REGULATION OF ECTODOMAIN SHEDDING OF RANKL

核因子-kappaB受体激活剂配体(RANKL)是一种跨膜糖蛋白，在破骨细胞的发育过程中起重要作用。RANKL胞外部分被蛋白质降解，产生可溶性RANKL，这一过程被称为胞外结构域脱落，但RANKL脱落的分子机制及其生理和病理作用尚不清楚。为了鉴定RANKL脱落酶的调控分子，我们建立了一种新的筛选系统，将编码分泌型胎盘碱性磷酸酶的表达载体与RANKL脱落酶的候选基因共转染小鼠RANKL。我们已经筛选了10万个克隆人。其中一个阳性克隆Capri的剪接变异体(Ca^&lt；2+&gt；-Promoted RAS失活因子)已被鉴定为Ras-GAP(GTP酶激活蛋白)的成员，被发现对RANKL的脱落有积极的作用。CAPRI是一种抑制R-…活性的分子更多的是以钙依赖的方式，但我们鉴定的其变体(ΔCapri)缺乏GAP相关结构域，因此它将是CAPRI的显性否定形式。在我们的检测系统中，RAS活性形式的表达载体刺激RANKL的脱落。我们还发现，基质金属蛋白酶(MMP14)和去整合素和金属蛋白酶(ADAM)10具有很强的RANKL脱落活性。在蛋白质印迹分析中，可溶性RANKL被检测为两种不同的相对分子质量产物，MMP14和ADAM10的RNA干扰分别导致较低和较高相对分子质量产物的减少。在原代成骨细胞中抑制MMP14导致膜结合RANKL增加，并在与骨髓细胞共培养中促进破骨细胞的形成。此外，MMP14基因缺陷小鼠的破骨细胞能有效地诱导破骨细胞生成，这与体内破骨细胞生成的增加是一致的。这些结果表明，RANKL的脱落是RANKL转录后调控的过程，下调局部破骨细胞的生成，而MMP14起关键作用。较少

Receptor activator of NF-kappa B ligand (RANKL) is a transmembrane glycoprotein, which plays an essential role in the development of osteoclasts. Extracellular portion of RANKL is cleaved proteolytically to produce soluble form of RANKL, the process called ectodomain shedding, but the molecular mechanism of RANKL shedding and its physiological and pathological role have not been clarified yet. To identify molecules involved in the regulation of RANKL shedding, we developed a novel screening system, in which expression plasmids encoding secreted placental alkaline phosphatase fused with mouse C-terminally truncated RANKL were co-transfected with candidate genes of RANKL sheddases. We have screened 100,000 clones. One of the positive clones, splice variant of CAPRI (Ca^<2+>-promoted Ras inactivator), which had been identified to be a member of RAS-GAP (GTPase-activating protein), was found to have a positive effect to RANKL shedding. CAPRI is a molecule which suppresses the activity of R … More as in Ca2+ dependent manner, but its variant which we identified (ΔCAPRI) lacks GAP related domain, then it will be a dominant negative form of CAPRI. Expression vector for active form of Ras stimulates shedding of RANKL in our assay system. We also found that matrix metalloproteinase (MMP) 14 and a disintegrin and metalloproteinase (ADAM) 10 had strong RANKL shedding activity. In western blot analysis, soluble RANKL was detected as two different molecular weight products, and RNA interference of MMP14 and ADAM10 resulted in reduction of the lower and the higher molecular weight products, respectively. Suppression of MMP14 in primary osetoblasts resulted in an increase in membrane-bound RANKL, and promoted osteoclastogenesis in cocultures with bone marrow cells. In addition, osteoclasts from MMP14-deficient mice efficiently induced osteoclastogenesis, which is consistent with the increased osteoclastogenesis in vivo. These results suggest that RANKL shedding is a process of post-transcriptional regulation of RANKL, which downregulates local osteoclastogenesis, and MMP14 plays a critical role. Less