VIP-induced gene expression in colonic smooth muscle cells

描述（申请人提供）：血管活性肠多肽（VIP）和垂体腺苷酸环化酶激活肽（PACAP）是肠壁中的抑制性神经递质。在过去的几十年里，这些神经递质在平滑肌松弛中的非基因组效应的机制已经被广泛研究。这些神经递质对平滑肌功能的基因组效应尚不清楚。VIP和PACAP与它们在平滑肌细胞上的受体结合，激活腺苷酸环化酶，产生介导平滑肌松弛的环3′-5′腺苷单磷酸（cAMP）。然而，cAMP也是一种众所周知的基因表达介质，通过转录因子CRE结合蛋白（CREB）与cAMP应答元件（CRE）在其靶基因启动子上的结合。我们已经获得了大量的初步数据，表明经典神经递质VIP和PACAP具有重要的新功能，可诱导人类结肠循环平滑肌细胞（HCCSMC）中l型Ca2+通道的成孔α 1c亚基的基因表达。Ca2+内流通过这些通道是兴奋-收缩耦合信号级联的直接早期步骤。我们的初步数据表明，VIP/PACAP也可能是抗炎神经肽，它可以对抗激活转录因子NF-KB的信号级联的启动，从而抑制炎症期间的细胞收缩性。基于这些初步数据，我们的具体目的是研究：1)VIP/ pacap通过cAMP/PKA信号通路诱导HCCSMC中alpha1C基因表达的增强。2)人类基因的顺、反调控通过VIP/ pacap诱导的转录因子CREB磷酸化。3) cAMP/PKA、MAPK、PKC和CaMKII信号通路在VIP和PACAP诱导alpha1C基因中的相互作用。4) VIP/PACAP在HCCSMC中rnyo保护作用的分子和表观遗传机制。本课题为肠抑制性运动神经元中两种重要且丰富的神经递质（VIP和PACAP）对平滑肌功能的基因组调控提供了新的研究方向。这一发现将为l型钙通道的表达调控提供基因组和分子视角，l型钙通道在正常状态和炎症期间的兴奋-收缩耦合中起着关键作用。

DESCRIPTION (provided by applicant): Vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase-activating peptide (PACAP) are the inhibitory neurotransmitters in the gut wall. The mechanisms of the non-genomic effects of these neurotransmitters in smooth muscle relaxation have been investigated extensively over the last several decades. The genomic effects of these neurotransmitters on smooth muscle function are not known. VIP and PACAP, on binding to their receptors on smooth muscle cells, activate adenylate cyclase to produce cyclic 3'-5' adenosine monophosphate (cAMP) that mediates smooth muscle relaxation. However, cAMP is also a well known mediator of gene expression through the binding of transcription factor CRE binding protein (CREB) to the cAMP response element (CRE) on the promoters of its target genes. We have obtained substantial preliminary data that suggest an important novel function of the classic neurotransmitters VIP and PACAP to induce gene expression of the pore-forming alpha1C subunit of L-type Ca2+ channels in human colonic circular smooth muscle cells (HCCSMC). Ca2+ influx through these channels is an immediate early step in the signaling cascade for excitation-contraction coupling. Our preliminary data indicate that VIP/PACAP may also be anti-inflammatory neuropeptides that counter the initiation of the signaling cascade that activates the transcription factor NF-KB resulting in the suppression of cell contractility during inflammation. Based on these preliminary data our specific aims are to investigate: 1) VIP/PACAP-induced enhancement of alpha1C gene expression through cAMP/PKA signaling pathway in HCCSMC. 2) The cis- and trans-regulation of human ?1C promoter by VIP/PACAP-induced phosphorylation of the transcription factor CREB. 3) The interactions between cAMP/PKA, MAPK, PKC and CaMKII signaling pathways for the induction of alpha1C gene by VIP and PACAP. 4) The molecular and epigenetic mechanisms of the rnyo-protective role of VIP/PACAP in HCCSMC. This grant proposal presents a novel direction of research in the genomic regulation of smooth muscle function by two prominent and abundant neurotransmitters (VIP and PACAP) of the enteric inhibitory motor neurons. The findings will provide genomic and molecular insights into regulation of the expression of L-type calcium channels that play a critical role in excitation-contraction coupling in the normal state and during inflammation.