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Endocrinology Vol. 148, No. 6 2806-2814
Copyright © 2007 by The Endocrine Society

Adhesion-Related Kinase Induction of Migration Requires Phosphatidylinositol-3-Kinase and Ras Stimulation of Rac Activity in Immortalized Gonadotropin-Releasing Hormone Neuronal Cells

Sheila M. Nielsen-Preiss, Melissa P. Allen, Mei Xu, Daniel A. Linseman, John E. Pawlowski, R. J. Bouchard, Brian C. Varnum, Kim A. Heidenreich and Margaret E. Wierman

Departments of Medicine (S.M.N.-P., M.P.A., M.X., J.E.P., M.E.W.), Physiology and Biophysics (M.E.W.), and Pharmacology (K.A.H.), The University of Colorado at Denver and Health Sciences Center, Aurora, Colorado 80045; Research Service (S.M.N.-P., M.X., D.A.L., J.E.P., R.J.B., K.A.H., M.E.W.), Veterans Affairs Medical Center, Denver, Colorado 80220; and Amgen (B.C.V.), Thousand Oaks, California 91320

Address all correspondence and requests for reprints to: Margaret E. Wierman, Veterans Affairs Medical Center, 1055 Clermont Street, Box 111H, Denver, Colorado 80220. E-mail: Margaret.Wierman{at}UCHSC.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
GnRH neurons migrate into the hypothalamus during development. Although migratory defects may result in disordered activation of the reproductive axis and lead to delayed or absent sexual maturation, specific factors regulating GnRH neuronal migration remain largely unknown. The receptor tyrosine kinase, adhesion-related kinase (Ark) (also known as Axl, UFO, and Tyro7), has been implicated in the migration of GnRH neuronal cells. Binding of its ligand, growth arrest-specific gene 6 (Gas6), promotes cytoskeletal remodeling and migration of NLT GnRH neuronal cells via Rac and p38 MAPK. Here, we examined the Axl effectors proximal to Rac in the signaling pathway. Gas6/Axl-induced lamellipodia formation and migration were blocked after phosphatidylinositol-3-kinase (PI3K) inhibition in GnRH neuronal cells. The p85 subunit of PI3K coimmunoprecipitated with Axl and was phosphorylated in a Gas6-sensitive manner. In addition, PI3K inhibition in GnRH neuronal cells diminished Gas6-induced Rac activation. Exogenous expression of a dominant-negative form of Ras also decreased GnRH neuronal lamellipodia formation, migration, and Rac activation. PI3K inhibition blocked Ras in addition to Rac activation and migration. In contrast, pharmacological blockade of the phospholipase C {gamma} effectors, protein kinase C or calcium/calmodulin protein kinase II, had no effect on Gas6/Axl signaling to promote Rac activation or stimulate cytoskeletal reorganization and migration. Together, these data show that the PI3K-Ras pathway is a major mediator of Axl actions upstream of Rac to induce GnRH neuronal cell migration.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GnRH IS THE hypothalamic releasing factor that controls gonadotropin gene expression and reproductive function. During development, GnRH neurons migrate through the nasal compartment and into the hypothalamus, where they ultimately promote reproductive competence (1, 2). Despite their pivotal role in regulating the reproductive axis, relatively little is known regarding the molecular basis of GnRH neuron motility. We and others have used immortalized murine GnRH neurons to identify and study factors involved in GnRH neuronal migration (3, 4, 5). One such protein, adhesion-related kinase (Ark; or its human homolog, Axl), is expressed in motile Gn10 and NLT GnRH neuronal cells but absent in nonmotile GT1–7 GnRH neuronal cells (6). In agreement with an in vivo role in migration, Axl mRNA is coexpressed with GnRH in regions of the brain along the route of GnRH neuronal migration during murine embryonic development (7).

Axl (also known as Ark in mouse, UFO, and Tyro7), Tyro3, and Mer define a unique subclass of receptor tyrosine kinases (RTKs) with diverse and cell-specific actions (8, 9, 10, 11, 12). The extracellular domains of these receptors are modular, containing immunoglobulin-like and fibronectin type III repeats similar to neural cell adhesion molecules. The intracellular domains encode a tyrosine kinase (12). Axl, Tyro3, and Mer are expressed in the immune, nervous, vascular, and reproductive systems where their physiological roles are under active investigation (9, 11, 13, 14, 15). Axl was originally identified as a transforming gene in chronic myelogenous leukemia (10). In addition, Axl is up-regulated and thought to have mitogenic and prosurvival roles in malignancies of the thyroid, kidney, breast, and ovary (16, 17, 18, 19).

