Goutam Ghosh Choudhury

University of Texas Health Science Center at San Antonio, San Antonio, Texas, United States

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Publications (125)626.62 Total impact

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    ABSTRACT: High glucose milieu inhibits PTEN expression to activate Akt kinase and induces glomerular mesangial cell hypertrophy and matrix protein expression in diabetic nephropathy. Specific mechanism by which high glucose inhibits PTEN expression is not clear. We found that high glucose increased the expression of the microRNA-26a (miR-26a) in mesangial cells. Using a sensor plasmid with 3'UTR-driven luciferase, we showed PTEN as a target of miR-26a in response to high glucose. Overexpression of miR-26a reduced the PTEN protein levels resulting in increased Akt kinase activity similar to high glucose treatment. In contrast, anti-miR-26a reversed high glucose-induced suppression of PTEN with concomitant inhibition of Akt kinase activity. Akt-mediated phosphorylation of tuberin and PRAS40 regulates mTORC1, which is necessary for mesangial cell hypertrophy and matrix protein expression. Inhibition of high glucose-induced miR-26a blocked phosphorylation of tuberin and PRAS40, which lead to suppression of phosphorylation of S6 kinase and 4EBP-1, two substrates of mTORC1. Furthermore, we show that expression of miR-26a induced mesangial cell hypertrophy and increased fibronectin and collagen I (α2) expression similar to that observed with the cells incubated with high glucose. Anti-miR-26a inhibited these phenomena in response to high glucose. Together our results provide the first evidence for the involvement of miR-26a in high glucose-induced mesangial cell hypertrophy and matrix protein expression. These data indicate the potential therapeutic utility of anti-miR-26a for the complications of diabetic kidney disease. Copyright © 2015. Published by Elsevier Inc.
    Cellular Signalling 03/2015; 27(7). DOI:10.1016/j.cellsig.2015.03.007 · 4.47 Impact Factor
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    ABSTRACT: Diabetes-induced kidney cell injury involves 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 if tadalafil, a phosphodiesterase 5 (PDE5) 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 mTORC1 and laminin γ1 accumulation in an AMP-activated protein kinase (AMPK)-dependent manner. Tadalafil increased AMPK phosphorylation by stimulating Calcium calmodulin kinase kinaseβ. Tadalafil rapidly increased the expression and activity of H2S generating enzyme, cystathionine-γ-lyase (CSE), by promoting its translation. DL-propargylglycine, a CSE inhibitor, and siRNA of CSE inhibited tadalafil-induced AMPK phosphorylation and abrogated tadalafil effect on high glucose stimulation of laminin γ1. In tadalafil treated podocytes, we examined the interaction between H2S and nitric oxide (NO). L-NAME and ODQ, 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; siRNA of 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 NO-H2S-AMPK axis leading to the inhibition of high glucose-induced mTORC1 activity and mRNA translation. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
    Journal of Biological Chemistry 03/2015; 290(19). DOI:10.1074/jbc.M114.615377 · 4.57 Impact Factor
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    ABSTRACT: 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 3β (GSK3β) in renal cells and in the kidneys of diabetic mice. We tested whether activation of GSK3β 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 GSK3β and inhibiting Akt, mTORC1, and events in mRNA translation regulated by mTORC1 and ERK. NONOate, an NO donor, also activated GSK3β, indicating that NO may mediate SNP stimulation of GSK3β. 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 GSK3β by activation of Src, Pyk2, Akt, and ERK; GSK3β inhibition activated mTORC1 and downstream events in mRNA translation in the kidney cortex. These reactions were abrogated by SNP. We conclude that activation of GSK3β by SNP ameliorates kidney injury induced by diabetes.
