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Division of Endocrinology and Metabolism, Department of Internal Medicine, National Institutes of Health Specialized Cooperative Center in Reproductive Research, University of Virginia Health Sciences Center (N.S., J.D.V.), Charlottesville, Virginia 22908; and Department of Cell Biology and Neuroscience, University of South Carolina School of Medicine (H.A.L.), Columbia, South Carolina 29208
Address all correspondence and requests for reprints to: Johannes D. Veldhuis, M.D., Endocrine Division, Box 202, Department of Internal Medicine, University of Virginia School of Medicine, Charlottesville, Virginia 22908. E-mail: jdv{at}virginia.edu
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Abstract |
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A stable cAMP analog, 8-bromo-cAMP (1 mM), and insulin/IGF-I also evoked supraadditive StAR promoter expression. To further explore the role of cAMP in LH-insulin (or IGF-I) actions, we cotransfected a Rous sarcoma virus (RSV)-driven minigene encoding the heat-stable inhibitor of the cAMP-dependent protein kinase (RSV/PKI) or a mutant plasmid (RSV/PKImut) along with the pStAR1423/luc promoter construct. Cotransfection of PKI, but not PKImut, with pStAR1423/luc significantly attenuated LH’s stimulation of luciferase activity and also reduced the magnitude of the transcriptional amplification exerted by LH and insulin or IGF-I. In corollary analyses of the protein kinase A (PKA) pathway, cotransfection of full-length pStAR1423/luc and a complementary DNA encoding a constitutively activated PKA catalytic subunit elevated basal and insulin (or IGF-I)-stimulated StAR promoter expression. LH and insulin (or IGF-I) also augmented steady state StAR transcript levels, as assessed by homologous RT-PCR, and StAR protein concentrations, as evaluated by Western blotting.
Together, these investigations document a significant role for insulin or IGF-I in enhancing LH-stimulated progesterone and cAMP biosynthesis and endogenous StAR message and protein accumulation and in augmenting cAMP-PKA-dependent transcriptional activation of the exogenous StAR promoter.
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Introduction |
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Several second messenger systems appear to participate in StAR
regulation, namely cAMP-dependent protein kinase A (PKA) (6, 8, 10), protein kinase C (PKC) (11, 12),
calcium-calmodulin (13), and growth factor signaling
pathways (8, 14). Transcriptional control of the StAR gene
has been the focus of several recent studies. For example,
steroidogenic factor-1 (SF-1), an orphan nuclear receptor appears to
play an important stimulatory role in StAR expression
(15). SF-1 controls endocrine glandular development in
part by regulating the expression of an array of genes involved in
steroidogenesis, including steroid hydroxylases (16, 17, 18)
and LHß (19). In addition, this transcription factor
influences expression of the ACTH receptor (20) and the
GnRH receptor (21). The porcine StAR promoter exhibits
three putative SF-1-binding sites, one or more of which may be
important for FSH/IGF-I-stimulated gene transcription
(22). Other transcription factors, such as
CCAAT/enhancer-binding proteins (C/EBP
and C/EBPß) and GATA-4, may
also act in combination and/or individually in governing StAR
transcription in some systems (23, 24, 25).
Both clinical and experimental data associate insulin or IGF-I with ovarian follicular growth and steroidogenic cytodifferentiation. Insulin and IGF-I stimulate granulosa cell steroidogenesis (26, 27, 28, 29). For example, insulinopenic animals and Igf1 mutant mice exhibit low ovarian weight, reduced follicular diameter, and impaired gonadotropin stimulation of estradiol and progesterone biosynthesis (30, 31, 32). Indeed, under some conditions insulin appears to function as a cogonadotropin to ovarian cells (29, 33, 34). However, precisely how gonadotropins such as LH interact with insulin or IGF-I at the level of gonadal cellular signaling and gene regulation is not known.
