Rho激酶抑制剂Y-27632对C3H10T1/2细胞增殖与成脂分化的作用

探究Rho激酶抑制剂Y-27632对间充质干细胞（mesenchymal stem cells,MSCs）C3H10T1/2增殖和成脂分化的影响.实验分为对照组、成脂诱导组和Y-27632处理组（Y-27632+成脂诱导）. 利用MTT检测细胞增殖情况,油红O染色,异丙醇萃取法检测细胞成脂分化情况,半定量RT-PCR检测过氧化物酶体增殖物激活受体γ(peroxisome proliferator activiated receptor γ, PPARγ)和CCAAT增强子结合蛋白α (CCAAT enhancer binding protein α, C/EBPα)基因表达. 结果表明,Y-27632能够显著抑制C3H10T1/2细胞的增殖(P<0.05),并呈一定的浓度依赖性;高浓度Y-27632对C3H10T1/2细胞成脂分化具有显著抑制作用（P<0.05）;半定量RT-PCR结果显示,成脂诱导处理组PPARγ和C/EBPα表达量在第3 d、5 d和7 d显著低于成脂诱导组（P<0.05）. 综上所述,Y-27632能够抑制C3H10T1/2细胞增殖与成脂分化.     

Thrombin induces MCP-1 expression through Rho-kinase and subsequent p38MAPK/NF-κB signaling pathway activation in vascular endothelial cells

Thrombin has been shown to increase expression of chemokines such as monocyte chemoattractant protein 1 (MCP-1) in endothelial cells, leading to the development of atherosclerosis. However, the precise mechanism of this induction remains unknown. In the present study, we investigated whether the small G protein RhoA, and its effector, Rho-kinase are involved in MCP-1 induction by thrombin in endothelial cells. Y-27632, a specific Rho-kinase inhibitor, potently inhibited MCP-1 induction by thrombin. Y-27632 significantly decreased the chemotactic activity of thrombin-stimulated supernatants of endothelial cells on monocytes. Importantly, fasudil, a specific Rho-kinase inhibitor, attenuated MCP-1 gene expression in the aorta of db/db mice. Y-27632 attenuated thrombin-mediated phosphorylation of p38MAPK and p65, indicating that Rho-kinase mediates thrombin-induced MCP-1 expression through p38MAPK and NF-κB activation. Our findings demonstrate that the Rho/Rho-kinase signaling pathway plays a critical role in thrombin-mediated MCP-1 expression and function, and suggest that Rho/Rho-kinase may be an important target in the development of new therapeutic strategies for atherosclerosis.     

Rho-kinase mediates TNF-α-induced MCP-1 expression via p38 MAPK signaling pathway in mesangial cells

Macrophage accumulation has been implicated in the pathogenesis of inflammatory glomerular disease. Monocyte chemoattractant protein-1 (MCP-1) plays a central role in recruiting monocytes to the glomeruli. Tumor necrosis factor-α (TNF-α) has been shown to induce MCP-1 expression in mesangial cells, although the precise mechanisms remain unclear. We previously demonstrated that RhoA and its effector, Rho-kinase (Rho-associated coiled-coil containing protein kinase, ROCK), are involved in the pathogenesis of diabetic nephropathy. However, its role in MCP-1 induction by TNF-α has not been elucidated. In the present study, we investigated whether the Rho/Rho-kinase signaling pathway regulates the TNF-α-mediated induction of MCP-1 in mesangial cells. Exposure of mouse mesangial cells (MES-13) to TNF-α resulted in an increase of MCP-1 expression (by RT-PCR) and secretion into the medium (by ELISA). Pull down and Western blot analysis revealed that TNF-α activated RhoA and Rho-kinase. Based on these observations, we speculated that the Rho/Rho-kinase signaling pathway may be involved in MCP-1 induction by TNF-α. In agreement with this concept, Y-27632, a specific Rho-kinase inhibitor, attenuated TNF-α-mediated induction of MCP-1. We demonstrated that Y-27632 inhibited TNF-α-mediated monocyte migration and attenuated TNF-α-mediated p38 MAPK activation. Based on these data we infer that Y-27632 inhibits TNF-α-induced MCP-1 expression, secretion and function through inhibition of Rho-kinase and p38 MAPK activity. Our study suggests that Rho/Rho-kinase is an important therapeutic target of monocyte recruitment and accumulation within the glomerulus in inflammatory renal disease.     

