High glucose-induced downregulation of PTEN-Long is sufficient for proximal tubular cell injury in diabetic kidney disease.

During the progression of diabetic kidney disease, proximal tubular epithelial cells respond to high glucose to induce hypertrophy and matrix expansion leading to renal fibrosis. Recently, a non-canonical PTEN has been shown to be translated from an upstream initiation codon CUG (leucine) to produce a longer protein called PTEN-Long (PTEN-L). Interestingly, the extended sequence present in PTEN-L contains cell secretion/penetration signal. Role of this non-canonical PTEN-L in diabetic renal tubular injury is not known. We show that high glucose decreases expression of PTEN-L. As a mechanism of its function, we find that reduced PTEN-L activates Akt-2, which phosphorylates and inactivate tuberin and PRAS40, resulting in activation of mTORC1 in tubular cells. Antibacterial agent acriflavine and antiviral agent ATA regulate translation from CUG codon. Acriflavine and ATA, respectively, decreased and increased expression of PTEN-L to altering Akt-2 and mTORC1 activation in the absence of change in expression of canonical PTEN. Consequently, acriflavine and ATA modulated high glucose-induced tubular cell hypertrophy and lamininγ1 expression. Importantly, expression of PTEN-L inhibited high glucose-stimulated Akt/mTORC1 activity to abrogate these processes. Since PTEN-L contains secretion/penetration signals, addition of conditioned medium containing PTEN-L blocked Akt-2/mTORC1 activity. Notably, in renal cortex of diabetic mice, we found reduced PTEN-L concomitant with Akt-2/mTORC1 activation, leading to renal hypertrophy and lamininγ1 expression. These results present first evidence for involvement of PTEN-L in diabetic kidney disease.

Oncoprotein DJ-1 interacts with mTOR complexes to effect transcription factor Hif1α-dependent expression of collagen I (α2) during renal fibrosis.

Proximal tubular epithelial cells respond to transforming growth factor ß (TGFß) to synthesize collagen I (α2) during renal fibrosis. The oncoprotein DJ-1 has previously been shown to promote tumorigenesis and prevent apoptosis of dopaminergic neurons; however, its role in fibrosis signaling is unclear. Here, we show TGFß-stimulation increased expression of DJ-1, which promoted noncanonical mTORC1 and mTORC2 activities. We show DJ-1 augmented the phosphorylation/activation of PKCßII, a direct substrate of mTORC2. In addition, coimmunoprecipitation experiments revealed association of DJ-1 with Raptor and Rictor, exclusive subunits of mTORC1 and mTORC2, respectively, as well as with mTOR kinase. Interestingly, siRNAs against DJ-1 blocked TGFß-stimulated expression of collagen I (α2), while expression of DJ-1 increased expression of this protein. In addition, expression of dominant negative PKCßII and siRNAs against PKCßII significantly inhibited TGFß-induced collagen I (α2) expression. In fact, constitutively active PKCßII abrogated the effect of siRNAs against DJ-1, suggesting a role of PKCßII downstream of this oncoprotein. Moreover, we demonstrate expression of collagen I (α2) stimulated by DJ-1 and its target PKCßII is dependent on the transcription factor hypoxia-inducible factor 1α (Hif1α). Finally, we show in the renal cortex of diabetic rats that increased TGFß was associated with enhanced expression of DJ-1 and activation of mTOR and PKCßII, concomitant with increased Hif1α and collagen I (α2). Overall, we identified that DJ-1 affects TGFß-induced expression of collagen I (α2) via an mTOR-, PKCßII-, and Hif1α-dependent mechanism to regulate renal fibrosis.

TGFß acts through PDGFRß to activate mTORC1 via the Akt/PRAS40 axis and causes glomerular mesangial cell hypertrophy and matrix protein expression.

