Arachidonic Acid Stimulates Cell Adhesion through a Novel p38 MAPK-RhoA Signaling Pathway That Involves Heat Shock Protein 27

Abstract

Rho GTPases are critical components of cellular signal transduction pathways. Both hyperactivity and overexpression of these proteins have been observed in human cancers and have been implicated as important factors in metastasis. We previously showed that dietary n-6 fatty acids increase cancer cell adhesion to extracellular matrix proteins, such as type IV collagen. Here we report that in MDA-MB-435 human melanoma cells, arachidonic acid activates RhoA, and inhibition of RhoA signaling with either C3 exoenzyme or dominant negative Rho blocked arachidonic acid-induced cell adhesion. Inhibition of the Rho kinase (ROCK) with either small molecule inhibitors or ROCK II-specific small interfering RNA (siRNA) blocked the fatty acid-induced adhesion. However, unlike other systems, inhibition of ROCK did not block the activation of p38 mitogen-activated protein kinase (MAPK); instead, Rho activation depended on p38 MAPK activity and the presence of heat shock protein 27 (HSP27), which is phosphorylated downstream of p38 after arachidonic acid treatment. HSP27 associated with p115RhoGEF in fatty acid-treated cells, and this association was blocked when p38 was inhibited. Furthermore, siRNA knockdown of HSP27 blocked the fatty acid-stimulated Rho activity. Expression of dominant negative p115-RhoGEF or p115RhoGEF-specific siRNA inhibited both RhoA activation and adhesion on type IV collagen, whereas a constitutively active p115RhoGEF restored the arachidonic acid stimulation in cells in which the p38 MAPK had been inhibited. These data suggest that n-6 dietary fatty acids stimulate a set of interactions that regulates cell adhesion through RhoA and ROCK II via a p38 MAPK-dependent association of HSP27 and p115RhoGEF.

The ability of tumor cells to metastasize to secondary sites is a hallmark of neoplastic disease. Unfortunately, this propensity to spread is the primary cause of morbidity and death in cancer patients (1). Metastasis is clearly a highly regulated, multistep process that occurs in a spatiotemporal manner (2–4). To escape the restrictive compartment boundaries characteristic of adult tissue, separate intravasation and extravasation steps requiring alterations in co-adhesion, adhesion, invasion, and migration must occur. Execution of these biological processes, involving multiple proteins and cellular organelles, require highly coordinated cell signaling mechanisms.

The Rho family of small GTPases regulates many facets of cytoskeletal rearrangements that facilitate cell attachment and migration (5–7). Rho GTPases act as molecular switches by changing from an inactive GDP-bound conformation to an active GTP-bound conformation, thereby regulating a signaling pathway. These proteins are directly regulated by Rho guanine nucleotide exchange factors (GEFs),² Rho GTPase activating proteins, and Rho GDP-dissociation inhibitors (8–12). RhoGEFs bind to the GTPase to catalyze the dissociation of GDP, allowing the binding of GTP and thereby promoting Rho activation (8). The RGS (regulators of G protein signaling) domain-containing RhoGEFs are a recently described family of GEFs. Currently, there are three members of this family, PDZ-RhoGEF, LARG, and p115RhoGEF (13–15), in which the RGS domains function as a heterotrimeric GTPase-activating domain (13, 15, 16). The RGS family of RhoGEFs has been shown to regulate Rho during several processes including cytoskeletal rearrangements, cell adhesion, and cancer progression (17–21).

There is significant interplay between the activity of small GTPases and signaling derived from fatty acid metabolism (22–28). Linoleic acid, which is metabolized to arachidonic acid, is an n-6 polyunsaturated fatty acid that is present at high levels in most western diets (29). In animal models, diets high in n-6 polyunsaturated fatty acids have been shown to enhance tumor progression and metastasis (30, 31). Additionally, arachidonic acid is stored in cell membranes and is made available by phospholipases under conditions of increased inflammatory response (32). Arachidonic acid is further metabolized by cyclooxygenases (COX), lipoxygenases (LOX), and cytochrome P450 monooxygenases to yield bioactive products that have myriad effects on cells, and altered metabolism of arachidonic acid by COX, LOX, and P450 has been implicated in cancer progression (31, 33–36).

We have studied mechanisms of cell adhesion using the MDA-MB-435 cells as a model of a highly metastatic human cancer cell line (37). These cells have been extensively studied for their ability to recapitulate the metastatic cascade in vivo and in vitro, although recent work indicates that the cells currently in use are most likely a human melanoma line (38). We initially observed that arachidonic acid (AA) enhanced adhesion of MDA-MB-435 cells to type IV collagen through specific integrin-mediated pathways (37). Exogenous AA led to the activation of mitogen-activated protein kinase (MAPK)-activated protein kinase 2 and the phosphorylation of heat shock protein 27 (HSP27) via a p38 MAPK-dependent process (39). Inhibition of p38 MAPK activation blocked cell adhesion as did function-blocking antibodies specific for subunits of the collagen receptor (40). More recently, we identified the key metabolite of AA (15-(S)- hydroxyeicosatetraenoic acid) and the upstream kinases (TAK1 and MKK6) that are responsible for activation of p38 MAPK in this system (41).