Suppression of lipin-1 expression increases monocyte chemoattractant protein-1 expression in 3T3-L1 adipocytes

Lipin-1 plays a crucial role in the regulation of lipid metabolism and cell differentiation in adipocytes. Expression of adipose lipin-1 is reduced in obesity, and metabolic syndrome. However, the significance of this reduction remains unclear. This study investigated if and how reduced lipin-1 expression affected metabolism. We assessed mRNA expression levels of various genes related to adipocyte metabolism in lipin-1-depleted 3T3-L1 adipocytes by introducing its specific small interfering RNA. In lipin-1-depleted adipocytes, mRNA and protein expression levels of monocyte chemoattractant protein-1 (MCP-1) were significantly increased, although the other genes tested were not altered. The conditioned media from the cells promoted monocyte chemotaxis. The increase in MCP-1 expression was prevented by treatment with quinazoline or salicylate, inhibitors of nuclear factor-κB activation. Because MCP-1 is related to adipose inflammation and systemic insulin resistance, these results suggest that a reduction in adipose lipin-1 in obesity may exacerbate adipose inflammation and metabolism.     

Cytokine profile and cytoskeletal changes after herpes simplex virus type 1 infection in human trabecular meshwork cells

Uveitis caused by herpes simplex virus (HSV)-1 is characterized by increased intraocular pressure (IOP) in the presence of anterior chamber inflammation. Despite their clinical significance, the pathogenic changes associated with HSV-1 infection in trabecular meshwork (TM) cells, the key cell type regulating IOP, have not been completely elucidated. In this study, cytokine array analyses showed a significant stepwise increase in monocyte chemoattractant protein (MCP)-1 expression upon HSV-1 infection in TM cells (p < 0.05). HSV-1 infection led to downregulation of fibrogenic molecules (fibronectin, α-smooth muscle actin, connective tissue growth factor and TGF-β1). Notably, HSV-1 infection caused a significant increase in actin stress fibres, with a twofold increase in active RhoA, which was enhanced by treatment with TGF-β1 and inhibited by treatment with the Rho-kinase inhibitor, Y-27632. TM cells treated with MCP-1 exhibited a dose-dependent increase in actin stress fibres compared to untreated TM cells. Our study suggests that HSV-1 infection in TM cells increases cell contractile activity rather than fibrotic changes in the extracellular matrix (ECM) components. Taken together, these observations demonstrate the enhanced expression of MCP-1 and TM cell contractile activity upon HSV-1 infection and events with potential implications for the pathobiology of abrupt IOP elevation in HSV-1 anterior uveitis.     

P2Y(1), P2Y(2), P2Y(4), and P2Y(6) receptors are coupled to Rho and Rho kinase activation in vascular myocytes

In the cardiovascular system, activation of ionotropic (P2X receptors) and metabotropic (P2Y receptors) P2 nucleotide receptors exerts potent and various responses including vasodilation, vasoconstriction, and vascular smooth muscle cell proliferation. Here we examined the involvement of the small GTPase RhoA in P2Y receptor-mediated effects in vascular myocytes. Stimulation of cultured aortic myocytes with P2Y receptor agonists induced an increase in the amount of membrane-bound RhoA and stimulated actin cytoskeleton organization. P2Y receptor agonist-induced actin stress fiber formation was inhibited by C3 exoenzyme and the Rho kinase inhibitor Y-27632. Stimulation of actin cytoskeleton organization by extracellular nucleotides was also abolished in aortic myocytes expressing a dominant negative form of RhoA. Extracellular nucleotides induced contraction and Y-27632-sensitive Ca(2+) sensitization in aortic rings. Transfection of Swiss 3T3 cells with P2Y receptors showed that Rho kinase-dependent actin stress fiber organization was induced in cells expressing P2Y(1), P2Y(2), P2Y(4), or P2Y(6) receptor subtypes. Our data demonstrate that P2Y(1), P2Y(2), P2Y(4), and P2Y(6) receptor subtypes are coupled to activation of RhoA and subsequently to Rho-dependent signaling pathways.     

