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Department of Biochemistry and Molecular Biology, University of Louisville School of Medicine, Louisville, Kentucky 40292
Address all correspondence and requests for reprints to: Dr. Barbara J. Clark, Department of Biochemistry and Molecular Biology, University of Louisville School of Medicine, Louisville, Kentucky 40292. E-mail: bjclark{at}louisville.edu
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Introduction |
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The stimulation of StAR synthesis and steroidogenesis by trophic hormones is mediated by cAMP in general; however, in the adrenal cortex, activation of divergent signaling pathways can independently stimulate adrenal steroidogenesis and StAR protein synthesis. Glucocorticoid synthesis by the adrenal zona fasiculata is regulated by ACTH via the cAMP-dependent protein kinase A (PKA) pathway, whereas the two major physiological regulators of aldosterone synthesis in the adrenal zona glomerulosa are angiotensin II (AII) and extracellular K+. AII binds to cell surface receptors that, in turn, activate phospholipase C, which results in the release of diacylglycerol and inositol 1,4,5-trisphosphate from phosphatidylinositol-4,5-bisphosphate (14). Diacylglycerol stimulates protein kinase C (PKC), whereas inositol 1,4,5-trisphosphate increases intracellular calcium levels ([Ca2+]i) via stimulating the release of Ca2+ from internal stores. AII also mediates an influx of external Ca2+ through dihydropyridine-insensitive Ca2+ channels in the plasma membrane. K+ action, on the other hand, is specifically associated with an influx of external Ca2+ via voltage-gated L- and T-type Ca2+ channels. Therefore, both agents act mainly by increasing the [Ca2+]i levels, although by distinct mechanisms.
The molecular mechanisms by which [Ca2+]i mediates an increase in steroidogenesis are only recently emerging (15, 16, 17, 18, 19). Early reports demonstrated that de novo protein synthesis was important in AII- and K+-stimulated steroidogenesis, as had been documented for the ACTH-cAMP-PKA pathway (6, 7, 20, 21). In terms of StAR expression, pp30 mitochondrial proteins (StAR) were shown to be radiolabeled in isolated bovine glomerulosa cells in an AII- or K+-dependent manner. Atrial natriuretic peptide, which blocks AII-stimulated steroidogenesis, blocked the AII-dependent increase in the pp30 proteins (22). Our previous studies in human adrenocortical H295R cells confirmed that increased mineralocorticoid synthesis by AII and K+ treatment is paralleled by an increase in StAR protein (15). Based on these data we proposed that StAR regulation may be a common mechanism by which the divergent signaling pathways control adrenal steroid production. In support of a role for increased [Ca2+]i mediating the AII or K+ signal, recent studies using calcium clamp techniques that elevate [Ca2+]i levels have shown that StAR synthesis is increased in bovine glomerulosa cells and that the effect is blocked by atrial natriuretic peptide, consistent with previous reports using AII and K+ (19). StAR messenger RNA (mRNA) levels were shown to be induced by increased [Ca2+]i (calcium clamp), and nuclear run-on assays indicated transcriptional activation as the mechanism for increased StAR expression.
In some cases, such as the human c-fos and human proenkephalin genes, the Ca2+-responsive element has been shown to be identical to the cAMP-responsive elements (CRE) (23, 24). For activation of c-fos, for example, the cAMP and Ca2+ pathways converge at the point of phosphorylation of the CRE-binding protein (24, 25). However, the StAR promoter lacks a consensus CRE as well as a classical phorbol ester element (26). 5'-Deletion analyses of the mouse, human, porcine, bovine, and rat StAR promoters indicate that the cAMP-responsive region is within 254, 150, 60, 100, and 342 bp of the transcriptional start site, respectively (26, 27, 28, 29). Inspection of these promoter regions indicates high sequence identity within the first 150 bp that includes conserved elements that have been shown to bind steroidogenic factor-1 (SF-1) (30). SF-1 is an orphan nuclear receptor that plays a critical role in organogenesis of the adrenal and gonads as well as the developmental and tissue-specific regulation of genes encoding the cytochrome P450 steroid hydroxylases (31, 32, 33). SF-1 has also been shown to trans-activate StAR promoter-dependent reporter gene activity in transiently transfected nonsteroidogenic cells (27, 28, 30, 34). In these studies, cAMP enhanced the SF-1-dependent response. Similarly, studies on reporter gene expression in human granulosa-lutein cells demonstrated that mutation of SF-1 elements located at -95 and -42 in the human StAR promoter greatly diminished basal expression as well as the cAMP-dependent response, although induction by forskolin was not abolished (35). On the other hand, mutation of SF-1 elements located at -42 and -135 in the mouse promoter did not affect cAMP induction in MA-10 mouse Leydig cells or Y1 adrenocortical cells (26). Clearly, more studies are required to determine the precise role of SF-1 in StAR gene activation. It may be that the cAMP-dependent response of StAR is dependent upon SF-1 in a tissue- or species-specific manner.