Interaction of transforming growth factor-ß (TGFß)-induced canonical signaling with the noncanonical kinase cascades regulates glomerular hypertrophy and matrix protein deposition, which are early features of glomerulosclerosis. However, the specific target downstream of the TGFß receptor involved in the noncanonical signaling is unknown. Here, we show that TGFß increased the catalytic loop phosphorylation of platelet-derived growth factor receptor ß (PDGFRß), a receptor tyrosine kinase expressed abundantly in glomerular mesangial cells. TGFß increased phosphorylation of the PI 3-kinase-interacting Tyr-751 residue of PDGFRß, thus activating Akt. Inhibition of PDGFRß using a pharmacological inhibitor and siRNAs blocked TGFß-stimulated phosphorylation of proline-rich Akt substrate of 40 kDa (PRAS40), an intrinsic inhibitory component of mTORC1, and prevented activation of mTORC1 in the absence of any effect on Smad 2/3 phosphorylation. Expression of constitutively active myristoylated Akt reversed the siPDGFRß-mediated inhibition of mTORC1 activity; however, co-expression of the phospho-deficient mutant of PRAS40 inhibited the effect of myristoylated Akt, suggesting a definitive role of PRAS40 phosphorylation in mTORC1 activation downstream of PDGFRß in mesangial cells. Additionally, we demonstrate that PDGFRß-initiated phosphorylation of PRAS40 is required for TGFß-induced mesangial cell hypertrophy and fibronectin and collagen I (α2) production. Increased activating phosphorylation of PDGFRß is also associated with enhanced TGFß expression and mTORC1 activation in the kidney cortex and glomeruli of diabetic mice and rats, respectively. Thus, pursuing TGFß noncanonical signaling, we identified how TGFß receptor I achieves mTORC1 activation through PDGFRß-mediated Akt/PRAS40 phosphorylation to spur mesangial cell hypertrophy and matrix protein accumulation. These findings provide support for targeting PDGFRß in TGFß-driven renal fibrosis.

ß2-Adrenergic receptor agonist induced hepatic steatosis in mice: modeling nonalcoholic fatty liver disease in hyperadrenergic states.

Nonalcoholic fatty liver disease (NAFLD) is a spectrum of disorders ranging from hepatic steatosis [excessive accumulation of triglycerides (TG)] to nonalcoholic steatohepatitis, which can progress to cirrhosis and hepatocellular carcinoma. The molecular pathogenesis of steatosis and progression to more severe NAFLD remains unclear. Obesity and aging, two principal risk factors for NAFLD, are associated with a hyperadrenergic state. ß-Adrenergic responsiveness in liver increases in animal models of obesity and aging, and in both is linked to increased hepatic expression of ß2-adrenergic receptors (ß2-ARs). We previously showed that in aging rodents intracellular signaling from elevated hepatic levels of ß2-ARs may contribute to liver steatosis. In this study we demonstrate that injection of formoterol, a highly selective ß2-AR agonist, to mice acutely results in hepatic TG accumulation. Further, we have sought to define the intrahepatic mechanisms underlying ß2-AR mediated steatosis by investigating changes in hepatic expression and cellular localization of enzymes, transcription factors, and coactivators involved in processes of lipid accrual and disposition-and also functional aspects thereof-in livers of formoterol-treated animals. Our results suggest that ß2-AR activation by formoterol leads to increased hepatic TG synthesis and de novo lipogenesis, increased but incomplete ß-oxidation of fatty acids with accumulation of potentially toxic long-chain acylcarnitine intermediates, and reduced TG secretion-all previously invoked as contributors to fatty liver disease. Experiments are ongoing to determine whether sustained activation of hepatic ß2-AR signaling by formoterol might be utilized to model fatty liver changes occurring in hyperadrenergic states of obesity and aging, and thereby identify novel molecular targets for the prevention or treatment of NAFLD.NEW & NOTEWORTHY Results of our study suggest that ß2-adrenergic receptor (ß2-AR) activation by agonist formoterol leads to increased hepatic TG synthesis and de novo lipogenesis, incomplete ß-oxidation of fatty acids with accumulation of long-chain acylcarnitine intermediates, and reduced TG secretion. These findings may, for the first time, implicate a role for ß2-AR responsive dysregulation of hepatic lipid metabolism in the pathogenetic processes underlying NAFLD in hyperadrenergic states such as obesity and aging.

Deacetylation of S6 kinase promotes high glucose-induced glomerular mesangial cell hypertrophy and matrix protein accumulation.