In this study we investigated the role of Rho activation in the MDA-MB-435 cells after exposure to arachidonic acid. Several aspects of the regulation of Rho signaling in these cells provide insights into the cross-talk between important signaling pathways.

EXPERIMENTAL PROCEDURES

Antibodies

Anti-Rho, anti-Rac, anti-HA, anti-p38, and anti-GAPDH were from Upstate (Charlottesville, VA). Anti-RhoA was from Cytoskeleton, Inc. (Denver, CO), anti-RhoB was from Bethyl Laboratories (Montgomery, TX), and anti-RhoC was from Santa Cruz (Santa Cruz, CA). Anti-p115RhoGEF was from Santa Cruz. Anti-V5 was from Invitrogen. Anti-ROCK I and ROCK II were from Pharmingen (San Diego, CA). Anti-phospho p38 (Tyr/Thr) and anti-Hsp27 were from Cell Signaling (Danvers, MA).

Materials

Cell-permeable C3 exoenzyme was from Cytoskeleton, Inc. The ROCK inhibitors Y27632 and H-1152 were from Calbiochem. GTPγS was from Upstate. Glutathione-Sepharose and ECL reagent were from Amersham Biosciences. Arachidonic acid was from Cayman Biochemicals (Ann Arbor, MI) and diluted in a KOH/NaCl solution as previously described (37). Collagen type IV and poly-d-lysine were from BD Biosciences. FuGENE 6 was from Roche Applied Science. Lipofectamine2000 was from Invitrogen. The p38 inhibitors PD169316, SB203580, and the inactive analog SB202474 were from Calbiochem.

Cell Culture

MDA-MB-435 cells were obtained from Janet Price (MD Anderson Cancer Center, Houston, TX). Cells were maintained at 37 °C under 5% CO₂ in Eagle's minimal essential medium (Invitrogen) supplemented with 5% fetal bovine serum (Hyclone, Logan, UT), 1 mm sodium pyruvate, 2 mm l-glutamine, and 2× minimal essential medium vitamin solution (Invitrogen).

DNA Constructs and siRNAs

The GST Rho binding domain of ROCK (GST-RBD) construct was a generous gift from John P. O'Bryan (University of Illinois College of Medicine). 3× HA-tagged wild-type RhoA, dominant negative RhoA (T17N), and constitutively active RhoA (G12V) were from the University of Missouri-Rolla cDNA Resource Center. Full-length, dominant negative, and constitutively active p115RhoGEF constructs carrying a V5-peptide tag (FLp115RhoGEF, 4Ap115RhoGEF, and ΔCp115RhoGEF) were generous gifts from Keith Burridge (University of North Carolina, Chapel Hill, NC). An siRNA that targets p115RhoGEF was from Dharmacon (Lafayette, CO). HSP27 siRNAs were from Cell Signaling. The H1 promoter-driven negative control and ROCK II short hairpin RNA constructs were from Upstate.

Adhesion Assays

Adhesion assays were carried out as described (37). Briefly, cells were harvested with Versene (Invitrogen), resuspended in serum-free medium at 3 × 10⁵ cells/ml, and allowed to recover for 30 min at 37 °C in 5% CO₂. When appropriate, the cells were treated with inhibitors H-1152 and Y27632 for 15 and 30 min, respectively. When C3 exoenzyme was used, cells were incubated with the inhibitor overnight before harvesting the cells. Arachidonic acid (30 μm) or vehicle (ethanol/KOH) was added to the cell suspension, and aliquots of the cells were added to a 96-well tissue culture plate previously coated with collagen type IV, poly-d-lysine, or bovine serum albumin. The plate was incubated for 45 min at 37 °C in 5% CO₂. The adherent cells were fixed, stained with crystal violet, and solubilized. The absorbance of the wells at 595 nm was measured, and collagen IV adhesion was calculated as a percentage of absorbance in poly-d-lysine-coated wells after the bovine serum albumin background was subtracted.

Small G Protein Activation Pulldown Assay

Cells were harvested, washed, and allowed to recover in serum-free medium for 30 min. After treatment with 30 μm arachidonic acid, cells were washed with ice-cold PBS and lysed with 50 mm HEPES, pH 7.5, 1.5 mm MgCl₂, 150 mm NaCl, 10% glycerol, 1% Triton X-100, 0.25% deoxycholate, 1 mm EGTA, 100 mm NaF, 10 μg/ml of each aprotinin and leupeptin, and 1 mm Na₃VO₄. Lysates were clarified and incubated on a rocker for 1 h at 4 °C with the GST fusion protein containing the Rho binding domain of ROCK bound to glutathione-Sepharose beads. Samples were washed three times with lysis buffer, and bound proteins were eluted by boiling in LDS NuPage® sample buffer (Invitrogen), separated by SDS-PAGE, and immunoblotted with appropriate antibodies, as described (42).

