Balachandar Venkatesan

Mesangial Cell Hypertrophy by High Glucose Is Mediated by Downregulation of the Tumor Suppressor PTEN

Diabetic nephropathy is characterized early in its course by glomerular hypertrophy and, importantly, mesangial hypertrophy, which correlate with eventual glomerulosclerosis. The mechanism of hypertrophy, however, is not known. Gene disruption of the tumor suppressor PTEN, a negative regulator of the phosphatidylinositol 3-kinase/Akt pathway, in fruit flies and mice demonstrated its role in size control in a cell-specific manner. Here, we investigated the mechanism of mesangial hypertrophy in response to high extracellular glucose. We link early renal hypertrophy with significant reduction in PTEN expression in the streptozotocin-induced diabetic kidney cortex and glomeruli, concomitant with activation of Akt. Similarly, exposure of mesangial cells to high concentrations of glucose also decreased PTEN expression and its phosphatase activity, resulting in increased Akt activity. Expression of PTEN inhibited high-glucose–induced mesangial cell hypertrophy, and expression of dominant-negative PTEN was sufficient to induce hypertrophy. In diabetic nephropathy, the hypertrophic effect of hyperglycemia is thought to be mediated by transforming growth factor-β (TGF-β). TGF-β significantly reduced PTEN expression in mesangial cells, with a reduction in its phosphatase activity and an increase in Akt activation. PTEN and dominant-negative Akt attenuated TGF-β–induced hypertrophy of mesangial cells. Finally, we show that inhibition of TGF-β signal transduction blocks the effect of high glucose on PTEN downregulation. These data identify a novel mechanism placing PTEN as a key regulator of diabetic mesangial hypertrophy involving TGF-β signaling.

WNT1-inducible signaling pathway protein-1 activates diverse cell survival pathways and blocks doxorubicin-induced cardiomyocyte death

The anthracycline antibiotic doxorubicin (DOX) is a potent cancer chemotherapeutic agent that exerts both acute and chronic cardiotoxicity. Here we show that in adult mouse cardiomyocytes, DOX activates (i) the pro-apoptotic p53, (ii) p38MAPK and JNK, (iii) Bax translocation, (iv) cytochrome c release, and (v) caspase 3. Further, it (vi) inhibits expression of anti-apoptotic Akt, Bcl-2 and Bcl-xL, and (vii) induces internucleosomal degradation and cell death. WNT1-inducible signaling pathway protein-1 (WISP1), a CCN family member and a matricellular protein, inhibits DOX-mediated cardiomyocyte death. WISP1 inhibits DOX-induced p53 activation, p38 MAPK and JNK phosphorylation, Bax translocation to mitochondria, and cytochrome c release into cytoplasm. Additionally, WISP1 reverses DOX-induced suppression of Bcl-2 and Bcl-xL expression and Akt inhibition. The pro-survival effects of WISP1 were recapitulated by the forced expression of mutant p53, wild-type Bcl-2, wild-type Bcl-xL, or constitutively active Akt prior to DOX treatment. WISP1 also induces the pro-survival factor Survivin via PI3K/Akt signaling. Overexpression of wild-type, but not mutant Survivin, blunts DOX cytotoxicity. Further, WISP1 stimulates PI3K-Akt-dependent GSK3beta phosphorylation and beta-catenin nuclear translocation. Importantly, WISP1 induces its own expression. Together, these results provide important insights into the cytoprotective effects of WISP1 in cardiomyocytes, and suggest a potential therapeutic role for WISP1 in DOX-induced cardiotoxicity.

WISP1, a Pro-mitogenic, Pro-survival Factor, Mediates Tumor Necrosis Factor-α (TNF-α)-stimulated Cardiac Fibroblast Proliferation but Inhibits TNF-α-induced Cardiomyocyte Death

