Myocardin, a coactivator of serum response factor (SRF), plays a critical role in the differentiation of vascular smooth muscle cells (SMCs). However, the molecular mechanisms regulating myocardin stability and activity are not well defined. Here we show that the E3 ligase C terminus of Hsc70-interacting protein (CHIP) represses myocardin-dependent SMC gene expression and transcriptional activity. CHIP interacts with and promotes myocardin ubiquitin-mediated degradation by the proteasome in vivo and in vitro. Furthermore, myocardin ubiquitination by CHIP requires its phosphorylation. Importantly, CHIP overexpression reduces the level of myocardin-dependent SMC contractile gene expression and diminishes arterial contractility ex vivo. These findings for the first time, to our knowledge, demonstrate that CHIP-promoted proteolysis of myocardin plays a key role in the physiological control of SMC phenotype and vessel tone, which may have an important implication for pathophysiological conditions such as atherosclerosis, hypertension, and Alzheimer's disease.Phenotypic modulation of vascular smooth muscle (SM) cells (SMCs) plays a pivotal role in vascular development and remodeling during diseases. It is well established that the SMC phenotype is regulated by a wide range of extracellular cues. In response to vascular injury, vascular SMCs (VSMCs) dedifferentiate to a proliferative phenotype that is required for the various pathological states in atherosclerosis, neointimal hyperplasia, and hypertension (27,28). VSMC differentiation is characterized by expressing the highest levels of SM contractile genes, whereas proliferating SMCs express reduced levels of these genes. Thus, changes in SM contractile gene expression are often used to mark SMC phenotypes. It has been known that most SM contractile genes such as SM ␣-actin, SM myosin heavy chain (SM-MHC), and SM22␣ are controlled by serum response factor (SRF), which binds to a sequence known as a CArG box (18, 28). The specific genes activated by SRF are determined by the intracellular signals, as well as the availability of positive and negative cofactors. One of these factors is the SRF cofactor myocardin, which activates SRF-dependent genes and functions as a "master regulator" of SMC differentiation during development (31,32,36).Myocardin is expressed specifically in SM and cardiac muscle lineages and belongs to a SAP (SAF-A/B, acinus, PIAS) superfamily which has been implicated in cardiovascular development and adaptation of the cardiovascular system to hemodynamic stress (18,28). Myocardin physically interacts with the MADS box transcription factor SRF, activating a subset of genes involved in cardiomyocyte and SMC differentiation in vivo (32). Recent reports from myocardin gain-and loss-offunction studies demonstrate that it is necessary and sufficient to initiate SMC differentiation (32). One study by Huang et al. shows that myocardin conditional mutant mice exhibit markedly diminished expression of SMC contractile proteins in the ductus arteriosus and a gen...

Atrogin-1/MAFbx is a major atrophy-related E3 ubiquitin ligase that is expressed specifically in striated muscle. Although the contribution of atrogin-1 to cardiac and muscle hypertrophy/atrophy has been examined extensively, it remains unclear whether atrogin-1 plays an essential role in the simulated ischemia/reperfusion-induced apoptosis of primary cardiomyocytes. Here we showed that atrogin-1 markedly enhanced ischemia/ reperfusion-induced apoptosis in cardiomyocytes via activation of JNK signaling. Overexpression of atrogin-1 increased phosphorylation of JNK and c-Jun and decreased phosphorylation of Foxo3a. In addition, atrogin-1 decreased Bcl-2, increased Bax, and enhanced the activation of caspases. Furthermore, JNK inhibitor SP600125 markedly blocked the effect of atrogin-1 on cell apoptosis and the expression of apoptotic-related proteins and caspases. Importantly, atrogin-1 induced sustained activation of JNK through a mechanism that involved degradation of MAPK phosphatase-1 (MKP-1) protein. Atrogin-1 interacted with and triggered MKP-1 for ubiquitin-mediated degradation. In contrast, proteasome inhibitors markedly blocked the degradation of MKP-1. Taken together, these results demonstrate that atrogin-1 promotes degradation of MKP-1 through the ubiquitin-proteasome pathway, thereby leading to persistent activation of JNK signaling and further cardiomyocyte apoptosis following ischemia/reperfusion injury.