Confocal Microscopy

Coverslips from Carolina Biological Supply Co. (Burlington, NC) were coated overnight with type IV collagen at a concentration of 6.4 μg/ml. Slides were blocked with 2% heat-denatured bovine serum albumin for 2 h and washed with serum-free Eagle's minimum essential medium. Cells were transfected for 24 h with Rho and p115RhoGEF constructs using FuGENE 6. Cells were harvested, washed, allowed to recover in serum-free media for 30 min, and treated with 30 μm arachidonic acid. Cells were placed on the collagen-coated coverslips at 2 × 10⁵ cells per coverslip. After treatment, cells were washed with PBS and fixed in 3.7% paraformaldehyde containing 0.1% Triton X-100 for 20 min at room temperature. Cells were washed 3 times with PBS, permeabilized with 0.5% Triton X-100 in PBS for 10 min, washed with PBS, and incubated for 45 min in 4% donkey serum in 0.1% gelatin blocking solution at room temperature. Cells were incubated with anti-HA or anti-V5 antibody (diluted 1:200). The slides were washed with PBS and incubated with Alexa Fluor®488-conjugated anti-mouse antibody (diluted 1:2000) and Alexa Fluor®594-labeled phalloidin (diluted 1:1000) for 1 h. The slides were washed with PBS and mounted using Prolong Gold anti-fade reagent (Invitrogen). Samples were analyzed with a Zeiss Pascal scanning confocal microscope (Carl Zeiss Microimaging, Inc., Thornwood, NY) using a 100 × 1.4 NA oil Plan Neofluor Apochromat objective lens.

Immunoprecipitation of HSP27

Cells were harvested as described for the G protein pulldown assays, exposed to vehicle or arachidonic acid for 5 min, and lysed in 50 mm Tris, pH 7.4, 150 mm NaCl, 0.25% deoxycholate, 1% IGEPAL, 1 mm EDTA, 1 mm NaF, 1 mm Na₃VO₄, 1 mm phenylmethylsulfonyl fluoride, and 10 μg/ml each of aprotinin, pepstatin, and leupeptin. The cell lysates were precleared with rabbit IgG-agarose beads (Santa Cruz) for 1 h at 4 °C and then incubated with a rabbit anti-HSP27 antibody overnight at 4 °C with constant rotation. The antibody complexes were pulled down using protein A/G-agarose beads (Santa Cruz) for 1 h, and the precipitates were washed 4 times with lysis buffer. The samples were denatured and immunoblotted as described for the small G protein pulldown assays.

RESULTS

Arachidonic Acid Increases RhoA Activity

Previously we showed that arachidonic acid increases adhesion of MDA-MB-435 cells to type IV collagen (37). This increased adhesion is accompanied by a variety of cytoskeletal rearrangements typical of increased adhesion and cell motility.³ These observations are similar to those seen in other cell types (e.g. Refs. 22, 24, and 43). As these structural and functional changes are frequently dependent on altered activity of Rho family small GTPases, we examined human tumor cells exposed to fatty acids for changes in the activity of Cdc42, Rac, and Rho. Rho activity in MDA-MB-435 cells significantly increased after arachidonic acid treatment, whereas total levels of Rho expression remained unchanged (Fig. 1, A and B). Several laboratories have shown that MDA-MB-435 cells exhibit significant levels of Rac activity (44, 45). We confirmed this result and observed a slight decrease in Rac activity upon arachidonic acid treatment (supplemental Fig. S1A). Untreated MDA-MB-435 cells did not exhibit detectable levels of Cdc42 activity with or without arachidonic acid treatment (supplemental Fig. S1B).

FIGURE 1.

Arachidonic acid induces an increase in active RhoA. A, active Rho was pulled down from MDA-MB-435 cell lysates prepared at various times after exposure to 30 μm arachidonic acid using the Rho binding domain of ROCK (GST-RBD); precipitated proteins were separated by SDS-PAGE and immunoblotted for Rho. Whole cell lysates used in the pulldown assay were immunoblotted for Rho to determine loading of total Rho into the assay. The data shown are representative of three independent experiments. B, quantitation of immunoblots from three independent experiments was performed using the Eastman Kodak ds^TM Image Station 440CF, and the -fold increase was obtained by normalization for Rho expression levels with the 0-min time point set to a value of 1. C, active Rho was pulled down as above from AA-treated (30 μm arachidonic acid for 20 min) and vehicle-treated (V) cells and immunoblotted with antibodies specific for RhoA, RhoB, and RhoC. Whole cell lysates used in the pulldown assay were probed for RhoA, RhoB, and RhoC. The data shown are representative of three independent experiments.

