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Molecular and Cellular Endocrinology
Rapid Paper The steroidogenic acute regulatory protein is induced by angiotensin II and K + in H295R adrenocortical cells
Barbara J. Clark*ª, Vincenzo Pezzib, Douglas M. Stoccoª, William E. Rainey“
ªDepartment of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, TX 79430, USA bHealth Center University of Calabria, Arcavacata di Rende (CS), Italy 87036
“Department of Obstetrics and Gynecology, The University of Texas Southwestern Medical Center, Dallas, TX 75235, USA
Received 28 July 1995; accepted 6 September 1995
Abstract
Adrenal steroid hormone biosynthesis can be activated by the protein kinase A pathway by ACTH, the protein kinase C pathway by angiotensin II (AII), or by increasing intracellular Ca2+ levels by AII or K +. Although their mechanisms of action are not known, each of these pathways is dependent upon the de novo synthesis of a protein that is required for the acute production of steroids. We have recently proposed the steroidogenic acute regulatory (StAR) protein as this required protein, therefore, we examined the effect of different agonists on StAR’s expression in H295R human adrenocortical carcinoma cells. (Bu)2CAMP, AII, K +, BAYK8644 (a calcium channel agonist) and TPA are all shown to induce StAR. Aldosterone synthesis was stimulated by all the agonists with the exception of TPA, indicating that AII-stimulated steroid production is mediated by increases in intracellular calcium. Thus, these data suggest that regulation of StAR expression may represent a common mechanism for divergent pathways to acutely control adrenal steroidogenesis.
Keywords: StAR; Steroidogenesis; Adrenal; Angiotensin; Potassium
1. Introduction
The acute response of steroidogenic cells to trophic hormone stimulation is a rapid increase in the rate of steroid hormone biosynthesis. The first enzymatic step in adrenal and gonadal steroid production is the con- version of cholesterol to pregnenolone via the action of cytochrome P450 cholesterol side-chain cleavage (P450scc), which is located in the inner mitochondrial membrane (Stone and Hechter, 1954). Present evidence, however, suggests that it is the delivery of cholesterol to the inner mitochondrial membrane rather than the P450scc activity which is the acutely regulated and rate-limiting step in this process (Simpson et al., 1972; Privalle et al., 1983; Jefcoate et al., 1987). Furthermore,
it has been well established that de novo synthesis of a putative regulatory protein is an absolute requirement for this intramitochondrial transfer of cholesterol (Fer- guson, 1963; Garren et al., 1965).
We have recently described a protein, the steroidogenic acute regulatory (StAR) protein, which is indispensible in the rate-limiting step in acute steroid production (Clark et al., 1994; Lin et al., 1995). Previ- ous studies on the hormone regulation of steroidogene- sis and StAR expression have mainly focused on cAMP-dependent protein kinase A (PKA) mechanisms (Krueger et al., 1983; Pon et al., 1986; Stocco and Sodeman, 1991; Clark et al., 1994), however, acute steroid hormone biosynthesis in the adrenal cortex ap- pears to be under the control of multiple second mes- senger pathways, most notably protein kinase C (PKC) and Ca2+ -dependent pathways stimulated by AII and K + (Spat, 1988). As with the PKA-dependent pathway, AII and K + -stimulated steroid production requires de
* Corresponding author, Deptartment of Biochemistry, University of Louisville, School of Medicine, Louisville, KY 40292, USA. Tel .: (502) 852 5217; Fax: (502) 852 6222.
novo protein synthesis (Saruta et al., 1972; Elliot and Goodfriend, 1984). Since StAR’s role in the stimulation of steroid hormones by alternate second messenger pathways is unknown, this study was undertaken to examine the effects of activation of the PKA, PKC and Ca2 +-dependent pathways on the expression of StAR and aldosterone production using human adrenocorti- cal H295R cells (Bird et al., 1993). These cells have been demonstrated to respond to treatment with (Bu)2CAMP, AII or K+ with an acute increase in aldosterone production (Bird et al., 1993; Rainey et al., 1994).
2. Materials and methods
2.1. Materials
[Val5]-Angiotensin II acetate salt, KCI, 12-O-tetrade- canoylphorbol-13-acetate (TPA) and d-aldosterone were obtained from Sigma Chemical Co. (St. Louis, MO). Dibutyryl cAMP (Bu)2cAMP) was obtained from Aldrich Chem. Co. (Milwaukee, WI) and BAYK8644 from Research Biochemicals International (Natick, MA).