Activation of Axl family RTKs is initiated via binding of the ligand, growth arrest-specific gene 6 (Gas6) (20, 21, 22). Gas6/Axl signaling has been shown to promote cell proliferation and protection from programed cell death in diverse cell types (23, 24, 25, 26, 27, 28). Mouse knockout studies have further implicated Axl, Tyro3, and Mer in cell survival. Mice null for all three receptors develop severe autoimmune disease due to excessive levels of antigen-presenting cells (15). In addition, the triple mutant mice have a spermatogenetic defect resulting from loss of receptor signaling in Sertoli cells, which normally promote survival of germ cells (14). Increased apoptosis was also documented in cells of the central nervous system, reproductive, and vascular tissues, arguing that signaling via this RTK family is essential for cell survival in a variety of tissues. In addition to promoting cell growth and survival, our studies have shown that Gas6/Axl signaling also represses GnRH mRNA expression when Axl is expressed in GT1–7 GnRH neuronal cells (7, 26, 29, 30).

Because of their homology to cell adhesion molecules, studies have also explored a role for Axl family receptors in cell adhesion and motility. Axl promotes cell-cell adhesion in fibroblasts via homophilic and heterophilic mechanisms (31, 32). A role for Axl in vascular smooth muscle cell migration has also been reported (33, 34). Both Axl and Gas6 are up-regulated in vascular tissue after injury, and Gas6 modulation of vascular smooth muscle cell chemotaxis may be important in vivo for vascular homeostasis.

Immortalized GnRH neurons have been useful to study factors involved in GnRH neuronal migration. NLT GnRH cells expressing endogenous Axl were used to dissect the role of Gas6/Axl signaling in GnRH neuronal migration (29). Gas6 promoted the development of lamellipodia, membrane ruffles, and chemotaxis of GnRH neuronal cells. The migration signaling pathway was orchestrated by Gas6 activation of Rac and its downstream effector, p38 MAPK. This was a novel observation because in many neuronal systems, p38 MAPK signaling promotes a prodeath pathway (35, 36, 37, 38). Downstream of p38 MAPK, MAPKAP kinase II and heat shock protein 25 were induced to trigger cytoskeletal remodeling and neuronal migration (29).

Having dissected the distal components of the pathway, the present study focused on defining the effectors proximal to the Axl receptor and upstream of p38 MAPK involved in the migratory signaling cascade. Together, the data show that Gas6/Axl-induced GnRH neuronal migration is mediated by the phosphatidylinositol-3-kinase (PI3K) and Ras pathway upstream of Rac.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
Recombinant human Gas6 was generated as described (20). The Y815F mutant Axl (pLXN) was a generous gift from P. Bellosta (New York University Medical Center, New York, NY). The pEGFP-N1 plasmid was obtained from Clontech Laboratories, Inc. (Mountain View, CA). Bisindolylmaleimide I [2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide; Bis), LY294002, KN93 [2-[N-(2-hydroxyethyl)]-N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methyl-benzylamine)], and PP2 [4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-D'pyrimidine] were purchased from Calbiochem (San Diego, CA). Rhodamine phalloidin was obtained from Molecular Probes (Eugene, OR). The Rac and Ras activation kits were purchased from Upstate Biotechnology (Charlottesville, VA). Transwell migration chambers (6.5-mm diameter, 8 µM pore size) were obtained from Costar (Corning, NY). Hybond polyvinylidene difluoride (PVDF) blotting membranes were purchased from Amersham Biosciences (Piscataway, NJ) or Whatman Schleicher and Schuell (Keene, NH). DMEM and antibiotics were from Life Technologies (Rockville, MD), and the fetal bovine serum (FBS) from Gemini Bio-Products (Calabasas, CA). The Vybrant Apoptosis Assay kit was from Molecular Probes. The Diff Quik staining kit was from Dade Behring (Newark, DE). Glass coverslips (12-mm circles) were purchased from Fisher Scientific (Pittsburgh, PA).

Cell culture
NLT GnRH neuronal cells (39) were grown in DMEM supplemented with 10% FBS at 37 C in humidified 5% CO2 unless otherwise indicated.

Actin localization
NLT GnRH neuronal cells (15,000) were seeded onto sterile glass coverslips. On the next day, the cells were washed once in PBS and incubated for 16–18 h in DMEM supplemented with 0.5% FBS. Cells were pretreated for 4 h with various signaling pathway inhibitors or dimethyl sulfoxide (DMSO, vehicle) and then stimulated with 400 ng/ml Gas6 for 10 min in the presence of inhibitor or vehicle. After Gas6 treatment, the cells were rinsed once in PBS, fixed in 4% paraformaldehyde/PBS for 30 min at room temperature (RT), and rinsed twice with PBS. The cells were permeabilized in PBS containing 5% BSA and 0.2% Triton X-100 for 30 min at RT. F actin was stained with 25 µl/ml rhodamine phalloidin in the permeabilization solution for 30 min at RT. The cells were rinsed three times with PBS, mounted onto slides, and viewed using a Zeiss Axioscope II microscope (Carl Zeiss GmbH, Jena, Germany). To quantify the percentage of cells that displayed membrane ruffles and/or lamellipodia, three fields were counted on each coverslip in each of three or more experiments. Cells that demonstrated membrane ruffling and/or lamellipodia by morphology and actin localization were considered positive and quantified relative to cells lacking these characteristics.

