Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.12.2 (MEK)
18,161 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Angiotensin II (Ang II) is a potent regulator of proximal tubule functions, including transport, metabolism, and cell proliferation. The opossum kidney (OK) cell line is a useful model of renal proximal tubule. Mitogen-activated protein (MAP) kinases are rapidly phosphorylated and activated in response to various agonists. We investigated Ang II effects on serine/threonine kinase cascades in OK cells. The major findings of the present study are that Ang II stimulated MAP kinase kinase (MAPKK), MAP kinase (MAPK), and S6 kinase activities, and that it increased phosphorylation of Raf-1 kinase and p42 MAP kinase in OK cells. These stimulations of kinases were dose-dependent (from 10(-6) to 10(-11) M). The time course of activation was sequential; the peak stimulation was reached at 5 to 10 minutes for Raf-1 kinase, MAPKK and MAPK, and at 20 minutes for S6 kinase. The activation of MAPK was inhibited by approximately 70% with prolonged 24-hour PMA pretreatment or in the presence of calphostin C or H-7. Tyrosine kinase inhibitors (genistein and herbimycin) did not inhibit AngII-induced MAPK activity. This activation of MAPK was also inhibited via AT1 receptor antagonist, Dup753 and pertussis toxin. This evidence suggests that the activation of serine/threonine cascades by Ang II is largely dependent on PMA-sensitive PKC, and is not dependent on tyrosine kinase and pertussis toxin.
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PMID:Sequential activation of MAP kinase cascade by angiotensin II in opossum kidney cells. 858 39

In our present studies utilizing a well characterized proximal tubule cell line, LLCPKcl4, we determined that all four EET regioisomers (5,6-EET, 8,9-EET, 11,12-EET, and 14,15-EET) stimulated [3H]thymidine incorporation, with 14,15-EET being the most potent. In contrast, no mitogenic effects were seen with arachidonic acid, other cP450 arachidonate metabolites (12R-hydroxyeicosatetraenoic acid (12R-HETE), 14,15-dihydroxyeicosatrienoic acid (14,15-DHET), or 20-HETE), or lipoxygenase metabolites (5S-HETE, leukotriene B4, or lipoxin A4). We found that their metabolically more stable sulfonimide (SI) analogs (11,12-EET-SI and 14,15-EET-SI) were also potent mitogens. In addition 14,15-EET-SI also increased cell proliferation as well as expression of both c-fos and egr-1 mRNA. The protein kinase C and A inhibitors, H-7 and H-8, or the cyclooxygenase inhibitor, indomethacin, had no effect upon 14, 15-EET-induced [3H]thymidine incorporation, but the selective tyrosine kinase inhibitor, genistein, significantly inhibited it. Immunoprecipitation and immunoblotting demonstrated increased tyrosine phosphorylation of PI3-kinase and epidermal growth factor receptor (EGFR) within 1 min of EET administration. EETs also stimulated association of PI3-kinase with EGFR. PI3-kinase inhibitors, wortmannin and LY 294002, markedly inhibited 14, 15-EET-SI-stimulated [3H]thymidine incorporation. In addition, 14, 15-EET-SI administration stimulated tyrosine phosphorylation of src homologous and collagen-like protein (SHC) and association of SHC with both growth factor receptor-binding protein (GRB2) and EGFR. Mitogen-activated protein kinase was also activated within 5 min. Pretreatment of the cells with the mitogen-activated protein kinase kinase inhibitor, PD98059, inhibited the 14,15-EET-SI-stimulated [3H]thymidine incorporation. Moreover, immunoblotting indicated that 14,15-EET stimulated tyrosine phosphorylation of the specific pp60(c-src) substrate p120 and c-Src association with EGFR. 14, 15-EET increased src kinase activity within 1 min. Our data indicate that EETs are potent mitogens for renal epithelial cells, and the mitogenic effects of the EETs are mediated, at least in part, by the activation of Src kinase and initiation of a tyrosine kinase phosphorylation cascade.
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PMID:Epoxyeicosatrienoic acids and their sulfonimide derivatives stimulate tyrosine phosphorylation and induce mitogenesis in renal epithelial cells. 978 38

