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Endocrinology Vol. 145, No. 6 2760-2766
Copyright © 2004 by The Endocrine Society

Requirement for Proximal Putative Sp1 and AP-2 cis-Deoxyribonucleic Acid Elements in Mediating Basal and Luteinizing Hormone- and Insulin-Dependent in Vitro Transcriptional Activation of the CYP17 Gene in Porcine Theca Cells

Gongqiao Zhang and Johannes D. Veldhuis

Division of Endocrinology, Department of Internal Medicine, National Institutes of Health Specialized Cooperative Center in Reproductive Research, University of Virginia School of Medicine (G.Z.), Charlottesville, Virginia 22908; and Division of Endocrinology and Metabolism, Department of Internal Medicine, Mayo Medical and Graduate Schools of Medicine, Endocrine Research Unit, Mayo Clinic (J.D.V.), Rochester, Minnesota 55905

Address all correspondence and requests for reprints to: Dr. Johannes D. Veldhuis, Division of Endocrinology and Metabolism, Department of Internal Medicine, Mayo Medical and Graduate Schools of Medicine, General Clinical Research Center, Mayo Clinic, Rochester, Minnesota 55905. E-mail: veldhuis.johannes{at}mayo.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The cytochrome P450 17{alpha}-hydroxylase/C17–20 lyase (CYP17) enzyme catalyzes the first committed step in androgen biosynthesis. In primary cultures of immature swine theca cells, LH and insulin induce CYP17 mRNA and incompletely processed heteronuclear RNA supraadditively over 2–6 h. To monitor in vitro transcriptional control by these two physiological signals, we cloned a –976 to +31-bp 5'-upstream region of the homologous CYP17 gene and fused it to a cytoplasmically targeted firefly luciferase minigene (CYP17/luc). LH and insulin individually stimulated transcriptional activity of transiently transfected CYP17/luc in theca cells by 2.7 ± 0.31- and 2.5 ± 0.24-fold over basal, respectively, at an optimal concentration (both 100 ng/ml) and time (6 h; both P < 0.01). Combined peptidyl agonists stimulated CYP17/luc by 6.6 ± 1.2-fold (P < 0.001). To identify possible LH- and insulin-sensitive cis-acting DNA regulatory regions, we prepared four deletional constructs, –839, –473, –174, and –75/+35 bp 5' upstream of the transcriptional start site. Deletion from –976 to –839 bp decreased basal transcriptional activity by 89% and that stimulated by LH, insulin, and both effectors by 82%, 91%, and 78%, respectively (each P < 0.01). Further deletion to –473 bp conferred partial responsiveness to combined hormone stimulation, suggesting an intervening inhibitory sequence. Truncation to –174 bp and more proximally reduced basal CYP17/luc activity and hormonal action by more than 95% (P < 0.001). The marked loss of combined LH and insulin stimulation caused by deleting the region between –473 and –175 bp suggested the possible relevance of partially overlapping specificity protein-1 (Sp1) and activating protein-2 (AP-2)-like binding sites located between –193 and –180 bp. Point mutation of the proximal Sp1-like element in full-length –976/+31 CYP17/luc impaired basal transcription minimally (by 21%; P = NS) and stimulation by LH (76%), insulin (67%), and combined peptides (54%) significantly (each P < 0.05 vs. wild type). Mutation of the AP-2 site alone decreased basal CYP17/luc activity nonsignificantly (by 25%), but repressed stimulated transcriptional responses prominently, viz. to LH (57%), insulin (77%), and both effectors (82%; each P < 0.025 vs. wild type). Mutation of both sites inhibited basal and hormonally stimulated CYP17/luc activity by more than 95% (P < 0.001). At the level of second messenger signaling, insulin did not potentiate LH-enhanced cAMP accumulation, whereas a stable cAMP analog mimicked LH action and augmented insulin’s stimulation of full-length and deletional fragments of CYP17/luc. In summary, LH and insulin stimulate transcriptional activity of a –976/+31 bp 5'-upstream cis-acting region of the (porcine) CYP17 gene individually and jointly in primary cultures of theca cells. Maximal transcriptional responsiveness to these peptide hormones requires proximal Sp1 and AP-2-like sequences –193 to –180 bp 5' upstream of the transcriptional start site. Exogenous cAMP mimics transcriptional up-regulation by LH and interacts analogously with insulin. These data are consistent with convergent drive of CYP17 gene expression by cAMP-protein kinase A and insulinsignaling pathways in untransformed theca cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CYTOCHROME P450 17{alpha}-hydroxylase/C17–20 lyase (CYP17) controls the entry of progesterone and pregnenolone into the androgen biosynthetic pathway (1, 2). CYP17 encodes a single protein, which catalyzes two biochemical reactions, 17{alpha}-hydroxylation of progesterone (and pregnenolone), and subsequent cleavage to androstenedione (and dehydroepiandrosterone sulfate). Androstenedione is secreted or converted to testosterone or estrone. We recently demonstrated that LH and insulin stimulate androstenedione and testosterone biosynthesis and induce CYP17 mRNA and incompletely processed heteronuclear RNA (hnRNA) individually and supraadditively in primary cultures of porcine theca cells (3).

