Wenyi Che

The hinge-helix 1 region of peroxisome proliferator-activated receptor γ1 (PPARγ1) mediates interaction with extracellular signal-regulated kinase 5 and PPARγ1 transcriptional activation: Involvement in flow-induced PPARγ activation in endothelial cells

ABSTRACT Peroxisome proliferator-activated receptors (PPAR) are ligand-activated transcription factors that form a subfamily of the nuclear receptor gene family. Since both flow and PPARγ have atheroprotective effects and extracellular signal-regulated kinase 5 (ERK5) kinase activity is significantly increased by flow, we investigated whether ERK5 kinase regulates PPARγ activity. We found that activation of ERK5 induced PPARγ1 activation in endothelial cells (ECs). However, we could not detect PPARγ phosphorylation by incubation with activated ERK5 in vitro, in contrast to ERK1/2 and JNK, suggesting a role for ERK5 as a scaffold. Endogenous PPARγ1 was coimmunoprecipitated with endogenous ERK5 in ECs. By mammalian two-hybrid analysis, we found that PPARγ1 associated with ERK5a at the hinge-helix 1 region of PPARγ1. Expressing a hinge-helix 1 region PPARγ1 fragment disrupted the ERK5a-PPARγ1 interaction, suggesting a critical role for hinge-helix 1 region of PPARγ in the ERK5-PPARγ interaction. Flow increased ERK5 and PPARγ1 activation, and the hinge-helix 1 region of the PPARγ1 fragment and dominant negative MEK5β significantly reduced flow-induced PPARγ activation. The dominant negative MEK5β also prevented flow-mediated inhibition of tumor necrosis factor alpha-mediated NF-κB activation and adhesion molecule expression, including vascular cellular adhesion molecule 1 and E-selectin, indicating a physiological role for ERK5 and PPARγ activation in flow-mediated antiinflammatory effects. We also found that ERK5 kinase activation was required, likely by inducing a conformational change in the NH2-terminal region of ERK5 that prevented association of ERK5 and PPARγ1. Furthermore, association of ERK5a and PPARγ1 disrupted the interaction of SMRT and PPARγ1, thereby inducing PPARγ activation. These data suggest that ERK5 mediates flow- and ligand-induced PPARγ activation via the interaction of ERK5 with the hinge-helix 1 region of PPARγ.

Activation of mitogen-activated protein kinases and p90 ribosomal S6 kinase in failing human hearts with dilated cardiomyopathy.

OBJECTIVE A new member of the MAP kinase family, big MAP kinase-1 (BMK1), has been recently identified to promote cell growth and attenuate apoptosis. P90 ribosomal S6 kinase (p90RSK), one of the potentially important substrates of extracellular signal regulated kinase (ERK), regulates gene expression in part via phosphorylation of CREB and the Na(+)/H(+) exchanger. Recently, we have demonstrated that the activity of BMK1, Src (the upstream regulator of BMK1) and p90RSK was increased in hypertrophied myocardium induced by pressure-overload in the guinea pig. However, the abundance and activity of these kinases in human hearts are unknown. METHODS In addition to the three classical MAP kinases (ERK, p38 kinase, and c-Jun NH(2)-terminal kinase (JNK)), we examined the protein expression and activity of Src, BMK1, and p90RSK in explanted hearts from patients with dilated cardiomyopathy (n=9). Normal donor hearts, which were not suitable for transplant for technical reasons, were used as controls (n=5). RESULTS There were no significant differences in the levels of protein expression of these kinases between normal and failing hearts. ERK1/2 and p90RSK were activated in heart failure compared to control (P<0.01 and P<0.03, respectively), while the activity of p38 kinase was decreased (P<0.05) and the activity of JNK was unchanged in heart failure. By contrast, the activities of Src and BMK1 were significantly reduced in end-stage heart failure compared to normal donor hearts (P<0.05). CONCLUSION These data suggest that multiple MAP kinases, p90RSK, and Src are differentially regulated in human failing myocardium of patients with idiopathic dilated cardiomyopathy and may be involved in the pathogenesis of this complex disease.

