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S Kartha

Publications and source records attributed to S Kartha.

At least 19 recordsLinked to original sources
PubMed2000

Regulation of cyclin D(1) expression and DNA synthesis by phosphatidylinositol 3-kinase in airway smooth muscle cells.

We have shown in bovine tracheal myocytes that extracellular signal-regulated kinase (ERK) and Rac1 function as upstream activators of transcription from the cyclin D(1) promoter. We now examine the role of phosphatidylinositol (PI) 3-kinase in this process. PI 3-kinase activity was increased by platelet-derived growth factor (PDGF) and attenuated by the PI 3-kinase inhibitors wortmannin and LY294002. These inhibitors also decreased cyclin D(1) promoter activity, protein abundance, and DNA synthesis. Overexpression of the active catalytic subunit of PI 3-kinase (p110(PI) (3-K)CAAX) was sufficient to activate the cyclin D(1) promoter. Wortmannin and LY294002 failed to attenuate PDGF-induced ERK activation, and overexpression of p110(PI) (3-K)CAAX was insufficient to activate ERK. p110(PI) (3-K)CAAX-induced cyclin D(1) promoter activity was not blocked by PD98059, an inhibitor of mitogen-activated protein kinase/ERK kinase. We next examined whether PI 3-kinase and the 21-kD guanidine triphosphatase Rac1 regulate cyclin D(1) promoter activity by similar mechanisms. p110(PI) (3-K)CAAX-induced cyclin D(1) promoter activity was decreased by two inhibitors of Rac1-mediated signaling, catalase and diphenylene iodonium. Further, PDGF, PI 3-kinase, and Rac1 each activated the cyclin D(1) promoter at the cyclic adenosine monophosphate response element binding protein (CREB)/activating transcription factor (ATF)-2 binding site, as evidenced by expression of a CREB/ATF-2 reporter plasmid. Finally, PI 3-kinase and Rac1-induced CREB/ATF-2 transactivation were each inhibited by catalase. Together, these data suggest that in airway smooth muscle (ASM) cells, PI 3-kinase regulates transcription from the cyclin D(1) promoter and DNA synthesis in an ERK-independent manner. Further, PI 3-kinase and Rac1 regulate ASM cell cycle traversal via a common cis-regulatory element in the cyclin D(1) promoter.

Activating Transcription Factor 2↗
PubMed1999

Forskolin inhibits cyclin D1 expression in cultured airway smooth-muscle cells.

Accumulation of intracellular cyclic adenosine monophosphate (cAMP) has been shown to inhibit the growth of cultured airway smooth-muscle cells, but the precise mechanism underlying the antimitogenic action of cAMP in these cells is unknown. We examined the effects of forskolin, an activator of adenylate cyclase, on DNA synthesis, cyclin D1 expression, and cAMP response element-binding protein (CREB) phosphorylation and DNA binding in bovine tracheal myocytes. DNA synthesis was assessed by measurement of [3H]thymidine incorporation. Cyclin D1 protein abundance and CREB phosphorylation were assessed by immunoblotting. Cyclin D1 promoter transcriptional activation was determined by measurement of luciferase activity in cells transiently cotransfected with complementary DNAs encoding the full-length cyclin D1 promoter subcloned into a luciferase reporter and beta-galactosidase (to normalize for transfection efficiency). The binding of nuclear proteins to the cyclin D1 promoter cAMP response element (CRE) was determined by electrophoretic mobility shift assay. We found that forskolin attenuated platelet-derived growth factor-induced DNA synthesis in a concentration-dependent manner. In addition, forskolin pretreatment decreased both cyclin D1 promoter activity and protein levels. Forskolin treatment induced the phosphorylation of CREB and increased the binding of nuclear protein to the cyclin D1 promoter CRE. Finally, addition of an antibody against CREB1 induced supershift of at least one protein-DNA complex. Together, these data suggest that cAMP suppresses cyclin D1 gene expression via phosphorylation and transactivation of CREB. Further studies are needed to determine whether this is the primary mechanism of cAMP-induced growth inhibition, or whether additional pathways are also involved.

Animals↗
PubMed1999

Partial characterization of a novel mitogen-activated protein kinase/extracellular signal-regulated kinase activator in airway smooth-muscle cells.