    Journal of Biological Chemistry 10/2014; 289(51). DOI:10.1074/jbc.M114.587840 · 4.57 Impact Factor
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    ABSTRACT: Enhanced TGFβ activity contributes to the accumulation of matrix proteins including collagen I (α2) by proximal tubular epithelial cells in progressive kidney disease. Although TGFβ rapidly activates its canonical Smad signaling pathway, it also recruits noncanonical pathway involving mTOR kinase to regulate renal matrix expansion. The mechanism by which chronic TGFβ treatment maintains increased mTOR activity to induce the matrix protein collagen I (α2) expression is not known. Deptor is an mTOR interacting protein that suppresses mTOR activity in both mTORC1 and mTORC2. In proximal tubular epithelial cells, TGFβ reduced deptor levels in a time-dependent manner with concomitant increase in both mTORC1 and mTORC2 activities. Expression of deptor abrogated activity of mTORC1 and mTORC2, resulting in inhibition of collagen I (α2) mRNA and protein expression via transcriptional mechanism. In contrast, neutralization of endogenous deptor by shRNAs increased activity of both mTOR complexes and expression of collagen I (α2) similar to TGFβ treatment. Importantly, downregulation of deptor by TGFβ increased the expression of Hif1α by increasing translation of its mRNA. TGFβ-induced deptor downregulation promotes Hif1α binding to its cognate hypoxia responsive element in the collagen I (α2) gene to control its protein expression via direct transcriptional mechanism. Interestingly, knockdown of raptor to specifically block mTORC1 activity significantly inhibited expression of collagen I (α2) and Hif1α while inhibition of rictor to prevent selectively mTORC2 activation did not have any effect. Critically, our data provide evidence for the requirement of TGFβ-activated mTORC1 only by deptor downregulation, which dominates upon the bystander mTORC2 activity for enhanced expression of collagen I (α2). Our results also suggest the presence of a safeguard mechanism involving deptor-mediated suppression of mTORC1 activity against developing TGFβ-induced renal fibrosis.
    PLoS ONE 10/2014; 9(10):e109608. DOI:10.1371/journal.pone.0109608 · 3.23 Impact Factor
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    ABSTRACT: High glucose-induced Akt acts as a signaling hub for mesangial cell hypertrophy and matrix expansion that are recognized as cardinal signatures for 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 high glucose-induced phosphorylation of Akt to suppress phosphorylation/inactivation of PRAS40 and mTORC1 activity. In contrast, dominant negative FoxO1 increased 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 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 production of ROS. Moreover, we show that catalase blocks high glucose-stimulated Akt phosphorylation to attenuate inactivation of FoxO1 and PRAS40, resulting in inhibition of mTORC1 and mesangial cell hypertrophy, and, fibronectin and PAI-1 expression. Finally, using kidney cortex 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 sustained activation of Akt involving inactivated FoxO1 and decrease in catalase expression, leading to increased ROS and mesangial cell hypertrophy and matrix protein expression.
    Journal of Biological Chemistry 10/2014; 289(47). DOI:10.1074/jbc.M114.605196 · 4.57 Impact Factor
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    ABSTRACT: 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 IKKβ, which activate NFκB-dependent transcription. Constitutively active (CA) Akt or CA IKKβ blocks PDCD4-mediated inhibition and restores renal cancer cell migration and invasion. PDCD4 inhibits mTORC1 activity, which was reversed by CA IKKβ. Moreover, CA mTORC1 restores cell migration and invasion inhibited by PDCD4- and dominant negative IKKβ. 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.
    Experimental Cell Research 07/2014; 328(1). DOI:10.1016/j.yexcr.2014.06.022 · 3.37 Impact Factor
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    ABSTRACT: Platelet-derived growth factor BB and its receptor (PDGFRβ) play pivotal role in development of renal glomerular mesangial cells. Their roles in increased mesangial cell proliferation during mesangioproliferative glomerulonephritis have long been noted; but the signaling mechanism 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 PI 3 kinase contributes to PDGF-induced proliferation of mesangial cells. Inhibition of PI 3 kinase blocked PDGF-induced phosphorylation of Erk5. Since PI 3 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 PI 3 kinase nodal point for PDGF-induced mesangial cell proliferation.