WNT1-inducible signaling pathway protein-1 (WISP1), a member of the CYR61/CTGF/Nov family of growth factors, can mediate cell growth, transformation, and survival. Previously we demonstrated that WISP1 is up-regulated in post-infarct heart, stimulates cardiac fibroblast proliferation, and is induced by the proinflammatory cytokine tumor necrosis factor-α (TNF-α). Here we investigated (i) the localization of TNF-α and WISP1 in post-infarct heart, (ii) the mechanism of TNF-α-mediated WISP1 induction in primary human cardiac fibroblasts (CF), (iii) the role of WISP1 in TNF-α-mediated CF proliferation and collagen production, and (iv) the effects of WISP1 on TNF-α-mediated cardiomyocyte death. TNF-α and WISP1 expressions were increased in the border zones and non-ischemic remote regions of the post-ischemic heart. In CF, TNF-α potently induced WISP1 expression in cyclic AMP response element-binding protein (CREB)-dependent manner. TNF-α induced CREB phosphorylation in vitro and DNA binding and reporter gene activities in vivo. TNF-α induced CREB activation via ERK1/2, and inhibition of ERK1/2 and CREB blunted TNF-α-mediated WISP1 induction. Most importantly, WISP1 knockdown attenuated TNF-α stimulated collagen production and CF proliferation. Furthermore, WISP1 attenuated TNF-α-mediated cardiomyocyte death, thus demonstrating pro-mitogenic and pro-survival effects for WISP1 in myocardial constituent cells. Our results suggest that a TNF-α/WISP1 signaling pathway may contribute to post-infarct cardiac remodeling, a condition characterized by fibrosis and progressive cardiomyocyte loss.

Downregulation of catalase by reactive oxygen species via PI 3 kinase/Akt signaling in mesangial cells

Reactive oxygen species (ROS) contribute to many glomerular diseases by targeting mesangial cells. ROS have been shown to regulate expression of many antioxidant enzymes including catalase. The mechanism by which the expression of catalase protein is regulated by ROS is not precisely known. Here we report that increased intracellular ROS level by hydrogen peroxide (H2O2) reduced the expression of catalase. H2O2 increased phosphorylation of Akt kinase in a dose‐dependent and sustained manner with a concomitant increase in the phosphorylation of FoxO1 transcription factor. Further analysis revealed that H2O2 promoted rapid activation of phosphatidylinositol (PI) 3 kinase. The PI 3 kinase inhibitor Ly294002 and expression of tumor suppressor protein PTEN inhibited Akt kinase activity, resulting in the attenuation of FoxO1 phosphorylation and preventing the downregulating effect of H2O2 on catalase protein level. Dominant negative Akt attenuated the inhibitory effect of H2O2 on expression of catalase. Constitutively active FoxO1 increased the expression of catalase. However, dominant negative FoxO1 inhibited catalase protein level. Catalase transcription was reduced by H2O2 treatment. Furthermore, expression of dominant negative Akt and constitutively active FoxO1 increased catalase transcription, respectively. These results demonstrate that ROS downregulate the expression of catalase in mesangial cells by PI 3 kinase/Akt signaling via FoxO1 as a target. J. Cell. Physiol. 211: 457–467, 2007.

Wisp1, a pro-mitogenic, pro-survival factor, mediates TNF-α stimulated cardiac fibroblast proliferation, but inhibits TNF- α-induced cardiomyocyte death

WNT1-inducible signaling pathway protein-1 (WISP1), a member of the CYR61/CTGF/Nov family of growth factors, can mediate cell growth, transformation, and survival. Previously we demonstrated that WISP1 is up-regulated in post-infarct heart, stimulates cardiac fibroblast proliferation, and is induced by the proinflammatory cytokine tumor necrosis factor-α (TNF-α). Here we investigated (i) the localization of TNF-α and WISP1 in post-infarct heart, (ii) the mechanism of TNF-α-mediated WISP1 induction in primary human cardiac fibroblasts (CF), (iii) the role of WISP1 in TNF-α-mediated CF proliferation and collagen production, and (iv) the effects of WISP1 on TNF-α-mediated cardiomyocyte death. TNF-α and WISP1 expressions were increased in the border zones and non-ischemic remote regions of the post-ischemic heart. In CF, TNF-α potently induced WISP1 expression in cyclic AMP response element-binding protein (CREB)-dependent manner. TNF-α induced CREB phosphorylation in vitro and DNA binding and reporter gene activities in vivo. TNF-α induced CREB activation via ERK1/2, and inhibition of ERK1/2 and CREB blunted TNF-α-mediated WISP1 induction. Most importantly, WISP1 knockdown attenuated TNF-α stimulated collagen production and CF proliferation. Furthermore, WISP1 attenuated TNF-α-mediated cardiomyocyte death, thus demonstrating pro-mitogenic and pro-survival effects for WISP1 in myocardial constituent cells. Our results suggest that a TNF-α/WISP1 signaling pathway may contribute to post-infarct cardiac remodeling, a condition characterized by fibrosis and progressive cardiomyocyte loss.

