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 mitogenactivated 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 GTPbound 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-c...

The Role of Rho/Rho-Kinase Pathway in the Expression of ICAM-1 by Linoleic Acid in Human Aortic Endothelial Cells

“…However, it has been known that there exists significant interplay between the activity of small GTPases including RhoA and signaling derived from fatty acid metabolism [36,37]. For example, arachidonic acid, one of the metabolites of LA, has been shown to activate Rho/ROCK pathway [38,39]. In addition, endothelium-derived prostaglandin, which can also be derived from LA, has been demonstrated to activate Rho/ROCK [40].…”

Kaposi's Sarcoma-Associated Herpesvirus G-Protein-Coupled Receptor Prevents AU-Rich-Element-Mediated mRNA Decay

“…At the same time, vGPCR associates with the G␣ 12/13 family of het-erotrimeric G proteins (43,57), which activate RhoA through the guanine nucleotide exchange factor p115RhoGEF (33,37). Furthermore, there is intriguing evidence that MK2 may indirectly regulate RhoA via the phosphorylation of Hsp27 and the formation of active p-Hsp27/p115RhoGEF complexes (27), suggesting that vGPCR may activate RhoA via multiple pathways. Recent work implicated RhoA in regulating PB assembly and function (62), which inspired us to investigate the consequences of vGPCR expression on these processes.…”

“…MK2 phosphorylates destabilizing AREbinding proteins like tristetraprolin (TTP), generating binding sites for cytoplasmic 14-3-3 scaffolding proteins that disrupt recruitment of a complex of endoand exonucleases known as the exosome (15,39). In addition, MK2 phosphorylates the heat shock protein 27 (Hsp27), which has been shown to form a complex with p115GEF to activate RhoA (27). In response to a variety of stresses, including viral infection, eukaryotic translation initiation factor 2␣ (eIF2␣) is phosphorylated, which prevents initiation by limiting the availability of eIF2-GTP-tRNA met .…”

Stimulation of Rho signaling by pathologic mechanical stretch is a “second hit” to Rho-independent lung injury induced by IL-6

“…This synergistic effect was dependent on Rho activity, inhibited by Y-27632, and resulted in exacerbation of IL-6-induced EC monolayer disruption, lung barrier dysfunction, and inflammation. Although cross talk between p38 MAPK, NF-B, and Rho signaling has been reported (17,25), the precise mechanisms and the hierarchy of these interactions remain to be investigated. Altogether, these signaling events contribute to IL-6-and CS-induced EC barrier disruption.…”