As LH and insulin/IGF-I act physiologically via individually distinct cell surface receptors and corresponding intracellular signaling pathways, their synergistic enhancement of granulosa cell progesterone synthesis probably entails important interactions at one or more regulatory loci. Here, we examine the actions of insulin/IGF-I on LH-stimulated cAMP accumulation, LH receptor (LHR) message levels, and the cAMP/PKA-dependent regulation of StAR gene expression. To this end, we investigate the control of endogenous StAR gene transcript and protein expression and exogenous StAR promoter activity under single and combined drive by LH/cAMP/PKA and insulin or IGF-I in primary cultures of porcine ovarian (granulosa-luteal) cells.
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Materials and Methods |
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Cell culture
Porcine ovaries from prepubertal (60- to 70-kg) gilts were
collected from an abattoir and transported in iced saline. The
aspirated granulosa cells were cultured as previously described
(8). Briefly, granulosa cells were isolated from small and
medium-sized (1–5 mm) antral follicles by fine needle aspiration under
sterile conditions. Cells were washed three times by low speed
centrifugation (3000 rpm) in Eagle’s MEM. Approximately 5 x
106 viable granulosa cells were plated in 12-well
culture dishes (Corning, Inc., Corning, NY) containing
bicarbonate-buffered MEM and 3% FBS (Life Technologies, Inc.) plus insulin (1 µg/ml), estradiol (0.5 µg/ml), and FSH
(5 ng/ml) to permit cell anchorage and initial luteinization. Cells
were allowed to attach to culture dishes for 48 h at 37 C and in
5% CO2. After attachment, monolayers were
maintained in serum-free MEM for 13–15 h. Thereafter, cells were
exposed to the indicated hormones in serum-free MEM. Replicate cultures
were terminated at 48 h for progesterone and cAMP assays of
culture medium, and the cells were harvested for RNA or protein
determination.
Progesterone and cAMP assays
Progesterone and cAMP concentrations in spent culture medium
were measured by RIA. Antibody-coated tubes manufactured by ICN Biomedicals, Inc. (Costa Mesa, CA), were used for progesterone
determinations. The RIA has a sensitivity of 0.15 ng/ml and less than
3% cross-reactivity with other relevant steroid hormones, including
estradiol, pregnenolone, and 17-hydroxyprogesterone. cAMP was
quantified in medium diluted in 0.1 N HCl followed by RIA
(35), which has a sensitivity of 0.65 pmol/ml.
Isolation and quantification of granulosa cell RNAs
Total RNA was isolated using TriReagent (Molecular Research Center, Inc., Cincinnati, OH) according to the
instructions of the manufacturer. RNA pellets were ethanol
precipitated, washed, and resuspended in sterile ribonuclease-free
water. Semiquantitative RT-PCR was used to assess the expression of
endogenous StAR and LHR gene transcripts. To denature secondary
structure, RNA was heated at 70 C for 3 min and then cooled to 4 C.
