Role of a TGF-B Regulated Gene in Human and Mouse Osteoblasts and Skeleton

描述（由申请人提供）：雌激素（E2）和TGFb在骨骼生长、成骨细胞（OB）分化和骨质疏松等骨骼疾病中发挥关键作用。尽管E2和TGFb在OB分化和骨重塑中发挥作用，但其在骨组织中的分子机制尚不完全清楚。在本实验室对E2和TGFb在OBs中的作用的研究中，我们发现并鉴定了作为Kruppel转录因子家族（KLF-10）成员的新型TGFb诱导早期基因-1 （TIEG）。结果表明，E2、TGFb和bmp可诱导OBs中TIEG的表达。在过去的资助期内，我们发现TIEG在介导TGFb信号传导中发挥重要作用，因为它抑制Smad 7的表达并诱导Smad 2的表达。为了更好地了解TIEG在骨骼中的功能，我们产生了TIEG-null （TIEG-/-）小鼠，并发现相对于野生型的幼崽，雌性而不是雄性的骨骼更小、更弱，表现为骨质减少。我们已经报道，从TIEG-/-小鼠分离的颅OBs骨矿化和支持破骨细胞发生的能力明显降低。进一步表征这些OB发现Runx2， osterix，碱性磷酸酶和其他重要OB标记基因的表达水平降低。最近，我们已经证明TIEG能够直接调节Runx2的转录，并且Runx2似乎至少在一定程度上负责TIEG-/- OB矿化中观察到的缺陷。我们的初步研究表明，E2以雌激素受体（ER）亚型特异性的方式诱导TIEG的表达。最后，E2在野生型OBs中也能诱导Runx2的表达，但在TIEG-/- OBs中则没有，这表明TIEG在骨中介导E2的作用中起重要作用。基于这些数据，我们的假设是，E2对TIEG的调控，以及随后TIEG对Runx2的调控，至少在一定程度上导致了OBs中观察到的缺陷，而这些缺陷反过来又导致了TIEG-/-小鼠的性别特异性骨减少表型。为了验证这一假设，我们计划确定：1)TIEG调控Runx2表达对TIEG-/- OB中观察到的缺陷的作用，2)E2调控TIEG表达对TIEG-/- OB表型的贡献，3)TIEG介导E2激活OBs中Runx2表达的作用，4)雄性和雌性TIEG-/-小鼠性腺切除术对骨骼表型的影响。这些研究的完成将有助于确定TIEG在OB功能以及骨骼发育和维持中的生物学作用。此外，这些研究将为E2和TIEG调控Runx2表达的机制及其在骨质疏松等骨病中的作用提供新的见解。

DESCRIPTION (provided by applicant): Estrogen (E2) and TGFb play pivotal roles in skeletal growth, osteoblast (OB) differentiation, and bone diseases such as osteoporosis. In spite of their role in OB differentiation and bone remodeling, the molecular mechanisms of E2 and TGFb action in bone tissue are not fully understood. During this laboratory's investigations of E2 and TGFb actions in OBs, we discovered and characterized the novel TGFb Inducible Early Gene-1 (TIEG) as a member of the Kruppel family of transcription factors (KLF-10). TIEG expression in OBs was shown to be induced by E2, TGFb, and BMPs. During the past funding period, we identified an important role for TIEG in mediating TGFb siganling as it represses Smad 7 expression and induces the expression of Smad 2. In order to better understand the function of TIEG in bone, we have generated TIEG-null (TIEG-/-) mice and have found that females, but not males, have smaller and weaker bones, characterized as osteopenic, relative to wild-type littermates. We have reported that calvarial OBs isolated from TIEG-/- mice have a markedly reduced capacity to mineralize bone and to support osteoclastogenesis. Further characterization of these OBs revealed decreased expression levels of Runx2, osterix, alkaline phosphatase, and other important OB marker genes. Recently, we have demonstrated that TIEG is capable of directly regulating the transcription of Runx2 and that Runx2 appears to be, at least in part, responsible for the observed defects in TIEG-/- OB mineralization. Our preliminary studies have shown that E2 induces the expression of TIEG in an estrogen receptor (ER) isoform specific manner. Finally, E2 also induces the expression of Runx2 in wild-type OBs, but not in TIEG-/- OBs, suggesting an important role for TIEG in mediating E2 action in bone. Based on these data, it is our hypothesis that the E2 regulation of TIEG, and the subsequent TIEG regulation of Runx2, is at least in part responsible for the observed defects in OBs which, in turn, leads to a gender-specific osteopenic phenotype in TIEG-/- mice. In order to test this hypothesis, we plan to determine: 1) the role that TIEG regulation of Runx2 expression has on the observed defects in TIEG-/- OBs, 2) the contribution of E2 regulation of TIEG expression to the TIEG-/- OB phenotype, 3) the role of TIEG in mediating E2 activation of Runx2 expression in OBs, and 4) the effects of gonadectomy of male and female TIEG-/- mice on the skeletal phenotype. The completion of these studies will help determine the biological role of TIEG in OB functions as well as skeletal development and maintenance. In addition, these studies will provide new insights into the mechanisms of E2 and TIEG regulation of Runx2 expression and their contribution to bone disease, including osteoporosis.