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-␤ (TGF-␤). TGF-␤ 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-␤-induced hypertrophy of mesangial cells. Finally, we show that inhibition of TGF-␤ 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-␤ signaling. Diabetes 55: [2115][2116][2117][2118][2119][2120][2121][2122][2123][2124][2125] 2006

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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 GSK3β phosphorylation and β-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.

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.

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-␣ (TNF-␣).

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/JNKdependent 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-κB, AP-1, CREB, and ATF-2 in cardiomyocytes, and induces IL-18 expression via Rac1-dependent PI3K/Akt/IKK/NF-κB and MKK7/JNK/AP-1 signaling. Moreover, EMMPRIN induces robust time-dependent induction of various MMP mRNA. 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.

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.

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 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/GSK3β/β-catenin/TCF/LEF and by Akt-dependent CREB activation. Further, Ang-II also activated CREB via superoxide-mediated p38MAPK and ERK activation. Continuous infusion of Ang-II for 7 days induced myocardial hypertrophy in rats, and was associated with increased Akt, phospho-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.