S6 kinase acts as a driver for renal hypertrophy and matrix accumulation, two key pathologic signatures of diabetic nephropathy. As a post-translational modification, S6 kinase undergoes acetylation at the C terminus. The role of this acetylation to regulate kidney glomerular cell hypertrophy and matrix expansion is not known. In mesangial cells, high glucose decreased the acetylation and enhanced phosphorylation of S6 kinase and its substrates rps6 and eEF2 kinase that lead to dephosphorylation of eEF2. To determine the mechanism of S6 kinase deacetylation, we found that trichostatin A, a pan-histone deacetylase (HDAC) inhibitor, blocked all high glucose-induced effects. Furthermore, high glucose increased the expression and association of HDAC1 with S6 kinase. HDAC1 decreased the acetylation of S6 kinase and mimicked the effects of high glucose, resulting in mesangial cell hypertrophy and expression of fibronectin and collagen I (α2). In contrast, siRNA against HDAC1 inhibited these effects by high glucose. A C-terminal acetylation-mimetic mutant of S6 kinase suppressed high glucose-stimulated phosphorylation of S6 kinase, rps6 and eEF2 kinase, and inhibited the dephosphorylation of eEF2. Also, the acetylation mimetic attenuated the mesangial cell hypertrophy and fibronectin and collagen I (α2) expression. Conversely, an S6 kinase acetylation-deficient mutant induced all the above effects of high glucose. Finally, in the renal glomeruli of diabetic rats, the acetylation of S6 kinase was significantly reduced concomitant with increased HDAC1 and S6 kinase activity. In aggregate, our data uncovered a previously unrecognized role of S6 kinase deacetylation in high glucose-induced mesangial cell hypertrophy and matrix protein expression.

microRNA-181a downregulates deptor for TGFß-induced glomerular mesangial cell hypertrophy and matrix protein expression.

TGFß contributes to mesangial cell hypertrophy and matrix protein increase in various kidney diseases including diabetic nephropathy. Deptor is an mTOR-interacting protein and suppresses mTORC1 and mTORC2 activities. We have recently shown that TGFß-induced inhibition of deptor increases the mTOR activity. The mechanism by which TGFß regulates deptor expression is not known. Here we identify deptor as a target of the microRNA-181a. We show that in mesangial cells, TGFß increases the expression of miR-181a to downregulate deptor. Decrease in deptor augments mTORC2 activity, resulting in phosphorylation/activation of Akt kinase. Akt promotes inactivating phosphorylation of PRAS40 and tuberin, leading to stimulation of mTORC1. miR-181a-mimic increased mTORC1 and C2 activities, while anti-miR-181a inhibited them. mTORC1 controls protein synthesis via phosphorylation of translation initiation and elongation suppressors 4EBP-1 and eEF2 kinase. TGFß-stimulated miR-181a increased the phosphorylation of 4EBP-1 and eEF2 kinase, resulting in their inactivation. miR-181a-dependent inactivation of eEF2 kinase caused dephosphorylation of eEF2. Consequently, miR-181a-mimic increased protein synthesis and hypertrophy of mesangial cells similar to TGFß. Anti-miR-181a blocked these events in a deptor-dependent manner. Finally, TGFß-miR-181a-driven deptor downregulation increased the expression of fibronectin. Our results identify a novel mechanism involving miR-181a-driven deptor downregulation, which contributes to mesangial cell pathologies in renal complications.

Reciprocal regulation of miR-214 and PTEN by high glucose regulates renal glomerular mesangial and proximal tubular epithelial cell hypertrophy and matrix expansion.

Aberrant expression of microRNAs (miRs) contributes to diabetic renal complications, including renal hypertrophy and matrix protein accumulation. Reduced expression of phosphatase and tensin homolog (PTEN) by hyperglycemia contributes to these processes. We considered involvement of miR in the downregulation of PTEN. In the renal cortex of type 1 diabetic mice, we detected increased expression of miR-214 in association with decreased levels of PTEN and enhanced Akt phosphorylation and fibronectin expression. Mesangial and proximal tubular epithelial cells exposed to high glucose showed augmented expression of miR-214. Mutagenesis studies using 3'-UTR of PTEN in a reporter construct revealed PTEN as a direct target of miR-214, which controls its expression in both of these cells. Overexpression of miR-214 decreased the levels of PTEN and increased Akt activity similar to high glucose and lead to phosphorylation of its substrates glycogen synthase kinase-3ß, PRAS40, and tuberin. In contrast, quenching of miR-214 inhibited high-glucose-induced Akt activation and its substrate phosphorylation; these changes were reversed by small interfering RNAs against PTEN. Importantly, respective expression of miR-214 or anti-miR-214 increased or decreased the mammalian target of rapamycin complex 1 (mTORC1) activity induced by high glucose. Furthermore, mTORC1 activity was controlled by miR-214-targeted PTEN via Akt activation. In addition, neutralization of high-glucose-stimulated miR-214 expression significantly inhibited cell hypertrophy and expression of the matrix protein fibronectin. Finally, the anti-miR-214-induced inhibition of these processes was reversed by the expression of constitutively active Akt kinase and hyperactive mTORC1. These results uncover a significant role of miR-214 in the activation of mTORC1 that contributes to high-glucose-induced mesangial and proximal tubular cell hypertrophy and fibronectin expression.

