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Regulation of interphotoreceptor retinoid-binding protein (IRBP) gene expression by cAMP in differentiated
A E El-Remessy1, A M Rabie, M M El-Shishtawy
1Department of Ophthalmology, Medical College of Georgia, Augusta, GA, USA.
Purpose:
To determine the mechanism of cyclic AMP (cAMP) regulation of the interphotoreceptor retinoid-binding protein (IRBP) gene in retinoblastoma cells.
Methods:
WERI-Rb1 cells pretreated with laminin or grown on poly-D-lysine-coated substratum for three days were treated with forskolin/3-isobutyl-1-methylxanthin (IBMX) or with dimethyl sulfoxide (DMSO). During a time course of 96 h, cell morphologies were determined by light microscopy, cellular cAMP levels measured by radioimmunoassay, and IRBP and b-actin gene expression determined by Northern blot and RNase protection analyses. IRBP expression and b-actin gene expression in these cells were also determined in the presence or absence of actinomycin D or cycloheximide.
Results:
After laminin treatment for 3 days, 27-34% of WERI-Rb1 cells differentiated into spindle shapes. Further forskolin treatment in the presence of IBMX for 5 days resulted in many cells exhibiting the formation of long, ramifying, neurite-like processes that were abolished by an inhibitor of protein kinase A. Cells grown on poly-D-lysine-coated substratum treated with forskolin remained undifferentiated. Treatment of laminin-pretreated cells with forskolin/IBMX, but not with DMSO/IBMX, raised the cAMP level 15-fold within the first hour of treatment. Northern blot analysis of these cells showed a rapid increase of IRBP mRNA, but not b-actin mRNA, reaching a maximum of about 3-fold at 6-8 h. A similar increase of IRBP mRNA was observed using RNase protection analysis except that the maximum was observed at 2-4 h. Actinomycin D blocked this IRBP mRNA induction. Cycloheximide had no effect in this induction.
Conclusions:
These results demonstrate that laminin induces WERI-Rb1 cell differentiation and cAMP provokes the formation of long ramifying neurite-like processes. Forskolin selectively induces IRBP gene expression in the laminin-treated cells through a cAMP-mediated pathway without de novo protein synthesis.