A role for src tyrosine kinase in regulating adrenal aldosterone production
R Sirianni1,2, B R Carr1, V Pezzi2 and W E Rainey1
1Department of Obstetrics and Gynecology, Division of Reproductive Endocrinology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas, USA
2 Department Pharmaco-Biology/Centro Sanitario, University of Calabria, Cosenza, Italy
(Requests for offprints should be addressed to W E Rainey, Department of Obstetrics and Gynecology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Rm. J6.114, Dallas, Texas 75390-9032, USA)
ABSTRACT
Adrenal aldosterone synthesis is influenced by a variety of factors. The major physiological regula- tors of aldosterone production are angiotensin II (Ang ) and potassium (K+). Ang stimulates aldosterone production through the activation of multiple intracellular signaling pathways. It has recently been demonstrated that Ang activates src tyrosine kinases in vascular smooth muscle cells. The src family of tyrosine kinases are widely distributed non-receptor kinases that influence several signal transduction pathways. In the present study we evaluated the effect of a selective src family inhibitor, PP2, on aldosterone production using a human adrenocortical carcinoma-derived (H295R) cell line. Treatments for 6 or 48 h with PP2 (0.3 µM-10 µM) inhibited basal, Ang II, K+ and dibutyryladenosine cyclic monophosphate (db- cAMP) stimulation of aldosterone production in a concentration-dependent manner. PP2 did not affect cell viability at any of the concentrations tested. Moreover, time course studies using PP2 (10 µM) for 6, 12, 24, and 48h revealed a time-dependent inhibition of aldosterone pro- duction. Inhibition by PP2 (0.3-10 uM) was also observed for the metabolism of 22R- hydroxycholesterol (22R-OHChol) to aldosterone in H295R cells. Since 22R-OHChol is a substrate for cytochrome P450 side-chain cleavage enzyme (CYP11A) that does not require steroidogenic acute regulatory (StAR) protein for transport to the inner mitochondrial membrane, these results suggest that
PP2 inhibition occurred beyond the rate-limiting step in aldosterone synthesis. Genistein, a non- specific tyrosine kinase inhibitor also blocked aldosterone production, but the inhibition was the result of a non-specific effect on 3ß-hydroxysteroid dehydrogenase (3ßHSD). In contrast, PP2 did not appear to act as a direct inhibitor of 36HSD activity. To further investigate the site of PP2 action, we examined its effect on H295R cell metabolism of [14C]progesterone using thin layer chromatography. PP2 treatment for 48 h caused an increase in the conversion of progesterone to 17a-hydroxyprogesterone. To determine if this apparent increase in 17a-hydroxylase activity was due to increased transcript, we examined the effect of PP2 on CYP17 mRNA. PP2 treatment caused an increase in CYP17 mRNA without an effect on 3ßHSD mRNA levels. Inhibition of protein synthesis with cycloheximide increased basal levels of CYP17 mRNA levels and blocked the induction observed by PP2. This suggests that new protein synthesis is a necessary part of PP2 induction of CYP17. Taken together these data suggest that the src tyrosine kinase inhibitor, PP2, is a potent inhibitor of aldosterone production. One mechan- ism for the inhibition is through an induction of CYP17 mRNA and enzyme activity. Src tyrosine kinases, therefore, may be involved with the promotion of a glomerulosa phenotype through the inhibition of CYP17 expression.
Journal of Molecular Endocrinology (2001) 26, 207-215
INTRODUCTION
Within the human adrenal, steroids are produced through the action of five forms of cytochrome P450
and 3ß-hydroxysteroid dehydrogenase (3ßHSD) (Simpson & Waterman 1992). It is the differential expression of these enzymes in the three adrenocor- tical zones that leads to the production of specific
steroids within each zone (Suzuki et al. 2000). Like all steroidogenic cells, the glomerulosa cell uses pregnenolone as a precursor for steroidogenesis. Newly formed pregnenolone can be metabolized by either 3ßHSD or 17a-hydroxylase, 17,20-lyase (CYP17) and it is the relative expression of these enzymes that influences the synthesis of aldosterone versus cortisol or C19 steroids (Conley & Bird 1997). High 3ßHSD expression combined with low CYP17 activity will favor aldosterone synthesis and oppose cortisol and adrenal androgen synthesis. In contrast, a high CYP17/3ßHSD ratio supports C19 steroid biosynthesis but would inhibit aldosterone synthesis. The proposed importance of the ratio of these enzymes is supported by the lack of expression of CYP17 within the glomerulosa (Sasano et al. 1988, Suzuki et al. 2000). Therefore, understanding the mechanisms that positively and negatively regulate CYP17 is of primary importance.
