Developmental and hormonal regulation of murine scavenger receptor, class B, type 1.

The scavenger receptor, class B, type I (SR-BI), is the predominant receptor that supplies plasma cholesterol to steroidogenic tissues in rodents. We showed previously that steroidogenic factor-1 (SF-1) binds a sequence in the human SR-BI promoter whose integrity is required for high-level SR-BI expression in cultured adrenocortical tumor cells. We now provide in vivo evidence that SF-1 regulates SR-BI. During mouse embryogenesis, SR-BI mRNA was initially expressed in the genital ridge of both sexes and persisted in the developing testes but not ovary. This sexually dimorphic expression profile of SR-BI expression in the gonads mirrors that of SF-1. No SR-BI mRNA was detected in the gonadal ridge of day 11.5 SF-1 knockout embryos. Both SR-BI and SF-1 mRNA were expressed in the cortical cells of the nascent adrenal glands. These studies directly support SF-1 participating in the regulation of SR-BI in vivo. We examined the effect of cAMP on SR-BI mRNA and protein in mouse adrenocortical (Y1-BS1) and testicular carcinoma Leydig (MA-10) cells. The time courses of induction were strikingly similar to those described for other cAMP- and SF-1-regulated genes. Addition of lipoproteins reduced SR-BI expression in Y1-BS1 cells, an effect that was reversed by administration of cAMP analogs. SR-BI mRNA and protein were expressed at high levels in the adrenal glands of knockout mice lacking the steroidogenic acute regulatory protein; these mice have extensive lipid deposits in the adrenocortical cells and high circulating levels of ACTH. Taken together, these studies suggest that trophic hormones can override the suppressive effect of cholesterol on SR-BI expression, thus ensuring that steroidogenesis is maintained during stress.     

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.     

Marked Cortisol Production by Intracrine ACTH in GIP-Treated Cultured Adrenal Cells in Which the GIP Receptor Was Exogenously Introduced

The ectopic expression of the glucose-dependent insulinotropic polypeptide receptor (GIPR) in the human adrenal gland causes significant hypercortisolemia after ingestion of each meal and leads to Cushing’s syndrome, implying that human GIPR activation is capable of robustly activating adrenal glucocorticoid secretion. In this study, we transiently transfected the human GIPR expression vector into cultured human adrenocortical carcinoma cells (H295R) and treated them with GIP to examine the direct link between GIPR activation and steroidogenesis. Using quantitative RT-PCR assay, we examined gene expression of steroidogenic related proteins, and carried out immunofluorescence analysis to prove that forced GIPR overexpression directly promotes production of steroidogenic enzymes CYP17A1 and CYP21A2 at the single cell level. Immunofluorescence showed that the transfection efficiency of the GIPR gene in H295R cells was approximately 5%, and GIP stimulation enhanced CYP21A2 and CYP17A1 expression in GIPR-introduced H295R cells (H295R-GIPR). Interestingly, these steroidogenic enzymes were also expressed in the GIPR (–) cells adjacent to the GIPR (+) cells. The mRNA levels of a cholesterol transport protein required for all steroidogenesis, StAR, and steroidogenic enzymes, HSD3β2, CYP11A1, CYP21A2, and CYP17A1 increased 1.2-2.1-fold in GIP-stimulated H295R-GIPR cells. These changes were reflected in the culture medium in which 1.5-fold increase in the cortisol concentration was confirmed. Furthermore, the levels of adenocorticotropic hormone (ACTH) receptor and ACTH precursor proopiomelanocortin (POMC) mRNA were upregulated 2- and 1.5-fold, respectively. Immunofluorescence showed that ACTH expression was detected in GIP-stimulated H295R-GIPR cells. An ACTH-receptor antagonist significantly inhibited steroidogenic gene expression and cortisol production. Immunostaining for both CYP17A1 and CYP21A2 was attenuated in cells treated with ACTH receptor antagonists as well as with POMC siRNA. These results demonstrated that GIPR activation promoted production and release of ACTH, and that steroidogenesis is activated by endogenously secreted ACTH following GIP administration, at least in part, in H295R cells.     

