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

描述(申请人提供)：对于部分出口梗阻，膀胱能够通过复杂的重塑过程经历肥大，从而适应新的工作负荷。这种重塑是由许多信号级联及其相应的转录因子驱动的。这项建议侧重于钙调神经磷酸酶途径及其相关转录因子，活化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NFAT的核输入有关。应用环孢素A(CsA)后，所致的膀胱肥大减轻，肌球蛋白重链(MHC)mRNA亚型逆转回到正常表达模式。我们建议利用含有NFAT-荧光素酶报告基因的转基因小鼠，在CsA存在和不同时间点给药的情况下，在部分膀胱出口梗阻小鼠模型中进一步研究这些观察结果。终点将包括膀胱质量、体外收缩和缩短速度的测定、形态和MHC亚型表达的分子分析。我们还寻求原位定位，其膀胱壁细胞群(S)显示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合成的部位，并确定这些DNA合成部位代表成纤维细胞、平滑肌细胞，还是两者兼而有之。这些体内实验与使用培养细胞的体外实验相补充，以评估机械变形对NFAT和MEF-2移位到细胞核的影响。这种方法将允许在体外筛选可以防止钙调神经磷酸酶激活的化合物，并将允许研究NFAT、MEF-2和平滑肌MHC启动子之间的相互作用。利用微阵列方法，我们建议在两个细胞群中鉴定NFAT和MEF-2反应靶基因：膀胱平滑肌细胞和膀胱成纤维细胞。当人们考虑到尿失禁和肾功能衰竭的社会破坏性后遗症时，这项拟议的工作与人类疾病的相关性是显而易见的，这可能是由于未经治疗的膀胱壁肥大造成的。 公共卫生相关性： 人类相关性--两种情况都可能导致儿童严重的膀胱壁肥大、后尿道瓣和脊柱裂。如果不治疗，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，可能会导致肾功能衰竭，因为膀胱失去了在低压下储存尿液的能力。保留和增强自然膀胱功能的新疗法将有助于避免终末期膀胱的发展。

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.