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PubMed2001

Activation of the Akt/protein kinase B signaling pathway is associated with granulosa cell survival.

Follicles from the hen ovary that have been selected into the preovulatory hierarchy are committed to ovulation and rarely become atretic under normal physiological conditions. In part, this is attributed to the resistance of the granulosa layer to apoptosis. The present studies were conducted to evaluate the role of the phosphatidylinositol (PI) 3-kinase/Akt signaling pathway in hen granulosa cell survival and, by implication, follicle viability. Cloning of the chicken akt2 homologue revealed a high degree of amino acid homology to its mammalian counterparts within the catalytic domain, plus complete conservation of the putative Thr(308) and Ser(474) phosphorylation sites. Treatment of granulosa cells from the three largest preovulatory follicles with insulin-like growth factor (IGF)-I and, to a lesser extent, transforming growth factor (TGF)-alpha induces rapid phosphorylation of Akt, and such phosphorylation is effectively blocked by the PI 3-kinase-inhibitor LY294006. Serum withdrawal from cultured cells for 33-44 h initiates oligonucleosome formation, an indicator of apoptotic cell death, whereas cotreatment with IGF-I prevents this effect. Moreover, treatment of cultured cells for 20 h with LY294006 induces apoptosis. The potential for nonspecific cell toxicity following LY294006 treatment is considered unlikely because of the ability of either LH or 8-bromo cAMP cotreatment to block LY294006-induced cell death. Finally, both IGF-I and TGF-alpha also activate mitogen-activated protein (MAP) kinase signaling, at least in part, through the phosphorylation of ERK: However, treatment with neither U0126 nor PD98059 (inhibitors of MAP kinase kinase) induced cell death in cultured granulosa cells, despite the ability of each inhibitor to effectively block Erk phosphorylation. Taken together, these results provide evidence for a role of the Akt signaling pathway in promoting cell survival within the preovulatory follicle granulosa layer. In addition, the data indicate the importance of an alternative survival pathway mediated via gonadotropins and protein kinase A independent of Akt signaling.

Animals↗
PubMed2005

The hedgehog signalling pathway in the gastrointestinal tract: implications for development, homeostasis, and disease.

The hedgehog signalling pathway is critical to normal mammalian gastrointestinal development. Through epithelial-mesenchymal interactions, hedgehog signalling ensures appropriate axial patterning of the embryonic gut. Congenital abnormalities, including malrotations, anorectal malformations, and tracheoesophageal fistula are associated with germ-line mutations/deletion of genes encoding hedgehog signalling components in man and present in genetically engineered animal models. In adults, there is evidence that the pathway plays a role in maintaining stem cell populations in the stomach and directing epithelial cell differentiation in the intestine. Recent data implicate hedgehog signalling in the formation and maintenance of a number of malignancies, including those of the upper gastrointestinal (GI) tract and pancreas, in which abrogation of the pathway offers a novel therapeutic approach in animal models. Most recently, evidence in vitro indicates that there is a recapitulation of embryonic hedgehog signalling in acute epithelial injury and chronic inflammation, a finding with key implications for inflammatory disorders of the intestine, such as inflammatory bowel diseases. This pathway may provide an important link between chronic inflammation and cancer. We summarize the available evidence demonstrating that this developmental pathway has continuing roles in adult homeostasis and is dysregulated in malignancy and inflammation of the gastrointestinal tract.

Animals↗
PubMedDate not supplied

Signalling pathways activated by 5-HT(1B)/5-HT(1D) receptors in native smooth muscle and primary cultures of rabbit renal artery smooth muscle cells.

