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的主要心血管表现（例如，马凡综合征（MFS）和埃勒斯-当洛斯综合征（EDS）），以及非综合征性瓣膜疾病。在人类中，二叶式主动脉瓣儿科患者的POR 4蛋白减少。在成人瓣膜疾病中，在TGF 2受体基因（综合征性瓣膜疾病）和SMAD 2相互作用基因Filamin-A（非综合征性瓣膜疾病）中发现突变。我们已经表明，骨膜蛋白（Postn）敲除小鼠表现出异常ECM重塑和增厚的阀门，并Postn蛋白在流出（OFT）和房室（AV）垫在Tgf 22的情况下下调。相反，Postn-/-小鼠表现出降低的pSmad 2，3水平和钝化的Tgf 2反应性，表明PO 4也可以增强TGF 2信号传导。此外，增厚的Tgfb 2-/-瓣膜中Postn的减少与Tgf 21配体基因表达的增加和Bmp信号的增加相关。从这些数据中不清楚的是Tgf 2和Bmp活性是否在Postn的上游或Tgf 21是否在Postn的下游。介导这些复杂相互作用的信号传导和干扰这些通路的功能结果将在目标1中进行检查。我们已经表明，Postn OFT转录元件位于3.9kb启动子内。与我们的模型一致，该序列包含Tgf 2（TRE）和Bmp（BRE）反应元件。该启动子将用于目的2，以测试在人类结缔组织病中观察到的异常瓣膜肌肉化是否受POR 4调节。谱系分析表明，3.9kb启动子标志着一个多能的OFT垫细胞亚群，也可以有助于AV瓣膜。我们已经将OFT增强子分离为304 bp-Postn最小序列，其包含TREs而不是BREs。尚不清楚额外的顺式元件是否是多能OFT 304 bp-Postn标记细胞的AV垫募集所必需的，以及它们的AV垫募集是否是Tgf 2依赖性的。这些问题将在目标3中回答。该基金的总体计划是确定Postn的上游和下游是什么（目标1），为Postn与人类问题相关的重要功能建立功能测试（目标2），并检查Postn表达细胞的命运以及Tgf 2调节在建立该命运中的作用（目标3）。总之，需要澄清POT与TGF 2和BMP信号传导以及POT功能之间的复杂关系，以增加对人类心脏结缔组织疾病的治疗干预的精确性。公共卫生相关性：细胞外基质和调节分化的关键作用在最近的一项研究中得到了强调，在该研究中，分泌的软骨调节素-I糖蛋白在小鼠中被删除，因为存活的无效心脏瓣膜的正常无血管性被废除，这导致血管内皮因子-A表达增强，血管生成和老化瓣膜钙化。因此，瓣膜甚至不必看起来异常就能引起临床问题（例如，马凡氏综合征、埃勒斯-当洛斯综合征、二尖瓣松弛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（POSTIN）在瓣膜基质重塑过程中发挥的作用;以及TGF 2和BMP信号传导在调节POSTIN中发挥的作用。

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.