D Senthil

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

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Publications (11)40.55 Total impact

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    ABSTRACT: Vascular endothelial growth factor (VEGF) increases protein synthesis and induces hypertrophy in renal tubular epithelial cells (Senthil, D., Choudhury, G. G., McLaurin, C., and Kasinath, B. S. (2003) Kidney Int. 64, 468-479). We examined the role of Erk1/2 MAP kinase in protein synthesis induced by VEGF. VEGF stimulated Erk phosphorylation that was required for induction of protein synthesis. VEGF-induced Erk activation was not dependent on phosphoinositide (PI) 3-kinase activation but required sequential phosphorylation of type 2 VEGF receptor, PLCgamma and c-Src, as demonstrated by inhibitors SU1498, U73122, and PP1, respectively. c-Src phosphorylation was inhibited by U73122, indicating it was downstream of phospholipase (PL)Cgamma. Studies with PP1/2 showed that phosphorylation of c-Src was required for tyrosine phosphorylation of Raf-1, an upstream regulator of Erk. VEGF also stimulated phosphorylation of Pyk-2; VEGF-induced phosphorylation of Pyk2, c-Src and Raf-1 could be abolished by BAPTA/AM, demonstrating requirement for induction of intracellular calcium currents. We examined the downstream events following the phosphorylation of Erk. VEGF stimulated phosphorylation of Mnk1 and eIF4E and induced Mnk1 to shift from the cytoplasm to the nucleus upon phosphorylation. VEGF-induced phosphorylation of Mnk1 and eIF4E required phosphorylation of PLCgamma, c-Src, and Erk. Expression of dominant negative Mnk1 abrogated eIF4E phosphorylation and protein synthesis induced by VEGF. VEGF-stimulated protein synthesis could be blocked by inhibition of PLCgamma by a chemical inhibitor or expression of a dominant negative construct. Our data demonstrate that VEGF-stimulated protein synthesis is Erk-dependent and requires the activation of VEGF receptor 2, PLCgamma, c-Src, Raf, and Erk pathway. VEGF also stimulates Erk-dependent phosphorylation of Mnk1 and eIF4E, crucial events in the initiation phase of protein translation.
    Journal of Biological Chemistry 09/2005; 280(31):28402-11. · 4.65 Impact Factor
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    ABSTRACT: Vascular endothelial growth factor (VEGF) is an important determinant of ocular complications of diabetes. Its potential role in diabetic renal disease has not been extensively studied. We employed mice with streptozotocin-induced type 1 diabetes and db/db mice with type 2 diabetes to study the regulation of renal VEGF. Studies of VEGF regulation of protein synthesis were performed using proximal tubular epithelial (MCT) cells in culture. A nearly three-fold increase of VEGF165 expression in the renal cortex was seen, coinciding with renal hypertrophy in mice with either type 1 or type 2 diabetes. VEGF increased de novo protein synthesis and induced significant hypertrophy in MCT cells. VEGF stimulation of protein synthesis was dependent on tyrosine phosphorylation of the type 2 VEGF receptor and phosphatidylinositol 3-kinase (PI 3-kinase) activity. Activity of Akt was increased two- to three-fold by VEGF. Expression of dominant-negative Akt showed that Akt activation was also needed for VEGF-induced protein synthesis and cell hypertrophy. As PI 3-kinase-Akt axis regulates initial events in protein translation, these events were examined in the context of VEGF regulation of protein synthesis. VEGF stimulated eukaryotic initiation factor 4E-binding protein (4E-BP1) phosphorylation, which was dependent on activation of PI 3-kinase and Akt. Stable transfection with 4E-BP1 Thr37,46-Ala37,46 mutant abolished the VEGF-induced de novo protein synthesis and cell hypertrophy. VEGF augments protein synthesis and induces hypertrophy in MCT cells in a PI 3-kinase- and Akt-dependent manner. Phosphorylation of Thr37,46 in 4E-BP1 is required for VEGF-induced protein synthesis and hypertrophy in MCT cells. These data suggest a role for VEGF in the pathogenesis of diabetic renal disease.
    Kidney International 09/2003; 64(2):468-79. · 8.52 Impact Factor