The potential of primary cultures of rabbit renal artery vascular smooth muscle cells (VSMCs) was assessed as a means to investigate the signalling pathways linked to 5-hydroxytryptamine (5-HT) 5-HT(1B)/5-HT(1D) receptors in native arteries. In renal artery segments denuded of endothelium, incubated with ketanserin and prazosin (each 1 microM), and prestimulated with 20 mM K(+) Krebs buffer, 5-HT and CP 93,129, a 5-HT(1B) receptor agonist, evoked concentration-dependent contractions. GR 127935, a 5-HT(1B)/5-HT(1D) receptor antagonist, significantly antagonised 5-HT-evoked contractions at nanomolar concentrations. Reverse transcription polymerase chain reaction (RT-PCR) of mRNA from smooth muscle cells from the isolated renal artery and from primary cultures of VSMCs from the same artery expressed mRNA transcripts for the 5-HT(1B) receptor and the 5-HT(1D) receptor in both preparations. The sequence of the PCR fragments corresponded to the known sequence for these receptors. Application of 5-HT evoked a concentration-dependent, pertussis toxin (PTx)-sensitive reduction in cyclic AMP in both cultured cells and intact artery (cyclic AMP concentration reduced by 65.53 +/- 3.33 and 52.65 +/- 5.34% from basal with 10 microM 5-HT, respectively). The effect of 10 microM 5-HT on cAMP was increased in the presence of 20 mM K(+) (reduced by 82.50 +/- 2.50 and 87.54 +/- 3.97%, respectively). In intact arteries, contraction through 5-HT(1B)/5-HT(1D) receptors was significantly attenuated by inhibitors of phosphatidylinositol 3-kinase (wortmannin) and activated mitogen-activated protein kinase (MAPK), MEK (U0126). In the cultured VSMCs, activated MAPK was identified by immunocytochemistry and immunoblotting after stimulation with 5-HT, but only if 20 mM K(+) was present at the onset of stimulation. These data provide the first direct evidence that 5-HT(1B)/5-HT(1B) receptors are linked to the activation of MAPK and indicate that primary cultures of renal VSMCs could provide a model system to study further the signalling pathways linked to these receptors.

Androstadienes↗
PubMed2005

Nitric oxide-cGMP-protein kinase G signaling pathway induces anoxic preconditioning through activation of ATP-sensitive K+ channels in rat hearts.

Nitric oxide (NO) plays an important role in anoxic preconditioning to protect the heart against ischemia-reperfusion injuries. The present work was performed to study better the NO-cGMP-protein kinase G (PKG) signaling pathway in the activation of both sarcolemmal and mitochondrial ATP-sensitive K+ (KATP) channels during anoxic preconditioning (APC) and final influence on reducing anoxia-reperfusion (A/R)-induced cardiac damage in rat hearts. The upstream regulating elements controlling NO-cGMP-PKG signal-induced KATP channel opening that leads to cardioprotection were investigated. The involvement of both inducible and endothelial NO synthases (iNOS and eNOS) in the progression of this signaling pathway was followed. Final cellular outcomes of ischemia-induced injury after different preconditioning in the form of lactate dehydrogenase release, DNA strand breaks, and malondialdehyde formation as indexes of cell injury and lipid peroxidation, respectively, were investigated. The lactate dehydrogenase and malondialdehyde values decreased in the groups that underwent preconditioning periods with specific mitochondrial KATP channels opener diazoxide (100 microM), nonspecific mitochondrial KATP channels opener pinacidil (50 microM), S-nitroso-N-acetylpenicillamine (SNAP, 300 microM), or beta-phenyl-1,N2-etheno-8-bromoguanosine-3',5'-cyclicmonophosphorothioate, Sp-isomer (10 microM) before the A/R period. Preconditioning with SNAP significantly reduced the DNA damage. The effect was blocked by glibenclamide (50 microM), 5-hydroxydecanoate (100 microM), NG-nitro-L-arginine methyl ester (200 microM), and beta-phenyl-1,N2-etheno-8-bromoguanosine-3',5'-cyclic monophosphorothioate, Rp-isomer (1 microM). The results suggest iNOS, rather than eNOS, as the major contributing NO synthase during APC treatment. Moreover, the PKG shows priority over NO as the upstream regulator of NO-cGMP-PKG signal-induced KATP channel opening that leads to cardioprotection during APC treatment.