Three Rho isoforms, RhoA, -B, and -C, have been shown to function in cell motility and adhesion (46). We investigated changes in these isoforms by means of Rho-GTP pulldown assays followed by immunoblotting with isoform-specific antibodies (Fig. 1C). Cells treated with arachidonic acid showed a significant increase in RhoA activity but not in RhoB or RhoC. A similar activation of RhoA by arachidonic acid occurred in the SK-BR-3 breast cancer cell line (supplemental Fig. S2).

RhoA Activation Is Necessary for Adhesion of MDA-MB-435 Cells to Collagen IV

Given the well described role of Rho in cell adhesion, we hypothesized that Rho activation is required for arachidonic acid-induced adhesion to type IV collagen. To test this hypothesis, we treated cells with cell-permeable C3 exoenzyme to block Rho signaling and measured arachidonic acid-induced cell adhesion to type IV collagen. C3 exoenzyme reduced arachidonic acid-induced adhesion to a level similar to that of untreated cells (Fig. 2A). To confirm these inhibitor results, cells were transfected with wild-type, constitutively active, or dominant negative RhoA-containing vectors and assayed for arachidonic acid-induced adhesion. Expression of a dominant negative RhoA completely blocked arachidonic acid-induced adhesion, whereas cells expressing wild-type RhoA showed a typical increase in adhesion to type IV collagen (Fig. 2B). Expression of a constitutively active RhoA also permitted arachidonic acid-induced adhesion (Fig. 2C) but did not increase the basal level of adhesion, indicating that active RhoA alone is not sufficient to enhance cell adhesion. This is consistent with our previous results showing that both MAPK and protein kinase C-dependent pathways must be activated, apparently independently, for induced cell adhesion by fatty acids (39, 40).

FIGURE 2.

Rho activity is necessary for arachidonic acid-induced cell adhesion to collagen IV. Cells were treated with vehicle (open) (ethanol) or 30 μm arachidonic acid (shaded) and assayed for adhesion to collagen IV as described. V, vehicle; C3, C3 exoenzyme (0.5 μg/ml). A, C3+AA-treated is significantly different from AA-treated (p < 0.5, Student's t test). Data shown are representative of three independent experiments. B, cells were transiently transfected with HA-tagged RhoA (WT) or HA-tagged dominant negative RhoAT17N (DN) then treated with vehicle or arachidonic acid and assayed for adhesion to collagen IV. DN+AA-treated is significantly different from AA-treated (p < 0.5, Student's t test). Data shown are representative of three independent experiments. C, cells were transiently transfected with HA- tagged RhoA (WT) or constitutively active Rho G12V (CA) then treated with vehicle or arachidonic acid and assayed for adhesion to collagen IV. Data shown are representative of three independent experiments. In each panel values that are significantly different from the vehicle-treated cells (V) (as determined by Student's t test (p < 0.05)) are indicated (*).

In cells treated with arachidonic acid, MDA-MB-435 cells show an increase in focal adhesions and an altered morphology (supplemental Fig. S3, A–F). Arachidonic acid-treated cells exhibited short cortical actin stress fibers accompanied by actin arcs (supplemental Fig. S3, D–F), a type of actin cytoskeleton structure previously shown to correspond with Rho activity (19). Cell morphology and actin structure were similar in both non-transfected and HA-tagged wild-type RhoA-expressing cells (supplemental Fig. S3, G–L). Additionally, a subset of HA-tagged RhoA was localized at the membrane, associated with cortical actin stress fibers and the tips of filapodial-like structures. Expression of dominant negative RhoA blocked the morphological and structural changes in response to fatty acid (supplemental Fig. S3, M–R).

ROCK Is Necessary for Adhesion of MDA-MB-435 Cells to Collagen IV

Rho GTPases frequently regulate cellular processes through downstream signals transduced by the Rho kinase, ROCK (47–49). Thus, we asked whether adhesion of cells to type IV collagen induced by arachidonic acid depended on ROCK activity. We used both a pharmacological inhibitor as well as a genetic approach to block ROCK. Two ROCK inhibitors blocked arachidonic acid-induced adhesion in a dose-dependent fashion (Fig. 3, A and B). There are two ROCK isoforms, ROCK I and II. We investigated which isoforms were expressed in the MDA-MB-435 cells and found that ROCK II is the main isoform in these cells (supplemental Fig. S4, A and B). Additionally, arachidonic acid-induced adhesion was blocked in cells transiently expressing two different short hairpin RNAs specifically targeting ROCK II (Fig. 3C). These results indicate that arachidonic acid-induced adhesion of MDA-MB-435 cells to type IV collagen requires both Rho activation and ROCK II signaling. Several proteins have been implicated as effectors of ROCK signaling, including myosin light chain (50), cofilin (51), and caveolin (52). We examined each of these in MDA-MB-435 cells and did not identify any ROCK-dependent changes in phosphorylation after treatment with arachidonic acid (data not shown), indicating that although Rho/ROCK is critical for the fatty acid-induced adhesion, the signaling apparently functions through other downstream effectors.