Bone Morphogenetic Protein-2 (BMP-2) Activates NFATc1 Transcription Factor via an Autoregulatory Loop Involving Smad/Akt/Ca2+ Signaling.

Bone remodeling is controlled by dual actions of osteoclasts (OCs) and osteoblasts (OBs). The calcium-sensitive nuclear factor of activated T cells (NFAT) c1 transcription factor, as an OC signature gene, regulates differentiation of OCs downstream of bone morphogenetic protein-2 (BMP-2)-stimulated osteoblast-coded factors. To analyze a functional link between BMP-2 and NFATc1, we analyzed bones from OB-specific BMP-2 knock-out mice for NFATc1 expression by immunohistochemical staining and found significant reduction in NFATc1 expression. This indicated a requirement of BMP-2 for NFATc1 expression in OBs. We showed that BMP-2, via the receptor-specific Smad pathway, regulates expression of NFATc1 in OBs. Phosphatidylinositol 3-kinase/Akt signaling acting downstream of BMP-2 also drives NFATc1 expression and transcriptional activation. Under the basal condition, NFATc1 is phosphorylated. Activation of NFAT requires dephosphorylation by the calcium-dependent serine/threonine phosphatase calcineurin. We examined the role of calcium in BMP-2-stimulated regulation of NFATc1 in osteoblasts. 1,2Bis(2aminophenoxy)ethaneN,N,N',N'-tetraacetic acid acetoxymethyl ester, an inhibitor of intracellular calcium abundance, blocked BMP-2-induced transcription of NFATc1. Interestingly, BMP-2 induced calcium release from intracellular stores and increased calcineurin phosphatase activity, resulting in NFATc1 nuclear translocation. Cyclosporin A, which inhibits calcineurin upstream of NFATc1, blocked BMP-2-induced NFATc1 mRNA and protein expression. Expression of NFATc1 directly increased its transcription and VIVIT peptide, an inhibitor of NFATc1, suppressed BMP-2-stimulated NFATc1 transcription, confirming its autoregulation. Together, these data show a role of NFATc1 downstream of BMP-2 in mouse bone development and provide novel evidence for the presence of a cross-talk among Smad, phosphatidylinositol 3-kinase/Akt, and Ca(2+) signaling for BMP-2-induced NFATc1 expression through an autoregulatory loop.

MicroRNA-214 Reduces Insulin-like Growth Factor-1 (IGF-1) Receptor Expression and Downstream mTORC1 Signaling in Renal Carcinoma Cells.

Elevated IGF-1/insulin-like growth factor-1 receptor (IGF-1R) autocrine/paracrine signaling in patients with renal cell carcinoma is associated with poor prognosis of the disease independent of their von Hippel-Lindau (VHL) status. Increased expression of IGF-1R in renal cancer cells correlates with their potency of tumor development and progression. The mechanism by which expression of IGF-1R is increased in renal carcinoma is not known. We report that VHL-deficient and VHL-positive renal cancer cells possess significantly decreased levels of mature, pre-, and pri-miR-214 than normal proximal tubular epithelial cells. We identified an miR-214 recognition element in the 3'UTR of IGF-1R mRNA and confirmed its responsiveness to miR-214. Overexpression of miR-214 decreased the IGF-1R protein levels, resulting in the inhibition of Akt kinase activity in both types of renal cancer cells. IGF-1 provoked phosphorylation and inactivation of PRAS40 in an Akt-dependent manner, leading to the activation of mTORC1 signal transduction to increase phosphorylation of S6 kinase and 4EBP-1. Phosphorylation-deficient mutants of PRAS40 and 4EBP-1 significantly inhibited IGF-1R-driven proliferation of renal cancer cells. Expression of miR-214 suppressed IGF-1R-induced phosphorylation of PRAS40, S6 kinase, and 4EBP-1, indicating inhibition of mTORC1 activity. Finally, miR-214 significantly blocked IGF-1R-forced renal cancer cell proliferation, which was reversed by expression of 3'UTR-less IGF-1R and constitutively active mTORC1. Together, our results identify a reciprocal regulation of IGF-1R levels and miR-214 expression in renal cancer cells independent of VHL status. Our data provide evidence for a novel mechanism for IGF-1R-driven renal cancer cell proliferation involving miR-214 and mTORC1.

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.

Hydrophobic motif site-phosphorylated protein kinase CßII between mTORC2 and Akt regulates high glucose-induced mesangial cell hypertrophy.