Animals↗
PubMed1996

Pharmacological targeting of signaling pathways in protein kinase C-stimulated superoxide generation in neutrophil-like HL-60 cells: effect of phorbol ester, arachidonic acid and inhibitors of kinase(s), phosphatase(s) and phospholipase A2.

The purpose of this investigation was to pharmacologically probe the signaling pathways thought to be involved in protein kinase C (PKC)-stimulated superoxide anion (O2-) generation in all-trans retinoic acid-treated human promyelocytic HL-60 cell line (HL-60), targeting PKC, mitogen-activated protein kinase (MAPK), MAPK kinase (MEK), protein serine-threonine phosphatase(s) (PSP), protein tyrosine kinase(s) (PTK) and phosphatase(s) (PTP), secretory phospholipase A2, cyclooxygenase (CO) and 5-lipoxygenase with selected inhibitors. The following agents inhibited phorbol 12-myristate 13-acetate-stimulated O2- generation significantly in the all-trans retinoic acid-treated HL-60 cells (expressed as percentage of control, P < .05): 1) PKC inhibitors: staurosporine (100 nM, 3 +/- 1%); Ro 31-8220 (1 microM, 3 +/- 2%); sphingosine (100 microM, 15 +/- 7%); 2) PSP 1 and 2a inhibitors, okadaic acid (10 microM, 35 +/- 1%); calyculin A (10 microM, 73 +/- 1%); 3) MAPK inhibitor: SB-203580 (100 microM, 62 +/- 1%); 4) PTP inhibitors: phenylarsine oxide (1 microM, 12 +/- 9%); diamide (1 mM, 21 +/- 11%); and 5) secretory phospholipase A2 inhibitors: manoalide (1 microM, 24 +/- 10%); scalaradial (1 microM, 11 +/- 4%). Exogenously added arachidonic acid-stimulated O2- generation in a time- and dose-dependent manner. The following inhibitors enhanced or did not significantly affect phorbol 12-myristate 13-acetate-stimulated O2- generation (expressed as percentage of control): 1) PTK inhibitors: genistein (100 microM, 69 +/- 12%); CGP 53716 (100 microM, 67 +/- 10%); herbimycin A (10 microM, 67.4 +/- 1%); 2) PSP 2b inhibitors: cyclosporin A (30 microM, 71 +/- 5%); FK506 (30 microM, 88 +/- 7%); 3) CO inhibitor: indomethacin (100 microM, 111 +/- 12%); 4) 5-lipoxygenase inhibitor: WY 50,295 (100 microM, 140 +/- 23%); 5) MEK inhibitor: PD98059 (100 microM, 94 +/- 6.7%); and 6) the PTP inhibitor: orthovanadate (100 microM, 131 +/- 25%). Our pharmacological study suggests that, in neutrophil-like HL-60 cells, the signaling pathways leading to PMA-stimulated O2- generation appear to involve PKC, MAPK, phospholipase A2, arachidonic acid, PSP 1 and 2a and PTP. Furthermore, PTK, MEK, CO, 5-lipoxygenase and PSP 2b do not appear to participate in the modulation of PKC-stimulated O2- generation.

Arachidonic Acid↗
PubMed2003

Cold exposure induces tissue-specific modulation of the insulin-signalling pathway in Rattus norvegicus.

