Bcr kinase activation by angiotensin II inhibits peroxisome-proliferator-activated receptor gamma transcriptional activity in vascular smooth muscle cells.

Bcr is a serine/threonine kinase activated by platelet-derived growth factor that is highly expressed in the neointima after vascular injury. Here, we demonstrate that Bcr is an important mediator of angiotensin (Ang) II and platelet-derived growth factor-mediated inflammatory responses in vascular smooth muscle cells (VSMCs). Among transcription factors that might regulate Ang II-mediated inflammatory responses we found that ligand-mediated peroxisome proliferator-activated receptor (PPAR)gamma transcriptional activity was significantly decreased by Ang II. Ang II increased Bcr expression and kinase activity. Overexpression of Bcr significantly inhibited PPARgamma activity. In contrast, knockdown of Bcr using Bcr small interfering RNA and a dominant-negative form of Bcr (DN-Bcr) reversed Ang II-mediated inhibition of PPARgamma activity significantly, suggesting the critical role of Bcr in Ang II-mediated inhibition of PPARgamma activity. Point-mutation and in vitro kinase analyses showed that PPARgamma was phosphorylated by Bcr at serine 82. Overexpression of wild-type Bcr kinase did not inhibit ligand-mediated PPARgamma1 S82A mutant transcriptional activity, indicating that Bcr regulates PPARgamma activity via S82 phosphorylation. DN-Bcr and Bcr small interfering RNA inhibited Ang II-mediated nuclear factor kappaB activation in VSMCs. DN-PPARgamma reversed DN-Bcr-mediated inhibition of nuclear factor kappaB activation, suggesting that PPARgamma is downstream from Bcr. Intimal proliferation in low-flow carotid arteries was decreased in Bcr knockout mice compared with wild-type mice, suggesting the critical role of Bcr kinase in VSMC proliferation in vivo, at least in part, via regulating PPARgamma/nuclear factor kappaB transcriptional activity.

The hinge-helix 1 region of peroxisome proliferator-activated receptor gamma1 (PPARgamma1) mediates interaction with extracellular signal-regulated kinase 5 and PPARgamma1 transcriptional activation: involvement in flow-induced PPARgamma activation in endothelial cells.

Peroxisome proliferator-activated receptors (PPAR) are ligand-activated transcription factors that form a subfamily of the nuclear receptor gene family. Since both flow and PPARgamma have atheroprotective effects and extracellular signal-regulated kinase 5 (ERK5) kinase activity is significantly increased by flow, we investigated whether ERK5 kinase regulates PPARgamma activity. We found that activation of ERK5 induced PPARgamma1 activation in endothelial cells (ECs). However, we could not detect PPARgamma phosphorylation by incubation with activated ERK5 in vitro, in contrast to ERK1/2 and JNK, suggesting a role for ERK5 as a scaffold. Endogenous PPARgamma1 was coimmunoprecipitated with endogenous ERK5 in ECs. By mammalian two-hybrid analysis, we found that PPARgamma1 associated with ERK5a at the hinge-helix 1 region of PPARgamma1. Expressing a hinge-helix 1 region PPARgamma1 fragment disrupted the ERK5a-PPARgamma1 interaction, suggesting a critical role for hinge-helix 1 region of PPARgamma in the ERK5-PPARgamma interaction. Flow increased ERK5 and PPARgamma1 activation, and the hinge-helix 1 region of the PPARgamma1 fragment and dominant negative MEK5beta significantly reduced flow-induced PPARgamma activation. The dominant negative MEK5beta also prevented flow-mediated inhibition of tumor necrosis factor alpha-mediated NF-kappaB activation and adhesion molecule expression, including vascular cellular adhesion molecule 1 and E-selectin, indicating a physiological role for ERK5 and PPARgamma 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 PPARgamma1. Furthermore, association of ERK5a and PPARgamma1 disrupted the interaction of SMRT and PPARgamma1, thereby inducing PPARgamma activation. These data suggest that ERK5 mediates flow- and ligand-induced PPARgamma activation via the interaction of ERK5 with the hinge-helix 1 region of PPARgamma.

Insulin-like growth factor-1 enhances inflammatory responses in endothelial cells: role of Gab1 and MEKK3 in TNF-alpha-induced c-Jun and NF-kappaB activation and adhesion molecule expression.