    AJP Cell Physiology 04/2014; 306(11). DOI:10.1152/ajpcell.00387.2013 · 3.67 Impact Factor
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    ABSTRACT: 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 if short duration of combined hyperglycemia and hyperinsulinemia, mimicking metabolic environment of pre-diabetes and early type 2 diabetes, induces kidney injury. Normal rats were subjected to either saline infusion (control, n=4) or seven hours 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 TGFβ and renal matrix proteins, laminin and fibronectin. This was associated with the activation of SMAD3, Akt, 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.
    AJP Cell Physiology 10/2013; 306(3). DOI:10.1152/ajpcell.00144.2013 · 3.67 Impact Factor
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    ABSTRACT: MicroRNAs regulate post-transcriptomic landscape in many tumors including renal cell carcinoma. We have recently shown significantly increased expression of miR-21 in renal tumors and that this miRNA contributes to the proliferation of renal cancer cells in culture. However, the mechanism by which miR-21 regulates renal cancer cells proliferation is poorly understood. Addiction to constitutive NFκB activity is hallmark of many cancers including renal cancer. Using miR-21 Sponge in renal cancer cells to block endogenous function of miR-21, we show inhibition of phosphorylation of p65 subunit of NFκB, IKKβ and IκB, which results in attenuation of NFκB transcriptional activity. Subtle reduction in the tumor suppressor PTEN has been linked to various malignancies. We showed previously that miR-21 targeted PTEN in renal cancer cells. Inhibition of PTEN by siRNAs restored miR-21 Sponge-induced suppression of phosphorylation of p65, IKKβ, IκB and NFκB transcriptional activity along with reversal of miR-21 Sponge-reduced phosphorylation of Akt. Expression of constitutively active Akt protected against miR-21 Sponge- and PTEN-mediated decrease in p65/IKKβ/IκB phosphorylation and NFκB transcriptional activity. Furthermore, IKKβ and p65 were required for miR-21-induced renal cancer cell proliferation. Interestingly, miR-21 controlled the expression of cyclin D1 through NFκB-dependent transcription. Finally, we demonstrate that miR-21-regulated renal cancer cell proliferation is mediated by cyclin D1 and CDK4. Together, our results establish a molecular order of a phosphatase-kinase couple involving PTEN/Akt/IKKβ and NFκB-dependent cyclin D1 expression for renal carcinoma cell proliferation by increased miR-21 levels.
    Cellular Signalling 08/2013; 25(12). DOI:10.1016/j.cellsig.2013.08.005 · 4.47 Impact Factor
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    ABSTRACT: Activation of glomerular mesangial cells (MCs) by angiotensin II (Ang II) leads to extracellular matrix accumulation. Here, we demonstrate that, in MCs, Ang II induces endothelial nitric oxide synthase (eNOS) uncoupling with enhanced generation of reactive oxygen species (ROS) and decreased production of nitric oxide (NO). Ang II promotes a rapid increase in 3-nitrotyrosine formation and uric acid attenuates Ang II-induced decrease in NO bioavailability, demonstrating that peroxynitrite mediates the effects of Ang II on eNOS dysfunction. Ang II rapidly up-regulates Nox4 protein. Inhibition of Nox4 abolishes the increase in ROS and peroxynitrite generation as well as eNOS uncoupling triggered by Ang II, indicating that Nox4 is upstream of eNOS. This pathway contributes to Ang II-mediated fibronectin accumulation in MCs. Ang II also elicits an increase in mitochondrial abundance of Nox4 protein and the oxidase contributes to ROS production in mitochondria. Overexpression of mitochondrial superoxide dismutase (MnSOD) prevents the stimulatory effects of Ang II on mitochondrial ROS production, loss of NO availability and MC fibronectin accumulation, whereas MnSOD depletion increases mitochondrial ROS, NO deficiency and fibronectin synthesis basally and in cells exposed to Ang II. This work provides the first evidence that uncoupled eNOS is responsible for Ang II-induced MC fibronectin accumulation and identifies Nox4 and mitochondrial ROS as mediators of eNOS dysfunction. These data shed light on molecular processes underlying the oxidative signaling cascade engaged by Ang II and identify potential targets for intervention to prevent renal fibrosis.