In this study we evaluated the effects of N6,2'-O-dibutyryl cAMP (Bt2cAMP), AII, and K+ on StAR mRNA levels in H295R cells to determine whether these physiological regulators of aldosterone synthesis mediate a transcriptional or posttranscriptional response for StAR synthesis and acute steroid production. A second objective was to compare the AII, K+, and Bt2cAMP responses to determine the point of convergence for these multiple second messengers on StAR expression. We demonstrate that StAR steady state mRNA levels are increased by Bt2cAMP and AII, but not by K+ or 12-O-tetradecanoylphorbol-13-acetate (TPA), treatment of H295R cells. Transient transfection assays using a luciferase reporter gene driven by the StAR promoter indicate that only Bt2cAMP and AII stimulate an increase in luciferase activity. Together these data suggest that the AII-mediated induction of StAR gene expression cannot be mimicked by K+ treatment alone. 5'-Deletion analysis of the StAR promoter indicates that both the cAMP and AII response elements are within 150 bases of the transcriptional start site. Mutation of an SF-1/AdBP4 element localized at -95 abolishes both Bt2cAMP- and AII-induced luciferase activity in these transient transfection assays. Thus, transcriptional activation of StAR gene by a SF-1-dependent mechanism may represent a common pathway for ACTH (PKA) and AII action in stimulating steroid production in adrenal zona fasciculata and glomerulosa cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-32P]Deoxy (d)-CTP was obtained from NEN Life Science Products (Wilmington, DE). RNAsin and
Taq DNA polymerase were purchased from Promega Corp. (Madison, WI).
H295R cell culture
H295R cells were a gift from Dr. William E. Rainey, University
of Texas Southwestern Medical Center (Dallas, TX). The cells were grown
and maintained in DMEM/F12 containing 2.5% NuSerum type I and 1% ITS
culture supplements (Collaborative Biomed Products, Bedford, MA) in
75-cm2 flasks at 37 C under a humid atmosphere of
5% CO2-95% air as previously detailed (15).
RT-PCR
H295R cells were grown in 60-mm tissue culture dishes to
70–80% confluence, then placed in serum-free medium for 24 h
before treatment. The cells were treated for 6 h with serum-free
medium alone (control) or containing Bt2cAMP (1
mM), AII (10 nM), K+ (16
mM), or TPA (10 nM). The medium was removed,
and total RNA was isolated using the Trizol reagent following the
instructions of the manufacturer (Life Technologies, Inc.). The RT and PCR reactions were performed as described
previously (36). The StAR oligonucleotide primer sequences are
5'-CCAGATGTGGGCAAGGTG-3' (forward; synthesized by Midland Certified
Reagent Co., Midland, TX) and 5'-CCTCTGCGCTTGGTACAGC-3' (reverse;
synthesized by Synthetic Genetics, San Diego, CA). These primers span
nucleotides 466–708 of the human (h) StAR complementary DNA (cDNA) to
generate a product of 242 bp. The 1 x Taq buffer
(Promega Corp.) and 2.5 mM MgCl2
concentrations were maintained for both the RT and amplification
reactions. Random hexamer oligonucleotides were used for the RT
reaction (20 µl), which contained dNTPs, RNAsin, MgCl2,
200 U Moloney murine leukemia virus reverse transcriptase, and 1.0 µg
total RNA. For amplification of the cDNA, the RT reaction was first
heat inactivated, then diluted to a final volume of 100 µl that
included 50 pmol of the primer pair for hStAR, 10 pmol of the human
ß-actin primer pair (CLONTECH, Palo Alto, CA), 2.5 µCi
[32P]dCTP, and Taq polymerase. In experiments
in which hStAR and ß-actin were amplified separately, the RT reaction
for an individual treatment was split (8 µl/reaction) and added to a
PCR reaction containing either the hStAR primer pair or the ß-actin
primer pair. After 25 cycles of PCR, the products were separated on a
2% agarose gel along with a 100-bp DNA ladder (GenSura Laboratories,
Del Mar, CA) and visualized by ethidium bromide staining. The gel was
dried and exposed to a phosphorscreen, and quantitation of the products
was performed using a Molecular Dynamics, Inc., PSF
PhosphorImager (Sunnyvale, CA). The gel was then exposed to x-ray film
and visualized by autoradiography.
