Multiple regulatory elements determine adrenocortical expression of steroid 21-hydroxylase

Steroid 21-hydroxylase (21-OHase) is specifically expressed at high levels in the adrenal cortex, where it is required for the synthesis of mineralocorticoids and glucocorticoids. In this study, we have investigated the regulatory elements in the 21-OHase promoter region which contribute to the expression of this gene in Y1 adrenocortical cells. Eight potential regulatory elements in the 5'-flanking region of the 21-OHase gene were identified by DNase I footprinting and gel mobility shift experiments. Some of these footprints were produced by nuclear extracts from many cell lines, whereas other interactions were seen only when using nuclear extracts from Y1 adrenocortical and MA-10 Leydig tumor cells. Mutation of most of the elements markedly decreased the expression of a 21-OHase gene transfected into Y1 cells, thus documenting their functional importance for expression. Moreover, oligonucleotides containing the sequences of two related elements at -65 and -210, which share the heptamer AGGTCAG, increased the activity of a heterologous promoter in a Y1 cell-specific manner. Collectively, these results demonstrate that expression of 21-OHase in Y1 adrenocortical cells requires interactions among multiple cis-acting elements and regulatory proteins.     

Promoter elements of the mouse 21-hydroxylase (Cyp-21) gene involved in cell-selective and cAMP-dependent gene expression

Cyp-21 (the mouse steroid 21-hydroxylase gene) is expressed exclusively in cells of the adrenal cortex, is induced by ACTH and cAMP, and is required for corticosteroid synthesis. This review examines the molecular basis for the regulated expression of Cyp-21 in the ACTH-responsive, mouse adrenocortical tumor cell line, Y1. We demonstrate that 330 bp of 5′-flanking DNA from the Cyp-21 gene are sufficient for cell-selective and ACTH-induced expression of Cyp-21, and that this promoter region comprises multiple, closely spaced enhancer elements each of which is required for promoter function. Within this promoter, we define three related elements that contain variations of an AGGTCA motif and that contribute to the cell-selective expression of Cyp-21. Variations of these same AGGTCA-bearing elements are also involved in the expression of Cyp 11a and Cyp 11b in Y1 adrenocortical cells. These elements interact with the same or closely related nuclear proteins found only in steroidogenic cell lines. Taken together, these results suggest that shared elements contribute to the adrenal cell-selective expression of at least three steroidogenic cytochrome P450 genes.

The element at −170 and the related elements at −65, −140 and −210 in the Cyp-21 promoter are not active as enhancers in the mutant Y1 cell line, Kin-8. Kin-8 cells contain a mutation in the regulatory subunit of the type 1 cAMP-dependent protein kinase that renders the enzyme resistant to activation by cAMP. Therefore, these elements appear to be selectively dependent upon an intact cAMP-dependent protein kinase for enhancer function. Individually, none of these elements confer cAMP-dependence to a reporter gene driven by a heterologous promoter. On the basis of these observations, we suggest that ACTH- and cAMP-dependent expression of Cyp-21 requires the combined actions of the element at −170, and the related elements at −140, −210 and −65.     

Salt-inducible kinase is involved in the ACTH/cAMP-dependent protein kinase signaling in Y1 mouse adrenocortical tumor cells.