Role of MAPK phosphatase-1 in the induction of monocyte chemoattractant protein-1 during the course of adipocyte hypertrophy

Monocyte chemoattractant protein-1 (MCP-1), an important chemokine whose expression is increased during the course of obesity, plays a role in macrophage infiltration into obese adipose tissue. This study was designed to elucidate the role of mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1) in the induction of MCP-1 during the course of adipocyte hypertrophy. We examined the time course of MKP-1 and MCP-1 mRNA expression and extracellular signal-regulated kinase (ERK) phosphorylation in the adipose tissue from mice rendered mildly obese by a short term high fat diet. We also studied the role of MKP-1 in the induction of MCP-1 in 3T3-L1 adipocytes during the course of adipocyte hypertrophy. MCP-1 mRNA expression was increased, followed by ERK activation and down-regulation of MKP-1, an inducible dual specificity phosphatase to inactivate ERK, in the adipose tissue at the early stage of obesity induced by a short term high fat diet, when macrophages are not infiltrated. Down-regulation of MKP-1 preceded ERK activation and increased production of MCP-1 in 3T3-L1 adipocytes in vitro during the course of adipocyte hypertrophy. Adenovirus-mediated restoration of MKP-1 in hypertrophied 3T3-L1 adipocytes reduced the otherwise increased ERK phosphorylation, thereby leading to the significant reduction of MCP-1 mRNA expression. This study provides evidence that the down-regulation of MKP-1 is critical for increased production of MCP-1 during the course of adipocyte hypertrophy.     

Role of the adipocyte-specific NF-κB activity in the regulation of IP-10 and T cell migration

Infiltration of immune cells into adipose tissue plays a central role in the pathophysiology of obesity-associated low-grade inflammation. The aim of this study was to analyze the role of adipocyte NF-κB signaling in the regulation of the chemokine/adipokine interferon-γ-induced protein 10 kDa (IP-10) and adipocyte-mediated T cell migration. Therefore, the regulation of IP-10 was investigated in adipose tissue of male C57BL/6J mice, primary human and 3T3-L1 preadipocytes/adipocytes. To specifically block the NF-κB pathway, 3T3-L1 cells stably overexpressing a transdominant mutant of IκBα were generated, and the chemical NF-κB inhibitor Bay117082 was used. Adipocyte-mediated T cell migration was assessed by a migration assay. It could be shown that IP-10 expression was higher in mature adipocytes compared with preadipocytes. Induced IP-10 expression and secretion were completely blocked by an NF-κB inhibitor in 3T3-L1 and primary human adipocytes. Stable overexpression of a transdominant mutant of IκBα in 3T3-L1 adipocytes led to an inhibition of basal and stimulated IP-10 expression and secretion. T cell migration was induced by 3T3-L1 adipocyte-conditioned medium, and both basal and induced T cell migration was strongly inhibited by stable overexpression of a transdominant IκBα mutant. In addition, with the use of an anti-IP-10 antibody, a significant decrease of adipocyte-induced T cell migration was shown. In conclusion, in this study, we could demonstrate that the NF-κB pathway is essential for the regulation of IP-10 in 3T3-L1 and primary human adipocytes. Adipocytes rather than preadipocytes contribute to NF-κB-dependent IP-10 expression and secretion. Furthermore, NF-κB-dependent factors and especially IP-10 represent novel signals from adipocytes to induce T cell migration.     

Epithelial�Cmesenchymal transition of rat peritoneal mesothelial cells via Rhoa/Rock pathway

The objective of this study was to investigate the role of the RhoA/Rock signaling pathway in the epithelial-mesenchymal transition (EMT) of rat peritoneal mesothelial cells (RPMCs). Primary SD rat peritoneal mesothelial cells were cultured in vitro. RPMCs were randomly assigned to four groups: group A (control), group B (TGF-β1, 10?μg/L), group C (10?μg/L TGF-β1?+?10?μmol/L Y-27632, an inhibitor of Rock that was pre-applied for 2?h before TGF-β1 stimulation), and group D (Y-27632 alone, 10?μmol/L). Our results were as follows: (1) TGF-β1 stimulation elicited a robust increase in RhoA activity in a time-dependent manner; the increase was 2.57?±?0.52 times larger than the activity observed for the control group (P?相似文献   