Transwell migration assay
NLT GnRH neuronal cells were rinsed once in PBS and incubated for 16–18 h in DMEM. The cells were removed from cell culture plates by trypsin digestion, pelleted by centrifugation at 2000 x g for 5 min, and resuspended in DMEM. Cells (30,000–50,000 per well) were plated into the upper chamber of Transwells and allowed to migrate for 18–20 h at 37 C in humidified 5% CO2. For chemotaxis experiments, vehicle or Gas6 (400 ng/ml) was added to the lower chamber containing the migration medium. For experiments using the various inhibitors, the reagents were added to both the upper and lower compartments of the Transwell. Inhibitors were diluted in DMSO (final concentration, 0.01%) and used at the following concentrations: LY294002, 25 µM; Bis, 5 µM; KN93, 20 µM; and PP2, 10 µM. After 18–20 h migration, the cells were fixed and stained using the Diff Quik kit. The number of migrated cells for each condition was determined by counting four fields at x200 magnification on each membrane. Duplicate membranes in three separate experiments were evaluated.

Apoptosis assay
NLT GnRH neuronal cells were washed once in PBS and incubated for 16–18 h in DMEM. The cells were then incubated with 25 µM LY294002 or DMSO for 24 h, rinsed once in PBS, and trypsinized. After serum inactivation of the trypsin, cells were counted and diluted to 1 x 106/ml. Cells were pelleted by centrifugation (2000 x g, 5 min, 4 C) and washed twice with PBS. The final cell pellet was resuspended in 1 ml of PBS and stained with the Vybrant Apoptosis Assay kit (Molecular Probes) according to the manufacturer’s instructions. Cells were incubated on ice for 20 min and then analyzed on a Beckman Coulter FC500 flow cytometer (Beckman Coulter, Fullerton, CA) by the University of Colorado at Denver and Health Sciences Center Core personnel.

Immunoblot and immunoprecipitation analysis
Protein aliquots in radioimmunoprecipitation assay buffer [150 mM NaCl, 1% NP40, 0.5% deoxycholate, 0.1% sodium dodecyl sulfate, 50 mM Tris (pH 8.0)] were quantified using the BCA system (Pierce, Rockford, IL) and resolved by electrophoresis through 10% SDS-PAGE. Gels were electrotransferred to PVDF membranes using the mini transblotter system (Bio-Rad Laboratories, Hercules, CA). The PVDF membranes were blocked in 5% nonfat dry milk containing 0.1% Tween 20 in Tris-buffered saline (TBST). Primary polyclonal antibodies [phospho-p85, 85 kDa; phospho-Akt (ser473), 60 kDa; total p85; and total Akt (Cell Signaling, Beverly, MA); Axl, 120–140 kDa (Affinity Bioreagents, Golden, CO); and monoclonal antibodies for Rac and Ras, 21 kDa (Upstate Biotechnology)] were diluted 1:1000 in blocking reagent, and membranes were incubated at 4 C overnight. Membranes were washed in TBST three times for 10 min each. Horseradish peroxidase-conjugated secondary antibody (Amersham Biosciences) was diluted 1:3000 in TBST and incubated on the PVDF membrane for 1 h at ambient temperature. The membranes were washed as above and visualized using chemiluminescence according to the manufacturer’s protocol (Kirkegaard & Perry Laboratories, Inc., Gaithersburg, MD). Blots probed for phospho-specific antibodies were stripped to reprobe with pan antibodies. The membranes were submerged in stripping buffer [110 mM ß-mercaptoethanol, 2% SDS, 100 mM Tris-HCl (pH 8.0)] and incubated at 50 C for 45 min with agitation. The membranes were then washed 3 x 10 min in TBST at RT.

For immunoprecipitation analysis, cell lysates (1.5 mg) were incubated with primary Axl antibody (6.4 µg) (20) overnight at 4 C with tumbling. Protein G-agarose was added, and incubation was continued for an additional hour. Immune complexes were washed three times with lysis buffer and subjected to immunoblot analysis with either the p85-specific antibody (Cell Signaling, Beverly, MA) or an affinity purified Axl-specific antibody (Affinity Bioreagents).

Rac and Ras activation assays
NLT GnRH neuronal cells were grown in DMEM supplemented with 10% FBS and penicillin (100 U/ml)/streptomycin (100 µg/ml)/amphotericin (0.25 µg/ml) to 80% confluence in 10-cm plates. The cells were rinsed once in PBS and incubated for 16–18 h in DMEM supplemented with 0.5% FBS. Cells were pretreated for 4 h with inhibitor (LY294002, 25 µM; Bis, 5 µM; and KN93, 20 µM) or DMSO (vehicle) and then stimulated with Gas6 (400 ng/ml) for 10 min. Lysis buffer (mammalian protein extraction reagent, 600 µl; Pierce) was added to each 10-cm plate to harvest cells on ice. A 30-µl aliquot of lysate was retained to be used for total Rac analysis. The remainder of the lysate was used for the substrate precipitation assays, where Rac or Ras in the GTP-bound state specifically interacted with PAK1 or Raf1, respectively, performed according to the manufacturer’s instructions (Upstate Biotechnology) and subjected to immunoblot analysis. Densitometric analysis was performed using the Bio-Rad Fluor-S Multi Imager and Quantity One software.

