Hakulinen Juha, Keski-Oja Jorma
Departments of Pathology and Virology, Haartman Institute and Biomedicum Helsinki, University of Helsinki and Helsinki University Hospital, Helsinki 00014, Finland.
Departments of Pathology and Virology, Haartman Institute and Biomedicum Helsinki, University of Helsinki and Helsinki University Hospital, Helsinki 00014, Finland.
J Biol Chem. 2006 Jul 28;281(30):21369-21376. doi: 10.1074/jbc.M602053200. Epub 2006 May 30.
Membrane cofactor protein CD46 controls complement activation on cells, is a receptor for several pathogens, and modulates immune responses by affecting CD8(+) T cells. Cells can release CD46 in an intact form on membrane vesicles and in a truncated form by a metalloproteolytic cleavage. The mechanism of shedding and its relationship to cell physiology has remained unclear. We have found using RNA interference analysis that a disintegrin and metalloproteinase (ADAM) 10 is responsible for the regulated shedding of the ectodomain of CD46 in apoptotic cells. The shedding of CD46 was initiated with staurosporine and UVB. Exposure of cell cultures to either UVB or staurosporine resulted in changes of cell morphology and detachment of cells from their matrices within 8-24 h. During this process CD46 was released both in apoptotic vesicles (vCD46) and proteolytically (sCD46) into the medium. Both the metalloproteinase inhibitor GM6001 and RNA interference of ADAM10 completely prevented the release of sCD46 and increased the expression of vCD46 on HaCaT cell vesicles, suggesting that ADAM10 releases sCD46 from the apoptotic vesicles. To explore whether the release of sCD46 is associated with apoptosis we analyzed the effects of caspase inhibitors. As expected, the inhibition of caspase activity attenuated the characteristic features of apoptosis and also decreased the release of sCD46. Our results reveal ADAM10 as an important regulator of CD46 expression during apoptosis. The ADAM10-mediated release of CD46 from apoptotic vesicles may represent a form of strategy to allow restricted complement activation to deal with modified self.
膜辅因子蛋白CD46可控制细胞上的补体激活，是多种病原体的受体，并通过影响CD8(+) T细胞来调节免疫反应。细胞能够以完整形式在膜泡上释放CD46，也能通过金属蛋白酶裂解以截短形式释放。其脱落机制及其与细胞生理学的关系仍不清楚。我们通过RNA干扰分析发现，解整合素和金属蛋白酶（ADAM）10负责凋亡细胞中CD46胞外域的调节性脱落。CD46的脱落由星形孢菌素和UVB引发。将细胞培养物暴露于UVB或星形孢菌素中，会在8 - 24小时内导致细胞形态改变以及细胞从其基质上脱离。在此过程中，CD46以凋亡小泡（vCD46）形式和通过蛋白水解（sCD46）释放到培养基中。金属蛋白酶抑制剂GM6001和ADAM10的RNA干扰均完全阻止了sCD46的释放，并增加了HaCaT细胞小泡上vCD46的表达，这表明ADAM10从凋亡小泡中释放sCD46。为了探究sCD46的释放是否与凋亡相关，我们分析了半胱天冬酶抑制剂的作用。正如预期的那样，半胱天冬酶活性的抑制减弱了凋亡的特征，同时也减少了sCD46的释放。我们的结果揭示了ADAM10是凋亡过程中CD46表达的重要调节因子。ADAM10介导的CD46从凋亡小泡中的释放可能代表了一种策略形式，以允许有限的补体激活来应对修饰的自身。