8-Phenylthio-adenines stimulate the expression of steroid hydroxylases, Cav3.2 Ca²⁺ channels, and cortisol synthesis by a cAMP-independent mechanism

The regulation of cortisol synthesis and the expression of genes coding for steroidogenic proteins by 8-substituted cAMP and 8-substituted adenine derivatives were studied in bovine adrenal zona fasciculata (AZF) cells. At concentrations of 10-50 μM, 8-(4-chlorophenylthio)-cAMP (8CPT-cAMP), but not the poorly hydrolyzable Sp-8CPT-cAMP, stimulated large increases in cortisol synthesis and CYP17 mRNA expression. Of the three Epac (exchange protein activated by cAMP)-specific cAMP analogs, 8CPT-2'-OMe-cAMP, but not 8HPT-2'-OMe-cAMP or 8MeOPT-2'-OMe-cAMP, induced mRNAs for CYP17 and CYP11a1 steroid hydroxylases and stimulated cortisol synthesis. 8-Substituted adenine derivatives (10-200 μM), including 8PT-adenine, 8MeOPT-adenine, and 8CPT-adenine, stimulated similar large, concentration-dependent, and reversible increases in cortisol synthesis and steroid hydroxylase gene expression, whereas 8Br-adenine was ineffective. The phenylthio-adenine derivatives produced additive effects on cortisol synthesis when applied to AZF cells in combination with 8Br-cAMP. In contrast, no additivity was observed for these three compounds when used in combination with ACTH. 8PT-adenine did not activate PKA or inhibit DNA synthesis by AZF cells. 8PT-adenine-stimulated cortisol secretion and CYP17 steroid hydroxylase mRNA expression were potently inhibited by diphenyl-butylpiperidine T-type Ca(2+) antagonists. In AZF cells, 8PT-adenine and 8MeOPT-adenine induced the expression of both CACNA1H mRNA and associated Ca(v)3.2 Ca(2+) current. These results indicate that 8-chloro (but not 8-hydroxy- or 8-methoxy-)-phenylthio-cAMP analogs are converted to an active metabolite, 8CPT-adenine, that induces the expression of genes coding for steroidogenic proteins in bovine AZF cells. Other PT-adenine analogs also potently stimulate cortisol synthesis through the same unidentified signaling pathway that requires the expression of functional Ca(v)3.2 Ca(2+) channels. These phenylthio-adenine compounds and ACTH may stimulate cortisol synthesis through the same cAMP-independent mechanism.     

The causal relationship between mutations in cAMP-dependent protein kinase and the loss of adrenocorticotropin-regulated adrenocortical functions.

The Y1 adrenocortical tumor cell mutants, Kin-7 and Kin-8, harbor point mutations in the regulatory subunit (RI) of the type 1 cAMP-dependent protein kinase (cAMPdPK) that render the enzyme resistant to activation by cAMP. These mutants also are resistant to many of the regulatory effects of ACTH and cAMP. In order to examine the causal relationships between the mutations in cAMPdPK and the resistance to ACTH and cAMP, the Kin mutants were transfected with expression vectors encoding wild type subunits of cAMPdPK in order to restore cAMP-responsive protein kinase activity. The transformants then were screened for the concomitant recovery of cellular responsiveness to ACTH and cAMP. In the mutant Kin-7, cAMP-responsive protein kinase activity was recovered after transfection with an expression vector encoding wild type mouse RI. Protein kinase activity in the mutant Kin-8 remained largely cAMP-resistant after transfection with the RI expression vector but could be rendered cAMP-responsive by transfection with an expression vector encoding the wild type catalytic subunit. The recovery of cAMP-responsive protein kinase activity was accompanied by the recovery of steroidogenic and morphological responses to ACTH and cAMP, suggesting that the cAMP-dependent signaling cascade plays an obligatory role in these actions of ACTH. The growth-regulatory effects of cAMP were not reversed with the recovery of cAMP-responsive protein kinase activity, suggesting that cAMP-resistant growth regulation results from second-site, adaptive mutations either in the original Kin mutant population or in the transformants. Studies on the conversion of 22(R)-hydroxycholesterol into steroid products in parent and mutant cells indicate that the Kin mutations reduce the steroidogenic capacity of the cell as well as inhibit the hormone- and cyclic nucleotide-dependent mobilization of substrate cholesterol.     

