Abstract
Abstract: :
Purpose: TNF–α has recently been identified to be a mediator of retinal ganglion cell (RGC) death. However, glial cells are relatively protected against this death stimulus. TNF–α signaling has been associated with different kinases, including MAPKs, and a transcription factor, NF–ΚB. To identify molecular mechanisms that control differential responses of RGCs and glial cells to TNF–α, we studied differential gene expression between RGCs and glial cells exposed to TNF–α, using pathway–specific cDNA array analysis of MAPK and NF–ΚB signaling. Methods: Primary cultures of rat RGCs and glial cells were incubated in a serum–free culture medium in the absence and presence of TNF–α (0.05 ng/ml) for 24 hours. Total RNA was prepared from cultured cells, labeled cDNA probes were synthesized using a primer mix and hybridized with the membranes containing pre–spotted gene–specific cDNA fragments. Using the GEArrayAnalyzer for data analysis, the relative expression level of each gene was determined after normalization to the signal of housekeeping genes. Results: We detected 129 genes out of 171 screened. 21 genes were significantly (more than 3–fold) up– or down–regulated in cells incubated with TNF–α compared to the vehicle treated controls. Approximately 50% of these genes were differentially up– or down–regulated in RGCs versus glia. The most prominently up–regulated genes in RGCs included transcription factors and adaptors (such as c–jun, p53, TRAF–1, and FADD) and rel–a, a component of NF–ΚB. In addition to some cell cycle and growth response genes, whose proteins are regulated by ERK, prominently up–regulated genes in glial cells included hsp27, NCAM–1, rel–a, and c–rel; while IΚB and VCAM–1 were down–regulated. RT–PCR confirmed the differential gene expression, and immunocytochemistry demonstrated gene products in cultured cells. Except for the up–regulation of some MAPK kinase kinases and p38 MAPK, different kinases studied (including MAPK kinases, MAPKs, and IΚKs) were not significantly up– or down–regulated. Conclusions: Findings of this study reveal some differences in the expression of genes associated with MAPK or NF–ΚB signaling between RGCs and glial cells exposed to TNF–α. Ongoing studies of differential protein expression and post–translational modifications should further define differences in the signaling pathways between RGCs and glial cells. Through the identification of signaling molecules differentially activated in RGCs and glial cells, our understanding of the differential cellular responses to glaucomatous stress can improve, and RGC survival can be manipulated for therapeutic gain.
Keywords: ganglion cells • gene microarray • signal transduction