Constitutive NF–kB Activity Renders C–abl Null Retina Resistant to Apoptosis

Abstract

Abstract: : Purpose: Mice deficient in the non–receptor tyrosine kinase c–abl are resistant to oxygen–induced retinopathy (OIR). The objective of this study was to identify differences in gene expression between wild type and c–abl null mice that could potentially explain why the c–abl null mice are protected from developing OIR. Methods: Gene expression profiling and semi–quantitative RT–PCR of cDNAs prepared from wt and c–abl null retinas and fibroblasts were performed for comparative analysis. To quantitate NF–kB levels, luciferase activity was measured using wt and c–abl null fibroblasts transiently transfected with a NF–kB–luciferase reporter. To determine if c–abl null fibroblasts were resistant to apoptosis stimulated by death receptor ligands, caspase 3 and TUNEL assays were performed on untreated wt and mutant fibroblasts, and on cells treated with TNF–alpha or IL–1. Results: Several known NF–kB target genes were upregulated in c–abl null retinas and fibroblasts compared to wild type tissue. NF–kB activity was elevated in untreated c–abl null fibroblasts compared to wild type cells. Moreover, NF–kB activity levels in c–abl–deficient fibroblasts did not increase in response to death receptor agonists whereas increased levels were observed in treated wild type cells. To measure the pro–apoptotic activity of TNF–alpha or IL–1, caspase 3 and TUNEL assays were performed on untreated and treated wild type and mutant cells. Unlike wild type cells, mutant fibroblasts did not exhibit elevated levels of caspase 3 activity or tunnel positive cells in response to TNF–alpha or IL–1. Conclusions: Gene expression profiling and NF–kB luciferase reporter experiments support the conclusion that NF–kB activity is constitutively elevated in c–abl–deficient fibroblasts when compared to wild type cells. NF–kB effects are reported to be anti–apoptotic as NF–kB regulates transcription of survival genes. We observed that mutant cells were resistant to apoptosis induced by death receptor ligands TNF–alpha or IL–1. We speculate that the NF–kB activity in mutant cells is protective against pro–apoptotic stimulation. Apoptosis of retinal capillary endothelial cells is observed in OIR and responsible for the capillary drop–out seen in retinopathy of prematurity. We hypothesize that NF–kB effects in the c–abl null retina are anti–apoptotic and attenuate OIR.

Keywords: apoptosis/cell death • gene microarray • signal transduction