scholarly journals TGFβ-induced RhoA activation and fibronectin production in mesangial cells require caveolae

2008 ◽  
Vol 295 (1) ◽  
pp. F153-F164 ◽  
Author(s):  
Fangfang Peng ◽  
Baifang Zhang ◽  
Dongcheng Wu ◽  
Alistair J. Ingram ◽  
Bo Gao ◽  
...  
Keyword(s):  
Transforming Growth Factor ◽  
Mesangial Cells ◽  
Rho Kinase ◽  
Glomerular Sclerosis ◽  
Cholesterol Depletion ◽  
Kinase Activation ◽  
Caveolin 1 ◽  
Rhoa Activation ◽  
Membrane Microdomain ◽  
Matrix Accumulation

Glomerular sclerosis of diverse etiologies is characterized by mesangial matrix accumulation, with transforming growth factor-β (TGFβ) an important pathogenic factor. The GTPase RhoA mediates TGFβ-induced matrix accumulation in some settings. Here we study the role of the membrane microdomain caveolae in TGFβ-induced RhoA activation and fibronectin upregulation in mesangial cells (MC). In primary rat MC, TGFβ1 time dependently increased RhoA and downstream Rho kinase activation. Rho pathway inhibition blocked TGFβ1-induced upregulation of fibronectin transcript and protein. TGFβ1-induced RhoA activation was prevented by disrupting caveolae with cholesterol depletion and rescued by cholesterol repletion. Compared with wild types, RhoA/Rho kinase activation was absent in MC lacking caveolae. Reexpression of caveolin-1 (and caveolae) restored these responses. Phosphorylation of caveolin-1 on Y14, effected by Src kinases, has been implicated in signaling responses. Overexpression of nonphosphorylatable caveolin-1 Y14A prevented TGFβ1-induced RhoA activation. TGFβ1 also activated Src, and its inhibition blocked RhoA activation. Furthermore, TGFβ1 led to association of RhoA and caveolin-1. This was prevented by Src or TGFβ receptor I inhibition, and by caveolin-1 Y14A overexpression. Last, fibronectin upregulation by TGFβ1 was blocked by Src inhibition, not seen in caveolin-1 knockout MC, and restored by caveolin-1 reexpression in the latter. TGFβ1-induced collagen I accumulation also required caveolae. TGFβ1-mediated Smad2/3 activation, however, did not require caveolae. We conclude that RhoA/Rho kinase mediates TGFβ-induced fibronectin upregulation. This requires caveolae and caveolin-1 interaction with RhoA. Interference with caveolin/caveolae or RhoA signaling thus represents a potential target for the treatment of fibrotic renal disease.

A potential role for caveolin-1 in VEGF-induced fibronectin upregulation in mesangial cells: involvement of VEGFR2 and Src

2013 ◽  
Vol 304 (6) ◽  
pp. F820-F830 ◽  
Author(s):  
Tingting Wu ◽  
Baifang Zhang ◽  
Feng Ye ◽  
Zeling Xiao
Keyword(s):  
Endothelial Cell ◽  
Matrix Protein ◽  
Mesangial Cells ◽  
Extracellular Matrix Protein ◽  
Glomerular Sclerosis ◽  
Cholesterol Depletion ◽  
Caveolin 1 ◽  
Rhoa Activation ◽  
Membrane Microdomain

VEGF is known to be an endothelial cell mitogen that stimulates angiogenesis by promoting endothelial cell survival, proliferation, migration, and differentiation. Recent studies have suggested that VEGF may play a pivotal role in glomerular sclerosis through extracellular matrix protein (ECM) accumulation, although the signaling mechanism is still unclear. The GTPase RhoA has been implicated in VEGF-induced type IV collagen accumulation in some settings. Here we study the role of different VEGF receptors and membrane microdomain caveolae in VEGF-induced RhoA activation and fibronectin upregulation in mesangial cells (MCs). In primary rat MC, VEGF time and dose dependently increased fibronectin production. Rho pathway inhibition blocked VEGF-induced fibronectin upregulation. VEGF-induced RhoA activation was prevented by disrupting caveolae with cholesterol depletion and rescued by cholesterol repletion. VEGF stimulation led to a markedly increased VEGFR2/caveolin-1 but failed to increase VEGFR1/caveolin-1 association. VEGF also increased caveolin-1/Src association and activated Src, and Src inhibitor blocked RhoA activation and fibronectin upregulation. Src-mediated phosphorylation of caveolin-1 on Y14 has also been implicated in signaling responses. Overexpression of nonphosphorylatable caveolin-1 Y14A prevented VEGF-induced RhoA activation and fibronectin upregulation. In vivo, although VEGFR1 and VEGFR2 protein levels were both increased in the kidney cortices of diabetic rats, VEGFR2/caveolin-1 association but not VEGFR1/caveolin-1 association was significantly increased. In conclusion, VEGF-induced RhoA activation and fibronectin upregulation require caveolae and caveolin-1 interaction with VEGFR2 and Src. Interference with caveolin/-ae signaling may provide new avenues for the treatment of fibrotic renal disease.

