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.

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.

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.

scholarly journals High Glucose Induces Mouse Mesangial Cell Overproliferation via Inhibition of Hydrogen Sulfide Synthesis in a TLR-4-Dependent Manner

2017 ◽  
Vol 41 (3) ◽  
pp. 1035-1043 ◽  
Author(s):  
Tao Ding ◽  
Wei Chen ◽  
Juan Li ◽  
Jiarong Ding ◽  
Xiaobin Mei ◽  
...  
Keyword(s):  
Diabetic Nephropathy ◽  
Hydrogen Sulfide ◽  
High Glucose ◽  
Mesangial Cell ◽  
Mesangial Cells ◽  
Specific Inhibitor ◽  
Protective Role ◽  
Akt Pathway ◽  
Dependent Manner ◽  
Respiration Sensor

Background/Aims: Overproliferation of mesangial cells was believed to play an important role in the progress of diabetic nephropathy, one of the primary complications of diabetes. Hydrogen sulfide (H2S), a well-known and pungent gas with the distinctive smell of rotten eggs, was discovered to play a protective role in diabetic nephropathy. Methods: MTT assay was used to examine the viability of mesangial cells. Small interfering RNA was used to knock down the expression of TLR4 while specific inhibitor LY294002 to suppress the function of PI3K. H2S generation rate was determined by a H2S micro-respiration sensor. Results: Glucose of 25mM induced significant mesangial cells proliferation, which was accomplished by significantly inhibited endogenous H2S synthesis. And exogenous H2S treatment by NaHS markedly mitigated the overproliferation of mouse mesangial cells. Furthermore, it was found that H2S deficiency could result in TLR4 activation. And H2S supplementation remarkably inhibited TLR4 expression and curbed the mesangial cell overproliferation. Besides, PI3K/Akt pathway inhibition also significantly ameliorated the cell overproliferation. Conclusion: High glucose (HG) induces mouse mesangial cell overproliferation via inhibition of hydrogen sulfide synthesis in a TLR-4-dependent manner. And PI3K/Akt pathway might also play a vital part in the HG-induced mesangial cell overproliferation.

scholarly journals Inhibition of Src Kinase Blocks High Glucose-Induced EGFR Transactivation and Collagen Synthesis in Mesangial Cells and Prevents Diabetic Nephropathy in Mice

Diabetes ◽  
2013 ◽  
Vol 62 (11) ◽  
pp. 3874-3886 ◽  
Author(s):  
K. Taniguchi ◽  
L. Xia ◽  
H. J. Goldberg ◽  
K. W. K. Lee ◽  
A. Shah ◽  
...  
Keyword(s):  
Diabetic Nephropathy ◽  
High Glucose ◽  
Collagen Synthesis ◽  
Mesangial Cells ◽  
Src Kinase ◽  
Egfr Transactivation

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

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.

scholarly journals Extracellular Signal-Regulated Kinase Mediates Stimulation of TGF-β1 and Matrix by High Glucose in Mesangial Cells

2000 ◽  
Vol 11 (12) ◽  
pp. 2222-2230
Author(s):  
MOTOHIDE ISONO ◽  
M. CARMEN IGLESIAS-DE LA CRUZ ◽  
SHELDON CHEN ◽  
SOON WON HONG ◽  
FUAD N. ZIYADEH
Keyword(s):  
High Glucose ◽  
Transforming Growth Factor ◽  
Mesangial Cells ◽  
Specific Inhibitor ◽  
Mrna Levels ◽  
Erk Activation ◽  
Extracellular Signal Regulated Kinase ◽  
Extracellular Signal ◽  
Tgf Β1 ◽  
Stimulation Of

Abstract. High ambient glucose exerts its injurious effects on renal cells through nonenzymatic and enzymatic pathways, including altered signal transduction and upregulation of the transforming growth factor-β (TGF-β) system. Extracellular signal-regulated kinase (ERK), a member of the mitogen-activated protein kinase (MAPK) cascade, is activated in mesangial cells cultured in high glucose and in glomeruli of diabetic rats. However, the biologic consequences of ERK activation in the kidney have not been investigated. To clarify the role of ERK activation, mouse mesangial cells were exposed to normal (5.5 mM) or high (25 mM) glucose with or without addition of PD98059, a specific inhibitor of MAPK/ERK kinase (MEK), an upstream kinase activator of ERK. Cells that were exposed to high glucose exhibited significant increases in ERK activity, TGF-β1 expression (total protein, mRNA levels, and promoter activity), [3H]-proline uptake, and α1(I) collagen and fibronectin mRNA levels. Treatment with PD98059 (up to 25 μM) significantly inhibited these parameters. In contrast, 25 μM PD98059 had no significant effect on any of the parameters measured in cells that were exposed to normal glucose. Overexpression of MAPK phosphatase CL 100 prevented TGF-β1 promoter activation by high glucose, confirming the involvement of the MEK-ERK pathway in response to high glucose. The conclusion is that activation of ERK in mesangial cells is responsible for high-glucose-induced stimulation of TGF-β1 and contributes to the increased extracellular matrix expression.

Sign in / Sign up

Export Citation Format

Share Document