Ischemia/reperfusion (I/R) injury induces both functional and morphological changes in the kidney. Necrosis, predominantly of the proximal tubule (PT), is the hallmark of this model of renal injury, whereas cells of the distal nephron survive, apparently intact. We examined whether differences in cellular outcome of the various regions of the nephron may be due to segmental variation in the activation of the mitogen-activated protein kinases (MAPKs) in response to I/R injury. Whereas c-Jun N-terminal kinase (JNK) is activated in both the cortex and inner stripe of the outer medulla, the extracellular regulated kinase (ERK) pathway is activated only in the inner stripe in which thick ascending limb (TAL) cells predominate. These studies are consistent with the notion that ERK activation is essential for survival. To test this hypothesis directly, we studied an in vitro system in which manipulation of these pathways and their effects on cellular survival could be examined. Oxidant injury was induced in mouse PT and TAL cells in culture by the catabolism of hypoxanthine by xanthine oxidase. PT cells were found to be more sensitive than TAL cells to oxidative stress as assessed by cell counting, light microscopy, propidium iodide uptake, and fluorescence-activated cell sorting (FACS) analysis. Immunoprecipitation/kinase analysis revealed that JNK activation occurred in both cell types, whereas ERK activation occurred only in TAL cells. We then examined the effect of PD-098059, a MAP kinase kinase (MEK)-1 inhibitor of the ERK pathway, on PT and TAL survival. In TAL cells, ERK inhibition reduced cell survival nearly fourfold (P < 0.001) after oxidant exposure. In PT cells, activation of the ERK pathway by insulin-like growth factor I (IGF-I) increased survival by threefold (P < 0.001), and this IGF-I-enhanced cell survival was inhibited by PD-098059. These results indicate that cell survival in the kidney after ischemia may be dependent on ERK activation, suggesting that this pathway may be a target for therapeutic treatment in I/R injury.
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PMID:MAPK activation determines renal epithelial cell survival during oxidative injury. 1044 73

The organic anion transport system in the proximal tubule of the kidney is of major importance for the excretion of a variety of endogenous and potentially toxic exogenous substances. Furthermore, the clearance of model substrates (e.g. para-aminohippurate) of this system is used for the determination of renal blood flow. We investigated regulation of organic anion secretion in a way that allowed us to examine simultaneously regulation of overall transepithelial secretion and to estimate the separate contributions of regulation of the basolateral and apical transport steps to this overall regulation. The data were verified by measurement of initial basolateral uptake rate and initial apical efflux rate. Opossum kidney cells were used as a suitable model system for proximal tubule cells, and [14C]para-aminohippurate was utilized as an organic anion. Stimulation of protein kinase C inhibited transepithelial secretion because of inhibition of both apical efflux and basolateral uptake. Inhibition of the mitogen-activated protein kinase (MAPK) kinase MEK reduced transepithelial secretion via inhibition of basolateral uptake and apical efflux. Epidermal growth factor (EGF) enhanced transepithelial secretion via stimulation of basolateral uptake but did not affect apical efflux. EGF induced stimulation of basolateral uptake was abolished by inhibition of MEK. EGF led to phosphorylation of ERK1/2, which was also abolished by inhibition of MEK. Thus, EGF stimulated basolateral uptake of organic anions via MAPKs. Transepithelial organic anion secretion can be regulated at two sites, at least: basolateral uptake and apical efflux. Both steps are under control of protein kinase C and MAPK. The pathophysiologically relevant growth factor EGF enhances transepithelial secretion via stimulation of basolateral uptake. EGF stimulates basolateral uptake via MEK and ERK1/2. Thus, renal organic anion extraction may be modulated, especially under pathophysiological conditions.
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PMID:Modulation of the basolateral and apical step of transepithelial organic anion secretion in proximal tubular opossum kidney cells. Acute effects of epidermal growth factor and mitogen-activated protein kinase. 1127 30

