Involvement of the extrinsic and intrinsic pathways in ultraviolet B-induced apoptosis of corneal epithelial cells.
Involvement of the extrinsic and intrinsic pathways in ultraviolet B-induced apoptosis of corneal epithelial cells.
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DOI：
10.1016/j.exer.2015.11.003
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发表时间：
2016-04
影响因子：
3.4
通讯作者：
Haarsma LD
中科院分区：
文献类型：
--
作者：
Ubels JL;Glupker CD;Schotanus MP;Haarsma LD
本研究的目的是阐明UVB引发人角膜缘上皮（HCLE）细胞K+外流和随后凋亡的途径。这项研究的最初重点是涉及Fas的外源性途径。将用Fas siRNA转染的HCLE细胞暴露于80-150 mJ/cm 2 UVB，并在含有5.5 mM K+的培养基中孵育。Fas的敲低导致UVB诱导的caspase-8和-3活性的有限降低。膜片钳记录显示Fas转染和对照细胞之间UVB诱导的标准化K+电流无差异。caspase-8的敲低对UVB暴露后caspase-3的活化没有影响，而caspase-8抑制剂完全消除了UVB对caspase-3的活化。这表明半胱天冬酶-8是一种稳健的酶，能够通过敲低后存在的残留半胱天冬酶-8激活半胱天冬酶-3，并且半胱天冬酶-8直接参与UVB激活半胱天冬酶-3。caspase-9的抑制显著降低了响应UVB的caspase-8和-3的活化。敲低Apaf-1，激活caspase-9所需的，导致UVB诱导的caspase-9，-8和-3的激活显着减少。敲低Apaf-1也抑制内在和UVB诱导的细胞凋亡水平，通过TUNEL法测定的DNA片段。在用半胱天冬酶-3抑制剂处理的UVB暴露培养物中，凋亡细胞的百分比降低至对照水平，证实了半胱天冬酶-3活化在DNA片段化中的必要性。Fas敲低对K+通道激活的影响的缺乏，以及对半胱天冬酶-8和-3的激活的有限影响，强烈地表明Fas和外源性途径在响应于UVB的HCLE细胞中的细胞凋亡的起始中不是主要重要的。抑制caspase-9后caspase-8和caspase-3的激活受到抑制，以及caspase-9、caspase-8和caspase-3的激活和DNA片段化响应于Apaf-1敲低而减少，这支持了这样的结论，即内源性途径在UVB诱导的HCLE细胞凋亡中更重要。
The goal of this study was to elucidate the pathway by which UVB initiates efflux of K+ and subsequently apoptosis in human corneal limbal epithelial (HCLE) cells. The initial focus of the study was on the extrinsic pathway involving Fas. HCLE cells transfected with Fas siRNA were exposed to 80–150 mJ/cm2 UVB and incubated in culture medium with 5.5 mM K+. Knock down of Fas resulted in limited reduction in UVB-induced caspase-8 and -3 activity. Patch-clamp recordings showed no difference in UVB-induced normalized K+ currents between Fas transfected and control cells. Knockdown of caspase-8 had no effect on the activation of caspase-3 following UVB exposure, while a caspase-8 inhibitor completely eliminated UVB activation of caspase-3. This suggests that caspase-8 is a robust enzyme, able to activate caspase-3 via residual caspase-8 present after knockdown, and that caspase-8 is directly involved in the UVB activation of caspase-3. Inhibition of caspase-9 significantly decreased the activation of caspases-8 and -3 in response to UVB. Knockdown of Apaf-1, required for activation of caspase-9, resulted in a significant reduction in UVB-induced activation of caspases-9, -8, and -3. Knockdown of Apaf-1 also inhibited intrinsic and UVB-induced levels of apoptosis, as determined by DNA fragmentation measured by TUNEL assay. In UVB exposed cultures treated with caspase-3 inhibitor, the percentage of apoptotic cells was reduced to control levels, confirming the necessity of caspase-3 activation in DNA fragmentation. The lack of effect of Fas knockdown on K+ channel activation, as well as the limited effect on activation of caspases-8 and -3, strongly suggest that Fas and the extrinsic pathway is not of primary importance in the initiation of apoptosis in response to UVB in HCLE cells. Inhibition of caspase-8 and -3 activation following inhibition of caspase-9, as well as reduction in activation of caspases-9, -8, and -3 and DNA fragmentation in response to Apaf-1 knockdown support the conclusion that the intrinsic pathway is more important in UVB-induced apoptosis in HCLE cells.