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BOR - Papers in Press, published online ahead of print April 14, 2004.
Biol Reprod 2004, 10.1095/biolreprod.104.027300
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BIOLOGY OF REPRODUCTION 71, 579–587 (2004)
DOI: 10.1095/biolreprod.104.027300
© 2004 by the Society for the Study of Reproduction, Inc.

Mechanisms of Hormone Action

Functional Luteinizing Hormone/Chorionic Gonadotropin Receptors in Human Adrenal Cortical H295R Cells

Ch.V. Rao1, X.L. Zhou2, and Z.M. Lei

Division of Research, Department of Obstetrics, Gynecology and Women's Health, University of Louisville Health Sciences Center, Louisville, Kentucky 40292


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Previous studies have suggested that activation of normal human adrenal and adrenal tumor luteinizing hormone (LH)/chorionic gonadotropin (hCG) receptors results in an increased secretion of steroid hormones. Since it is not feasible to test this suggestion on normal human adrenal cells, we used human adrenal cortical carcinoma H295R cells, which are similar in some respects to normal adrenal cortical cells. These cells contained LH/hCG receptor transcripts and receptor protein that can bind 125I-hCG in a hormone-specific manner. Culturing the cells with highly purified hCG resulted in a time- and dose-dependent significant increase in dehydroepiandrosterone sulfate (DHEAS) secretion as compared with the controls. The DHEAS response was hormone as well as steroid specific. Since hCG treatment did not increase DHEA secretion, we suspected that the hCG might increase DHEA sulfotransferase (ST). Consistent with this possibility, hCG treatment increased steady-state DHEA-ST mRNA levels. The hCG effects require its receptors, as inhibition of their synthesis by treatment with antisense phosphorothioate oligodeoxynucleotides (ODN) made from the LH/hCG receptor sequence resulted in loss of DHEA-ST and DHEAS responses. The findings that 1) hCG treatment increased cAMP levels and activated protein kinase A (PKA), 2) 8-bromo cAMP mimicked hCG, and 3) blocking PKA activation prevented hCG as well as 8-bromo cAMP from increasing both DHEA-ST mRNA and DHEAS levels suggested that cAMP/PKA signaling was involved in the hCG actions. In conclusion, H295R cells contain LH/hCG receptors, which are coupled to increasing DHEAS secretion through upregulating the ST enzyme mRNA level. This action is mediated by the cAMP/PKA signaling pathway. These findings support the concept that adrenal function in normal and pathological conditions could be influenced by LH and hCG.

adrenal, cyclic adenosine monophosphate, human chorionic gonadotropin, luteinizing hormone, steroid hormones


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Luteinizing hormone (LH) receptor, which also binds human chorionic gonadotropin (hCG), is a member of the G-protein-coupled receptor superfamily [1, 2]. It is a transmembrane glycoprotein with an extracellular hormone binding region, seven transmembrane-spanning regions, and a short cytoplasmic tail coupled to G-protein-coupled signal transduction pathways [1, 2]. Contrary to the widely held belief until about 15 yr ago, low levels of functional LH/hCG receptors have been found to be widely distributed in the body [3]. Human adrenals are among the nongonadal tissues that contain these receptors [4–7]. Activation of nongonadal LH/hCG receptors results in tissue-specific responses that, in the case of adrenals, could be associated with an increase in the secretion of steroid hormones. Consistent with this possibility, human fetal adrenals, which also contain LH/hCG receptors [8], are a target of hCG regulation in increasing dehydroepiandrosterone sulfate (DHEAS) secretion [9–11]. The role of LH and hCG in adult human adrenals is unknown. However, circumstantial evidence suggests that they also may be involved in regulating the secretion of steroid hormones. For example, although adrenocorticotropic hormone (ACTH) can also increase adrenal androgen secretion, there are conditions in which adrenal androgen secretion diverges from cortisol secretion [12]. The conditions are chronic anovulation with or without polycystic ovarian disease (PCOD) and adrenarche in which serum adrenal androgen levels are elevated when ACTH levels are in the normal range [13–17]. These elevated levels can be decreased by treatment with GnRH agonists [18–20]. Pregnancy is another condition in which serum and urinary adrenal steroid hormone levels increase during pregnancy when ACTH levels are low and hCG levels are high [21]. All these findings suggest that LH and hCG may also increase the secretion of adrenal steroid hormones. Consistent with this possibility, adrenal LH/hCG receptors are present primarily in zona reticularis and the deeper portion of zona fasciculata, both of which secrete DHEAS [4]. Despite this circumstantial evidence, more direct data for LH and hCG increasing DHEAS secretion in adult human adrenals were not available. Since it is not feasible to obtain this evidence using fresh adult human adrenal tissue or cells, we chose human adrenocortical carcinoma H295R cells that were derived from an invasive primary andrenocortical carcinoma from a patient who showed symptoms of mineralocorticoid, glucocorticoid, and androgen excess and that have been in culture for more than 10 yr [22]. These cells secrete steroid hormones similar to those secreted by normal human adrenal cortical cells and retained responsiveness to ACTH treatment and the protein kinase A (PKA) signaling pathway [22–24]. Thus, this cell line is a reasonable alternative for normal human adrenal cortical cells to test our hypothesis that adrenal LH/hCG receptors are functionally coupled to increasing DHEAS secretion.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture