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    ABSTRACT: Protein synthesis is required for renal hypertrophy, and proximal tubular epithelial cells are an important cell type involved in this process. We examined IGF-I regulation of protein synthesis in murine proximal tubular epithelial (MCT) cells. We focused on initial events in protein translation and the signaling events involved. Translation of capped mRNAs is under the control of eukaryotic initiation factor 4E (eIF4E). In the resting cell, eIF4E is normally kept in an inactive state by binding to 4E-BP1, its binding protein. Phosphorylation of 4E-BP1 results in dissociation of the eIF4E-4E-BP1 complex allowing eIF4E to initiate peptide synthesis. IGF-I stimulated protein synthesis, augmented phosphorylation of 4E-BP1 and promoted the dissociation of eIF4E from 4E-BP1. IGF-I stimulated the activities of phosphatidylinositol (PI) 3-kinase, Akt, and ERK1/2-type MAPK in MCT cells. IGF-I-induced phosphorylation of 4E-BP1, dissociation of the 4E-BP1-eIF4E complex, and increase in protein synthesis required activation of both PI 3-kinase and ERK pathways. Furthermore, ERK activation by IGF-I was also PI 3-kinase dependent. Transfection with the Thr37,46-->Ala37,46 mutant of 4E-BP1 showed that phosphorylation of Thr37,46 residues was required for IGF-I induction of protein synthesis in MCT cells. Our observations reveal the importance of initial events in protein translation in IGF-I-induced protein synthesis in MCT cells and identify the regulatory signaling pathways involved.
    American journal of physiology. Renal physiology 01/2003; 283(6):F1226-36. · 3.61 Impact Factor
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    ABSTRACT: Vascular endothelial growth factor (VEGF) isoforms exert their biological effects through receptors that possess intrinsic tyrosine kinase activity. Whether VEGF binding to its receptors recruits insulin receptor substrate (IRS) family of docking proteins to the receptor is not known. Following incubation of mouse kidney proximal tubular epithelial cells with VEGF, we observed an increase in tyrosine phosphorylation of several proteins, including one of approximately 200 kDa, suggesting possible regulation of phosphorylation of IRS proteins. VEGF augmented tyrosine phosphorylation of IRS-1 in kidney epithelial cells and rat heart endothelial cells in a time-dependent manner. In the epithelial cells, association of IRS-1 with type 2 VEGF receptor was promoted by VEGF. VEGF also increased association of IRS-1 with the p85 regulatory subunit of phosphoinositide 3-kinase (PI 3-kinase), and PI 3-kinase activity in IRS-1 immunoprecipitates was increased in VEGF-treated cells. Incubation of epithelial cells with antisense IRS-1 oligonucleotide, but not sense oligonucleotide, reduced expression of the protein and VEGF-induced PI 3-kinase activity in IRS-1 immunoprecipitates. Additionally, VEGF-induced protein synthesis was also impaired by antisense but not sense IRS-1 oligonucleotide. These data provide the first evidence that binding of VEGF to its type 2 receptor promotes association of IRS-1 with the receptor complex. This association may account for some of the increase in VEGF-induced PI 3-kinase activity, and the increase in de novo protein synthesis seen in renal epithelial cells.
    Biochemical Journal 12/2002; 368(Pt 1):49-56. · 4.65 Impact Factor
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    ABSTRACT: Interaction between angiotensin II, which binds a G-protein-coupled receptor, and insulin, a ligand for receptor tyrosine kinase, was examined in renal proximal tubular epithelial cells. Augmented protein translation by insulin involves activation of eukaryotic initiation factor 4E (eIF4E) which follows the release of the factor from a heterodimeric complex by phosphorylation of its binding protein, 4E-BP1. Angiotensin II (1 nM) or insulin (1 nM) individually stimulated 4E-BP1 phosphorylation. However, pre-incubation with angiotensin II abrogated insulin-induced phosphorylation of 4E-BP1, resulting in persistent binding to eIF4E. Although angiotensin II and insulin individually activated phosphoinositide 3-kinase and extracellular signal-regulated kinase (ERK)-1/-2-type mitogen-activated protein (MAP) kinase, pre-incubation with angiotensin II abolished insulin-induced stimulation of these kinases, suggesting more proximal events in insulin signalling may be intercepted. Pretreatment with angiotensin II markedly inhibited insulin-stimulated tyrosine phosphorylation of insulin-receptor beta-chain and insulin-receptor substrate 1. Losartan prevented angiotensin II inhibition of insulin-induced ERK-1/-2-type MAP kinase activation and 4E-BP1 phosphorylation, suggesting mediation of the effect of angiotensin II by its type 1 receptor. Insulin-stimulated de novo protein synthesis was also abolished by pre-incubation with angiotensin II. These data show that angiotensin II inhibits 4E-BP1 phosphorylation and stimulation of protein synthesis induced by insulin by interfering with proximal events in insulin signalling. Our data provide a mechanistic basis for insulin insensitivity induced by angiotensin II.
