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.

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.

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.

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.

Tadalafil Integrates Nitric Oxide-Hydrogen Sulfide Signaling to Inhibit High Glucose-induced Matrix Protein Synthesis in Podocytes.

Diabetes-induced kidney cell injury involves an increase in matrix protein expression that is only partly alleviated by current treatment, prompting a search for new modalities. We have previously shown that hydrogen sulfide (H2S) inhibits high glucose-induced protein synthesis in kidney podocytes. We tested whether tadalafil, a phosphodiesterase 5 inhibitor used to treat erectile dysfunction, ameliorates high glucose stimulation of matrix proteins by generating H2S in podocytes. Tadalafil abrogated high glucose stimulation of global protein synthesis and matrix protein laminin γ1. Tadalafil inhibited high glucose-induced activation of mechanistic target of rapamycin complex 1 and laminin γ1 accumulation in an AMP-activated protein kinase (AMPK)-dependent manner. Tadalafil increased AMPK phosphorylation by stimulating calcium-calmodulin kinase kinase ß. Tadalafil rapidly increased the expression and activity of the H2S-generating enzyme cystathionine γ-lyase (CSE) by promoting its translation. dl-Propargylglycine, a CSE inhibitor, and siRNA against CSE inhibited tadalafil-induced AMPK phosphorylation and abrogated the tadalafil effect on high glucose stimulation of laminin γ1. In tadalafil-treated podocytes, we examined the interaction between H2S and nitric oxide (NO). N(ω)-Nitro-L-arginine methyl ester and 1H-[1,2,4]-oxadiazolo-[4,3-a]-quinoxalin-1-one, inhibitors of NO synthase (NOS) and soluble guanylyl cyclase, respectively, abolished tadalafil induction of H2S and AMPK phosphorylation. Tadalafil rapidly augmented inducible NOS (iNOS) expression by increasing its mRNA, and siRNA for iNOS and 1400W, an iNOS blocker, inhibited tadalafil stimulation of CSE expression and AMPK phosphorylation. We conclude that tadalafil amelioration of high glucose stimulation of synthesis of proteins including matrix proteins in podocytes requires integration of the NO-H2S-AMPK axis leading to the inhibition of high glucose-induced mechanistic target of rapamycin complex 1 activity and mRNA translation.

Activation of glycogen synthase kinase 3ß ameliorates diabetes-induced kidney injury.

Increase in protein synthesis contributes to kidney hypertrophy and matrix protein accumulation in diabetes. We have previously shown that high glucose-induced matrix protein synthesis is associated with inactivation of glycogen synthase kinase 3ß (GSK3ß) in renal cells and in the kidneys of diabetic mice. We tested whether activation of GSK3ß by sodium nitroprusside (SNP) mitigates kidney injury in diabetes. Studies in kidney-proximal tubular epithelial cells showed that SNP abrogated high glucose-induced laminin increment by stimulating GSK3ß and inhibiting Akt, mTORC1, and events in mRNA translation regulated by mTORC1 and ERK. NONOate, an NO donor, also activated GSK3ß, indicating that NO may mediate SNP stimulation of GSK3ß. SNP administered for 3 weeks to mice with streptozotocin-induced type 1 diabetes ameliorated kidney hypertrophy, accumulation of matrix proteins, and albuminuria without changing blood glucose levels. Signaling studies showed that diabetes caused inactivation of GSK3ß by activation of Src, Pyk2, Akt, and ERK; GSK3ß inhibition activated mTORC1 and downstream events in mRNA translation in the kidney cortex. These reactions were abrogated by SNP. We conclude that activation of GSK3ß by SNP ameliorates kidney injury induced by diabetes.

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.

Combined acute hyperglycemic and hyperinsulinemic clamp induced profibrotic and proinflammatory responses in the kidney.

Increase in matrix protein content in the kidney is a cardinal feature of diabetic kidney disease. While renal matrix protein content is increased by chronic hyperglycemia, whether it is regulated by acute elevation of glucose and insulin has not been addressed. In this study, we aimed to evaluate whether short duration of combined hyperglycemia and hyperinsulinemia, mimicking the metabolic environment of prediabetes and early type 2 diabetes, induces kidney injury. Normal rats were subjected to either saline infusion (control, n = 4) or 7 h of combined hyperglycemic-hyperinsulinemic clamp (HG+HI clamp; n = 6). During the clamp, plasma glucose and plasma insulin were maintained at about 350 mg/dl and 16 ng/ml, respectively. HG+HI clamp increased the expression of renal cortical transforming growth factor-ß (TGF-ß) and renal matrix proteins, laminin and fibronectin. This was associated with the activation of SMAD3, Akt, mammalian target of rapamycin (mTOR) complexes, and ERK signaling pathways and their downstream target events in the initiation and elongation phases of mRNA translation, an important step in protein synthesis. Additionally, HG+HI clamp provoked renal inflammation as shown by the activation of Toll-like receptor 4 (TLR4) and infiltration of CD68-positive monocytes. Urinary F2t isoprostane excretion, an index of renal oxidant stress, was increased in the HG+HI clamp rats. We conclude that even a short duration of hyperglycemia and hyperinsulinemia contributes to activation of pathways that regulate matrix protein synthesis, inflammation, and oxidative stress in the kidney. This finding could have implications for the control of short-term rises in blood glucose in diabetic individuals at risk of developing kidney disease.