2.2. Cell culture
H295R cells (ATCC, Rockville, MD) were main- tained in a 1:1 mixture of Dulbecco’s modified Eagle’s and Ham’s F12 medium (DME/F12 containing pyri- doxine HCI, L-glutamine and 15 mM Hepes (Gibco BRL, Gaithersberg, MD). The media was supple- mented with insulin (6.25 µg/ml), transferrin (6.25 µg/ ml), selenium (6.25 ng/ml), linoleic acid (5.35 µg/ml) (1% ITS plus), 2.5% NuSerum type I Culture Supple- ment (Collaborative Biomed Prod., Bedford, MA), and antibiotics. Cells were maintained and grown in 75-cm2 flasks at 37°℃ under an atmosphere of 5% CO2/95% air, and were subcultured into 12-well plates 48 h prior to use in experiments.
2.3. Aldosterone synthesis
Cells were maintained for 24 h in DME/F12 medium containing 0.2% calf serum, 0.01% BSA and antibiotics (low serum). Fresh low serum medium containing the indicated agonists were added to the cells and incu- bated at 37℃ for the specified times. The media was recovered and aldosterone measured by RIA as de- scribed previously (Bird et al., 1993). Results are ex- pressed as pmol aldosterone per mg cell protein. Statistical analysis of the data was accomplished using analysis of variance, followed by Student-Newman- Keuls multiple comparison analysis.
2.4. Protein determination and immunoblot analysis Cells were solubilized in lysis buffer and protein
concentrations determined as previously described (Bird et al., 1993). Equivalent amounts of total cell protein were separated by SDS-PAGE (12.5%), electrophoreti- cally transfered to a polyvinylidene difluoride mem- brane and analyzed by immunoblot for StAR protein as described previously (Clark et al., 1994). StAR was detected by chemiluminescence (Renaissance kit, Du- Pont NEN) and the StAR-specific bands were quanti- tated using the Biolmage Visage 2000 computer-assisted image analysis system (BioImage, Ann Arbor, MI). Different exposure times were used to insure linearity.
3. Results
The agonists used in this study were selected to activate different second messenger pathways. (Bu)2cAMP directly activates PKA while AII activates phosphoinositidase-C which releases diacylglycerol and inositol 1,4,5-trisphosphate. Diacylglycerol, in turn, di- rectly activates PKC while inositol 1,4,5-trisphosphate increases intracellular Ca2+ levels. To separate the AII-induced effects of PKC activation from increased Ca2+ levels, the cells were treated with the phorbol ester, TPA, which only activates PKC. Finally, K +, which opens the plasma membrane voltage-dependent
| Sample | (pmol aldosterone/mg protein) | ||
|---|---|---|---|
| Expt. 1 (4 h) | Expt. 2 (6 h) | Expt. 3 (6 h) | |
| Aldosterone production | |||
| Cont | 2.91 ± 0.33 | 1.16±0.08 | 1.11 ± 0.06 |
| (Bu)2 | 5.90 ± 0.57 | 3.86 ±0.18 | 4.13 ±0.38 |
| AII | 3.95 ±0.59 | 3.30 ± 0.12 | 3.21 ± 0.25 |
| K + | 5.06 ± 0.28 | 2.62 ±0.17 | 2.83 ±0.21 |
| TPA | 3.12±0.11 | 1.60 ±0.21 | 1.58 ±0.08 |
| BAYK | nd | 2.70 ±0.08 | 3.53 ±0.41 |
| Relative integrated optical density | |||
| StAR induction | |||
| Cont | 1.0 | 1.0 | 1.0 |
| (Bu)2 | 2.9 | 5.0 | 7.7 |
| AII | 1.8 | 4.5 | 7.4 |
| K + | 2.2 | 2.9 | 4.5 |
| TPA | 2.7 | 5.1 | 5.7 |
| BAYK | nd | 5.2 | 6.8 |
MA10
(Bu)2CAMP
AII
K+
TPA
BAY K
C
Ca2+ channels, and BAYK, a calcium channel ago- nist, were used to increase intracellular Ca2+. Our data show that treatment of H295R cells with either (Bu)2CAMP, AII, K+ or BAYK resulted in 2-3-fold increases in aldosterone synthesis (Table 1). TPA, on the other hand, did not increase steroid production over basal levels. StAR expression, as determined by immunoblot analysis, was induced similarly by each of the agonists, including TPA (Fig. 1 and Table 1). The potency of induction was as follows; (Bu)2CAMP ≥ AII ≥ TPA, BAYK > K+, with K+ being approximately 1.5-fold lower (Fig 1, Table 1). Thus, PKA ((Bu)2cAMP) and Ca2+ (AII, K+ and BAYK) can activate both steroid production and StAR syn- thesis. The immunocross-reactive band of approxi- mately 26 kDa (Fig. 1) has also been detected in cell lysates from MA-10 cells and is not observed when isolated mitochondrial protein from H295R cells is used for immunoblot analysis (data not shown). While we do not know the origin or nature of this protein at this time, it is not localized to the mito- chondria and it is not hormonally regulated, therefore, we suggest it is a cross-reactive protein not related to StAR.