Interleukin-18 induces EMMPRIN expression in primary cardiomyocytes via JNK/Sp1 signaling and MMP-9 in part via EMMPRIN and through AP-1 and NF-κB activation

IL-18 and the extracellular matrix metalloproteinase (MMP) inducer (EMMPRIN) stimulate the expression of proinflammatory cytokines and MMPs and are elevated in myocardial hypertrophy, remodeling, and failure. Here, we report several novel findings in primary cardiomyocytes treated with IL-18. First, IL-18 activated multiple transcription factors, including NF-κB (p50 and p65), activator protein (AP)-1 (cFos, cJun, and JunD), GATA, CCAAT/enhancer-binding protein, myocyte-specific enhancer-binding factor, interferon regulatory factor-1, p53, and specific protein (Sp)-1. Second, IL-18 induced EMMPRIN expression via myeloid differentiation primary response gene 88/IL-1 receptor-associated kinase/TNF receptor-associated factor-6/JNK-dependent Sp1 activation. Third, IL-18 induced a number of MMP genes, particularly MMP-9, at a rapid rate as well as tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-3 at a slower rate. Finally, the IL-18 induction of MMP-9 was mediated in part via EMMPRIN and through JNK- and ERK-dependent AP-1 activation and p38 MAPK-dependent NF-κB activation. These results suggest that the elevated expression of IL-18 during myocardial injury and inflammation may favor EMMPRIN and MMP induction and extracellular matrix degradation. Therefore, targeting IL-18 or its signaling pathways may be of potential therapeutic benefit in adverse remodeling.

Akt kinase targets association of CBP with SMAD 3 to regulate TGFβ-induced expression of plasminogen activator inhibitor-1

Transforming growth factor‐β (TGFβ) controls expression of plasminogen activator inhibitor type 1 (PAI‐1), which regulates degradation of extracellular matrix proteins in fibrotic diseases. The TGFβ receptor‐specific Smad 3 has been implicated in the PAI‐1 expression. The mechanism by which non‐Smad signaling contributes to this process is not known. We studied the cross‐talk between Smad 3 and PI 3 kinase/Akt signaling in TGFβ‐induced PAI‐1 expression in renal mesangial cells. Inhibition of PI 3 kinase and Akt kinase blocked TGFβ‐ and Smad 3‐mediated expression of PAI‐1. In contrast, constitutively active PI 3 kinase and Akt kinase increased PAI‐1 expression, similar to TGFβ. Inhibition of PI 3 kinase and Akt kinase had no effect on TGFβ‐induced Smad 3 phosphorylation and its translocation to the nucleus. Notably, inhibition of PI 3 kinase‐dependent Akt kinase abrogated TGFβ‐induced PAI‐1 transcription, without affecting binding of Smad 3 to the PAI‐1 Smad binding DNA element. However, PI 3 kinase inhibition and dominant negative Akt kinase antagonized the association of the transcriptional coactivator CBP with Smad 3 in response to TGFβ, resulting in inhibition of Smad 3 acetylation. Together our findings identify TGFβ‐induced PI 3 kinase/Akt signaling as a critical regulator of Smad 3‐CBP interaction and Smad 3 acetylation, which cause increased PAI‐1 expression. J. Cell. Physiol. 214: 513–527, 2008.

EMMPRIN activates multiple transcription factors in cardiomyocytes, and induces interleukin-18 expression via Rac1-dependent PI3K/Akt/IKK/NF-κB andMKK7/JNK/AP-1 signaling

The transmembrane glycoprotein extracellular matrix metalloproteinase inducer (EMMPRIN), and the pleiotropic proinflammatory cytokine interleukin (IL)-18, play critical roles in myocardial remodeling, by inducing matrix degrading metalloproteinases (MMPs). Previously we showed that IL-18 induces EMMPRIN expression in cardiomyocytes via MyD88/IRAK4/TRAF6/JNK-dependent Sp1 activation. Here in reciprocal studies we demonstrate that EMMPRIN is a potent inducer of IL-18 transcription, protein expression and protein secretion in primary mouse cardiomyocytes. We show for the first time that EMMPRIN stimulates the activation of NF-kappaB, AP-1, CREB, and ATF-2 in cardiomyocytes, and induces IL-18 expression via Rac1-dependent PI3K/Akt/IKK/NF-kappaB and MKK7/JNK/AP-1 signaling. Moreover, EMMPRIN induces robust time-dependent induction of various MMP mRNAs. EMMPRIN also induces the mRNA of TIMPs 1 and 3, but in a delayed fashion. These results suggest that IL-18-induced EMMPRIN expression may favor net MMP expression and ECM destruction, and thus identify both as potential therapeutic targets in countering adverse myocardial remodeling.