Dominant-negative form of Ras (N17Ras) transfection
NLT GnRH neuronal cells were grown to 80% confluence in a six-well plate. Cells were transiently transfected with 1 µg N17Ras dominant-negative construct (Upstate Biotechnology) in the pUSE vector, or 1 µg of the empty vector as control, using the Lipofectamine Plus reagents as per the manufacturer’s instructions (Invitrogen, Carlsbad, CA). Cells were evaluated in functional assays 24–48 h posttransfection. Cells were cotransfected with the eGFP-N1 vector, revealing consistent transfection efficiency (40%) in the presence of both the pUSE and N17Ras vectors.

Statistical analyses
All results are shown as mean ± SEM. Differences between groups were analyzed for statistical significance using one-way ANOVA with post hoc Tukey’s all pairs comparison. P values < 0.05 were considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
PI3K modulates cytoskeletal remodeling and migration in GnRH neuronal cells
Cell movement is facilitated by actin cytoskeletal remodeling, where the leading edge of the cell must extend forward, and the rear must retract. The forward extension of a lamellipodia is often regulated by Rac, whereas the rear retraction is mediated by Rho (40, 41). Accordingly, our previous studies showed that Gas6/Axl signaling promotes lamellipodial extension, membrane ruffling, and migration of NLT GnRH neuronal cells via activation of Rac (7). Based on work in other systems, we hypothesized that Axl-mediated lamellipodial advance and migration of GnRH neuronal cells may require intermediate signaling via PI3K. Therefore, PI3K activity was inhibited using LY294002, and effects on Gas6/Axl-induced actin cytoskeletal reorganization were measured (Fig. 1Go). NLT neuronal cells developed membrane ruffles and lamellipodial extensions within 10 min of Gas6 treatment as visualized by F actin staining (29.1 ± 1.8% of cells displayed morphological changes, Fig. 2AGo). In contrast, NLT neuronal cells treated with LY294002 developed only baseline levels (13.1 ± 0.7% of the cells displayed morphology) of lamellipodial extensions and membrane ruffles in the presence of Gas6 (Fig. 2AGo). There was no discernible effect of LY294002 on the actin cytoskeleton in the absence of agonist (Figs. 1Go, lower left, and 2AGo).


Figure 1
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  		FIG. 1. Gas6/Axl-induced actin cytoskeletal remodeling of NLT GnRH neurons in the presence of the PI3K inhibitor, LY294002. NLT cells (15,000 cells per coverslip) were pretreated for 4 h with LY294002 (25 µM) or DMSO (control), stimulated with Gas6 (400 ng/ml, 10 min), and then stained with rhodamine phalloidin. The data are representative of four independent experiments (scale bar, 20 µm). Arrows, Lamellipodia and membrane ruffles.

 

Figure 2
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  		FIG. 2. Gas6/Axl-induced lamellipodia formation and GnRH neuronal migration in the presence of LY294002. A, Quantitation of lamellipodia and membrane ruffles from Fig. 1Go; control, cells treated with vehicle, DMSO. One hundred cells were analyzed per experiment. Gas6-induced lamellipodia formation (control vs. Gas6; *, n = 4; P < 0.0001). LY294002 decreases Gas6-induced lamellipodia formation (Gas6 vs. LY + Gas6; **, n = 4; P < 0.0001) to baseline levels (control vs. LY + Gas6; n = 4; P = 0.92). B, NLT GnRH neuronal cells (50,000 cells/well) were subjected to the transwell migration assay in the presence of vehicle (DMSO control) or LY294002 (25 µM) as described in Materials and Methods. The data represent the fold increase in migration in the presence of Gas6 (400 ng/ml). Gas6 induced migration (control vs. Gas6; *, n = 8; P < 0.0001). LY294002 diminished Gas6-induced migration (Gas6 vs. LY + Gas6; **, n = 5; P < 0.0001) to baseline levels (control vs. LY + Gas6, n = 5, P = 0.25).

 
We then examined whether inhibition of PI3K modulated GnRH neuronal migration in a transwell migration assay. Pharmacological blockade of PI3K by LY294002 significantly reduced Gas6/Axl-mediated GnRH neuronal migration from 7.0- ± 0.7-fold to 2.2- ± 0.5-fold increase (Fig. 2BGo). There was no effect of LY294002 treatment on basal migration of GnRH neuronal cells. Together, these data indicate that signals from PI3K are necessary for Gas6/Axl-mediated actin reorganization and migration of NLT GnRH neuronal cells.