Oligo(deoxythymidine)15 and/or 18S ribosomal RNA
(rRNA) reverse primers were used for the RT reaction. The later
contained 1 mM each of deoxy (d)-TTP, dATP, dCTP, and dGTP
(Roche Molecular Biochemicals, Mannheim, Germany); 2.5
mM MgCl2; 1 x PCR buffer II (pH
8.3); 20 U ribonuclease inhibitor (Perkin-Elmer Corp.,
Norwalk, CT); 25 U murine leukemia virus reverse transcriptase
(Perkin-Elmer Corp.); and 1 µg total RNA in a 20-µl
reaction. AmpliWax PCR beads (Perkin-Elmer Corp.) were
used to form the moisture barrier. The reaction was incubated at 42 C
for 30 min and then terminated at 99 C for 5 min before being cooled to
4 C.
PCR amplifications of StAR, LHR, and 18S complementary DNAs (cDNAs) were performed in separate parallel 100-µl reactions containing of 2.5 mM MgCl2, 100 pmol each of forward and reverse gene-specific primers, 1 x PCR buffer II (pH 8.3), 50 µM of each dNTP, 1 µM of each gene-specific primer, 2.5 U AmpliTaq Gold DNA polymerase (Perkin-Elmer Corp.), and 5 µl RT cDNA product (see above). AmpliWax PCR beads (Perkin-Elmer Corp.) were used to form the moisture barrier. Using a thermocycler (Thermolyne Amplitron I, Barnstead/Thermolyne Corp., Dubuque, IA), all PCR reactions were heated to 95 C for 10 min to activate the DNA polymerase. The number of cycles and the optimal temperatures for linear cDNA amplification of each primer set were determined using experimental granulosa-luteal cell RNA in preliminary optimization experiments. StAR and LHR were amplified for 35 and 27 cycles, respectively, with denaturation at 94 C for 30 sec, annealing at 60 C for 30 sec, and extension at 72 C for 1 min. After a final extension step at 72 C for 10 min, the reaction was rapidly cooled to 4 C. The program used to amplify 18S cDNA consisted of 20 cycles of denaturation at 94 C for 30 sec, annealing at 58 C for 30 sec, and extension at 72 C for 1 min. After a final extension step at 72 C for 10 min, the reaction was rapidly cooled to 4 C. The presence of specific PCR products was confirmed by visualization on a 1.2% agarose gel stained with ethidium bromide. A positive (corpus luteum) and a negative (yeast) control RNA were included in all RT-PCR reactions. The identities of the cDNAs derived by RT-PCR were confirmed in multiple (at least two) independent samples from different batches of ovaries by automated DNA sequencing (ABI model, Biomolecular Research Facility, University of Virginia, Charlottesville, VA). PCR products were quantified using the PicoGreen fluorescent dye assay (Molecular Probes, Inc., Eugene, OR), with a sensitivity of 25 pg DNA.
PCR oligonucleotide primer pairs were designed based on the known cDNA sequences of the various target genes, so that the expected PCR product would be 393 bp for the porcine StAR cDNA, 343 bp for the porcine LHR, and 315 bp for 18S rRNA. The sense and antisense primers used for RT-PCR were: porcine StAR: sense, 5'-GGTTCTCAGCTGGAAGACAC-3' (300–319 bp); and antisense, 5'-GGTACAGCCGACACTCACAA-3' (673–692 bp; GenBank accession no. U53020); porcine LHR: sense, 5'-AGCCGACTATCACTCACCTA-3' (218–237 bp); and antisense, 5'-CTGGTACGGTGGTTATGTGT-3' (541–560 bp; GenBank accession no. M29525); 18S: sense, 5'-CGATGCTCTTAGCTGAGTGT-3' (862–881 bp); and antisense, 5'-GGAACTACGACGGTATCTGA-3' (1157–1176 bp; GenBank accession no. M10098). As porcine 18S rRNA sequence was not available from GenBank, we identified fully conserved sequences among the human, rat, mouse, and rabbit. PCR products were expressed as a ratio of specific gene product amplified to that of 18S rRNA.
PicoGreen assay for gene-specific PCR products
PCR products were quantified using the sensitive PicoGreen
(Molecular Probes, Inc.) fluorescent dye assay for double
stranded DNA as previously described (36). Ten microliters
of the PCR product and 90 µl 1 x buffer were mixed thoroughly
and added to 100 µl PicoGreen dye [diluted 1:200 in TE buffer (20
mM Tris-HCl, 1 mM EDTA, pH 7.5)] in
96-well plates. The reaction was incubated for 2–5 min
(37), and fluorometry was performed via a FluorImager 595
Optical Scanner (version 5.01, Molecular Dynamics, Inc.,
Sunnyvale, CA) to allow final quantitation using ImageQuant software
(version 5.0, Molecular Dynamics, Inc.).
Western blot analysis
Granulosa-luteal cell cultures were treated with control
solvent, LH, and/or insulin/IGF-I, and total cellular protein was
isolated to monitor StAR protein expression as described previously
(6). Briefly, equal (100-µg) amounts of protein were
subjected to 10% SDS-PAGE and transferred by electroblotting to
nitrocellulose membranes to allow detection with human StAR antibody.
The rabbit polyclonal antibody against recombinant human StAR protein
was provided by Jerome F. Strauss III (University of Pennsylvania,
Philadelphia, PA) and was described previously (38).
