Sestrin2 regulates monocyte activation through AMPK-mTOR nexus under high-glucose and dyslipidemic conditions.

The vicious cycle between hyperinsulinemia and insulin resistance results in the progression of atherosclerosis in the vessel wall. The complex interaction between hyperglycemia and lipoprotein abnormalities promotes the development of atherogenesis. In the early phase of atherosclerosis, macrophage-derived foam cells play an important role in vascular remodeling. Mechanistic target of rapamycin (mTOR) signaling pathway has been identified to play an essential role in the initiation, progression, and complication of atherosclerosis. Recently sestrin2, an antioxidant, was shown to modulate TOR activity and thereby regulating glucose and lipid metabolism. But the role of sestrin2 in monocyte activation is still not clearly understood. Hence, this study is focussed on investigating the role of sestrin2 in monocyte activation under hyperglycemic and dyslipidemic conditions. High-glucose and oxidized low-density lipoprotein (LDL) treatments mediated proinflammatory cytokine production (M1) with a concomitant decrease in the anti-inflammatory cytokine (M2) levels in human monocytic THP1 cells. Both glucose and oxidized LDL (OxLDL) in a dose and time-dependent manner increased the mTOR activation with a marked reduction in the levels of pAMPK and sestrin2 expression. Both high-glucose and OxLDL treatment increased foam cell formation and adhesion of THP1 cells to endothelial cells. Experiments employing activator or inhibitor of adenosine monophosphate kinase (AMPK) as well as overexpression or silencing of sestrin2 indicated that high-glucose mediated monocyte polarization and adhesion of monocytes to the endothelial cells were appeared to be programmed via sestrin2-AMPK-mTOR nexus. Our results evidently suggest that sestrin2 plays a major role in regulating monocyte activation via the AMPK-mTOR-pathway under diabetic and dyslipidemic conditions and also AMPK regulates sestrin2 in a feedback mechanism.

PDGF receptor-ß uses Akt/mTORC1 signaling node to promote high glucose-induced renal proximal tubular cell collagen I (α2) expression.

Increased expression of PDGF receptor-ß (PDGFRß) has been shown in renal proximal tubules in mice with diabetes. The core molecular network used by high glucose to induce proximal tubular epithelial cell collagen I (α2) expression is poorly understood. We hypothesized that activation of PDGFRß by high glucose increases collagen I (α2) production via the Akt/mTORC1 signaling pathway in proximal tubular epithelial cells. Using biochemical and molecular biological techniques, we investigated this hypothesis. We show that high glucose increases activating phosphorylation of the PDGFRß, resulting in phosphorylation of phosphatidylinositol 3-kinase. A specific inhibitor, JNJ-10198409, and small interfering RNAs targeting PDGFRß blocked this phosphorylation without having any effect on MEK/Erk1/2 activation. We also found that PDGFRß regulates high glucose-induced Akt activation, its targets tuberin and PRAS40 phosphorylation, and finally, mTORC1 activation. Furthermore, inhibition of PDGFRß suppressed high glucose-induced expression of collagen I (α2) in proximal tubular cells. Importantly, expression of constitutively active Akt or mTORC1 reversed these processes. As a mechanism, we found that JNJ and PDGFRß knockdown inhibited high glucose-stimulated Hif1α expression. Furthermore, overexpression of Hif1α restored expression of collagen I (α2) that was inhibited by PDGFRß knockdown in high glucose-stimulated cells. Finally, we show increased phosphorylation of PDGFRß and its association with Akt/mTORC1 activation, Hif1α expression, and elevated collagen I (α2) levels in the renal cortex of mice with diabetes. Our results identify PDGFRß as a driver in activating Akt/mTORC1 nexus for high glucose-mediated expression of collagen I (α2) in proximal tubular epithelial cells, which contributes to tubulointerstitial fibrosis in diabetic nephropathy.

Angiotensin-II type 1 receptor and NOX2 mediate TCF/LEF and CREB dependent WISP1 induction and cardiomyocyte hypertrophy.