FIGURE 3.

ROCK activity is necessary for arachidonic acid-induced cell adhesion to collagen IV. A, cells were pretreated with 10 μm of ROCK inhibitor (Y-27632) for 30 min, then treated with vehicle (V, open) or arachidonic acid (shaded) and assayed for adhesion to collagen IV. Assays were performed in triplicate. Y267632+AA-treated is significantly different from AA-treated (p < 0.5, Student's t test). Data shown are representative of three independent experiments. B, cells were pretreated with the ROCK inhibitor (H-1152) and treated with either vehicle (■) or arachidonic acid (▲). Assays were performed in triplicate, and data shown are representative of three independent experiments. C, cells were mock-transfected (V and AA) and transfected with a negative control short hairpin RNA vector (NC) and two short hairpin RNAs targeting ROCK II (K1 and K2) for 72 h. The cells were treated with vehicle (V) (open) or AA (shaded) and assayed for adhesion to collagen IV. K1+AA-treated and K2+AA-treated are significantly different from NC+AA-treated (p < 0.5, Student's t test). Data shown are representative of three independent experiments. Inset, cell lysates were separated by SDS-PAGE and immunoblotted (IB) for ROCK II and GAPDH to determine knockdown efficiency. The numbers between the panels indicate the ratio of ROCK II protein in the samples to that in the mock-transfected after normalization to the GAPDH signal. In all panels values that are significantly different from the vehicle (as determined by Student's t test (p < 0.05)) are indicated (*).

p38 MAPK Mediates Arachidonic Acid-induced RhoA Activity through HSP27

A number of studies have shown that the activation of Rho isoforms can lead to signaling through the MAPK pathways. We have shown that arachidonic acid induces phosphorylation of p38 MAPK and that its activity is required for adhesion to type IV collagen (39, 40). Thus, we asked if p38 MAPK functions upstream or downstream of Rho signaling after arachidonic acid exposure in these cells. Pharmacological inhibition (Y27632) of ROCK had no effect on arachidonic acid-induced phosphorylation of p38 (Fig. 4A). In contrast, a p38 inhibitor, PD169316, blocked activation of RhoA in arachidonic acid-treated cells, whereas the inactive analog did not (Fig. 4B). These data are consistent with p38 MAPK functioning upstream of Rho activation in arachidonic acid-induced cell adhesion but do not define the mechanism.

FIGURE 4.

p38 MAPK is upstream of arachidonic acid-induced Rho signaling. A, whole cell lysates were prepared from AA-treated and vehicle-treated (V) cells, separated by SDS-PAGE, and probed for phospho (p)-p38 and total p38. Where indicated, cells were pretreated with 10 μm ROCK inhibitor, Y27632. The data shown are representative of three independent experiments. B, cells were pretreated with 10 μm p38 inhibitor (PD169316) or the inactive analog (SB202474) for 30 min. Active Rho was pulled down from arachidonic acid-treated (+) (30 μm arachidonic acid for 20 min) and vehicle-treated (−) cells and immunoblotted (IB) with antibodies specific for RhoA. Whole cell lysates were immunoblotted for total Rho.

We (39, 41) (supplemental Fig. S5) and others (53–55) have previously shown that phosphorylation of HSP27 occurs downstream of p38 MAPK activation. HSP27 has been proposed to play a critical role in the regulation of actin filament polymerization and invasion (56–58). We asked whether HSP27 was critical for arachidonic acid-induced RhoA activity by using an siRNA targeting HSP27 to decrease its expression. Cells transfected with the HSP27 siRNA exhibited a reduction in arachidonic acid-induced RhoA activity when compared with untransfected and control siRNA-transfected cells (Fig. 5A). Because the process of actin filament formation is regulated by Rho and its effectors, we asked whether arachidonic acid induced an association between HSP27 and factors influencing Rho activity. Immunoprecipitations of HSP27 from cells that had been treated with arachidonic acid showed a significant increase in association with p115RhoGEF (Fig. 5B), a Rho guanine nucleotide exchange factor that regulates RhoA signaling (19, 21, 59). Because inhibition of p38 blocked RhoA activity, we asked whether the HSP27·p115RhoGEF association was dependent on p38 activation. Indeed, the fatty acid-stimulated increase in HSP27·p115RhoGEF association was blocked by a p38 inhibitor (Fig. 5B), thus suggesting that the increase in phospho-HSP27 is the critical feature in the formation of the HSP27·p115 complex. However, we cannot rule out the possibility that other factors are also activated and critical for the HSP27·p115 association.

FIGURE 5.