Regulation of the mesangial cell myofibroblast phenotype by actin polymerization

Mesangial cells in diverse glomerular diseases become myofibroblast-like, characterized by activation of smooth muscle alpha-actin (alpha-SMA) expression. In cultured mesangial cells, serum-deprivation markedly increases alpha-SMA expression, cell size, and stress fiber formation. Since stress fibers are assembled from actin monomers, we investigated the hypothesis that alterations in stress fiber formation regulate alpha-SMA expression and hypertrophy. Human mesangial cells were treated with agents that disrupt or stabilize actin stress fibers. Depolymerization of actin stress fibers in serum-deprived cells with actin-depolymerizing agents, cytochalasin B (CytB) and latrunculin B (LatB), or with inhibitors of Rho-kinase, Y-27632 and HA-1077 decreased alpha-SMA mRNA as judged by Northern blot analysis. Western blot analysis showed that CytB also reduced alpha-SMA protein levels. In serum-fed cells, agents that stabilized actin stress fibers, jasplakinolide (Jas) and phalloidin, increased alpha-SMA mRNA and protein. Treatment of human or rat mesangial cells with CytB, LatB, or Y-27632 decreased alpha-SMA promoter activity. In contrast, Jas increased promoter activity 5.6-fold in rat mesangial cells. The presence of an RNA polymerase inhibitor blocked degradation of alpha-SMA mRNA in cells treated with CytB suggesting that destabilization of this message is dependent on a newly transcribed or rapidly degraded factor. Inhibition of actin polymerization by CytB, LatB, Y-27623, and HA-1077 inhibited incorporation of (3)[H]-leucine into newly synthesized protein. Additionally, CytB and LatB decreased cell volume as determined by flow cytometry. Collectively, these results indicate that the state of polymerization of the actin cytoskeleton regulates alpha-SMA expression, hypertrophy, and myofibroblast differentiation in mesangial cells.     

H-89 potentiates adipogenesis in 3T3-L1 cells by activating insulin signaling independently of protein kinase A

Among four kinds of protein kinase A (PKA) inhibitors tested, H-89 exhibited a unique action to remarkably enhance adipocyte differentiation of 3T3-L1 cells, whereas the other three PKA inhibitors, PKA inhibitor Fragment 14-22 (PKI), Rp-cAMP, and KT 5720, did not enhance adipocyte differentiation. H-85, which is an inactive form of H-89, exhibited a similar enhancing effect on adipocyte differentiation. H-89 also potentiated the phosphorylation of Akt and extracellular signal-regulated kinase (ERK) 1/2 in 3T3-L1 cells, which function as downstream signaling of insulin. Phosphoinositide 3-kinase (PI3K) inhibitor wortmannin and mitogen-activated protein kinase kinase (MEK) inhibitor PD 98059 suppressed both the H-89-induced promotion of adipocyte differentiation and the H-89-induced potentiation of phosphorylation of Akt and ERK1/2. Rho kinase inhibitor Y-27632 also promoted the phosphorylation of both Akt and ERK1/2 and enhanced adipocyte differentiation, although its effect was somewhat less than that of H-89. Even when cells were treated with a mixture of Y-27632 and H-89, the additive enhancing effects on both the insulin signaling and adipocyte differentiation were not detected. Therefore, it is suggested that the major possible mechanism whereby H-89 potentiates adipocyte differentiation of 3T3-L1 cells is activation of insulin signaling that is elicited mostly by inhibiting Rho/Rho kinase pathway.     

Oxidized LDL and lysophosphatidylcholine stimulate plasminogen activator inhibitor-1 expression through reactive oxygen species generation and ERK1/2 activation in 3T3-L1 adipocytes

Plasminogen activator inhibitor-1 (PAI-1) is secreted from adipose tissue and is considered to be a risk factor for both atherosclerosis and insulin resistance. Here we report for the first time that PAI-1 expression is enhanced by oxidized low-density lipoprotein (OxLDL) and its lipid component lysophosphatidylcholine (LPC) in mouse 3T3-L1 adipocytes. In fully differentiated 3T3-L1 cells, OxLDL treatment increased the mRNA expression and protein secretion of PAI-1 in a dose- and time-dependent manner, whereas native LDL had no effect. The addition of an anti-CD36 antibody suppressed OxLDL-stimulated PAI-1 expression by 50%, suggesting that adipose-derived CD36 contributes to roughly half of the PAI-1 expression stimulated by OxLDL. In addition, pharmacological experiments showed that the OxLDL-stimulated enhancement in PAI-1 expression was mediated through the generation of reactive oxygen species (ROS) and phosphorylation of extracellular signal-regulated kinase 1/2. Furthermore, LPC, a major lipid component of OxLDL, was responsible for the enhanced expression of PAI-1 as phospholipase A(2)-treated acetyl LDL, which generates LPC, strongly stimulated PAI-1 expression, whereas acetyl LDL itself had no such activity. These data demonstrate that the uptake of OxLDL and, in particular, its lipid component LPC into adipocytes triggers aberrant ROS-mediated PAI-1 expression, which may be involved in the pathogenesis of metabolic syndrome.