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.

Female Protection Against Diabetic Kidney Disease Is Regulated by Kidney-Specific AMPK Activity.

Reduced kidney AMPK activity is associated with nutrient stress-induced chronic kidney disease (CKD) in male mice. In contrast, female mice resist nutrient stress-induced CKD. The role of kidney AMPK in sex-related organ protection against nutrient stress and metabolite changes was evaluated in diabetic kidney tubule-specific AMPKγ2KO (KTAMPKγ2ΚΟ) male and female mice. In wild-type (WT) males, diabetes increased albuminuria, urinary kidney injury molecule-1, hypertension, kidney p70S6K phosphorylation, and kidney matrix accumulation; these features were not exacerbated with KTAMPKγ2ΚΟ. Whereas WT females had protection against diabetes-induced kidney injury, KTAMPKγ2ΚΟ led to loss of female protection against kidney disease. The hormone 17ß-estradiol ameliorated high glucose-induced AMPK inactivation, p70S6K phosphorylation, and matrix protein accumulation in kidney tubule cells. The mechanism for female protection against diabetes-induced kidney injury is likely via an estrogen-AMPK pathway, as inhibition of AMPK led to loss of estrogen protection to glucose-induced mTORC1 activation and matrix production. RNA sequencing and metabolomic analysis identified a decrease in the degradation pathway of phenylalanine and tyrosine resulting in increased urinary phenylalanine and tyrosine levels in females. The metabolite levels correlated with loss of female protection. The findings provide new insights to explain evolutionary advantages to females during states of nutrient challenges.

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.

Hydrogen sulfide inhibits high glucose-induced matrix protein synthesis by activating AMP-activated protein kinase in renal epithelial cells.

Hydrogen sulfide, a signaling gas, affects several cell functions. We hypothesized that hydrogen sulfide modulates high glucose (30 mm) stimulation of matrix protein synthesis in glomerular epithelial cells. High glucose stimulation of global protein synthesis, cellular hypertrophy, and matrix laminin and type IV collagen content was inhibited by sodium hydrosulfide (NaHS), an H(2)S donor. High glucose activation of mammalian target of rapamycin (mTOR) complex 1 (mTORC1), shown by phosphorylation of p70S6 kinase and 4E-BP1, was inhibited by NaHS. High glucose stimulated mTORC1 to promote key events in the initiation and elongation phases of mRNA translation: binding of eIF4A to eIF4G, reduction in PDCD4 expression and inhibition of its binding to eIF4A, eEF2 kinase phosphorylation, and dephosphorylation of eEF2; these events were inhibited by NaHS. The role of AMP-activated protein kinase (AMPK), an inhibitor of protein synthesis, was examined. NaHS dose-dependently stimulated AMPK phosphorylation and restored AMPK phosphorylation reduced by high glucose. Compound C, an AMPK inhibitor, abolished NaHS modulation of high glucose effect on events in mRNA translation as well as global and matrix protein synthesis. NaHS induction of AMPK phosphorylation was inhibited by siRNA for calmodulin kinase kinase ß, but not LKB1, upstream kinases for AMPK; STO-609, a calmodulin kinase kinase ß inhibitor, had the same effect. Renal cortical content of cystathionine ß-synthase and cystathionine γ-lyase, hydrogen sulfide-generating enzymes, was significantly reduced in mice with type 1 diabetes or type 2 diabetes, coinciding with renal hypertrophy and matrix accumulation. Hydrogen sulfide is a newly identified modulator of protein synthesis in the kidney, and reduction in its generation may contribute to kidney injury in diabetes.

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.

miR-21 is targeted by omega-3 polyunsaturated fatty acid to regulate breast tumor CSF-1 expression.

Increasing evidence shows the beneficial effects of fish oil on breast cancer growth and invasion in vitro and in animal models. Expression of CSF-1 (colony stimulating factor-1) by breast cancer cells acts as potent activator of malignancy and metastasis. In this report, we used two human breast cancer cell lines, MDA-MB-231 and MCF-7, to show that the bioactive fish oil component DHA (docosahexaenoic acid) inhibits expression of CSF-1 and its secretion from these cancer cells. We found that the tumor suppressor protein PTEN regulates CSF-1 expression through PI 3 kinase/Akt signaling via a transcriptional mechanism. The enhanced abundance of microRNA-21 (miR-21) in breast cancer cells contributes to the growth and metastasis. Interestingly, DHA significantly inhibited expression of miR-21. miR-21 Sponge, which derepresses the miR-21 targets, markedly decreased expression of CSF-1 and its secretion. Furthermore, miR-21-induced upregulation of CSF-1 mRNA and its transcription were prevented by expression of PTEN mRNA lacking 3'-untranslated region (UTR) and miR-21 recognition sequence. Strikingly, miR-21 reversed DHA-forced reduction of CSF-1 expression and secretion. Finally, we found that expression of miR-21 as well as CSF-1 was significantly attenuated in breast tumors of mice receiving a diet supplemented with fish oil. Our results reveal a novel mechanism for the therapeutic function of fish oil diet that blocks miR-21, thereby increasing PTEN levels to prevent expression of CSF-1 in breast cancer.

