transfected with lentivirus for 24–48 h. The shRNA-treated cells were allowed to recover for 24 h before the migration assay.

qPCR Total RNA from brain tissue homogenates of the cerebral parenchyma in the peri-ischemic region and the contralateral hemisphere in the cerebral cortex of MCAO rats was extracted with the Tripure extraction reagent for FKN quantification. A brain that received the same MCAO surgical operation but without insertion of the monofilament nylon suture was used as the control sample. For CX3CR1 analysis, 5 9 106 BMSCs treated with H-R and s-FKN stimulation were collected. cDNA was generated from 1 lg of total RNA with the PrimeScript RT reagent kit (TaKaRa, Japan), and amplified with primers targeting FKN, CX3CR1 and glyceraldehyde-3phosphate dehydrogenase, as reported in our previous study [14]. Reaction data were collected with an ABI 7500 system (ABI, USA) and quantified with the 2 DDCt method.

Protein expression and activity – western blotting, immunoprecipitation, and phosphatase assays Three isotypes of Jak and five isotypes of Stat, Akt, PTEN, Fak, Pax and ICAM-1, as well as their phosphorylated forms, were extensively investigated to determine the signal transduction and cytoskeletal activation events related to the response of BMSCs to FKN. Total protein was extracted from 5 9 106 BMSCs with RIPA buffer [20 mM Tris/HCl (pH 7.5), 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% NP-40, 1% sodium deoxycholate, 2.5 mM sodium pyrophosphate, 1 mM b-glycerophosphate, 1 mM Na3VO4, 1 lg
mL 1 leupeptin, 1 mM PMSF]. A total of 30 lg of protein was separated by SDS/PAGE and transferred to poly(vinylidene difluoride) membranes (Millipore, USA). The proteins were sequentially hybridized with different primary antibodies and horseradish peroxidase-labeled secondary antibodies (Zhongshan, China). Protein bands were visualized with an enhanced chemiluminescence method (Amersham, USA). BMSCs were incubated with FKN (100 ng
mL 1) for 15 min or 30 min for immunoprecipitation assays. The cells were lysed in radioimmunoprecipitation assay buffer. Antibodies against Stat5a and Stat5b were added, and the cells were gently rotated at 4 °C overnight. Protein– agarose beads (Invitrogen, USA) were then added to the reaction, and this was followed by rotation for 3 h at 4 °C. After washing and centrifugation (12 000 g, 10 min), the pellet was resuspended, denatured, and run

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on a NuPAGE gel for immunoblotting with antibodies against Stat5 phosphorylated on tyrosine. To determine the protein phosphatase activity of PTEN, PTEN protein was immunoprecipitated from total protein derived from BMSC lysate with an antibody against PTEN and antibody-coupled magnetic beads, according to the manufacturer’s instructions (Miltenyi Biotechnology, MA, USA). As controls, we used an IgG isoantibody targeting nonspecific protein and the absence of any antibody. The dephosphorylation was initiated in phosphatase reaction buffer [100 mM Tris/HCl (pH 8.0), 10 mM DTT, 200 mM diC16-phosphatidylinositol 3,4,5-triphosphate] for 2 h at 37 °C. The resulting samples (100 lL) were then transferred to a 96-well plate and incubated with a phosphatidylinositol 4,5-bisphosphate detector and then with a secondary detector. The production of phosphate from the substrate was measured by a colorimetric method with absorbance at 450 nm in a Thermo system (Varioskam Flash, USA). The theoretical amount of free phosphate was calculated from the standard curve line-fit data.

Integrin reassembly and cytoskeletal reorganization CLSM was employed to examine integrin remodeling and cytoskeletal reorganization [30]. Briefly, BMSCs were cultured in 96-well plates until they reached 70–80% confluence. The cells were treated either with r-FKN (100 ng
mL 1, 30 min) alone, or with AG490 (100 lM)/ shRNA overnight followed by r-FKN stimulation. After treatment with glutaraldehyde and Triton X-100 at 4 °C for 15 min and 2 min, respectively, immunofluorescence staining was performed with a goat primary mAb against integrin a5b1 (1: 200) and rhodamine-conjugated secondary antibodies (1 : 100, Zhongshan, China). Finally, nuclei were counterstained with Hoechst-33342, and fluorescence images were obtained via CLSM at 550 nm (Leica, Germany).

Silencing of CX3CR1 and Jak2 with lentivirus shRNA in vivo shRNA-expressing lentivirus gene transfer vectors targeting CX3CR1 (CX3CR1 shRNA) and Jak2 (Jak2 shRNA) and a control (control shRNA) containing a scrambled sequence were purchased from Genechem (Shanghai, China). The interference expression constructs were cotransfected into HEK293T cells with Lipofectamine 2000 (Invitrogen, CA, USA). Lentivirus particles were collected after 48 h, and BMSCs were transduced at 37 °C in 5% CO2 for 24–48 h. The transduction efficiency was measured on the basis of the frequency of GFP positivity, and knockdown efficacy was confirmed by screening mRNA and protein levels following virus infection.

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Fractalkine-induced chemotaxis via Jak2 pathway

BMSC transplantation and immunohistochemistry At 24 h after the onset of cerebral I-R, 2 9 106 BMSCs transfected with either Jak2 shRNA or control shRNA were injected slowly into the tail vein. Rats were killed under deep anesthesia at 24 h or 72 h after transplantation. Frozen blocks of coronal brain sections were analyzed with immunohistochemistry, and the GFP label delivered in the lentivirus enabled the monitoring of the transplanted cells at 480 nm. A negative sham control for MCAO was established by performing the same surgical operation but without insertion of the monofilament nylon suture. To determine the distribution of transplanted BMSCs (GFP-positive cells), cell sections were incubated first with a goat polyclonal antibody against GFP overnight (1 : 200), and then with FITC-conjugated rabbit anti-(goat IgG) (1 : 100). The colocalization of FKN-positive and GFP-positive cells was determined by additionally staining the sections with an antibody against FKN (1 : 200) and a secondary antibody against Cy3 (1 : 500). All sections were assessed with CLSM. To determine the number of GFP-positive cells in the ischemic brain, the average number of cells in five equally spaced sections (at ~ 200-lm intervals) was determined in each brain block, and the number of GFP-positive cells was counted within the seven 2-mm-thick blocks. To reduce bias introduced by the sampling parameters, all of the sections obtained from the rats were stained for GFP simultaneously. The analysis was conducted by observers who were blinded to the treatment conditions.

Statistics All quantitative assays were performed in triplicate, and the results are expressed as the means  standard errors. Statistical significance was evaluated with an unpaired Student’s t-test for comparisons between two groups, and ANOVA followed by Tukey’s post hoc test for multiple comparisons. The significance level was set to 0.05 throughout the analysis.

Acknowledgements This work was supported by the Nature Science Foundation of China (81000507), and the Chongqing Natural Science Committee (CSTC201JJA10011 and CSTC2011JJA10079).

Author contributions Jie Zhu: designed the study. Yuan Zhang, Jian Zheng, Jie Zhu, Zhujuan Zhou, and Zili Gong: performed research. Yuan Zhang and Jie Zhu: analyzed the data

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Fractalkine-induced chemotaxis via Jak2 pathway

and conducted the statistical analyses. Yuan Zhang and Jie Zhu: wrote the article. 12

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