Regulation of Periostin by TGFbeta and BMP in Heart Development

描述（由申请人提供）：心脏瓣膜增厚是结缔组织疾病（如马凡氏综合征（MFS）和ehers - danlos综合征（EDS））和非综合征性瓣膜疾病的主要心血管表现。在人类中，患有二尖瓣主动脉瓣的儿童患者中，POSTN蛋白减少。在成人瓣膜疾病中，TGF2受体基因（综合征性瓣膜疾病）和smad2相互作用基因丝蛋白a（非综合征性瓣膜疾病）中发现突变。我们已经证明，骨膜蛋白（Postn）敲除小鼠表现出异常的ECM重塑和瓣膜增厚，并且在缺乏Tgf22的情况下，流出液（OFT）和房室（AV）缓冲中Postn蛋白下调。相反，Postn-/-小鼠表现出psmad2,3水平降低和Tgf2反应性减弱，表明Postn也可能增强Tgf2信号传导。此外，增厚的Tgfb2-/-瓣膜中Postn的减少与Tgf21配体基因表达增加和Bmp信号传导增加相关。从这些数据中尚不清楚的是，Tgf2和Bmp活性是位于Postn的上游，还是Tgf21位于Postn的下游。介导这些复杂相互作用的信号传导和干扰这些途径的功能结果将在Aim 1中进行研究。我们已经证明，Postn OFT转录元件位于一个3.9kb的启动子内。与我们的模型一致，该序列包含Tgf2 （TRE）和Bmp （BRE）响应元素。该启动子将在Aim 2中用于测试人类结缔组织疾病中观察到的异常瓣膜肌肉化是否受POSTN调节。谱系分析表明，3.9kb的启动子标志着OFT缓冲细胞的多能亚群，它也可以促进房室瓣膜的形成。我们将OFT增强子分离为304bp-Postn最小序列，该序列包含TREs而不包含BREs。目前尚不清楚多能OFT 304bp-Postn标记细胞的AV缓冲募集是否需要额外的顺式元件，以及它们的AV缓冲募集是否依赖于tgf2。这些问题将在Aim 3中得到解答。该资助的总体计划是确定什么是Postn的上游和下游（目标1），为与人类问题相关的Postn的重要功能建立功能测试（目标2），并检查表达Postn的细胞的命运和tgf2调节在确定命运中的作用（目标3）。总之，POSTN与TGF2和BMP信号以及POSTN功能之间的复杂关系需要澄清，以增加对人类心脏结缔组织疾病治疗干预的准确性。公共卫生相关性：最近的一项研究强调了细胞外基质和调节分化的关键作用，该研究在小鼠中删除了分泌的软骨调节素- 1糖蛋白，因为可存活的空心脏瓣膜的正常无血管性被废除，导致血管内皮因子- a表达增强，血管生成和钙化老化瓣膜。因此，即使瓣膜看起来不正常也会引起临床问题（如马凡氏综合征、埃勒斯-丹洛斯综合征、二尖瓣软陷综合征和二尖瓣脱垂）。这些拟议的研究将主要集中在确定分泌的细胞外基质蛋白骨膜蛋白（POSTN）在瓣膜基质重塑中起什么作用；以及TGF2和BMP信号在调控POSTN中的作用。

DESCRIPTION (provided by applicant): Cardiac valve thickening is a major cardiovascular manifestation of connective tissue disorders (e.g., Marfan syndrome (MFS) and Ehlers-Danlos syndrome (EDS)), and non-syndromic valvular diseases. In humans POSTN protein is reduced in pediatric patients with bicuspid aortic valve. In adult valvular diseases mutations are found in TGF2 receptor genes (syndromic valve disease) and in the SMAD2-interacting gene Filamin-A (non-syndromic valve disease). We have shown that Periostin (Postn) knockout mice exhibit anomalous ECM remodeling and thickened valves, and that Postn protein is down-regulated in both outflow (OFT) and atrioventricular (AV) cushions in the absence of Tgf22. Conversely, Postn-/- mice exhibit reduced pSmad2,3 levels and blunted Tgf2 responsiveness, indicating that POSTN may also augment TGF2 signaling. Moreover, reduced Postn in the thickened Tgfb2-/- valves correlates with increased Tgf21 ligand gene expression and increased Bmp signaling. What is unclear from these data is whether the Tgf2 and Bmp activities are upstream of Postn or whether Tgf21 is downstream of Postn. The signaling mediating these complex interactions and the functional outcomes of interfering with those pathways will be examined in Aim 1. We have shown that Postn OFT transcriptional elements are located within a 3.9kb promoter. Consistent with our model, this sequence contains both Tgf2 (TRE) and Bmp (BRE) response elements. That promoter will be used in Aim 2 to test whether the aberrant valve muscularization seen in human connective tissue disease is regulated by POSTN. Lineage analysis demonstrates that the 3.9kb promoter marks a multipotent subpopulation of OFT cushion cells which can also contribute to the AV valves. We have isolated the OFT enhancer to a 304bp-Postn minimal sequence that contains the TREs and not the BREs. It is unclear whether additional cis-elements are necessary for AV cushion recruitment of the multipotent OFT 304bp-Postn marked cells, and whether their AV cushion recruitment is Tgf2-dependent. These questions will be answered in Aim 3. The overall scheme of the grant is to determine what is up- and down-stream of Postn (Aim 1), to set up a functional test for an important function of Postn that has relevance to a human problem (Aim 2), and to examine the fate of Postn-expressing cells and the role of Tgf2-regulation in establishing that fate (Aim 3). In summary, the complex relationship between POSTN and TGF2 & BMP signaling, and POSTN function, need clarification to add precision to therapeutic intervention in human connective tissue disorders of the heart. PUBLIC HEALTH RELEVANCE: The pivotal role of extracellular matrix and regulated differentiation is highlighted in a recent study in which the secreted chondromodulin-I glycoprotein was deleted in mice, as the normal avascularity of viable null cardiac valves was abrogated which resulted in enhanced Vascular endothelial factor-A expression, angiogenesis and calcification in aged valves. Thus, valves do not even have to look abnormal to cause clinical problems (e.g. Marfan's syndrome, Ehlers-Danlos syndrome, floppy mitral valve syndrome and mitral valve prolapse). These proposed studies will largely focus on determining what role the secreted extracellular matrix protein Periostin (POSTN) plays during valve matrix remodeling; and the part that TGF2 and BMP signaling plays in regulating POSTN.