PRAS40 ACTS AS A NODAL REGULATOR OF HIGH GLUCOSE-INDUCED TORC1 ACTIVATION IN GLOMERULAR MESANGIAL CELL HYPERTROPHY

Diabetic nephropathy manifests aberrant activation of TORC1, which senses key signals to modulate protein synthesis and renal hypertrophy. PRAS40 has recently been identified as a raptor‐interacting protein and is a component and a constitutive inhibitor of TORC1. The mechanism by which high glucose stimulates TORC1 activity is not known. PRAS40 was identified in the mesangial cells in renal glomeruli and in tubulointerstitium of rat kidney. Streptozotocin‐induced diabetic renal hypertrophy was associated with phosphorylation of PRAS40 in the cortex and glomeruli. In vitro, high glucose concentration increased PRAS40 phosphorylation in a PI 3 kinase‐ and Akt‐dependent manner, resulting in dissociation of raptor–PRAS40 complex in mesangial cells. High glucose augmented the inactivating and activating phosphorylation of 4EBP‐1 and S6 kinase, respectively, with concomitant induction of protein synthesis and hypertrophy. Expression of TORC1‐nonphosphorylatable mutant of 4EBP‐1 and dominant‐negative S6 kinase significantly inhibited high glucose‐induced protein synthesis and hypertrophy. PRAS40 knockdown mimicked the effect of high glucose on phosphorylation of 4EBP‐1 and S6 kinase, protein synthesis, and hypertrophy. To elucidate the role of PRAS40 phosphorylation, we used phosphorylation‐deficient mutant of PRAS40, which in contrast to PRAS40 knockdown inhibited phosphorylation of 4EBP‐1 and S6 kinase, leading to reduced mesangial cell hypertrophy. Thus, our data identify high glucose‐induced phosphorylation and inactivation of PRAS40 as a central node for mesangial cell hypertrophy in diabetic nephropathy. J. Cell. Physiol. 225: 27–41, 2010. � 2010 Wiley‐Liss, Inc.

Resveratrol inhibits PDGF receptor mitogenic signaling in mesangial cells: role of PTP1B.

Mesangioproliferative glomerulonephritis is associated with overactive PDGF receptor signal transduction. We show that the phytoalexin resveratrol dose dependently inhibits PDGF‐induced DNA synthesis in mesangial cells with an IC50 of 10 µM without inducing apoptosis. Remarkably, the increased SIRT1 deacetylase activity induced by resveratrol was not necessary for this inhibitory effect. Resveratrol significantly blocked PDGF‐stimulated c‐Src and Akt kinase activation, resulting in reduced cyclin D1 expression and attenuated pRb phosphorylation and cyclin‐dependent kinase‐2 (CDK2) activity. Furthermore, resveratrol inhibited PDGFR phosphorylation at the PI 3 kinase and Grb‐2 binding sites tyrosine‐751 and tyrosine‐716, respectively. This deficiency in PDGFR phosphorylation resulted in significant inhibition of PI 3 kinase and Erk1/2 MAPK activity. Interestingly, resveratrol increased the activity of protein tyrosine phosphatase PTP1B, which dephosphorylates PDGF‐stimulated phosphorylation at tyrosine‐751 and tyrosine‐716 on PDGFR with concomitant reduction in Akt and Erk1/2 kinase activity. PTP1B significantly inhibited PDGF‐induced DNA synthesis without inducing apoptosis. These results for the first time provide evidence that the stilbene resveratrol targets PTP1B to inhibit PDGFR mitogenic signaling.—Venkatesan, B., Ghosh‐Choudhury, N., Das, F., Mahimainathan, L., Kamat, A., Kasinath, B. S., Abboud, H. E., Choudhury, G. G. Resveratrol inhibits PDGF receptor mitogenic signaling in mesangial cells: role of PTP1B. FASEB J. 22, 3469–3482 (2008)