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 cholesterol side-chain cleavage enzyme (P-450_scc, resulting in complete elimination of the adrenal gene expression and causing congenital adrenal hyperplasia in the rabbit. Using the rabbit congenital adrenal hyperplasia model, we investigated the wild type (wt) P-450_scc gene dose effect on gene expression in three P-450_scc genotype animals [wt/wt, wt/mutant (mt), mt/mt] identified by Southern blot analysis. Northern blots using a rabbit P-450_scc cDNA probe revealed 1 no detectable P-450_scc mRNA in individual adrenals of animals with congenital adrenal hyperplasia (mt/mt) and approximately half or slightly less than half the levels of the mRNA in the pooled adrenals of five heterozygous (wt/mt) newborn animals compared with the mRNA levels in the pooled adrenals of five homozygous normal (wt/wt) newborn animals. Identical P-450_scc mRNA levels were found individual adrenals of adult animals with regard to the P-450_scc genotype, although at a higher expression level than in the newborn animals of the same genotype. Control Northern blots using human CPY21-B cDNA and cytoplasmic actin cDNA probes confirmed the accuracy and integrity of RNA. Western immu-noblotting using antiovine P-450_scc antibody revealed decreased P-450_scc protein in the adrenals of wt/mt animals at approximately half the level of the P-450_scc protein in the adrenals of the wt/wt animals. Baseline and ACTH-stimulated serum corticosterone (B) levels in vivo were similar between the age-matched wt/mt and wt/wt animals, whereas ACTH-stimulated B levels in adult animals were higher than those in the newborn animals irrespective of P-450_scc genotype. These data demonstrated for the first time the presence of a wt steroidogenic enzyme gene dose effect on gene expression in vivo at the levels of the mRNA and enzyme protein. With age, the gene dose-related gene expression was maintained, although at higher expression levels. The normal adrenal B-pro-ducing capacity in heterozygous, wt/mt animals despite the markedly decreased P-450_scc gene expression suggests the presence of either a mechanism compensating the availability of mitochondrial cholesterol substrate to overcome the decreased P-450_scc or rate-limiting effect of P-450_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 irrespective of P-450_scc genotype therefore is likely from the greater adrenal receptor capacity for ACTH rather than from the greater P-450_scc gene expression level.