PKCßII controls the pathologic features of diabetic nephropathy, including glomerular mesangial cell hypertrophy. PKCßII contains the COOH-terminal hydrophobic motif site Ser-660. Whether this hydrophobic motif phosphorylation contributes to high glucose-induced mesangial cell hypertrophy has not been determined. Here we show that, in mesangial cells, high glucose increased phosphorylation of PKCßII at Ser-660 in a phosphatidylinositol 3-kinase (PI3-kinase)-dependent manner. Using siRNAs to downregulate PKCßII, dominant negative PKCßII, and PKCßII hydrophobic motif phosphorylation-deficient mutant, we found that PKCßII regulates activation of mechanistic target of rapamycin complex 1 (mTORC1) and mesangial cell hypertrophy by high glucose. PKCßII via its phosphorylation at Ser-660 regulated phosphorylation of Akt at both catalytic loop and hydrophobic motif sites, resulting in phosphorylation and inactivation of its substrate PRAS40. Specific inhibition of mTORC2 increased mTORC1 activity and induced mesangial cell hypertrophy. In contrast, inhibition of mTORC2 decreased the phosphorylation of PKCßII and Akt, leading to inhibition of PRAS40 phosphorylation and mTORC1 activity and prevented mesangial cell hypertrophy in response to high glucose; expression of constitutively active Akt or mTORC1 restored mesangial cell hypertrophy. Moreover, constitutively active PKCßII reversed the inhibition of high glucose-stimulated Akt phosphorylation and mesangial cell hypertrophy induced by suppression of mTORC2. Finally, using renal cortexes from type 1 diabetic mice, we found that increased phosphorylation of PKCßII at Ser-660 was associated with enhanced Akt phosphorylation and mTORC1 activation. Collectively, our findings identify a signaling route connecting PI3-kinase to mTORC2 to phosphorylate PKCßII at the hydrophobic motif site necessary for Akt phosphorylation and mTORC1 activation, leading to mesangial cell hypertrophy.

High glucose forces a positive feedback loop connecting Akt kinase and FoxO1 transcription factor to activate mTORC1 kinase for mesangial cell hypertrophy and matrix protein expression.

High glucose-induced Akt acts as a signaling hub for mesangial cell hypertrophy and matrix expansion, which are recognized as cardinal signatures for the development of diabetic nephropathy. How mesangial cells sustain the activated state of Akt is not clearly understood. Here we show Akt-dependent phosphorylation of the transcription factor FoxO1 by high glucose. Phosphorylation-deficient, constitutively active FoxO1 inhibited the high glucose-induced phosphorylation of Akt to suppress the phosphorylation/inactivation of PRAS40 and mTORC1 activity. In contrast, dominant negative FoxO1 increased the phosphorylation of Akt, resulting in increased mTORC1 activity similar to high glucose treatment. Notably, FoxO1 regulates high glucose-induced protein synthesis, hypertrophy, and expression of fibronectin and PAI-1. High glucose paves the way for complications of diabetic nephropathy through the production of reactive oxygen species (ROS). We considered whether the FoxO1 target antioxidant enzyme catalase contributes to sustained activation of Akt. High glucose-inactivated FoxO1 decreases the expression of catalase to increase the production of ROS. Moreover, we show that catalase blocks high glucose-stimulated Akt phosphorylation to attenuate the inactivation of FoxO1 and PRAS40, resulting in the inhibition of mTORC1 and mesangial cell hypertrophy and fibronectin and PAI-1 expression. Finally, using kidney cortices from type 1 diabetic OVE26 mice, we show that increased FoxO1 phosphorylation is associated with decreased catalase expression and increased fibronectin and PAI-1 expression. Together, our results provide the first evidence for the presence of a positive feedback loop for the sustained activation of Akt involving inactivated FoxO1 and a decrease in catalase expression, leading to increased ROS and mesangial cell hypertrophy and matrix protein expression.

microRNA-21-induced dissociation of PDCD4 from rictor contributes to Akt-IKKß-mTORC1 axis to regulate renal cancer cell invasion.