To ensure that the diminished migration was not due to LY294002-induced apoptosis, NLT GnRH neuronal cells were cultured in serum-free conditions in the presence of inhibitor or vehicle for 18–20 h as in the migration assay. The percentage of cells undergoing apoptosis was determined by flow cytometry. After trypsinization, rates of apoptosis were similar among cells that were untreated (17%), DMSO treated (13.5%), and those exposed to 25 µM LY294002 for 24 h (18%, data not shown). Thus, the inhibition of GnRH neuronal motility in the presence of the PI3K blockade LY294002 was not due to increased rates of programed cell death.

PI3K and Axl interact in a Gas6-sensitive manner to induce Rac activity
PI3K consists of two subunits, the catalytic subunit, p110, and the adapter subunit, p85 (42). PI3K activation occurs via interaction with RTKs and subsequent phosphorylation of the p85 subunit. Phosphorylation of the p85 subunit increased (by 8-fold) after Gas6 treatment of NLT GnRH neuronal cells (Fig. 3AGo). Furthermore, an interaction was identified between the p85 subunit of PI3K and Axl, which increased greater than 2-fold in the presence of ligand (Fig. 3BGo). To confirm that Gas6/Axl promotes PI3K activation, an Axl receptor harboring a mutation in one of the PI3K binding sites was tested. GT1–7 cells, which do not express Axl protein endogenously, were transfected with Ark Y815F or WT receptor. To determine whether PI3K activation was compromised in the Ark Y815F cells, the activation of a downstream PI3K effector, Akt, was evaluated. Gas6/Axl-mediated Akt activation decreased from 2.3- to 1.3-fold in WT vs. Y815F transfected cells, respectively (data not shown). Together, these data confirmed that Gas6 promotes Axl interaction with, and activation of, PI3K.


Figure 3
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  		FIG. 3. Axl association with PI3K and activation of Rac upon Gas6 stimulation. A, p85 subunit of PI3K is phosphorylated in response to Gas6 stimulation. NLT GnRH neuronal cells were stimulated with Gas6 (400 ng/ml, 10 min). Cell lysates were subjected to immunoblot analysis with a phospho-specific p85 antibody (85 kDa). The blots were stripped and reprobed with a pan-p85 antibody, n = 3. B, Axl and PI3K interact in a coimmunoprecipitation assay. NLT GnRH neuronal cells were stimulated with Gas6 (400 ng/ml, 10 min) and then lysed. The cell lysate was immunoprecipitated with an Axl antibody and subjected to immunoblot analysis with antibodies against either Axl (120–140 kDa) or p85, n = 3. C, LY294002 blocks Gas6-induced Rac activation. NLT GnRH neurons were incubated with inhibitor or vehicle (DMSO) for 4 h and then stimulated with Gas6 (400 ng/ml) for 10 min followed by a Rac activation assay. The more than 2-fold increase in Rac-GTP was calculated relative to the total Rac (21 kDa) present in each sample. The data are representative of three independent experiments.

 
Because Rac activation is required for Gas6/Axl-induced lamellipodial extension and migration, experiments tested whether Axl activation of Rac was mediated by PI3K. Rac is active when bound to GTP and inactive when bound to GDP. NLT GnRH neuronal cells were pretreated with LY294002 or vehicle (DMSO) for 4 h, stimulated with Gas6 for 10 min, and subjected to a Rac activation assay. Gas6 increased the levels of active Rac (2.2-fold, Fig. 3CGo). Inhibition of PI3K by LY294002 resulted in a 3.6-fold reduction in Rac activation to near-baseline levels. These data confirmed a role for PI3K in the Gas6-induced Rac activation associated with lamellipodia formation and migration in the NLT GnRH neurons.

Phospholipase C (PLC) {gamma} effectors, protein kinase C (PKC) and calcium/calmodulin protein kinase II (CaMK), do not alter Rac activity, GnRH neuronal cell morphology, or migration
PLC{gamma} has been implicated in migratory signaling programs initiated by growth factors, such as epidermal growth factor and platelet-derived growth factor (43, 44). To evaluate whether the PLC{gamma} effectors, PKC or CaMK II, also contribute to GnRH neuronal migration via Gas6/Axl/Rac signaling, NLT cells were exposed to pharmacological inhibitors of these kinases and tested in cytoskeletal remodeling, migration, and Rac activation assays. NLT neuronal cells incubated with Bis or KN93, to inhibit PKC or CaMK II, respectively, developed membrane ruffles and lamellipodia similarly to control cells when treated with Gas6. Quantitation of the effects of the inhibitors demonstrated no significant changes in Gas6-stimulated actin modifications (Gas6, 29.1 5 ± 1.8%; Bis + Gas6, 30.9 ± 2.5%; Gas6 + KN93, 23.5 ± 0.5%, Fig. 4AGo). In addition, Bis and KN93 had no significant effect on Gas6-induced GnRH neuronal migration (Gas6, 6.2- ± 0.7-fold; Bis + Gas6, 7.4- ± 2.7-fold; Gas6 + KN93, 5.6- ± 0.1-fold, Fig. 4BGo) or Gas6 induction of Rac activity (Fig. 4CGo). Thus, although Axl has been shown to couple to PLC{gamma} in fibroblasts (45), the PLC{gamma} effectors PKC and CaMK II are not essential for Gas6/Axl activation of GnRH neuronal cell migration.