Cold exposure provides a reproducible model of improved glucose turnover accompanied by reduced steady state and glucose-induced insulin levels. In the present report we performed immunoprecipitation and immunoblot studies to evaluate the initial and intermediate steps of the insulin-signalling pathway in white and brown adipose tissues, liver and skeletal muscle of rats exposed to cold. Basal and glucose-induced insulin secretion were significantly impaired, while glucose clearance rates during a glucose tolerance test and the constant for glucose decay during a 15 min insulin tolerance test were increased, indicating a significantly improved glucose turnover and insulin sensitivity in rats exposed to cold. Evaluation of protein levels and insulin-induced tyrosine (insulin receptor, insulin receptor substrates (IRS)-1 and -2, ERK (extracellular signal-related kinase)) or serine (Akt; protein kinase B) phosphorylation of proteins of the insulin signalling cascade revealed a tissue-specific pattern of regulation of the molecular events triggered by insulin such that in white adipose tissue and skeletal muscle an impaired molecular response to insulin was detected, while in brown adipose tissue an enhanced response to insulin was evident. In muscle and white and brown adipose tissues, increased 2-deoxy-D-glucose (2-DG) uptake was detected. Thus, during cold exposure there is a tissue-specific regulation of the insulin-signalling pathway, which seems to favour heat-producing brown adipose tissue. Nevertheless, muscle and white adipose tissue are able to take up large amounts of glucose, even in the face of an apparent molecular resistance to insulin.

Adaptation, Physiological↗
PubMed1998

Activation of alpha1-adrenergic receptor during Ca2+ pre-conditioning elicits strong protection against Ca2+ overload injury via protein kinase C signaling pathway.

The objective was to test the hypothesis that transient activation of the alpha1-adrenergic receptor mimics the beneficial effects of Ca2+ preconditioning on the Ca2+ paradox (Ca2+ PD) injury in rat hearts, and that the protection is mediated by protein kinase C (PKC) signaling pathway. Langendorff-perfused rat hearts were subjected to the Ca2+ PD (10 min of Ca2+ depletion followed by 10 min of Ca2+ repletion). The effects of alpha1-adrenergic receptor activation and other interventions on functional, biochemical and pathological changes were assessed. In hearts pretreated with 50 micromol/l phenylephrine, left ventricular end-diastolic pressure and coronary flow were significantly preserved after Ca2+ PD; furthermore, peak loss of lactate dehydrogenase was significantly decreased while ATP was significantly preserved. A remarkable preservation of cell structure was observed in phenylephrine-treated hearts in contrast to non-treated Ca2+ PD hearts. However, pre-conditioning elicited by phenylephrine caused only a mild improvement in left ventricular developed pressure (LVDP) as opposed to its impressive recovery of left ventricular end-diastolic pressure (LVEDP), heart rate (HR), or coronary flow (CF). The salutary effects of phenylephrine on the Ca2+ PD injury were almost similar to those observed in hearts which underwent Ca2+ pre-conditioning (CPC) or were pretreated with 1-stearoyl-2-arachidonoyl-glycerol (SAG), a potent PKC activator. In phenylephrine pretreated hearts, PKC isoform-alpha was localized in the sarcolemma and nucleus, while PKC-delta and PKC-epsilon were localized in the cell membrane, and intercalated disk respectively. Prazosin, a specific alpha1-adrenergic receptor antagonist completely abolished the beneficial effects of phenylephrine on the Ca2+ PD and blocked translocation of PKC isoforms. In addition, prazosin (1 micromol/l) also reversed salutary effects of CPC. Moreover, the beta-adrenergic antagonist, propranolol, had no effect on the protection provided by phenylephrine against the Ca2+ PD injury. This study suggests that the activation of the alpha1-adrenergic receptor confers protection against the lethal injury of the Ca2+ PD via PKC-mediated signaling pathways. The protection is shared by stimuli common with calcium pre-conditioning.

Animals↗
PubMed2005

Endogenous zinc can be a modulator of glycinergic signaling pathway in the rat retina.