LH induces CYP17 gene expression in significant measure via the cAMP-protein kinase A (PKA) pathway (4, 5). Insulin and IGFs further amplify gonadotropin-stimulated steroidogenesis by granulosa and theca cells (6). The basic mechanisms mediating such potentiating interactions are not well defined. Insights into this issue may be relevant to understanding the clinical association between excessive LH and insulin concentrations and biochemical hyperandrogenism in the anovulatory syndrome of polycystic ovarian disease (7, 8, 9). Accordingly, the present study explores single and combined control by LH and insulin of CYP17 transcriptional activity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
Ovine LH (NIDDK oLH-26; potency, 2.3 x NIH oLH-S1) was obtained from the National Hormone and Pituitary Program, NIH (Bethesda, MD); porcine insulin and 8-bromo-cAMP (8Br-cAMP) were purchased from Sigma-Aldrich Corp. (St. Louis, MO); and DMEM, penicillin/streptomycin, and Lipofectamine reagent were obtained from Life Technologies, Inc. (Grand Island, NY). The Dual-Luciferase Reporter Assay System was purchased from Promega Corp. (Madison, WI).

Porcine theca-cell culture
Porcine ovaries were collected from prepubertal (60–70 kg) gilts at an abattoir and transported in iced saline. Theca cells were collected and purified as previouysly described (3, 10). Briefly, combined theca-granulosa membranes were removed from immature follicles (diameter, <5 mm) using forceps, and granulosa cells were detached by mechanical agitation in tissue culture medium for 3 min. Theca-basal lamina linings were allowed to settle at unit gravity, and the supernatant containing granulosa cells was discarded. Residual tissue was digested in collagenase (1 mg/ml) and DNase (10 µg/ml), and debris was removed by filtration (185 µm, nylon; Millipore Corp., Bedford, MA). Cells were washed three times in Ham’s F-12/DMEM (1:1) by centrifugation for 5 min at 1000 rpm and were purified further using Percoll density gradient (3). Theca cells were resuspended in Ham’s F-12/DMEM (1:1) with 2.5% serum and incubated in 96-well plates (Costar, Cambridge, MA) at a density of 1.5 x 105 cells/well in humidified 95% air and 5% CO2 at 37 C. After overnight culture to allow anchorage, cultures were washed in serum-free culture medium and exposed to transfection solution (below).

Construction of CYP17 promoter-containing firefly-luciferase reporter plasmid
Based on the reported porcine CYP17 gene sequence (accession no. Z11854, GenBank), we designed forward (5'-AGATCTAGCTGCCAGCCTATGCCAGA) and reverse (5'-AGATCTAGGCAGGACAGACAGCTCTG) primers targeted to the putative 5'-upstream CYP17 promoter region. Primers were engineered with BglII restriction sites (AGATCT) at the 5' end to facilitate subsequent subcloning of PCR products into a firefly luciferase reporter vector (pGL3-Basic). A PCR product containing 1007 bp of CYP17 sequence flanked by 6-bp BglII restriction recognition sites was generated by PCR (95 C for 12 min; 28 cycles at 95 C for 35 sec, 62 C for 30 sec, 67 C for 1 min and 30 sec, and 67 C for 10 min). PCR mixtures (100 µl final volume) contained 2.5 mM MgCl2, 100 pmol forward and reverse primers, 0.2 mM of each deoxy-NTP, 10 µl 10x PCR buffer, 1 µg porcine genomic DNA isolated from theca cells as template, and 2.5 U AmpliTaq Gold DNA polymerase. The putative CYP17 sequence region was ligated into the BglII-restricted pGL3-Basic plasmid according to the protocol provided by the manufacturer. DNA sequencing was used to delineate orientation and identity of a –976/+31 bp sequence 5' upstream of the transcriptional start site (11).