Renal cancer metastasis may result from oncogenic forces that contribute to the primary tumor. We have recently identified microRNA-21 as an oncogenic driver of renal cancer cells. The mechanism by which miR-21 controls renal cancer cell invasion is poorly understood. We show that miR-21 directly downregulates the proapoptotic protein PDCD4 to increase migration and invasion of ACHN and 786-O renal cancer cells as a result of phosphorylation/activation of Akt and IKKß, which activate NFκB-dependent transcription. Constitutively active (CA) Akt or CA IKKß blocks PDCD4-mediated inhibition and restores renal cancer cell migration and invasion. PDCD4 inhibits mTORC1 activity, which was reversed by CA IKKß. Moreover, CA mTORC1 restores cell migration and invasion inhibited by PDCD4 and dominant negative IKKß. Moreover, PDCD4 negatively regulates mTORC2-dependent Akt phosphorylation upstream of this cascade. We show that PDCD4 forms a complex with rictor, an exclusive component of mTORC2, and that this complex formation is reduced in renal cancer cells due to increased miR-21 expression resulting in enhanced phosphorylation of Akt. Thus our results identify a previously unrecognized signaling node where high miR-21 levels reduce rictor-PDCD4 interaction to increase phosphorylation of Akt and contribute to metastatic fitness of renal cancer cells.

A positive feedback loop involving Erk5 and Akt turns on mesangial cell proliferation in response to PDGF.

Platelet-derived growth factor BB and its receptor (PDGFRß) play a pivotal role in the development of renal glomerular mesangial cells. Their roles in increased mesangial cell proliferation during mesangioproliferative glomerulonephritis have long been noted, but the operating logic of signaling mechanisms regulating these changes remains poorly understood. We examined the role of a recently identified MAPK, Erk5, in this process. PDGF increased the activating phosphorylation of Erk5 and tyrosine phosphorylation of proteins in a time-dependent manner. A pharmacologic inhibitor of Erk5, XMD8-92, abrogated PDGF-induced DNA synthesis and mesangial cell proliferation. Similarly, expression of dominant negative Erk5 or siRNAs against Erk5 blocked PDGF-stimulated DNA synthesis and proliferation. Inhibition of Erk5 attenuated expression of cyclin D1 mRNA and protein, resulting in suppression of CDK4-mediated phosphorylation of the tumor suppressor protein pRb. Expression of cyclin D1 or CDK4 prevented the dominant negative Erk5- or siErk5-mediated inhibition of DNA synthesis and mesangial cell proliferation induced by PDGF. We have previously shown that phosphatidylinositol 3-kinase (PI3-kinase) contributes to PDGF-induced proliferation of mesangial cells. Inhibition of PI3-kinase blocked PDGF-induced phosphorylation of Erk5. Since PI3-kinase acts through Akt, we determined the role of Erk5 on Akt phosphorylation. XMD8-92, dominant negative Erk5, and siErk5 inhibited phosphorylation of Akt by PDGF. Interestingly, we found inhibition of PDGF-induced Erk5 phosphorylation by a pharmacological inhibitor of Akt kinase and kinase dead Akt in mesangial cells. Thus our data unfold the presence of a positive feedback microcircuit between Erk5 and Akt downstream of PI3-kinase nodal point for PDGF-induced mesangial cell proliferation.

c-Abl-dependent molecular circuitry involving Smad5 and phosphatidylinositol 3-kinase regulates bone morphogenetic protein-2-induced osteogenesis.

Skeletal remodeling consists of timely formation and resorption of bone by osteoblasts and osteoclasts in a quantitative manner. Patients with chronic myeloid leukemia receiving inhibitors of c-Abl tyrosine kinase often show reduced bone remodeling due to impaired osteoblast and osteoclast function. BMP-2 plays a significant role in bone generation and resorption by contributing to the formation of mature osteoblasts and osteoclasts. The effects of c-Abl on BMP-2-induced bone remodeling and the underlying mechanisms are not well studied. Using a pharmacological inhibitor and expression of a dominant negative mutant of c-Abl, we show an essential role of this tyrosine kinase in the development of bone nodules containing mature osteoblasts and formation of multinucleated osteoclasts in response to BMP-2. Calvarial osteoblasts prepared from c-Abl null mice showed the absolute requirement of this tyrosine kinase in maturation of osteoblasts and osteoclasts. Activation of phosphatidylinositol 3-kinase (PI 3-kinase)/Akt signaling by BMP-2 leads to osteoblast differentiation. Remarkably, inhibition of c-Abl significantly suppressed BMP-2-stimulated PI 3-kinase activity and its downstream Akt phosphorylation. Interestingly, c-Abl regulated BMP-2-induced osteoclastogenic CSF-1 expression. More importantly, we identified the requirements of c-Abl in BMP-2 autoregulation and the expressions of alkaline phosphatase and osterix that are necessary for osteoblast differentiation. c-Abl contributed to BMP receptor-specific Smad-dependent transcription of CSF-1, osterix, and BMP-2. Finally, c-Abl associates with BMP receptor IA and regulates phosphorylation of Smad in response to BMP-2. We propose that activation of c-Abl is an important step, which induces into two signaling pathways involving noncanonical PI 3-kinase and canonical Smads to integrate BMP-2-induced osteogenesis.

