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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The role and regulation of signal transduction pathways in proliferation and differentiation of intestinal epithelial cells are still poorly understood. However, growing evidences have been recently accumulated demonstrating that mitogen-activated protein kinases (MAPKs) play a pivotal function in the normal development of intestine. We have investigated, in the intestinal cell line HT-29, the regulation (namely activity and phosphorylation degree) of MAP kinases ERK 1 (p44) and ERK 2 (p42) during differentiation. Addition of fetal calf serum to HT-29 undifferentiated resting cells caused a rapid phosphorylation of both ERKs and an increase of their specific kinase activity. Moreover, nuclear translocation of ERK 1 and ERK 2 occurred concurrently to their activation, leading to the conclusion that ERK 1 and ERK 2 are classically regulated when quiescent HT-29 cells are induced to proliferate. Butyrate addition to the intestinal cell line resulted in terminal differentiation and in a selective down-regulation of ERK 2 activity (and phosphorylation degree) without any effect on ERK 1. Conversely, when HT-29 cells were differentiated by repeated passages in a glucose-free medium, we observed a progressive dephosphorylation and inactivation of p42 and p44 kinases along with the failure of serum to activate both the enzymes. Our findings suggest that, during the differentiation of intestinal cells, remarkable changes occur in ERK 1 and ERK 2 control mechanisms leading to an unresponsiveness of MAP kinase pathway.
Mol Cell Biochem 2002 Feb
PMID:Down-regulation of ERK1 and ERK2 activity during differentiation of the intestinal cell line HT-29. 1195 64

The cellular localization, agonist-mediated internalization, and desensitization properties of the alpha(1)-adrenoceptor (alpha(1)-AR) subtypes conjugated with green fluorescent protein (alpha(1)-AR/GFP) were assessed using real-time imaging of living, transiently transfected human embryonic kidney (HEK) 293 cells. The alpha(1B)-AR/GFP fluorescence was detected predominantly on the cell surface. Stimulation of the alpha(1B)-AR with phenylephrine led to an increase in extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation and promoted rapid alpha(1B)-AR/GFP internalization. Long-term exposure (15 h) to phenylephrine resulted in desensitization of the alpha(1B)-AR-mediated activation of ERK1/2 phosphorylation. Alpha(1A)-AR/GFP fluorescence was detected not only on the cell surface but also intracellularly. The rate of internalization of the cell surface population alpha(1A)-AR/GFPs was slower than that seen for the alpha(1B)-AR. Agonist exposure also resulted in desensitization of the alpha(1A)-AR-mediated increase in ERK1/2 phosphorylation. The alpha(1D)-AR/GFP fluorescence was detected mainly intracellularly, and this localization was unaffected by exposure to phenylephrine. Phenylephrine treatment of alpha(1D)-AR/GFP expressing cells increased ERK1/2 phosphorylation. However, this increase was not significant. Cotransfection with beta-arrestin 1 did not increase the rate or extent of agonist-stimulated alpha(1A)- or alpha(1B)-AR/GFP internalization. However, a dominant-negative form of the beta-arrestin 1, beta-arrestin 1 (319-418), blocked agonist-mediated internalization of both the alpha(1A)- and alpha(1B)-ARs. These data show that transfected alpha(1)-AR/GFP fusion proteins are functional, that there are differences in the cellular distribution and agonist-mediated internalization between the alpha(1)-ARs, and that agonist-mediated alpha(1)-AR internalization is dependent on arrestins and can be desensitized by long-term exposure to an agonist. These differences could contribute to the diversity in physiologic responses regulated by the alpha(1)-ARs.
Mol Pharmacol 2002 May
PMID:Differences in the cellular localization and agonist-mediated internalization properties of the alpha(1)-adrenoceptor subtypes. 1196 Nov 18