ERK5 Activation Inhibits Inflammatory Responses via Peroxisome Proliferator-activated Receptor δ (PPARδ) Stimulation

Peroxisome proliferator-activated receptors (PPAR) decrease the production of cytokine and inducible nitric-oxide synthase (iNOS) expression, which are associated with aging-related inflammation and insulin resistance. Recently, the involvement of the induction of heme oxygenase-1 (HO-1) in regulating inflammation has been suggested, but the exact mechanisms for reducing inflammation by HO-1 remains unclear. We found that overexpression of HO-1 and [Ru(CO)3Cl2]2, a carbon monoxide (CO)-releasing compound, increased not only ERK5 kinase activity, but also its transcriptional activity measured by luciferase assay with the transfection of the Gal4-ERK5 reporter gene. This transcriptional activity is required for coactivation of PPARδ by ERK5 in C2C12 cells. [Ru(CO)3Cl2]2 activated PPARδ transcriptional activity via the MEK5/ERK5 signaling pathway. The inhibition of NF-κB activity by ERK5 activation was reversed by a dominant negative form of PPARδ suggesting that ERK5/PPARδ activation is required for the anti-inflammatory effects of CO and HO-1. Based on these data, we propose a new mechanism by which CO and HO-1 mediate anti-inflammatory effects via activating ERK5/PPARδ, and ERK5 mediates CO and HO-1-induced PPARδ activation via its interaction with PPARδ.

Activation of big MAP kinase 1 (BMK1/ERK5) inhibits cardiac injury after myocardial ischemia and reperfusion.

Big MAP kinase 1 (BMK1/ERK5) plays a critical role in pre‐natal development of the cardiovascular system and post‐natal eccentric hypertrophy of the heart. Of the two isoforms upstream of MAPK‐kinase 5 (MEK5) known to exist, only the longer MEK5α isoform potently activates BMK1. We generated cardiac‐specific constitutively active form of the MEK5α (CA‐MEK5α transgenic (Tg) mice), and observed a 3 to 4‐fold increase in endogenous BMK1 activation and hyperphosphorylation of connexin 43 in the ventricles of the Tg compared to wild‐type mice. The CA‐MEK5α‐Tg‐mice demonstrated a profoundly accelerated recovery of left ventricular developed pressure after ischemia/reperfusion. We propose a novel role for BMK1 in protecting the heart from ischemia/reperfusion‐induced cardiac injury.

Inhibition of Tumor Necrosis Factor-α–Induced SHP-2 Phosphatase Activity by Shear Stress A Mechanism to Reduce Endothelial Inflammation

Objectives—Atherosclerosis preferentially occurs in areas of turbulent flow, whereas laminar flow is atheroprotective. Inflammatory cytokines have been shown to stimulate adhesion molecule expression in endothelial cells that may promote atherosclerosis, in part, by stimulating c-Jun N-terminal kinase (JNK) and nuclear factor (NF)-&kgr;B transcriptional activity. Methods and Results—Because Src kinase family and Src homology region 2-domain phosphatase-2 (SHP-2) may regulate JNK activation, we studied the effect of shear stress on endothelial inflammation and JNK. Human umbilical vein endothelial cells preexposed to flow showed decreased tumor necrosis factor (TNF)-&agr;–induced c-Jun and NF-&kgr;B transcriptional activation. TNF-&agr;–mediated JNK, c-Jun, and NF-&kgr;B activation required Src and SHP-2 activity. Shear stress significantly inhibited SHP-2 phosphatase activity without affecting TNF-&agr;–induced Src family kinase activation. Because MEKK3 and Gab1 are critical for TNF-&agr;–induced c-Jun and NF-&kgr;B activation, we determined the role of SHP-2 phosphatase activity in MEKK3 signaling. A catalytically inactive form of SHP-2 increased MEKK3/Gab1 interaction and inhibited MEKK3 (but not MEKK1)-mediated c-Jun and NF-&kgr;B activation. Conclusions—These results suggest that SHP-2 is a key mediator for the inhibitory effects of shear stress on TNF-&agr; signaling in part via regulating MEKK3/Gab1 interaction, MEKK3 signaling, and subsequent adhesion molecule expression.

The novel role of the C-terminal region of SHP-2. Involvement of Gab1 and SHP-2 phosphatase activity in Elk-1 activation.

