Lei Wei

Activation of Rho-associated coiled-coil protein kinase 1 (ROCK-1) by caspase-3 cleavage plays an essential role in cardiac myocyte apoptosis

Rho-associated coiled-coil protein kinase 1 (ROCK-1) is a direct cleavage substrate of activated caspase-3, which is associated with heart failure. In the course of human heart failure, we found marked cleavage of ROCK-1 resulting in a 130-kDa subspecies, which was absent in normal hearts and in an equivalent cohort of patients with left ventricular assist devices. Murine cardiomyocytes treated with doxorubicin led to enhanced ROCK-1 cleavage and apoptosis, all of which was blocked by a caspase-3 inhibitor. In addition, a bitransgenic mouse model of severe cardiomyopathy, which overexpresses Gq protein and hematopoietic progenitor kinase-/germinal center kinase-like kinase, revealed the robust accumulation of the 130-kDa ROCK-1 cleaved fragment. This constitutively active ROCK-1 subspecies, when expressed in cardiomyocytes, led to caspase-3 activation, indicating a positive feed-forward regulatory loop. ROCK-1-dependent caspase-3 activation was coupled with the activation of PTEN and the subsequent inhibition of protein kinase B (Akt) activity, all of which was attenuated by siRNA directed against ROCK-1 expression. Similarly, ROCK-1-null mice (Rock-1−/−) showed a marked reduction in myocyte apoptosis associated with pressure overload. These data suggest an obligatory role for ROCK-1 cleavage in promoting apoptotic signals in myocardial hypertrophy and/or failure.

RhoA Signaling via Serum Response Factor Plays an Obligatory Role in Myogenic Differentiation

Serum response factor (SRF) plays a central role during myogenesis, being required for the expression of striated α-actin genes. As shown here, the small GTPase RhoA-dependent activation of SRF results in the expression of muscle-specific genes, thereby promoting myogenic differentiation in myoblast cell lines. Co-expression of activated V14-RhoA and SRF results in an approximately 10-fold activation of the skeletal α-actin promoter in replicating myoblasts, while SRFpm1, a dominant negative SRF mutant, blocks RhoA dependent skeletal α-actin promoter activity. Serum withdrawal further potentiates RhoA- and SRF-mediated activation of α-actin promoter to about 30-fold in differentiated myotubes. In addition, the proximal SRE1 in the skeletal α-actin promoter is sufficient to mediate RhoA signaling via SRF. Furthermore, SRFpm1 and to a lesser extent dominant negative N19-RhoA inhibit myoblast fusion, postreplicative myogenic differentiation, and expression of direct SRF targets such as skeletal α-actin and indirect targets such as myogenin and α-myosin heavy chain. Moreover, RhoA also stimulates the autoregulatable murine SRF gene promoter in myoblasts, and the expression level of SRF is reduced in myoblasts overexpressing N19-RhoA. Our study supports the concept that RhoA signaling via SRF serves as an obligatory muscle differentiation regulatory pathway.

Inhibitory Cardiac Transcription Factor, SRF-N, Is Generated by Caspase 3 Cleavage in Human Heart Failure and Attenuated by Ventricular Unloading

Background—Knowledge about molecular mechanisms leading to heart failure is still limited, but reduced gene activities and modest activation of caspase 3 are hallmarks of end-stage heart failure. We postulated that serum response factor (SRF), a central cardiac transcription factor, might be a cleavage target for modest activated caspase 3, and this cleavage of SRF may play a dominant inhibitory role in propelling hearts toward failure. Methods and Results—We examined SRF protein levels from cardiac samples taken at the time of transplantation in 13 patients with end-stage heart failure and 7 normal hearts. Full-length SRF was markedly reduced and processed into 55- and 32-kDa subfragments in all failing hearts. SRF was intact in normal samples. In contrast, the hearts of 10 patients with left ventricular assist devices showed minimal SRF fragmentation. Specific antibodies to N- and C-terminal SRF sequences and site-directed mutagenesis revealed 2 alternative caspase 3 cleavage sites, so that 2 fragments were detected of each containing either the N- or C-terminal SRF. Expression of SRF-N, the 32-kDa fragment, in myogenic cells inhibited the transcriptional activity of &agr;-actin gene promoters by 50% to 60%, which suggests that truncated SRF functioned as a dominant-negative transcription factor. Conclusions—Caspase 3 activation in heart failure sequentially cleaved SRF and generated a dominant-negative transcription factor, which may explain the depression of cardiac-specific genes. Moreover, caspase 3 activation may be reversible in the failing heart with ventricular unloading.

β1 integrin and organized actin filaments facilitate cardiomyocyte-specific RhoA-dependent activation of the skeletal α-actin promoter

