Central pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) decrease the baroreflex sensitivity in trout

Although PACAP and VIP exert diverse actions on heart and blood vessels along the vertebrate phylum, no information is currently available concerning the potential role of these peptides on the regulation of the baroreflex response, a major mechanism for blood pressure homeostasis. Consequently, the goal of this study was to examine in our experimental model, the unanesthetized rainbow trout Oncorhynchus mykiss, whether PACAP and VIP are involved in the regulation of the cardiac baroreflex sensitivity (BRS). Cross-spectral analysis techniques using a fast Fourier transform algorithm were employed to calculate the coherence, phase and gain of the transfer function between spontaneous fluctuations of systolic arterial blood pressure and R-R intervals of the electrocardiogram. The BRS was estimated as the mean of the gain of the transfer function when the coherence between the two signals was high and the phase negative. Compared with vehicle, intracerebroventricular (ICV) injections of trout PACAP-27 and trout VIP (25-100 pmol) dose-dependently reduced the cardiac BRS to the same extent with a threshold dose of 50 pmol for a significant effect. When injected intra-arterially at the same doses as for ICV injections, only the highest dose of VIP (100 pmol) significantly attenuated the BRS. These results suggest that the endogenous peptides PACAP and VIP might be implicated in the central control of cardiac baroreflex functions in trout.     

Hypoxia regulation of expression and angiogenic effects of vasoactive intestinal peptide (VIP) and VIP receptors in LNCaP prostate cancer cells

Vascular endothelial growth factor (VEGF) is a main factor promoting neovascularization (angiogenesis) of solid tumours as prostate carcinoma. Hypoxia stimulates VEGF gene expression by activating the hypoxia-inducible factor-1 (HIF-1alpha). In the present study, the hypoxia-mimicking agent Ni(2+) induced vasoactive intestinal peptide (VIP) expression at both mRNA and peptide levels but it did not modify the expression of VIP receptors (VPAC(1), VPAC(2) and PAC(1) receptors) in androgen-dependent human LNCaP prostate cancer cells. VIP increased the mRNA levels of VPAC(1) and PAC(1) receptors whereas it decreased VPAC(2) receptor mRNA level. These features support that hypoxia up-regulation of VIP gene expression in prostatic carcinoma may lead to VIP regulation of the expression of its receptors by means of autocrine/paracrine mechanisms. Either VIP or hypoxia mimetics with Ni(2+) increased VEGF expression whereas both conditions together resulted in an additive response. It suggests two independent mechanisms for the observed pro-angiogenic activities of VIP and hypoxia. VIP did not stimulate HIF-1alpha mRNA expression but increased the translocation of HIF-1alpha from the cytosolic compartment to the cell nucleus. Moreover, VIP was unable to modify the expression of the HIF-1alpha inhibitor FIH-1 discarding the possibility of an indirect effect of VIP on HIF-1 transactivation.     

Thrombopoietin-induced activation of the mitogen-activated protein kinase (MAPK) pathway in normal megakaryocytes: role in endomitosis.

Thrombopoietin (TPO) plays a critical role in megakaryocyte proliferation and differentiation. Using various cultured cell lines, several recent studies have implicated the mitogen-activated protein kinase (MAPK) pathway in megakaryocyte differentiation. In the study reported here, we examined the role played by thrombopoietin-induced MAPK activity in a cytokine-dependent cell line (BAF3/Mpl) and in primary murine megakaryocytes. In both systems, extracellular signal-regulated protein kinase (ERK) 1 and 2 MAPK phosphorylation was rapidly induced by TPO stimulation. To identify the Mpl domain responsible for MAPK activation, BAF3 cells expressing truncated forms of the Mpl receptor were studied. Phosphorylation of ERKs did not require elements of the cytoplasmic signaling domain distal to Box 2 and was not dependent on phosphorylation of the adapter protein Shc. ERK activation in murine megakaryocytes was maximal at 10 minutes and was markedly decreased over the subsequent 3 hours. Next, the physiologic consequences of MAPK inhibition were studied. Using the MAPK kinase (MEK) inhibitor, PD 98059, blockade of MAPK activity substantially reduced TPO-dependent proliferation in BAF3/Mpl cells and markedly decreased mean megakaryocyte ploidy in cultures. To exclude an indirect effect of MAPK inhibition on stromal cells in whole bone marrow, CD41(+) cells were selected and then cultured in TPO. The number of polyploid megakaryocytes derived from the CD41-selected cells was also significantly reduced by MEK inhibition, as was their geometric mean ploidy. These studies show an important role for MAPK in TPO-induced endomitosis and underscore the value of primary cells when studying the physiologic effects of signaling pathways.     

