Differential Regulation of Adhesion Complex Turnover by ROCK1 and ROCK2

ROCK1 and ROCK2 are serine/threonine kinases that function downstream of the small GTP-binding protein RhoA. Rho signalling via ROCK regulates a number of cellular functions including organisation of the actin cytoskeleton, cell adhesion and cell migration.In this study we use RNAi to specifically knockdown ROCK1 and ROCK2 and analyse their role in assembly of adhesion complexes in human epidermal keratinocytes. We observe that loss of ROCK1 inhibits signalling via focal adhesion kinase resulting in a failure of immature adhesion complexes to form mature stable focal adhesions. In contrast, loss of ROCK2 expression results in a significant reduction in adhesion complex turnover leading to formation of large, stable focal adhesions. Interestingly, loss of either ROCK1 or ROCK2 expression significantly impairs cell migration indicating both ROCK isoforms are required for normal keratinocyte migration.ROCK1 and ROCK2 have distinct and separate roles in adhesion complex assembly and turnover in human epidermal keratinocytes

Early activation of p160(ROCK) by pressure overload in rat heart

We investigated the effects of acute pressure overload on activation of p160(ROCK) in rat myocardium. Constriction of transverse aorta, controlled to increase peak systolic pressure of ascending aorta by similar to40 mmHg, induced a rapid association of RhoA with Dbl-3 and p160(ROCK). The binding of p160(ROCK) to RhoA was rapidly increased, peaking at 30 min ( similar to 3.5- fold), but reduced to lower levels ( similar to 1.9- fold) by 60 min of pressure overload. The activity of immunoprecipitated p160(ROCK) toward myosin light chain increased similar to 2.5-fold within 10 min but decreased to lower levels ( similar to 1.6-fold) after 60 min of pressure overload. Confocal microscopic analysis indicated that pressure overload induced the formation of aggregates of p160(ROCK) and RhoA along the longitudinal axis of cardiac myocytes. Immunoelectron microscopic analysis showed that pressure overload induced the association of p160(ROCK) and RhoA to Z-line, T-tubule, and subsarcolemmal areas. The rapid activation of p160(ROCK) by pressure overload and its aggregation in subcellular structures involved in transmission of mechanical force suggest a role for this enzyme in the mechanobiochemical transduction in the myocardium.o TEXTO COMPLETO DESTE ARTIGO, ESTARÁ DISPONÍVEL À PARTIR DE AGOSTO DE 2015.2846C1411C141

RhoA/ROCK signaling is critical to FAK activation by cyclic stretch in cardiac myocytes

Focal adhesion kinase (FAK) has been shown to be activated in cardiac myocytes exposed to mechanical stress. However, details of how mechanical stimuli induce FAK activation are unknown. We investigated whether signaling events mediated by the RhoA/Rho-associated coiled coil-containing kinase (ROCK) pathway are involved in regulation of stretch-induced FAK phosphorylation at Tyr(397) in neonatal rat ventricular myocytes (NRVMs). Immunostaining showed that RhoA localized to regions of myofilaments alternated with phalloidin (actin) staining. The results of coimmunoprecipitation assays indicated that FAK and RhoA are associated in nonstretched NRVMs, but cyclic stretch significantly reduced the amount of RhoA recovered from anti-FAK immunoprecipitates. Cyclic stretch induced rapid and sustained (up to 2 h) increases in phosphorylation of FAK at Tyr(397) and ERK1/2 at Thr(202)/Tyr(204). Blockade of RhoA/ROCK signaling by pharmacological inhibitors of RhoA (Clostridium botulinum C3 exoenzyme) or ROCK (Y-27632, 10 mu mol/l, 1 h) markedly attenuated stretch-induced FAK and ERK1/2 phosphorylation. Similar effects were observed in cells treated with the inhibitor of actin polymerization cytochalasin D. Transfection of NRVMs with RhoA antisense oligonucleotide attenuated stretch-induced FAK and ERK1/2 phosphorylation and expression of beta-myosin heavy chain mRNA. Similar results were seen in cells transfected with FAK antisense oligonucleotide. These findings demonstrate that RhoA/ROCK signaling plays a crucial role in stretch-induced FAK phosphorylation, presumably by coordinating upstream events operationally linked to the actin cytoskeleton.o TEXTO COMPLETO DESTE ARTIGO, ESTARÁ DISPONÍVEL À PARTIR DE AGOSTO DE 2015.2894H1488H149

