scholarly journals Progesterone Induces RhoA Inactivation in Male Rat Aortic Smooth Muscle Cells Through Up-Regulation of p27kip1

Endocrinology ◽  
2014 ◽  
Vol 155 (11) ◽  
pp. 4473-4482 ◽  
Author(s):  
Hui-Chen Wang ◽  
Wen-Sen Lee
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Molecular Mechanisms ◽  
Kinase Inhibitor ◽  
Muscle Cells ◽  
Mitogen Activated Protein Kinase ◽  
Male Rat ◽  
Migration Inhibition ◽  
Aortic Smooth Muscle Cells ◽  
Aortic Smooth Muscle

Abstract Previously, we showed that progesterone (P4) at physiologic concentrations (5nM–500nM) inhibits proliferation and migration of rat aortic smooth muscle cells (RASMCs). The P4-induced migration inhibition in RASMC was resulted from Rat sacroma homolog gene family, member A (RhoA) inactivation induced by activating the cSrc/AKT/ERK 2/p38 mitogen-activated protein kinase-mediated signaling pathway. We also demonstrated that up-regulation of cyclin-dependent kinase inhibitor 1B (p27kip1) is involved in the P4-induced migration inhibition in RASMC. Because P4 can increase formation of the p27kip1-RhoA complex in RASMC, this finding led us to hypothesize that the P4-induced inactivation in RhoA might be caused by up-regulation of p27kip1. Here, we showed that P4 increased phosphorylation of p27kip1 at Ser10 in the nucleus, which in turn caused p27kip1 translocation from the nucleus to the cytosol, subsequently increasing formation of the p27kip1-RhoA complex. These effects were blocked by knocking-down kinase-interacting stathmin (KIS) using KIS small interfering RNA. Knock-down of p27kip1 abolished the P4-induced decreases in the level of RhoA protein in RASMC. However, pretreatment of RASMC with the proteasome inhibitor, N-(benzyloxycarbonyl)leucinylleucinylleucinal (MG132), prevented the P4-induced degradation of p27kip1 and RhoA. Taken together, our investigation of P4-induced migration inhibition in RASMC showed a sequence of associated intracellular events that included 1) increase in formation of the KIS-p27kip1 complex in the nucleus; 2) phosphorylated nuclear p27kip1 at Ser10; 3) increased cytosolic translocation of p27kip1 and formation of the p27kip1-RhoA complex in the cytosol; and 4) degradation of p27kip1 and RhoA through the ubiquitin-proteasome pathway. These findings highlight the molecular mechanisms underlying P4-induced migration inhibition in RASMC.

Dual effect of thapsigargin on cell death in porcine aortic smooth muscle cells

2007 ◽  
Vol 292 (1) ◽  
pp. C383-C395 ◽  
Author(s):  
Ting-Yu Chin ◽  
Hsiu-Chen Lin ◽  
Ju-Ping Kuo ◽  
Sheau-Huei Chueh
Keyword(s):  
Cell Death ◽  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Er Stress ◽  
Kinase Inhibitor ◽  
Muscle Cells ◽  
Mitogen Activated Protein Kinase ◽  
Mapk Activation ◽  
Aortic Smooth Muscle Cells ◽  
Aortic Smooth Muscle

