Differential effects of kinase inhibitors on erythropoietin and vascular endothelial growth factor gene expression in rat hepatocytes

Abstract

This study sought to investigate whether a common protein kinase activity is involved in the sequence of events by which oxygen controls the expression of the genes for erythropoietin (EPO) and for vascular endothelial growth factor (VEGF) in rat hepatocytes. To this end we examined the influence of the non-specific kinase inhibitor staurosporine and of the tyrosine kinase inhibitor genistein on EPO and VEGF mRNA levels in primary cultures of rat hepatocytes kept at either high (20% O₂) or low (1% O₂) oxygen tension. We found that 3 h of exposure to the low O₂ tension increased EPO mRNA levels about 20-fold and the three VEGF (−180, −164, −120) mRNA levels, on average, about fourfold. Staurosporine did not change EPO and VEGF mRNA levels at 20% O₂, but in a concentration-dependent manner, decreased EPO and VEGF mRNA at 1% O₂ with IC₅₀ values of 30 nM and 1000 nM, respectively. In the presence of 1 % O₂, genistein decreased EPO mRNA and VEGF mRNA levels with IC₅₀ values of about 36 and 360 μM, respectively. Although mRNA levels for glycerine aldehyde phosphatehydrogenase (GAPDH) were not changed, staurosporine and genistein inhibited uridine incorporation into total RNA with IC₅₀ values of about 1 μM and 100 μM, respectively. Comparison with the transcription inhibitor actinomycin D suggested that the effects of both kinase inhibitors on VEGF mRNA but not on EPO mRNA levels could be attributed to the non-specific inhibition of transcription in hepatocytes. These findings suggest that a kinase activity is specifically involved in the O₂-dependent control of EPO gene expression but not of VEGF gene expression in hepatocytes.