Suppression of cAMP-mediated signal transduction in mouse adrenocortical cells which express apolipoprotein E.

We have reported previously that expression of the human apolipoprotein E (apoE) gene in mouse Y1 adrenocortical cells suppresses basal and adrenocorticotropin (ACTH)-stimulated steroidogenesis. To understand the mechanism of this suppression, we have examined the integrity of cAMP regulated events required for adrenal steroidogenesis. Both acute and chronic responses to ACTH or cAMP are suppressed in Y1 cells which express apoE (Y1-E cells) as compared with parental Y1 cells. Acute morphologic changes in response to cAMP and acute induction of steroidogenesis by cAMP are suppressed in the Y1-E cell lines. Constitutive expression of P450-cholesterol side chain cleavage enzyme mRNA, the rate-limiting enzyme in steroid hormone synthesis, is reduced up to 11-fold in the Y1-E cell lines. The level of mRNA encoding P450-cholesterol side chain cleavage correlates directly with the reduction in basal steroid production observed in the individual Y1-E cell lines. Expression of P450-11 beta-hydroxylase mRNA, although readily detectable in Y1 parent cells, is absent or reduced in the Y1-E cell lines. Inhibition of cAMP-regulated gene expression is not restricted to genes required for steroid synthesis, since cAMP induction of ornithine decarboxylase mRNA is also inhibited in the Y1-E cell lines. These data indicate that suppression of steroidogenesis in Y1-E cells is due, at least in part, to inhibition of cAMP-regulated gene expression. These effects are not due to a defective cAMP-dependent protein kinase, since kinase activity in vitro and activation in vivo are unaltered in the Y1-E cell lines. These results suggest that expression of apoE in Y1 cells blocks cAMP-mediated signal transduction at a point distal to activation of cAMP-dependent protein kinase.     

Physiological functions and hormonal regulation of mouse vas deferens protein (AKR1B7) in steroidogenic tissues

The MVDP (mouse vas deferens protein) gene encodes an aldose reductase-like protein (AKR1B7) highly expressed in vas deferens epithelium and zona fasciculata of the adrenal cortex. Recombinant MVDP showed kinetic properties distinct from those of aldose reductase, including its spectrum of substrates, cofactor preference and sensitivity to inhibitors. We demonstrate that in adrenocortical cells, MVDP, rather than aldose reductase, is the principal reductase for isocaproaldehyde (a product of side-chain cleavage of cholesterol) and 4-hydroxynonenal (a lipid peroxidation product). In steroidogenic tissues MVDP expression is regulated by pituitary trophic hormones, namely ACTH in adrenals, FSH in ovaries, and LH in testicular Leydig cells.     

Physiological functions and hormonal regulation of mouse vas deferens protein (AKR1B7) in steroidogenic tissues

The MVDP (mouse vas deferens protein) gene encodes an aldose reductase-like protein (AKR1B7) highly expressed in vas deferens epithelium and zona fasciculata of the adrenal cortex. Recombinant MVDP showed kinetic properties distinct from those of aldose reductase, including its spectrum of substrates, cofactor preference and sensitivity to inhibitors. We demonstrate that in adrenocortical cells, MVDP, rather than aldose reductase, is the principal reductase for isocaproaldehyde (a product of side-chain cleavage of cholesterol) and 4-hydroxynonenal (a lipid peroxidation product). In steroidogenic tissues MVDP expression is regulated by pituitary trophic hormones, namely ACTH in adrenals, FSH in ovaries, and LH in testicular Leydig cells.     