Angiotensin-II (Ang-II) plays a key role in myocardial hypertrophy, remodeling and failure. We investigated whether Ang-II-induced cardiomyocyte hypertrophy is dependent on WNT1 inducible signaling pathway protein 1 (WISP1), a pro growth factor. Ang-II induced hypertrophy and WISP1 expression in neonatal rat cardiomyocytes (NRCM), effects that were significantly inhibited by pre-treatment with the AT1 antagonist losartan and by WISP1 knockdown. Further, Ang-II induced WISP1 was superoxide-dependent, and inhibited by DPI, an inhibitor of NADPH oxidases, and by knockdown of NOX2. AT1 was physically associated with NOX2 both in vitro and in vivo, and Ang-II increased this interaction in vivo. Ang-II induced WISP1 expression via superoxide/Akt/GSK3ß/ß-catenin/TCF/LEF and by Akt-dependent CREB activation. Further, Ang-II also activated CREB via superoxide-mediated p38 MAPK and ERK activation. Continuous infusion of Ang-II for 7days induced myocardial hypertrophy in rats, and was associated with increased Akt, p-Akt, p-p38 MAPK, p-ERK1/2, and WISP1 expression. These results demonstrate that Ang-II induced cardiomyocyte hypertrophy is mediated through AT1, NOX2 and the induction of WISP1, and may involve the direct interaction of AT1 with NOX2. Thus targeting both WISP1 and NOX2 may have a therapeutic potential in improving cardiomyocyte survival and growth following myocardial injury and remodeling. This article is part of a Special Issue entitled 'Possible Editorial'.

EMMPRIN activates multiple transcription factors in cardiomyocytes, and induces interleukin-18 expression via Rac1-dependent PI3K/Akt/IKK/NF-kappaB 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.

TSC2 deficiency increases PTEN via HIF1alpha.

Substantial evidence suggests roles of TSC2 and PTEN in the development of cancer predisposition syndromes. Loss of TSC2 results in benign tumors, neurological disorders, and angiomyolipomas. We found that PTEN mRNA and protein levels are elevated in Tsc2(-/-) mouse embryo fibroblasts with concomitant reduction in Akt phosphorylation. Reconstitution of TSC2 in Tsc2(-/-) mouse embryo fibroblasts decreases PTEN levels. Interestingly, increased HIF1alpha activity present in Tsc2 null cells is required for PTEN transcription and protein expression. We identified a canonical hypoxia-responsive element in the PTEN promoter, which regulates the transcription of this tumor suppressor protein in a TSC2-dependent manner. Finally, we demonstrate a positive correlation between expression of HIF1alpha and PTEN in renal angiomyolipomas from TSC patients. Our results reveal a unique function of HIF1alpha in up-regulation of PTEN and provide a new mechanism of reduced Akt phosphorylation in Tsc2 null cells. These data suggest that PTEN may safeguard against developing malignant tumors in patients with TSC deficiency.

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.

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-kappaB 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.

Resveratrol blocks interleukin-18-EMMPRIN cross-regulation and smooth muscle cell migration.