HSP27 is critical for arachidonic acid activation of Rho and pulls down a RhoGEF. A, cells were either untransfected or transfected with 50 nm control siRNA or with 50 nm HSP27-specific siRNA and plated for 48 h, then were treated with ethanol vehicle (V) or 30 μm arachidonic acid and assayed for Rho activity. Whole cell lysates were immunoblotted for HSP27, total RhoA, and GAPDH. Data shown are representative of two independent experiments. B, cells were treated either with the p38 inhibitor SB203580 (10 μm) or the solvent vehicle (DMSO) for 30 min, then exposed to arachidonic acid (30 μm) or vehicle (NaCl/KOH) for 5 min. Whole cell lysates were immunoprecipitated (IP) for HSP27 and immunoblotted (IB) for p115RhoGEF and HSP27. Data shown are representative of four independent experiments.

p38 MAPK and p115RhoGEF Are Required for RhoA Activation Leading to Adhesion on Type IV Collagen

A variety of RhoGEFs exist that can regulate the activity of Rho. Because HSP27 appears to associate with the p115RhoGEF, we hypothesized that p115RhoGEF associates with Rho and mediates arachidonic acid-dependent RhoA activity in these cells. Immunoblots revealed that p115RhoGEF was pulled down with active Rho in arachidonic acid-treated cells but was not detected in pulldown assays from vehicle-treated cells (Fig. 6A). Inhibition of p38 MAPK blocked both the activation of RhoA and the association of p115RhoGEF (Fig. 6A). To determine whether p115RhoGEF is required for RhoA activation, we transfected cells with wild-type p115RhoGEF (FLp115RhoGEF) or a dominant negative form (4Ap115RhoGEF) of p115RhoGEF (19). RhoA activity was induced by arachidonic acid in cells transfected with wild-type p115RhoGEF, but this activation was blocked in cells transfected with the dominant negative GEF (Fig. 6B). Moreover, a significant reduction of p115RhoGEF by siRNA targeting this GEF also blocked fatty acid-induced RhoA activity (Fig. 6C). Both the dominant negative (4Ap115RhoGEF) (Fig. 6D) and the p115-specific siRNA blocked arachidonic acid-induced adhesion on collagen IV (Fig. 6, E and F). Collectively, these data indicate that p115RhoGEF is critical for both arachidonic acid-induced RhoA activation and adhesion.

FIGURE 6.

p115RhoGEF is required for arachidonic acid-induced p38 MAPK mediated adhesion on type IV collagen. A, cells were pretreated with 10 μm of p38 inhibitor (PD169316) or the inactive analog (SB202474). Active Rho was pulled down from AA-treated (30 μm arachidonic acid for 20 min) and vehicle-treated (V) cells and immunoblotted with antibodies specific for RhoA. Whole cell lysates used in the pulldown assay were separated by SDS-PAGE and probed for RhoA or p115RhoGEF. The data shown are representative of three independent experiments. B, MDA-MB-435 cells were untransfected, mock-transfected, or transfected with either V5-tagged p115RhoGEF (FLp115) or dominant negative p115RhoGEF (4Ap115) for 24 h. GST-RBD was used to pull down active Rho in cell lysates that were treated with ethanol vehicle (V) or AA for 20 min. The precipitates were immunoblotted for the V5 tag and RhoA. Whole cell lysates used in the pulldown assay were separated by SDS-PAGE and immunoblotted for RhoA and GAPDH. Data shown are representative of three independent experiments. C, active Rho was pulled down from cells that were mock-transfected or transfected with 50 nm concentrations of either control siRNA or 50 nm p115RhoGEF siRNA for 48 h and treated with vehicle (V) or AA. Cell lysates were separated by SDS-PAGE and immunoblotted for p115RhoGEF and GAPDH to determine a knockdown efficiency of 75%. Data shown are representative of two independent experiments. D, cells transfected as in B were treated with ethanol vehicle (open) or 30 μm arachidonic acid (shaded) and assayed for adhesion to collagen IV. 4A+AA-treated is significantly different from AA-treated (p < 0.5, Student's t test). Data shown are representative of three independent experiments. M, mock-transfected. FL, full-length. E, cells were untransfected (N), transfected with 50 nm control siRNA (C), or transfected with 50 nm p115RhoGEF siRNA (P) and plated for 48 h. Cells were then treated with ethanol vehicle (open) or 30 μm arachidonic acid (shaded) and assayed for adhesion to collagen IV. Cell lysates were separated by SDS-PAGE and immunoblotted for p115RhoGEF and GAPDH to determine knockdown efficiency. The numbers between the panels indicate the ratio of p115RhoGEF protein in the samples to that of the untransfected cells after normalization to the GAPDH signal. Data shown are representative of two independent experiments. P+AA-treated is significantly different from AA-treated (p < 0.5, Student's t test). Values in panels C and D that are significantly different from the vehicle-treated (V) (Student's t test (p < 0.05) are indicated (*). F, whole cell lysates from cells treated as in panel E were separated by SDS gel electrophoresis and immunoblotted for either p115RhoGEF or GAPDH.