A sustained increase in the cytosolic Ca2+ concentration ([Ca2+]i) can cause cell death. In this study, we found that, in cultured porcine aortic smooth muscle cells, endoplasmic reticulum (ER) stress, triggered by depletion of Ca2+ stores by thapsigargin (TG), induced an increase in the [Ca2+]i and cell death. However, the TG-induced death was not related to the [Ca2+]i increase but was mediated by targeting of activated Bax to mitochondria and the opening of mitochondrial permeability transition pores (PTPs). Once the mitochondrial PTPs had opened, several events, including collapse of the mitochondrial membrane potential, cytochrome c release, and caspase-3 activation, occurred and the cells died. TG-induced cell death was completely inhibited by the pan-caspase inhibitor Z-VAD-fmk and was enhanced by the Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane- N,N,N′,N′-tetraacetic acid (BAPTA), suggesting the existence of a Ca2+-dependent anti-apoptotic mechanism. After TG treatment, Ca2+-sensitive mitogen-activated protein kinase (MAPK) activation was induced and acted as a downstream effector of phosphatidylinositol 3-kinase (PI 3-kinase). The protective effect of Z-VAD-fmk on TG-induced cell death was reversed by BAPTA, PD-098059 (an MAPK kinase inhibitor), or LY-294002 (a PI 3-kinase inhibitor). Taken together, our data indicate that ER stress simultaneously activate two pathways, the mitochondrial caspase-dependent death cascade and the Ca2+-dependent PI 3-kinase/MAPK anti-apoptotic machinery. The Bax activation and translocation, but not the [Ca2+]i increase, may activate mitochondrial PTPs, which, in turn, causes activation of caspases and cell death, whereas Ca2+-dependent MAPK activation counteracts death signaling; removal of Ca2+ activated a second caspase-independent death pathway.

scholarly journals Calcium-sensing receptor modulates extracellular Ca2+ entry via TRPC-encoded receptor-operated channels in human aortic smooth muscle cells

2011 ◽  
Vol 301 (2) ◽  
pp. C461-C468 ◽  
Author(s):  
Jimmy Y. C. Chow ◽  
Christine Estrema ◽  
Tiffany Orneles ◽  
Xiao Dong ◽  
Kim E. Barrett ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Molecular Mechanisms ◽  
Muscle Cells ◽  
Hill Coefficient ◽  
Maximal Response ◽  
Trpc Channels ◽  
Pkc Inhibitor ◽  
Aortic Smooth Muscle Cells ◽  
Aortic Smooth Muscle

Ca-sensing receptor (CaSR), a member of the G protein-coupled receptor family, regulates the synthesis of parathyroid hormone in response to changes in serum Ca2+ concentrations. The functions of CaSR in human vascular smooth muscle cells are largely unknown. Here we sought to study CaSR activation and the underlying molecular mechanisms in human aortic smooth muscle cells (HASMC). Extracellular Ca2+ ([Ca2+]o) dose-dependently increased free cytosolic Ca2+ ([Ca2+]cyt) in HASMC, with a half-maximal response (EC50) of 0.52 mM and a Hill coefficient of 5.50. CaSR was expressed in HASMC, and the [Ca2+]o-induced [Ca2+]cyt rise was abolished by dominant negative mutants of CaSR. The CaSR-mediated increase in [Ca2+]cyt was also significantly inhibited by pertussis toxin, the phospholipase C inhibitor U-73122, or the general protein kinase C (PKC) inhibitor chelerythrine, but not by the conventional PKC inhibitor, Gö6976. Depletion of membrane cholesterol by pretreatment with methyl-β-cyclodextrin markedly decreased CaSR-induced increase in [Ca2+]cyt. Blockade of TRPC channels with 2-aminoethoxydiphenyl borate, SKF-96365, or La3 significantly inhibited [Ca2+]o entry, whereas activation of TRPC6 channels with flufenamic acid potentiated [Ca2+]o entry. Neither cyclopiazonic acid nor caffeine or ionomycin had any effect on [Ca2+]cyt in [Ca2+]o-free solutions. TRPC6 and PKCε mRNA and proteins were detected in HASMC, and [Ca2+]o induced PKCε phosphorylation, which could be prevented by chelerythrine. Our data suggest that CaSR activation mediates [Ca2+]o entry, likely through TRPC6-encoded receptor-operated channels that are regulated by a PLC/PKCε cascade. Our study therefore provides evidence not only for functional expression of CaSR, but also for a novel pathway whereby it regulates [Ca2+]o entry in HASMC.

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