    Biochemical Journal 12/2001; 360(Pt 1):87-95. · 4.65 Impact Factor
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    ABSTRACT: In utero ethanol exposure causes abnormal fetal brain development that may partly be due to enhanced cell death. The mechanisms underlying this remain to be defined, but ethanol-induced oxidative stress may play a role. The following studies investigated the effects of short-term in utero ethanol exposure on fetal brain mitochondrial events that are known to elicit apoptotic cell death. Evidence is presented suggesting that 4-hydroxynonenal (HNE), a toxic product of lipid oxidation, is a causal factor in the observed mitochondrial damage. Mitochondria were isolated from control and ethanol-exposed fetal brains (days 17 and 18 of gestation). Permeability transition was determined spectrophotometrically, and cytochrome c and apoptosis-inducing factor (AIF) release were assessed by Western blotting. Caspase-3 activity and DNA fragmentation were determined both as markers for mitochondrially mediated apoptosis and as consequences of cytochrome c and AIF release. Maternal ethanol intake caused an increase in mitochondrial permeability transition, and this was accompanied by cytochrome c and AIF release from fetal brain mitochondria that exceeded control values by 62 and 25%, respectively (p < 0.05). In utero ethanol exposure resulted in a 30% increase in caspase-3 activity and a 25% increase in DNA fragmentation (p < 0.05) in the fetal brain. HNE levels were increased by 23% (p < 0.05) in mitochondria by in vivo ethanol exposure. In vitro treatment of fetal brain mitochondria with HNE (25-100 microM) also caused increases in mitochondrial permeability transition, as well as dose-dependent releases of cytochrome c and AIF. These studies illustrate that in utero ethanol exposure can elicit a cascade of events in the fetal brain that are consistent with mitochondrially mediated apoptotic cell death. Additionally, the increase in mitochondrial content of HNE after ethanol intake and the ability of HNE added to fetal brain mitochondria to mimic these effects of in vivo ethanol exposure support a potential role for HNE in the proapoptotic responses to ethanol.
    Alcoholism Clinical and Experimental Research 06/2001; 25(6):862-71. · 3.42 Impact Factor
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    ABSTRACT: Background: In utero ethanol exposure causes abnormal fetal brain development that may partly be due to enhanced cell death. The mechanisms underlying this remain to be defined, but ethanol-induced oxidative stress may play a role. The following studies investigated the effects of short-term in utero ethanol exposure on fetal brain mitochondrial events that are known to elicit apoptotic cell death. Evidence is presented suggesting that 4-hydroxynonenal (HNE), a toxic product of lipid oxidation, is a causal factor in the observed mitochondrial damage.Methods: Mitochondria were isolated from control and ethanol-exposed fetal brains (days 17 and 18 of gestation). Permeability transition was determined spectrophotometrically, and cytochrome c and apoptosis-inducing factor (AIF) release were assessed by Western blotting. Caspase-3 activity and DNA fragmentation were determined both as markers for mitochondrially mediated apoptosis and as consequences of cytochrome c and AIF release.Results: Maternal ethanol intake caused an increase in mitochondrial permeability transition, and this was accompanied by cytochrome c and AIF release from fetal brain mitochondria that exceeded control values by 62 and 25%, respectively (p < 0.05). In utero ethanol exposure resulted in a 30% increase in caspase-3 activity and a 25% increase in DNA fragmentation (p < 0.05) in the fetal brain. HNE levels were increased by 23% (p < 0.05) in mitochondria by in vivo ethanol exposure. In vitro treatment of fetal brain mitochondria with HNE (25–100 μM) also caused increases in mitochondrial permeability transition, as well as dose-dependent releases of cytochrome c and AIF.Conclusions: These studies illustrate that in utero ethanol exposure can elicit a cascade of events in the fetal brain that are consistent with mitochondrially mediated apoptotic cell death. Additionally, the increase in mitochondrial content of HNE after ethanol intake and the ability of HNE added to fetal brain mitochondria to mimic these effects of in vivo ethanol exposure support a potential role for HNE in the proapoptotic responses to ethanol.
