Interplay between Cell Migration and Neurite Outgrowth Determines SH2B1β-Enhanced Neurite Regeneration of Differentiated PC12 Cells

The regulation of neurite outgrowth is crucial in developing strategies to promote neurite regeneration after nerve injury and in degenerative diseases. In this study, we demonstrate that overexpression of an adaptor/scaffolding protein SH2B1β promotes neurite re-growth of differentiated PC12 cells, an established neuronal model, using wound healing (scraping) assays. Cell migration and the subsequent remodeling are crucial determinants during neurite regeneration. We provide evidence suggesting that overexpressing SH2B1β enhances protein kinase C (PKC)-dependent cell migration and phosphatidylinositol 3-kinase (PI3K)-AKT-, mitogen activated protein kinase (MAPK)/extracellular signal-regulated protein kinase (ERK) kinase (MEK)-ERK-dependent neurite re-growth. Our results further reveal a cross-talk between pathways involving PKC and ERK1/2 in regulating neurite re-growth and cell migration. We conclude that temporal regulation of cell migration and neurite outgrowth by SH2B1β contributes to the enhanced regeneration of differentiated PC12 cells.


Introduction
Neuronal injury and degeneration are responsible for various neurological diseases. The limited regeneration capacity restricts the recovery of neuronal damage. Thus, better understanding of the mechanisms for neuronal repair will facilitate clinical application of therapy toward neurological disorders. Peripheral nerve transection (axotomy) is often used as a neuronal injury model. During regeneration of the peripheral nervous system (PNS), cell body of the neurons must to receive appropriate signals to sustain intrinsic growth to ensure successful regeneration. Thus, the regulation of signaling cascades and downstream gene expression often determines the regeneration outcome [1,2,3]. For instance, axonal injury induces local activation and retrograde transport of extracellular signal-regulated protein kinase (ERK) [4,5,6] and c-Jun N-terminal kinase (JNK) [7,8]. A study showed that mitogen activated protein kinase (MAPK)/ERK kinase (MEK) kinase 1 (MEKK1) controls neurite re-growth by balancing ERK1/2 and JNK2 signaling after experimental injury [9]. These studies suggest that activation of JNK and ERK and their interaction with the dynein/dynactin retrograde molecular motors is required for regeneration [1,5,7]. Moreover, overexpression of constitutively activated AKT has been shown to protect motor neurons from injury-induced cell death and thus promotes axonal regeneration [10,11]. By intraperitoneally administrating vanadium compounds to stimulate the activation of phosphatidylinositol 3-kinase (PI3K)-AKT and MEK-ERK1/2 pathways, neurogenesis as well as newborn cells are increased in response to brain ischemia [12].
Cell migration is instrumental for injury-induced neurogenesis and tissue regeneration [26,27,28,29,30]. It has been shown that progenitor cells from periventricular region proliferate and migrate into the hippocampus to regenerate new neurons after ischemia, thus reduce neurological deficits in vivo [31]. Evidence suggests that activities of PLC and PKC, and intracellular Ca 2+ levels are important regulators of cell migration in the developing brain [32]. PLC is the key enzyme which mediates the balance between intracellular Ca 2+ and cellular phosphoinositide. PLC isozymes mainly localize in cytosol, and translocate to plasma membrane, where PLC hydrolyzes phosphatidylinositol 4,5bisphosphate (PIP2) into inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG), as stimulated by hormones or neurotransmitters [33]. The produced second messenger IP3 increases endoplasmic reticulum-released Ca 2+ and DAG regulates the activation of PKC. One of the PLC isozymes mainly regulated by receptor and cytosolic tyrosine kinase is PLCc1 [34,35], which expresses in lung, thymus, and highly expresses in neurons of adult rat brain [36]. In addition to growth factors-mediated chemotaxis, a study indicates that PLCc1 is an essential molecule for cell motility in integrin signaling [37], suggesting the general role of PLCc1 in cell motility. PKC isoforms are known to influence cell morphogenesis by remodeling microfilaments [38]. They have also been implicated in diverse signal propagation [39]. The ability of neurons to respond to injury, integrate signals, and remodel actin cytoskeleton determines the recovery outcome. Increasing evidence suggests that protein scaffolds are involved in temporally and spatially organizing signaling events to downstream transcriptional activity and actin remodeling.