Zinc is a modulator of glutamatergic inputs in the hippocampus. In the retina, however, we previously reported that endogenous zinc is present in the non-glutamatergic neural processes and earlier electrophysiological studies suggest that zinc is a modulator of inhibitory signaling pathways, which are mediated by glycine and GABA. AII amacrine cells, a subpopulation of glycinergic amacrine cells, are identified by selective immunoreactivity for parvalbumin in the rat retina. In the present study, therefore, we focused on whether zinc is present in AII amacrine cells using silver amplification combined with immunohistochemistry in the rat retina. We also examined whether zinc modulate glycine response in the rat retina by the patch clamp technique. Association of silver precipitates with the parvalbumin-immunoreactive neural processes was observed at the ultrastructural level. We also found that zinc existed in the neural processes which were not parvalbumin-immunoreactive. Glycine-induced responses were augmented when the concentration of Zn(2+) was below 10 microM, but inhibited at Zn(2+) concentrations of 50 microM or more. Our results suggest the notion that zinc in neural processes of retinal neurons modulates the inhibitory signaling pathway, particularly that mediated by glycine receptors in AII amacrine cells.

Animals↗
PubMed2006

Differential regulation of interleukin 5-stimulated signaling pathways by dynamin.

Through the yeast two-hybrid screen we have identified dynamin-2 as a molecule that interacts with the alpha subunit of the interleukin (IL) 5 receptor. Dynamin-2 is a GTPase that is critical for endocytosis. We have shown that dynamin-2 interacts with the IL-5 receptor-associated tyrosine kinases, Lyn and JAK2, in eosinophils. Tyrosine phosphorylation of dynamin is markedly enhanced upon IL-5 stimulation. The inhibition of tyrosine kinases results in complete abolition of ligand-induced receptor endocytosis. Inhibition of dynamin by a dominant-negative mutant or by small interfering RNA results in enhancement of IL-5-stimulated ERK1/2 signaling and cell proliferation. In contrast, the absence of a functional dynamin does not affect STAT5 or AKT phosphorylation or cell survival. Thus, we have identified specific functions for dynamin in the IL-5 signaling pathway and demonstrated its role in receptor endocytosis and termination of the ERK1/2 signaling pathway.

Cell Proliferation↗
PubMed1999

Latent membrane protein 1 associated signaling pathways are important in tumor cells of Epstein-Barr virus negative Hodgkin's disease.

The latent membrane protein 1 (LMP1) oncogene of Epstein-Barr virus (EBV) is selectively expressed in the Reed-Sternberg (RS) cells of EBV-associated Hodgkin's disease (HD). However, no differences in clinical presentation and course are found between EBV positive and EBV negative forms of HD suggesting a common pathogenetic mechanism. We have studied the LMP1 associated signaling pathways and their dominant negative inhibition in the myelomonocytic HD-MyZ and the B-lymphoid L-428 HD cell lines. In both EBV negative cell lines expression of LMP1 is associated with the formation of multinuclear RS cells. Dominant negative inhibition of NF-kappa B mediated signaling at the step of I kappa B-alpha phosphorylation results in increased cell death with only a few typical RS cells resistant to overexpression of the dominant negative inhibitor I kappa B-alpha-N delta 54. However, dominant negative inhibition of NF-kappa B mediated signaling at the early step of TRAF2 interaction results in the formation of multinuclear cells in both cell lines and, in addition, in clusters of small mononuclear cells in the HD-MyZ cell line. In HD-MyZ cells overexpression of the powerful JBD-inhibitor of the JNK signal transduction pathway is restricted to small cells and never observed in RS cells. These small cells undergo apoptosis as shown by the TUNEL technique. Apoptosis of small cells is still observed after co-transfection of JBD and LMP1 but in addition a few apoptotic HD-MyZ cells with large fused nuclear masses are identified suggesting that specific inhibition of JNK leads also to apoptosis of LMP1 induced RS cells. Thus, activation of the JNK signaling pathway is also important in the formation of Reed-Sternberg cells. Our findings are consistent with a model where all three LMP1 associated functions, i.e. NF-kappa B mediated transcription, TRAF2 dependent signaling, and c-Jun activation act as a common pathogenetic denominator of both EBV negative and EBV positive HD.