    Alcoholism Clinical and Experimental Research 05/2001; 25(6):862 - 871. · 3.42 Impact Factor
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    ABSTRACT: Sodium pentosan polysulphate (SPP) has been shown to inhibit calcium oxalate crystallization in vitro. Here, the effect of SPP was studied on in vivo crystallization. Ammonium oxalate was used as the inducer of calcium oxalate crystallization in rats and the effect of SPP on in vivo crystallization factor is reported. The increased excretions of stone forming constituents associated with urinary marker enzymes are the salient features observed in experimental rats. Sodium pentosan polysulphate administration brought about a significant reduction in urinary stone forming constituents. It also decreased the extent of renal tubular damage as evidenced from the reduced level of marker enzymes in urine. These observations highlight the inhibitory capacity of SPP against in vivo calcium oxalate crystallization.
    Renal Failure 08/1998; 20(4):573-80. · 0.94 Impact Factor
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    ABSTRACT: To study the effect of L-cysteine on urinary risk factors in hyperoxaluric urolithiasis in rats. Twenty-four rats were divided into four groups of six rats each; those in Group I served as controls, those in Group II had hyperoxaluria induced using sodium glycollate, those in Group III were given cysteine alone and those in Group IV were given sodium glycollate and cysteine. Samples of 24 h urine were collected and analysed to determine the content of calcium, oxalate, phosphorus and magnesium, and the organic acids, glycollic, glyoxylic and citric acid. Hyperoxaluric rats treated with cysteine had significantly lower excretion rates of calcium, oxalate, phosphorus, glycollic and glyoxylic acid than the glycollate fed rats. When cysteine was given, the excretion of urinary citric acid and magnesium were also increased. Cysteine may be a useful agent in preventing oxalate urolithiasis.
    British Journal of Urology 08/1996; 78(1):22-4.
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    ABSTRACT: Calcium oxalate crystal growth and aggregation leads to the formation of renal calculi. It is known to be inhibited by several compounds both in vitro and in vivo conditions. The present study highlights the inhibitory potential of sodium pentosan polysulphate (SPP), a semi-synthetic glycosaminoglycan (GAG) on calcium oxalate crystal growth in vitro. Its efficacy was compared with those of known inhibitors like pyrophosphate, heparin and chondroitin-4-sulphate. Of the above compounds pyrophosphate was found to be the most potent inhibitor. Among the GAGs, SPP exhibited 80% inhibitory activity as compared to heparin. A lesser degree of inhibition was observed with chondroitin-4-sulphate.
    Molecular and Cellular Biochemistry 04/1996; 156(1):31-5. · 2.33 Impact Factor
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    ABSTRACT: Oxalate, the major stone-forming constituent induces lipid peroxidation during lithogenesis. In experimental condition oxalate formation was induced by the administration of its precursor glycollate. Glycollate-fed rats showed increased susceptibility to lipid peroxidation in the presence of promoters. In addition, antioxidant enzymes-catalase, superoxide dismutase and glutathione peroxidase also showed decreased activity. Reduced glutathione, total thiols and ascorbic acid were also significantly decreased. On the other hand, an increased xanthine oxidase and decreased glucose-6-phosphate dehydrogenase activity was also observed upon glycollate administration. Cysteine, a sulphydryl compound, is known to inhibit free radical toxicity in various pathologies. Cysteine administration to glycollate-fed rats brought about a significant decrease in the peroxidative level, with an increase in the antioxidant status.
    Pharmacological Research 10/1995; 32(3):165-9. · 4.35 Impact Factor

Publication Stats

264 Citations
40.55 Total Impact Points

Institutions

  • 2001–2005
    • University of Texas Health Science Center at San Antonio
      • Division of Hospital Medicine
      San Antonio, TX, United States
  • 1995–1998
    • University of Madras
      • • Department of Biochemistry
      • • Department of Medical Biochemistry
      Chennai, State of Tamil Nadu, India