Figure 4
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  		FIG. 4. Gas6-induced lamellipodia formation and migration in the presence of inhibitors of PLC{gamma} effectors. A, NLT GnRH neuronal cells (15,000 cells per coverslip) were pretreated for 4 h with Bis (5 µM), KN93 (20 µM), or DMSO (control), stimulated with Gas6 (400 ng/ml, 10 min), and then stained with rhodamine phalloidin. Lamellipodia and membrane ruffles were quantitated. Gas6 stimulation resulted in significant increases in the number of cells displaying actin modifications in the absence of inhibitor (control vs. Gas6; *, n = 3; P < 0.0001) in the presence of Bis (Bis vs. Bis + Gas6; **, n = 3; P < 0.0001) or in the presence of KN93 (KN93 vs. KN93 + Gas6; ***, n = 3; P = 0.0002). There were no statistically significant differences among Gas6-treated samples. B, NLT GnRH neuronal cells (30,000 cells/well) were subjected to the migration assay in the presence of vehicle (DMSO, control), Bis, or KN93 as described in Materials and Methods. The data shown represent the fold increase in migration in the presence of Gas6 (400 ng/ml). Gas6 induced migration in the presence of inhibitor (control vs. Gas6, *, n = 3, P = 0.03; Bis vs. Bis + Gas6, **, n = 3, P = 0.05; KN93 vs. KN93 + Gas6, ***, n = 3, P < 0.001). There were no statistically significant differences between any of the Gas6-treated samples, with or without inhibitor, P = 0.69–0.96. C, Gas6/Axl-induced Rac activation is unaffected by inhibitors of PLC{gamma}. NLT GnRH neurons were incubated with inhibitor or vehicle (DMSO) for 4 h and then stimulated with Gas6 (400 ng/ml) for 10 min followed by a Rac activation assay.

 
Ras modulates Rac activation and GnRH neuronal cell migration
Studies were then performed to determine whether Ras contributed to the activation of Rac as described in other systems (46, 49, 54). Consistent with a role for Ras, NLT neuronal cells expressing a dominant-negative form of Ras, N17Ras (47), had a diminished capacity to activate Rac (1.6-fold reduction, Fig. 5AGo) and developed significantly fewer lamellipodial extensions and membrane ruffles in the presence of Gas6 (pUSE, 10.4 ± 2.6%; pUSE + Gas6, 45.7 ± 3.0%; N17Ras + Gas6, 15.4 ± 5.9%, Fig. 5Go, B and C). In addition, expression of the dominant-negative Ras decreased Gas6/Axl-mediated GnRH neuronal cell migration from 6.8- ± 1.5- to 3.5- ± 0.3-fold.


Figure 5
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  		FIG. 5. Expression of a dominant-negative form of Ras diminishes Gas6/Axl-induced Rac activation, lamellipodia formation, and migration. A, NLT GnRH neurons were transfected with pUSE or the pN17RasUSE construct. Twenty-four hours after transfection, the cells were stimulated with Gas6 (400 ng/ml) for 10 min and subjected to a Rac activation assay. The data are representative of three independent experiments. B, NLT GnRH neuronal cells were transfected with pUSE or the pN17RasUSE construct. Twenty-four hours after transfection, cells were trypsinized and seeded at 15,000 cells per coverslip. The cells were cultured in serum-deprivation conditions for 18–20 h and then stimulated with Gas6 (400 ng/ml) for 10 min. Cells were fixed in 4% paraformaldehyde and then stained with rhodamine phalloidin to visualize lamellipodia at x400 magnification. Arrows, Lamellipodia or membrane ruffles. C, Quantitation of lamellipodia and membrane ruffles from B. Gas6-induced lamellipodia formation in vector-transfected cells (pUSE vs. pUSE + Gas6; *, n = 3; P < 0.02). N17Ras transfection decreases Gas6-induced lamellipodia formation (pUSE + Gas6 vs. N17Ras + Gas6; **, n = 3; P = 0.03) to baseline levels (pUSE vs. N17Ras + Gas6, n = 3, P = 0.74). D, NLT GnRH neuronal cells were transfected with vector or the N17Ras construct. Twenty-four hours after transfection, cells (50,000 cells/well) were subjected to the migration assay as described in Materials and Methods. The data shown represent the fold change in migration in response to Gas6 in the absence (pUSE vs. pUSE + Gas6; *, n = 3; P = 0.008) or presence (pUSE vs. N17Ras + Gas6; **, n = 3; P < 0.0001) of N17Ras.