CYP17 reporter deletional fragments
To identify the general region of hormone-sensitive DNA regulatory elements in the putative promoter, four deletional constructs were made (Fig. 1Go). Forward primers (CYP17F, 870 bp; CYP17F, 504 bp; CYP17F, 205 bp; CYP17F, 106 bp) used to amplify porcine CYP17 gene deletional fragments from the full-length (1007 bp) 5'-upstream regulatory sequence were 5'-GAGTCTACCGGATCCTTAACCCACTG-3', 5'-GAGTCTTCATTACCGTCTTGTGTGACC-3', 5'-GAGTCTGCTTGCCTGAAGTTGAGCCA-3', and 5'-GAGTCTAAGGCAAGCGATAACATAAA-3'. Forward primers correspond to deletional constructs –839/+31, –473/+31, –174/+31, and –75/+31, respectively. Each was engineered with a SacI restriction site (GAGTCT) at the 5' end. Each forward primer was adapted with the single reverse primer (5'-AGATCTAGGCAGGACAGACAGCTCTG-3' with a 5'-end BglII restriction site (AGATCT) used to synthesize the 1007-bp CYP17 promoter fragment (above). The PCR mixture and cycling mode were as noted above, except for including pGL3-CYP17–1007 bp as template instead of genomic DNA. The PCR products containing the deletional fragments were restricted by SacI and BglII, ligated into SacI/BglII-cut pGL3-Basic, and sequenced.



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  		FIG. 1. Schema of deletional segments used in the porcine 5'upstream CYP17 gene.

 
Site-directed mutagenesis
To mutate a proposed specificity protein-1 (Sp1)/activating protein-2 (AP-2) overlap region, 5'-CCTAGCCCCACCCC-3' (–193/–180; Sp1, 5'-CCTAGCCCC-3'; AP-2, 5'-CCCACCCC-3'; Fig. 1Go), three pairs of primers were designed to contain one mutation site each (10, 11). The preparation of mutants was carried out using the QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA) based on the manufacturer’s procedure. The latter entailed denaturation of the pGL3-CYP17–1007 bp and annealing with the oligonucleotide primers containing the desired mutation. Using the nonstrand-displacing action of Pfu Turbo DNA polymerase, mutagenic primers were extended and incorporated into the resulting nicked circular strands. The methylated nonmutated parental DNA template was digested with DpnI, and the circular nicked double-stranded DNA was transformed into XL1-Blue supercompetent cells to repair nicks. Mutated plasmids were sequenced to confirm mutations.

Transfection of porcine theca cells
Transfection conditions were optimized by testing a suitable matrix of Lipofectamine concentrations, plasmid/Lipofectamine ratio, transfection duration, and initial plating conditions. Consistent transfection was achieved by plating 1.5 x 105 theca cells/well overnight in 96-well plates in tissue culture medium with 2.5% serum. The next morning, after a single wash in serum-free medium, cells were exposed to tissue-culture medium plus a ratio of total DNA to Lipofectamine of 0.5 µg:1.5 µl/well for 4 h. Then the medium was changed to include effectors, and incubations were continued at 37 C for the indicated times. Transfections were performed with full-length (–976/+31 bp) CYP17/luc and deletional constructs at constant total DNA concentrations by variable addition of the promoterless luciferase construct, pGL3/luc, which exhibited no significant responsiveness. To normalize responses for transfection efficiency, 30 ng pRL-TK DNA containing the Renilla luciferase-coding sequence was cotransfected with pGL3-CYP17. The activities of CYP17-driven firefly and constitutive Renilla luciferase were quantitated in a Turner TD-20e luminometer (Turner Designs, Sunnyvale, CA) in 40 µl cell lysate containing 100 µl luciferase substrate buffer and Stop & Glow solution (Promega Corp. protocol).