Northern analysis
Treatment of H295R cells and RNA isolation were performed as
described above for RT-PCR. Thirty micrograms of total RNA were
separated on an agarose-formaldehyde gel (1.25%:3%) as previously
detailed (37). The RNA was transferred to Hybond-N+ nylon
membrane and fixed by UV cross-linking (StrataLinker,
Stratagene, La Jolla, CA). The blot was probed with the
hStAR PCR product that was purified and radiolabeled using
[-32P]dCTP and the Prime-A-Gene kit (Promega Corp.). The RNA was sized using a 500-bp fluorescein ruler
(Bio-Rad Laboratories, Inc., Hercules, CA) and an
end-labeled 1-kb DNA ladder. The blot was exposed to a phosphorscreen,
and the products were quantitated using a Molecular Dynamics, Inc., PSF PhosphorImager, then exposed to x-ray film and
visualized by autoradiography. The blot was stripped and reprobed with
human ß-actin PCR product that was purified and radiolabeled using
[-32P]dCTP and the Prime-A-Gene kit and detected as
described above.
Plasmids and transient transfection of H295R cells
Plasmids containing DNA for human StAR promoter in
pGL2 vector were a gift from Dr. Jerome F. Strauss III,
University of Pennsylvania (Philadelphia, PA), and are the constructs
described and used in previous manuscripts (27, 35). pGL2 containing
-1521 bp of the CYP11B2 promoter was a gift from Dr. William E.
Rainey, University of Texas Southwestern Medical Center (Dallas, TX)
(18). H295R cells were plated onto 24-well tissue culture dishes at
300,000 cells/well the day before transfection. The cells were
cotransfected with the indicated luciferase reporter gene construct (2
µg/ml) and pCMVß-galactosidase expression vector (2 µg/ml) using
lipofectamine reagent (20 µg/ml) and serum-free, antibiotic-free
DMEM/F12 following the instructions of the manufacturer (Life Technologies, Inc.) as previously described (9). The cells were
treated with the lipid/DNA mixture for 6 h, then the medium was
replaced with complete DMEM/F12 for 16 h. Cells were placed in
serum-free medium for 24 h, then treated for 6 h with either
Bt2cAMP (1 mM), AII (10 nM),
K+ (16 mM), or TPA (10 nM). The
luciferase assay system (Promega Corp.) was used to
prepare cell lysates and measure luciferase activity (Lumat LB 9507
luminometer, Wallac, Inc., Gaithersburg, MD).
ß-Galactosidase activity was determined using
ß-D-galactopyranoside (Roche Molecular Biochemicals, Indianapolis, IN) as a substrate and monitoring
the absorbance at 595 nm. Each treatment was performed in triplicate,
and the luciferase (relative light units) and ß-galactosidase
activities were corrected for background activity (nontransfected cell
lysates), then the relative light units were normalized to
ß-galactosidase. The means ± SD were determined for
each treatment group, then the data were expressed relative to the
control value (unstimulated sample). To determine a statistical
significance, the mean ± SEM for the relative fold
induction from each experiment was determined and used in Student’s
pooled t test to calculate the confidence interval.
P < 0.05 was considered statistically significant.
Immunoblot analysis
H295R cells were grown in 60-mm tissue culture dishes to
70–80% confluence, then placed in serum-free medium for 24 h
before treatment. The cells were treated for 6 h with serum-free
medium alone (control) or medium containing Bt2cAMP (1
mM), AII (10 nM), K+ (16
mM), or TPA (10 nM). The cells were washed with
PBS, and the cells were collected in 0.25 M sucrose, 10
mM Tris-HCl (pH 7.4), and 0.1 mM EDTA plus
protease inhibitors as described previously (9). The cells were lysed
by repeated freeze-thaw cycles, and the protein concentration of
postnuclear supernatant (800 x g supernatant) was
determined by the method of Bradford using Bio-Rad Laboratories, Inc., reagent (Bio-Rad Laboratories, Inc.,
Hercules, CA). Fifty micrograms of cell lysate were separated on 12.5%
SDS-PAGE using standard procedures as described previously (9), and the
proteins were transferred to polyvinylidene difluoride membranes
(Bio-Rad Laboratories, Inc., Hercules, CA) using 20
mM Tris-HCl (pH 7.4), 150 mM glycine, 10%
methanol, and 0.01% SDS. Immunoblot analysis was performed as
described previously, except that the primary antibody used was a
rabbit anti-StAR polyclonal antibody raised against purified mouse StAR
protein and was provided by Drs. Dale B. Hales and Karen Held-Hales,
University of Illinois (Chicago, IL) (9, 36). The secondary antibody
was a donkey antirabbit IgG conjugated with horseradish peroxidase
(Amersham Pharmacia Biotech, Arlington Heights, IL), and
the signal was detected by chemiluminescence using the Renaissance kit
from NEN Life Science Products (Boston, MA). In some
cases the proteins were isolated from the phenol-ethanol supernatant
saved from the RNA isolations with Trizol reagent following the
instructions of the manufacturer.