Vascular smooth muscle cell (SMC) migration is an important mechanism in atherogenesis and postangioplasty arterial remodeling. Previously, we demonstrated that the proinflammatory cytokine interleukin (IL)-18 is a potent inducer of SMC migration. Since extracellular matrix metalloproteinase inducer (EMMPRIN) stimulates ECM degradation and facilitates cell migration, we investigated whether IL-18 and EMMPRIN regulate each other's expression, whether their cross talk induces SMC migration, and whether the phytoalexin resveratrol inhibits IL-18-EMMPRIN signaling and SMC migration. Our studies demonstrate that 1) IL-18 induces EMMPRIN mRNA and protein expressions and stimulates EMMPRIN secretion from human aortic SMCs; 2) IL-18 stimulates EMMPRIN expression via oxidative stress and phosphatidylinositol 3-kinase (PI3K)-Akt-ERK signaling; 3) IL-18-stimulated SMC migration is significantly blunted by EMMPRIN knockdown, EMMPRIN function-blocking antibodies, or adenoviral transduction of mutant EMMPRIN; 4) conversely, EMMPRIN stimulates IL-18 expression and secretion via PI3K, Akt, and ERK; and 5) resveratrol attenuates IL-18- and EMMPRIN-mediated PI3K, Akt, and ERK activations; blunts IL-18-mediated oxidative stress; blocks IL-18-EMMPRIN cross-regulation; and inhibits SMC migration. Collectively, our results demonstrate that the coexpression and regulation of IL-18 and EMMPRIN in the vessel wall may amplify the inflammatory cascade and promote atherosclerosis and remodeling. Resveratrol, via its antioxidant and anti-inflammatory properties, has the potential to inhibit the progression of atherosclerosis by blocking IL-18 and EMMPRIN cross-regulation and SMC migration.

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 IC(50) of 10 microM 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.

Raptor-rictor axis in TGFbeta-induced protein synthesis.

Transforming growth factor-beta (TGFbeta) stimulates pathological renal cell hypertrophy for which increased protein synthesis is critical. The mechanism of TGFbeta-induced protein synthesis is not known, but PI 3 kinase-dependent Akt kinase activity is necessary. We investigated the contribution of downstream effectors of Akt in TGFbeta-stimulated protein synthesis. TGFbeta increased inactivating phosphorylation of Akt substrate tuberin in a PI 3 kinase/Akt dependent manner, resulting in activation of mTOR kinase. mTOR activity increased phosphorylation of S6 kinase and the translation repressor 4EBP-1, which were sensitive to inhibition of both PI 3 kinase and Akt. mTOR inhibitor rapamycin and a dominant negative mutant of mTOR suppressed TGFbeta-induced phosphorylation of S6 kinase and 4EBP-1. PI 3 kinase/Akt and mTOR regulated dissociation of 4EBP-1 from eIF4E to make the latter available for binding to eIF4G. mTOR and 4EBP-1 modulated TGFbeta-induced protein synthesis. mTOR is present in two multi protein complexes, mTORC1 and mTORC2. Raptor and rictor are part of mTORC1 and mTORC2, respectively. shRNA-mediated downregulation of raptor inhibited TGFbeta-stimulated mTOR kinase activity, resulting in inhibition of phosphorylation of S6 kinase and 4EBP-1. Raptor shRNA also prevented protein synthesis in response to TGFbeta. Downregulation of rictor inhibited serine 473 phosphorylation of Akt without any effect on phosphorylation of its substrate, tuberin. Furthermore, rictor shRNA increased phosphorylation of S6 kinase and 4EBP-1 in TGFbeta-independent manner, resulting in increased protein synthesis. Thus mTORC1 function is essential for TGFbeta-induced protein synthesis. Our data also provide novel evidence that rictor negatively regulates TORC1 activity to control basal protein synthesis, thus conferring tight control on cellular hypertrophy.

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

Transforming growth factor-beta (TGFbeta) controls expression of plasminogen activator inhibitor type 1 (PAI-1), which regulates degradation of extracellular matrix proteins in fibrotic diseases. The TGFbeta 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 TGFbeta-induced PAI-1 expression in renal mesangial cells. Inhibition of PI 3 kinase and Akt kinase blocked TGFbeta- and Smad 3-mediated expression of PAI-1. In contrast, constitutively active PI 3 kinase and Akt kinase increased PAI-1 expression, similar to TGFbeta. Inhibition of PI 3 kinase and Akt kinase had no effect on TGFbeta-induced Smad 3 phosphorylation and its translocation to the nucleus. Notably, inhibition of PI 3 kinase-dependent Akt kinase abrogated TGFbeta-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 TGFbeta, resulting in inhibition of Smad 3 acetylation. Together our findings identify TGFbeta-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.

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-beta (TGF-beta). TGF-beta 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-beta-induced hypertrophy of mesangial cells. Finally, we show that inhibition of TGF-beta 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-beta signaling.