The above data indicate that p115RhoGEF is required. If this GEF functions downstream of p38 MAPK, then expression of a constitutively active form of the Rho-associated factor should restore cell adhesion after inhibition of p38 activity. If it functions upstream of p38, the constitutively active p115 would not restore cell adhesion in the presence of a p38 inhibitor. We found that both arachidonic acid-induced RhoA activity and cell adhesion on type IV collagen, when blocked by the p38 MAPK inhibitor, were restored by the expression of a constitutively active p115RhoGEF construct (ΔCp115RhoGEF) (Fig. 7, A and B). Furthermore, although this constitutively active GEF activates RhoA in the absence of fatty acid, it does not activate p38 nor does it induce increased cell adhesion to collagen IV.

FIGURE 7.

RhoGEF is downstream of p38 MAPK. A, MDA-MB-435 cells were transfected with V5-tagged full-length p115RhoGEF (FLp115RhoGEF) or constitutively active p115RhoGEF (ΔCp115RhoGEF) for 24 h. Active Rho was pulled down in cell lysates that were pretreated with DMSO vehicle (V) or PD169316 for 30 min and treated with either ethanol vehicle or 30 μm arachidonic acid. The proteins were separated by SDS-PAGE and immunoblotted for RhoA. Whole cell lysates used in the Rho assay were separated by SDS-PAGE and immunoblotted for p-p38 MAPK, RhoA, and the V5 tag. Data shown are representative of two experiments. B, MDA-MB-435 cells were transfected with V5-tagged full-length p115RhoGEF (FLp115RhoGEF) or constitutively active p115RhoGEF (ΔCp115RhoGEF) for 24 h and pretreated with PD169316 p38 MAPK inhibitor for 30 min. The cells were then treated with ethanol vehicle (open) or 30 μm arachidonic acid (shaded) and assayed for adhesion to collagen IV. FLp115RhoGEF expressing cells exposed to PD169316 and arachidonic acid were significantly different from AA-treated alone (p < 0.5, Student's t test). Data shown are representative of three independent experiments.

p115RhoGEF Is Required for Arachidonic Acid-induced Cell Morphology and Cytoskeleton Structural Changes on Type IV Collagen

Several studies have shown that p115RhoGEF is critical for changes in actin dynamics resulting from adhesion to extracellular matrices (19). We hypothesized that p115RhoGEF is critical for RhoA-mediated actin cytoskeletal changes in these tumor cells. As previously observed, arachidonic acid exposure of MDA-MB-435 cells led to a change in morphology and in altered cytoskeletal structures (Fig. 8, A–C versus D–F). FLp115RhoGEF-transfected cells that were treated with arachidonic acid (Fig. 8, J–L) exhibited a similar change in both morphology and actin filaments. Additionally, a subset of FLp115RhoGEF was localized at the plasma membrane of the cell where it colocalized with cortical actin filaments (Fig. 8L). In contrast, cells expressing the dominant negative 4Ap115RhoGEF exhibited no change in actin cytoskeleton architecture when treated with arachidonic acid (Fig. 8, P–R), indicating that the arachidonic acid-induced Rho-dependent changes in morphology and cytoskeleton depend on the presence of active p115RhoGEF.

FIGURE 8.

p115RhoGEF is critical for arachidonic acid-induced cell spreading on collagen IV. MDA-MB-435 cells (A–F), cells expressing FLp115RhoGEF (G–L), or 4Ap115RhoGEF (M–R) cells were treated with vehicle (V) or AA and seeded onto type IV collagen-coated coverslips for 45 min. Cells were fixed and probed for mouse anti-HA (green) followed by Alexa Fluor® 488 donkey anti-mouse and phalloidin (red). Samples were stained with 4′,6-diamidino-2-phenylindole (blue) to mark the cell nuclei. Co-localization of HA-tagged constructs and actin filaments was determined in a merged image as yellow (arrows). Data shown are representative of three independent experiments.

DISCUSSION

The aim of the present study was to delineate the role that Rho GTPases play in fatty acid-induced adhesion and cell signaling and to define the components of the pathway. We found that the guanine nucleotide exchange factor p115RhoGEF is critical for arachidonic acid-induced RhoA activity as well as changes in morphology and adhesion on type IV collagen. Furthermore, p38 and one of its downstream targets, HSP27, are critical upstream components for this induction of RhoA activity, whereas in most previously described systems (22, 24, 43) p38 MAPK has been found downstream of RhoA. The mechanism for this fatty acid-induced activation appears to involve a complex that includes HSP27 and p115RhoGEF, thereby providing a previously undescribed link between p38 MAPK and p115RhoGEF/RhoA. Consistent with prior studies (22, 24, 43), arachidonic acid induces an increase in the activity of RhoA, leading to increased adhesion to type IV collagen, changes in cell morphology, and rearrangement of cytoskeletal architecture.