Herpesvirus 4, Human↗
PubMed2003

Biomimetic peptides that engage specific integrin-dependent signaling pathways and bind to calcium phosphate surfaces.

Many important matrix proteins involved in bone remodeling contain separate domains that orient the protein on hydroxyapatite and interact with target cell receptors, respectively. We have designed two synthetic peptides that mimic the dual activities of these large, complex proteins by binding to calcium phosphate minerals and by engaging integrin-dependent signaling pathways in osteoblasts. The addition of either PGRGDS from osteopontin or PDGEA from collagen type I to the HAP-binding domain of statherin (N15 domain) did not alter its alpha-helical structure or diminish its affinity for hydroxyapatite. Immobilized N15-PGRGDS bound MC3T3-E1 osteoblasts predominantly via the alpha v beta 3 integrin and induced focal adhesion kinase (FAK) phosphorylation at comparable levels to immobilized osteopontin. Immobilized N15-PDGEA bound MC3T3-E1 osteoblasts predominantly through the alpha 2 beta 1 integrin and induced similar levels of FAK phosphorylation. Although both peptides induced FAK phosphorylation with similar time courses, only the N15-PDGEA peptide induced ERK1/2 phosphorylation, showing that these peptides are also capable of engaging integrin-specific signaling pathways. This peptide system can be used to study adhesion-dependent control of signaling in the context of the relevant biomineral surface and may also be useful in biomaterial and tissue engineering applications.

Adsorption↗
PubMed2004

Contribution of MEK/MAPK and PI3-K signaling pathway to the malignant behavior of Ewing's sarcoma cells: therapeutic prospects.

Insulin-like growth factor receptor I (IGF-I)-mediated circuit is a major autocrine loop for Ewing's sarcoma (ES) cells and appears to be particularly important in the pathogenesis of this tumor. In this study, we analyzed the contribution of the 2 major pathways of the intracellular IGF-IR signaling cascade to the overall effects elicited by IGF-I in ES. Both the mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-kinase (PI3-K) signaling pathways appeared to be constitutively activated in ES, likely due to the presence of the IGF-IR-mediated autocrine loop. We demonstrated that both MEK/MAPK (PD98059 or U0126) and PI3-K inhibitors (LY294002) profoundly impaired ES cell growth in monolayer and soft agar basal conditions. Both PD98059 and LY294002 inhibited ES cell cycle progression by inducing G1 blockage, whereas only LY294002 significantly affected the survival of ES cells. Exogenous IGF-I completely reverted LY294002-induced growth inhibition by abrogating antiproliferative and proapoptotic effects of the PI3-K inhibitor. By contrast, IGF-I could not rescue cells from growth inhibition induced by PD98059. MEK/MAPK blockade also significantly reduced the migratory ability of ES cells, both in basal and IGF-I-induced conditions, and increased chemosensitivity to doxorubicin, a leader drug in the treatment of ES patients. Our findings therefore identify MAPK pathway as a promising target for pharmacologic intervention in ES.

Antineoplastic Agents↗
PubMed2006

The redox regulation of thiol dependent signaling pathways in cancer.

Reactive oxygen species (ROS) play a central role as second messengers in many signal transduction pathways, where they can post-translationally modify proteins via the oxidation of redox sensitive cysteine residues. The range of cellular processes under redox regulation is extensive and includes both the proliferative and apoptotic pathways. Control of the cellular redox environment is therefore essential for normal physiological function and perturbations to this redox balance are characteristic of many pathological states. Oxidative stress is particularly prevalent in cancer, where many malignant cell types possess an abnormal redox metabolism involving down-regulation of antioxidant enzymes and impaired mitochondrial function. This provides a major opportunity to design therapeutic strategies to selectively target cancer cells based on their redox profile. This review will provide a background to this emerging field by summarizing the known redox biochemistry of ROS signaling. The mechanisms of ROS generation by the action of oxidoreductases and nitric oxide synthases will be discussed in conjunction with the cell's major antioxidant defenses, with special emphasis placed on the subcellular location of these redox reactions. The effect of ROS on proliferation and apoptosis will be examined by looking at interactions with transcription factors and the Akt, TNF and MAPK signaling pathways. The review will also outline the major differences in redox metabolism between cancer cells and their non-malignant counterparts. Although the full extent of the ROS regulation of signaling pathways is only beginning to be mapped, early indications are that this paradigm will provide new therapeutic targets for cancer therapy.