Aldosterone assay
The medium was saved from the transient transfection experiments
or RNA isolation experiments, and aldosterone was measured directly in
the cell medium by RIA using a commercial kit from Diagnostics Systems Laboratories, Inc. (Webster, TX). The assays were
performed in duplicate, and data were expressed as picograms of
aldosterone per ml medium. The mean ± SD for each
treatment (triplicate samples for transient transfection or duplicate
samples for RNA) were determined, and the data were normalized to
control values. The relative fold induction was then determined for
each experiment, and the mean ± SEM for all
experiments were determined.
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Results |
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) was used to
determine whether steady state levels of StAR mRNA are increased in
H295R cells treated with Bt2cAMP, AII, K+, and
TPA. Activation of the PKA pathway by Bt2cAMP resulted in a
1.8-fold increase in StAR mRNA compared with the control value, and AII
activation of the Ca2+ and PKC pathways resulted in a
1.5-fold induction within 6 h of treatment (Table 1). Although an increase in mRNA levels
was observed in each experiment for K+ treatment of cells,
the fold induction over control levels was not statistically
significant. TPA had no effect on StAR mRNA levels, and cotreatment
with both agents, K+ and TPA, was indistinguishable from
K+ alone (data not shown). Northern analysis (Fig. 2) demonstrated that two major StAR
transcripts of 1.7 and 2.7 kb are present in H295R cells. In addition,
the increase in StAR steady state mRNA levels detected by RT-PCR
appears to be due mainly to an increase in the 1.7-kb transcript.
Induction levels determined by Northern analysis were consistent with
the RT-PCR data (Table 1). As the AII response was only 50% increased
over the control level, we tested the response of the promoter by
transient transfection of H295R cells with a luciferase reporter gene
driven by 1.3 kb of the 5'-flanking region of the human StAR gene. The
data support the results of the endogenous StAR gene expression;
luciferase activity was induced 3.9-fold by Bt2cAMP and
2.4-fold by AII, whereas K+ and TPA resulted in 1.6- and
1.3-fold changes, respectively (Fig. 3).
Neither the K+ nor the TPA response was significantly
increased over the control value within each individual experiment.
However, fold induction from all experiments indicates that
K+, but not TPA, can modestly induce transcriptional
activity of the StAR promoter in transient transfection assays. These
data clearly discriminate between induction of StAR by AII signaling
and K+ depolarization of H295R cells.
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Compared with that in unstimulated cells, K+ treatment
resulted in a 3.7-fold increase in aldosterone levels, which verifies
that these cells exhibit a glomerulosa-specific response. As observed
previously, the potency of induction of steroid synthesis was
Bt2cAMP > AII > K+, and TPA did not
stimulate aldosterone production. K+ treatment of H295R
cells transiently transfected with a CYP11B2-luciferase reporter gene
construct resulted in a 1.8-fold increase in luciferase activity (Fig. 4), and the Bt2cAMP and AII
responses were 3- and 2.7-fold increased over untreated levels,
respectively. These data are consistent with the previous report for
CYP11B2 and verify that the cells are steroidogenically responsive to
K+ treatment (18). Western blot analysis demonstrated that
StAR protein was induced by Bt2cAMP, AII, K+,
and TPA treatment of H295R cells (Fig. 5). Comparison of the induction of StAR
mRNA, protein, and steroidogenesis by these agonists indicates that the
steroidogenic responsiveness of the cells correlates with the relative
level of agonist-induced StAR protein expression
(Bt2cAMP > AII > K+) with the noted
exception of TPA. The increases in StAR protein and aldosterone
synthesis by K+ treatment were approximately 62% and 69%
of the AII response, respectively (Table 1), which is consistent with
our previous report (15). Our data indicate that StAR protein levels
and steroid production are markedly increased above control levels in
response to K+ treatment, whereas there is no statistically
significant increase in endogenous StAR mRNA levels. Similarly,
TPA-induced increases in StAR protein occur without concomitant
increases in steady state mRNA levels. Together these data suggest that
a posttranscriptional mechanism(s) contributes to increased StAR
expression in response to K+ and TPA. The AII response, on
the other hand, has a transcriptional component that results in full
responsiveness of the cells to this agonist.
