A novel RhoA/ROCK-CPI-17-MEF2C signaling pathway regulates vascular smooth muscle cell gene expression

Differentiation of vascular smooth muscle cells (VSMC) is a fundamental aspect of normal development and vascular disease. During contraction, VSMCs modulate calcium sensitivity through RhoA/ROCK-mediated inhibition of the myosin light chain phosphatase complex (MLCP). Previous studies have demonstrated that this signaling pathway functions in parallel to increase the expression of smooth muscle genes through the myocardin-family of co-activators. MEF2C fulfills a critical role in VSMC differentiation and regulates myocardin expression, leading us to investigate whether the RhoA/ROCK signaling cascade might regulate MEF2 activity. Depolarization-induced calcium signaling increased the expression of myocardin, which was sensitive to ROCK and p38 MAPK inhibition. We previously identified protein phosphatase 1α (PP1α), a known catalytic subunit of the MLCP in VSMCs, as a potent repressor of MEF2 activity. PP1α inhibition resulted in increased expression of myocardin, while ectopic expression of PP1α inhibited the induction of myocardin by MEF2C. Consistent with these data, shRNA-mediated suppression of a PP1α inhibitor, CPI-17, reduced myocardin expression and inhibited VSMC differentiation, suggesting a pivotal role for CPI-17 in regulating MEF2 activity. These data constitute evidence of a novel signaling cascade that links RhoA-mediated calcium sensitivity to MEF2-dependent myocardin expression in VSMCs through a mechanism involving p38 MAPK, PP1α, and CPI-17.     

病毒性心肌炎与支原体肺炎患儿心肌损伤标志物水平的检验意义

目的:探讨病毒性心肌炎与支原体肺炎患者心肌损伤标志物水平检测意义。方法:回顾性分析医院收治的病毒性心肌炎患儿53例和肺炎支原体肺炎患儿49例分别作为病毒性心肌炎组和支原体肺炎组,选取同期体检正常儿童50例作为对照组,分别检测心肌酶指标和心肌蛋白指标。结果:病毒性心肌炎组心肌肌钙蛋白I(c Tnl)、肌红蛋白(MYO)显著高于支原体肺炎组、对照组,差异显著(P0.05);支原体肺炎组和对照组组间差异显著,具有统计学意义(P0.05)。病毒性心肌炎组肌酸激酶(CK)、肌酸激酶同工酶(CK-MB)、门冬氨酸氨基转移酶(AST)、乳酸脱氢酶(LDH)均显著高于支原体肺炎组、对照组,差异显著(P0.05);支原体肺炎组、对照组组间对比差异显著,具有统计学意义(P0.05)。2组入院10 d c Tnl、MYO均低于入院第1 d,具有统计学意义(P0.05);病毒性心肌炎组入院第10 d c Tnl、MYO显著高于支原体肺炎组,具有统计学意义(P0.05)。2组入院10 d CK、CK-MB、AST、LDH均低于入院第1 d,具有统计学意义(P0.05);病毒性心肌炎组入院第10 d CK、CK-MB、AST显著高于支原体肺炎组,差异具有统计学意义(P0.05)。根据ROC曲线分析临床性能,c Tnl、MYO、CK、CK-MB、AST、LDH的临界值分别为0.38μg/L、56.2μg/L、236.58 U/L、32.8 U/L、71.6 U/L、232.8 U/L,灵敏度分别为82.7%、85.4%、84.8%、89.6%、90.2、79.8%。结论:心肌损伤标志物可作为诊断病毒性心肌炎和支原体肺炎的重要指标,应用ROC回归曲线确定各指标的临界值,还可对两种疾病进行鉴别诊断。     

Distinct Effects of Voltage- and Store-dependent Calcium Influx on Stretch-induced Differentiation and Growth in Vascular Smooth Muscle

Stretch of the vascular wall stimulates smooth muscle hypertrophy by activating the MAPK and Rho/Rho kinase (ROK) pathways. We investigated the role of calcium in this response. Stretch-stimulated expression of contractile and cytoskeletal proteins in mouse portal vein was inhibited at mRNA and protein levels by blockade of voltage-dependent Ca²⁺ entry (VDCE). In contrast, blockade of store-operated Ca²⁺ entry (SOCE) did not affect smooth muscle marker expression but decreased global protein synthesis. Activation of VDCE caused membrane translocation of RhoA followed by phosphorylation of its downstream effectors LIMK-2 and cofilin-2. Stretch-activated cofilin-2 phosphorylation depended on VDCE but not on SOCE. VDCE was associated with increased mRNA expression of myocardin, myocyte enhancer factor (MEF) -2A and -2D, and smooth muscle marker genes, all of which depended on ROK activity. SOCE increased ERK1/2 phosphorylation and c-Fos expression but had no effect on phosphorylation of LIMK-2 and cofilin-2 or on myocardin and MEF2 expression. Knockdown of MEF2A or -2D eliminated the VDCE-induced activation of myocardin expression and increased basal c-Jun and c-Fos mRNA levels. These results indicate that MEF2 mediates VDCE-dependent stimulation of myocardin expression via the Rho/ROK pathway. In addition, SOCE activates the expression of immediate-early genes, known to be regulated by MEF2 via Ca²⁺-dependent phosphorylation of histone deacetylases, but this mode of Ca²⁺ entry does not affect the Rho/ROK pathway. Compartmentation of Ca²⁺ entry pathways appears as one mechanism whereby extracellular and membrane signals influence smooth muscle phenotype regulation, with MEF2 as a focal point.     

Effects of cyclic stretch on the molecular regulation of myocardin in rat aortic vascular smooth muscle cells

Background

The expression of myocardin, a cardiac-restricted gene, increases during environmental stress. How mechanical stretch affects the regulation of myocardin in vascular smooth muscle cells (VSMCs) is not fully understood. We identify the mechanisms and pathways through which mechanical stretch induces myocardin expression in VSMCs.

Results

Rat VSMCs grown on a flexible membrane base were stretched to 20% of maximum elongation, at 60 cycles per min. An in vivo model of aorta-caval shunt in adult rats was also used to investigate myocardin expression. Cyclic stretch significantly increased myocardin and angiotensin II (AngII) expression after 18 and 6 h of stretch. Addition of extracellular signal-regulated kinases (ERK) pathway inhibitor (PD98059), ERK small interfering RNA (siRNA), and AngII receptor blocker (ARB; losartan) before stretch inhibited the expression of myocardin protein. Gel shift assay showed that myocardin-DNA binding activity increased after stretch. PD98059, ERK siRNA and ARB abolished the binding activity induced by stretch. Stretch increased while myocardin-mutant plasmid, PD98059, and ARB abolished the promoter activity. Protein synthesis by measuring [³H]proline incorporation into the cells increased after cyclic stretch, which represented hypertrophic change of VSMCs. An in vivo model of aorta-caval shunt also demonstrated increased myocardin protein expression in the aorta. Confocal microscopy showed increased VSMC size 24 h after cyclic stretch and VSMC hypertrophy after creation of aorta-caval shunt for 3 days.

Conclusions

Cyclic stretch enhanced myocardin expression mediated by AngII through the ERK pathway in cultured rat VSMCs. These findings suggest that myocardin plays a role in stretch-induced VSMC hypertrophy.