One such scaffolding protein, SH2B1, belongs to the SH2B adaptor protein family [40,41]. SH2B1b (b splice variant) has been shown to bind to multiple receptor tyrosine kinases, including receptors for NGF, FGF, and GDNF [42,43,44,45,46,47]. SH2B1 is necessary for survival of sympathetic neurons [45]. It positively regulates the Akt/Forkhead pathway and has recently been shown to reduce oxidative stress-induced cell death in PC12 cells, hippocampal neurons and Drosophila [22,48,49]. Overexpressing SH2B1b also enhances both NGF-, FGF1-and GDNF-induced neuronal differentiation of PC12 cells [42,43,44,45]. Given its ability to promote neurite outgrowth and survival, we hypothesize that SH2B1b may play a positive role in neuronal regeneration. This study investigated the role of SH2B1b in the regeneration of injured differentiated PC12 cells.

Neuronal injury experiments and cell migration tracking
For wound healing assays, cells were seeded on collagen-coated plates at 30% confluency and differentiated using NGF (50 ng/ml) for 8 days. The medium was changed every other day. The definition of neuronal differentiation for PC12 cells is that the length of neurites is at least twice the diameter of the cell body. On day 8, cells were wounded by scraping cells with a P10 pipette tip. Live cell images of cells were taken everyday using an inverted Zeiss Axiover 135 fluorescence microscope or the Carl Zeiss Observer Z1. The averaged width of the wounded gaps was measured from 12 different locations per wounded gap per time point (also see Figure S1). At least 6 wounded gaps were quantified from three independent experiments. The width of the remaining wounded gaps was averaged and divided by the width of initial wounded gaps. The percentage of wound closure was calculated as (100%-remaining wounded gaps%). The tracking function of AxioVision software (Zeiss) was used to track cell migration of a specific group of cells. For each wound healing experiment, at least 8 groups of cells per field were tracked for each time point. Position of cells was imaged and the net cell migration every 24 h was calculated for at least 6 days. Accumulated distance is the total distance that cells migrate over time (0-144 h). Cell motility is defined as the accumulated distance/time.
For re-plating experiments, PC12-GFP and PC12-SH2B1b cells were seeded on 10-cm collagen-coated plates at 30% confluency and differentiated with NGF (50 ng/ml). On day 8, cells were rinsed using 16 PBS three times, trypsinization, and centrifuged (2356g, 1 min at RT). Cells were re-suspended by pipetting up and down to remove the neurites, and then re-plated cells in 25 ng/ml NGF-containing differentiation medium. To examine neurite re-growth of differentiated PC12 cells, live cell images of random fields of at least 80 re-plated cells were taken using Carl Zeiss Observer Z1 microscope. The percentage of differentiated cells is defined as the numbers of cells with neurite length at least twice the diameter of cell bodies divided by the numbers of total cells counted.

Immunoblotting and immunoprecipitation
Cells were harvested into RIPA (50 mM Tris, pH 7.5, 1% Triton X-100, 150 mM NaCl, 2 mM EGTA) containing 1 mM Na 3 VO 4 , 1 mM phenylmethanesulphonylfluoride (PMSF), 10 ng/ml aprotinin and 10 ng/ml leupeptin. Protein concentration of each sample was determined. Equal amounts of proteins were loaded to and resolved by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE), followed by western blotting analysis using the indicated antibodies. For immunoprecipitation of PKC, cells were lysed in RIPA buffer containing 1 mM Na 3 VO 4 , 1 mM PMSF, 10 ng/ml aprotinin and 10 ng/ml leupeptin. Cell lysates were incubated with monoclonal PKC antibodies (1:200) overnight at 4uC. Immuno-precipitated proteins were subjected to SDS-PAGE and Western blotting. The immunoblots were detected using either IRDyeconjugated IgG and the Odyssey Infrared Imaging System (LI- COR Biosciences) or the horseradish peroxidase-conjugated IgG and ECL system.

Immunofluorescence staining
Cells were seeded on the collagen-coated plates at 30% confluency and differentiated with 50 ng/ml NGF in the differentiation medium for 8 days. On day 8, cells were wounded by a P10 pipette tip and fixed by 4% paraformaldehyde, permeabilized, and incubated with anti-neuronal tubulin antibodies, followed by incubation with the Alexa Fluor 555-conjugated secondary antibody for visualization. DAPI staining was used to visualize the nucleus. Images were taken by an upright fluorescent microscope Zeiss/Axioskop 2 mot plus and the Carl Zeiss Observer Z1.

