Peroxisome proliferator-activated receptors (PPAR) are ligand-activated transcription factors that form a subfamily of the nuclear receptor gene family. Since both flow and PPAR␥ have atheroprotective effects and extracellular signal-regulated kinase 5 (ERK5) kinase activity is significantly increased by flow, we investigated whether ERK5 kinase regulates PPAR␥ activity. We found that activation of ERK5 induced PPAR␥1 activation in endothelial cells (ECs). However, we could not detect PPAR␥ phosphorylation by incubation with activated ERK5 in vitro, in contrast to ERK1/2 and JNK, suggesting a role for ERK5 as a scaffold. Endogenous PPAR␥1 was coimmunoprecipitated with endogenous ERK5 in ECs. By mammalian two-hybrid analysis, we found that PPAR␥1 associated with ERK5a at the hinge-helix 1 region of PPAR␥1. Expressing a hinge-helix 1 region PPAR␥1 fragment disrupted the ERK5a-PPAR␥1 interaction, suggesting a critical role for hinge-helix 1 region of PPAR␥ in the ERK5-PPAR␥ interaction. Flow increased ERK5 and PPAR␥1 activation, and the hinge-helix 1 region of the PPAR␥1 fragment and dominant negative MEK5␤ significantly reduced flow-induced PPAR␥ activation. The dominant negative MEK5␤ also prevented flow-mediated inhibition of tumor necrosis factor alpha-mediated NF-B activation and adhesion molecule expression, including vascular cellular adhesion molecule 1 and E-selectin, indicating a physiological role for ERK5 and PPAR␥ activation in flow-mediated antiinflammatory effects. We also found that ERK5 kinase activation was required, likely by inducing a conformational change in the NH 2 -terminal region of ERK5 that prevented association of ERK5 and PPAR␥1. Furthermore, association of ERK5a and PPAR␥1 disrupted the interaction of SMRT and PPAR␥1, thereby inducing PPAR␥ activation. These data suggest that ERK5 mediates flow-and ligand-induced PPAR␥ activation via the interaction of ERK5 with the hinge-helix 1 region of PPAR␥.Peroxisome proliferator-activated receptors (PPAR) are ligand-activated transcription factors that form a subfamily of the nuclear receptor gene family. Among PPAR family members, the expression of PPAR␣ and PPAR␥ has been reported in endothelial cells (ECs). Recently, Pasceri et al. reported that PPAR␥ activators inhibit expression of vascular cellular adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1) in activated ECs and significantly reduce monocyte/macrophage homing to atherosclerotic plaques (23). Mitogen-activated protein (MAP) kinase signaling pathways have been shown to phosphorylate PPAR␥ and to decrease PPAR␥ transcriptional activity (7, 13). The NH 2 -terminal domain of PPAR␥ contains a consensus MAP kinase site in a region conserved between PPAR␥1 and PPAR␥2 isoforms (7, 13). Phosphorylation of PPAR␥2 Ser112 (13) and PPAR␥1 Ser82 (7) significantly inhibits both ligand-independent and ligand-dependent transcriptional activation by PPAR␥. Phosphorylation-mediated transcriptional repression is due to a diminished ability of PPAR␥ to become transcriptionally a...

Endothelial cell (EC) migration contributes to reendothelialization after angioplasty or rupture of atherosclerotic plaques. Extracellular signal-regulated kinase (ERK)1/2 translocates to the nucleus and activates transcription factors such as Ets-like transcription factor-1 and early growth response factor-1 (Egr-1) during reendothelialization. Because ERK1/2 does not possess a nuclear localization signal (NLS), its mechanism of translocation and accumulation in the nucleus remains unclear. Because Gab1 has a putative NLS in its N-terminal region, and Gab1 associates with phosphorylated ERK1/2, we hypothesized that Gab1 participates in ERK1/2 and Egr-1 nuclear accumulation. Using regenerating EC as a model system, we found that endogenous growth factor receptor-bound protein 2-associated binder-1 (Gab1) translocates into the nucleus in migrating EC. Wild-type red fluorescent protein-tagged Gab1 could be observed in both nucleus and cytoplasm, whereas the putative NLS deletion mutant (⌬NLS-Gab1) specifically localized in the cytoplasm. In addition, reduction of Gab1 expression by antisense Gab1 oligos or overexpression of ⌬NLS-Gab1 inhibited serum-induced ERK1/2 and Egr-1 nuclear accumulation, suggesting a functional role for the NLS of Gab1 and a role for Gab1-ERK1/2 interactions in ERK1/2-Egr-1 nuclear accumulation. To investigate whether Gab1-ERK1/2 interaction is critical for ERK1/2 and Egr-1 nuclear accumulation, we created a dominant-negative Gab1 construct that consisted of the c-Met binding domain (amino acids 442-536) of Gab1. We found that overexpression of the c-Met binding domain of Gab1 disrupted serum-induced Gab1-ERK1 interaction and inhibited ERK1 and Egr-1 nuclear accumulation. These data suggest that Gab1-ERK1/2 binding and their nuclear translocation play a crucial role in Egr-1 nuclear accumulation. Endothelial cell (EC)1 migration is important for angiogenesis, wound repair, tumor growth, and re-endothelialization after angioplasty or rupture of atherosclerotic plaques. Wound repair after arterial injury involves migration and proliferation of EC and is associated with increased expression of plateletderived growth factor (PDGF)-A and -B, tissue factor, and fibroblast growth factor-2 (1-3). It has been reported that extracellular signal-regulated kinase (ERK)1/2 and its downstream transcription factor early growth response (Egr)-1 regulate expression of PDGF-A and PDGF-B as well as fibroblast growth factor-2 and tissue factor (1-3). Khachigian et al. (2) have demonstrated an immediate and transient increase in the expression of the transcription factor Egr-1 exclusively in EC at a wound edge after gentle injury of the rat aorta. Because Egr-1 expression and its nuclear translocation are dependent upon ERK1/2 activation (4 -6), the activation of ERK1/2 and Egr-1 seems to be critical for regulating the expression of several growth factors, cytokines, and coagulation factors in ECs at a wound edge (7).Although ERK1/2 is widely involved in eukaryotic signal transduction, the mechanism by which...