Mouse Models to Delineate a Unique Metabolic and Skeletal Network

描述（由申请人提供）：在当前的RO1 AR54604中，我们正在研究骨骼获得与骨髓肥胖之间的相互作用，并测试小鼠6号染色体上一系列基因控制的共享骨骼和代谢网络的假设。这一竞争性修订扩展了我们正在进行的工作，基于我们的合作者Jorge Plutzky最近的发现，维甲酸（RA）前体视黄醛（Rald）在脂肪中作为一种新的生物活性介质，通过抑制PPAR3来决定代谢表型。Rosen/Horowitz实验室一直在对基因（如B6）进行表征。C3H- 6t)和近交系小鼠（C57BL6和C3H/HeJ），并确定骨量和IGF-I的遗传决定因素。我们在小鼠Chr6的中间区域发现了一些基因，这些基因作为一个调节单元一起起作用，决定了低骨量、成骨细胞（OB）分化受损、低循环IGF-I和骨髓肥胖增强的6T小鼠的骨骼表型。虽然6T小鼠在高脂肪饮食（HFD）挑战后会失去骨骼，但它们的代谢特征是保护它们免受饮食诱导的胰岛素抵抗和肥胖。值得注意的是，这个小基因组簇（4Mb）位于小鼠Chr 6上，包括4个在脂肪形成中重要的基因：过氧化物酶体增殖激活受体- γ (PPAR3)、Alox5、SDF-1和FSP 27。最近，我们在PPAR3基因的3'UTR内发现了几个多态性，并研究了这种“功能获得”对IGF-I等靶标的下游影响。这些数据为研究骨髓间质细胞成脂分化机制提供了重要的理论依据。Plutzky小组报道了Rald在体内抑制PPAR3-RXR并抑制脂肪形成后，我们开始合作确定Rald在骨中的作用机制。此前，Rald在视网膜外没有任何记载。我们分析了视黄醛脱氢酶缺失（Raldh1-/-）小鼠的骨骼表型，发现与B6相比，Raldh1-/-小鼠在12周的股骨面骨密度显著增加。此外，与对照组相比，空白小鼠的股骨体积分数增加了两倍以上。这些证据现在允许我们测试Rald如何通过RXR- PPAR复合体，或独立地在基质细胞命运中发挥关键的调节作用，并最终在骨形成中发挥作用。我们将通过充分表征常规和高脂肪饮食以及使用和不使用罗格列酮的Raldh1-/-小鼠的骨骼表型来实现这一目标。我们将探讨成骨细胞和破骨细胞的分子机制，以确定Rald和Raldh1的存在以及类视黄醛轴对骨髓基质细胞分配和破骨细胞发生的影响。

DESCRIPTION (provided by applicant): In the current RO1 AR54604, we are studying the interaction between skeletal acquisition and bone marrow adiposity, and testing the hypothesis that there are shared skeletal and metabolic networks controlled by a series of genes situated on mouse chromosome 6. This competitive revision expands our ongoing work by building on the recent identification by our collaborator, Jorge Plutzky, that the retinoic acid (RA) precursor, retinaldehyde (Rald), functions as a novel biologically active mediator in fat where it determines metabolic phenotypes by repressing PPAR3. The Rosen/Horowitz laboratories have been characterizing congenic (e.g. B6.C3H-6T) and inbred strains of mice (C57BL6 and C3H/HeJ) and identifying genetic determinants of bone mass and IGF-I. We found genes in the mid region of mouse Chr6 that function together as a regulatory unit to determine a skeletal phenotype in 6T mice of low bone mass, impaired osteoblast (OB) differentiation, low circulating IGF-I, and enhanced bone marrow adiposity. Although 6T mice lose bone after a high fat diet (HFD) challenge, their metabolic profile is characterized by protection from diet-induced insulin resistance and obesity. Remarkably, the location of this small genomic cluster (4Mb) on mouse Chr 6 includes 4 genes important in adipogenesis: peroxisome proliferator-activated receptor-gamma (PPAR3), Alox5, SDF-1, and FSP 27. Recently we identified several polymorphisms within the 3'UTR of the PPAR3 gene, and examined the downstream effects of this 'gain of function' on targets such as IGF-I. These data provided an important rationale for studying the mechanisms of adipogenic differentiation in marrow stromal cells. After the Plutzky group reported that Rald, which previously had no documented role outside of the retina, inhibited PPAR3-RXR and repressed adipogenesis in vivo, we began a collaboration to determine Rald's mechanism of action in bone. We analyzed skeletal phenotypes from retinaldehyde dehydrogenase null (Raldh1-/-) mice and found markedly increased femoral areal bone mineral density in 12 week Raldh1-/- mice vs B6. In addition, there was more than twice the femoral bone volume fraction in the null mice compared to controls. These lines of evidence now permit us to test how Rald, either through the RXR- PPAR complex, or independently, has a critical regulatory role in stromal cell fate, and ultimately in bone acquisition. We will accomplish this by fully characterizing the skeletal phenotype of the Raldh1-/- mice on regular and high fat diets as well as with and without rosiglitazone. We will interrogate the molecular mechanisms in osteoblasts and osteoclasts to determine the presence of Rald and Raldh1 and the effects of the retinoid axis on bone marrow stromal cell allocation and osteoclastogenesis. PUBLIC HEALTH RELEVANCE: The importance of nuclear receptor networks in bone marrow differentiation programs has been firmly established and in our ongoing grant, AR54604, we identified several genetic polymorphisms in the PPAR3 gene which we identified as a key determinant of adipocyte and bone cell fate. We now propose to study retinaldehyde and its effect on RXR which partners with PPAR3, to form an activated transcriptional complex to regulate gene expression and ultimately to influence bone mass acquisition. A more comprehensive understanding of the regulatory inputs that determine PPAR3 responses in bone has important clinical implications, particularly after the discovery that the thiazolidinediones, drugs that enhance insulin sensitivity and are exogenous ligands for PPAR3, cause bone loss and fractures.