ACTH-induced caveolin-1 tyrosine phosphorylation is related to podosome assembly in Y1 adrenal cells

Y1 adrenocortical cells respond to ACTH with a characteristic rounding-up that facilitates cAMP signaling, critical for transport of cholesterol to the mitochondria and increase in steroid secretion. We here demonstrate that caveolin-1 participates in coupling activation of protein kinase A (PKA) to the control of cell shape. ACTH/8-Br-cAMP induced reorganization of caveolin-1-positive structures in correlation with the cellular rounding-up. Concomitant with this change, there was an increase in the phosphorylation of caveolin-1 (Tyr-14) localized at focal adhesions (FA) with reorganization of FA to rounded, ringlike structures. Colocalization with phalloidin showed that phosphocaveolin is present at the edge of actin filaments and that after ACTH stimulation F-actin dots at the cell periphery become surrounded by phosphocaveolin-1. These observations along with electron microscopy studies revealed these structures as podosomes. Podosome assembly was dependent on both PKA and tyrosine kinase activities because their formation was impaired after treatment with specific inhibitors [myristoylated PKI (mPKI) or PP2, respectively] previous to ACTH/8-Br-cAMP stimulation. These results show for the first time that ACTH induces caveolin-1 phosphorylation and podosome assembly in Y1 cells and support the view that the morphological and functional responses to PKA activation in steroidogenic cells are related to cytoskeleton dynamics.     

Ca2+ signal stimulates the expression of steroidogenic acute regulatory protein and steroidogenesis in bovine adrenal fasciculata-reticularis cells

Adrenal glucocorticoid synthesis is stimulated by ACTH or its nitrophenylsulphenyl derivative, NPS-ACTH. Acute stimulation of steroid hormone biosynthesis is highly dependent on the expression of steroidogenic acute regulatory (StAR) protein. To determine the regulatory mechanism of StAR expression in bovine fasciculata/reticularis cells, we analyzed the second messenger systems involved in StAR protein expression using cultured cells activated by ACTH and NPS-ACTH. We concluded that cAMP is not the essential second messenger for StAR protein expression, since NPS-ACTH activated StAR protein expression more than ACTH without increase in cellular cAMP. A 15-lipoxygenase metabolite(s) of arachidonic acid stimulated steroidogenesis without increase in StAR protein expression, since AA-861, a lipoxygenase inhibitor, inhibited steroidogenesis without affecting StAR protein expression. Stimulation of StAR protein expression and the corresponding increase in the steroidogenesis were inhibited by nicardipine in cells treated with ACTH or NPS-ACTH. These data indicate that the dominant second messenger for the stimulation of StAR protein expression is Ca2+. Calmodulin-dependent kinase II inhibitors KN-93 and KN-62 suppressed steroidogenic activity without affecting StAR expression. The protein kinase C inhibitor Ro 31-8220 did not show any effects on StAR expression and steroidogenesis. Calmodulin-dependent kinase II and protein kinase C can therefore be concluded not to be involved in StAR protein expression in bovine cells.     

