CUL4B在调控间充质干细胞成骨／成脂分化中的作用及其机制研究

CUL4B is a scaffold protein in the Cullin 4B-RING E3 ligase complex (CRL4B). Our previous studies have shown that loss-of-function mutations in CUL4B gene cause developmental defects and embryonic lethality in human and mice, respectively. Using tissue-specific knockout mouse models, we have demonstrated that CUL4B participates in a broad variety of biological processes including neurogenesis, hematopoiesis, and adipogenesis as well as pancreatic δ cell paracrine. The fact that short stature and obesity are common features of the patients with CUL4B mutations implicates that CUL4B could be involved in the regulation of the adipo-osteogenic differentiation of mesenchymal stem cells (MSCs). To test this hypothesis, we will generate MSC-specific Cul4b knockout mice, and determine the effects of CUL4B deficiency in MSCs on bone formation and bone homeostasis as well as the relationship between bone marrow adiposity and age-related bone loss. We will study the underlying mechanisms and define the key molecules/pathways that regulate osteo-adipogenic differentiation of MSCs. The results obtained from this study would not only reveal the novel function of CUL4B, but also be important both for understanding the novel mechanisms and for developing new therapeutic methods for the diseases caused by dysregulation of the osteo-adipogenic differentiation of MSCs, such as osteoporosis and obesity.

CUL4B是泛素连接酶复合物的骨架蛋白，我们的前期研究发现CUL4B丧失功能突变导致人类发育异常（X连锁智力低下综合征）和小鼠胚胎致死。通过特异性敲除特定组织中Cul4b基因，我们发现CUL4B在调控小鼠神经发育、血液细胞分化、脂肪细胞分化和胰岛δ细胞分泌功能等方面发挥重要作用。由于间充质干细胞是成骨细胞、软骨细胞和脂肪细胞共同的前体细胞源，CUL4B突变可能通过改变间充质干细胞分化而引起患者身材矮小、短指和肥胖等表型。为此，本课题将在构建间充质干细胞特异性敲除Cul4b基因小鼠模型的基础上，分析敲除间充质干细胞Cul4b基因对骨骼生长发育、骨稳态维持、脂肪发育和成骨/成脂分化平衡维持的影响及其作用机制，发现CUL4B调控间充质干细胞成骨/成脂分化的关键分子及其调控机制，为理解CUL4B在骨骼生长发育异常、年龄相关性骨丢失和肥胖等疾病发生中的作用提供依据。

CUL4B是泛素连接酶复合物CRL4B的骨架蛋白，我们的前期研究发现CUL4B丧失功能突变导致人类身材矮小、短指和肥胖，脂肪组织特异性敲除Cul4b小鼠表现为脂肪细胞分化增多，易患高脂饮食诱导的肥胖。由于间充质干细胞是成骨细胞和脂肪细胞共同的前体细胞，CUL4B突变可能通过改变间充质干细胞分化而引起患者身材矮小、短指和肥胖等表型。为此，本课题我们建立了间充质干细胞特异性敲除Cul4b小鼠模型，分析间充质干细胞缺失CUL4B对骨骼生长发育、骨稳态维持、脂肪发育和成骨/成脂分化的影响及其作用机制。取得了以下结果：1）间充质干细胞缺失Cul4b导致发育期小鼠骨骼发育迟缓；2）间充质干细胞缺失CUL4B加重骨质疏松和骨髓脂肪细胞沉积；3）间充质干细胞缺失CUL4B显著降低骨强度； 4）CUL4B促进间充质细胞成骨分化、抑制成脂分化；5）通过转录组分析筛选出CUL4B调控成骨成脂分化的关键转录因子Klf4和Cebpd; 发现CUL4B负调控Klf4和Cebpd表达； 6）CUL4B通过调控Klf4和Cepbd表达而调控间充质干细胞成骨/成脂分化；7）CUL4B复合物直接结合并表观抑制Klf4和Cebpd转录。以上结果揭示了CUL4B在调控间充质干细胞成骨/成脂分化中的关键作用，也为年龄相关的骨质疏松治疗研究奠定了基础。除此之外，我们还发现缺失CUL4B的间充质干细胞具有更强的免疫抑制能力，表现在：具有更强的抑制T细胞增殖能力、对ConA诱导的肝损伤的保护能力、具有更强的促进肿瘤生长能力，增加的免疫抑制能力是通过增加iNOS产生实现的。