We demonstrated previously that in bovine tracheal myocytes, pretreatment with either forskolin or histamine significantly reduces both platelet-derived growth factor (PDGF)- and epidermal growth factor- induced Raf-1 activation but fails to inhibit extracellular signal-regulated kinase (ERK) activation substantially, evidence of a Raf-1-independent ERK activation pathway. To identify Raf-1-independent upstream signaling intermediates of mitogen-activated protein kinase/ERK kinase-1 (MEK1), the dual-function kinase required and sufficient for ERK activation in these cells, lysates from forskolin and PDGF-treated bovine tracheal myocytes were resolved using ion exchange chromatography. Kinase activity for MEK1 was assessed by in vitro phosphorylation assay. In all experiments, the major peak of MEK1 phosphorylation activity was detected in fractions 18 through 26 (80 to 160 mM NaCl), with the peak fraction eluting at a NaCl concentration of 140 mM. The ability of these fractions to activate MEK1 was confirmed by examining the phosphorylation of myelin basic protein, a known substrate for ERKs, in the presence of functional MEK1 and ERK1. Fractions containing kinase activity were also probed with antibodies against MEK kinase-1, Raf-1, A-Raf, B-Raf, Mos, and Tpl-2. None of these proteins was detected in fractions containing peak kinase activity, suggesting the presence of a novel PDGF-stimulated, forskolin-insensitive MEK1 kinase. Further separation of fractions holding peak MEK phosphorylation activity by gel filtration suggested an apparent molecular mass of 40 to 45 kD. We conclude that PDGF-induced activation of MEK1 in bovine tracheal myocytes is mediated at least in part by a novel kinase.

Animals↗
PubMed1998

Hydrogen peroxide activates extracellular signal-regulated kinase via protein kinase C, Raf-1, and MEK1.

We have previously demonstrated that hydrogen peroxide (H2O2) treatment of bovine tracheal myocytes increases the activity of extracellular signal-regulated kinases (ERK), serine/threonine kinases of the mitogen-activated protein (MAP) kinase superfamily thought to play a key role in the transduction of mitogenic signals to the cell nucleus. Moreover, H2O2-induced ERK activation was partially reduced by pretreatment with phorbol 12,13-dibutyrate, which depletes protein kinase C (PKC). In this study, we further examined the signaling intermediates responsible for ERK activation by H2O2 in airway smooth muscle, focusing on MAP kinase/ERK kinase (MEK), a dual-function kinase which is required and sufficient for ERK activation in bovine tracheal myocytes; Raf-1, a serine/threonine kinase known to activate MEK; and PKC. Pretreatment of cells with inhibitors of MEK (PD98059), Raf-1 (forskolin), and PKC (chelerythrine) each reduced H2O2-induced ERK activity. In addition, H2O2 treatment significantly increased both MEK1 and Raf-1 activity. No activation of MEK2 was detected. Together these data suggest that H2O2 may stimulate ERK via successive activation of PKC, Raf-1, and MEK1.

Animals↗
PubMed1995

Differential gene expression in migrating renal epithelial cells after wounding.

An in vitro model of wound healing was used to study cell migration that is independent of proliferation during renal regeneration after acute tubular necrosis. Monolayer cultures of high-density, quiescent renal epithelial cells of the BSC-1 line were subjected to scrape wounding and then Northern blot analysis was employed to identify genes that mediate cell migration. After wounding the monolayer, there is maximal induction of the immediate-early genes Egr-1, c-fos, NAK-1, and gro at 1 hour, followed by peak induction of connective tissue growth factor (CTGF) and c-myc at 4 hours. Message levels of urokinase-type plasminogen activator (u-PA) and its inhibitor (PAI-1) and heat shock protein (HSP)-70 are markedly raised 4-8 hours after wounding. Constitutive expression is repressed at 1 hour for transcripts that encode receptors for fibronectin (FN), epidermal growth factor, and hepatocyte growth factor (c-met), and the secreted proteins FN and osteopontin. Expression of genes encoding transforming growth factor (TGF)-beta 1 and -beta 2, retinoic acid receptor alpha, int-1, int-2, and gap junction protein which can play a role in cell movement, appeared unchanged after wounding. Differential expression of genes was a function of cell location relative to the wound; NAK-1, PAI-1, and HSP-70 were induced or stimulated only in cells at the wound edge, u-PA was stimulated in cells away from the wound, and CTGF was induced in each of these populations suggesting that cell-to-cell communication may regulate gene expression after wounding. Adenosine diphosphate, a potent stimulator of cell migration which enhances expression of u-PA and PAI-1 in nonwounded cultures, additively stimulates these genes after wounding and may thereby potentiate wound healing. Thus scrape wounding of renal epithelial cells is followed by induction, stimulation, or repression of specific genes with distinct responses in different populations of cells.