Mechanisms of digoxin and digitoxin on the production of corticosterone in zona fasciculata-reticularis cells of ovariectomized rats

Previous studies have indicated that digoxin (DG) inhibits testosterone production by rat testicular interstitial cells through both in vivo and in vitro experiments. DG and digitoxin (DT), but not ouabain, inhibit the progesterone, pregnenolone, and corticosterone secretion by rat granulosa cells, luteal cells, and zona fasciculata-reticularis (ZFR) cells, respectively. However, the effect of DG and DT on the enzyme kinetics of cytochrome P450 side chain cleavage enzyme (P450scc), the protein expression of P450scc and steroidogenic acute regulatory protein (StAR), and mRNA expression of StAR are unclear. ZFR cells were prepared from adrenocortical tissues of ovariectomized rats, and then challenged with adrenocorticotropin (ACTH), 8-Br-cAMP, forskolin, A23187, cyclopiazonic acid (CPA), nicotinic acid adenine dinucleotide phosphate (NAADP), trilostane, 25-OH-Cholesterol, progesterone, or deoxycorticosterone in the presence of DG, DT, or ouabain for 1 h. Enzyme kinetics of P450scc, protein expression of acute regulatory protein (StAR) and P450scc, and mRNA expression of StAR were investigated. DG and DT but not ouabain suppressed basal and other evoked-corticosterone release significantly. DG and DT also inhibited pregnenolone production. The Vmax of the DG and DT group was the same as the control group, but the Km was higher in DG- and DT-treated group than in control group. DT and ouabain significant suppressed mRNA expression of StAR. DG and DT had no effect on the P450scc and StAR protein expression at basal state, but diminished ACTH-induced StAR protein expression to basal level. These results indicated that DG and DT have an inhibitory effect on corticosterone production via a Na+, K+-ATPase-independent mechanism by diminishing actions on cAMP-, Ca2+-pathway, competitive inhibition of P450scc enzyme and reduction of StAR mRNA expression.     

Exon/intron organization and transcription units of the human acyl-CoA synthetase 4 gene

Acyl-CoA synthetase 4 (ACS4) is an arachidonate-preferring isozyme of ACS family predominantly expressed in steroidogenic tissues. Isolation and characterization of genomic clones encoding human ACS4 revealed that the genomic organization of the gene. The human ACS4 gene spans approximately 90 kb and consists of 16 exons. Sequence inspection of the 5'-flanking region revealed potential DNA elements including GATAs, p300, AP-4, SRY, CREB and MyoD. A minimal promoter region required for the expression of ACS4 in HeLa S3 cells was determined. The human ACS4 gene was mapped between the STS markers, WI-17685 and CHLC.GATA81B07 on Xq22-23 region.     

Altered cyclic AMP-dependent protein kinase activity in a mutant adrenocortical tumor cell line

A somatic cell genetic approach has been used to evaluate the role of cyclic AMP-dependent protein kinase in ACTH action on adrenal steroidogenesis. A mutant clone, 8BrcAMP^r-1, previously was isolated from an ACTH-sensitive adrenocortical tumor cell line (clone Y1) following mutagenesis and selective growth in 8-bromoadenosine 3′, 5′-monophosphate. This study demonstrates that the 8BrcAMP⁴-1 cells have an altered cyclic AMP-dependent protein kinase. The protein kinase in the cytosol of the mutant characteristically requires, for half-maximal activity, concentrations of cyclic AMP 7-fold higher than those required by the enzyme in preparations from the parent. The cytosolic cyclic AMP-dependent protein kinases of Y1 and 8BrcAMP^r-1 cells chromatograph similarly on columns of DEAE-cellulose. From each cell line, a major peak of activity (≥ 70% of recovered activity), designated as Peak I, elutes with 0.04–0.06 M NaCl; a second peak of activity, designated as Peak II, elutes with 0.12–0.14 M NaCl. Protein kinase activity in the Peak I fraction of mutant cells has a decreased apparent affinity (4-fold) for cyclic AMP relative to the corresponding fraction of parental Y1 cells. The protein kinase activities present in Peak II fractions from Y1 and mutant cells are indistinguishable. The protein kinase mutant exhibits poor steroidogenic responses to added ACTH and cyclic AMP; and as shown previously does not display the growth arrest and morphological changes produced in Y1 by these agents. These results suggest that cyclic AMP-dependent protein kinase is important in the regulation of adrenal steroidogenesis, morphology and growth by ACTH.