Expression of Three Negative Regulators of CYP17 Gene Transcription in Adrenocortical Cells

Abstract

The expression of negative regulators of CYP17 gene expression: DAX-1, COUP-TF and N-CoR was investigated in bovine adrenocortical cells in primary culture. The cells were incubated for 6 hours in a defined medium, containing ACTH (10 nM) or forskolin (25 μM). Total RNA was isolated, the concentration of CYP17 gene transcript was determined by hybridization analysis, and polyA RNA was reverse-transcribed to obtain cDNA. Fragments of CYP17, DAX-1, COUP-TF, N-CoR and β-actin cDNA were amplified by PCR and the amplified cDNAs were analyzed by agarose gel electrophoresis followed by densitometry.

It was found that DAX-1 and COUP-TF mRNA levels were unaffected by treatment with ACTH or forskolin. On the contrary, both compounds enhanced the level of mRNA encoding N-CoR. Under the same conditions, ACTH or forskolin substantially stimulated CYP17 mRNA accumulation.

Our results suggest that in the adrenocortical cells in culture, the expression of DAX1 and COUP-TF is constitutive, whereas N-CoR expression might be inducible.