High glucose-induced RhoA activation requires caveolae and PKCβ1-mediated ROS generation

2012 ◽  
Vol 302 (1) ◽  
pp. F159-F172 ◽  
Author(s):  
Y. Zhang ◽  
F. Peng ◽  
B. Gao ◽  
A. J. Ingram ◽  
J. C. Krepinsky
Keyword(s):  
Diabetic Nephropathy ◽  
Nadph Oxidase ◽  
High Glucose ◽  
Transforming Growth Factor ◽  
Mesangial Cells ◽  
Ros Generation ◽  
Caveolin 1 ◽  
Rhoa Activation ◽  
Rhoa Signaling ◽  
Tgf Β1

Glomerular matrix accumulation is a hallmark of diabetic nephropathy. We previously showed that RhoA activation by high glucose in mesangial cells (MC) leads to matrix upregulation (Peng F, Wu D, Gao B, Ingram AJ, Zhang B, Chorneyko K, McKenzie R, Krepinsky JC. Diabetes 57: 1683–1692, 2008). Here, we study the mechanism whereby RhoA is activated. In primary rat MC, RhoA activation required glucose entry and metabolism. Broad PKC inhibitors (PMA, bisindolylmaleimide, Gö6976), as well as specific PKCβ blockade with an inhibitor and small interfering RNA (siRNA), prevented RhoA activation by glucose. PKCβ inhibition also abrogated reactive oxygen species (ROS) generation by glucose. The ROS scavenger N-acetylcysteine (NAC) or NADPH oxidase inhibitors apocynin and DPI prevented glucose-induced RhoA activation. RhoA and some PKC isoforms localize to caveolae. Chemical disruption of these microdomains prevented RhoA and PKCβ1 activation by glucose. In caveolin-1 knockout cells, glucose did not induce RhoA and PKCβ1 activation; these responses were rescued by caveolin-1 reexpression. Furthermore, glucose-induced ROS generation was significantly attenuated by chemical disruption of caveolae and in knockout cells. Downstream of RhoA signaling, activator protein-1 (AP-1) activation was also inhibited by disrupting caveolae, was absent in caveolin-1 knockout MC and rescued by caveolin-1 reexpression. Finally, transforming growth factor (TGF)-β1 upregulation, mediated by AP-1, was prevented by RhoA signaling inhibition and by disruption or absence of caveolae. In conclusion, RhoA activation by glucose is dependent on PKCβ1-induced ROS generation, most likely through NADPH oxidase. The activation of PKCβ1 and its downstream effects, including upregulation of TGF-β1, requires caveolae. These microdomains are thus important mediators of the profibrogenic process associated with diabetic nephropathy.

O-linked β-N-acetylglucosamine supports p38 MAPK activation by high glucose in glomerular mesangial cells

2011 ◽  
Vol 301 (4) ◽  
pp. E713-E726 ◽  
Author(s):  
Howard Goldberg ◽  
Catharine Whiteside ◽  
I. George Fantus
Keyword(s):  
Diabetic Nephropathy ◽  
P38 Mapk ◽  
High Glucose ◽  
Transforming Growth Factor ◽  
Kinase Inhibitor ◽  
Mesangial Cells ◽  
Mapk Signaling ◽  
Mitogen Activated Protein Kinase ◽  
Glomerular Mesangial Cells ◽  
Matrix Accumulation