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, Bt2cAMP or AII treatment of H295R cells that were
transiently transfected with a luciferase reporter gene construct
containing 1.3 of the StAR 5'-flanking region resulted in a 4.3 ±
0.4-fold or 2.4 ± 0.1-fold increase in luciferase activity,
respectively. Deletion to -885 bp did not affect either basal or
Bt2cAMP induction, but further deletion to -235 resulted
in increased basal and Bt2cAMP-dependent luciferase
activities. Nevertheless, the induction by Bt2cAMP or AII
was not affected by deletion of 1065 bp of the promoter. However,
deletion of an additional 85 bp resulted in a 2-fold increase in the
Bt2cAMP response, suggesting the potential for a negative
regulatory element between -150 and -235 bp. The final reporter gene
construct, containing -85 to +39 bp of the StAR promoter, was not
responsive to either Bt2cAMP or AII, suggesting that the
region of the StAR promoter required for the AII-dependent increase in
luciferase activity colocalizes with the cAMP-dependent region (-85 to
-150).
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). These data
confirm the importance of the SF-1 element located at -95 for cAMP
responsiveness of the human StAR gene in an acutely hormonally
responsive human cell line. Furthermore, these studies are the first to
demonstrate that the AII response is also dependent upon the SF-1
element located at -95 bp from the transcriptional start site.
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Discussion |
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The role of increased intracellular Ca2+ in the AII response of the adrenal glomerulosa to produce mineralocorticoids is well established. However, the AII response cannot be mimicked by K+ depolarization of the plasma membrane (16, 43, 44). Although intracellular [Ca2+]i was not determined in the experiments herein, previous studies by Bird et al. demonstrated a 2-fold increase in [Ca2+]i in K+-treated H295R cells (45). In these studies the K+ response was 64% of the AII response in terms of increased intracellular Ca2+, and nifedipine blocked the K+-mediated increase, but not the initial AII response. Similar effects on the differential sensitivity of the AII and K+ responses to nicardipin, another Ca2+ channel antagonist, have been shown for bovine adrenal glomerulosa cells (43). AII-stimulated steroid production was not blocked by [Ca2+]i channel antagonists, but it was partially diminished (40%). On the other hand, the dependence on extracellular Ca2+ for the stimulatory action of K+ is demonstrated by the inhibition of extracellular Ca2+ influx and steroid production by nifedipine or nicardipin (43, 45). These studies indicate that the influx of extracellular Ca2+ in response to AII is distinct from K+ for steroidogenesis. Our data also support the proposal that the Ca2+ pools contributing to the steroidogenic response of AII are distinct from the K+ response in terms of StAR regulation. To this end, we first confirmed that K+ treatment of H295R cells results in a 4.6-fold increase in StAR protein (15). However, the increase in protein observed with K+ treatment is not paralleled by an increase in StAR steady state mRNA levels, as determined by Northern analysis and RT-PCR. The K+ induction of StAR protein was approximately 62% of the AII response, consistent with the K+-stimulated aldosterone production, which was approximately 69% of the AII-stimulated steroid production. Similarly, the greater increases in StAR protein as a result of Bt2cAMP and AII treatment are correlated with greater increases in steroid production. These data support the premise of a direct relationship between the levels of StAR protein and the steroidogenic capacity of the cell. The exception to this rule is that TPA stimulates an increase in StAR protein without stimulating adrenal steroid production in H295R cells (15). This point will be discussed below.