 
Ras and PI3K have been often, but not exclusively, found to function in the same signaling pathway (48, 49). Therefore, we evaluated whether PI3K and Ras were functioning in the same pathway and, if so, which molecule was upstream. NLT GnRH neurons expressing dominant-negative Ras displayed a Gas6-dependent phosphorylation of both the p85 subunit of PI3K and Akt comparable with that of cells transfected with vector alone, suggesting that Ras activation was downstream of PI3K (Fig. 6AGo). In contrast, Gas6-induced Ras activation was reduced to baseline levels in NLT GnRH neurons treated with the PI3K inhibitor, LY294002 (Fig. 6BGo). Together, these data suggest that Ras activation is dependent on and therefore downstream of PI3K and is consistent with the immunoprecipitation data showing that Axl and PI3K directly interact (Fig. 3Go).


Figure 6
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  		FIG. 6. Gas6-dependent Ras activation is diminished by PI3K inhibition. A, NLT GnRH neurons were transfected with pUSE or the pN17RasUSE construct. Twenty-four hours after transfection, the cells were stimulated with Gas6 (400 ng/ml) for 10 min. Cell lysates were subjected to immunoblot analyses with a phospho-specific p85 or Akt antibody (60 kDa) and a total Ras antibody (21 kDa). B, NLT GnRH neurons were incubated with LY294002 (25 µM) or vehicle (DMSO) for 4 h and then stimulated with Gas6 (400 ng/ml) for 10 min followed by a Ras activation assay and compared with the expression of total Ras.

 
Src does not contribute to Gas6-induced and Rac-dependent migration
Because Axl has been shown to activate Src in fibroblasts (27), and this kinase is a known regulator of cell migration, we also evaluated its contribution to GnRH neuronal migration. Pharmacological inhibition of Src by PP2 dramatically reduced both basal (77% reduction from control, data not shown) and Gas6/Axl-stimulated migration (Gas6, 3.0- ± 0.6-fold; Gas6 + PP2, 1.1- ± 0.2-fold, Fig. 7AGo) but had no inhibitory affects on lamellipodia formation (Gas6, 69.0 ± 6.1%; Gas6 + PP2, 89.0 ± 2.7%, Fig. 7BGo). Although treatment with PP2 attenuated Gas6/Axl-induced migration, there was no effect of this inhibitor on Gas6 activation of Rac, the focus of the current studies (Fig. 7CGo).


Figure 7
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  		FIG. 7. Src inhibition abolishes Gas6-induced GnRH migration by a Rac-independent mechanism. A, NLT GnRH neuronal cells (30,000 cells/well) were subjected to the migration assay in the presence of vehicle (DMSO) or PP2 (10 µM) as described in Materials and Methods. The data shown represent the fold change in Gas6-induced migration in the presence (*, n = 3; P < 0.05) or absence (**, n = 3; P < 0.05) of PP2. B, NLT GnRH neuronal cells (15,000 cells per coverslip) were pretreated for 4 h with PP2 (10 µM) or DMSO (control), stimulated with Gas6 (400 ng/ml, 10 min), and then stained with rhodamine phalloidin. Lamellipodia and membrane ruffles were quantitated. The data are representative of at least two independent experiments. *, n = 2; P < 0.05. C, Gas6/Axl-induced Rac activation is unaffected by Src inhibition. NLT GnRH neurons were incubated with inhibitor or vehicle (DMSO) for 4 h and then stimulated with Gas6 (400 ng/ml) for 10 min followed by a Rac activation assay. Each sample contained a similar amount of Rac protein. The data are representative of three independent experiments.

 
Based on these data, we propose the model for Gas6/Axl-mediated GnRH neuronal migration described in Fig. 8Go. Gas6 binding to Axl results in an interaction with, and subsequent activation of, PI3K. PI3K facilitates the activation of Ras, which, in turn, is instrumental in the activation of Rac, a required step for GnRH neuronal migration. PKC and CaMK II are not required for Gas6/Axl-induced GnRH neuronal migration. Although Src is clearly important for efficient migration, it functions in a Rac-independent pathway.


Figure 8
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  		FIG. 8. Schematic illustrating the contribution of various signaling cascades downstream of Gas6/Axl during GnRH neuronal migration.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Because of the structural similarity to neuronal cell adhesion molecules, Axl family RTKs have been proposed to regulate both cell adhesion and migration (12). Using a GnRH neuronal cell line (NLT) that endogenously expresses Axl, we have implicated Axl in the migration of NLT GnRH neurons (29). Our studies showed that Gas6-induced actin cytoskeletal reorganization and migration of GnRH neuronal cells required Axl activation of Rac and the downstream activation of a p38 MAPK, MAPKAP kinase II, heat shock protein 25 pathway. Given that GnRH neurons are known to migrate to the hypothalamus during development, our studies suggest a potential role for Axl family receptors in this process.

