小鼠骨保护素配基胞外片段的表达、纯化及生物活性分析

骨保护素配基(OPGL)是调节破骨细胞分化和成熟的核心细胞因子。由小鼠骨组织提取总RNA,RTPCR扩增得到小鼠OPGL胞外片段(sOPGL)cDNA,以特定策略克隆人表达载体pET-42a( ),以便使未来表达产物的融合标签序列能够完全被因子Xa切除。重组载体在大肠杆菌中诱导表达可获得高水平的47kD产物,Western blotting证实它可被OPGL抗体识别。经Glutathione-sepharose 4B亲和层析,除融合蛋白外,还有一约30 kD蛋白与层析柱发生了特异性亲和。该30kD蛋白可被GST-IGF-I多克隆抗体识别,但不能被OPGL抗体识别,提示它的产生乃由于融合蛋白在融合位点附近发生裂解。融合蛋白经Xa因子裂解和进一步纯化,得到分子量约17．5kD的sOPGL。生物活性分析证明,重组sOPGL可以促进OLC的生成,并呈现剂量依赖关系。     

Detection and characterization of RANK ligand and osteoprotegerin in the thyroid gland

Receptor activator of NF-kappaB (RANK) ligand (RANKL) and osteoprotegerin (OPG) play essential roles in bone metabolism and immune responses. RANKL activates RANK, which is expressed by osteoclasts and dendritic cells (DC), whereas OPG acts as its decoy receptor. The role of RANKL and OPG in thyroid physiology is unclear. Northern analysis revealed pronounced OPG mRNA levels in normal human thyroid. By contrast, RANKL mRNA levels were most abundant in lymph node and appendix, and low in the thyroid. In the human thyroid follicular cell line XTC and in primary human thyroid follicular cells, OPG mRNA levels and protein secretion were upregulated by interleukin (IL)-1beta (33-fold), tumor necrosis factor (TNF)-alpha (eightfold), and thyrotropin (TSH) (threefold). RANKL mRNA was stimulated in XTC by IL-1beta and TNF-alpha, but inhibited by TSH. Conditioned medium harvested from IL-1beta-treated XTC (containing high concentrations of OPG) inhibited RANKL-induced CD40 upregulation and cluster formation of DC. OPG mRNA levels were three times more abundant in surgical thyroid specimens of Graves' disease as compared to other thyroid diseases. Our data suggest that RANKL and OPG are produced in the thyroid gland by thyroid follicular cells, are regulated by cytokines and TSH, and are capable of modulating dendritic cell functions. Thus, these cytokines may represent important local immunoregulatory factors involved in the pathogenesis of autoimmune thyroid diseases.     

The effects of fatty acids consumption on OPG/RANKL/RANK system in cardiovascular diseases: Current status and future perspectives for the impact of diet-gene interaction

Cardiovascular disease (CVD) is an overall term that comprises a number of related pathologies, these include peripheral arterial disease, cerebrovascular disease, coronary heart disease (CHD), venous thromboembolism, and rheumatic and congenital heart diseases. Fatty acids in the diet have been reported to affect CVD. The OPG/RANKL/RANK system appears to have a role in CVD outcomes. However, there have been few studies on the impact of diet-gene interaction for effects of fatty acids consumption on the OPG/RANKL/RANK system in CVD. This review focuses on the effects of fatty acids on OPG/RANKL/RANK in CVD.     

Bone morphogenetic protein signaling in prostate cancer cell lines

Prostate cancer is the most commonly diagnosed malignancy in men and is often associated with bone metastases. Prostate cancer bone lesions can be lytic or schlerotic, with the latter predominating. Bone morphogenetic proteins (BMPs) are a family of growth factors, which may play a role in the formation of prostate cancer osteoblastic bone metastases. This study evaluated the effects of BMPs on prostate cancer cell lines. We observed growth inhibitory effects of BMP-2 and -4 on LNCaP, while PC-3 was unaffected. Flow cytometric analysis determined that LNCaP cell growth was arrested in G(1) after bone morphogenetic protein-2 treatment. Treatment of LNCaP and PC-3 with BMP-2 and -4 activated downstream signaling pathways involving SMAD-1, up-regulation of p21(CIP1/WAF1) and changes in retinoblastoma (Rb) phosphorylation. Interestingly, bone morphogenetic protein-2 treatment stimulated a 2.7-fold increase in osteoprotegerin (OPG), a molecule, which inhibits osteoclastogenesis, production in PC-3.     

