EVIDENCE OF A STEROIDOGENIC ENZYME GENE DOSE-EFFECT ON ADRENAL GENE-EXPRESSION IN HEREDITARY RABBIT CONGENITAL ADRENAL-HYPERPLASIA

We previously reported the gene deletion encoding cytochrome P-450 cho lesterol side-chain cleavage enzyme (P-450(S)CC), resulting in complet e elimination of the adrenal gene expression and causing congenital ad renal hyperplasia in the rabbit. Using the rabbit congenital adrenal h yperplasia model, we investigated the wild type (wt) P-450(SCC) gene d ose effect on gene expression in three P-450(SCC) genotype animals [wt /wt, wt/mutant (mt), mt/mt] identified by Southern blot analysis. Nort hern blots using a rabbit P-450(SCC) cDNA probe revealed no detectable P-450(SCC) mRNA in individual adrenals of animals with congenital adr enal hyperplasia (mt/mt) and approximately half or slightly less than half the levels of the mRNA in the pooled adrenals of five heterozygou s (wt/mt) newborn animals compared with the mRNA levels in the pooled adrenals of five homozygous normal (wt/wt) newborn animals. Identical P450(SCC) mRNA levels were found individual adrenals of adult animals with regard to the P-450(SCC) genotype, although at a higher expressio n level than in the newborn animals of the same genotype. Control Nort hern blots using human CPY21-B cDNA and cytoplasmic actin cDNA probes confirmed the accuracy and integrity of RNA. Western immunoblotting us ing antiovine P450(SCC) antibody revealed decreased P-450(SCC) protein in the adrenals of wt/mt animals at approximately half the level of t he P450(SCC) protein in the adrenals of the wt/wt animals. Baseline an d ACTH-stimulated serum corticosterone (B) levels in vivo were similar between the age-matched wt/mt and wt/wt animals, whereas ACTH-stimula ted B levels in adult animals were higher than those in the newborn an imals irrespective of P-450(SCC) genotype. These data demonstrated for the first time the presence of a wt steroidogenic enzyme gene dose ef fect an gene expression in vivo at the levels of the mRNA and enzyme p rotein. With age, the gene dose-related gene expression was maintained , although at higher expression levels. The normal adrenal B-producing capacity in heterozygous, wt/mt animals despite the markedly decrease d P-450(SCC) gene expression suggests the presence of either a mechani sm compensating the availability of mitochondrial cholesterol substrat e to overcame the decreased P-450(SCC) or rate-limiting effect of P-45 0(SCC) in the conversion of excess cholesterol substrate available in the adrenal mitochondria. The greater B-producing capacity under ACTH stimulation in the adult animals compared with the newborn animal irre spective of P-450(SCC) genotype therefore is likely from the greater a drenal receptor capacity far ACTH rather than from the greater P-450(S CC) gene expression level.