Protection against ischemic kidney injury is afforded by 24 h of ureteral obstruction (UO) applied 6 or 8 days prior to the ischemia. Uremia or humoral factors are not responsible for the protection, since unilateral UO confers protection on that kidney but not the contralateral kidney. Prior UO results in reduced postischemic outer medullary congestion and leukocyte infiltration. Prior UO results in reduced postischemic phosphorylation of c-Jun N-terminal stress-activated protein kinase 1/2 (JNK1/2), p38, mitogen-activated protein kinase (MAPK) kinase 4 (MKK4), and MKK3/6. Very few cells stain positively for proliferating cell nuclear antigen after obstruction, indicating that subsequent protection against ischemia is not related to proliferation with increased numbers of newly formed daughter cells more resistant to injury. UO increases the expression of heat shock protein (HSP)-25 and HSP-72. The increased HSP-25 expression persists for 6 or 8 days, whereas HSP-72 does not. HSP-25 expression is increased in the proximal tubule cells in the outer stripe of the outer medulla postobstruction, prior to, and 24 h after ischemia. In LLC-PK(1) renal epithelial cells, adenovirus-expressed human HSP-27 confers resistance to chemical anoxia and oxidative stress. Increased HSP-27 expression in LLC-PK(1) cells results in reduced H(2)O(2)-induced phosphorylation of JNK1/2 and p38. In conclusion, prior transient UO renders the kidney resistant to ischemia. This resistance to functional consequences of ischemia is associated with reduced postischemic activation of JNK, p38 MAP kinases, and their upstream MAPK kinases. The persistent increase in HSP-25 that occurs as a result of UO may contribute to the reduction in phosphorylation of MAPKs that have been implicated in adhesion molecule up-regulation and cell death.
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PMID:Prevention of kidney ischemia/reperfusion-induced functional injury, MAPK and MAPK kinase activation, and inflammation by remote transient ureteral obstruction. 1169 40

Aminoglycoside antibiotics (AGAs) are nephrotoxic, with most of the damage confined to the proximal tubule, but the mechanism for cellular toxicity is not clear. It has been previously shown that the extracellular-calcium sensing receptor (CaR) is expressed in intact rat proximal tubule and can be stimulated by the AGA neomycin. To investigate whether CaR could contribute to AGA-induced nephrotoxicity, the acute responses to various AGAs in the proximal tubule-derived opossum kidney (OK) cell line were examined. The presence in OK cells of CaR-related transcripts and protein was demonstrated by northern analyses, reverse transcriptase-PCR, immunocytochemistry, and immunoblotting. OK cells responded to elevated extracellular calcium (Ca(2+)(o)) and neomycin but also to gentamicin and tobramycin with an increase in cytosolic [Ca(2+)]. Ca(2+)(o), neomycin, and gentamicin also activated the extracellular signal-regulated kinases, ERK1 and ERK2. Neomycin-induced ERK activation was both dose- and time-dependent and was attenuated by inhibitors of phosphatidylinositol 3-kinase, phosphatidylinositol bisphosphate (PIP(2))-specific phospholipase C, and MEK1, but not of protein kinase C. Thus, proximal tubular OK cells express a CaR that mediates Ca(2+)(i) mobilization and PIP(2)-PLC-dependent ERK activation in response to AGAs and thus could play a role in AGA-induced nephrotoxicity.
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PMID:Aminoglycosides increase intracellular calcium levels and ERK activity in proximal tubular OK cells expressing the extracellular calcium-sensing receptor. 1203 77