Adenosine Diphosphate↗
PubMed1994

Glyceraldehyde-3-phosphate dehydrogenase modifier protein is associated with microtubules in kidney epithelial cells.

After exposure of monkey kidney epithelial cells to a reduced concentration of K, a known mitogenic signal, the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (G3PD) is activated by a cytosolic protein whose function appears to be novel. A monospecific antibody was used as an immunoprobe to study the contribution of this G3PD modifier protein (MP) to signal transduction. Raising the extracellular Na concentration as well as lowering the K concentration of the medium increased the amount of MP in cytosol and also activated G3PD. Metabolic labeling of cells followed by preparation of detergent-soluble (cytosolic) and detergent-resistant (cytoskeletal) fractions, immunoprecipitation, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of radiolabeled immune precipitates suggested that the protein was also associated with cytoskeleton. Depolymerization of the microtubules with colchicine or nocodazole increased cytosolic immunoreactive MP, whereas cytochalasin D had no effect. Taxol, which stabilizes microtubules, blocked the effects of colchicine or nocodazole. When tubulin, actin, and intermediate filament fractions of the cytoskeleton were prepared, blotted, and probed with specific antibodies, MP was found in the tubulin fraction. These observations suggest that MP is associated with the microtubules and can be displaced into the cytosol, wherein it could activate G3PD and thereby stimulate glycolytic production of ATP during mitogenic signal transduction.

Animals↗
PubMed1992

Cytokeratin reorganization induced by adenosine diphosphate in kidney epithelial cells.

Exogenous adenosine diphosphate (ADP), the most potent mitogen for nontransformed African green monkey kidney epithelial cells of the BSC-1 line, rapidly alters the appearance of the cell monolayer. Examination of the cells with indirect immunofluorescence using monoclonal antibodies reveals a considerable reorganization of cytokeratin filaments without a major change in the pattern of microtubules or microfilaments. In untreated confluent cells, cytokeratin filaments are predominantly confined to a star-like spot in the perinuclear area, but these can be seen to begin to spread within 2 min after addition of ADP. The effect is particularly notable using anti-cytokeratin 8 antibodies. At 6 h this process is complete and produces a well-developed filamentous network throughout the cell. By 12 h, the network appears to collapse, so that the filaments again form a spot in the perinuclear area, a process that is complete by 24 h. Immunoblotting of total cellular proteins reveals no apparent alterations in the amounts of several species of cytokeratins, including cytokeratin 8 and 18, at 3 or 24 h after exposure to ADP. Other purine and pyrimidine nucleotides which do not stimulate DNA synthesis in these cells fail to alter cytokeratin organization, and there is no apparent alteration in the distribution of vimentin, another intermediate filament protein. The rapid ADP-induced cytokeratin reorganization appears to coincide with the induction of early growth-response gene transcription in these cells and may be correlated with the capacity of ADP to subsequently initiate DNA synthesis. This dramatic and reversible cytokeratin reorganization immediately after exposure to ADP may be an important step in the mitogenic signal transduction pathway.

Adenosine Diphosphate↗
PubMed1992

Adenine nucleotides stimulate migration in wounded cultures of kidney epithelial cells.

Adenine nucleotides speed structural and functional recovery when administered after experimental renal injury in the rat and stimulate proliferation of kidney epithelial cells. As cell migration is a component of renal regeneration after acute tubular necrosis, we have used an in vitro model of wound healing to study this process. High density, quiescent monkey kidney epithelial cultures were wounded by mechanically scraping away defined regions of the monolayer to simulate the effect of cell loss after tubular necrosis and the number of cells that migrated into the denuded area was counted. Migration was independent of cell proliferation. Provision of adenosine, adenine nucleotides, or cyclic AMP increased the number of migrating cells and accelerated repair of the wound. Other purine and pyrimidine nucleotides were not effective. Arginine-glycine-aspartic acid-serine peptide, which blocks the binding of extracellular fibronectin to its cell surface receptor, completely inhibited migration in the presence or absence of ADP. Very low concentrations of epidermal growth factor (K0.5 approximately 0.3 ng/ml) stimulated migration, whereas transforming growth factor-beta 2 was inhibitory (Ki approximately 0.2 ng/ml). Thus, adenosine and/or adenine nucleotides released from injured or dying renal cells, or administered exogenously, may stimulate surviving cells in the wounded nephron to migrate along the basement membrane, thereby rapidly restoring tubular structure and function.

Adenine Nucleotides↗
PubMed1991

Signals that release growth factors from renal epithelial cells.

