Serine Protease HTRA1 Antagonizes Transforming Growth Factor-β Signaling by Cleaving Its Receptors and Loss of HTRA1 <i>In Vivo</i> Enhances Bone Formation

HTRA1 is a member of the High Temperature Requirement (HTRA1) family of serine proteases, which play a role in several biological and pathological processes. In part, HTRA1 regulation occurs by inhibiting the TGF-β signaling pathway, however the mechanism of inhibition has not been fully defined. Previous studies have shown that HTRA1 is expressed in a variety of tissues, including sites of skeletal development. HTRA1 has also been implicated in the process of bone formation, although the precise manner of regulation is still unknown. This study investigated how HTRA1 regulates TGF-β signaling and examined the in vivo effects of the loss of HTRA1. We demonstrated that recombinant HTRA1 was capable of cleaving both type II and type III TGF-β receptors (TβRII and TβRIII) in vitro in a dose-dependent manner, but it did not affect the integrity of TβRI or TGF-β. Overexpression of HTRA1 led to decreased levels of both TβRII and III on the cell surface but had no effect on TβRI. Silencing HTRA1 expression significantly increased TGF-β binding to the cell surface and TGF-β responsiveness within the cell. To examine the role of HTRA1 in vivo, we generated mice with a targeted gene deletion of HTRA1. Embryonic fibroblasts isolated from these mice displayed an increase in TGF-β-induced expression of several genes known to promote bone formation. Importantly, the loss of HTRA1 in the knockout mice resulted in a marked increase in trabecular bone mass. This study has identified a novel regulatory mechanism by which HTRA1 antagonizes TGF-β signaling, and has shown that HTRA1 plays a key role in the regulation of bone formation.HTRA1属于丝氨酸蛋白酶高温需求蛋白（High Temperature Requirement, HTRA1）家族，该家族成员参与多种生理及病理过程。HTRA1的调控部分通过抑制转化生长因子-β（Transforming Growth Factor-β，TGF-β）信号通路实现，但其具体抑制机制尚未完全阐明。既往研究显示，HTRA1可在多种组织中表达，包括骨骼发育相关区域。HTRA1也被证实参与骨形成过程，但其精确的调控方式仍未明确。本研究探究了HTRA1调控TGF-β信号通路的具体机制，并检测了HTRA1缺失后的体内生物学效应。本研究证实，重组HTRA1在体外可呈剂量依赖性切割Ⅱ型及Ⅲ型TGF-β受体（TβRII和TβRIII），但不会影响Ⅰ型TGF-β受体（TβRI）或TGF-β本身的结构完整性。过表达HTRA1会导致细胞表面TβRII与TβRIII的表达水平显著降低，但对TβRI无影响。沉默HTRA1的表达可显著提升细胞表面TGF-β的结合能力以及细胞内TGF-β的信号响应性。为探究HTRA1在体内的作用，我们构建了HTRA1基因靶向敲除小鼠。从该小鼠中分离的胚胎成纤维细胞，其经TGF-β诱导的多种已知促骨形成基因的表达水平显著升高。尤为重要的是，敲除小鼠中HTRA1的缺失导致骨小梁质量显著增加。本研究揭示了HTRA1拮抗TGF-β信号通路的全新调控机制，并证实HTRA1在骨形成的调控中发挥关键作用。