We have previously demonstrated that angio- tensin II (Ang II) promotes the production of aldosterone not only through acute actions (Clark et al. 1995) but also by increasing the expression of 3ßHSD and inhibiting CYP17 expression (Bird et al. 1992, 1996a,b). Ang II signal transduction occurs through the action of calcium/calmodulin- dependent protein kinases, protein kinase C, and a number of tyrosine kinases (Bird et al. 1990, Quinn & Williams 1992, Berk & Corson 1997). Recent evidence suggests that Ang II can activate the src family of cytoplasmic tyrosine kinases (Ishida et al. 1995, Berk & Corson 1997, Sayeski et al. 1999). The availability of inhibitors of the src tyrosine kinases (Hanke et al. 1996) has allowed us to examine the role of src in adrenal cell production of aldosterone. We demonstrate that inhibition of src blocks the production of aldosterone. Part of the inhibition of aldosterone production appears to result from an increase in CYP17 activity and mRNA expression. This increase in CYP17 effectively removes sub- strate from the pathway leading to aldosterone. Experiments will be needed in the future to further define the role of the src kinases in adrenocortical function.
MATERIALS AND METHODS
Cell culture
H295R cells were cultured in Dulbecco’s modified Eagle’s and Ham’s F-12 (DME/F12) medium (GIBCO BRL, Gaithersburg, MD, USA), supple- mented with 2% Ultroser G (Biosepra SA, Villeneuve la Garenne Cedex, France), 1% ITS Plus (insulin, 6.25 µg/ml; transferrin, 6.25 µg/ml; selenium, 6.25 ng/ml; bovine serum albumin,
1.25 mg/ml; linoleic acid, 5.35 µg/ml) (Collaborative Research, Bedford, MT, USA) and antibiotics as previously described (Bird et al. 1993). Cells were subcultured onto 12-well culture dishes for steroid assay, 3ßHSD activity assay and for thin layer chromatography experiments (5 × 105 cells/well). For experiments designed to study cellular RNA and pp60c-src activity, cells were plated onto 100 mm dishes (7 × 106 cells/plate) and used for experiments 48 h later.
Stimulation of steroid secretion and analysis of steroids
Prior to experiments, cells were maintained over- night in DME/F12 medium containing 0.1% Ultroser G and antibiotics (low serum medium). Where indicated, cells were then preincubated with PP2 (Calbiochem-Novabiochem Corporation, San Diego, CA, USA) for 30 min at 37 ℃ in fresh low serum medium. Ang II, K+ and dibutyryladenosine cyclic monophosphate (dbcAMP) (Sigma-Aldrich, St Louis, MO, USA) were added to the cells and the incubation carried out at 37 ℃ for the indicated times. The aldosterone content of medium recov- ered from each well was determined against aldosterone standards prepared in low serum medium using an aldosterone radioimmunoassay kit (Diagnostic System Laboratories, Webster, TX, USA). Results of aldosterone assay were normalized to the cellular protein content per well and expressed as pmol per mg cell protein.
Protein determination
Cells were solubilized in Tris-HC1 (50 mM, pH 7.4) containing NaCl (150 mM), SDS (1%), EGTA (5 mM), MgCl2 (0.5 mM), MnCl2 (0.5 mM) and phenylmethylsulfonylfluoride (PMSF, 0.2 mM). The protein contents of samples were then determined by the bicinchoninic acid protein assay, using the BCA assay kit (Pierce, Rockford, IL, USA).
