Regulation of gut smooth muscle contraction and relaxation by cytokines

描述（由申请人提供）：已知促炎介质，如IL-1 β和tnf - α，部分通过直接作用于平滑肌来抑制平滑肌收缩性。我们的假设是，这些介质通过诱导或抑制介导收缩和舒张的信号通路中关键靶点的表达来抑制收缩性。在对照和TNBS结肠炎肌条以及新鲜分散和培养的肌肉细胞的初步研究中，我们发现了六种新的细胞因子（IL-1 β）介导的效应，反映了特定信号靶点的表达和/或活性的变化。这包括：(i)上调RGS4和RGS12的表达，导致Gq和gi偶联受体激动剂分别抑制初始Ca2+依赖性收缩；（ii）内源性MLC磷酸酶抑制剂CPI-17下调，导致rhoa依赖性持续收缩受到抑制；（iii）分别调节Ca2+摄取和Ca2+释放的SERCA2和ip3r - 1上调；（iv） PDE4D5和PDE5A表达上调，分别导致cAMP和cGMP的降解增强；(v)通过PKG/ jnk依赖的转录后机制下调可溶性鸟苷环化酶（sGC）的表达，从而抑制cGMP的合成；（vi）通过inos依赖性亚硝基化使腺苷酸环化酶（AC）失活，从而抑制cAMP合成。到目前为止，IL-1 β处理的结肠肌肉所引起的影响也在TNBS结肠炎的结肠肌肉中观察到。具体目的是：(1)表征信号靶点（RGS4/RGS12, CPI-17， SERCA2和IP3R-I）表达和活性的变化，介导初始Ca2+依赖性和持续Ca2+非依赖性收缩；(2)表征调节松弛的信号靶点（PDE3A、PDE4D5、PDE5A、sGC和AC）的表达和活性变化；(3) NF-kappa B和调节激酶（p38 MAP激酶、ERK1/2和PI 3激酶/Akt/ gsk3 β通路）对RGS4/RGS12和CPI-17的转录调控，以及通过PKG/JNK/AP-1/HuR通路对sGC表达的转录后调控。这些研究将在体外和已建立的结肠炎症模型中全面分析炎症细胞因子在细胞水平上抑制收缩性的机制。

DESCRIPTION (provided by applicant): Pro-inflammatory mediators, such as IL-1 beta and TNF-alpha, are known to inhibit smooth muscle contractility in part by acting directly on smooth muscle. Our hypothesis is that these mediators inhibit contractility by inducing or suppressing the expression of critical targets in the signaling pathways mediating contraction and relaxation. In preliminary studies on control and TNBS colitis muscle strips, and on freshly dispersed and cultured muscle cells, we have identified six novel cytokine (IL-1 beta)-mediated effects reflecting changes in the expression and/or activity of specific signaling targets. These include: (i) up-regulation of RGS4 and RGS12 expression, which leads to inhibition of initial Ca2+-dependent contraction by Gq and Gi-coupled receptor agonists, respectively; (ii) down-regulation of the endogenous MLC phosphatase inhibitor, CPI-17, which leads to inhibition of sustained RhoA-dependent contraction; (iii) up-regulation of SERCA2 and IP3R-I, which regulate Ca2+ uptake and Ca2+ release, respectively; (iv) up-regulation of PDE4D5 and PDE5A expression, which leads to greater degradation of cAMP and cGMP, respectively; (v) down-regulation of soluble guanylyl cyclase (sGC) expression via a PKG/JNK-dependent post-transcriptional mechanism, which leads to inhibition of cGMP synthesis; and (vi) inactivation of adenylyl cyclase (AC) via iNOS-dependent nitrosylation, which leads to inhibition of cAMP synthesis. Where examined so far, the effects elicited in IL-1 beta-treated colonic muscle were observed also in colonic muscle from TNBS colitis. The specific aims are to: (1) characterize the changes in the expression and activity of signaling targets (RGS4/RGS12, CPI-17, SERCA2 and IP3R-I) mediating initial Ca2+-dependent and sustained Ca2+-independent contraction; (2) characterize the changes in expression and activity of signaling targets (PDE3A, PDE4D5, PDE5A; sGC and AC) that regulate relaxation; and (3) characterize the transcriptional regulation of RGS4/RGS12 and CPI-17 by NF-kappa B and modulatory kinases (p38 MAP kinase, ERK1/2 and the PI 3-kinase/Akt/GSK3beta pathway), and the post-transcriptional regulation of sGC expression via a PKG/JNK/AP-1/HuR pathway. These studies will provide a comprehensive analysis of the mechanisms by which inflammatory cytokines inhibit contractility at cellular level, both in vitro and in an established model of colonic inflammation.