Simvastatin prevents skeletal metastasis of breast cancer by an antagonistic interplay between p53 and CD44.

Substantial data from clinical trials and epidemiological studies show promising results for use of statins in many cancers, including mammary carcinoma. Breast tumor primarily metastasizes to bone to form osteolytic lesions, causing severe pain and pathological fracture. Here, we report that simvastatin acts as an inhibitor of osteolysis in a mouse model of breast cancer skeletal metastasis of human mammary cancer cell MDA-MB-231, which expresses the mutant p53R280K. Simvastatin and lovastatin attenuated migration and invasion of MDA-MB-231 and BT-20 breast tumor cells in culture. Acquisition of phenotype to express the cancer stem cell marker, CD44, leads to invasive potential of the tumor cells. Interestingly, statins significantly decreased the expression of CD44 protein via a transcriptional mechanism. shRNA-mediated down-regulation of CD44 markedly reduced the migration and invasion of breast cancer cells in culture. We identified that in the MDA-MB-231 cells, simvastatin elevated the levels of mutated p53R280K, which was remarkably active as a transcription factor. shRNA-derived inhibition of mutant p53R280K augmented the expression of CD44, leading to increased migration and invasion. Finally, we demonstrate an inverse correlation between expression of p53 and CD44 in the tumors of mice that received simvastatin. Our results reveal a unique function of statins, which foster enhanced expression of mutant p53R280K to prevent breast cancer cell metastasis to bone.

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.

Ribosomal biogenesis induction by high glucose requires activation of upstream binding factor in kidney glomerular epithelial cells.

Diabetes promotes protein synthesis to induce kidney hypertrophy and increase renal matrix proteins. Increased capacity for mRNA translation by way of ribosomal biogenesis facilitates sustained stimulation of protein synthesis. We tested the hypothesis that high glucose induces ribosomal biogenesis as indicated by an increase in rRNA synthesis in the setting of augmented protein synthesis. High glucose (30 mM) increased global protein synthesis, expression of matrix proteins, laminin γ1 and fibronectin, and rDNA transcription in glomerular epithelial cells (GECs) compared with 5 mM glucose. High glucose induced Ser388 phosphorylation of upstream binding factor (UBF), an rDNA transcription factor, along with increased phosphorylation of Erk and p70S6 kinase. Inactivation of Erk and p70S6 kinase either by their respective chemical inhibitors or by expression of their inactive mutant constructs blocked high-glucose-induced UBF phosphorylation. High glucose reduced nuclear content of p19ARF and promoted dissolution of inactive UBF-p19ARF complex. High glucose also promoted association of UBF with RPA194, a subunit of RNA polymerase I. Inhibition of Erk, p70S6 kinase, and UBF1 by transfecting GECs with their respective inactive mutants abolished laminin γ1 synthesis, protein synthesis, and rDNA transcription. Renal cortex from type 1 diabetic rats and type 2 diabetic db/db mice showed increased phosphorylation of UBF, Erk, and p70S6 kinase coinciding with renal hypertrophy and onset of matrix accumulation. Our data suggest that augmented ribosome biogenesis occurs in an UBF-dependent manner during increased protein synthesis induced by high glucose in the GECs that correlates with UBF activation and renal hypertrophy in rodents with type 1 and type 2 diabetes.

The NADPH oxidase subunit p22phox inhibits the function of the tumor suppressor protein tuberin.

Mutations in the von Hippel-Lindau (VHL) gene give rise to renal cell carcinoma. Reactive oxygen species, generated by Nox oxidases, are involved in tumorigenesis. We have previously demonstrated that in VHL-deficient cells, p22(phox)-dependent Nox1 and Nox4 oxidases maintain hypoxia inducible factor-2alpha (HIF-2alpha) protein expression through an Akt-dependent translational pathway. Phosphorylation of tuberin, by Akt, results in its inactivation. Here we show that diphenyleneiodonium chloride, an inhibitor of Nox oxidases, and small-interfering RNA-mediated down-regulation of p22(phox) inhibit Akt-dependent phosphorylation of tuberin and stabilizes tuberin protein levels in VHL-deficient renal carcinoma cells. p22(phox)-mediated inactivation of tuberin is associated with an increase in ribosomal protein S6 kinase 1 and eukaryotic initiation factor 4E-binding protein-1 (4E-BP1) phosphorylation as well as HIF-2alpha stabilization. Importantly, we find that marked up-regulation of p22(phox) in human renal cell carcinoma correlates with increased tuberin phosphorylation, decreased tuberin protein levels, and increased phosphorylation of 4E-BP1. Our data provide the first evidence that p22(phox)-based Nox oxidases maintain HIF-2alpha protein expression through inactivation of tuberin and downstream activation of ribosomal protein S6 kinase 1/4E-BP1 pathway.