Osteoprotegerin disrupts peripheral adhesive structures of osteoclasts by modulating Pyk2 and Src activities

Osteoprotegerin has previously been shown to modulate bone mass by blocking osteoclast maturation and function. The detailed mechanisms of osteoprotegerin-induced disassembly of podosomes, disruption of adhesive structures and modulation of adhesion-related proteins in osteoclasts, however, are not well characterized. In this study, tartrate-resistant acidic phosphatase staining demonstrated that osteoprotegerin inhibited differentiation of osteoclasts. The use of scanning electron microscopy, real-time cell monitoring and confocal microscopy indicated that osteoclasts responded in a time and dose-dependent manner to osteoprotegerin treatments with retraction of peripheral adhesive structures and detachment from the extracellular substrate. Combined imaging and Western blot studies showed that osteoprotegerin induced dephosphorylation of Tyr 402 in Pyk2 and decreased its labeling in peripheral adhesion regions. osteoprotegerin induced increased intracellular labeling of Tyr 402 in Pyk2, Tyr 416 in Src, increased dephosphorylation of Tyr 527 in Src, and increased Pyk2/Src association in the central region of osteoclasts. This evidence suggests that Src may function as an adaptor protein that competes for Pyk2 and relocates it from the peripheral adhesive zone to the central region of osteoclasts in response to osteoprotegerin treatment. Osteoprotegerin may induce podosome reassembly and peripheral adhesive structure detachment by modulating phosphorylation of Pyk2 and Src and their intracellular distribution in osteoclasts.     

破骨细胞形成抑制因子TNFR结构区在大肠杆菌中表达、抗体制备及活性测定

破骨细胞形成抑制因子(OPG)对骨的重建与再吸收有重要调节作用,其TNFR结构区行使抑制破骨细胞形成与活性的功能。通过PCR将该区基因片段克隆出来,插入表达载体PET-28a质粒多克隆位点,重组质粒转入大肠杆菌BL21中进行表达,表达产物以包涵体形式存在,包涵体经变性复性后,亲合层析获得重组蛋白。纯化的产物作为抗原免疫兔,得到较高特异性的兔源多克隆抗体。利用小鼠降血钙实验检测变性复性后产物的活性,结果表明该重组蛋白有一定的生物活性。     

Update on Wnt signaling in bone cell biology and bone disease

For more than a decade, Wnt signaling pathways have been the focus of intense research activity in bone biology laboratories because of their importance in skeletal development, bone mass maintenance, and therapeutic potential for regenerative medicine. It is evident that even subtle alterations in the intensity, amplitude, location, and duration of Wnt signaling pathways affects skeletal development, as well as bone remodeling, regeneration, and repair during a lifespan. Here we review recent advances and discrepancies in how Wnt/Lrp5 signaling regulates osteoblasts and osteocytes, introduce new players in Wnt signaling pathways that have important roles in bone development, discuss emerging areas such as the role of Wnt signaling in osteoclastogenesis, and summarize progress made in translating basic studies to clinical therapeutics and diagnostics centered around inhibiting Wnt pathway antagonists, such as sclerostin, Dkk1 and Sfrp1. Emphasis is placed on the plethora of genetic studies in mouse models and genome wide association studies that reveal the requirement for and crucial roles of Wnt pathway components during skeletal development and disease.     

Retinoid metabolism and its effects on the vasculature

Retinoids, the metabolically-active structural derivatives of vitamin A, are critical signaling molecules in many fundamental biological processes including cell survival, proliferation and differentiation. Emerging evidence, both clinical and molecular, implicates retinoids in atherosclerosis and other vasculoproliferative disorders such as restenosis. Although the data from clinical trials examining effect of vitamin A and vitamin precursors on cardiac events have been contradictory, this data does suggest that retinoids do influence fundamental processes relevant to atherosclerosis. Preclinical animal model and cellular studies support these concepts. Retinoids exhibit complex effects on proliferation, growth, differentiation and migration of vascular smooth muscle cells (VSMC), including responses to injury and atherosclerosis. Retinoids also appear to exert important inhibitory effects on thrombosis and inflammatory responses relevant to atherogenesis. Recent studies suggest retinoids may also be involved in vascular calcification and endothelial function, for example, by modulating nitric oxide pathways. In addition, established retinoid effects on lipid metabolism and adipogenesis may indirectly influence inflammation and atherosclerosis. Collectively, these observations underscore the scope and complexity of retinoid effects relevant to vascular disease. Additional studies are needed to elucidate how context and metabolite-specific retinoid effects affect atherosclerosis. This article is part of a Special Issue entitled: Retinoid and Lipid Metabolism.     

显微CT在骨微结构研究中的应用

该文对OPG基因敲除小鼠进行micro-CT扫描,进行全身骨骼三维重建,并分析股骨结构。结果发现OPG基因敲除纯合子小鼠全身骨骼出现弥漫性蜂窝状低密度影。OPG基因敲除小鼠的骨密度、骨结构模型指数、骨小梁厚度、骨体积分数、骨表面积与体积比、连接密度以及皮质骨厚度等指标均明显小于同窝野生型小鼠,而两基因型小鼠在骨小梁数目、骨小梁分离度及各项异性度等方面无明显差异。以上数据结果表明,OPG基因敲除导致小鼠出现骨质疏松。应用micro-CT可以对小鼠骨进行无创微结构分析,较体视学方法具有一定的优势。