Hyperglycemia augments flux through the hexosamine biosynthetic pathway and subsequent O-linkage of single β- N-acetyl-d-glucosamine moieties to serine and threonine residues on cytoplasmic and nuclear proteins ( O-GlcNAcylation). Perturbations in this posttranslational modification have been proposed to promote glomerular matrix accumulation in diabetic nephropathy, but clear evidence and mechanism are lacking. We tested the hypothesis that O-GlcNAcylation enhances profibrotic signaling in rat mesangial cells. An adenovirus expressing shRNA directed against O-GlcNAc transferase (OGT) markedly reduced basal and high-glucose-stimulated O-GlcNAcylation. Interestingly, O-GlcNAc depletion prevented high-glucose-induced p38 mitogen-activated protein kinase (MAPK) and c-Jun NH2-terminal kinase phosphorylation. Downstream of p38, O-GlcNAc controlled the expression of plasminogen activator inhibitor-1, fibronectin, and transforming growth factor-β, important factors in matrix accumulation in diabetic nephropathy. Treating mesangial cells with thiamet-G, a highly selective inhibitor of O-GlcNAc-specific hexosaminidase ( O-GlcNAcase), increased O-GlcNAcylation and p38 phosphorylation. The high-glucose-stimulated kinase activity of apoptosis signal-regulating kinase 1 (ASK1), an upstream MAPK kinase kinase for p38 that is negatively regulated by Akt, was inhibited by OGT shRNA. Akt Thr308 and Ser473 phosphorylation were enhanced following OGT shRNA expression in high-glucose-exposed mesangial cells, but high-glucose-induced p38 phosphorylation was not attenuated by OGT shRNA in cells pretreated with the phosphatidylinositol 3-kinase inhibitor LY-294002. OGT shRNA also reduced high-glucose-stimulated reactive oxygen species (ROS) formation. In contrast, diminished O-GlcNAcylation caused elevated ERK phosphorylation and PKCδ membrane translocation. Thus, O-GlcNAcylation is coupled to profibrotic p38 MAPK signaling by high glucose in part through Akt and possibly through ROS.

Akt and RhoA activation in response to high glucose require caveolin-1 phosphorylation in mesangial cells

2014 ◽  
Vol 306 (11) ◽  
pp. F1308-F1317 ◽  
Author(s):  
Su-Zhen Wu ◽  
Fang-Fang Peng ◽  
Jia-Lin Li ◽  
Feng Ye ◽  
Shao-Qing Lei ◽  
...  
Keyword(s):  
Renal Disease ◽  
High Glucose ◽  
Mesangial Cells ◽  
Src Kinase ◽  
Specific Inhibitor ◽  
Membrane Translocation ◽  
Caveolin 1 ◽  
Rhoa Activation ◽  
Diabetic Renal Disease ◽  
Src Activation

Glomerular matrix accumulation is a hallmark of diabetic renal disease. Serine/threonine kinase PKC-β1 mediates glucose-induced Akt S473 phosphorylation, RhoA activation, and transforming growth factor (TGF)-β1 upregulation and finally leads to matrix upregulation in mesangial cells (MCs). It has been reported that glucose-induced PKC-β1 activation is dependent on caveolin-1 and the presence of intact caveolae in MCs; however, whether activated PKC-β1 regulates caveolin-1 expression and phosphorylation are unknown. Here, we showed that, although the caveolin-1 protein level had no significant change, the PKC-β-specific inhibitor LY-333531 blocked caveolin-1 Y14 phosphorylation in high glucose (HG)-treated MCs and in the renal cortex of diabetic rats. The Src-specific inhibitor SU-6656 prevented the HG-induced association between PKC-β1 and caveolin-1 and PKC-β1 membrane translocation, whereas PKC-β1 small interfering RNA failed to block Src activation, indicating that Src kinase is upstream of PKC-β1 activation. Although LY-333531 blocked PKC-β1 membrane translocation, it had no effect on the PKC-β1/caveolin-1 association, suggesting that PKC-β1 activation requires the interaction of caveolin-1 and PKC-β1. PKC-β1-mediated Akt S473 phosphorylation, RhoA activation, and fibronectin upregulation in response to HG were prevented by SU-6656 and nonphosphorylatable mutant caveolin-1 Y14A. In conclusion, Src activation by HG mediates the PKC-β1/caveolin-1 association and PKC-β1 activation, which assists in caveolin-1 Y14 phosphorylation by Src kinase. The downstream effects, including Akt S473 phosphorylation, RhoA activation, and fibronectin upregulation, require caveolin-1 Y14 phosphorylation. Caveolin-1 is thus an important mediator of the profibrogenic process in diabetic renal disease.

scholarly journals ROCK2 regulates TGF-β-induced expression of CTGF and profibrotic genes via NF-κB and cytoskeleton dynamics in mesangial cells

2019 ◽  
Vol 317 (4) ◽  
pp. F839-F851 ◽  
Author(s):  
Yosuke Nagai ◽  
Keiichiro Matoba ◽  
Daiji Kawanami ◽  
Yusuke Takeda ◽  
Tomoyo Akamine ◽  
...  
Keyword(s):  
Kidney Disease ◽  
Diabetic Kidney Disease ◽  
Transforming Growth Factor ◽  
Nuclear Translocation ◽  
Mesangial Cells ◽  
Rho Kinase ◽  
Small Gtpase ◽  
Actin Dynamics ◽  
Diabetic Glomerulosclerosis ◽  
Diabetic Kidney