Transforming growth factor ß integrates Smad 3 to mechanistic target of rapamycin complexes to arrest deptor abundance for glomerular mesangial cell hypertrophy.

In many renal diseases, transforming growth factor ß (TGFß)-stimulated canonical Smad 3 and noncanonical mechanistic target of rapamycin (mTOR) promote increased protein synthesis and mesangial cell hypertrophy. The cellular underpinnings involving these signaling molecules to regulate mesangial cell hypertrophy are not fully understood. Deptor has recently been identified as an mTOR interacting protein and functions as an endogenous inhibitor of the kinase activity for both TORC1 and TORC2. Prolonged incubation of mesangial cells with TGFß reduced the levels of deptor concomitant with an increase in TORC1 and TORC2 activity. Sustained TGFß activation was required to inhibit association of deptor with mTOR, whereas rapid activation had no effect. Using the mTOR inhibitor PP242, we found that TGFß-induced both early and sustained activation of TORC1 and TORC2 was necessary for deptor suppression. PP242-induced reversal of deptor suppression by TGFß was associated with a significant inhibition of TGFß-stimulated protein synthesis and hypertrophy. Interestingly, expression of siRNA against Smad 3 or Smad 7, which blocks TGFß receptor-specific Smad 3 signaling, prevented TGFß-induced suppression of deptor abundance and TORC1/2 activities. Furthermore, overexpression of Smad 3 decreased deptor expression similar to TGFß stimulation concomitant with increased TORC1 and TORC2 activities. Finally, knockdown of deptor reversed Smad 7-mediated inhibition of protein synthesis and mesangial cell hypertrophy induced by TGFß. These data reveal the requirement of both early and late activation of mTOR for TGFß-induced protein synthesis. Our results support that TGFß-stimulated Smad 3 acts as a key node to instill a feedback loop between deptor down-regulation and TORC1/2 activation in driving mesangial cell hypertrophy.

Unrestrained mammalian target of rapamycin complexes 1 and 2 increase expression of phosphatase and tensin homolog deleted on chromosome 10 to regulate phosphorylation of Akt kinase.

Tuberous sclerosis complex 2 (TSC2) and phosphatase and tensin homolog deleted on chromosome 10 (PTEN) function to block growth factor-induced mammalian target of rapamycin (mTOR) signaling and are mutated in autosomal dominant hamartoma syndromes. mTOR binds to a spectrum of common and different proteins to form TOR complex 1 (TORC1) and TORC2, which regulate cell growth, division, and metabolism. TSC2 deficiency induces constitutive activation of mTOR, leading to a state of insulin resistance due to a negative feedback regulation, resulting in reduced Akt phosphorylation. We have recently described an alternative mechanism showing that in TSC2 deficiency, enhanced PTEN expression contributes to reduced Akt phosphorylation. To explore the mechanism of PTEN regulation, we used rapamycin and constitutively active mTOR to show that TORC1 increases the expression of PTEN mRNA and protein. We found that in TSC2(-/-) mouse embryonic fibroblasts expression of a kinase-dead mutant of mTOR, which inhibits both TORC1 and TORC2, decreases the expression of PTEN via transcriptional mechanism. Furthermore, kinase-dead mTOR increased and decreased phosphorylation of Akt at catalytic loop site Thr-308 and hydrophobic motif site Ser-473, respectively. Moreover, inhibition of deregulated TORC1 in TSC2-null mouse embryonic fibroblasts or in 293 cells by down-regulation of raptor decreased the levels of the transcription factor Hif1α and blocked PTEN expression, resulting in enhanced phosphorylation of Akt at Thr-308 and Ser-473. Finally, knockdown of rictor or mSin1 attenuated the expression of Hif1α, which decreased transcription of PTEN. These results unravel a previously unrecognized cell-autonomous function of TORC1 and TORC2 in the up-regulation of PTEN, which prevents phosphorylation of Akt and may shield against the development of malignancy in TSC patients.

TGFß-induced PI 3 kinase-dependent Mnk-1 activation is necessary for Ser-209 phosphorylation of eIF4E and mesangial cell hypertrophy.