Antigen-antibody complexes were detected with donkey antirabbit Ig,
horseradish peroxidase, and the enhanced chemiluminescence Western
blotting analysis system (Amersham Pharmacia Biotech,
Piscataway, NJ). StAR protein bands with a nominal molecular mass of 30
kDa were quantified by laser densitometry using ImageQuant software
(Molecular Dynamics, Inc.).
Transfection of porcine granulosa-luteal cells
Our laboratory previously cloned a 1553-bp fragment (-1423 to
+130 bp) of the porcine StAR promoter and generated 5'-nested deleted
constructs upstream of a cytoplasmically targeted promoterless firefly
luciferase cDNA (22). Transient transfections of these
reporter constructs were carried out using Lipofectamine (Life Technologies, Inc.). Granulosa cells were plated in 12-well
plates and were allowed to attach and cytodifferentiate for 48 h
(as above). Media were changed after the first 24 h, and 24 h
later granulosa-luteal cell cultures were rinsed with serum-free MEM
without antibiotics for 20–30 min before transfection. Transfection
medium (1 ml/well) consisted of serum-free MEM without antibiotics with
2 µg total plasmid DNA and 12 µl Lipofectamine. After transfection
for 6 h (based on preliminary optimization experiments), the
medium was replaced with serum-free MEM containing antibiotics and the
indicated hormone treatments or vehicle. Expression was allowed to
proceed for 24 h, which was optimal to define the relative effects
of LH and or insulin/IGF-I on expression of the full-length promoter
construct, pStAR1423/luc. At the end of the treatment period, cells
were rinsed once at room temperature with Dulbecco’s PBS, lysed in 100
µl 1 x lysis buffer (Luciferase Assay System, Promega Corp., Madison, WI), and stored at -70 C until later assay.
Luciferase activity was measured using 100 µl luciferin substrate
(Promega Corp.)/20 µl cellular lysate, in a Turner
TD-20e luminometer (Turner Designs, Sunnyvale, CA). Protein
concentrations from the lysate were measured using protein dye reagent
(Bio-Rad Laboratories, Inc., Hercules, CA).
Transfections with 5'-deletional fragments were performed using equimolar concentration of pStAR/luc constructs in a total of 2 µg DNA. A promoterless luciferase construct, p0/luc, which exhibits no significant activity in response to any treatment, was used to adjust total DNA to 2 µg when necessary. Cotransfection consisted of 1.5 µg pStAR1423/luc with 0.2 µg Rous sarcoma virus (RSV)/ßPKA or 0.2 µg RSV/PKA inhibitor (PKI) or 0.2 µg RSV/PKImut or 0.2 µg p0/luc. The expression vector for RSV/PKI (251-bp DNA fragment encoding the complete amino acid sequence of rabbit muscle), RSV/PKImut (replaced the oligonucleotides coding for glycine rather than arginine at positions 20 and 21 of the protein), and RSV/ßPKA were described previously (39, 40). A negative control for RSV promoter activity consisted of 0.2 µg RSV/ß-galactosidase and 1.8 µg pStAR1423/luc. The RSV/ßPKA, RSV/PKI, and RSV/PKImut were provided by Dr. Richard Day (University of Virginia, Charlottesville, VA).
Statistical methods
Data are presented as the mean ± SEM of three
or more independent experiments, using separate batches of 200–300
ovaries on each occasion to confirm the reproducibility of results.
Data were subjected to ANOVA. Means were contrasted using the
post-hoc Tukey multiple comparison test. P
< 0.05 was considered significant.
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Results |
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monolayers of granulosa cells after anchorage and biochemical
luteinization were exposed to LH (100 ng/ml) and/or insulin or IGF-I
(100 ng/ml each) for 48 h in serum-free MEM. Figure 1, A and B,
summarizes the effects of these stimuli on progesterone and cAMP
accumulation in the medium. LH alone elevated progesterone and cAMP
accumulation compared with that in untreated (control) and
insulin/IGF-I treated granulosa cells (P < 0.01). Both
insulin and IGF-I augmented LH-induced progesterone and cAMP
accumulation synergistically (P < 0.001), which was
2-fold compared with the effect of LH alone. Insulin and IGF-I alone
did not significantly alter 48-h progesterone and cAMP
concentrations.