Induction of low-density lipoprotein (LDL) receptor transcription in response to depletion of cellular sterols in animal cells is well established. The intracellular signal or signals involved in regulating this process, however, remain unknown. Using a specific inhibitor of protein kinase C (PKC), calphostin C, we show the requirement of this kinase in the induction process in human hepatoma HepG2 cells. Overexpression of PKC epsilon, but not PKC alpha, -gamma, -delta, or -zeta was found to dramatically induce (approximately 18-fold) LDL receptor promoter activity. Interestingly, PKC epsilon-mediated induction was found to be sterol resistant. To further establish that PKC epsilon is involved in the sterol regulation of LDL receptor gene transcription, endogenous PKC epsilon was specifically inhibited by transfection with antisense PKC epsilon phosphorothionate oligonucleotides. Antisense treatment decreased endogenous PKC epsilon protein levels and completely blocked induction of LDL receptor transcription following sterol depletion. PKC epsilon-induced LDL receptor transcription is independent of the extracellular signal-regulated kinase 1 and 2 (p42/44(MAPK)) cascade, because the MEK-1/2 inhibitor, PD98059 did not inhibit, even though it blocked p42/44(MAPK) activation. Finally, photoaffinity labeling studies showed an isoform-specific interaction between PKC epsilon and sterols, suggesting that sterols may directly modulate its function by hampering binding of activators. This was confirmed by PKC activity assays. Altogether, these results define a novel signaling pathway leading to induction of LDL receptor transcription following sterol depletion, and a model is proposed to account for a new function for PKC epsilon as part of a sterol-sensitive signal transduction pathway in hepatic cells.
Mol Cell Biol 2002 Jun
PMID:Critical role of diacylglycerol- and phospholipid-regulated protein kinase C epsilon in induction of low-density lipoprotein receptor transcription in response to depletion of cholesterol. 1199 13

Over-expression of alpha7 nicotinic acetylcholine receptor (alpha7nAChR) in PC12 cells, independent of agonistic stimulation, induces marked neurite outgrowth and high capacity for migration and adherence (differentiation-like transformation), and increases tolerance against cell damage. In the present study, we investigated the effects of alpha7nAChR over-expression and nicotine on ERK phosphorylation and N-cadherin expression by comparing 3 groups of cells: PC12 cells transfected with alpha7 subunit cDNA (alpha7pCMV cells); untransfected PC12 cells exposed to 50 microM nicotine (PC12 cells+nicotine); and PC12 cells transfected with vector only (pCMV cells). alpha7 subunit protein was detected in alpha7pCMV cells at 24 to 72 h after transfection. alpha7pCMV cells exhibited sustained expression of phospho-ERKs (p42 and p44) at 24 to 72 h after transfection, and differentiation-like transformation at 72 h after transfection. PC12 cells+nicotine exhibited transient expression of phospho-ERKs at 48 h after addition of nicotine, but did not exhibit differentiation-like transformation. Neither ERK phosphorylation nor differentiation-like transformation was observed in pCMV cells. Expression of surface N-cadherin increased at 72 h after transfection on alpha7pCMV cells, but did not increase on PC12 cells+nicotine or pCMV cells. These findings suggest that, in PC12 cells, over-expression of alpha7nAChR induces sustained activation of ERK, which probably promotes N-cadherin expression and differentiation-like transformation.
Brain Res Mol Brain Res 2002 Oct 15
PMID:Over-expression of alpha7 nicotinic acetylcholine receptor induces sustained ERK phosphorylation and N-cadherin expression in PC12 cells. 1239 68