Forkhead transcription factors (FoxOs) play a pivotal role in controlling cellular proliferation and survival. The cellular level of these factors is tightly regulated through the phosphoinositide 3-kinase/Akt and ubiquitin-mediated degradation. However, the ubiquitin ligases responsible for the degradation of FoxO1 and the relevance of this regulation to smooth muscle cell (SMC) proliferation and survival have not been fully identified. Here we showed that overexpression of C terminus of Hsc70-interacting protein (CHIP) promoted ubiquitination and degradation of FoxO1 in SMCs in response to tumor necrosis factor-␣. Both the U-box (containing ubiquitin ligase activity) and the charged (essential for FoxO1 binding) domains within CHIP were required for CHIP-mediated FoxO1 down-regulation. Moreover, interaction and ubiquitination of FoxO1 by CHIP depended on phosphorylation of FoxO1 at Ser-256. Furthermore, overexpression of CHIP repressed FoxO1-mediated transactivation and its proapoptotic function following tumor necrosis factor-␣ treatment. In contrast, knockdown of CHIP by small interfering RNA enhanced FoxO1-mediated transactivation and its effect on SMC proliferation and survival. Taken together, our data indicate that CHIP is a negative regulator of FoxO1 activity through ubiquitin-mediated degradation, and inhibition of CHIP may serve as a potential therapeutic target for reducing proliferative arterial diseases.The atherosclerotic lesion is characterized by the endothelial dysfunction, inflammation, and accumulation of vascular smooth muscle cells (SMCs), 3 foam cells, and matrix protein and lipids in the intima (1, 2). Although the pathogenic mechanisms of atherosclerosis and restenosis are complex, the balance between proliferation and apoptosis of vascular SMCs seems to be a major factor in the progression of these diseases (1, 2). Various growth factors and cytokines are known to be involved in these processes (3-6). One important pleiotropic cytokine is tumor necrosis factor-␣ (TNF-␣), which is believed to play a key role in modulating SMC proliferation, migration, survival, or apoptosis (3-6). TNF-␣ has been shown to promote cellular proliferation and survival via phosphoinositide 3-kinase (PI3K)/Akt and nuclear factor-B signaling pathways in several cell types (7-10). Activated Akt phosphorylates and regulates a number of downstream proapoptotic proteins, among which are the forkhead factors FoxO1, FoxO3a, and FoxO4 (formally known as FKHR, FKHRL1, and AFX, respectively) (11). The FoxO proteins are multifunctional transcription factors, which have important roles in regulating cellular differentiation, proliferation, survival in various cell lines, including cancer cells, fibroblasts, myoblasts, endothelial cells, and SMCs (3-6). Activated FoxO proteins modulate apoptosis through regulation of a number of proapoptotic proteins, inducing Bim, the TNF-related death inducing ligand, Fas ligand, and TNF-R1-associated death domain, which all are involved in apoptotic signaling (11)(12)(13)(14). Recent s...

Epigallocatechin-3-gallate (EGCG), the major constituent of green tea, has been shown to promote apoptosis in cancer cells. However, the role of EGCG in endothelial cells following ischemia/reperfusion (I/R) injury remains unclear. In the present study, we investigated the mechanisms by which EGCG enhances I/R-induced cell growth inhibition and apoptosis in human umbilical vein endothelial cells (HUVECs). Our results showed that EGCG treatment caused cell proliferation inhibition during I/R injury, and this effect was associated with increased p27 and p21 levels and reduced cyclin D1 level. Moreover, treatment of cells with EGCG resulted in increase of caspase-3 and Bax and decrease of Bcl-2, enhancing I/R-induced apoptosis. Interestingly, EGCG decreased I/R-induced phosphorylation of AKT and its downstream substrates Foxo1 and Foxo3a and ERK1/2. In contrast, EGCG increased JNK1/2 and c-Jun phosphorylation. Furthermore, both wortamannin (PI3K inhibitor) and U0126 (MEK1/2 inhibitor) markedly enhanced EGCG-induced apoptosis during I/R, whereas SP600125 (JNK inhibitor) attenuated the action of EGCG. Taken together, our study for the first time suggest that EGCG is able to enhance growth arrest and apoptosis of HUVECs during I/R injury, at least in part, through inhibition of AKT and ERK1/2 and activation of JNK1/2 signaling pathways.

Nuclear factor of activated T cells (NFATc4) has been implicated as a critical regulator of the cardiac development and hypertrophy. However, the mechanisms for regulating NFATc4 stability and transactivation remain unclear. We showed that NFATc4 protein was predominantly ubiquitinated through the formation of Lysine 48-linked polyubiquitin chains, and this modification decreased NFATc4 protein levels and its transcriptional activity. Furthermore, activation of GSK3b markedly enhanced NFATc4 ubiquitination and decreased its transactivation, whereas inhibition of GSK3b had opposite effects. Importantly, ubiquitination and phosphorylation induced by GSK3b repressed NFATc4-dependent cardiac-specific gene expression. These results demonstrate that the ubiquitin-proteasome system plays an important role in regulating NFATc4 stability and transactivation.