Suppressor of cytokine signaling 3 is induced by angiotensin II in heart and isolated cardiomyocytes, and participates in desensitization

Angiotensin II (Ang II) exerts a potent growth stimulus on the heart and vascular wall. Activation of the Janus kinase/signal transducer and activator of transcription (JAK/STAT) intracellular signaling pathway by Ang II mediates at least some of the mitogenic responses to this hormone. In other signaling systems that use the JAK/STAT pathway, proteins of the suppressor of cytokine signaling (SOCS) family participate in signal regulation. In the present study it is demonstrated that SOCS3 is constitutively expressed at a low level in rat heart and neonatal rat ventricular myocytes. Ang II at a physiological concentration enhances the expression of SOCS3 mRNA and protein, mainly via AT1 receptors. After induction, SOCS3 associates with JAK2 and impairs further activation of the JAK2/STAT1 pathway. Pretreatment of rats with a specific phosphorthioate antisense oligonucleotide to SOCS3, reverses the desensitization to angiotensin signaling, as detected by a fall in c-Jun expression after repetitive infusions of the hormone. Thus, SOCS3 is induced by Ang II in rat heart and neonatal rat ventricular myocytes and participates in the modulation of the signal generated by this hormone.o TEXTO COMPLETO DESTE ARTIGO, ESTARÁ DISPONÍVEL À PARTIR DE AGOSTO DE 2015.144104586459

Early activation of the multicomponent signaling complex associated with focal adhesion kinase induced by pressure overload in the rat heart

Mechanical overload elicits functional and structural adaptive mechanisms in cardiac muscle. Signaling pathways Linked to integrin/cytoskeleton complexes may have a function in mediation of the effects of mechanical stimulus in myocardial cells. We investigated the tyrosine phosphorylation and the assembly of the multicomponent signaling complex associated with focal adhesion kinase (Fak) and the actin cytoskeleton in the overloaded myocardium of rats. Pressure overload induced a 3-fold increase in Fak tyrosine phosphorylation within 3 minutes after a 60-mm Hg rise in aortic pressure. A pressure stimulus that lasted for 60 minutes was accompanied by a 5-fold increase in the amount of tyrosine-phosphorylated Fak, and a stimulus as low as 10 mm Hg doubled the amount of tyrosine-phosphorylated Fak in the myocardium within 10 minutes. Pressure overload also induced a time-dependent association of actin with Fak and an increase in the amount of Fak detected in the cytoskeletal fraction of the myocardium. These events were paralleled by c-Src activation and binding to Fak and by an association of Grb2 and p85 subunit of phosphatidylinositol 3-kinase with Fak. Erk1/2 and Akt, two possible downstream effecters of Fak via Grb2 and phosphatidylinositol 3-kinase, were also shown to be activated in parallel with Fak. These findings show that pressure overload induced a rapid activation of the Fak multiple signaling complex in the myocardium of rats, which Suggests that this mechanism may have a role in mechanotransduction in the myocardium.O TEXTO COMPLETO DESTE ARTIGO, ESTARÁ DISPONÍVEL À PARTIR DE FEVEREIRO DE 2015.87755856

Central leptin action improves skeletal muscle AKT, AMPK, and PGC1 alpha activation by hypothalamic PI3K-dependent mechanism

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Central leptin action requires PI3K activity to modulate glucose homeostasis and peripheral metabolism. However, the mechanism behind this phenomenon is not clearly understood. We hypothesize that hypothalamic PI3K activity is important for the modulation of the AMP-activated protein kinase (AMPK)/acetyl-CoA carboxylase (ACC) pathway, PGC1 alpha, and AKT in skeletal muscle (SM). To address this issue, we injected leptin into the lateral ventricle of rats. Hypothalamic JAK2 and AKT were activated by intracerebroventricular (ICV) injection of leptin in a time-dependent manner. Central leptin improved tolerance to glucose (GTT), increased PGC1 alpha expression, and AKT, AMPK, ACC and JAK2 phosphorylation in the soleus muscle. Previous ICV administration of either LY294002 or propranolol (IP) blocked these effects. We concluded that the activation of the hypothalamic PI3K pathway is important for leptin-induced AKT phosphorylation, as well as for active catabolic pathway through AMPK and PGC1 alpha in SM. Thus, a defective leptin signalling PI3K pathway in the hypothalamus may contribute to peripheral resistance to insulin associated to diet-induced obesity. (C) 2009 Elsevier Ireland Ltd. All rights reserved.31416269Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Citrate diminishes hypothalamic acetyl-CoA carboxylase phosphorylation and modulates satiety signals and hepatic mechanisms involved in glucose homeostasis in rats