1,25-Dihydroxyvitamin D3 (1,25D3) exhibits potent antitumor activity in the murine squamous cell carcinoma (SCC) SCCVII/SF, and the combination of 1,25D3 with cisplatin (1,25D3/cisplatin) demonstrates even greater activity. Because these agents possess different mechanisms of cytotoxicity, studies were initiated to define the mechanism by which the combination displays enhanced activity. Median dose-effect analysis demonstrates that 1,25D3 and cisplatin act synergistically to inhibit SCC growth. When SCC cells were treated with 1,25D3 (10 nM) and/or cisplatin (0.5 microg/ml), greater caspase-3 activation was observed for the combination than for either agent alone. This suggests that the enhanced cytotoxicity is, at least in part, due to greater induction of apoptosis. No alterations in cellular platinum concentration or platinum-DNA adducts were observed for 1,25D3/cisplatin cotreatment compared with cisplatin treatment alone. Effects of the combination on cisplatin and 1,25D3 signaling pathways in adherent (nonapoptotic) and floating (apoptotic) cells were explored. Cisplatin induced p53 and its downstream targets, p21(Cip1) (p21) and Bax, in both cell populations. In contrast, 1,25D3 reduced p53, p21, and Bax to nearly undetectable levels in adherent cells. In the floating cells, 1,25D3 reduced levels of p53 and p21, but Bax expression was maintained at control levels. Expression of these proteins in cells treated with 1,25D3/cisplatin was similar to treatment with 1,25D3 alone. The two agents also had divergent effects on survival and stress signaling pathways. Phospho-extracellular signal-regulated kinase 1/2 and phospho-Jun levels increased after treatment with cisplatin but decreased after treatment with 1,25D3 and 1,25D3/cisplatin. Moreover, cisplatin decreased levels of mitogen-activated protein kinase kinase kinase (MEKK-1), whereas 1,25D3 up-regulated MEKK-1, and 1,25D3/cisplatin further up-regulated MEKK-1. We propose that the increased cytotoxicity for 1,25D3/cisplatin results from cisplatin enhancement of 1,25D3-induced apoptotic signaling through MEKK-1.
Mol Cancer Ther 2002 Aug
PMID:Cisplatin potentiates 1,25-dihydroxyvitamin D3-induced apoptosis in association with increased mitogen-activated protein kinase kinase kinase 1 (MEKK-1) expression. 1249 15

Hydrogen peroxide mediates vasodilation, but the mechanisms responsible for this process remain undefined. We examined the effect of H(2)O(2) on nitric oxide (NO*) production and the signaling events involved. NO* release from bovine aortic endothelial cells was detected with an NO*-specific microelectrode. The addition of H(2)O(2) caused a potent dose-dependent increase in NO* production. This was partially Ca(2+)-dependent because BAPTA/AM reduced NO* production at low (<50 microM) but not high (>100 microM) concentrations of H(2)O(2). Phosphatidylinositol (PI) 3-kinase inhibition [with wortmannin or 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride], infection with a dominant-negative mutant of Akt, or mitogen-activated protein kinase kinase/extracellular signal-regulated kinase 1/2 (MEK/ERK1/2) inhibition (with PD98059 or U0126) partially attenuated, whereas inhibition of both PI 3-kinase and MEK1/2 abolished H(2)O(2)-dependent NO* production. ERK1/2 seemed necessary for NO* production early (<5 min) after H(2)O(2) addition, whereas PI 3-kinase/Akt was more important at later time points. Phosphorylation of endothelial nitric-oxide synthase (eNOS) at serine 1179 was observed >10 min after the addition of H(2)O(2), and this was prevented by wortmannin but not by PD98059. c-Src family tyrosine kinase(s) was found to be upstream of H(2)O(2)-dependent Akt and eNOS serine 1179 phosphorylation and subsequent NO* production. In summary, H(2)O(2) causes endothelial NO* release mediated by cooperative effects between PI 3-kinase/Akt-dependent eNOS serine 1179 phosphorylation and activation of MEK/ERK1/2. This may represent an acute cellular adaptation to an increase in oxidant stress.
Mol Pharmacol 2003 Feb
PMID:Akt-dependent phosphorylation of serine 1179 and mitogen-activated protein kinase kinase/extracellular signal-regulated kinase 1/2 cooperatively mediate activation of the endothelial nitric-oxide synthase by hydrogen peroxide. 1252 3

