The role of connexin43 on the cell death induced by staurosporine

Abstract

gap junction protein이 apoptosis에 어떻게 작용하는지 알아보기 위해 wild type HeLa (Wt), CX43 full frame을 overexpressing하는 HeLa (Bonnie)와 C terminal truncated CX43을 overexpressing하는 HeLa (M257)에서 ST과 EDTA에 의해 유도되는 cell death를 측정하였다. Bonnie에서는 ST에의한 death가 Wt과 M257에 비해 크게 증가된 반면에 EDTA에 의해서는 세 종류의 cell line에서 비슷한 세포사멸 양상을 보여주었다. Bonnie에서 ST에 더 민감한 이유를 조사하기 위해 anti CX43 antibody를 이용해서 CX43 distribution과phosphorylation을 조사하였다. ST이 처리된 Bonnie에서는 CX43의 phosphorylation이 크게 감소했으나 EDTA에 의해서는 큰 변화를 보이지 않았다. 더욱이 ST이 처리된 Bonnie에서 CX43은 주로 세포막에 존재한 반면 EDTA를 처리한 경우에는 세포질 내에 위치했다. 반면에 CX43 phosphorylation site가 없는 M257에서는 ST과 EDTA를 처리한 경우 모두 CX43이 세포질 내에 존재했다. ST을 처리한 Bonnie의 세포막에 위치한 CX43에 의해 형성된 hemichannel이 channel activity를 갖는지 알아보기 위해 ST을 처리한 후 carboxyfluorescein을 이용해서 dye uptake를 조사하였다. Wt과 M257에서는 ST의 처리에 관계없이 dye uptake율에 변화가 없었던 반면, ST을 처리한 Bonnie에서는 시간이 경과함에 따라 dye uptake가 11배까지 증가했다. 이상의 결과는 ST을 처리한 Bonnie에서 CX43 hemichannel과 hemichannel activity의 증가는 ST에 의한 Bonnie에서의 크게 증가된 세포사멸을 설명하는 뒷받침이 될 수 있다. ; To address the role of gap junction on apoptosis, the cell death induced by staurosporine (ST) and EDTA was measured in wild type (Wt), full frame connexin (CX) 43 transfected (Bonnie), and C terminal truncated CX43 transfected (M257) HeLa cells. The ST-mediated cell death was accelerated in Bonnie compared to Wt and M257. In contrast all three cell lines treated with EDTA revealed similar pattern of cell death. To figure out why Bonnie cells were more susceptible to ST, the localization and phosphorylation of CX43 protein were examined using anti-CX43 antibody. The phosphorylated form of CX43 was remarkably reduced in Bonnie treated with ST not in Bonnie treated with EDTA. Moreover, CX43 in ST treated Bonnie was mainly observed in the plasma membrane while that in the EDTA treated cell was located in the cytosol. In contrast C terminal truncated CX43 which does not have phosphorylation sites was observed in the cytosol when M257 cells were treated with either ST or EDTA. To address the hemichannels formed by CX43 localized to the plasma membrane of ST treated Bonnie have functional channel activities, dye uptake using carboxyfluorescein was examined in ST treated cells. The number of fluorescent dye labeled cells did not change in Wt and M257 whether ST was treated or not, while that of the ST treated Bonnie increased time dependently up to 11 fold. These results indicate that the increase of CX43 hemichannels and hemichannel activities in ST treated Bonnie could be a reason for the accelerated cell death of Bonnie to ST.