Transferrin receptor 2 and HFE regulate furin expression via mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/Erk) signaling. Implications for transferrin-dependent hepcidin regulation

Background

Impaired regulation of hepcidin in response to iron is the cause of genetic hemochromatosis associated with defects of HFE and transferrin receptor 2. However, the role of these proteins in the regulation of hepcidin expression is unclear.

Design and Methods

Hepcidin expression, SMAD and extracellular signal-regulated kinase (Erk) phosphorylation and furin expression were analyzed in hepatic HepG2 cells in which HFE and transferrin receptor 2 were down-regulated or expressed, or furin activity specifically inhibited. Furin expression was also analyzed in the liver of transferrin receptor 2 null mice.

Results

We showed that the silencing of HFE and transferrin receptor 2 reduced both Erk phosphorylation and furin expression, that the exogenous expression of the two enhanced the induction of phosphoErk1/2 and furin by holotransferrin, but that this did not occur when the pathogenic HFE mutant C282Y was expressed. Furin, phosphoErk1/2 and phosphoSMAD1/5/8 were down-regulated also in transferrin receptor 2-null mice. Treatment of HepG2 cells with an inhibitor of furin activity caused a strong suppression of hepcidin mRNA, probably due to the inhibition of bone morphogenic protein maturation.

Conclusions

The data indicate that transferrin receptor 2 and HFE are involved in holotransferrin-dependent signaling for the regulation of furin which involved Erk phosphorylation. Furin in turn may control hepcidin expression.     

VEGF通过MAPK途径上调MC3T3-E1及C2C12细胞中Cbfal基因的表达

目的 研究生长因子VEGF对Cbfal的调节作用及其与MAPK信号传导通路的关系.方法 采用瞬时转染技术和RT-PCR方法.结果 在MC3T3-E1细胞中,VEGF(1×10-8～1×10-6g·L-1)作用24小时能够增加Cbfal基因启动子活性,相对荧光素酶活性分别为对照组的1.29(P<0.05),1.28(P<0.01)和1.40(P<0.05).而加入PD98059(10μmol/L)后不但抑制了Cbfal基因启动子活性的基础表达,而且完全阻断了VEGF所诱导的Cbfal基因启动子活性的增加(P<0.05).C2C12细胞中不同浓度的VEGF处理组,其相对荧光素酶活性与对照组相比未见显著性差异.MC3T3-E1细胞中,VEGF处理前后,MC3T3-E1细胞中Cbfal基因mRNA的表达水平未见显著变化.C2C12细胞中,VEGF处理后,第2天和第3天Cbfal基因mRNA的表达由处理前的32.63±4.41,分别增加至46.56±2.70(P<0.01)和50.35 ±5.62(P<0.05).PD98059阻断了C2C12细胞中VEGF对Cbfal基因mRNA表达的上调作用.结论 VEGF部分通过MAPK信号传导通路增加MC3T3-E1和C2C12细胞中小鼠Cbfal基因启动子活性及mRNA的表达.     

肺癌患者丝裂原活化蛋白激酶1(MAPK1)表达水平及临床意义

目的检测肺癌患者血浆中丝裂原活化蛋白激酶1(MAPK1/ERK2)表达水平及探讨其与肺癌类型、临床分期、TNM分期的关系。方法用ELISA法分别检测肺癌组(n=78),良性肺部疾病组(n=27),健康对照组(n=14)血浆中MAPK1/ERK2的水平含量。结果肺癌患者血浆中MAPK1/ERK2的含量高于良性肺部疾病组和健康对照组(P<0.05),良性肺部疾病组高于健康对照组(P<0.05)。肺癌患者血浆中MAPK1/ERK2的表达与性别,年龄,吸烟状况,肿瘤病理类型,临床分期,TNM分期无显著性差异。结论 MAPK1/ERK2对肺癌的辅助诊断有一定的临床价值,可作为一项新的肺癌生物标志物应用。     

Clostridium difficile toxin A-induced colonocyte apoptosis involves p53-dependent p21(WAF1/CIP1) induction via p38 mitogen-activated protein kinase

