Hui-Hua Li

Atrogin-1/muscle atrophy F-box inhibits calcineurin-dependent cardiac hypertrophy by participating in an SCF ubiquitin ligase complex

Calcineurin, which binds to the Z-disc in cardiomyocytes via alpha-actinin, promotes cardiac hypertrophy in response to numerous pathologic stimuli. However, the endogenous mechanisms regulating calcineurin activity in cardiac muscle are not well understood. We demonstrate that a muscle-specific F-box protein called atrogin-1, or muscle atrophy F-box, directly interacts with calcineurin A and alpha-actinin-2 at the Z-disc of cardiomyocytes. Atrogin-1 associates with Skp1, Cul1, and Roc1 to assemble an SCF(atrogin-1) complex with ubiquitin ligase activity. Expression of atrogin-1 decreases levels of calcineurin A and promotes its ubiquitination. Moreover, atrogin-1 attenuates agonist-induced calcineurin activity and represses calcineurin-dependent transactivation and NFATc4 translocation. Conversely, downregulation of atrogin-1 using adenoviral small interfering RNA (siRNA) expression enhances agonist-induced calcineurin activity and cardiomyocyte hypertrophy. Consistent with these cellular observations, overexpression of atrogin-1 in hearts of transgenic mice reduces calcineurin protein levels and blunts cardiac hypertrophy after banding of the thoracic aorta. These studies indicate that the SCF(atrogin-1) ubiquitin ligase complex interacts with and represses calcineurin by targeting calcineurin for ubiquitin-mediated proteolysis, leading to inhibition of cardiac hypertrophy in response to pathologic stimuli.

CHIP activates HSF1 and confers protection against apoptosis and cellular stress.

Induction of molecular chaperones is the characteristic protective response to environmental stress, and is regulated by a transcriptional program that depends on heat shock factor 1 (HSF1), which is normally under negative regulatory control by molecular chaperones Hsp70 and Hsp90. In metazoan species, the chaperone system also provides protection against apoptosis. We demonstrate that the dual function co‐chaperone/ubiquitin ligase CHIP (C‐terminus of Hsp70‐interacting protein) regulates activation of the stress‐chaperone response through induced trimerization and transcriptional activation of HSF1, and is required for protection against stress‐induced apoptosis in murine fibroblasts. The consequences of this function are demonstrated by the phenotype of mice lacking CHIP, which develop normally but are temperature‐sensitive and develop apoptosis in multiple organs after environmental challenge. CHIP exerts a central and unique role in tuning the response to stress at multiple levels by regulation of protein quality control and transcriptional activation of stress response signaling.

Muscle-specific RING finger 1 is a bona fide ubiquitin ligase that degrades cardiac troponin I

Muscle-specific RING finger protein 1 (MuRF1) is a sarcomere-associated protein that is restricted to cardiac and skeletal muscle. In skeletal muscle, MuRF1 is up-regulated by conditions that provoke atrophy, but its function in the heart is not known. The presence of a RING finger in MuRF1 raises the possibility that it is a component of the ubiquitin–proteasome system of protein deg-radation. We performed a yeast two-hybrid screen to search for interaction partners of MuRF1 in the heart that might be targets of its putative ubiquitin ligase activity. This screen identified troponin I as a MuRF1 partner protein. MuRF1 and troponin I were found to associate both in vitro and in vivo in cultured cardiomyocytes. MuRF1 reduced steady-state troponin I levels when coexpressed in COS-7 cells and increased degradation of endogenous troponin I protein in cardiomyocytes. The degradation of troponin I in cardiomyocytes was associated with the accumulation of ubiquitylated intermediates of troponin I and was proteasome-dependent. In vitro, MuRF1 functioned as a ubiquitin ligase to catalyze ubiquitylation of troponin I through a RING finger-dependent mechanism. In isolated cardiomyocytes, MuRF1 reduced indices of contractility. In cardiomyocytes, these processes may determine the balance between hypertrophic and antihypertrophic signals and the regulation of myocyte contractile responses in the setting of heart failure.

Atrogin-1 inhibits Akt-dependent cardiac hypertrophy in mice via ubiquitin-dependent coactivation of Forkhead proteins

Cardiac hypertrophy is a major cause of human morbidity and mortality. Although much is known about the pathways that promote hypertrophic responses, mechanisms that antagonize these pathways have not been as clearly defined. Atrogin-1, also known as muscle atrophy F-box, is an F-box protein that inhibits pathologic cardiac hypertrophy by participating in a ubiquitin ligase complex that triggers degradation of calcineurin, a factor involved in promotion of pathologic hypertrophy. Here we demonstrated that atrogin-1 also disrupted Akt-dependent pathways responsible for physiologic cardiac hypertrophy. Our results indicate that atrogin-1 does not affect the activity of Akt itself, but serves as a coactivator for members of the Forkhead family of transcription factors that function downstream of Akt. This coactivator function of atrogin-1 was dependent on its ubiquitin ligase activity and the deposition of polyubiquitin chains on lysine 63 of Foxo1 and Foxo3a. Transgenic mice expressing atrogin-1 in the heart displayed increased Foxo1 ubiquitylation and upregulation of known Forkhead target genes concomitant with suppression of cardiac hypertrophy, while mice lacking atrogin-1 displayed the opposite physiologic phenotype. These experiments define a role for lysine 63-linked ubiquitin chains in transcriptional coactivation and demonstrate that atrogin-1 uses this mechanism to disrupt physiologic cardiac hypertrophic signaling through its effects on Forkhead transcription factors.