The hypothalamic AMP-activated protein kinase (AMPK)/acetyl-CoA carboxylase (ACC) pathway is known to play an important role in the control of food intake and energy expenditure. Here, we hypothesize that citrate, an intermediate metabolite, activates hypothalamic ACC and is involved in the control of energy mobilization. Initially, we showed that ICV citrate injection decreased food intake and diminished weight gain significantly when compared to control and pair-fed group results. In addition, we showed that intracerebroventricular (ICV) injection of citrate diminished (80% of control) the phosphorylation of ACC, an important AMPK substrate. Furthermore, citrate treatment inhibited (75% of control) hypothalamic AMPK phosphorylation during fasting. In addition to its central effect, ICV citrate injection led to low blood glucose levels during glucose tolerance test (GTT) and high glucose uptake during hyperglycemic-euglycemic clamp. Accordingly, liver glycogen content was higher in animals given citrate (ICV) than in the control group (23.3 +/- 2.5 vs. 2.7 +/- 0.5 mu g mL(-1) mg(-1), respectively). Interestingly, liver AMPK phosphorylation was reduced (80%) by the citrate treatment. The pharmacological blockade Of beta(3)-adrenergic receptor (SR 59230A) blocked the effect of ICV citrate and citrate plus insulin on liver AMPK phosphorylation. Consistently with these results, rats treated with citrate (ICV) presented improved insulin signal transduction in liver, skeletal muscle, and epididymal fat pad. Similar results were obtained by hypothalamic administration of ARA-A, a competitive inhibitor of AMPK. Our results suggest that the citrate produced by mitochondria may modulate ACC phosphorylation in the hypothalamus, controlling food intake and coordinating a multiorgan network that controls glucose homeostasis and energy uptake through the adrenergic system. (c) 2008 Elsevier Inc. All rights reserved.8225-261262127

Focal adhesion kinase is activated and mediates the early hypertrophic response to stretch in cardiac myocytes

Previously we reported that the rapid activation of the Fak/Src multicomponent signaling complex mediates load-induced activation of growth and survival signaling pathways in adult rat heart. In this study, we report that 5% to 20% (10-minute) cyclic stretch (1 Hz) of neonatal rat ventricular myocytes (NRVMs) was paralleled by increases of Fak phosphorylation at Tyr-397 (from 1.5- to 2.8-fold), as detected by anti-Fak-pY(397) phosphospecific antibody. Moreover, 15% cyclic stretch lasting from 10 to 120 minutes increased Fak phosphorylation at Tyr-397 by 2.5- to 3.5-fold. This activation was accompanied by a dramatic change in Fak localization in NRVMs from densely concentrated in the perinuclear regions in nonstretched cells to aggregates regularly distributed along the myofilaments in stretched cells. Furthermore, a 4-hour cyclic stretch enhanced the activity of an atrial natriuretic factor (ANF) promoter-luciferase reporter gene by 2.7-fold. Disrupting endogenous Fak/Src signaling either by expression of a dominant-negative Fak mutant with phenylalanine substituted for Tyr-397 or by treatment with a c-Src pharmacological inhibitor (PP-2) markedly attenuated stretch-induced Fak activation and clustering at myofilaments and inhibited stretch-induced ANF gene activation. On the other hand, overexpression of wild-type Fak potentiated the stretch-induced Fak phosphorylation but did not enhance either baseline or stretch-induced ANF promoter-luciferase reporter gene activity compared with the responses of nontransfected NRVMs. These findings identify Fak as an important element in the early responses induced by stretch in cardiac myocytes, indicating that it may coordinate the cellular signaling machinery that controls gene expression program associated with load-induced cardiac myocyte hypertrophy