H295R cells (American Type Culture Collection, Rockville, MD) were maintained in a 1:1 mixture of Dulbecco modified Eagle medium nutrient mixture F-12 Ham (Sigma Chemical Co., St. Louis, MO) containing 0.03 µg/ml pyridoxine HCl, 0.365 mg/ml glutamine, and 15 mM Hepes. The media were supplemented with 6.25 µg/ml each of insulin, transferrin, and selenium; 5.35 µg/ml linoleic acid added in the form of 1% ITS plus 2.5% Nu-serum type I culture supplement (both BD Biosciences, Bedford, MA); and 0.5% antibiotic-antimycotic mixture (Life Technologies Inc., Grand Island, NY). Cells were maintained and grown in 75-cm2 flasks at 37°C under an atmosphere of 5% CO2/95% air.

Northern Blotting

Total RNA was isolated from H295R cells and then 30-µg aliquots were heat denatured for 5 min at 100°C. The denatured RNA was resolved on formaldehyde-agarose gels and blotted onto Gene Screen Plus membranes [25]. The RNA was then cross-linked to the membranes by irradiation for 2.5 min under ultraviolet light and baked for 10 min at 70°C. The nonspecific binding sites were blocked by exposure to salmon sperm DNA, and the blots were sequentially hybridized overnight with 0.5–1 x 107 cpm/ml of 32P-labeled LH/hCG receptor riboprobe (210-base complementary to +819 to +1029 spanning exons 9–12) transcribed from human receptor cDNA (a gift from Dr. Aaron J.W. Hsueh at the Stanford University Medical Center, Palo Alto, CA) with an in vitro transcription kit (Promega Corp., Madison, WI) or either with 0.8–1 x 10 7 cpm/ml of 32P-labeled dehydroepiandrosterone sulfotransferase (DHEA-ST) cDNA (a gift from Dr. Richard Weinshilboum at Mayo Clinic, Rochester, MN) or with 2–5 x 106 cpm/ml of 32P-labeled glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA prepared by random priming using a kit (Promega). Hybridization temperature of 65°C for LH/hCG receptors and 42°C for DHEAS-ST and GAPDH were used. After washing twice with 2x SSC (1x SSC = 150 mM sodium chloride and 15 mM sodium citrate, pH 7.4) containing 0.1% sodium dodecyl sulfate (SDS) and then two more washes with 0.1x SSC 0.1% SDS (LH/hCG receptor) at corresponding temperatures, the membranes were exposed for 48 to 72 h at –70°C to Kodak XAR-5 film (Eastman Kodak, Rochester, NY) with intensifying screens. The molecular size of the transcripts was determined by running a RNA ladder in an adjacent lane. A major transcript of 1.8 kb and a minor transcript of 1.1 kb of DHEA-ST were detected. Since the detection of minor transcript was not consistent, it was not included in the measurement of the relative optical density.

Western Blotting

The H295R cells were homogenized in buffer containing 200 mM phenylmethylsufonyl fluoride/20 mM leupeptin (Sigma) to inhibit the endogenous proteases activity. Then 40-µg protein aliquots were separated by discontinuous 10% SDS-polyacrylamide gel electrophoresis under reducing conditions [26]. The separated proteins were electroblotted to immobilon-P membranes [27]. After blocking the nonspecific binding sites with 5% nonfat dry milk in 5 mM Tris-HCl, pH 7.4, 136 mM NaCl, 0.1% Tween-20 (TBST buffer), the blots were incubated for 2 h at 22°C with 1:2000 dilution of polyclonal rat LH/hCG receptor antibody raised against a synthetic N-terminus amino acid sequence of 15–38 (a gift from Dr. Patrick Roche, who is now at Ventana Medical Systems, Tucson, AZ) or 1:1000 dilution of monoclonal ß-tubulin antibody (Sigma), washed two times for 10 min each with TBST buffer and once for 15 min and then for 30 min with TBST containing 40 mM NaN3. The washed blots were either reincubated for 1 h at 22°C with 1:4000 dilution of horseradish peroxidase-labeled anti-rabbit immunoglobulin G (Life Technologies) for the detection of LH/hCG receptor or for 1 h at 22°C with 1:1000 dilution of horseradish peroxidase-labeled anti-mouse immunoglobulin G for the detection of ß-tubulin. Then the blots were washed the same as the first time. The LH/hCG receptor and ß-tubulin antibody binding was detected by an enhanced chemiluminescence Western blotting detection kit (Amersham Life Sciences, Arlington Heights, IL). The molecular size of the LH/ hCG receptor and ß-tubulin protein was determined by running molecular size marker proteins in an adjacent lane. In the procedural control, either the primary antibody was preabsorbed with excess peptide (LH/hCG receptor) or nonspecific IgG was used in place of primary antibody (ß-tubulin).