The small GTPase Rho and its effector Rho kinase (ROCK) are involved in the pathogenesis of diabetic kidney disease. Rho kinase has two isoforms: ROCK1 and ROCK2. However, it remains unclear which is mainly involved in the progression of diabetic glomerulosclerosis and the regulation of profibrotic mediators. Glomeruli isolated from type 2 diabetic db/ db mice demonstrated increased gene expression of transforming growth factor (TGF)-β and its downstream profibrotic mediators. Chemical inhibition of ROCK suppressed the expression of profibrotic mediators in both isolated glomeruli and cultured mesangial cells. An investigation of mechanisms underlying this observation revealed activated ROCK functions through the phosphorylation of JNK and Erk and the nuclear translocation of NF-κB via actin dynamics. Knockdown by siRNA against ROCK1 and ROCK2 showed that ROCK2 but not ROCK1 controls this fibrotic machinery. Further in vivo experiments showed that ROCK2 activity in the renal cortex of db/ db mice was elevated compared with control db/ m mice. Importantly, oral administration of ROCK2 inhibitor attenuated renal ROCK2 activity, albuminuria, and glomerular fibrosis in db/ db mice. These observations indicate that ROCK2 is a key player in the development of diabetic renal injury. Glomerular ROCK2 may be a potential therapeutic target for the treatment of diabetic kidney disease.

scholarly journals RhoA Activation in Mesangial Cells by Mechanical Strain Depends on Caveolae and Caveolin-1 Interaction

2006 ◽  
Vol 18 (1) ◽  
pp. 189-198 ◽  
Author(s):  
Fangfang Peng ◽  
Dongcheng Wu ◽  
Alistair J. Ingram ◽  
Baifang Zhang ◽  
Bo Gao ◽  
...  
Keyword(s):  
Mesangial Cells ◽  
Mechanical Strain ◽  
Caveolin 1 ◽  
Rhoa Activation

scholarly journals Upregulation of MUC5AC by VEGF in human primary bronchial epithelial cells: implications for asthma

2019 ◽  
Vol 20 (1) ◽  
Author(s):  
Sung-Ho Kim ◽  
Qing-Mei Pei ◽  
Ping Jiang ◽  
Juan Liu ◽  
Rong-Fei Sun ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Bronchial Epithelial Cells ◽  
Cholesterol Depletion ◽  
Vegf Receptor ◽  
Dependent Manner ◽  
Bronchial Epithelial ◽  
Caveolin 1 ◽  
Protein Levels ◽  
Rhoa Activation ◽  
Primary Bronchial Epithelial Cells

Abstract Background Airway mucus hypersecretion is an important pathophysiological feature in asthma. Mucins are glycoproteins that are mainly responsible for the viscoelastic property of mucus, and MUC5AC is a major mucin glycoprotein that is overproduced in asthma. Vascular endothelial growth factor (VEGF) has been implicated in inflammatory and airway blood vessel remodeling in asthmatics. Therefore, we sought to investigate the effect of VEGF on MUC5AC expression and study the underlying mechanisms. Methods In order to elucidate the precise mechanism underlying the effect of VEGF on MUC5AC expression, we tested the effects of VEGF on RhoA activation and the association of caveolin-1 and VEGFR2 in Primary Bronchial Epithelial Cells. Results VEGF up-regulated MUC5AC mRNA and protein levels in a dose- and time-dependent manner, and activated RhoA. Additionally, VEGF-induced MUC5AC expression and RhoA activation were enhanced by disrupting caveolae with cholesterol depletion and reversed by cholesterol repletion, and inhibited by a selective VEGF receptor 2 (VEGFR2) inhibitor SU1498. Furthermore, phospho-VEGFR2 expression was decreased via overexpression of caveolin-1. VEGF treatment reduced the association of caveolin-1 and VEGFR2. Conclusion Collectively, our findings suggest that VEGF up-regulates MUC5AC expression and RhoA activation by interaction with VEGFR2, and this phenomenon was related with the association of caveolin-1 and VEGFR2. Further studies on these mechanisms are needed to facilitate the development of treatments for asthma.