SHP-2, a nontransmembrane-type protein-tyrosine phosphatase that contains two Src homology 2 (SH2) domains, is thought to participate in growth factor signal transduction pathways via SH2 domain interactions. To determine the role of each region of SHP-2 in platelet-derived growth factor signaling assayed by Elk-1 activation, we generated six deletion mutants of SHP-2. The large SH2 domain deletion SHP-2 mutant composed of amino acids 198–593 (SHP-2-(198–593)), but not the smaller SHP-2-(399–593), showed significantly higher SHP-2 phosphatase activity in vitro. In contrast, SHP-2-(198–593) mutant inhibited wild type SHP-2 phosphatase activity, whereas SHP-2-(399–593) mutant increased activity. To understand these functional changes, we focused on the docking protein Gab1 that assembles signaling complexes. Pull-down experiments with Gab1 suggested that the C-terminal region of SHP-2 as well as the SH2 domains (N-terminal region) associated with Gab1, but the SHP-2-(198–593) mutant did not associate with Gab1. SHP-2-(1–202) or SHP-2-(198–593) inhibited platelet-derived growth factorinduced Elk-1 activation, but SHP-2-(399–593) increased Elk-1 activation. Co-expression of SHP-2-(1–202) with SHP-2-(399–593) inhibited SHP-2-(399–593)/Gab1 interaction, and the SHP-2-(399–593) mutant induced SHP-2 phosphatase and Elk-1 activation, supporting the autoinhibitory effect of SH2 domains on the C-terminal region of SHP-2. These data suggest that both SHP-2/Gab1 interaction in the C-terminal region of SHP-2 and increased SHP-2 phosphatase activity are important for Elk-1 activation. Furthermore, we identified a novel sequence for SHP-2/Gab1 interactions in the C-terminal region of SHP-2.

19 BCR SERINE/THREONINE KINASE ENHANCES ANGIOTENSIN II-MEDIATED NUCLEAR FACTOR κB TRANSCRIPTIONAL ACTIVATION IN VASCULAR SMOOTH MUSCLE CELLS VIA INHIBITION OF PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR γ.

Bcr is a serine/threonine kinase and is highly expressed in the neointima after vascular injury. Because angiotensin II (Ang II) and inflammation play important roles in the development of intimal proliferation, we hypothesized that Bcr is an important regulator of Ang II-mediated nuclear factor (NF)-κB activation. By transfecting vascular smooth muscle cells (VSMCs) with dominant negative Bcr (DN-Bcr), we demonstrated that DN-Bcr inhibited Ang II-mediated NF-κB activation in VSMCs (82 ± 10% inhibition, n = 3, p < .01). Similarly, we demonstrated that Bcr siRNA inhibited Ang II-mediated NF-κB activation in VSMCs (79 ± 7%, n = 3, p < .01). In a survey of nuclear factors that might regulate NF-κB activation, we found that wild-type (WT) Bcr decreased ligand-mediated peroxisome proliferator-activated γ (PPARγ) activity (86 ± 12% inhibition, p < .01), whereas DN-Bcr significantly enhanced ligand-mediated PPARγ activity (2.4-fold, p < .01). Assessment by in vitro kinase assay demonstrated that Bcr phosphorylates PPARγ. Overexpression of WT-Bcr kinase did not inhibit ligand-mediated PPARγ1 S82A or S82D mutant transcriptional activity, suggesting that Bcr regulates PPARγ activity via S82 phosphorylation. To assess the physiologic role of Bcr, we examined the effect of Ang II on Bcr expression and transcriptional activation. Using VSMCs, we demonstrated that Ang II only weakly increases Bcr kinase activity but significantly increased its expression after 6 hours of Ang II stimulation (4.3 ± 0.8-fold increase, p < .01). In addition, we found that whereas Ang II inhibits PPARγ activation, Bcr siRNA reverses Ang II inhibition of PPARγ. To determine the role of Bcr/PPARγ on Ang II-mediated NF-κB activation, we cotransfected VSMCs with DN-Bcr and DN-PPARγ. We found that DN-PPARγ reversed DN-Bcr-mediated inhibition of NF-κB activation. In addition, WT-Bcr-induced NF-κB activation was inhibited by PPARγ1 S82A mutant but not WT-PPARγ1, strongly suggesting that Bcr-induced inhibition of PPARγ activity reciprocally increases NF-κB activation and regulates Ang II-mediated NF-κB activation. Finally, our preliminary data suggest a decrease in intima to media ratio (0.31 vs 0.73) in Bcr knockout mice compared with controls in a partial ligation model of the left carotid artery. Given our findings that Bcr kinase inhibits PPARγ activation and enhances Ang II-mediated NF-κB transcription, we believe that Bcr acts as an important regulator of the sensitivity of VSMCs to inflammatory stimuli.