Although a modest increase in StAR gene expression was detected by luciferase reporter gene activity, the changes in endogenous StAR gene activity, as measured by steady state mRNA levels, clearly discriminate between the effects of AII and K+ or TPA on StAR expression. Recently, StAR was shown to be transcriptionally activated by increased [Ca2+]i in bovine adrenal glomerulosa cells (19). In these studies cells were treated with ionomycin to maintain intracellular [Ca2+]i of 600–700 nM. StAR mRNA was increased 1.9- and 1.7-fold over control levels, as determined by Northern and nuclear-run on assays, respectively, whereas protein levels were increased 2-fold. Comparison of data from the bovine studies with our data suggests that the ionomycin treatment mimics the AII pathway for StAR transcriptional regulation. The lack of a K+-mediated increase in StAR mRNA in H295R cells could be due to the level of [Ca2+]i; 600–700 nM is approximately 2-fold greater than the levels measured in H295R cells with K+ treatment (45). However, localization of Ca2+ within the cell has been reported to be dependent upon the stimulus/agonist (44, 46, 47). Therefore, it is likely that the mechanism of entry of Ca2+ into the cell and its cellular localization will dictate the response to AII compared with that to K+ or ionomycin. In our studies the response is StAR expression, and our results suggest posttranscriptional regulation of StAR by the influx of [Ca2+]i via voltage-gated Ca2+ channels (K+ mediated), whereas AII mediates both transcriptional and posttranscriptional responses. As TPA treatment did not induce StAR mRNA levels, it is likely that the transcriptional component of AII action is mediated by a Ca2+-dependent signaling pathway. However, further studies are required to determine the transcriptional and posttranscriptional mechanisms for AII- and AII/K+-mediated increases in StAR.
To elucidate possible transcriptional regulatory mechanisms, we have used reporter gene assays to identify the AII-responsive region(s) in the 5'-flanking region of the hStAR promoter. The hStAR promoter lacks a consensus CRE as well as a phorbol ester response element, but 5'-deletion analysis narrowed the Bt2cAMP-responsive region to -150 bp of the start site of transcription. Site-directed mutagenesis of a SF-1 element at -95 bp demonstrated the importance of SF-1 in StAR gene expression and in the cAMP-dependent response (27, 48). We show that the same SF-1 element at -95 bp is required for the Bt2cAMP and AII responses of StAR. This is consistent with previous studies that have demonstrated that the Ca2+-responsive region colocalizes with the CRE for the human proenkephalin and c-fos genes as well as the hCYP11B2 gene (18, 23, 24). CYP11B2 encodes aldosterone synthase and is expressed in the adrenal glomerulosa. Analysis of the CYP11B2 promoter in H295R cells indicated that the cAMP-, K+-, and AII-responsive regions colocalize to a common responsive element, indicating the potential for cross-talk among multiple signaling pathways (18). It is possible that phosphorylation of SF-1 mediates the PKA and Ca2+ responses for StAR gene regulation in H295R cells (49, 50). However, as we used AII, it is also possible that Ca2+ is not the direct mediator, but activates another second messenger, such as cAMP (44).
Potential mediators of the Ca2+ signal in adrenal glomerulosa cells are the Ca2+-binding protein calmodulin (CaM) as well as the CaM-binding proteins, calcineurin and Ca2+/CaM-dependent protein kinase II (CaMKII) (51, 52, 53, 54). Calmidizolium and KN93, antagonists of calmodulin and CaMKII, respectively, inhibit AII- and K+-induced aldosterone production in H295R cells (52). Previously, we demonstrated that AII- or K+-induced StAR protein expression is not inhibited by KN93, suggesting that CaMKII is not required for StAR expression (52). More recently, KN93 was shown to block CYP11B2 mRNA induction by K+, but had no effect on AII induction of gene expression (53). Thus, a difference in Ca2+ signaling pathways may contribute to the AII vs. K+ responsiveness of CYP11B2. This may also explain the difference in transcriptional vs. posttranscriptional regulation of StAR by AII and K+.
Although CaMKII is not required for StAR expression, a CaMKII-dependent phosphorylation of StAR may be required to regulate the function of StAR. In support of this proposal, previous studies showed that mutation of Ser194 to Ala, which is within a consensus PKA and CaMKII phosphorylation motif, decreased StAR function 60% in transient transfection assays in COS-1 cells (55). Furthermore, StAR was shown to be synthesized, but not phosphorylated, in MA-10 cells or rat adrenal glomerulosa cell treated with TPA. In these cells, TPA also failed to stimulate steroid production (56, 57). Although a potential PKC phosphorylation site at Thr249 is conserved between mouse and human StAR, the functional significance of this site has not been experimentally tested. Herein we confirmed that activation of the PKC pathway with TPA treatment resulted in increased StAR protein without stimulating aldosterone synthesis (15). Furthermore, the effect of PKC appears to be at the posttranscriptional level, as no increase in StAR gene transcription was detected.
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Acknowledgments |
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Footnotes |
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Received May 10, 1999.
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-hydroxylase/c17–20 lyase). Mol Endocrinol 10:147–158[Abstract]
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