TGFbeta intercepts nuclear glycogen synthase kinase 3beta to inhibit PDGF-induced DNA synthesis in mesangial cells.

Here, we demonstrate a mechanism of TGFbeta-mediated inhibition of PDGF-induced DNA synthesis in mesangial cells. TGFbeta significantly inhibited nuclear Akt phosphorylation without any effect on PDGF-stimulated phosphorylation of PDGFR at PI 3 kinase binding site (Tyr-751). Remarkably, TGFbeta inhibited cyclin D1 and cyclin E expression with concomitant decrease in CDK2 activity induced by PDGF. More importantly, we demonstrate that TGFbeta significantly abolished Akt-mediated serine-9 phosphorylation of glycogen synthase kinase 3beta (GSK3beta), thus prevented its inactivation. Expression of inactive GSK3betaK85R mutant increased cyclin D1 expression and DNA synthesis similar to PDGF. These results provide the first evidence that TGFbeta intercepts Akt kinase activity in the nucleus to block inactivation of GSK3beta, leading to attenuation of PDGF-induced CDK2 activity and DNA synthesis.

c-Src couples PI 3 kinase/Akt and MAPK signaling to PDGF-induced DNA synthesis in mesangial cells.

Platelet-derived growth factor BB (PDGF) and PDGF receptor-beta (PDGFR) play critical roles in mesangial cell proliferation during embryonic development and in mesangioproliferative glomerulonephritis. We have shown previously that phosphatidylinositol (PI) 3 kinase/Akt and Erk1/2 mitogen-activated protein kinase (MAPK) contribute to PDGF-dependent proliferation of mesangial cells, but the mechanism by which these two enzyme cascades are activated by PDGFR signaling is not precisely known. We examined the role of c-Src tyrosine kinase in this process. PDGF increased phosphorylation of c-Src in a time-dependent manner indicating its activation. A pharmacologic inhibitor of c-Src, PP1, blocked PDGF-induced DNA synthesis with concomitant inhibition of c-Src phosphorylation. Immune-complex kinase assays of c-Src and PDGFR demonstrated inhibition of c-Src tyrosine kinase activity by PP1, without an effect on PDGFR tyrosine phosphorylation. Both PP1 and expression of dominant negative c-Src inhibited PDGF-induced PI 3 kinase, resulting in attenuation of Akt kinase activity. Expression of constitutively active c-Src increased Akt activity to the same extent as with PDGF. Constitutively active c-Src augmented PDGF-induced Akt activity, thus contributing to Akt signaling. Inhibition of c-Src tyrosine kinase blocked PDGF-stimulated MAPK activity and resulted in attenuation of c-fos gene transcription with concomitant prevention of Elk-1 transactivation. Furthermore, inhibition of c-Src increased p27(Kip1) cyclin kinase inhibitor, and attenuated PDGF-induced pRb phosphorylation and CDK2 activity. These data provide the first evidence in mesangial cells that PDGF-activated c-Src tyrosine kinase relays signals to PI 3 kinase/Akt and MAPK. Furthermore our results demonstrate that c-Src integrates signals into the nucleus to activate CDK2, which is required for DNA synthesis.

PI 3 kinase-dependent Akt kinase and PKCepsilon independently regulate interferon-gamma-induced STAT1alpha serine phosphorylation to induce monocyte chemotactic protein-1 expression.