Activation of RhoA GTPase causes actin filament bundling into stress fibers, integrin clustering, and focal adhesion formation through its action on actin cytoskeleton organization. RhoA also regulates transcriptional activity of serum response factor (SRF). Recent studies in NIH 3T3 fibroblasts have shown that SRF activation by RhoA does not require an organized cytoskeleton and may be regulated by G‐actin level. In cardiac myocytes, the organization of actin fibers into myofibrils is one of the primary characteristics of cardiac differen¬tiation and hypertrophy. The primary purpose of this study was to examine if RhoA regulates SRF‐dependent gene expression in neonatal cardiomyocytes in a manner different from that observed in fibroblasts. Our results show that RhoA‐dependent skeletal α‐actin promoter ac¬tivation requires βl integrin and a functional cytoskeleton in cardiomyocytes but not in NIH 3T3 fibroblasts. Activa¬tion of the α‐actin promoter by RhoA is greatly potenti¬ated (up to 15‐fold) by co‐expression of the integrin βlA or βlD isoform but is significantly reduced by 70% with a co‐expressed dominant negative mutant of βl integrin. Furthermore, clustering of βl integrin with anti‐βl integrin antibodies potentiates synergistic RhoA and βl integrin activation of the α‐actin promoter. Cytochalasin D and latrunculin B, inhibitors of actin polymerization, significantly reduced RhoA‐induced activation of the α‐actin promoter. Jasplakinolide, an actin polymerizing agent, mimics the synergistic effect of RhoA and βl integrin on the actin promoter. These observations sup¬port the concept that RhoA regulates SRF‐dependent cardiac gene expression through cross‐talk with βl integrin signal pathway via an organized actin cytoskeleton.— Wei, L., Wang, L., Carson, J. A., Agan, J. E., ImanakaYoshida, K., and Schwartz, R. J. βl integrin and organized actin filaments facilitate cardiomyocyte‐specific RhoA‐de¬pendent activation of the skeletal α‐actin promoter. FASEBJ. 15, 785‐796 (2001)

Signaling Mechanism of Renal Fibrosis in Unilateral Ureteral Obstructive Kidney Disease in ROCK1 Knockout Mice

It has been shown that blockade of Rho kinase with pharmacologic inhibitors inhibits renal fibrosis. This study examined the role of Rho kinase in renal fibrosis in the unilateral ureteral obstruction (UUO) model in mice that do not express the ROCK1 gene, a critical downstream mediator of Rho GTPase. Unexpected, real-time PCR, Western blot, and immunohistochemistry demonstrated that, compared with the wild-type mice, mice with ROCK1 knockout (KO) were not protected against renal fibrosis at both the early (day 5) and late (day 10) UUO, as determined by histology and expression of both mRNA and protein levels of alpha-smooth muscle actin, collagen types I and III, and fibronectin within the diseased kidney. Then the mechanisms of loss of protective effect on renal fibrosis in ROCK1 KO mice were investigated. It is interesting that mice that lacked ROCK1 did not have altered expression of ROCK2 but significantly increased TGF-beta expression and Smad2/3 activation (phosphorylation and nuclear translocation) in the diseased kidney at day 5, which remained high at day 10 of UUO. Similarly, primary cultures of kidney fibroblasts that were obtained from both ROCK1 wild-type and KO mice showed that deletion of ROCK1 did not prevent TGF-beta-induced activation of Smad2/3 and collagen I expression. This also was observed in the presence of Rho kinase inhibitor Y-27632. Taken together, results from this study suggest that Rho/Rho kinase may not be a necessary or a central pathway for renal fibrosis in the UUO model. The interplay between the Rho/Rho kinase pathway and the Smad signaling pathway may be a key mechanism by which loss of ROCK1 does not prevent renal fibrosis in the UUO model.

Disruption of Rho signaling results in progressive atrioventricular conduction defects while ventricular function remains preserved

Recent studies suggest that RhoA and Rac1 mediate hypertrophic signals in cardiac myocyte hypertrophy. However, effects on cardiac function caused by inhibition of their activity in the heart have yet to be evaluated. Cardiac‐specific inhibition of Rho family protein activities was achieved by expressing Rho GDIα, an endogenous specific GDP dissociation inhibitor for Rho family proteins, using the α‐myosin heavy‐chain promoter. Increased expression of Rho GDIα led to atrial arrhythmias and mild ventricular hypertrophy in adult mice (4–7 months). However, left ventricular systolic and diastolic function was largely preserved before and after the development of cardiac hypertrophy, indicating that Rho GTPases are not required to maintain ventricular contractile function under basal physiological condition. Electrocardiography and intracardiac electrophysiological studies revealed first‐degree atrioventricular (AV) block in the transgenic heart at 1 week of age, which further progressed into second‐degree AV block at 4 weeks of age before the development of cardiac hypertrophy. Expression of connexin 40 dramatically decreased from 1 week to 4 weeks of age in the transgenic heart, which may contribute in part to the conduction defects in the transgenic mice. This study provides novel evidence for an important role of Rho GTPases in regulating AV conduction.

Amelioration of Albuminuria in ROCK1 Knockout Mice with Streptozotocin-Induced Diabetic Kidney Disease

Background: Although blockade of Rho kinase with pharmacologic inhibitors ameliorates renal fibrosis and diabetic kidney disease (DKD), the underlined mechanisms remain largely unclear. The present study tested the hypothesis that ROCK1 may regulate the early development of albuminuria via the megalin/cubilin-dependent mechanism. Methods: A DKD model was induced in ROCK1 knockout and wild-type mice by streptozotocin (STZ). The effect of deleted ROCK1 on urinary albumin excretion and the expression of megalin/cubilin were examined. In addition, the effect of blocking ROCK activities with an inhibitor (Y-27632) on tubular albumin reabsorption was tested in a normal rat tubular epithelial cell line (NRK52E) under high-glucose conditions. Expression of transforming growth factor (TGF)-β1, interleukin-1β and collagen-1 was also been examined. Results: Urinary albumin excretion was significantly increased in ROCK1 WT mice at 8 weeks after STZ injection. In contrast, mice lacking ROCK1 gene were protected against the development of albuminuria. This was associated with the protection against the loss of megalin/cubilin and an increase in TGF-β1, IL-1β, and fibrosis in the kidney. In vitro, we also found that blockade of Rho kinase with inhibitor Y-27632 prevented high-glucose-induced loss of megalin expression and an increase of TGF-β1, thereby increasing the absorption rate of FITC-labeled albumin by tubular epithelial cells. Conclusion: ROCK1 may play a role in the development of albuminuria in DKD by downregulating the endocytosis receptors complex – megalin/cubilin.