Animals↗
PubMed2006

Monoclonal pathogenic antibodies to the thyroid-stimulating hormone receptor in Graves' disease with potent thyroid-stimulating activity but differential blocking activity activate multiple signaling pathways.

The thyroid target Ag for disease-inducing autoantibodies in Graves' disease is the receptor for thyroid-stimulating hormone (TSH), but little is known about the molecular basis of this pathogenic Ab response. We describe the characteristics of two high- affinity mAbs developed from an experimental murine model of hyperthyroid Graves' disease that exhibit potent thyroid-stimulating activity. Nanogram concentrations of the IgG mAbs KSAb1 and KSAb2 and their Fab induce full stimulation of the TSH receptor that is matched by the ligand TSH and, thus, act as full agonists for the receptor. However, KSAb1 and KSAb2 display differential activities in their ability to block TSH-mediated stimulation of the receptor, indicating subtle differences in their biological properties. In displacement studies, IgG and Fabs of KSAb1 and KSAb2 compete with Graves' disease autoantibodies as well as thyroid-blocking Abs present in some hypothyroid patients, indicating a close relationship between these autoimmune determinants on the receptor. In passive transfer studies, single injections of microgram quantities of KSAb1 or KSAb2 IgG led to rapid elevation of serum thyroxine and a hyperthyroid state that was maintained for a number of days. The thyroid glands showed evidence of cell necrosis, but there was no accompanying mononuclear cell infiltrate. In studying their receptor activation pathways, both KSAb1 and KSAb2 provoked phosphorylation of the intracellular ERK1/2 pathway in primary thyrocytes, indicating that multiple signaling pathways may participate in the pathogenesis of Graves' disease. In summary, our findings emphasize the similarities of the experimental mouse model in reproducing the human disorder and provide improved means for characterizing the molecular basis of this pathogenic response.

Animals↗
PubMed2005

Matrix metalloproteinase-1 up-regulation by hepatocyte growth factor in human dermal fibroblasts via ERK signaling pathway involves Ets1 and Fli1.

In this study, we clarified the molecular mechanism(s) underlying the regulation of matrix metalloproteinase (MMP)-1 gene by hepatocyte growth factor (HGF) in cultured human dermal fibroblasts. HGF induced MMP-1 protein as well as mRNA at a transcriptional level via extracellular signal-regulated kinase (ERK) signaling pathway. The region in the MMP-1 promoter mediating the inducible responsiveness to HGF, defined by the transient transfection analysis of the serial 5' deletion constructs, contained an Ets binding site. Mutation of this Ets binding site abrogated the HGF-inducible promoter activity. Ets1 up-regulated the expression of MMP-1 promoter activity, whereas Fli1 had antagonistic effects on them. After HGF treatment, the protein level and the binding activity of Ets1 was increased and those of Fli1 was decreased, which were canceled by PD98059. These results suggest that HGF up-regulates MMP-1 expression via ERK signaling pathway through the balance of Ets1 and Fli1, which may be a novel mechanism of regulating MMP-1 gene expression.

DNA-Binding Proteins↗
PubMed2004

Contribution of the calcineurin signaling pathway to overload-induced skeletal muscle fiber-type transition.