Monocyte chemotactic protein-1 (MCP-1) recruits activated phagocytes to the site of tissue injury. Interferon-gamma (IFN-gamma) present in the microenvironment of glomerulus acts on mesangial cells to induce local production of MCP-1. The mechanism by which IFN-gamma stimulates expression of MCP-1 is not clear. We therefore examined the role of PI 3 kinase signaling in regulating the IFN-gamma-induced MCP-1 expression in mesangial cells. Blocking PI 3 kinase activity with Ly294002 attenuated IFN-gamma-induced MCP-1 protein and mRNA expression. IFN-gamma increased Akt kinase activity in a PI 3 kinase-dependent manner. Expression of dominant negative Akt kinase inhibited serine phosphorylation of STAT1alpha, without any effect on its tyrosine phosphorylation, and decreased IFN-gamma-induced expression of MCP-1. These data for the first time indicate a role for PI 3 kinase-dependent Akt kinase in MCP-1 expression. We have recently shown that along with Akt, PKCepsilon is a downstream target of PI 3 kinase in IFN-gamma signaling. Similar to dominant negative Akt kinase, dominant negative PKCepsilon also inhibited serine phosphorylation of STAT1alpha without any effect on tyrosine phosphorylation. Dominant negative PKCepsilon also abrogated MAPK activity, resulting in decrease in IFN-gamma-induced MCP-1 expression. Furthermore, Akt and PKCepsilon are present together in a signaling complex. IFN-gamma had no effect on this complex formation, but did increase PKCepsilon-associated Akt kinase activity. PKCepsilon did not regulate IFN-gamma-induced Akt kinase. Finally, expression of dominant negative Akt kinase blocked IFN-gamma-stimulated MAPK activation. These data provide the first evidence that PI 3 kinase-dependent Akt and PKCepsilon activation independently regulate MAPK activity and serine phosphorylation of STAT1alpha to increase expression of MCP-1.

Association of circulatory asymmetric dimethylarginine (ADMA) with diabetic nephropathy in Asian Indians and its causative role in renal cell injury.

AIM: Asymmetric dimethylarginine (ADMA) is involved in the regulation of nitric oxide synthesis and in the maintenance of vascular tone and structure. But the role and status of ADMA in diabetes induced kidney injury is not clear. Hence this study is investigating the role of ADMA in the progression of kidney injury and its circulatory status in Asian Indians with and without diabetic nephropathy. METHODS: Recruited study subjects were divided into normal glucose tolerance (NGT), type 2 diabetes mellitus (T2DM) and T2DM with micro or macroalbuminuria. Albuminuria was calculated using urinary albumin and creatinine ratio (UACR). ADMA was measured using ELISA. Kidney cell damage in terms of fibrotic markers and ADMA metabolism in terms of DDAH activity were investigated in kidney fibroblasts and mesangial cells. RESULTS: There was a significant elevation in plasma ADMA levels in micro and macroalbuminuric diabetic patients. We found a significant positive correlation between ADMA and UACR, serum creatinine, HbA1C and fasting plasma glucose. A cut-off value of ADMA, 0.666µM/l had a sensitivity and specificity of 70.0% and 65.6%, respectively for detecting diabetic nephropathy. DDAH activity was significantly decreased and fibrotic markers such as fibronectin and α-SMA were significantly increased upon high glucose and ADMA treatment. CONCLUSION: We are suggesting a causative role of ADMA in the development of kidney injury in terms of renal fibrosis and also a cut point of 0.666µM/l of plasma ADMA level appears to be a predictive risk threshold for diabetic nephropathy in south Asian Indian population.

CIKS (Act1 or TRAF3IP2) mediates high glucose-induced endothelial dysfunction.