Regulation of the Cytoplasmic Quality Control Protein Degradation Pathway by BAG2

The cytoplasm is protected against the perils of protein misfolding by two mechanisms: molecular chaperones (which facilitate proper folding) and the ubiquitin-proteasome system, which regulates degradation of misfolded proteins. CHIP (carboxyl terminus of Hsp70-interacting protein) is an Hsp70-associated ubiquitin ligase that participates in this process by ubiquitylating misfolded proteins associated with cytoplasmic chaperones. Mechanisms that regulate the activity of CHIP are, at present, poorly understood. Using a proteomics approach, we have identified BAG2, a previously uncharacterized BAG domain-containing protein, as a common component of CHIP holocomplexes in vivo. Binding assays indicate that BAG2 associates with CHIP as part of a ternary complex with Hsc70, and BAG2 colocalizes with CHIP under both quiescent conditions and after heat shock. In vitro and in vivo ubiquitylation assays indicate that BAG2 is an efficient and specific inhibitor of CHIP-dependent ubiquitin ligase activity. This activity is due, in part, to inhibition of interactions between CHIP and its cognate ubiquitin-conjugating enzyme, UbcH5a, which may in turn be facilitated by ATP-dependent remodeling of the BAG2-Hsc70-CHIP heterocomplex. The association of BAG2 with CHIP provides a cochaperone-dependent regulatory mechanism for preventing unregulated ubiquitylation of misfolded proteins by CHIP.

Muscle ring finger protein-1 inhibits PKCΕ activation and prevents cardiomyocyte hypertrophy

Much effort has focused on characterizing the signal transduction cascades that are associated with cardiac hypertrophy. In spite of this, we still know little about the mechanisms that inhibit hypertrophic growth. We define a novel anti-hypertrophic signaling pathway regulated by muscle ring finger protein-1 (MURF1) that inhibits the agonist-stimulated PKC-mediated signaling response in neonatal rat ventricular myocytes. MURF1 interacts with receptor for activated protein kinase C (RACK1) and colocalizes with RACK1 after activation with phenylephrine or PMA. Coincident with this agonist-stimulated interaction, MURF1 blocks PKCε translocation to focal adhesions, which is a critical event in the hypertrophic signaling cascade. MURF1 inhibits focal adhesion formation, and the activity of downstream effector ERK1/2 is also inhibited in the presence of MURF1. MURF1 inhibits phenylephrine-induced (but not IGF-1–induced) increases in cell size. These findings establish that MURF1 is a key regulator of the PKC-dependent hypertrophic response and can blunt cardiomyocyte hypertrophy, which may have important implications in the pathophysiology of clinical cardiac hypertrophy.

Macrophage-stimulated cardiac fibroblast production of IL-6 is essential for TGF β/Smad activation and cardiac fibrosis induced by angiotensin II.

Interleukin-6 (IL-6) is an important cytokine participating in multiple biologic activities in immune regulation and inflammation. IL-6 has been associated with cardiovascular remodeling. However, the mechanism of IL-6 in hypertensive cardiac fibrosis is still unclear. Angiotensin II (Ang II) infusion in mice increased IL-6 expression in the heart. IL-6 knockout (IL-6-/-) reduced Ang II-induced cardiac fibrosis: 1) Masson trichrome staining showed that Ang II infusion significantly increased fibrotic areas of the wild-type mouse heart, which was greatly suppressed in IL-6-/- mice and 2) immunohistochemistry staining showed decreased expression of α-smooth muscle actin (α-SMA), transforming growth factor β1 (TGF-β1) and collagen I in IL-6-/- mouse heart. The baseline mRNA expression of IL-6 in cardiac fibroblasts was low and was absent in cardiomyocytes or macrophages; however, co-culture of cardiac fibroblasts with macrophages significantly increased IL-6 production and expression of α-SMA and collagen I in fibroblasts. Moreover, TGF-β1 expression and phosphorylation of TGF-β downstream signal Smad3 was stimulated by co-culture of macrophages with cardiac fibroblasts, while IL-6 neutralizing antibody decreased TGF-β1 expression and Smad3 phosphorylation in co-culture of macrophage and fibroblast. Taken together, our results indicate that macrophages stimulate cardiac fibroblasts to produce IL-6, which leads to TGF-β1 production and Smad3 phosphorylation in cardiac fibroblasts and thus stimulates cardiac fibrosis.