    Journal of Biological Chemistry 08/2013; 288(40). DOI:10.1074/jbc.M113.470971 · 4.57 Impact Factor
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    ABSTRACT: Transforming growth factorβ (TGFβ)-induced canonical signal transduction is involved in glomerular mesangial cell hypertrophy; however, the role played by the noncanonical TGFβ signaling remains largely unexplored. TGFβ 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 TGFβ-induced protein synthesis and mesangial cell hypertrophy. Expression of constitutively active MEK was sufficient to induce protein synthesis and hypertrophy similar to those induced by TGFβ. 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 TGFβ. Dominant negative Mnk-1 significantly inhibited TGFβ-stimulated protein synthesis and hypertrophy. Interestingly, inhibition of mTORC1 activity, which blocks dissociation of eIF4E-4EBP-1 complex, decreased TGFβ-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 TGFβ. 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 TGFβ-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. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
    Journal of Cellular Physiology 07/2013; 228(7). DOI:10.1002/jcp.24327 · 3.87 Impact Factor
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    ABSTRACT: Podocyte apoptosis is a critical mechanism for excessive loss of urinary albumin that eventuates in kidney fibrosis. Pharmacological doses of the mTOR inhibitor rapamycin reduce albuminura in diabetes. We explored the hypothesis that mTOR mediates podocyte injury in diabetes. High glucose (HG) induces apoptosis of podocytes, inhibits AMPK activation, inactivates tuberin and activates mTOR. HG also increases the levels of Nox4 and Nox1 and NADPH oxidase activity. Inhibition of mTOR by low dose rapamycin decreases HG-induced Nox4 and Nox1, NADPH oxidase activity and podocyte apoptosis. Inhibition of mTOR had no effect on AMPK or tuberin phosphorylation indicating that mTOR is downstream of these signaling molecules. In isolated glomeruli of OVE26 mice, there is similar decrease in the activation of AMPK and tuberin and activation of mTOR with increase in Nox4 and NADPH oxidase activity. Inhibition of mTOR by small dose of rapamycin reduces podocyte apoptosis, attenuates glomerular injury and albuminuria. Our data provide evidence for a novel function of mTOR in Nox4-derived ROS generation and podocyte apoptosis that contributes to urinary albumin excretion in type 1 diabetes. Thus mTOR and or NADPH oxidase inhibition may represent a therapeutic modality of diabetic kidney disease.
    Diabetes 04/2013; 62(8). DOI:10.2337/db12-1504 · 8.47 Impact Factor
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    ABSTRACT: In many renal diseases, TGFβ-stimulated canonical Smad 3 and noncanonical 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 TGFβ reduced the levels of deptor concomitant with increase in TORC1 and TORC2 activity. Sustained TGFβ activation was required to inhibit association of deptor with mTOR while rapid activation had no effect. Using mTOR inhibitor PP242, we found that TGFβ-induced both early and sustained activation of TORC1 and TORC2 were necessary for deptor suppression. PP242-induced reversal of deptor suppression by TGFβ was associated with significant inhibition of TGFβ-stimulated protein synthesis and hypertrophy. Interestingly, expression of siRNA against Smad 3 or Smad 7, which blocks TGFβ receptor-specific Smad 3 signaling, prevented TGFβ-induced suppression of deptor abundance and TORC1/2 activities. Furthermore, overexpression of Smad 3 decreased deptor expression similar to TGFβ 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 TGFβ. These data reveal the requirement of both early and late activation of mTOR for TGFβ-induced protein synthesis. Our results support that TGFβ-stimulated Smad 3 acts as a key node to instill a feed back loop between deptor downregulation and TORC1/2 activation in driving mesangial cell hypertrophy.