Statistics
Data are presented as the ratio of hormonally treated firefly to Renilla luciferase activity to that of untreated control transfected with the full-length promoter. Reported values are the mean ± SEM determined in three or more independent experiments using separate batches of ovaries. Logarithmically transformed ratios were compared by two-tailed two-sample t test (control vs. intervention) or one-way ANOVA (for three or more comparisons). Post hoc comparisons were made using Tukey’s procedure. Significance was construed for P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Preliminary time-course optimization experiments showed that LH (100 ng/ml) and insulin (100 ng/ml) are significant individual agonists of –976/+31 bp CYP17/luc activity over control solvent (basal) across the interval 2–6 h (P < 0.01, by ANOVA). Combined hormonal stimulation exceeded that of LH or insulin alone at 4 h (P < 0.05) and 6 h (P < 0.001), and then waned gradually as basal expression rose slowly. Therefore, agonist concentration-dependent stimulation was evaluated at 6 h (Fig. 2Go). LH and insulin increased transcriptional activity in a concentration-dependent fashion (P < 0.01, by ANOVA). The maximal effect of LH and insulin occurred at a concentration of 100 ng/ml each, which yielded elevations 2.7 ± 0.31- and 2.5 ± 0.24-fold over basal, respectively (both P < 0.01). The response to combined stimulation by LH and insulin was 6.6 ± 1.2-fold (P < 0.001 vs. basal, P < 0.02 vs. individual effects of LH or insulin).



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  		FIG. 2. Concentration-dependent stimulation of –976/+31 CYP17/luc activity by LH and/or insulin in swine theca cells incubated for 6 h beginning 4 h after Lipofectamine-mediated transfection. Firefly luciferase activity was normalized for coexpression of pRL-TK Renilla luciferase activity (see Materials and Methods). Data are the mean ± SEM (three independent experiments, each conducted with triplicate incubations).

 
Based on these data, further analyses were performed at 6 h using LH and insulin concentrations of 100 ng/ml. The results of successive 5'-upstream deletion of –976/+31 bp CYP17/luc are shown in Fig. 3Go. Compared with (full length) –976/+31 CYP17/luc, –839/+31 bp CYP17/luc exhibited low basal activity (P < 0.01 vs. control), no detectable incremental response to insulin, a 2.5-fold response to LH (P < 0.05), and a 7-fold response (over the low basal level) to combined LH and insulin stimulation (P < 0.01). The –473/+31-bp deletional fragment exhibited equal basal activity to –976/+31 and greater basal activity than –839/+31-bp CYP/luc (P < 0.05) and retained partial responsiveness to combined, but not individual, LH and insulin drive (P < 0.01). The –174/+31 and –75/+31-bp fragments manifested a 95% decrease in basal activity and unresponsiveness to LH, insulin, and both agonists (P < 0.001). The pGL3 plasmid lacking CYP17 sequences was inactive.



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  		FIG. 3. Individual and concerted stimulation by LH and insulin (each 100 ng/ml) of successive 5'-upstream CYP17 gene deletional fragments compared with –976/+31 CYP17/luc and empty pGL3 plasmid in primary cultures of porcine theca cells. Responses were monitored at 6 h. Data are presented as described in Fig. 2Go.

 
Inspection of the CYP17 5'-upstream region revealed proximal Sp1 and AP-2-like binding sites between –193 and –180 bp. Relevance was suggested by 50% reduction in basal and marked loss of agonist-stimulated transcriptional activity of the deletional construct –174/+31 bp (Fig. 2Go). Site-directed mutagenesis was used to assess the possible functional role of one or both sequences. Figure 4Go demonstrates that mutation of either the Sp1- or AP-2-like site did not reduce basal –976/+31-bp CYP17/luc transcriptional activity significantly (viz. by 22–25%; P = NS), but inhibited transcription stimulated by LH, insulin, and combined LH/insulin by 76%, 67%, and 54% (Sp1; P < 0.05) and 57%, 77%, and 82% (AP-2; P < 0.025). Simultaneous mutation of the presumptive Sp1 and AP-2 sites decreased basal and hormonally induced transcriptional activity by more than 95% (P < 0.001).



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  		FIG. 4. Site-directed mutagenesis of Sp1- and/or AP-2-like sequence contained in the proximal –193/-180 bp region of porcine –976/+31 CYP17/luc impairs hormonally stimulated transcriptional activity. Data are from theca cells incubated with vehicle, LH (100 ng/ml), insulin (100 ng/ml), or both for 6 h. See Fig. 2Go for data presentation.