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, A and B, summarizes the results of
RT-PCR analysis of StAR and LHR messenger RNA (mRNA) accumulation in
granulosa-luteal cells after the administration of LH alone or in
combination with insulin or IGF-I. Relative quantification was possible
by normalizing LHR and StAR transcript signals for simultaneously
estimated values of 18S rRNA, as the amount of this reference gene
remained unaffected by any of our in vitro hormonal
treatments. LHR mRNA transcript accumulation rose significantly
(P < 0.05) after the administration of insulin or
IGF-I alone for 48 h compared with either basal or LH treatment.
In other studies in rat granulosa-luteal cultures, LH receptor mRNA
transcript half-life increased significantly after treatment with IGF-I
(41). Granulosa-luteal cells treated with LH with or
without insulin (or with or without IGF-I) also showed a small, albeit
significant, rise in LHR mRNA concentrations over the control value
(P < 0.05). Normalized StAR mRNA levels were increased
superadditively (P < 0.05) by the combination of LH
and insulin or IGF-I compared with LH alone. LH exposure significantly
increased StAR mRNA expression compared with untreated control
granulosa cells (P < 0.05). However, neither insulin
nor IGF-I alone altered StAR mRNA accumulation under the current
culture conditions.
Western blotting was used to test whether LH and/or insulin or IGF-I
elevates the expression of StAR protein in our porcine granulosa-luteal
cell cultures. As shown in Fig. 3, combined hormone stimulation (LH plus insulin or IGF-I) increased StAR
protein synergistically (P < 0.001) over the effect of
LH, insulin, or IGF-I alone. Individual exposure to LH, insulin, or
IGF-I did not significantly enhance StAR protein expression over the
basal level. Under these conditions, we observed primarily (>85%)
functionally mature 30-kDa StAR proteins (1, 38).
To examine LH and/or insulin/IGF-I-driven transcriptional activation of
the StAR gene, we quantified exogenous StAR promoter activation after
transient transfection of a full-length (-1423 to +130 bp) or
5'-deleted fragment(s) of the cloned upstream region of the porcine
StAR gene (22). Preliminary time-course data identified
24 h of expression as optimal for monitoring the actions of LH
with or without insulin or IGF-I. Figure 4, A and B, illustrates the stimulatory
impact of exposure to LH (100 ng/ml) alone on pStAR1423/luc reporter
activity. Insulin or IGF-I alone had no effect on the activity of this
full-length (or various deleted) StAR promoter fragment(s). Combining
LH and insulin (or IGF-I) enhanced pStAR1423/luc activity significantly
over that stimulated by LH alone. Deletional analysis suggested
transcriptional dependence within the upstream region of -219 to -31
bp. A construct containing -31 to +130 bp of the StAR promoter was
unresponsive to LH and/or insulin/IGF-I.
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. As shown in Fig. 6, expression of the RSV-driven PKA
ß-catalytic subunit activated pStAR1423/luc promoter activity, and
this effect was heightened significantly by coadministration of insulin
or IGF-I. In parallel cultures in the same experiment, granulosa-luteal
cells cotransfected with RSV/ß-galactosidase (0.2 µg/well) and
pStAR1423/luc exhibited no response to insulin or IGF-I. Cotransfection
of cells with a plasmid containing the protein kinase inhibitor cDNA
(RSV/PKI) repressed LH/insulin-induced StAR promoter expression (Fig. 7). Cotransfection of PKImut
allowed 60–80% expression of pStAR1423/luc expression, which was
otherwise attenuated by the wild-type protein kinase inhibitor.