Inhibitor and activator assays
Cells were seeded on the collagen-coated 6-well plates at 30% confluency and differentiated with 50 ng/ml NGF in the differentiation medium for 8 days. On day 8, cells were pretreated with DMSO (Mock), 20 mM U0126, 20 mM LY294002 or 0.1-2.5 mM Bis for 1 h before wounding. Images were taken by the Carl Zeiss Observer Z1. For the PKC activation assay, wounded cells were treated with 162 nM PMA or DMSO (Mock). Images of cells were taken using the inverted Zeiss Axiover 135 fluorescence microscope or the Carl Zeiss Observer Z1. Images of wounded cells were tracked using the tracking function of Axio Vision software (Zeiss).

Statistical analysis
Most of the comparisons were determined using paired Student's t-test or one-way ANOVA. Significant difference is defined as P value less than 0.05.

Overexpression of SH2B1b promotes wound healing of differentiated PC12 cells
To determine the role of SH2B1b in neurite regeneration, two stable cell lines, PC12 cells stably expressing GFP (PC12-GFP) and PC12 cells stably expressing GFP-SH2B1b (PC12-SH2B1b), were established [42]. Wound healing (scraping) assays were performed to recapitulate injury and recovery. These two stable cell lines were differentiated into neurons by NGF (50 ng/ml) treatment for 8 days. On day 8, cells were subjected to mechanical scraping ( Figure 1A and C). Differentiated PC12 cells depend on NGF for maintaining neuronal status. Thus, NGF was present at all time during healing (regeneration). The percentage of wound closure during healing was measured as described in the Materials and Methods, and in Figure S1. After healing for 5 days, differentiated PC12-SH2B1b cells healed better than PC12-GFP cells ( Figure 1B and D). The quantified results revealed that the percentages of wound closure were higher for differentiated PC12-SH2B1b cells compared to those for PC12-GFP cells after healing day 4 ( Figure 1E). On healing day 5, wound closure was 70% for differentiated PC12-GFP cells whereas it was 85% for differentiated PC12-SH2B1b cells. The percentage of wound closure for differentiated PC12-SH2B1b cells continued to increase over time while the healing rate was slower for PC12-GFP cells. To verify that the enhanced percentage of wound closure is SH2B1dependent, knocking down SH2B1 seems to be the best approach to address the question. However, knocking down SH2B1 significantly reduces neuronal differentiation. Thus, two loss-offunction mutants of SH2B1b, SH2B1b(D150-200) and SH2B1b(D200-268), were used to examine the effect on neurite regeneration. SH2B1b(D150-200) lacks nuclear localizing sequences whereas SH2B1b(D200-268) lacks nuclear export signal. As shown in Figure 1F, PC12 cells stably expressing SH2B1b(D150-200) or SH2B1b(D200-268) reduced % of wound closure compared to cells expressing SH2B1b. These results suggest that the enhanced regeneration is SH2B1b-dependent. Notably, the wounded gaps were filled with neurites and a few cell bodies ( Figure 1B and D). These results implicate the involvement of neurite re-growth and cell migration during the healing process. In addition, SH2B1b may promote neurite re-growth and/or cell migration during neuronal regeneration.
Overexpression of SH2B1b promotes MEK-ERK1/2-and PI3K-AKT-dependent neurite re-growth To examine whether SH2B1b could promote neurite regrowth, cell re-plating experiments were performed on the differentiated PC12 cells. PC12-GFP and PC12-SH2B1b cells were differentiated, trypsinized to detach cells, pipetted up and down to remove neurites and then re-plated. Neurite re-growth was monitored over time. As shown in Figure 2A, all neurites were removed after trypsinization. Neurite re-growth occurred as early as 2 h after re-plating. The percentages of differentiated cells (defined in the Materials and Methods) for PC12-SH2B1b cells at 6 and 24 h after re-plating were around 3.5-and 1.8-fold of those for PC12-GFP cells respectively ( Figure 2B). Together with results from Figure 1, overexpression of SH2B1b enhances neurite regrowth in response to injury.
