Guo Sha, Peng Min, Zhao Qing, Zhang Wei
Department of Immunology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100005, China.
Chin Med Sci J. 2012 Jun;27(2):73-9.
To investigate the main proteinases responsible for CD16b shedding under different stimulators.
HEK293 cell line stably expressing CD16b was constructed by lentivirus system. The cell line was then overexpressed with a disintegrin and metalloproteinase 10 (ADAM10) or ADAM17, suppressed with short hairpin RNA of ADAM10 or ADAM17, and reconstituted with ADAM10 or ADAM17, respectively. After each treatment, the cell line was stimulated with ionomycin or phorbol 12-myristate- 13-acetate (PMA) for 12 hours. The soluble CD16b released from cell membrane was detected by immunoprecipition and immunoblot. Quantitation was then implemented to compare the amount of soluble CD16b in cell supernatant after stimulation.
HEK293 cell line stably expressing CD16b was successfully established. When CD16b expressing cell line was overexpressed with ADAM10, shedding of CD16b was increased after stimulation with ionomycin but not PMA; when the cell line overexpressed with ADAM17, shedding of CD16b was increased after stimulation with PMA but not ionomycin. Similarly, when ADAM10 was suppressed by short hairpin RNA, CD16b shedding was decreased after stimulation with ionomycin; when ADAM17 was suppressed by short hairpin RNA, CD16b shedding was decreased after stimulation with PMA. The shedding of CD16b was increased again when CD16b expressing cell line was reconstituted with ADAM10 and stimulated by ionomycin or reconstituted with ADAM17 and stimulated by PMA.
Both ADAM10 and ADAM17 could shed CD16b, but they possess differed preferences. ADAM10 is the main sheddase under stimulation of ionomycin, while ADAM17 is the main sheddase under stimulation of PMA.
研究在不同刺激物作用下负责CD16b脱落的主要蛋白酶。
利用慢病毒系统构建稳定表达CD16b的HEK293细胞系。然后分别用整合素金属蛋白酶10（ADAM10）或ADAM17对该细胞系进行过表达，用ADAM10或ADAM17的短发夹RNA进行抑制，并分别用ADAM10或ADAM17进行重组。每次处理后，用离子霉素或佛波醇12 -肉豆蔻酸酯-13 -乙酸酯（PMA）刺激该细胞系12小时。通过免疫沉淀和免疫印迹检测从细胞膜释放的可溶性CD16b。然后进行定量分析，比较刺激后细胞上清液中可溶性CD16b的量。
成功建立了稳定表达CD16b的HEK293细胞系。当用ADAM10对表达CD16b的细胞系进行过表达时，离子霉素刺激后CD16b的脱落增加，但PMA刺激后无增加；当用ADAM17对细胞系进行过表达时，PMA刺激后CD16b的脱落增加，但离子霉素刺激后无增加。同样，当用短发夹RNA抑制ADAM10时，离子霉素刺激后CD16b的脱落减少；当用短发夹RNA抑制ADAM17时，PMA刺激后CD16b的脱落减少。当用ADAM10对表达CD16b的细胞系进行重组并用离子霉素刺激或用ADAM17进行重组并用PMA刺激时，CD16b的脱落再次增加。
ADAM10和ADAM17均可使CD16b脱落，但它们具有不同的偏好。ADAM10是离子霉素刺激下的主要脱落酶，而ADAM17是PMA刺激下的主要脱落酶。
