NFAT and MEF-2 Choreograph Bladder Wall Remodeling Following Partial Outlet Obstr

描述（由申请人提供）：为了应对部分出口梗阻，膀胱能够通过复杂的重塑过程而肥大，从而使其适应新的工作负荷。这种重塑是由许多信号级联及其相应的转录因子驱动的。该提案重点关注钙调磷酸酶途径及其相关转录因子激活 T 细胞核因子 (NFAT) 和肌细胞增强因子 2 (MEF-2) 的作用。初步数据支持我们的中心假设，即部分膀胱出口梗阻与细胞内钙稳态失调和钙调神经磷酸酶激活以及随后的 NFAT 核输入有关。服用环孢素 A (CSA) 后，所产生的膀胱肥大会减少，并且肌球蛋白重链 (MHC) mRNA 亚型会逆转回正常表达模式。我们建议在部分膀胱出口梗阻的小鼠模型中进一步研究这些观察结果，使用含有 NFAT-荧光素酶报告基因构建体的转基因小鼠，在不同时间点施用和不施用 CSA 的情况下。终点包括膀胱质量、体外收缩性和缩短速度、形态学测定以及 MHC 同工型表达的分子分析。我们还寻求原位定位，哪些膀胱壁细胞群显示出钙调神经磷酸酶激活的证据。我们还试图定位膀胱壁内发生 DNA 合成的位点，并确定这些 DNA 合成位点是否代表成纤维细胞、平滑肌细胞或两者。这些体内实验得到了使用培养细胞的体外实验的补充，以评估机械变形对 NFAT 和 MEF-2 易位到细胞核的影响。这种方法将允许体外筛选可以阻止钙调神经磷酸酶激活的化合物，并允许研究 NFAT、MEF-2 和平滑肌 MHC 启动子之间的相互作用。我们建议使用微阵列方法来鉴定两个细胞群内的 NFAT 和 MEF-2 响应靶基因；膀胱平滑肌和膀胱成纤维细胞。当人们考虑到未经治疗的膀胱壁肥大可能导致尿失禁和肾衰竭的社会破坏性后遗症时，这项拟议工作与人类疾病的相关性是显而易见的。 公共卫生相关性： 人类相关性 - 有两种情况可能导致儿童严重的膀胱壁肥大、后尿道瓣膜和脊柱裂。如果不及时治疗，膀胱壁肥大会导致尿失禁的社会破坏性后遗症，在极端情况下，由于膀胱失去在低压下储存尿液的能力，可能会导致肾衰竭。保留和增强天然膀胱功能的新疗法将有助于避免末期膀胱的发展。

DESCRIPTION (provided by applicant): In response to partial outlet obstruction, the urinary bladder is capable of undergoing hypertrophy via a complex remodeling process that allows it to adapt to its new workload. This remodeling is driven by a number of signaling cascades and their corresponding transcription factors. This proposal focuses on the role of the calcineurin pathway and its associated transcription factors the Nuclear Factor of Activated T Cells (NFAT) and Myocyte Enhancing Factor 2 (MEF-2). Preliminary data support our central hypothesis that partial bladder outlet obstruction is associated with dysregulation of intracellular calcium homeostasis and activation of calcineurin and the subsequent nuclear importation of NFAT. With administration of cyclosporine A (CSA), the resulting bladder hypertrophy is diminished, and there is a reversal of the myosin heavy chain (MHC) mRNA isoforms back towards a normal expression pattern. We propose to further study these observations in a murine model of partial bladder outlet obstruction using a transgenic mouse containing an NFAT-luciferase reporter construct in the presence and absence of CSA administered of varying time points. End points will include bladder mass, in vitro determinations of contractility and shortening velocity, morphology, and a molecular analysis of MHC isoform expression. We also seek to localize in situ, which bladder wall cell population(s) show evidence of calcineurin activation. We also seek to localize the sites where DNA synthesis is taking place within the bladder wall and determine whether these sites of DNA synthesis represent fibroblasts, smooth muscle cells, or both. These in vivo experiments are complemented by in vitro experiments using cultured cells to assess the impact of mechanical deformation upon NFAT and MEF- 2 translocation to the nucleus. This approach will allow for in vitro screening of compounds that can prevent calcineurin activation, and will allow for study of the interaction between NFAT, MEF-2 and the smooth muscle MHC promoter. Using microarray methods we propose to identify the NFAT and MEF-2 responsive target genes within two cell populations; bladder smooth muscle and bladder fibroblasts. The relevance of this proposed work to human disease is apparent when one considers the socially devastating sequelae of urinary incontinence and renal failure that may result from untreated bladder wall hypertrophy. PUBLIC HEALTH RELEVANCE: Human Relevance - Two conditions can result in severe bladder wall hypertrophy in children, posterior urethral valves, and spinal bifida. If left untreated bladder wall hypertrophy leads to the socially devastating sequelae of urinary incontinence and in extreme cases renal failure may result as the bladder loses its ability to store urine at low pressures. New therapy to preserve and enhance native bladder function will help avoid the development of the end stage bladder.