pp60c-src immune complex kinase assay
Src assay was carried out as previously described (Ishida et al. 1995). Cells were incubated with Ang II for 5 min. After a wash with phosphate-buffered saline, ice-cold cells were lysed in RIPA buffer (USBiological, Swampscott, MA, USA), scraped off the plate and centrifuged at 9000 r.p.m. in a microfuge (4 °℃ for 10 min); protein concentrations of the supernatants were determined as explained above. Lysates containing the same amount of soluble proteins were incubated overnight at 4 ℃
with the src antibody mAb327 (Oncogene Science Inc., Boston, MA, USA) and protein G-agarose (GIBCO BRL) to determine antibody complex precipitation. Precipitates were washed three times in buffer containing 50 mM Tris, pH 7-4; 150 mM NaCl; 0.1% Triton-X 100; 1 mM PMSF; 10 µg/ml leupeptin and 10 µg/ml aprotinin and twice in a buffer containing 20 mM PIPES, pH 7.0 and 10 mM MnCl2. The precipitates were then sus- pended in the kinase reaction buffer (20 mM PIPES, pH 7.0; 10 mM MnCl2 and 50 uM ATP) with 5 µg acid-denaturated (with 25 mM sodium acetate, pH 3·3, 30 ℃, 5 min) rabbit muscle enolase (Sigma-Aldrich). The kinase reaction was started by the addition of 10 µCi [y-32P]ATP (3000 mCi/ mmol) (Amersham) in 50 ul volume and run at 30 ℃ for 10 min. Reaction was finished by the addition of LDS-PAGE sample buffer (Novex, San Diego, CA, USA), samples were warmed 5 min at 65 ℃ and subjected to SDS-PAGE using a precast Novex gel electrophoresis system with 4-12% bis-tris NuPage gels (Novex). Dried gels were exposed to film and the amount of radioactivity was determined using phosphorimaging analysis (425E, Molecular Dynamics, Amersham Pharmacia Biotech Inc., Piscataway, NJ, USA).
Analysis of 3ßHSD activity
3ßHSD enzymatic activity was determined as pre- viously described (Bird et al. 1996). Briefly, cells were rinsed with plain medium and incubated at 37 ℃ with 1.5 ml of a serum-free DME/F12 medium supplemented with 1 uM dehydroepian- drosterone (DHEA) and 100 000 d.p.m. [3a- 3H]DHEA. Radiolabel and kinase inhibitors were added simultaneously, and at the indicated times 100 ul of the medium were removed from each well and the volume was brought to 1 ml by adding 900 ul water. Radiolabeled steroids were then extracted by mixing with chloroform (3 ml), and phase separation achieved by brief centrifugation. An aliquot (750 ul) of the upper phase was recovered and mixed with an equal volume of charcoal/dextran (5%/0.5% w/v). Following centrifugation to pellet the charcoal, 1 ml of the aqueous phase was removed and radioactivity was determined in a ß-counter. The 3ßHSD enzy- matic activity was then calculated after appropriate correction for volume, and expressed as pmol/mg cell protein.
Thin layer chromatographic identification of [14C]progesterone metabolites
Cells treated for 48 h without (basal) or with PP2 (10 µM) were incubated for 6 h with serum-free
medium containing [4-14C]progesterone (150 000 d.p.m./well) (NEN Life Science Products Inc., Boston, MA, USA) and 0.5 uM unlabeled proges- terone. At the end of the incubation, medium was extracted twice with dichloromethane (3 ml), dried and redissolved in 100 ul dichloromethane. The organic extracts were applied to silica gel plates (Keiselgel 60, F254, EM Industries, Hawthorne, NY, USA) and developed twice in chloroform-ethyl acetate, 90:10, vol/vol. Lanes containing radio- labeled samples were then scanned using a Bioscan detector (Bioscan Inc., Washington DC, USA). Peaks were identified by comparison to authenti- cated standards that were run on the same plate. Results are shown as percentage of total counts.
RNA extraction and Northern analysis
RNA was extracted from cells using Ultraspec RNA isolation system (Biotecx Laboratories Inc., Houston, TX, USA). Purity and integrity of the RNA were checked spectroscopically and by gel electrophoresis prior to use. Samples of RNA (10 µg) were separated by electrophoresis on gels containing 1% agarose in the presence of formalde- hyde. RNA was transferred to a nylon membrane (Hybond-N+, Amersham Pharmacia Biotech Inc., Piscataway, NJ, USA) by overnight blotting at 10 V and was cross-linked under UV light. Prehybridiz- ation was carried out at 42 ℃ for 6 h as previously described (Bird et al. 1995). Hybridizations were performed in the same composition buffer at 42 ℃ for 16 h, using antisense RNA probes. A 174 bp DNA fragment of the human 3@HSDII gene (GenBank accession no. M77144, nucleotides 7907- 8080) and a 302 bp fragment of the human CYP17 gene (GenBank accession no. M14564, nucleo- tides 48-349) were cloned into pBluescript KS (Stratagene, La Jolla, CA, USA). To generate a radioactive riboprobe complementary to the cloned 3ßHSD fragment, the plasmid was linearized with KpnI and used in a transcription reaction with [32P]UTP (3000 Ci/mmol) (Amersham Pharmacia Biotech Inc.) and T7 RNA polymerase following the protocol from the MAXIscript T7/T3 kit (Ambion Inc., Austin, TX, USA). To generate a radioactive riboprobe complementary to the cloned CYP17 fragment, the plasmid was linearized with SacI and transcripted as above using T3 RNA polymerase. After hybridization, blots were washed in 2 × SSC containing 0.1% SDS at room tempera- ture for 30 min, then in 0.1 x SSC containing 0.1% SDS at 37 ℃ for 30 min and at 42 ℃ for 15 min. Quantification of bound probe was determined using a PhosphorImager analysis (425E, Molecular Dynamics). Blots were subsequently exposed to film.