    Journal of Biological Chemistry 01/2013; 288(11). DOI:10.1074/jbc.M113.455782 · 4.57 Impact Factor
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    ABSTRACT: We explored molecular events associated with aging-induced matrix changes in the kidney. C57BL6 mice were studied in youth, middle age, and old age. Albuminuria and serum cystatin C level (an index of glomerular filtration) increased with aging. Renal hypertrophy was evident in middle-aged and old mice and was associated with glomerulomegaly and increase in mesangial fraction occupied by extracellular matrix. Content of collagen types I and III and fibronectin was increased with aging; increment in their mRNA varied with the phase of aging. The content of ZEB1 and ZEB2, collagen type I transcription inhibitors, and their binding to the collagen type Iα2 promoter by ChIP assay also showed age-phase-specific changes. Lack of increase in mRNA and data from polysome assay suggested decreased degradation as a potential mechanism for kidney collagen type I accumulation in the middle-aged mice. These changes occurred with increment in TGFβ mRNA and protein and activation of its SMAD3 pathway; SMAD3 binding to the collagen type Iα2 promoter was also increased. TGFβ-regulated microRNAs (miRs) exhibited selective regulation. The renal cortical content of miR-21 and miR-200c, but not miR-192, miR-200a, or miR-200b, was increased with aging. Increased miR-21 and miR-200c contents were associated with reduced expression of their targets, Sprouty-1 and ZEB2, respectively. These data show that aging is associated with complex molecular events in the kidney that are already evident in the middle age and progress to old age. Age-phase-specific regulation of matrix protein synthesis occurs and involves matrix protein-specific transcriptional and post-transcriptional mechanisms.
    Aging cell 09/2012; 11(6). DOI:10.1111/acel.12008 · 5.94 Impact Factor
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    ABSTRACT: Kidney injury is one of the most serious complications of diabetes. Current therapies only slow down but do not stop its progression to end stage kidney failure; thus, there is a need for novel approaches. Accumulation of extracellular matrix proteins, e.g., laminin, fibronectin, contributes to the loss of functioning parenchyma and progressive loss of kidney function in diabetes. We have recently reported that H2S inhibits high glucose-induced increase in synthesis of proteins including matrix proteins (H.J. Lee et al., J. Biol. Chem. 287 (2012) 4451). We wish to identify H2S generating agents that can be used in humans to treat diabetic kidney disease. We hypothesized that tadalafil, a H2S donor and phosphodiesterase 5 (PDE5) inhibitor, inhibits high glucose stimulation of synthesis of proteins including ECM proteins by generating H2S in mouse kidney podocytes. High glucose (30 mM), but not equimolar mannitol, significantly promoted global protein synthesis, cellular hypertrophy and expression of laminin-γ1 and fibronectin. These events were inhibited by 10 μM tadalafil. Because mRNA translation is rate-limiting for protein synthesis, we examined tadalafil effect on events in translation. Tadalafil abrogated high glucose-induced mTORC1 activation as indicated by phosphorylation of p70S6 kinase and 4E-BP1; both these events are important for the initiation phase of translation. Tadalafil reversed high glucose-induced changes in the phosphorylation of eEF2 and eEF2 kinase, which regulate the elongation phase of translation. Upstream regulators of mTORC1 were investigated. Tadalafil stimulated activating phosphorylation of AMPK in a dose dependent manner, peaking at 10 μM. Pre-incubation with Compound C, an AMPK inhibitor, completely abolished tadalafil effect on high glucose stimulation of events in mRNA translation as well as global protein and laminin synthesis. Tadalafil-induced AMPK phosphorylation was abolished by dl-propargylglycine (PAG), an inhibitor of cystathionine γ lyase (CSE), a H2S generating enzyme. Tadalafil rapidly increased CSE protein expression in 1 h which was inhibited by cycloheximide but not actinomycin-D, suggesting regulation at the level of mRNA translation. In an enzymatic assay, PAG abrogated tadalafil stimulation of generation of H2S. PAG completely abolished tadalafil effect on high glucose stimulation of events in mRNA translation as well as global protein and laminin synthesis. Tadalafil is a PDE5 inhibitor that increases cGMP content. ODQ, an inhibitor of guanylyl cyclase that generates cGMP, abolished tadalafil induction of AMPK phosphorylation. ODQ abrogated tadalafil inhibition of high glucose-induced increase in laminin expression. Renal cortical content of cystathionine β synthase (CBS) and CSE, H2S generating enzymes, was significantly reduced in the kidney in mice with type 1 or type 2 diabetes. Our data show that tadalafil stimulates H2S-AMPK axis to inhibit mTORC1 and mRNA translation leading to amelioration of high glucose-induced global and matrix protein increment in renal podocytes. It is likely that tadalafil stimulation of H2S is cGMP-dependent in the podocyte. Future work is needed to define whether tadalafil can ameliorate kidney injury in animal models of diabetes.