To determine what signaling molecules are involved in SH2B1b-mediated neurite re-growth, activity of MEK-ERK1/2 and PI3K-AKT pathways during neuronal regeneration were examined. PC12-GFP and PC12-SH2B1b cells were differentiated for 8 days and then pre-treated with U0126 (MEK inhibitor) or LY294002 (PI3K inhibitor) for 1 h before wounding. In the present of U0126, the phosphorylation of ERK1/2 was inhibited ( Figure 2C). The percentages of wound closure were reduced 70-85% for PC21-GFP cells and 58-86% for PC12-SH2B1b cells in the presence of U0126 ( Figure 2D). These results suggest that ERK1/2 activity is essential for neurite re-growth as well as SH2B1b-mediated functions.
PI3K-AKT pathway contributes to both cell survival and neurite outgrowth [11,23,50]. The effects of inhibiting phosphorylated AKT during wound healing were also examined. Tuj1 staining results showed that neurite re-growth was better for differentiated PC12-SH2B1b cells compared to PC12-GFP cells on healing day 7. In addition, LY294002 treatment reduced neurite re-growth ( Figure 3A). Similar with the inhibition of pERK1/2, neurite re-growth was reduced 37 to 77% in the present of LY294002 for both cell lines ( Figure 3B). These results demonstrated that both MEK-ERK1/2 and PI3K-AKT pathways Overexpression of SH2B1b correlates with enhanced cell migration and elevated phosphorylation of PLCc1 and PKC In addition to regenerated neurites, cell bodies also appeared in the wounded gaps for both PC12-GFP and PC12-SH2B1b cells on healing day 5, indicative of cell migration to the wounded gaps ( Figure 1B and D). We thus examined whether overexpression of SH2B1b would enhance cell migration to promote regeneration. PC12-GFP and PC12-SH2B1b cells were differentiated and subjected to wound healing protocol. Migration of cells from healing time 0 (blue color) to 144 h (red color) were tracked and analyzed ( Figure 4A). During 6 days of healing, the accumulated distance that PC12-SH2B1b cells migrated was 170 mm in average, compared to 90 mm by PC12-GFP cells ( Figure 4B, left panel), suggesting that their motility was higher ( Figure 4B, right panel). Based on the known effects of PKC on cell motility, we next examined whether SH2B1b increases cell migration through PLCc and/or PKC. To this end, cell lysates were collected from differentiated PC12-GFP and PC12-SH2B1b cells before wounding (U) and during healing days 0-5. Phosphorylation of PLCc1 was determined via western blotting. The overall phosphorylation levels of PLCc1 in differentiated PC12-SH2B1b cells were around 1.4-to 3.1-fold of those in PC12-GFP cells. In addition, the levels of pPLCc1 were reduced on healing day 5 ( Figure 4C-D). The phosphorylation of PKC, a downstream effector of PLCc1, was also examined. Total PKC was immunoprecipitated and its phosphorylation level was determined. The relative PKC phosphorylations at serine and threonine residues for differentiated PC12-SH2B1b cells were around 1.7-and 2.6-fold of those in PC12-GFP cells respectively ( Figure 4E). These results are consistent with our hypothesis that SH2B1b promotes cell migration through PLCc-PKC pathway. To determine whether the enhanced cell migration is SH2B1-dependent, cell migration after scraping injury was determined using PC12-(D150-200) and PC12-(D200-268) cells that express two loss-of-function mutants of SH2B1b. Cell migration for PC12-(D150-200) and PC12-(D200-268) cells during healing was reduced compared to PC12-SH2B1b cells and comparable to control PC12-GFP cells ( Figure 5). Along this line, cell migration for PC12-SH2B1b cells was reduced around 20% by 1 mM PKC inhibitor, Bisindolyleimide I (Bis) ( Figure 6). Dose-dependent inhibition of cell migration by 0-2.5 mM Bis was determined ( Figure S2). To reduce the possibility that inhibiting PKC may affect normal physiology of neuron cells, low dosage of 1 mM Bis was used for this study. The fact that cell migration of PC12-GFP cells was not inhibited by 1 mM Bis could be due to lower motility or PKC activity in PC12-GFP cells ( Figure 4). Together, these results implicate that PLCc-PKC pathway is involved in SH2B1b-mediated cell migration.