Interleukin (IL)-9 is a pleiotropic cytokine that has been proposed as a candidate gene for asthma. As IL-9 expression is correlated with airway hyperresponsiveness in animals, we examined the effects of IL-9 on cultured human airway smooth muscle (HASM) cells. IL-9 alone had no effect on IL-8 release, but at concentrations of > or =30 ng/ml, IL-9 significantly increased IL-8 release induced by TNF-alpha. IL-9 increased phosphorylation of extracellular signal-regulated protein kinase (ERK, p42 and p44) in a concentration- and time-dependent fashion, and U-0126 (10 micro M), which inhibits ERK phosphorylation, abolished the synergism between TNF-alpha and IL-9 on IL-8 release. IL-9 alone had no effect on eotaxin release into HASM cell supernatants but at concentrations of > or =10 ng/ml caused an approximately 50% increase in release of eotaxin evoked by IL-13 (10 ng/ml). U-0126 blocked the synergism between IL-9 and IL-13 on eotaxin release. IL-9 had no effect on cyclooxygenase-2 (COX-2) expression or PGE(2) release and did not augment the COX-2 expression that was induced by IL-1beta. Our results indicate that airway smooth muscle is a target for IL-9 and that IL-9 amplifies the potential for these cells to recruit eosinophils and neutrophils into the airways by a mechanism involving ERK.
Am J Physiol Lung Cell Mol Physiol 2003 Jun
PMID:Interleukin-9 influences chemokine release in airway smooth muscle: role of ERK. 1258 3

Norepinephrine (NE) is involved in many cardiovascular diseases such as congestive heart failure. We have recently reported that NE had a comitogenic effect in isolated cardiac fibroblasts, and that it activated p42/p44 mitogen activated protein kinase (MAPK). This study was designed to characterize a possible mechanism involved in the proliferative effect of NE. Isolated rat cardiac fibroblasts were exposed to NE (10 microM) for up to 8 h, and interleukin-6 (IL-6) expression was measured by Ribonuclease Protection Assay and Western blotting. The activity of p42/p44MAPK was analyzed by Western blotting. Cell number was assessed by use of a Coulter Counter. IL-6/GAPDH mRNA was increased by NE in a time-dependent manner reaching 23 fold stimulation after 1 h compared to untreated samples. Immunoreactivity to IL-6 was not found in controls. After 16 h of exposure to NE, IL-6 protein was detected. It further increased up to 48 h. The effect of NE on IL-6 mRNA was abolished by the beta-adrenoceptor blockers propranolol, metoprolol (beta1) and ICI 118.551 (beta2), but not by the alpha-adrenoceptor blockers prazosin (alpha1) and yohimbine (alpha2). The MAPK-inhibitor PD98059 suppressed the NE-induced MAPK activation in a concentration-dependent fashion after 5 min, attenuated the NE-induced IL-6 expression after 2 h, and suppressed the proliferative effect of NE from 53 to 18% after 48 h. Recombinant IL-6 caused an increase in proliferation by 31% after 48 h. Simultaneous application of the IL-6 antibody reduced the NE-induced proliferation to 34%, and completely prevented the IL-6 induced effect. These results suggest that NE induces proliferation of rat cardiac fibroblasts in part by increasing the expression of IL-6 through regulation of MAPK.
Mol Cell Biochem 2003 Jan
PMID:Regulation of norepinephrine-induced proliferation in cardiac fibroblasts by interleukin-6 and p42/p44 mitogen activated protein kinase. 1261 90