    Nitric Oxide 09/2012; 27:S18–S19. DOI:10.1016/j.niox.2012.08.017 · 3.18 Impact Factor
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    ABSTRACT: Transforming growth factor-β (TGFβ) promotes glomerular hypertrophy and matrix expansion, leading to glomerulosclerosis. MicroRNAs are well suited to promote fibrosis because they can repress gene expression, which negatively regulate the fibrotic process. Recent cellular and animal studies have revealed enhanced expression of microRNA, miR-21, in renal cells in response to TGFβ. Specific miR-21 targets downstream of TGFβ receptor activation that control cell hypertrophy and matrix protein expression have not been studied. Using 3'UTR-driven luciferase reporter, we identified the tumor suppressor protein PTEN as a target of TGFβ-stimulated miR-21 in glomerular mesangial cells. Expression of miR-21 Sponge, which quenches endogenous miR-21 levels, reversed TGFβ-induced suppression of PTEN. Additionally, miR-21 Sponge inhibited TGFβ-stimulated phosphorylation of Akt kinase, resulting in attenuation of phosphorylation of its substrate GSK3β. Tuberin and PRAS40, two other Akt substrates, and endogenous inhibitors of mTORC1, regulate mesangial cell hypertrophy. Neutralization of endogenous miR-21 abrogated TGFβ-stimulated phosphorylation of tuberin and PRAS40, leading to inhibition of phosphorylation of S6 kinase, mTOR and 4EBP-1. Moreover, downregulation of miR-21 significantly suppressed TGFβ-induced protein synthesis and hypertrophy, which were reversed by siRNA-targeted inhibition of PTEN expression. Similarly, expression of constitutively active Akt kinase reversed the miR-21 Sponge-mediated inhibition of TGFβ-induced protein synthesis and hypertrophy. Furthermore, expression of constitutively active mTORC1 prevented the miR-21 Sponge-induced suppression of mesangial cell protein synthesis and hypertrophy by TGFβ. Finally, we show that miR-21 Sponge inhibited TGFβ-stimulated fibronectin and collagen expression. Suppression of PTEN expression and expression of both constitutively active Akt kinase and mTORC1 independently reversed this miR-21-mediated inhibition of TGFβ-induced fibronectin and collagen expression. Our results uncover an essential role of TGFβ-induced expression of miR-21, which targets PTEN to initiate a non-canonical signaling circuit involving Akt/mTORC1 axis for mesangial cell hypertrophy and matrix protein synthesis.
    PLoS ONE 08/2012; 7(8):e42316. DOI:10.1371/journal.pone.0042316 · 3.23 Impact Factor
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    ABSTRACT: 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.
