REGULATION OF ECTODOMAIN SHEDDING OF RANKL

nf - κ B配体受体激活因子（Receptor activator of nf - κ B ligand， 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的胞外部分被蛋白水解裂解产生可溶性RANKL，这一过程被称为外域脱落，但RANKL脱落的分子机制及其生理和病理作用尚不清楚。为了鉴定参与RANKL脱落调控的分子，我们开发了一种新的筛选系统，将编码分泌的胎盘碱性磷酸酶与小鼠c端截断的RANKL融合的表达质粒与RANKL脱落酶的候选基因共转染。我们已经筛选了10万个克隆体。其中一个阳性克隆CAPRI （Ca^<2+>- promoting Ras inactivator）的剪接变体，已被鉴定为Ras - gap （GTPase-activating protein）的成员，对RANKL的脱落有积极作用。CAPRI是一种以Ca2+依赖的方式抑制R活性的分子，但我们发现其变体（ΔCAPRI）缺乏GAP相关结构域，那么它将是CAPRI的显性阴性形式。在我们的检测系统中，活性Ras表达载体刺激RANKL的脱落。我们还发现基质金属蛋白酶（MMP） 14和崩解素和金属蛋白酶（ADAM） 10具有较强的RANKL脱落活性。在western blot分析中，可溶性RANKL被检测为两种不同分子量的产物，RNA干扰MMP14和ADAM10分别导致低分子量和高分子量产物的减少。MMP14在原代成骨细胞中的抑制导致膜结合RANKL的增加，并促进与骨髓细胞共培养的破骨细胞的发生。此外，来自mmp14缺陷小鼠的破骨细胞有效诱导破骨细胞生成，这与体内破骨细胞生成增加一致。这些结果表明，RANKL脱落是RANKL转录后调控的过程，RANKL下调局部破骨细胞生成，MMP14在其中起关键作用。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