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Discussion |
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In the testis and ovary, gonadotropins activate cAMP-dependent PKA signaling and up-regulate multiple key steps in steroidogenesis, including StAR gene expression (42). In addition to the pituitary gonadotropic hormones, insulin and IGF-I can facilitate basal and FSH/LH-stimulated steroidogenesis in granulosa (6, 8, 14, 43) and thecal cells (44). Insulinomimetic effects appear to be mediated via the IGF-I or insulin receptor (reviewed in Ref. 29) and possibly also via the inositol-glycan signaling pathway (45). Both receptor autophosphorylation (46) and in situ binding studies (47) indicate that insulin as well as IGF-I receptors (48) are expressed on swine granulosa cells. Other studies of swine granulosa-luteal cultures show that 100 ng/ml insulin has negligible cross-reactivity with IGF-I receptors (49). Although we did not repeat IGF/insulin binding studies in our culture conditions, insulin may be acting via its own receptor at this concentration.
Although the complex basis for the transcriptional amplification observed here between LH and insulin (or IGF-I) is not yet clear, LH, exogenous cAMP, and the transfected ß-catalytic subunit of PKA all synergized with insulin/IGF-I in enhancing StAR promoter activity. Conversely, a transiently transfected PKI minigene opposed this amplification by LH and insulin/IGF-I. These and earlier analyses show that insulin, IGF-I, and IGF-II alone do not stimulate StAR promoter-luciferase construct expression in immature or more differentiated granulosa-luteal cells (present data and Refs. 7 and 22). Such observations for the StAR gene differ from those for the porcine P450scc promoter, which can be activated independently by IGF-I alone (50). The foregoing distinction could indicate that insulinomimetic peptides exert their effects on the StAR promoter only in the presence of gonadotropin. For example, the latter interaction might require cAMP/PKA-dependent facilitation of promoter responsiveness to insulin/IGF-I, possibly via coactivation of cAMP-responsive regions (51, 52). Thus, we infer that whereas insulin/IGF-I can stimulate LHR message and enhance LH-stimulated cAMP accumulation (present data), the fundamental mechanism of synergistic induction of StAR gene transcription can be reproduced by distal (post-cAMP/PKA) signaling interactions.
LH and insulin/IGF-I synergistic stimulation of StAR gene transcription, as inferred by exogenous transient transfection assays, was corroborated by parallel induction of endogenous granulosa-luteal cell StAR mRNA and protein along with enhanced progesterone biosynthesis. In contrast, we showed previously that LH and/or insulin do not induce sterol carrier protein-2 gene expression in pig granulosa-luteal cells (8), thus defining the specificity of this response. Analogous gonadotropin/insulin synergistic interactions on StAR gene expression are not evident in cultured human granulosa-luteal cells (7), but can be demonstrated in studies of porcine and bovine granulosa-luteal cells (6, 14, 43) and rat Leydig cells (53). In addition to species differences, the endocrine status of the donor, the relative species dependence on IGF-II vs. IGF-I actions (54), and the experimental conditions (e.g. short-term vs. longer-term cultures) may account for the foregoing apparent discrepancy between the human and other mammalian species.
The synergism between LH and insulin/IGF-I was reproduced at the exogenous promoter level for all 5'-deletional fragments capable of responding to LH alone. Preliminary inspection of the porcine StAR gene 5'-flanking sequence reveals two putative SF-1, two C/EBP, and a single GATA sequence(s) within -120 bp of the transcriptional start site, as is also evident in the human, ovine, rat, and mouse promoters (24). Whereas deletion of the proximal putative SF-1 site in the present study abolished all remaining LH- plus insulin/IGF-inducible StAR promoter activity, other regulatory elements within the StAR promoter may contribute to maximal responsiveness. For example, two regulatory regions (-129 to -117 and -96 to -85) display a high degree of homology with the consensus binding element for members of the CCAAT/enhancer binding protein family of transcription factors (55). In this regard, LH/hCG promotes C/EBPß gene expression in granulosa cells obtained from rat antral follicles (56, 57). Transgenic mice harboring a targeted homozygous deletion of the C/EBPß gene exhibit reproductive defects, and adult females are sterile (31, 32, 57). Our 5'-deletional analysis of the porcine StAR promoter showed that deleting one C/EBPß site (within the region of -115 bp), moderately decreased hormone-inducible promoter expression, whereas further deletion of the second putative C/EBPß site (-96/-85) and two SF-1 (-111/-102 and -48/-41) sites abolished promoter expression, driven by either LH or LH and insulin/IGF-I. Further targeted mutational analysis of these implicated regions will be helpful to confirm their roles in the gonadotropin/insulin/IGF responsiveness of the StAR gene.
