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

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.