Membranes were than stripped by repeated washing in 0.1 x SSC and 0.5% SDS at 80 ℃ and checked for lack of radioactivity before reprobing. Finally, membranes were probed with a 598 bp DNA segment from the human glyceraldehyde-3- phosphate dehydrogenase (G3PDH) gene (Gen- Bank accession no. M17851, nucleotides 185-782). Probe was generated with the Rediprime II DNA Labelling System (Amersham Pharmacia Biotech Inc.) and used in a hybridization reaction. Bound probe was quantified as described above. G3PDH transcript was used to normalize data for CYP17 and 3ßHSD mRNA. For the cycloheximide (CX) experiments, CX (35 uM) was added to the plates 10 min before the addition of PP2. Because of the inhibitory effect of CX on G3PDH expression, loading was normalized by probing for 28S ribosomal RNA. 28S probe was prepared by incubation of the oligonucleotide 5’-AAA CGA TCA GAG TAG TGG TAT TTC ACC G-3’ with polynucleotide kinase in the presence of [y-32P]ATP (3000 mCi/mmol). Prehybridization and hybridiz- ation were performed exactly as described above.
RESULTS
Previous studies have shown that Ang II can activate the src signaling pathway (Hishida et al. 1995). Treatment of H295R cells for 5 min with Ang II stimulated the phosphorylation of the src substrate, rabbit muscle enolase, by 1.59 ± 0.13-fold (mean ± s.E. from three independent experiments). To determine if src tyrosine kinases play a role in aldosterone synthesis, the src kinase inhibitor PP2 (Hanke et al. 1996) was pre-incubated with H295R cells prior to stimulation with Ang II, K+, or dbcAMP. As previously demonstrated, the H295R cell line responds to Ang , K+, and dbcAMP by increasing production of aldosterone (Bird et al. 1993) (Fig. 1). Treatment of cells for 6 h with Ang II (100 nM), K+ (20 mM), or dbcAMP (1 mM) increased aldosterone by 2-fold, 2-fold, and 4-fold respectively (Fig. 1, upper panel). Chronic treat- ment (48 h) of the H295R cells increased the expression of enzymes involved in aldosterone and therefore had a greater fold increase in steroid production (Ang II, 6-fold; K+, 4.7-fold; dbcAMP, 3-fold) (Fig. 1, lower panel). PP2 caused a concentration-dependent inhibition of aldosterone production under basal, Ang II, K+, and dbcAMP stimulation. Inhibition was observed at both 6 and 48 h of treatment (Fig. 1). Half-maximal inhibition of aldosterone production was observed using between 1 and 3 M PP2 for cell treatments. In H295R cells aldosterone production occurred in a
16
6 hours
14
basal
- Ang II
Aldosterone production pmol/mg protein
12
K+
dbcAMP
10
8
6
4
2
48 hours
80
Aldosterone production pmol/mg protein
60
40
20
.
0
0.3
1
3
10
[PP2] UM
time-dependent manner under basal and Ang II treatment (Fig. 2). Inclusion of PP2 significantly inhibited basal and Ang II-stimulated aldosterone production throughout the times examined (6, 12, 24, and 48 h).
The first step in the synthesis of aldosterone is the conversion of cholesterol to pregnenolone. This step occurs in the mitochondria and is rate-limiting. To determine if PP2 inhibited aldosterone pro- duction at the rate limiting step, we examined its effects on metabolism of 22R-hydroxycholesterol (22R-OHChol) (Fig. 3). 22R-OHChol enters the mitochondria of steroidogenic cells by-passing the normal transport mechanisms needed for
30
平
25
basal
PP2
Aldosterone production pmol/mg protein
Ang II
Ang Il+PP2
20
15
愛
10
5
€
0
6
12
24
48
Time (h)
50
40
Aldosterone production pmol/mg protein
30
20
10
[PP2] µM
0
0
0.3
1
3
10
0
[22R-OHChol] uM 10
10
10
10
10
0
cholesterol. Treatment with PP2 for 6 h inhibited the conversion of 22R-OHChol to aldosterone. The inhibition was concentration-dependent with a decrease of aldosterone by 61% observed at 10 uM
PP2. These data suggested that PP2 inhibited aldosterone production in a manner beyond the regulation of cholesterol transport to the inner mitochondria membrane.