Aquaporin-1 (AQP1) is a water channel that is induced by hypertonicity. The present study was undertaken to clarify the osmoregulation mechanism of AQP1 in renal medullary cells. In cultured mouse medullary (mIMCD-3) cells, AQP1 expression was significantly induced by hypertonic treatment with impermeable solutes, whereas urea had no effect on AQP1 expression. This result indicates the requirement of a hypertonic gradient. Hypertonicity activated ERK, p38 kinase, and JNK in mIMCD-3 cells. Furthermore, all three MAPKs were phosphorylated by the upstream activation of MEK1/2, MKK3/6, and MKK4, respectively. The treatments with MEK inhibitor U0126, p38 kinase inhibitor SB203580, and JNK inhibitor SP600125 significantly attenuated hypertonicity-induced AQP1 expression in mIMCD-3 cells. In addition, hypertonicity-induced AQP1 expression was significantly reduced by both the dominant-negative mutants of JNK1- and JNK2-expressing mIMCD-3 cells. NaCl-inducible activity of AQP1 promoter, which contains a hypertonicity response element, was attenuated in the presence of U0126, SB203580, and SP600125 in a dose-dependent manner and was also significantly reduced by the dominant-negative mutants of JNK1 and JNK2. These data demonstrate that the activation of ERK, p38 kinase, and JNK pathways and the hypertonicity response element in the AQP1 promoter are involved in hypertonicity-induced AQP1 expression in mIMCD-3 cells.
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PMID:Hypertonicity-induced aquaporin-1 (AQP1) expression is mediated by the activation of MAPK pathways and hypertonicity-responsive element in the AQP1 gene. 1260 Sep 99

Endogenous cardiotonic steroids (ECS) are putative ligands of the inhibitory binding site of the membrane sodium pump (Na+, K+-ATPase). There is growing evidence that cardiotonic steroids may promote the growth of cardiac and vascular myocytes, including evidence indicating growth stimulation at concentrations in the same range as circulating ECS concentrations. We investigated four parameters to determine whether ouabain, a proposed ECS, promotes growth of immortalized rat proximal tubule epithelial cells: cell count by hemocytometer; metabolic activity as reflected in the mitochondrial conversion of the tetrazolium salt, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, to its formazan product (MA); DNA synthesis reflected as bromodeoxyuridine incorporation (DNA); and mitosis reflected as histone phosphorylation state detected using anti-phosphohistone 3 antibody (HP). Maximum stimulatory responses were observed at 1 nm ouabain (MA, 20.3% increase, p < 0.01; DNA, 28.4% increase, p < 0.001; HP, maximum response at 0.5 h, 50% increase, p < 0.001). We observed that growth stimulation was associated with stimulation of ERK1/2 phosphorylation (ERK-P), and both growth and ERK-P could be blocked by the MEK inhibitor (U0126, 100 nm). Western blot analysis revealed that the only alpha isoform of Na+, K+-ATPase that could be detected in these cultures was the highly ouabain-resistant alpha1 isoform. Measurement of ouabain inhibition of ion transport in these cultures using 86Rb+ uptake revealed the predominance of the expected ouabain-resistant isoform (IC50 = 24 microm) and an additional minor ( approximately 15%) ouabain-sensitive inhibition with IC50 approximately 30 pm. Similar bimodal transport inhibition curves were obtained in freshly dissected rat proximal tubules. These results indicate that renal epithelial cells may be a sensitive target of the ERK1/2-activating and growth-promoting effects of ouabain even in the presence of ouabain-resistant Na+, K+-ATPase.
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PMID:Ouabain is a potent promoter of growth and activator of ERK1/2 in ouabain-resistant rat renal epithelial cells. 1273 49