PKC activation promotes SH2B1b-enhanced cell migration
Since PKC activity is involved in SH2B1b-enhanced cell migration during wound healing, we next determined whether activation of PKC contributes to SH2B1b-mediated migration. Differentiated PC12-GFP and PC12-SH2B1b cells were wounded, mock-treated or treated with PKC activator -phorbol 12myristate 13-acetate (PMA), and the effect on cell migration was assessed. PMA activates PKC because of its similar structure to DAG. The distance PC12-SH2B1b cells migrated was around 2fold of that by PC12-GFP cells in mock-treated conditions (Figure 7). PMA treatment significantly enhanced cell migration of PC12-SH2B1b cells after healing for 6 days (Figure 7). The fact that PMA stimulation promoted obvious cell migration for PC12-SH2B1b cells but not so much for PC12-GFP cells suggests that overexpression of SH2B1b may recruit PMA substrates closer to plasma membrane where PMA acts. To confirm that the activation of PKC was induced after the treatment of PMA, the differentiated PC12-GFP and PC12-SH2B1b cells were treated with or without PMA. Relative phosphoserine (pSer) of PKC was increased approximately 60% after 6 h of PMA treatment for PC12-GFP but not for PC12-SH2B1b cells ( Figure S3). Relative phosphothreonine (pThr) of PKC, on the other hand, was induced 1.1-to 3.4-fold for both cell lines ( Figure S3). PMA-induced PKC phosphorylation peaked at 6 h and then reduced by 24 h. Consistent with the results in Figure 4E, basal phosphorylation of PKC was higher in PC12-SH2B1b cells compared to PC12-GFP cells. Interestingly, PMA treatment also increased the expression of PKC. The increased PKC levels in differentiated PC12-SH2B1b cells were 1.3-to 1.6-fold of those in PC12-GFP cells ( Figure S3). This may explain why the relative phosphory-lation of PKC (after normalized to total PKC levels) in response to PMA stimulation was not higher for PC12-SH2B1b cells ( Figure S3). A study showed that PMA promoted degradation of PKC in human osteosarcoma cells [51]. However, it was not clear which isozyme was referred to. Our data suggested that PMA promoted degradation of PKCd (data not shown) but increased synthesis of pan-PKCs ( Figure S3). Thus, PMA-increased expression and phosphorylation of PKC likely account for the increased cell migration of PC12-SH2B1b cells.
Fine balance between cell migration and neurite regrowth warrants better wound healing As PMA treatment promotes cell migration during wound healing, it unexpectedly interfered with wound closure. Treatment of PMA reduced 10% wound closure for differentiated PC12-GFP cells and 25% for differentiated PC12-SH2B1b cells ( Figure 8A). This result raises a possibility that high motility of cells may impede neurite outgrowth. Because neurite re-growth is largely dependent on pERK1/2 (Figure 2), we thus examine whether pERK1/2 levels would be affected by PMA treatment. Cell lysates were collected from differentiated cells before wounding (U) and during healing days 0-5. Phosphorylation of ERK1/2 was examined via western blotting. As shown in Figure 8B-C, pERK1/2 levels were significantly reduced after the treatment of PMA for both PC12-GFP and PC12-SH2B1b cells. The downregulation of pERK1/2 levels likely contributes to the reduced neurite re-growth and wound healing. This result also reveals an antagonistic role of cell motility and neurite outgrowth. As cell migration is enhanced by PMA treatment, neurite re-growth is reduced. If this is true, higher cell motility of PC12-SH2B1b cells may not accompany with better neurite re-growth. Taking a closer look at cell migration each day for both cell lines, cell motility for PC12-SH2B1b cells was higher during healing days 0-4 and reduced after healing day 4 ( Figure 9A, left panel). Cell migration during healing day 7-8 was also reduced compared to healing day 0-1 for PC12-SH2B1b cells ( Figure 9A, right panel). On the other hand, pERK1/2 levels were lower for un-wounded PC12-SH2B1b cells compared to PC12-GFP cells ( Figure 9B, left panel), whereas they were higher on healing day 7 ( Figure 9B, right panel). The reverse correlation between cell migration and pERK1/2 suggests that overexpression of SH2B1b significantly promoted cell migration toward wounded area before healing day 4 and the neurite outgrowth after healing day 4. These results suggest that a fine balance between cell motility and neurite regrowth warrants better healing.
Together, results from this study demonstrated that overexpression of SH2B1b promoted regeneration of differentiated PC12 cells, in response to scraping injury, after healing day 4. Rate of SH2B1benhanced cell migration was faster during healing days 0-4 and pERK1/2-mediated neurite re-growth was increased after healing day 4. The fact that activated PKC increased cell migration but reduced neurite re-growth suggests that cell migration and neurite re-growth do not occur concomitantly during regeneration.