    Carcinogenesis 06/2012; 33(10):1897-908. DOI:10.1093/carcin/bgs198 · 5.27 Impact Factor
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    ABSTRACT: Metastatic renal cancer manifests multiple signatures of gene expression. Deviation in expression of mature miRNAs has been linked to human cancers. Importance of miR-21 in renal cell carcinomas is proposed from profiling studies using tumor tissue samples. However, the role of miR-21 function in causing renal cancer cell proliferation and invasion has not yet been shown. Using cultured renal carcinoma cells, we demonstrate enhanced expression of mature miR-21 along with pre-and pri-miR-21 by increased transcription compared to normal proximal tubular epithelial cells. Overexpression of miR-21 Sponge to quench endogenous miR-21 levels inhibited proliferation, migration and invasion of renal cancer cells. In the absence of mutation in the PTEN tumor suppressor gene, PTEN protein levels are frequently downregulated in renal cancer. We show that miR-21 targets PTEN mRNA 3'untranslated region to decrease PTEN protein expression and augments Akt phosphorylation in renal cancer cells. Downregulation of PTEN as well as overexpression of constitutively active Akt kinase prevented miR-21 Sponge-induced inhibition of renal cancer cell proliferation and migration. Moreover, we show that miR-21 Sponge inhibited the inactivating phosphorylation of the tumor suppressor protein tuberin and attenuated TORC1 activation. Finally, we demonstrate that expression of constitutively active TORC1 attenuated miR-21 Sponge-mediated suppression of proliferation and migration of renal cancer cells. Our results uncover a layer of post-transcriptional regulation of PTEN by transcriptional activation of miR-21 to force the canonical oncogenic Akt/TORC1 signaling conduit to drive renal cancer cell proliferation and invasion.
    PLoS ONE 06/2012; 7(6):e37366. DOI:10.1371/journal.pone.0037366 · 3.23 Impact Factor
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    ABSTRACT: The study was undertaken to explore the effect of rapamycin, an anti-inflammatory agent, on the metabolic profile of type 2 diabetic mice. Seven-month-old diabetic db/db mice and their lean littermate non-diabetic controls (db/m) were randomized to receive control chow or chow mixed with rapamycin (2.24 mg/kg/day) (each group n =20, males and females) for 4 months and sacrificed. Serum samples were analyzed for the measurement of glucose, creatinine, blood urea nitrogen (BUN), alkaline phosphatase (ALP), alanine aminotransferase (ALT), total cholesterol, total triglyceride, and total protein, using the automated dry chemistry analysis. Rapamycin elevated serum glucose in female diabetic mice. Serum creatinine tended to be higher in diabetic mice but was not affected by rapamycin; there was no difference in BUN levels among the groups. Serum ALP was elevated in diabetic mice and rapamycin lowered it only in female diabetic mice; serum ALT levels were increased in female diabetic mice, unaffected by rapamycin. Serum total protein was elevated in diabetic mice of both genders but was not affected by rapamycin. Diabetic mice from both genders had elevated serum cholesterol and triglycerides; rapamycin did not affect serum cholesterol but decreased serum total triglycerides in male diabetic mice. We conclude that rapamycin elicits complex metabolic responses in aging diabetic mice, worsening hyperglycemia in females but improving ALP in female diabetic and total triglycerides in male diabetic mice, respectively. The metabolic effects of rapamycin should be considered while performing studies with rapamycin in mice.
    04/2012; 2. DOI:10.3402/pba.v2i0.15896
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    ABSTRACT: 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 β, but not LKB1, upstream kinases for AMPK; STO-609, a calmodulin kinase kinase β inhibitor, had the same effect. Renal cortical content of cystathionine β-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.
    Journal of Biological Chemistry 02/2012; 287(7):4451-61. · 4.57 Impact Factor

Publication Stats

4k Citations
626.62 Total Impact Points

Institutions

  • 1989–2015
    • University of Texas Health Science Center at San Antonio
      • • Division of Hospital Medicine
      • • Department of Medicine
      • • Department of Pathology
      • • Division of Nephrology
      • • Department of Cellular and Structural Biology
      San Antonio, Texas, United States
  • 2009
    • University of Texas Health Science Center at Tyler
      Tyler, Texas, United States
  • 2008
    • Ludwig-Maximilians-University of Munich
      München, Bavaria, Germany
  • 1990–2007
    • University of Texas at San Antonio
      San Antonio, Texas, United States
  • 1992
    • Roswell Park Cancer Institute
      • Department of Human Genetics
      Buffalo, New York, United States