Ventilator-induced lung injury syndromes are characterized by profound increases in vascular leakiness and activation of inflammatory processes. To explore whether excessive cyclic stretch (CS) directly causes vascular barrier disruption or enhances endothelial cell sensitivity to edemagenic agents, human pulmonary artery endothelial cells (HPAEC) were exposed to physiologically (5% elongation) or pathologically (18% elongation) relevant levels of strain. CS produced rapid (10 min) increases in myosin light chain (MLC) phosphorylation, activation of p38 and extracellular signal-related kinase 1/2 MAP kinases, and actomyosin remodeling. Acute (15 min) and chronic (48 h) CS markedly enhanced thrombin-induced MLC phosphorylation (2.1-fold and 3.2-fold for 15-min CS at 5 and 18% elongation and 2.1-fold and 3.1-fold for 48-h CS at 5 and 18% elongation, respectively). HPAEC preconditioned at 18% CS, but not at 5% CS, exhibited significantly enhanced thrombin-induced reduction in transendothelial electrical resistance but did not affect barrier protective effect of sphingosine-1-phosphate (0.5 microM). Finally, expression profiling analysis revealed a number of genes, including small GTPase rho, apoptosis mediator ZIP kinase, and proteinase activated receptor-2, to be regulated by CS in an amplitude-dependent manner. Thus our study demonstrates a critical role for the magnitude of CS in regulation of agonist-mediated pulmonary endothelial cell permeability and strongly suggests phenotypic regulation of HPAEC barrier properties by CS.
Am J Physiol Lung Cell Mol Physiol 2003 Oct
PMID:Magnitude-dependent regulation of pulmonary endothelial cell barrier function by cyclic stretch. 1295 24

CD40, a tumor necrosis factor receptor superfamily member, is up-regulated on intraheptatic endothelial cells (IHEC) and epithelial cells during inflammatory liver disease, and there is evidence that the functional outcome of CD40 ligation differs between cell types. Ligation of CD40 on cholangiocytes or hepatocytes results in induction of Fas-mediated apoptosis, whereas ligation of IHEC CD40 leads to enhanced chemokine secretion and adhesion molecule expression. We now report that differential activation of two transcription factors, nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1), in primary human hepatocytes or IHEC, is associated with and may explain, in part, the different responses of these cell types to CD40 ligation. CD40 ligation induced a rise in NF-kappaB activity in hepatocytes,which peaked at 2 h and returned to baseline by 24 h; however, IHEC CD40 ligation resulted in a sustained up-regulation of NF-kappaB (>24 h). In hepatocytes, CD40 ligation led to sustained up-regulation of AP-1 activity >24 h associated with increased protein levels of RelA (p65), c-Jun, and c-Fos, whereas no induction of AP-1 activity was observed in IHECs. Analysis of mitogen-activated protein kinase phosphorylation (phospho-extracellular signal-regulated kinase 1/2 and phospho-c-Jun NH(2)-terminal kinase 1/2) and expression of inhibitor kappaBalpha were entirely consistent, and thus confirmed the profiles of NF-kappaB and AP-1 signaling and the effects of the selective inhibitors assessed using electrophoretic mobility shift assay or Western immunoblotting. CD40 ligation resulted in induction of apoptosis in hepatocytes after 24 h, but on IHECs, CD40 ligation resulted in proliferation. Inhibition of (CD40-mediated) NF-kappaB activation prevented IHEC proliferation and led to induction of apoptosis. Selective extracellular signal-regulated kinase and c-Jun NH(2)-terminal kinase inhibitors reduced levels of apoptosis in (CD40-stimulated) hepatocytes by approximately 50%. We conclude that differential activation of these two transcription factors in response to CD40 ligation is associated with differences in cell fate. Transient activation of NF-kappaB and sustained AP-1 activation is associated with apoptosis in hepatocytes, whereas prolonged NF-kappaB activation and a lack of AP-1 activation in IHECs result in proliferation.
Mol Biol Cell 2003 Apr
PMID:Differential induction of nuclear factor-kappaB and activator protein-1 activity after CD40 ligation is associated with primary human hepatocyte apoptosis or intrahepatic endothelial cell proliferation. 1268 91


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