Treatments for 24 h with the general tyrosine kinase inhibitor, genistein, also inhibited aldoster- one production when used alone (73%) or in association with Ang II (96%) (Fig. 4A). One report has suggested that genistein has a direct (albeit non-specific) effect on the activity of the enzyme 3ßHSD (Wong & Keung 1999). To determine if either genistein or PP2 inhibited H295R 3ßHSD activity, cells were incubated with PP2 or genistein and activity was determined. As shown in Fig. 4B, genistein completely inhibited 3ßHSD activity while PP2 reduced the enzyme activity by no more than 25% at all times examined.
To determine if the overall metabolism of steroid precursor was modified by PP2, we examined the metabolism of radiolabeled progesterone (Fig. 5). H295R cells were incubated with PP2 (10 µM) for 36 h. Cells were then incubated with [14C]progesterone (0.5 uM) for 6 h. Under basal conditions, very little progesterone was metabo- lized. However, treatment with PP2 caused a dramatic increase in progesterone metabolism to 17a-hydroxyprogesterone. These data suggest that PP2 increases the level of 17a-hydroxylase activity.
To determine the effect of PP2 on the expression of CYP17 mRNA, we carried out Northern analysis (Fig. 6). RNA was isolated from H295R cells under basal conditions as well as from cells treated with PP2 (10 µM), Ang II (100 nM), dbcAMP (1 mM) or Ang II (100 nM) plus PP2 (10 µM). As previously reported, Ang II and dbcAMP increased expression of 3ßHSD. In addition, CYP17 tran- script levels were increased by dbcAMP. However, PP2 caused a specific induction of CYP17 without an effect on 3ßHSD mRNA. These data suggest that PP2 may inhibit aldosterone production, in part, by affecting the ratio of 3@HSD/CYP17 expression specifically by stimulating levels of CYP17 expression.
The transcription of some steroid-metabolizing enzymes is influenced by labile proteins and newly synthesized proteins. Therefore, the levels of steroidogenic enzyme transcripts can be affected by treatment with protein synthesis inhibitors such as cycloheximide (CX). We examined the action of CX on the PP2-induced expression of CYP17 (Fig. 7). As previously shown, CX increased mRNA levels of CYP17 compared with basal levels (Bird et al. 1998). Interestingly, CX blocked the PP2 induction of CYP17 mRNA suggesting that the PP2 effects rely on newly synthesized protein.
A
50
Aldosterone production pmol/mg protein
40
30
20
10
-
0
basal
PP2
Genistein
Ang II
Ang II + PP2
Ang II + Genistein
B
14
12
basal
3ßHSD activity nmol/mg protein
10
PP2
8
6
4
2
0
Genistein
0.5
1.0
2.0
4.0
Time (h)
DISCUSSION
In this study, we demonstrate that inhibition of the src family of tyrosine kinases blocks aldosterone
100
90
1. 11-Deoxycortisol/Corticosterone
4
basal
% of Total Counts
80
2. 17aOH-Progesterone
PP2 10uM
70
3. 11-Deoxycorticosterone
60
4. Progesterone
50
40
2
30
origin
20
10
1
3
0
0.0
2.0
4.0
6.0
8.0
10.0
12.0
14.0
16.0 18.0 20.0
Distance (cm)
synthesis. The inhibition, in part, resulted from the induction of 17a-hydroxylase activity and mRNA levels, which effectively removed substrate away from the pathway leading to aldosterone. These data suggest an important role for src in the regulation of adrenal steroidogenesis and particularly in the regulation of CYP17 levels.