Covalent Receptor Cross-Linking

For this procedure, 100-µg H295R cell homogenate protein aliquots were incubated for 30 min at 37°C with 1–2 x 106 cpm/ml of 125I-hCG in the presence or absence of 5 µg unlabeled human thyroid stimulating hormone (TSH; AFP-4314C; 15 IU/mg), human follicle stimulating hormone (FSH; AFP-87929B; 1683 IU/mg), {alpha} (CR-125) and ß (CR-125; 129 IU/mg) subunits of hCG, dimer hCG (CR-127; 14 900 IU/mg), and LH (AFP-0264B; 4015 IU/mg) (gifts from NIDDK's National Hormone and Pituitary Program and Dr. A.F. Parlow, Torrance, CA) [28]. The hCG was radioiodinated by a lactoperoxidase technique that yielded a specific activity of 81.2 µCi/µg [29]. The receptor bound 125I-hCG was cross-linked for 1 h at room temperature with 100 mM bis (sulfosuccimidyl) suberate (Pierce Chemical Co., Rockford, IL) dissolved in 5 mM sodium citrate buffer, pH 5.0. The reaction was then stopped by the addition of 1M Tris-HCl, pH 7.5, and centrifuged at 27 000 x g for 30 min at 4°C. The pellets containing 125I-hCG-receptor complexes were solubilized with 1% Triton X-100 and diluted with sample buffer (125 mM Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, and 10% mercaptoethanol) and finally separated on 8% SDS-PAGE under nonreducing conditions. The gels were fixed, dried and exposed to Kodak XAR-5 film with intensifying screens at –70°C for 1–3 days. The molecular sizes of the 125I-hCG-receptor complexes and the unbound 125I-hCG were determined by running molecular weight marker proteins in an adjacent lane.

Measurement of Steroid Hormone Levels

The levels of cortisol, androstenedione, DHEA, and DHEAS in 50-µl media aliquots were quantified in duplicate by Coat-a-Count RIA kits (Diagnostic Products Corp., Los Angeles, CA). The antisera are highly specific with a relatively low cross-reactivity to other naturally occurring steroids. The intrassay and interassay coefficients of variations were less than 10%. The detection limits of the assay were 0.2 µg/dl for cortisol, 0.04 ng/ml for androstenedione, 0.04 ng/ml for DHEA, and 2.1 µg/dl for DHEAS.

Measurement of cAMP Levels

The cAMP levels in 50-µl media aliquots were quantified by an enzyme immunoassay kit (Cayman Chemical Co., Ann Arbor, MI). The specificity of cAMP antibody was 100% for acetylated cAMP, 0.3% for cAMP, 0.05% for acetylated cGMP, and 0.01% or less for cGMP, acetylated adenosine, cytidine, guanosine, and uridine. The intra- and interassay coefficients of variation were less than 10%. The detection limit of the assay was 1.1 pmol/ml.

Measurement of PKA and PKC Activities

Cells were lysed by sonication at 4°C in 200 µl of 25 mM Tris-HCl buffer, pH 7.5, containing 1 mM EDTA, 1 mM dithiothreitol, 20 mM NaCl, 0.5 mM PMSF, 1 µM aprotinin, and 50 µM leupeptin. PK activities were determined by pretag nonradioactive PKA and PKC measurement kits (Promega). Briefly, 5–7 µg lysate protein were incubated for 30 min at 30°C with fluorescent-labeled A1 peptide for the PKA assay and fluorescent labeled C1 peptide for the PKC assay. Then nonphosphorylated and phosphorylated fluorescent peptides were separated on 0.8% agarose gels. Phosphorylated fluorescent peptide bands were excised and eluted, and the optical density at 570 nm was measured using a 96-well plate reader. PK activities were calculated from the densitometric values using instructions provided by the kit manufacturer. Positive and negative controls supplied in the kits were assayed at the same time as H295R cell samples.