Members of the family of Rho-GTPases have been shown to regulate cell adhesion, and our previous studies show that dietary fatty acids induce adhesion of the MDA-MB-435 cells to type IV collagen (7, 40). Our current study provides a link between Rho-GTPase activity and arachidonic acid-induced adhesion to type IV collagen. Concomitant with the increase in Rho activity, there was a decrease in Rac activity. This counter activity of Rac and Rho, observed in multiple systems, is key to cell migration and, therefore, may be necessary for tumor cell metastasis (60–63). The cross-talk regulating the counter activity of Rho and Rac remains unclear (64–66), although recent work suggests that proteins Rho GTPase-activating proteins may be involved (67).

The various Rho isoforms have been shown to play a role in tumor metastasis, with RhoC being most frequently associated with malignant phenotypes (46). RhoB expression has been shown to be anti-metastatic and to decrease with disease progression (68, 69). We found that there was very little RhoB in the MDA-MB-435 cells and no detectable change in its activation with arachidonic acid treatment. In contrast, RhoA and RhoC overexpression are indicative of a pro-metastatic phenotype, suggesting that the activation of these proteins may be critical for metastasis (70, 71). Only RhoA was activated in the arachidonic acid-treated cells, and inhibition of RhoA activation blocked arachidonic acid-induced adhesion on type IV collagen. These data indicate that arachidonic acid may promote processes critical for certain steps in metastasis through RhoA signaling.

Tumor cell migration requires a set of complex adhesion and de-adhesion steps concomitant with dynamic changes in the cellular architecture (72). Previous work has shown that RhoA and p115RhoGEF function as regulators of the cytoskeleton leading to cell adhesion and spreading (e.g. Ref. 19). Arachidonic acid-induced adhesion is accompanied by a variety of cytoskeletal rearrangements typical of increased adhesion and cell motility.³ We show here that RhoA and its regulator p115RhoGEF are required for arachidonic acid-induced changes in cell morphology and cytoskeleton. These data indicate that arachidonic acid-induced Rho signaling is critical both for cell adhesion to collagen IV and for the regulation of actin filament formation.

p38 MAPK activity is a critical component of arachidonic acid-induced signaling, leading to adhesion on type IV collagen (39, 40). Studies in other cell systems have found that p38 functions downstream of RhoA activity (73, 74). Surprisingly, we found that p38 inhibition blocked RhoA-GTP formation after exposure to arachidonic acid. Because a p115RhoGEF-specific siRNA blocks the arachidonic acid stimulation, the rescue of p38 inhibition with a constitutively active p115RhoGEF suggests that the p115 is both necessary and sufficient to function in a p38-RhoA pathway, and the simplest model is that p38 functions upstream of p115RhoGEF-RhoA signaling in these cells. The inability of a constitutively active p115RhoGEF, which activates RhoA in this cell line, to induce phosphorylation of p38 is consistent with this model. We initially hypothesized that p38 might phosphorylate targets associated with Rho and examined p115RhoGEF for serine/threonine phosphorylation after arachidonic acid treatment of the cells but did not detect any change in phosphorylation (data not shown). Thus, we examined the possibility that p38 MAPK mediated an interaction between a downstream effector and the Rho activating proteins. We previously showed MAPK-activated protein kinase 2 is activated to phosphorylate HSP27 after arachidonic acid treatment, leading to an alteration in the phosphorylation state of HSP27 in vivo (39). Our current study now reveals a p38-dependent interaction of this small heat shock protein with the p115RhoGEF that is critical for arachidonic acid-induced Rho activation and adhesion to extracellular matrix. The data suggest that phospho-HSP27 is the critical species involved in the complex, but we cannot rule out that p38 activation modulates the activity of another factor that serves to facilitate formation of an HSP27·p115 complex. The presence of HSP27 is consistent with and complementary to other findings that suggest HSP27 can serve a scaffolding function for various kinase complexes (75, 76). Thus, our data are compatible with a model (Fig. 9) in which HSP27 interacts with p115RhoGEF or a complex on actin filaments containing the RhoGEF to activate RhoA, leading to changes in the cellular cytoskeleton and adhesion. These results suggest a new paradigm for activation of RhoA, at least in a well studied human melanoma cell line. It will be interesting to see if other cells use this or alternate pathways for generating complexes between small scaffolding proteins such as HSP27 and critical regulators of cytoskeletal change. Identification of these type of complexes support the concept (77) that the small heat shock proteins are an interesting subject for therapeutic intervention to reduce activities critical for tumor cell metastasis.

FIGURE 9.

Model for arachidonic acid-induced activation of RhoA/ROCK in MDA-MB-435 cells. 15-(S)-(HETE),15-(S)- hydroxyeicosatetraenoic acid.