The regulation of CYP17 is a key determinant in defining the steroids produced in adrenocortical cells (Conley & Bird 1997). In the glomerulosa, this enzyme is not expressed therefore allowing sub- strate to move in the direction of aldosterone. In contrast, the fasciculata and reticularis express high levels of CYP17, as its activity is necessary for cortisol and adrenal androgen synthesis. The mechanisms that block expression of 17a- hydroxylase in the glomerulosa are not clearly understood. We and others, using several adrenal model systems, have shown that Ang II is a potent inhibitor of CYP17 expression (McAllister & Hornsby 1988, Rainey et al. 1991, Bird et al. 1992, 1996a,b, Mason et al. 1995). These data suggest that Ang II stimulation of aldosterone production occurs not only through the activation of aldosterone synthase expression, but also by the inhibition of CYP17 expression. In addition to Ang II, epidermal growth factor (EGF) has been shown to inhibit CYP17 levels and increase the expression of 3ßHSD (Kim et al. 1998, Doi et al. 2000). Both Ang II and EGF have been shown to activate multiple signaling pathways that include the activation of src (Sayeski et al. 1999). Recently, an EGF-like protein (Pref-1) (Halder et al. 1998) was cloned that localizes to the glomerulosa of the adrenal. The role of Pref-1 in adrenal function has yet to be determined, however
Journal of Molecular Endocrinology (2001) 26, 207-215
Ang II + PP2
basal
PP2
Ang II
dbcAMP
CYP17
3ßHSD
G3PDH
the similarity with EGF suggests that src could be part of its signal pathway. In addition, previous studies have shown that aldosterone production is inhibited by the non-specific tyrosine kinase inhibitor, genistein (Bodart et al. 1995, Aptel et al. 1999, Wong & Keung 1999). Recent reports have suggested that the effects of genistein are non- specific and may relate to a direct inhibition of the activity of 3HSD (Wong & Keung 1999). Herein, we confirm that genistein is a potent inhibitor of aldosterone production. However, this inhibitor was also a potent inhibitor of 3ßHSD activity in H295R cells. Thus, our results further question the use of genistein in the study of the role of tyrosine kinases in steroidogenic cells.
The development of more specific inhibitors of the tyrosine kinase families has allowed us to examine the role of src tyrosine kinases in adrenal aldosterone production. Src is the best understood member of a family of eight tyrosine kinases that regulate cellular responses to extracellular stimuli
basal
PP2+CX
dbcAMP
PP2
CX
CYP17
28S
(Brown & Cooper 1996, Abram & Courtneidge 2000). In mammals, this family consists of Src, Fyn, and Yes that are ubiquitously expressed, whereas other members Lck, Hck, Fgr, Lyn, Blk and Yrk have more tissue restricted expression mainly in hematopoietic cells. Src is involved in the signaling of many receptors including receptor tyrosine kinases (i.e. EGF and platelet-derived growth factor), integrins, and some G-protein coupled receptors (i.e. AT1). PP1 and PP2 are recently developed inhibitors that show specificity for the src family of kinases (Hanke et al. 1996, Liu et al. 1999). Detailed studies of these inhibitors have shown that they rely on a single region revolving around Thr338 (Liu et al. 1999) that is found in all src family members. Detailed analysis of the inhibitors have confirmed that they are powerful tools to study the roles of src tyrosine kinases in differentiation and cell division.
There are currently no studies investigating the role of src in adrenocortical cells. Our study made use of PP2 to determine if src activity was important for aldosterone synthesis. PP2 potently inhibited basal, Ang II, K+ and dbcAMP stimulation of aldosterone synthesis. The ability of PP2 to inhibit basal and agonist-stimulated aldosterone production suggests effects on either viability or expression of steroid metabolizing enzymes. We observed no effect on cell viability and the PP2-increased conversion of progesterone to 17a- hydroxyprogesterone suggests that cell integrity was maintained. As this conversion could occur as a
result of increased expression of CYP17, we examined CYP17 mRNA by Northern analysis. These data confirmed that PP2 is an activator of CYP17 expression. Importantly, PP2 did not affect basal expression of 3ßHSD or inhibit its activity. This shift in the ratio of CYP17 to 36HSD effectively removed substrate from the pathway leading to aldosterone synthesis. Thus, one mechan- ism for PP2 inhibition of aldosterone production would be the induction of CYP17.
The exact role of the src family of tyrosine kinases in adrenal cell steroidogenesis will need further study. However, the ability of Ang II to increase src activity suggests that src signaling is yet another pathway that will influence steroid hormone production in adrenal cells. Indeed, the data presented herein support the idea that src signaling has an important role in the regulation of aldosterone production. Finally, because the inhi- bition of src increased CYP17 expression, it can be hypothesized that src tyrosine kinase may influence steroidogenesis by regulating the expression of steroid-metabolizing enzymes.
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RECEIVED IN FINAL FORM 29 January 2001 ACCEPTED 16 February 2001