Serum-Glucocorticoid Regulated Kinase 1 Regulates Alternatively Activated Macrophage Polarization Contributing to Angiotensin II–Induced Inflammation and Cardiac Fibrosis

Objective—Inflammatory responses play a pivotal role in the pathogenesis of hypertensive cardiac remodeling. Macrophage recruitment and polarization contribute to the development of cardiac fibrosis. Although serum-glucocorticoid regulated kinase 1 (SGK1) is a key mediator of fibrosis, its role in regulating macrophage function leading to cardiac fibrosis has not been investigated. We aimed to determine the mechanism by which SGK1 regulates the cardiac inflammatory process, thus contributing to hypertensive cardiac fibrosis. Methods and Results—After angiotensin II infusion in mice, cardiac hypertrophy and fibrosis developed in wild-type but not SGK1 knockout mice, with equal levels of hypertension in both groups. Compared with wild-type hearts, SGK1 knockout hearts showed less infiltration of leukocytes and macrophages. Importantly, SGK1 deficiency led to decreased proportion of alternatively activated (M2) macrophages and increased levels of profibrotic cytokines. Angiotensin II infusion induced phosphorylation and nuclear localization of signal transducer and activator of transcription 3 (STAT3) whereas SGK1 knockout hearts showed this effect attenuated. In a 3-dimensional peptide gel culture, inhibition of STAT3 suppressed differentiation into M2 macrophages. Coculture of macrophages with cardiac fibroblasts in 3-dimensional peptide gel stimulated the expression of &agr;-smooth muscle actin and collagen in cardiac fibroblasts. However, SGK1 knockout mice with macrophage deficiency showed reduced fibroblast-to-myofibroblast transition. Conclusion—SGK1 may play an important role in macrophage recruitment and M2 macrophage activation by activating the STAT3 pathway, which leads to angiotensin II–induced cardiac fibrosis.

Comparative Effects of Paclitaxel and Rapamycin on Smooth Muscle Migration and Survival Role of Akt-Dependent Signaling

Objective—Advances in stent technology have enabled the delivery of drugs to improve outcomes after stent deployment. However, the optimal payloads for stents are not clear, and the appropriate stent-based therapies for high-risk patients, such as diabetics, have not been clearly established. Methods and Results—We used smooth muscle cell culture models to compare the activities of rapamycin and paclitaxel. Smooth muscle cells were grown in normal or high glucose to induce insulin resistance. Both paclitaxel and rapamycin activate mitogen-activated protein kinase pathways similarly. However, rapamycin potently activates AKT-dependent signaling, an effect that overrides the downregulation of this pathway by insulin resistance and that causes phosphorylation of the AKT-dependent transcription factor FOXO1. This effect is associated with attenuation of the anti-migratory effects of rapamycin under high glucose conditions that are not observed with paclitaxel, as well as with increased protection against ceramide-induced cytotoxicity, both of which are dependent on FOXO1 phosphorylation. Conclusions—Differences between the ability of rapamycin and paclitaxel to activate AKT may account for their differential cell survival and antichemotactic activities. These observations may provide a basis for understanding clinical differences between rapamycin- and paclitaxel-coated stents. The approaches used in these studies can be expanded to other candidate stent payloads as a method for triage in preclinical studies.

Osteopontin stimulates autophagy via integrin/CD44 and p38 MAPK signaling pathways in vascular smooth muscle cells

Osteopontin (OPN) exerts pro‐inflammatory effect and is associated with the development of abdominal aortic aneurysm (AAA). However, the molecular mechanism underlying this association remains obscure. In the present study, we compared gene expression profiles of AAA tissues using microarray assay, and found that OPN was the highest expressed gene (>125‐fold). Furthermore, the expression of LC3 protein and autophagy‐related genes including Atg4b, Beclin1/Atg6, Bnip3, and Vps34 was markedly upregulated in AAA tissues. To investigate the ability of OPN to stimulate autophagy as a potential mechanism involved in the pathogenesis of this disease, we treated vascular smooth muscle cells (SMCs) with OPN, and found that OPN significantly increased the formation of autophagosomes, expression of autophagy‐related genes and cell death, whereas blocking the signal by anti‐OPN antibody markedly inhibited OPN‐induced autophagy and SMC death. Furthermore, inhibition of integrin/CD44 and p38 MAPK signaling pathways markedly abrogated the biological effects of OPN on SMCs. These data for the first time demonstrate that OPN sitmulates autophagy directly through integrin/CD44 and p38 MAPK‐mediated pathways in SMCs. Thus, inhibition of OPN‐induced autophagy might be a potential therapeutic target in the treatment of AAA disease. J. Cell. Physiol. 227: 127–135, 2012.