BACKGROUND & AIMS: Clostridium difficile toxin A causes marked apoptosis of colonocytes in vivo and in vitro, which contributes to the formation of ulcers and pseudomembranes. We investigated the role of p53-dependent pathways and p38 mitogen-activated protein kinase (p38) in toxin A-induced colonocyte apoptosis. METHODS: The effects of the activation of p53 and p53-dependent pathways including p21(WAF1/CIP1) were assessed in nontransformed human colonic NCM460 epithelial cells exposed to toxin A. Phosphorylation of p53 protein by p38 was measured by in vitro kinase assay, whereas p21 induction by activated p53 was determined by gel shift assays and RNA silencing (small interfering RNA). The relationship between colonocyte apoptosis and p38/p53-dependent pathways was studied in intact mice. RESULTS: Toxin A stimulated p38 and p53 activation and induced cell cycle arrest (G(2)-M) with persistent expression of p21(WAF1/CIP1). Blockage of p38 by SB203580 inhibited p53 phosphorylation and induction of p21(WAF1/CIP1). In intact mice, p38 blockade suppressed toxin A-mediated destruction of intestinal villi, p21(WAF1/CIP1) expression, and enterocyte apoptosis. In addition, toxin A-mediated p21(WAF1/CIP1) and Bak induction, cytochrome c release, and caspase-3 activation were markedly attenuated in p53-silenced colonocytes, despite active p38. Overexpression of p21(WAF1/CIP1) triggered apoptosis and increased toxin A-associated colonocyte apoptosis. CONCLUSIONS: The signaling pathway for colonocyte apoptosis following toxin A exposure involves p38-dependent activation of p53 and subsequent induction of p21(WAF1/CIP1), resulting in cytochrome c release and caspase-3 activation through Bak induction.     

Tumor necrosis factor alpha rapidly activates the mitogen-activated protein kinase (MAPK) cascade in a MAPK kinase kinase-dependent, c-Raf-1-independent fashion in mouse macrophages.

Tumor necrosis factor alpha (TNF alpha) is bound by two cell surface receptors, CD120a (p55) and CD120b (p75), that belong to the TNF/nerve growth factor receptor family and whose signaling is initiated by receptor multimerization in the plane of the plasma membrane. The initial signaling events activated by receptor crosslinking are unknown, although activation of the mitogen-activated protein kinase (MAPK) cascade occurs shortly after ligand binding to CD120a. In this study, we investigated the upstream kinases that mediate the activation of the 42-kDa MAPK p42mapk/erk2 following crosslinking of CD120a in mouse macrophages. Exposure of mouse macrophages to TNF alpha stimulated a time-dependent increase in the activity of MAPK/ERK kinase (MEK) that temporally preceded peak activation of p42mapk/erk2. MEKs, dual-specificity threonine/tyrosine kinases, act as a convergence point for several signaling pathways including Ras/Raf, MEK kinase (MEKK), and Mos. Incubation of macrophages with TNF alpha was found to transiently stimulate a MEKK that peaked in activity within 30 sec of exposure and progressively declined toward basal levels by 5 min. By contrast, under these conditions, activation of either c-Raf-1 or Raf-B was not detected. These data suggest that the activation of the MAPK cascade in response to TNF alpha is mediated by the sequential activation of a MEKK and a MEK in a c-Raf-1- and Raf-B-independent fashion.     

Over-expression of the mitogen-activated protein kinase (MAPK) kinase (MEK)-MAPK in hepatocellular carcinoma: Its role in tumor progression and apoptosis

Background  

Hepatocellular carcinoma (HCC) is one of the most common malignancies in South East Asia. Although activation of the MEK-MAPK is often associated with cellular growth, the role of MEK-MAPK in growth and survival of hepatocarcinoma cells has not been established.     

Vasoactive intestinal polypeptide and pituitary adenylate cyclase-activating polypeptides stimulate mitogen-activated protein kinase in the pituitary cell line GH4C1 by a 3',5'-cyclic adenosine monophosphate pathway

Vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP38) regulate anterior pituitary cell secretion and proliferation. In the somatolactotrope GH4C1 cell line, these effects are mediated through the type-II-like PACAP receptor (VPAC2) coupled to the cAMP pathway. In this study, the control of the extracellularly responsive kinases (ERKs) by VIP and PACAP38 was investigated in GH4C1 cells. VIP and PACAP38 increased ERK1 and ERK2 phosphorylation and were equipotent stimulators of both kinases. ERK activation was mimicked by cholera toxin, forskolin and 8bromo-cAMP. VIP and PACAP38 activation of ERK2 was blocked by the protein kinase A inhibitor H89, whereas the protein kinase C inhibitor GF109203X, or prior PMA-induced depletion of the protein kinases C, failed to inhibit VIP and PACAP38 activation of ERK2. In contrast, thyrotropin-releasing hormone (TRH) elicited ERK activation by a PKC-dependent process. ERK activation by VIP or PACAP38 and TRH were additive and both sensitive to the MEK inhibitors PD98059 and U0126. In parallel, U0126 reduced prolactin (PRL) mRNA levels induced by VIP. These results demonstrate for the first time that VIP and PACAP38 activate ERK in GH4C1 cells. Cyclic AMP increase is sufficient to elicit ERK activation in these cells and thus likely to represent the transduction pathway underlying VIP- and PACAP38-dependent ERK activation. This mechanism seems to be involved in VIP-induced PRL gene regulation.