Identification and Characterization of Two Upstream Elements that Regulate Adrenocortical Expression of Steroid 11β-Hydroxylase

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Abstract
We have studied the mechanisms that regulate the expression of the mouse gene encoding steroid 11.beta.-hydroxylase (11.beta.-OHase), a steroidogenic cytochrome P450 enzyme that is expressed only in the adrenal cortex. DNase I footprinting and gel-mobility shift analyses revealed potential regulatory elements at -370 and -310 in the 11.beta.-OHase promoter region. To determine the contributions of these elements to expression, we altered their sequences by site-selected mutagenesis and studied promoter activity transfection into Y1 mouse adrenocortical tumor cells. Mutation of either element markedly decreased basal promoter activity but did not affect the response to treatment with 8-bromo cAMP. These experiments thus document the functional roles of these elements, within the context of the intact promoter, in constitutive expression of 11.beta.-OHase. Moreover, addition of either of these elements to p-40GH, a 5''-deletion plasmid containing 11.beta.-OHase sequences from -40 to +8 upstream of a growth hormone reporter gene, significantly increased promoter activity but did not confer cAMP responsiveness. Finally, increased expression was seen after transfection of Y1 derivatives deficient in cAMP-dependent protein kinase, indicating that neither element required cAMP-dependent protein kinase activity. These studies thus define two regulatory elements that play important roles in 11.beta.-OHase expression.