In the current report, we sought to define the effectors proximal to Rac in the migratory signaling cascade initiated by Gas6/Axl. The Axl family member UFO has been shown to interact with a variety of downstream effectors through a C-terminal multisubstrate docking site (45). Specifically, tyrosine residue 821 binds the p85{alpha} and ß subunits of PI3K, Grb-2, PLC{gamma}, lck, Src, and lower affinity binding sites for PLC{gamma} at tyrosine 866 and p85 proteins at tyrosine 779. The PI3K p85ß subunit was also shown to associate with Axl after Gas6 treatment of NIH3T3 cells (27). Given that PI3K, PLC{gamma}, and Src had previously been implicated upstream of the Rho family GTPases during cell motility (50, 51, 52, 53), we investigated their involvement in Gas6/Axl-induced migration of NLT GnRH neurons. Gas6 treatment of NLT GnRH neuronal cells resulted in an increased association of PI3K with the Axl receptor and subsequent phosphorylation of its p85 subunit. Moreover, pharmacological inhibition of PI3K resulted in a marked decrease in Gas6 activation of Rac, confirming that Axl couples to PI3K to promote Rac activation. Similarly, agonist-induced lamellipodia extension, membrane ruffling, and migration of GnRH neuronal cells were attenuated in the presence of LY294002. These data support a role for PI3K in Axl-mediated cell motility. Although Src is necessary for Gas6/Axl-mediated migration in these cells, its activity is not required upstream of Rac. Potential Rac-independent pathways contributing to GnRH neuronal migration are currently under investigation.

The contribution of Ras in the activation of Rac has been broadly documented during cytoskeletal rearrangement and migration in epithelial cells (46, 54) and fibroblasts (49, 55); therefore, we investigated whether Ras may also contribute to Rac activation during migration of GnRH neuronal cells. Expression of a dominant-negative Ras diminished Gas6/Axl-induced Rac activity and partially inhibited neuronal migration similarly to that observed upon PI3K inhibition. Ras activity was modulated by inhibition of PI3K, suggesting that Ras is downstream of PI3K and upstream of Rac. Thus, Gas6/Axl-induced GnRH neuronal migration is governed by a serial signaling pathway involving PI3K and Ras functioning upstream of Rac.

Although additional signaling pathways are likely relevant, PI3K and Rac are clearly central regulators downstream of the Gas6/Axl ligand/receptor complex. PI3K is also obligatory for the survival activities induced by Gas6 binding to Ark/Axl. Our previous studies demonstrated that Gas6 prevented serum withdrawal-induced apoptosis of GnRH neuronal cells via PI3K activation of Akt (26). Gas6 also prevents apoptosis of NIH3T3 fibroblasts, but in that system, both PI3K and Src-dependent pathways are involved (27). Notably, the Gas6 survival pathway requires PI3K activation of Rac and the downstream Rac-specific effector protein, PAK, in nonneuronal cells (24). The mitogenic activity of Gas6/Axl in 3T3 fibroblast cells has been coupled to PI3K as well (27). In addition to migration, cell growth, and survival, PI3K and Rac have recently been implicated in Gas6/Axl-regulated gene expression. Repression of GnRH gene expression involves an Axl, Rac, Erk, MAPK cascade (30). Moreover, in vascular smooth muscle cells, Gas6 induces class A scavenger receptor expression via PI3K and Akt, suggesting a role for Gas6/Axl signaling in atherogenesis (56). Given that PI3K and Rac are also key players in the migratory pathways described here and the regulation of gene expression, future studies will focus on defining the downstream effectors required to achieve cell-specific physiological responses initiated by the Gas6/Axl complex in GnRH neurons.


   Footnotes
 
First Published Online March 1, 2007

Abbreviations: Ark, Axl, adhesion-related kinase; Bis, bisindolylmaleimide; CaMK II, calcium/calmodulin protein kinase II; DMSO, dimethylsulfoxide; FBS, fetal bovine serum; Gas6, growth arrest specific gene-6; N17Ras, dominant-negative form of Ras; PI3K, phosphatidylinositol-3-kinase; PKC, protein kinase C; PLC, phospholipase C; PVDF, polyvinylidene diflouride; RT, room temperature; RTK, receptor tyrosine kinase; TBST, 0.1% Tween 20 in Tris-buffered saline.

This work was supported by the National Institutes of Health (Grants HD08667 to M.P.A., HD31191 to M.E.W., N538619 to K.A.H.), by the Veterans Affairs Merit Review (grants to M.E.W. and K.A.H.), and by the Research Enhancement Award Program (to D.A.L.).

S.M.N.-P., M.P.A., M.X., D.A.L., J.E.P., R.J.B., and K.A.H. have nothing to disclose. B.C.V. is employed by Amgen, has stock options in Amgen, and a patent on Gas6, but no commercial interest. M.E.W. is on the speakers bureau for Merck and Pfizer.

Received January 12, 2007.

Accepted for publication February 22, 2007.


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