Transforming growth factorß (TGFß)-induced canonical signal transduction is involved in glomerular mesangial cell hypertrophy; however, the role played by the noncanonical TGFß signaling remains largely unexplored. TGFß time-dependently stimulated eIF4E phosphorylation at Ser-209 concomitant with enhanced phosphorylation of Erk1/2 (extracellular signal regulated kinase1/2) and MEK (mitogen-activated and extracellular signal-regulated kinase kinase) in mesangial cells. Inhibition of Erk1/2 by MEK inhibitor or by expression of dominant negative Erk2 blocked eIF4E phosphorylation, resulting in attenuation of TGFß-induced protein synthesis and mesangial cell hypertrophy. Expression of constitutively active (CA) MEK was sufficient to induce protein synthesis and hypertrophy similar to those induced by TGFß. Pharmacological or dominant negative inhibition of phosphatidylinositol (PI) 3 kinase decreased MEK/Erk1/2 phosphorylation leading to suppression of eIF4E phosphorylation. Inducible phosphorylation of eIF4E at Ser-209 is mediated by Mnk-1 (mitogen-activated protein kinase signal-integrating kinase-1). Both PI 3 kinase and Erk1/2 promoted phosphorylation of Mnk-1 in response to TGFß. Dominant negative Mnk-1 significantly inhibited TGFß-stimulated protein synthesis and hypertrophy. Interestingly, inhibition of mTORC1 activity, which blocks dissociation of eIF4E-4EBP-1 complex, decreased TGFß-stimulated phosphorylation of eIF4E without any effect on Mnk-1 phosphorylation. Furthermore, mutant eIF4E S209D, which mimics phosphorylated eIF4E, promoted protein synthesis and hypertrophy similar to TGFß. These results were confirmed using phosphorylation deficient mutant of eIF4E. Together our results highlight a significant role of dissociation of 4EBP-1-eIF4E complex for Mnk-1-mediated phosphorylation of eIF4E. Moreover, we conclude that TGFß-induced noncanonical signaling circuit involving PI 3 kinase-dependent Mnk-1-mediated phosphorylation of eIF4E at Ser-209 is required to facilitate mesangial cell hypertrophy.

TGFß instructs mTORC2 to activate PKCßII for increased TWIST1 expression in proximal tubular epithelial cell injury.

The plasticity of proximal tubular epithelial cells in response to TGFß contributes to the expression of TWIST1 to drive renal fibrosis. The mechanism of TWIST1 expression is not known. We show that both PI3 kinase and its target mTORC2 increase TGFß-induced TWIST1 expression. TGFß enhances phosphorylation on Ser-660 in the protein kinase C ßII (PKCßII) hydrophobic motif site. Remarkably, phosphorylation-deficient PKCßIIS660A, kinase-dead PKCßII, and PKCßII knockdown blocked TWIST1 expression by TGFß. Inhibition of TWIST1 arrested TGFß-induced tubular cell hypertrophy and the expression of fibronectin, collagen I (α2), and α-smooth muscle actin. By contrast, TWIST1 overexpression induced these pathologies. Interestingly, the inhibition of PKCßII reduced these phenomena, which were countered by the expression of TWIST1. These results provide the first evidence for the involvement of the mTORC2-PKCßII axis in TWIST1 expression to promote tubular cell pathology.

MicroRNA-21 orchestrates high glucose-induced signals to TOR complex 1, resulting in renal cell pathology in diabetes.

Hyperglycemia induces a wide array of signaling pathways in the kidney that lead to hypertrophy and matrix expansion, eventually culminating in progressive kidney failure. High glucose-induced reduction of the tumor suppressor protein phosphatase and tensin homolog deleted in chromosome 10 (PTEN) contributes to renal cell hypertrophy and matrix expansion. We identified microRNA-21 (miR-21) as the molecular link between high glucose and PTEN suppression. Renal cortices from OVE26 type 1 diabetic mice showed significantly elevated levels of miR-21 associated with reduced PTEN and increased fibronectin content. In renal mesangial cells, high glucose increased the expression of miR-21, which targeted the 3'-UTR of PTEN mRNA to inhibit PTEN protein expression. Overexpression of miR-21 mimicked the action of high glucose, which included a reduction in PTEN expression and a concomitant increase in Akt phosphorylation. In contrast, expression of miR-21 Sponge, to inhibit endogenous miR-21, prevented down-regulation of PTEN and phosphorylation of Akt induced by high glucose. Interestingly, high glucose-stimulated miR-21 inactivated PRAS40, a negative regulator of TORC1. Finally, miR-21 enhanced high glucose-induced TORC1 activity, resulting in renal cell hypertrophy and fibronectin expression. Thus, our results identify a previously unrecognized function of miR-21 that is the reciprocal regulation of PTEN levels and Akt/TORC1 activity that mediate critical pathologic features of diabetic kidney disease.