Analyses by other laboratories indicate that the candidate dosage-sensitive sex reversal gene, DAX-1, binds to hairpin structures in the human StAR promoter formed by DNA sequences immediately upstream of the TTAAA box (58). Overexpression of DAX-1 antagonizes SF-1-stimulated expression of the gene encoding Mullerian inhibiting substance in the testis (59) and also diminishes SF-1 mRNA expression. The latter inhibition of SF-1 activity by DAX-1 can impair T3-stimulated StAR gene expression in mouse Leydig tumor cells (13) and in cAMP analog-stimulated Y-1 adrenal cells (60). On the other hand, bovine luteal cells expressed SF-1 constitutively and equally in small and large luteal cells (43). Thus, posttranslational, rather than transcriptional, activation of SF-1 may be the relevant mode of regulation of SF-1 efficacy. In this regard, the mitogen-activated protein kinase-associated signaling pathway appears to be able to activate SF-1 by phosphorylating this transcription factor (61). Thus, a plausible speculation is that insulin/IGF-I enhance LH’s stimulation of StAR promoter activity by facilitating one or more interactions between SF-1, C/EBPß, GATA, and/or another as yet unidentified regulatory factor(s) (24). In principle, this could occur in part via posttranslational modification of SF-1 by the insulinomimetic growth factor-stimulated mitogen-activated protein kinase signaling pathway. Further studies are needed to elucidate the insulin/IGF signaling mechanism in this context.
Cotransfection of porcine granulosa-luteal cells with selected PKA modulatory expression vectors documented a role for endogenous PKA in mediating the LH/insulin supraadditive drive of the exogenous pStAR1423/luc promoter. Transfection of a RSV/PKI, but not a RSV/PKImut, expression vector inhibited LH/insulin amplification markedly. PKI minigene transfection is believed to achieve highly specific inhibition of PKA by redistribution of the PKA catalytic subunit from the active nuclear compartment to the cytoplasm (62). The PKI expression vector also impeded exogenous cAMP-stimulated expression of the endogenous PRL gene and its exogenous promoter, whereas PKImut did not alter transcription of the latter (39). In the present study, cotransfection of PKImut did not significantly inhibit pStAR1423/luc expression. In conjunction with the facilitative effects of exogenous cAMP and expression of the ß- catalytic subunit of PKA (above), the available data suggest in aggregate that the combined actions of LH and insulin/IGF-I may be mediated in part via signaling interactions between cAMP/PKA and insulin/IGF-I pathways.
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Acknowledgments |
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Footnotes |
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Received April 4, 2000.
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References |
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-hydroxylase/c17–20 lyase). Mol Endocrinol 10:147–158 is modulated by the negative
transcription factor DAX-1. Endocrine 10:83–91[CrossRef][Medline]
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L. Devoto, P. Kohen, R. R. Gonzalez, O. Castro, I. Retamales, M. Vega, P. Carvallo, L. K. Christenson, and J. F. Strauss III Expression of Steroidogenic Acute Regulatory Protein in the Human Corpus Luteum throughout the Luteal Phase J. Clin. Endocrinol. Metab., November 1, 2001; 86(11): 5633 - 5639. [Abstract] [Full Text] [PDF]
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N. Sekar and J. D. Veldhuis Concerted Transcriptional Activation of the Low Density Lipoprotein Receptor Gene by Insulin and Luteinizing Hormone in Cultured Porcine Granulosa-Luteal Cells: Possible Convergence of Protein Kinase A, Phosphatidylinositol 3-Kinase, and Mitogen-Activated Protein Kinase Signaling Pathways Endocrinology, July 1, 2001; 142(7): 2921 - 2928. [Abstract] [Full Text] [PDF]
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