Monkey kidney epithelial cells of the nontransformed BSC-1 line have been used as a model system to investigate growth control. Renal growth in K depletion nephropathy was studied in culture by reducing the K concentration of the medium, which accelerated cell proliferation. This response appeared to be mediated by release of a growth-promoting activity that has an apparent molecular weight of 12,000 to 30,000. Growth stimulation was also observed when the Na concentration of the medium was reduced and was associated with the appearance of two growth-promoting factors (apparent molecular weight, 6,200 and 9,000) that exhibited cell-type specificity. Thus, modest reductions in the extracellular concentration of K or Na result in rapid appearance of autocrine factors that could modulate cell function along the nephron. The most powerful mitogen for BSC-1 cells is adenosine diphosphate (ADP). This nucleotide stimulates expression of several cell cycle-specific genes and proto-oncogenes, and induces secretion of a platelet-derived growth factor-like protein that is not mitogenic for BSC-1 cells. Release of this growth factor by renal epithelial cells in vivo would represent a paracrine mechanism to initiate proliferation of neighboring stromal or vascular cells.

Animals↗
PubMed1990

Kidney epithelial cells release growth factors in response to extracellular signals.

The growth of nontransformed monkey kidney epithelial cells in culture appears to be regulated by the interplay of positive and negative autocrine growth factors. Reduction of the potassium or sodium concentration of the medium induces rapid release of novel growth-promoting activities, whereas addition of the mitogen adenosine diphosphate stimulates the appearance of a platelet-derived growth factor-like protein which could function in a paracrine manner. These observations suggest that autocrine and paracrine growth factors could play an important role in physiological and pathological states in the kidney.

Epithelial Cells↗
PubMed1990

A human early response gene homologous to murine nur77 and rat NGFI-B, and related to the nuclear receptor superfamily.

When a human fetal muscle cDNA library was screened with the human thyroid hormone receptor alpha 2 cDNA at low stringency, we found a weakly hybridizing cDNA. The sequence of the insert was 2498 basepairs, with an open reading frame of 1794 basepairs encoding a protein of 598 amino acids and a predicted molecular mass of 64 kDa. The DNA-binding domain and the ligand-binding domain are similar to those of steroid and thyroid hormone receptors. Moreover, this cDNA is highly homologous to mouse nur77 and rat NGFI-B, which are early response genes induced by nerve growth factor and other serum growth factors. We designated this gene NAK1. The modulation of expression of NAK1 during stimulation of cell growth was studied. The mRNA of NAK1 was induced rapidly and transiently by growth-stimulating agents, such as adenosine diphosphate, in monkey kidney cells (BSC-1), by phytohemagglutinin in human lymphocytes, and by serum stimulation of arrested fibroblasts. It is expressed in human fetal muscle and adult liver, brain, and thyroid. NAK1 could be a nuclear receptor. It will be of great interest to determine the ligand for NAK1 and the genes that are regulated by it.

Amino Acid Sequence↗
PubMed1988

An alternate method utilizing small quantities of ligand for affinity purification of monospecific antibodies.

An alternate method was designed to couple a limited quantity of protein to an affinity support when a conventional technique was unsuccessful. This was achieved through the introduction of a small number of sulfhydryl groups to the ligand by reaction with 2-iminothiolane which resulted in a limited number of reactive sites on the protein. Amino groups on an AH-Sepharose 4B matrix were linked to sulfhydryl groups on the ligand using the heterobifunctional agent m-maleimidobenzoyl sulfosuccinimide ester (sulfo-MBS). This method was employed to prepare an affinity support using a cytosolic protein that activates glyceraldehyde-3-phosphate dehydrogenase as a ligand. Monospecific antibody purified from the affinity column recognized only this protein on a Western blot of a cytosolic extract of kidney epithelial cells.

Animals↗
PubMed1988

Sequence of a human kidney cDNA clone encoding thymosin beta 10.

We have isolated a cDNA clone encoding the 43 amino acid protein, thymosin beta 10, from an adult human kidney cDNA library. A comparison of human and rat thymosin beta 10 cDNA sequences revealed 100% identity for the deduced amino acid sequence and 95% nucleotide identity for the coding region. The thymosin beta 10 cDNA clone was used to probe RNA isolated from monkey kidney epithelial cells of the BSC-1 line. The probe recognized 850 bp and 2.4 kb transcripts in quiescent cells which indicates that the gene is constitutively expressed. The abundance of the 2.4 kb transcript was markedly increased one hour after serum stimulation and declined progressively to a basal value at 24 hours.

Adult↗