Insulin-like growth factor (IGF)-1 and the type I IGF-1 receptor are important regulators of vascular function that may contribute to cardiovascular disease. We hypothesized that IGF-1 causes endothelial cell dysfunction and expression of neutrophil and monocyte adhesion molecules by enhancing pro-inflammatory cytokine signal transduction. Long-term IGF-1 treatment of endothelial cells potentiated c-Jun and nuclear factor NF-kappaB activation by tumor necrosis factor (TNF)-alpha and enhanced TNF-alpha-mediated adhesion molecule expression. In response to IGF-1 treatment, the expression of kinases in the c-Jun/c-Jun NH(2)-terminal kinase signaling pathway (MEKK1, MEK4, and JNK1/2) was unchanged, but expressions of insulin receptor substrate-1 and Grb2-associated binder-1 (Gab1) were significantly decreased. Because Gab1 is involved in both c-Jun and NF-kappaB activation by TNF-alpha, we focused on Gab1-dependent signaling. Gab1 inhibited c-Jun and NF-kappaB transcriptional activation by TNF-alpha. Interestingly, Gab1 inhibited c-Jun transcriptional activity induced by MEKK3 but not MEKK1 and MEK4. Gab1 associated with MEKK3, and a catalytically inactive form of MEKK3 inhibited TNF-alpha-induced c-Jun and NF-kappaB transcriptional activation, suggesting a critical role for Gab1 and MEKK3 in TNF-alpha signaling. These data demonstrate that Gab1 and MEKK3 play important roles in endothelial cell inflammation via regulating the activation of c-Jun and NF-kappaB. Furthermore, the IGF-1-mediated downregulation of Gab1 expression represents a novel mechanism to promote vascular inflammation and atherosclerosis.

Inhibition of tumor necrosis factor-[alpha]-induced SHP-2 phosphatase activity by shear stress: a mechanism to reduce endothelial inflammation.

OBJECTIVE: 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)-kappaB 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)-alpha-induced c-Jun and NF-kappaB transcriptional activation. TNF-alpha-mediated JNK, c-Jun, and NF-kappaB activation required Src and SHP-2 activity. Shear stress significantly inhibited SHP-2 phosphatase activity without affecting TNF-alpha-induced Src family kinase activation. Because MEKK3 and Gab1 are critical for TNF-alpha-induced c-Jun and NF-kappaB 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-kappaB activation. CONCLUSIONS: These results suggest that SHP-2 is a key mediator for the inhibitory effects of shear stress on TNF-alpha signaling in part via regulating MEKK3/Gab1 interaction, MEKK3 signaling, and subsequent adhesion molecule expression.

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.

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 MEK5alpha isoform potently activates BMK1. We generated cardiac-specific constitutively active form of the MEK5alpha (CA-MEK5alpha 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-MEK5alpha-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.

ERK5 activation inhibits inflammatory responses via peroxisome proliferator-activated receptor delta (PPARdelta) 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)(3)Cl(2)](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 PPARdelta by ERK5 in C2C12 cells. [Ru(CO)(3)Cl(2)](2) activated PPARdelta transcriptional activity via the MEK5/ERK5 signaling pathway. The inhibition of NF-kappaB activity by ERK5 activation was reversed by a dominant negative form of PPARdelta suggesting that ERK5/PPARdelta 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/PPARdelta, and ERK5 mediates CO and HO-1-induced PPARdelta activation via its interaction with PPARdelta.

Mitochondrial Dok-4 recruits Src kinase and regulates NF-kappaB activation in endothelial cells.

The downstream of kinase (Dok) family of adapter proteins consists of at least five members structurally characterized by an NH2-terminal tandem of conserved pleckstrin homology and phosphotyrosine binding domains linked to a unique COOH-terminal region. To determine the role of the novel adapter protein Dok-4 in endothelial cells, we first investigated the cell localization of Dok-4. Most surprisingly, immunofluorescence microscopy, cell fractionation studies, and studies with enhanced green fluorescent protein chimeras showed that wild type Dok-4 (Dok-4-WT) specifically localized in mitochondria. An NH2-terminal deletion mutant of Dok-4 (Dok-4-(deltaN11-29)), which lacks the mitochondrial targeting sequence, could not accumulate in mitochondria. Co-immunoprecipitation revealed an interaction of c-Src with Dok-4-WT in endothelial cells. Most interestingly, overexpression of Dok-4-WT, but not Dok-4-(deltaN1-99), increased mitochondrial c-Src expression, whereas knock-down of endogenous Dok-4 with a small interfering RNA vector greatly inhibited mitochondrial localization of c-Src, suggesting a unique function for Dok-4 as an anchoring protein for c-Src in mitochondria. Dok-4-WT significantly decreased 39-kDa subunit complex I expression. PP2, a specific Src kinase inhibitor, prevented the Dok-4-mediated complex I decrease, suggesting the involvement of Src kinase in regulation of complex I expression. Dok-4-WT enhanced tumor necrosis factor-alpha (TNF-alpha)-mediated reactive oxygen species (ROS) production, supporting the functional relevance of a Dok-4-Src-complex I/ROS signaling pathway in mitochondria. Finally, Dok-4 enhanced TNF-alpha-mediated NF-kappaB activation, whereas this was inhibited by transfection with Dok-4 small interfering RNA. In addition, Dok-4-induced NF-kappaB activation was also inhibited by transfection of a dominant negative form of c-Src. These data suggest a role for mitochondrial Dok-4 as an anchoring molecule for the tyrosine kinase c-Src, and in turn as a regulator of TNF-alpha-mediated ROS production and NF-kappaB activation.