 
To assess possible second messenger signaling interactions between LH and insulin, we tested whether insulin augments LH-stimulated cAMP accumulation. As summarized in Table 2Go, LH, but not insulin, increased cAMP accumulation at the end of 6-h incubations. The stable cAMP analog, 8Br-cAMP, was used as an indirect probe to explore possible facilitation of cAMP signaling by insulin. Figure 5Go demonstrates that combining an optimized concentration of 8Br-cAMP (0.5 mM) with insulin (100 ng/ml) increased transcriptional activity of –976/+31 CYP17/luc comparably to that achieved by LH and insulin. Deletional analyses revealed that the –839/+31 CYP17/luc construct retained less than 10% basal luciferase activity and showed minimal responsiveness to 8Br-cAMP (0.5 mM), insulin (100 ng/ml), and both agonists (P < 0.001 vs. full-length control). The –473/+31-bp fragment retained partial luciferase activity basally and in response to stimulation by 8Br-cAMP, insulin, and both effectors (P < 0.05 vs. –893/+31 CYP17/luc). Proximal –174/+31 and –75/+31 CYP17/luc maintained low (< 15%) basal luciferase activity and no responsiveness to cAMP and/or insulin (P < 0.001).


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  		TABLE 2. Concentration-dependent stimulation by LH, but not insulin, of cAMP accumulation in porcine theca cells

 


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  		FIG. 5. Stimulation by 8Br-cAMP (0.5 mM), alone or in combination with insulin (100 ng/ml), of full-length –976/+31 bp and successive deletional CYP17/luc constructs transiently transfected into monolayer cultures of theca cells. Data at 6 h are presented, as described in Fig. 2Go.

 
Figure 6Go summarizes the effects of mutating proximal Sp1- and/or AP-2-like site(s) on transcriptional activity of –976/+31 CYP17/luc stimulated by 8Br-cAMP and/or insulin. The outcomes of single and combined point mutations mimicked those observed in response to LH and/or insulin (see above). An interesting specific exception was that single mutation of the AP-2-like sequence impaired stimulation by 8Br-cAMP more than that by LH (P < 0.05).



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  		FIG. 6. Individual and combined point mutations of proximal Sp1- and/or AP-2-like sequences inhibit 8Br-cAMP (0.5 mM)- and/or insulin-stimulated transcriptional activity of –976/+31 CYP17/luc. Analyses were performed in primary cultures of theca cells incubated with the indicated effectors for 6 h. Data are presented as defined in Fig. 2Go.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The current analyses extend insights into the mechanisms by which LH and insulin induce CYP17 mRNA and hnRNA supraadditively in primary cultures of porcine theca cells (3). The synergy may be relevant to understanding how hypersecretion of LH and hyperinsulinism in polycystic ovarian syndrome predispose to elevated androgen secretion (3, 7, 8, 9, 10, 11, 12, 13, 14, 15). The capability of the combined agonists to stimulate incompletely processed hnRNA (containing both intronic and exonic sequences) points to cooperative transcriptional drive. To explore this hypothesis further, we cloned a –976/+31-bp 5'-upstream region of the homologous CYP17 gene and optimized in vitro conditions for transient reporter transfection and hormonal responsiveness of theca cells. LH and insulin stimulated –976/+31-bp CYP17/luc activity by 2.5- to 2.7-fold individually and by 6.5-fold together. Combined transcriptional up-regulation was markedly impaired by 5'-upstream deletion to –174 bp and to a lesser extent by truncation to –473/+31 bp. Sequence analysis of the intervening –473/–174-bp region revealed contiguous Sp1 and AP-2-like elements in the –193/–180-bp interval. Single mutation of these putative regulatory sites in the full-length –976/+31-bp CYP17 sequence did not alter basal expression, but diminished transcriptional responsiveness to LH and/or insulin by more than 50%. Simultaneous mutation of both the Sp1- and AP-2-like regions suppressed basal and hormonally stimulated CYP17/luc activity by more than 95%, thus precluding quantitation of effector regulation. Replacement of the LH stimulus with a stable cAMP analog yielded analogous interactions with insulin in the full-length and deletional reporter constructs. Mimicry included requirements for proximal Sp-1- and AP-2-like elements for maximal stimulation. Accordingly, to the extent that in vitro transfection outcomes mirror in situ mechanisms of transcriptional control, the present data indicate that LH and insulin amplify CYP17 gene expression in theca cells via cAMP- and insulin-related signaling pathways that (at least) converge on key proximal Sp-1- and AP-2-like cis-acting DNA elements.

