Activation of Caspase‐8/BID Pathway in Dexamethasone‐Induced Apoptosis in a Human Lens Epithelial Cell Line

Abstract

BackgroundProlonged glucocorticoid steroid usage is capable of inducing changes to the transcription of genes in lens epithelial cells and is associated with the development of several ocular diseases, including cataract formation. Mechanisms of cataract formation, however, are not well known, although apoptosis has been implicated. We therefore investigated glucocorticoid dexamethasone‐induced apoptosis in a human lensepithelial cell line.MethodsHuman lens epithelial cells were exposed to dexamethasone and apoptosis was evaluated using flow cytometry for AnnexinV expression, chromogenic substrates for Caspase activity measurements, Western blotting for Bax andBCL‐2 expression as well as PARP cleavage, and agarose gel electrophoresis for detection of DNA fragmentation. Mitochondrial pathway activity was determined by measuring the levels of Cytochrome C release, Bax translocation to the mitochondria, and BID cleavage.ResultsA significant induction of early apoptosis was observed by the AnnexinV/PI dual staining flow cytometric analyses. Increased expression ratios of pro‐apoptotic protein Bax toanti‐apoptotic protein BCL‐2 following dexamethasone exposure demonstrated apro‐apoptotic tendency. Sequential activation of caspase‐8 and caspase‐3, and BID cleavage with Cytochrome C extrusion from mitochondria, indicated an extrinsic apoptotic pathway. Nuclear changes of DNA fragmentation and PARP cleavage, both indicative of late stage apoptosis, were detected by electrophoresis only after dexamethasone exposure, despite lack of increased late stage apoptotic detection using Flow cytometry.ConclusionResults suggest that Dexamethasone exposure induces an extrinsic apoptotic pathway in a human lensepithelial cell line which follows along the activation of caspase‐8/BIDpathway and supports the hypothesis that apoptosis may be an important mechanism involved in steroid‐induced cataract formation.Support or Funding InformationIntramural Funding from the Department of Defense