The involvement of salt-inducible kinase, a recently cloned protein serine/threonine kinase, in adrenal steroidogenesis was investigated. When Y1 mouse adrenocortical tumor cells were stimulated by ACTH, the cellular content of salt-inducible kinase mRNA, protein, and enzyme activity changed rapidly. Its level reached the highest point in 1-2 h and returned to the initial level after 8 h. The mRNA levels of cholesterol side-chain cleavage cytochrome P450 and steroidogenic acute regulatory protein, on the other hand, began to rise after a few hours, reaching the highest levels after 8 h. The salt-inducible kinase mRNA level in ACTH-, forskolin-, or 8-bromo-cAMP-treated Kin-7 cells, mutant Y1 with less cAMP-dependent PKA activity, remained low. However, Kin-7 cells, when transfected with a PKA expression vector, expressed salt-inducible kinase mRNA. Y1 cells that overexpressed salt-inducible kinase were isolated, and the mRNA levels of steroidogenic genes in these cells were compared with those in the parent Y1. The level of cholesterol side-chain cleavage cytochrome P450 mRNA in the salt-inducible kinase-overexpressing cells was markedly low compared with that in the parent, while the levels of Ad4BP/steroidogenic factor-1-, ACTH receptor-, and steroidogenic acute regulatory protein-mRNAs in the former were similar to those in the latter. The ACTH-dependent expression of cholesterol side-chain cleavage cytochrome P450- and steroidogenic acute regulatory protein-mRNAs in the salt-inducible kinase-overexpressing cells was significantly repressed. The promoter activity of the cholesterol side-chain cleavage cytochrome P450 gene was assayed by using Y1 cells transfected with a human cholesterol side-chain cleavage cytochrome P450 promoter-linked reporter gene. Addition of forskolin to the culture medium enhanced the cholesterol side-chain cleavage cytochrome P450 promoter activity, but the forskolin-dependently activated promoter activity was inhibited when the cells were transfected with a salt-inducible kinase expression vector. This inhibition did not occur when the cells were transfected with a salt-inducible kinase (K56M) vector that encoded an inactive kinase. The salt-inducible kinase's inhibitory effect was also observed when nonsteroidogenic, nonAd4BP/steroidogenic factor-1 -expressing, NIH3T3 cells were used for the promoter assays. These results suggested that salt-inducible kinase might play an important role(s) in the cAMP-dependent, but Ad4BP/steroidogenic factor-1-independent, gene expression of cholesterol side-chain cleavage cytochrome P450 in adrenocortical cells.     

Salt-inducible kinase-mediated regulation of steroidogenesis at the early stage of ACTH-stimulation

Salt-inducible kinase (SIK), expressed in Y1 mouse adrenocortical tumor cells at an early stage of adrenocorticotropic hormone (ACTH)-stimulation, represses the cAMP-responsive element (CRE)-dependent gene expression of CYP11A and StAR by acting on bZIP domain of CRE-binding protein. ACTH induced the SIK’s nuclear to cytosolic translocation in a PKA-dependent manner. A mutant SIK in which the PKA-dependently phosphorylatable Ser577 had been replaced with Ala could not move out of the nucleus. The degree of CRE-reporter repression by SIK was strong as long as SIK was present in the nucleus. These indicated that intracellular translocation of SIK might be an important factor to determine the time-dependent change in the level of steroidogenic gene expression in ACTH-stimulated cells. Promoter analyses suggested that SIK repressed gene expressions not only of CYP11A and StAR but also of CYP11B1, CYP11B2 and SIK itself. We propose here that SIK is one of important molecule regulating expression of steroidogenic genes in the early phase of ACTH treatment.     

Identification and characterization of two upstream elements that regulate adrenocortical expression of steroid 11 beta-hydroxylase

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 after 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.     

Regulation of ferredoxin gene in steroidogenic and nonsteroidogenic cells

Ferredoxin is an electron transport intermediate for all the mitochondrial cytochromes P450. It is especially abundant in steroidogenic organs where it functions in steroid biosynthesis. The regulation of ferredoxin gene expression was studied in both steroidogenic and nonsteroidogenic cell lines. In steroidogenic cell line Y1, the expression of ferredoxin was stimulated by cAMP and repressed slightly by angiotensin II and phorbol ester PMA. These drugs exhibited the same effect on the basal promoter of the ferredoxin gene, which includes one TATA box and an SP1 site. In human adrenocortical cell line H295, the stimulation of the ferredoxin gene by cAMP was blocked by cycloheximide, as observed in bovine adrenocortical cell culture. In nonsteroidogenic cell lines such as HeLa and COS-1, the stimulation of ferredoxin gene expression by cAMP was not observed, although basal expression was strong. Transfection studies showed that the ferredoxin promoter could not be stimulated by cAMP in nonsteroidogenic cells. Therefore the steroidogenic cell-specific regulation and the general expression pattern appears to be a property unique to the ferredoxin gene.