Hyperglycemia-induced endothelial dysfunction is characterized by enhanced inflammatory cytokine and adhesion molecule expression, and endothelial-monocyte adhesion. The adapter molecule CIKS (connection to IKK and SAPK/JNK; also known as Act1 or TRAF3IP2) is an upstream regulator of NF-κB and AP-1, and plays a role in inflammation and injury. Here we show that high glucose (HG; 25mM vs. 5mM d-glucose)-induced endothelial-monocyte adhesion and inhibition of endothelial cell (EC) migration were both reversed by CIKS knockdown. In EC, HG induced CIKS mRNA and protein expression via DPI-inhibitable Nox4-dependent ROS generation. Further, HG induced CIKS transcription and enhanced CIKS promoter-dependent reporter gene activation via Nox4, ROS, AP-1 and C/EBP. Coimmunoprecipitation and immunoblotting revealed CIKS/IKKß/JNK physical association under basal conditions that was enhanced by HG treatment. Importantly, CIKS knockdown inhibited HG-induced (i) IKKß and JNK phosphorylation, (ii) p65 and c-Jun nuclear translocation, and (iii) NF-κB- and AP-1-dependent proinflammatory cytokine, chemokine, and adhesion molecule expression. Similar to HG, the deleterious metabolic products of chronic hyperglycemia, AGE-HSA, AOPPs-HSA and oxLDL, also induced CIKS-dependent endothelial dysfunction. Notably, aortas from streptozotocin-induced and the autoimmune type 1 diabetic NOD and Akita mice showed enhanced DPI-inhibitable ROS generation and CIKS expression. Since CIKS mediates high glucose-induced NF-κB and AP-1-dependent inflammatory signaling and endothelial dysfunction, targeting CIKS may delay progression of vascular diseases during diabetes mellitus and atherosclerosis.

High glucose upregulation of early-onset Parkinson's disease protein DJ-1 integrates the PRAS40/TORC1 axis to mesangial cell hypertrophy.

The Akt kinase signaling pathway is frequently deregulated in many human diseases including cancer, autoimmune disease and diabetes. In nephropathy, associated with diabetes, increased Akt signal transduction results in glomerular especially mesangial cell hypertrophy. The mechanism of Akt activation by elevated glucose is poorly understood. The oncogene DJ-1 prevents oxidative damage and apoptosis of dopaminergic neurons in animal models of Parkinson's disease and in culture. We identified DJ-1 to increase in response to high glucose in renal glomerular mesangial cells concomitant with an increase in phosphorylation of Akt in a time-dependent manner. Plasmid-derived overexpression as well as downregulation of DJ-1 by siRNA showed the requirement of this protein in high glucose-stimulated Akt phosphorylation. The tumor suppressor protein PTEN acts as a negative regulator of Akt activation. Interestingly, DJ-1 was associated with PTEN and this interaction was significantly increased in response to high glucose. High glucose-induced increase in DJ-1 promoted phosphorylation of the PRAS40, a negative regulator of TORC1 kinase activity, resulting in activating and inactivating phosphorylation of S6 kinase and 4EBP-1, respectively. Furthermore, DJ-1 increased protein synthesis and hypertrophy of mesangial cells. Our results provide evidence for a unique mechanism whereby DJ-1 induces Akt/PRAS40/TORC1-mediated hypertrophy in response to high glucose.

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-alpha (TNF-alpha)-stimulated cardiac fibroblast proliferation but inhibits TNF-alpha-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-alpha (TNF-alpha). Here we investigated (i) the localization of TNF-alpha and WISP1 in post-infarct heart, (ii) the mechanism of TNF-alpha-mediated WISP1 induction in primary human cardiac fibroblasts (CF), (iii) the role of WISP1 in TNF-alpha-mediated CF proliferation and collagen production, and (iv) the effects of WISP1 on TNF-alpha-mediated cardiomyocyte death. TNF-alpha and WISP1 expressions were increased in the border zones and non-ischemic remote regions of the post-ischemic heart. In CF, TNF-alpha potently induced WISP1 expression in cyclic AMP response element-binding protein (CREB)-dependent manner. TNF-alpha induced CREB phosphorylation in vitro and DNA binding and reporter gene activities in vivo. TNF-alpha induced CREB activation via ERK1/2, and inhibition of ERK1/2 and CREB blunted TNF-alpha-mediated WISP1 induction. Most importantly, WISP1 knockdown attenuated TNF-alpha stimulated collagen production and CF proliferation. Furthermore, WISP1 attenuated TNF-alpha-mediated cardiomyocyte death, thus demonstrating pro-mitogenic and pro-survival effects for WISP1 in myocardial constituent cells. Our results suggest that a TNF-alpha/WISP1 signaling pathway may contribute to post-infarct cardiac remodeling, a condition characterized by fibrosis and progressive cardiomyocyte loss.