A positive feedback loop of phosphodiesterase 3 (PDE3) and inducible cAMP early repressor (ICER) leads to cardiomyocyte apoptosis.

cAMP plays crucial roles in cardiac remodeling and the progression of heart failure. Recently, we found that expression of cAMP hydrolyzing phosphodiesterase 3A (PDE3A) was significantly reduced in human failing hearts, accompanied by up-regulation of inducible cAMP early repressor (ICER) expression. Angiotensin II (Ang II) and the beta-adrenergic receptor agonist isoproterenol (ISO) also induced persistent PDE3A down-regulation and concomitant ICER up-regulation in vitro, which is important in Ang II- and ISO-induced cardiomyocyte apoptosis. We hypothesized that interactions between PDE3A and ICER may constitute an autoregulatory positive feedback loop (PDE3A-ICER feedback loop), and this loop would cause persistent PDE3A down-regulation and ICER up-regulation. Here, we demonstrate that ICER induction repressed PDE3A gene transcription. PDE3A down-regulation activated cAMP/PKA signaling, leading to ICER up-regulation via PKA-dependent stabilization of ICER. With respect to Ang II, the initiation of the PDE3A-ICER feedback loop depends on activation of Ang II type 1 receptor (AT1R), classical PKC(s), and CREB (cAMP response element binding protein). We further show that the PDE3A-ICER feedback loop is essential for Ang II-induced cardiomyocyte apoptosis. ISO and PDE3 inhibitors also induced the PDE3A-ICER feedback loop and subsequent cardiomyocyte apoptosis, highlighting the importance of this PDE3A-ICER feedback loop and cAMP signaling in cardiomyocyte apoptosis. Our findings may provide a therapeutic paradigm to prevent cardiomyocyte apoptosis and the progression of heart failure by inhibiting the PDE3A-ICER feedback loop.

Differential role of MEK5alpha and MEK5beta in BMK1/ERK5 activation.

Big mitogen-activated protein kinase 1/extracellular-regulated kinase 5 (BMK1/ERK5) is regulated sequentially by a series of upstream MAP kinase kinases (MEKs) in a signaling cascade. MEKs activate their downstream MAPK by phosphorylation of threonine and tyrosine in the T- X-Y motif. MEK5 is the upstream BMK1 kinase and exists as naturally occurring splice variants, MEK5alpha and MEK5beta. The full-length MEK5 (MEK5alpha) is 89 amino acids longer than MEK5beta at the N terminus, but the precise functional difference between the two splice variants is not known. Dual phosphorylation site mutation of MEK5alpha (Ser-311 --> Asp and Thr- 315 --> Asp; MEK5alpha(S311D/T315D)) activated BMK1, but the corresponding dual phosphorylation sites mutant of MEK5beta could not induce BMK1 kinase activation or nuclear translocation. Furthermore, MEK5beta inhibited epidermal growth factor-induced BMK1 activation and MEK5alpha(S311D/T315D)-induced MEF2 transcriptional activity. Both MEK5alpha and MEK5beta individually co-immunoprecipitated with BMK1, but the presence of MEK5beta prevented association of MEK5alpha with BMK1 suggesting a mechanistic basis for the dominant-negative behavior of MEK5beta on BMK1 activation. The ratio of MEK5alpha to MEK5beta expression was higher in cancer cell lines, and overexpression of MEK5beta-inhibited serum-induced DNA synthesis. These data suggest that alternative splicing of MEK5alpha and MEK5beta may play a critical role in BMK1 activation and subsequent cell proliferation.

Suppression of estrogen receptor-mediated transcription and cell growth by interaction with TR2 orphan receptor.

The transcriptional activity of the estrogen receptor (ER) is known to be highly modulated by the character and amount of coregulator proteins present in the cells. TR2 orphan receptor (TR2), a member of the nuclear receptor superfamily without identified ligands, is found to be expressed in the breast cancer cell lines and to function as a repressor to suppress ER-mediated transcriptional activity. Utilizing an interaction blocker, ER-6 (amino acids 312-340), responsible for TR2 interaction, the suppression of ER by TR2 could be reversed, suggesting that this suppression is conferred by the direct protein-protein interaction. Administration of antisense TR2, resulting in an enhancement of ER transcriptional activity in MCF7 cells, indicates that endogenous TR2 normally suppresses ER-mediated signaling. To gain insights into the molecular mechanism by which TR2 suppresses ER, we found that TR2 could interrupt ER DNA binding via formation of an ER-TR2 heterodimer that disrupted the ER homodimerization. The suppression of ER transcription by TR2 consequently caused the inhibition of estrogen-induced cell growth and G(1)/S transition in estrogen-dependent breast cancer cells. Taken together in addition to the potential roles in spermatogenesis and neurogenesis, our data provide a novel biological function of TR2 that may exert an important repressor in regulating ER activity in mammary glands.

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.

ERK1/2 associates with the c-Met-binding domain of growth factor receptor-bound protein 2 (Grb2)-associated binder-1 (Gab1): role in ERK1/2 and early growth response factor-1 (Egr-1) nuclear accumulation.

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 (deltaNLS-Gab1) specifically localized in the cytoplasm. In addition, reduction of Gab1 expression by antisense Gab1 oligos or overexpression of deltaNLS-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.