Bcl-2 protects cells from apoptosis initiated by a variety of stimuli including loss of cell adhesion. Bcl-2 -/- mice develop renal hypoplastic/cystic dysplasia with renal cyst formation coinciding with renal maturation in normal mice. To gain a better understanding of the role cell-adhesive mechanisms play during renal maturation, we generated proximal tubule and collecting duct cell lines from postnatal day 10 (P10) and P20 bcl-2 +/+ and bcl-2 -/- mice. Very little is known about the role cell-adhesive and migratory mechanisms play during renal maturation. We observed that modulation of cell-adhesive properties, which normally occur in a nephron segment-specific manner during renal maturation, and cell migration were altered in cells from bcl-2 -/- mice. Enhanced migration of bcl-2 -/- proximal tubule cells in a scratch wound assay was completely inhibited by incubation with PP1 (Src inhibitor) and moderately affected by incubation with SB-203580 (p38 inhibitor). These cells expressed increased levels of fibronectin and had numerous central focal adhesions. P20 bcl-2 -/- proximal tubule cells adhered to fibronectin but adhered poorly to collagen, vitronectin, or laminin. Collecting duct cells, similar to proximal tubule cells from bcl-2 -/- mice, demonstrated enhanced migration in a scratch wound assay that was inhibited by incubation with PP1. Migration of these cells was moderately affected by incubation with PD-98059 (MEK inhibitor) or LY-294002 (PI3 kinase inhibitor), whereas incubation with SB-203580 had no effect. P10 bcl-2 -/- collecting duct cells also expressed increased levels of fibronectin but decreased levels of thrombospondin-1 and demonstrated precocious binding to fibronectin and vitronectin compared with bcl-2 +/+ cells. The ability of P20 bcl-2 +/+ collecting duct cells to adhere to fibronectin and vitronectin corresponded with a decline in thrombospondin-1 expression. Therefore, alterations in cell-adhesive and migratory characteristics may be an early indicator of aberrant renal epithelial cell differentiation.
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PMID:Alterations in cell-adhesive and migratory properties of proximal tubule and collecting duct cells from bcl-2 -/- mice. 1529 44

Parathyroid hormone (PTH) inhibits Na+-K+-ATPase activity by serine phosphorylation of the alpha1 subunit through protein kinase C (PKC)- and extracellular signal-regulated kinase (ERK)-dependent pathways. Based on previous studies we postulated that PTH regulates sodium pump activity through isoform-specific PKC-dependent activation of ERK. In the present work utilizing opossum kidney cells, a model of renal proximal tubule, PTH stimulated membrane translocation of PKCalpha by 102 +/- 16% and PKCbetaI by 41 +/- 7% but had no effect on PKCbetaII and PKCzeta. Both PKCalpha and PKCbetaI phosphorylated the Na+-K+-ATPase alpha1 subunit in vitro. PTH increased the activity of PKCalpha but not PKCbetaI. Coimmunoprecipitation assays demonstrated that treatment with PTH enhanced the association between Na+-K+-ATPase alpha1 subunit and PKCalpha, whereas the association between Na+-K+-ATPase alpha1 subunit and PKCbetaI remained unchanged. A PKCalpha inhibitory peptide blocked PTH-stimulated serine phosphorylation of the Na+-K+-ATPase alpha1 subunit and inhibition of Na+-K+-ATPase activity. Pharmacologic inhibition of MEK-1 blocked PTH-stimulated translocation of PKCalpha, whereas transfection of constitutively active MEK-1 cDNA induced translocation of PKCalpha and increased phosphorylation of the Na+-K+-ATPase alpha1 subunit. In contrast, PTH-stimulated ERK activation was not inhibited by pretreatment with the PKCalpha inhibitory peptide. Inhibition of PKCalpha expression by siRNA did not inhibit PTH-mediated ERK activation but significantly reduced PTH-mediated phosphorylation of the Na+-K+-ATPase alpha1 subunit. Pharmacologic inhibition of phosphoinositide 3-kinase blocked PTH-stimulated ERK activation, translocation of PKCalpha, and phosphorylation of the Na+-K+-ATPase alpha1 subunit. We conclude that PTH stimulates Na+-K+-ATPase phosphorylation and decreases the activity of Na+-K+-ATPase by ERK-dependent activation of PKCalpha.
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PMID:Parathyroid hormone-mediated regulation of Na+-K+-ATPase requires ERK-dependent translocation of protein kinase Calpha. 1563 80


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