Thrombospondin-2 therapy ameliorates experimental glomerulonephritis via inhibition of cell proliferation, inflammation, and TGF-β activation

2009 ◽  
Vol 297 (5) ◽  
pp. F1299-F1309 ◽  
Author(s):  
Christoph Daniel ◽  
Andrea Wagner ◽  
Bernd Hohenstein ◽  
Christian Hugo
Keyword(s):  
Extracellular Matrix ◽  
Cell Proliferation ◽  
Inflammatory Response ◽  
Time Course ◽  
Transforming Growth Factor ◽  
Mesangial Cells ◽  
Cell Number ◽  
Thigh Muscle ◽  
Glomerular Cell ◽  
Matrix Accumulation

We recently identified thrombospondin-2 (TSP-2) as an endogenous regulator of matrix remodelling and inflammation in experimental kidney disease by studying TSP-2-deficient mice. In this study, we asked whether systemic TSP-2 overexpression via thigh muscle transfection is able to ameliorate the time course of the anti-Thy1 glomerulonephritis model. After induction of anti-Thy1 nephritis, rats were transfected either with an overexpression plasmid for TSP-2 or lacZ as a control. Biopsies, urine, and blood samples were taken on days 1, 3, and 6 after disease induction. Muscular overexpression of TSP-2 reduced glomerular transforming growth factor (TGF)-β activation and glomerular extracellular matrix formation as determined by collagen IV and fibronectin. In addition, activation of mesangial cells to the myofibroblast-like phenotype was also significantly decreased in TSP-2-overexpressing animals. TSP-2 overexpression inhibited both glomerular endothelial and mesangial cell proliferation, resulting in a reduced glomerular cell number and glomerular tuft area. The inflammatory response, as monitored by T cells and antigen-presenting cells, was reduced significantly by TSP-2 overexpression, but influx of macrophages was unchanged. These data demonstrate TSP-2 as a potential therapeutic agent to inhibit the glomerular proliferative and inflammatory response as well as TGF-β activation and extracellular matrix accumulation in experimental mesangial proliferative glomerulonephritis.

scholarly journals Caveolin-1 in mesangial cells suppresses MAP kinase activation and cell proliferation induced by bFGF and PDGF

2004 ◽  
Vol 66 (5) ◽  
pp. 1794-1804 ◽  
Author(s):  
Yutaka Fujita ◽  
Shoichi Maruyama ◽  
Hiroshi Kogo ◽  
Seiichi Matsuo ◽  
Toyoshi Fujimoto
Keyword(s):  
Cell Proliferation ◽  
Map Kinase ◽  
Mesangial Cells ◽  
Kinase Activation ◽  
Caveolin 1

scholarly journals Mechanism of RhoA/Rho kinase activation in endothelin-1- induced contraction in rabbit basilar artery

2002 ◽  
Vol 283 (3) ◽  
pp. H983-H989 ◽  
Author(s):  
Liyan Miao ◽  
Yun Dai ◽  
John Zhang
Keyword(s):  
Tyrosine Kinase ◽  
Basilar Artery ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Rho Kinase ◽  
Kinase Activation ◽  
Rhoa Activity ◽  
Rhoa Activation ◽  
Rho Kinase Inhibitor ◽  
Mlc Phosphorylation

This study was undertaken to demonstrate the role of the RhoA/Rho kinase pathway in endothelin-1 (ET-1)-induced contraction of the rabbit basilar artery. Isometric tension and Western blot were used to examine ET-1-induced contraction and RhoA activation. The upstream effect on ET-1-induced RhoA activity was determined by using ETA and ETB receptor antagonists, protein kinase C (PKC), tyrosine kinase, and phosphatidylinositol-3 kinase inhibitors. The downstream effect of ET-1-induced contraction and RhoA activity was studied in the presence of the Rho kinase inhibitor Y-27632. The effect of Rho kinase inhibitor on ET-1-induced myosin light chain (MLC) phosphorylation was investigated by using urea-glycerol-PAGE immunoblotting. We found 1) ET-1 increased RhoA activity (membrane binding RhoA) in a concentration-dependent manner; 2) ETA, but not ETB, receptor antagonist abolished the effect of ET-1 on RhoA activation; 3) phosphodylinositol-3 kinase inhibitor, but not PKC and tyrosine kinase inhibitors, reduced ET-1-induced RhoA activation; 4) Rho kinase inhibitor Y-27632 (10 μM) inhibited ET-1-induced contraction; and 5) ET-1 increased the level of MLC phosphorylation. Rho kinase inhibitor Y-27632 reduced the effect of ET-1 on MLC phosphorylation. This study demonstrated that RhoA/Rho kinase activation is involved in ET-1-induced contraction in the rabbit basilar artery. Phosphodylinositol-3 kinase and MLC might be the upstream and downstream factors of RhoA activation.

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