Skeletal muscle is highly adaptable, being capable of undergoing changes in its structural and functional properties in response to physiological stimuli. The fast-to-slow muscle fiber-type transition is evoked by increased motor nerve activity. Recently, the calcineurin (CaN) signaling pathway has been implicated in the transcriptional regulation of slow muscle fiber genes. Here we investigated the effect of treatment with a CaN-specific inhibitor, FK506, on skeletal muscle fiber-type transition in functionally overloaded muscles. The overloaded plantaris muscle showed fast-to-slow muscle fiber type transition, i.e., a decrease in myosin heavy chain (MHC) IIb, an increase in MHCIIa+d/x, and new expression of MHCI. In the FK506-administered group, however, overload-induced muscle fiber-type transition was completely prevented. We have demonstrated, therefore, that the CaN signaling pathway is required for fast-to-slow skeletal muscle fiber-type transition. Furthermore, we also confirmed that the protein expression levels of downstream effectors of CaN signaling exhibit a transient increase in the early phase of the overloaded condition.

Animals↗
PubMed2004

Ethanol-inhibited [3H]thymidine incorporation via protein kinase C-p44/42 mitogen-activated protein kinase/phospholipase A2 signal pathway in renal proximal tubule cells.

BACKGROUND: Ethanol exposure leads to changes of cell proliferation in a variety of cell types. However, how ethanol affects the proliferation of renal proximal tubule cells is not known. METHODS: To examine the effect of ethanol on cell proliferation and its related signaling pathway, [H]thymidine incorporation, release of [H]arachidonic acid (AA), and Western blotting of protein kinase C (PKC)/mitogen-activated protein kinase (MAPK) were performed in primary cultured rabbit renal proximal tubule cells. RESULTS: Ethanol inhibited [H]thymidine incorporation in a time- and dose-dependent manner. An inhibitory effect of ethanol on [H]thymidine incorporation was predominantly observed after 12 hr of treatment with 100 mM ethanol. Ethanol increased AA release and prostaglandin E2 production. In addition, ethanol-induced inhibition of [H]thymidine incorporation was blocked by phospholipase A2 inhibitors and was significantly blocked by PKC inhibitors. Indeed, ethanol induced a PKC translocation from the cytosolic to the membrane fraction. In addition, ethanol-induced inhibition of [H]thymidine incorporation was blocked by PD 98059 (a p44/42 MAPK inhibitor), but not by SB 203580 (a p38 MAPK inhibitor), and ethanol increased the phosphorylation of p44/42 MAPK. Results of phosphorylated p44/42 MAPK by ethanol were consistent with those of [H]thymidine incorporation and [H]AA-release experiments. CONCLUSIONS: Ethanol inhibited [H]thymidine incorporation via PKC, p44/42 MAPK, and phospholipase A2 signaling pathways in primary cultured renal proximal tubule cells.

Animals↗
PubMed2000

Patched represses the Hedgehog signalling pathway by promoting modification of the Smoothened protein.

Hedgehog (Hh) signalling plays a central role in many developmental processes in both vertebrates and invertebrates [1]. The multipass membrane-spanning proteins Patched (Ptc) [2-4] and Smoothened (Smo) [5-7] have been proposed to act as subunits of a putative Hh receptor complex. According to this view, Smo functions as the transducing subunit, the activity of which is blocked by a direct interaction with the ligand-binding subunit, Ptc [8]. Activation of the intracellular signalling pathway occurs when Hh binds to Ptc [8-11], an event assumed to release Smo from Ptc-mediated inhibition. Evidence for a physical interaction between Smo and Ptc is so far limited to studies of the vertebrate versions of these proteins when overexpressed in tissue culture systems [8,12]. To test this model, we have overexpressed the Drosophila Smo protein in vivo and found that increasing the levels of Smo protein per se was not sufficient for activation of the pathway. Immunohistochemical staining of wild-type and transgenic embryos revealed distinct patterns of Smo distribution, depending on which region of the protein was detected by the antibody. Our findings suggest that Smo is modified to yield a non-functional form and this modification is promoted by Ptc in a non-stoichiometric manner.

Animals↗