Angiotensin II-induced mitogen-activated protein kinase phosphatase-1 expression in bovine adrenal glomerulosa cells: implications in mineralocorticoid biosynthesis.

Angiotensin II (AngII) stimulates aldosterone biosynthesis in the zona glomerulosa of the adrenal cortex. AngII also triggers the MAPK pathways (ERK1/2 and p38). Because ERK1/2 phosphorylation is a transient process, phosphatases could play a crucial role in the acute steroidogenic response. Here we show that the dual specificity (threonine/tyrosine) MAPK phosphatase-1 (MKP-1) is present in bovine adrenal glomerulosa cells in primary culture and that AngII markedly increases its expression in a time- and concentration-dependent manner (IC(50) = 1 nm), a maximum of 548 +/- 10% of controls being reached with 10 nm AngII after 3 h (n = 3, P < 0.01). This effect is completely abolished by losartan, a blocker of the AT(1) receptor subtype. Moreover, this AngII-induced MKP-1 expression is reduced to 250 +/- 35% of controls (n = 3, P < 0.01) in the presence of U0126, an inhibitor of ERK1/2 phosphorylation, suggesting an involvement of the ERK1/2 MAPK pathway in MKP-1 induction. Indeed, shortly after AngII-induced phosphorylation of ERK1/2 (220% of controls at 30 min), MKP-1 protein expression starts to increase. This increase is associated with a reduction in ERK1/2 phosphorylation, which returns to control values after 3 h of AngII challenge. Enhanced MKP-1 expression is essentially due to a stabilization of MKP-1 mRNA. AngII treatment leads to a 53-fold increase in phosphorylated MKP-1 levels and a doubling of MKP-1 phosphatase activity. Overexpression of MKP-1 results in decreased phosphorylation of ERK1/2 and aldosterone production in response to AngII stimulation. These results strongly suggest that MKP-1 is the specific phosphatase induced by AngII and involved in the negative feedback mechanism ensuring adequate ERK1/2-mediated aldosterone production in response to the hormone.

A novel mutation in the steroidogenic acute regulatory protein gene promoter leading to reduced promoter activity.

We have identified a novel cytosine/thymidine polymorphism of the human steroidogenic acute regulatory (StAR) gene promoter located 3 bp downstream of the steroidogenic factor-1 (SF-1)-binding site and 9 bp upstream of the TATA box (ATTTAAG). Carriers of this mutation have a high prevalence of primary aldosteronism. In transfection experiments, basal StAR promoter activity was unaltered by the mutation in murine Y-1 cells and human H295R cells. In Y-1 cells, forskolin (25 microM, 6 h) significantly increased wild-type promoter activity to 230+/-33% (P<0.05, n=4). In contrast, forskolin increased mutated promoter activity only to 150+/-27%, with a significant 35% reduction compared to wild type (P<0.05, n=3). In H295R cells, angiotensin II (AngII; 10 nM) increased wild-type StAR promoter activity to 265+/-22% (P<0.01, n=3), while mutated StAR promoter activity in response to AngII only reached 180+/-29% of controls (P< 0.01, n=3). Gel mobility shift assays show the formation of two additional complexes with the mutated promoter: one with the transcription repressor DAX-1 and another with a yet unidentified factor, which strongly binds the SF-1 response element. Thus, this novel mutation in the human StAR promoter is critically involved in the regulation of StAR gene expression and is associated with reduced promoter activity, a finding relevant for adrenal steroid response to physiological stimulators.

Expression and modulation of steroidogenic acute regulatory protein messenger ribonucleic acid in rat cardiocytes and after myocardial infarction.

We examined whether the mRNA for steroidogenic acute regulatory (StAR) protein, a crucial factor in the rate-limiting step of aldosterone biosynthesis, is expressed and regulated in rat heart. We performed quantitative RT-PCR for StAR mRNA in an in vitro and an in vivo model: purified rat neonatal cardiomyocytes in primary culture and myocardial infarction (MI) in the rat. StAR mRNA was expressed in cultured cardiomyocytes, and angiotensin II (10 nM) increased it in a time-dependent fashion (132 +/- 2.7% of controls after 24 h; n = 3; P < 0.05). Concomitantly, angiotensin II stimulated aldosterone production in the culture medium from 32.6 +/- 6.1 to 54 +/- 12.7 fmol/mg protein after 24 h (n = 8; P < 0.05). StAR mRNA levels in cardiomyocytes were dramatically reduced after 24-h treatment with dexamethasone in a concentration-dependent manner (50% inhibitory concentration, 10 nM); maximal inhibition (to 15 +/- 6% of control; P < 0.001; n = 6) was achieved with 100 nM dexamethasone. This inhibition was prevented by RU486. In the rat MI model, StAR mRNA was also present in control heart tissue and was increased 2.4-fold in the noninfarcted area of the left ventricle after MI (n = 6; P < 0.01). This effect was completely prevented by treatment with losartan (8 mg/kg per d) and spironolactone (80 mg/kg per d), which reduced StAR mRNA levels to values not different from those in non-MI controls. Thus, the mRNA for an indispensable factor in aldosterone biosynthesis, the StAR protein, is expressed in the rat heart and is up-regulated after MI. These results support the view of a local synthesis of aldosterone in the heart and of intracrine/paracrine deleterious effects of the mineralocorticoid in heart failure.