Insulin induces rapid autophosphorylation of the ßsubunit of the cognate membrane-associated receptor in porcine granulosa and theca cells (11, 12). Insulin may also stimulate ovarian cells in part via the inositolglycan pathway (13, 14). The present outcomes extend these concepts by demonstrating that whereas insulin does not enhance LH-driven theca-cell cAMP accumulation, it potentiates 8Br-cAMP-stimulated transcriptional activity of –976/+31 CYP17/luc and more proximal sequences. Thus, collective data indicate that insulinomimetic peptides amplify cAMP-dependent induction of CYP17 as well as CYP11A, steroidogenic acute regulatory protein, and low density lipoprotein receptor gene expression in steroidogenic ovarian cells (15, 16, 17, 18, 19).

Promoter deletional analysis revealed marked reduction in CYP17 transcriptional activity associated with –839/+31 vs. –976/+31-bp CYP17/luc. Further 5'-upstream truncation to –473 bp restored partial transcriptional responsiveness to combined stimulation with LH and insulin. In principle, this finding might reflect unknown inhibitory activity mediated by the excluded (–839/–473 bp) region. Additional analyses will be required to explore the latter idea. By way of potential precedence, a considerably more proximal (chicken oviduct upstream promoter-transcription factor) region in the bovine CYP17 gene appears to confer repression by phorbol esters (20).

Loss of detectable hormonal responsiveness upon deletion of the 5'-upstream region between –473/–174 of CYP17/luc suggested the operation of stimulatory elements therein. Sequence analysis identified an apparent Sp1/AP-2 overlap region –193 to –180 bp 5' upstream of the transcriptional start site. Individual mutation of the putative Sp-1- and AP-2-like elements diminished single and concerted hormonal stimulation of CYP17 promoter activity substantially (by 57–82%). Inactivation of the AP-2-like element abolished responsiveness to an exogenous cAMP stimulus, but not to LH. The latter distinction could indicate that LH drives CYP17 in part via non-cAMP/PKA-dependent paths, such as by way of phospholipase C and protein kinase C activation (21, 22, 23). Simultaneous mutations eliminated basal and stimulated –976/+31 CYP17/luc expression. Together these data indicate that proximal Sp-1- and AP-2-like sequences in the porcine CYP17 gene are necessary for maximal individual, and detectable combined, transcriptional drive by LH or cAMP and insulin. Albeit not yet evaluated for CYP17 regulation in untransformed gonadal cells, Sp1- and AP-2-like elements mediate interactive transcriptional control of other genes via cAMP and tyrosine kinase receptors, such as IGF-binding protein-3 (24, 25). Interspecies generality in the case of CYP17 is suggested by the existence of an Sp1 site in the same location in the porcine and bovine promoters and putative Sp1 and AP-2 sites in the rat CYP17 promoter (26, 27).

In summary, the present experiments delineate concentration- and time-dependent stimulation by LH and insulin of transcriptional activity of a –976/+31-bp 5'-upstream cis-DNA region of the porcine CYP17 gene in primary cultures of swine theca cells. Proximal Sp1- and AP-2-like sequences overlapping within a region –193 to –180/+31 bp 5' upstream of the transcriptional start site are individually required for maximal up-regulation of CYP17 by LH or cAMP and insulin and are jointly necessary to maintain basal CYP17 expression. At the level of second messengers, insulin does not augment LH-induced cAMP accumulation in theca cells, but amplifies exogenous cAMP-stimulated CYP17/luc transcriptional activity. Based on these data and earlier studies of in situ gene expression in porcine theca cells, we hypothesize that LH-induced cAMP/PKA and insulin receptor-related signals drive transcription of the CYP17 gene via convergent actions that require at least two proximal Sp1- and AP-2-like sites.


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  		TABLE 1. Primer-based mutations of the –976/+31 CYP17 5'-upstream regulatory sequence

 

   Acknowledgments
 
We thank Jean Plote for excellent assistance with manuscript preparation, and Dr. Sekar Natesampillai for prior critical review.


   Footnotes
 
This work was supported in part by NICHHD/NIH through Cooperative Agreement U54-HD-28934 as part of the Specialized Cooperative Centers Program in Reproduction Research and NIH Grant RO1-HD-16393.

Abbreviations: AP-2, Activating protein-2; 8Br-cAMP, 8-bromo-cAMP; CYP17, cytochrome P450 17{alpha}-hydroxylase/C17–20 lyase; hnRNA, heteronuclear RNA; PKA, protein kinase A.; Sp1, specificity protein-1.

Received November 12, 2003.

Accepted for publication February 17, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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