Discussion
In this study, we demonstrated that overexpression of a signaling adaptor protein SH2B1b promotes neurite regeneration of differentiated PC12 cells. During neurite regeneration, overexpression of SH2B1b increases both cell motility and neurite re-growth (Figures 1, 2 and 4). Neurite re-growth is significantly blocked in the presence of MEK or PI3K inhibitor for both PC12-GFP and PC12-SH2B1b cells (Figures 2 and 3). No obvious effect on cell migration was found in the presence of these two inhibitors. These results are in line with published results that both ERK1/2 and AKT contribute to neurite outgrowth [10,25].
Accumulating evidence from behavior, genetic and pharmacological studies have implicated PKC in an array of neuronal functions, including release of neurotransmitters, synaptic functions, learning and memory processes [52,53,54]. However, the effect of PKC activation on neurite outgrowth has been controversial. For example, PKC inhibitors have been reported to block neurite outgrowth in retinal axons, dorsal root ganglion neurons, sympathetic neurons, PC12 cells, and hippocampal cultures [55,56,57,58,59]. PKC inhibitors have also been shown to promote dendritic growth of Purkinjie cells in cerebellar slice cultures and to extend filopodia in DRG [60,61]. The addition of dopamine causes neurite retraction of retinal neurons via activating PKC, and eicosanoid activation of PKC also serves as growth cone repellent signaling [62,63]. The discrepancy of these results could simply be due to various types of neurons from peripheral or central nervous systems. Alternatively, as suggested in this study, temporal and possibly spatial control of PKC activity during wound healing lead to diverse cell fates or healing outcomes. Through inhibitor and activator assays, we further demonstrated that PLCc-PKC pathway was involved in SH2B1benhanced cell migration (Figures 6-7). Although PMA is a wellknown activator of PKC, Src has been suggested to be one of the downstream targets and has been implicated in cell migration [64,65,66]. To address this possibility, Src phosphorylation (pSrc) with or without PMA treatment was investigated. As shown in Figure S4, with PMA treatment, levels of pSrc were significantly increased around 55 and 67% for PC12-GFP cells on healing days 1 and 4, respectively. However, cell migration of PC12-GFP cells was not significantly affected by PMA ( Figure 7C). For PC12-SH2B1b cells, pSrc was significantly increased by PMA only on healing day 5 ( Figure S4). However, PMA treatment increased migration of PC12-SH2B1b cells before healing day 5 ( Figure 7). Thus, it seems unlikely that PMA stimulates SH2B1b-mediated cell migration through increasing pSrc.
We found that cell motility was reversely correlated with pERK1/2-dependent neurite re-growth. After healing day 4, cell motility was reduced for PC12-SH2B1b cells whereas the wound closure was better (Figures 1 and 9). Nonetheless, cell motility is not necessary further reduced afterwards because, at a later healing stage -healing day 23, more cell bodies were found at wounded gaps suggesting another phase of increased cell migration ( Figure S5). Thus, we think that as both cell migration and neurite re-growth contribute to neuronal regeneration, these processes may occur in a pulsatile manner. An intriguing question raised here is what signals cell to reduce or increase cell migration. We found that PMA treatment increased the levels of SH2B1b ( Figure S6), phosphorylation of PKC ( Figure S3), and cell migration (Figure 7). Although SH2B1b has predicted PKC phosphorylation site, exactly how PKC activity and SH2B1b regulate each other during neuronal regeneration and whether putative regulators are involved remain to be elucidated.
Evidence suggests that injury can induce stem cells migrating to the injured sites to aid wound healing [67,68,69,70]. Thus, promoting migration of neural stem cells or neural progenitor cells to injured sites can enhance regeneration. As we demonstrated that overexpression of SH2B1b promoted cell migration, we cannot discern whether SH2B1b promotes differentiation of PC12 cells and then their migration to the wounded area, or SH2B1b promotes cell migration to the wounded area and then enhances their differentiation. Unfortunately, it is difficult to track undifferentiated cells from a distance to the wounded area in our system.
The current study reveals a novel function of SH2B1b in promoting neuronal regeneration. MEK-ERK1/2 and PI3K-AKT pathways are necessary for SH2B1b-mediated neurite regrowth during wound healing. Increased SH2B1b level associates with elevated phosphorylation of PLCc and PKC, and thus promotes cell migration during regeneration. We further demonstrate that a cross-talk between PKC and ERK1/2 pathways contributes to temporal regulation of cell migration, neurite regrowth and thus the outcome of regeneration.