We next examined if the induction of StAR protein correlated with the aldosterone synthesis in a time-de- pendent manner. As shown in Fig. 2, both (Bu)2CAMP and AII stimulated aldosterone production and StAR expression in parallel. These data provide yet another example of the direct link between StAR expression and steroidogenesis and strongly support the hypothe- sis that the expression of StAR may act as the com- mon mechanism regulating steroid hormone biosynthesis by both the PKA and Ca2+-dependent pathways.
4. Discussion
This study has examined the effects of alternate second messenger pathways on the regulation of StAR protein expression and steroidogenesis in H295R hu- man adrenocortical cells. First we have demonstrated that StAR protein expression and aldosterone produc- tion in H295R cells are acutely regulated via the cAMP-dependent PKA pathway. The maximal induc- tion of steroidogenesis in H295R cells were observed after 2 h of stimulation, similar to what we have reported for the MA-10 cells, thus it appears the acute response is somewhat delayed in this system (Clark et al., 1995). However, the parallel time courses of induc- tion for StAR protein and aldosterone indicate that cAMP mediates the concominant regulation of StAR
A. (Bu)2 CAMP
pmol aldosterone/mg protein
8
aldosterone
8
StAR Protein
4
Integrated Optical Density
6
6
4
4
2
2
0
0
1
2
4
6
8
12
time in hours
B. Angiotensin II
7
pmol aldosterone/mg protein
7
aldosterone
StAR Protein
0
6
6
5
5
4
4
3
3
2
2
Integrated Optical Density
1
1
1
2
4
6
8
12
time in hours
and steroid production. These data confirm the close association between hormone-induced StAR expression and steroidogenesis in a second hormonally regulated steroidogenic cell type and strengthen our proposal that induction of StAR protein acutely regulates steroidoge- nesis. These data are consistent with the early studies of Orme-Johnson and colleagues in which similar protein(s) to StAR, termed pp30 proteins, were first described and characterized as ACTH-induced mito- chondrial phosphoproteins in rat adrenal cells (Krueger et al., 1983; Pon et al., 1986). Thus, it is highly likely that pp30 and StAR are homologous proteins.
Angiotensin II acutely increases aldosterone synthesis in adrenal glomerulosa cells through activation of PKC and calcium second messenger pathways, while K + and BAYK both function to increase intracellular Ca2+. We have demonstrated that all these agonists increase the level of StAR protein and aldosterone production in H295R cells. These results are in keeping with a previous report of the appearance of pp30-like mito- chondrial proteins in bovine adrenal glomerulosa cells in response to AII and K + stimulation (Elliot et al., 1993). We now extend those observations to show that AII acutely regulates the induction of StAR and steroidogenesis in a temporally coordinated manner. Furthermore, our data indicate that it is the increases in intracellular Ca2+ by AII which plays a major role in the acute regulation of steroid hormone biosynthesis in the H295R adrenocortical cells. The finding that TPA, which activates PKC, had no effect on aldosterone production but induced StAR expression in H295R cells is similar to an earlier observation in which we demonstrated TPA-induced synthesis of the 30-kDa (StAR) proteins in MA-10 Leydig cells but did not stimulate steroidogenesis (Chaudhary and Stocco, 1991). This observation coupled to the finding that K + and BAYK can activate both StAR synthesis and al- dosterone production suggests that a crucial Ca2 + -me- diated event may occur at a step prior to StAR, perhaps the delivery of cholesterol to the mitochondria. Alternatively, it is possible that StAR induced by TPA is in a non-functional form. It has been previously proposed that phosphorylation of the pp30 proteins is required for their function (Pon et al., 1986). To sup- port this hypothesis we did not detect significant amounts of the phosphorylated forms of the 30-kDa (StAR) proteins in MA-10 cells induced by TPA, which indicates that phosphorylation of StAR is not mediated by PKC in this cell line (Chaudhary and Stocco, 1991). This observation that StAR is not phosphorylated in response to TPA was confirmed recently in rat adrenal glomerulosa cells which are also not steroidogenically active in response to TPA (Hartigan et al., 1995). However, the authors further demonstrate that, in bovine adrenal fasciculata cells, which synthesize steroids in response to TPA, the pp30 proteins are
phosphorylated. Thus, these data strengthen the pro- posed link between phosphorylation of StAR and steroidogenesis.
Regardless of which second messenger pathway is activated, the acute regulation of steroid hormone biosynthesis is dependent upon the synthesis of a regu- latory protein that functions to facilitate the transloca- tion of cholesterol from the outer to the inner mitochondrial membrane and the P450scc. To date, the biochemical and genetic evidence support StAR as this regulatory protein. We have now demonstrated that StAR expression is regulated by several second messen- gers which stimulate steroidogenesis. Together these data suggest that the regulation of StAR expression is a common mechanism utilized by multiple pathways for the acute regulation of steroid hormone biosynthesis.
Acknowledgements
This work was supported by N.I.H. Grants HD 07688 (BJC), HD 17481 (DMS), and DK 43140 (WER).
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