Culturing with Phosphorothioate Antisense and Sense Oligodeoxynucleotides Synthesized from Human LH/ hCG Receptor cDNA Sequence

H295R cells were plated at a density of 5 x 10 5 cells/well in six-well plates and cultured for 24 h in serum and phenol red-free medium containing 21 mer phosphorothioate 5 µM antisense (5'-GCC GAG AAC CGC TGC TTC ATG-3') or sense (5'-CAT GAA GCA GCG GTT CTC GGC-3') oligodeoxynucleotides (ODNs) synthesized from human LH/ hCG receptor cDNA sequence covering ATG translation initiation codon. Then Western blot for LH/hCG receptors was performed on some cells, whereas other cells were cultured again for 2 h (cAMP), 4 h (DHEA-ST mRNA), or 8 h (DHEAS) in the presence or absence of 100 ng/ml of hCG.

Densitometry

A Z-gel computer was used for determining the optical densities in a linear range of autoradiographic bands of DHEA-ST and GAPDH mRNAs, LH/hCG receptors, and ß-tubulin proteins.

Replication of Experiments and Statistical Analysis

All the experiments were performed in duplicate and repeated at least three times on different occasions. All the data points were pooled for the calculation of means and their standard errors and for one-way analysis of variance and Duncan's multiple comparison test [30].


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
LH/hCG Receptors in Human Adrenal Cortical Cells

We first tested whether H295R cells contain LH/hCG receptors. Northern blotting detected multiple LH/hCG receptor transcripts (major 4.3 kb and minor 2.4 and 1.8 kb), and Western blotting showed an 80-kDa protein that was not detected when the receptor antibody was preabsorbed with the receptor peptide (Fig. 1). Covalent receptor cross-linking demonstrated that the 80-kDa protein can bind 125I-hCG, and this binding was inhibited in the presence of excess unlabeled hCG and LH but not FSH, TSH, or isolated {alpha} and ß subunits of hCG (Fig. 1).



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  		FIG. 1. Northern blot (lane 1), Western blot (lanes 2 and 3), and covalent receptor cross-linking (lanes 4–10) for LH/hCG receptors in human H295R cells. Lane 3 is receptor antibody preabsorption control. Lane 4 contained only 125I-hCG, whereas lanes 5–10 also contained excess unlabeled TSH, FSH, {alpha}, ß subunits of hCG, dimer hCG, and LH, respectively. The 125 kDa represents the size of 125I-hCG-receptor complex, and 45 kDa represents the size of unbound 125I-hCG. The 80-kDa size difference represents the molecular size of free receptor protein

Effect of hCG on Adrenal Steroids

We cultured H295R cells with highly purified hCG to determine whether it could stimulate the secretion of steroid hormones. The results showed indeed that hCG treatment significantly increased DHEAS secretion in a time- and dose-dependent manner (Fig. 2). The effect was hormone specific, as LH mimicked hCG, whereas the other members of the glycoprotein hormone family, such as TSH and FSH, had no effect (Fig. 3). Isolated {alpha} and ß subunits had no effect, suggesting that dimer conformation of hCG was required for the effect.



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  		FIG. 2. The effect of hCG on DHEAS secretion by H295R cells. In time-dependency experiments, cells were cultured for increasing lengths of time in the presence or absence of 100 ng/ml of hCG. In dose-dependency experiments, cells were cultured for 8 h in the presence or absence of increasing concentrations of hCG. Then the media DHEAS levels were measured, which in the control cultures (49.0 ± 4.2 µg/dl) were considered as 100% for the calculation of changes in the others. In this figure and in all the others, means and their standard errors are presented. Asterisks indicate significant differences at P < 0.05 as compared with the corresponding controls



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  		FIG. 3. The hormone specificity of the hCG effect on DHEAS secretion by H295R cells. The cells were cultured for 8 h in the presence or absence 100 ng/ml of various hormones. Then the media DHEAS levels were measured, which in the control cultures (47.1 ± 3.8 µg/dl) were considered as 100% for the calculation of changes in the others. Asterisks indicate significant differences at P < 0.05 as compared with the control

The hCG effect on DHEAS secretion was steroid specific, as incubation of H295R cells for increasing lengths of time with 100 ng/ml hCG or for 8 h with increasing hCG concentrations had no effect on androstenedione, cortisol, or DHEA secretion (Fig. 4).



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  		FIG. 4. The effect of hCG on androstenedione, cortisol, and DHEA secretion by H295R cells. In time-dependency experiments, the cells were cultured for increasing lengths of time in the presence or absence of 100 ng/ml hCG. In dose-dependency experiments, cells were cultured for 8 h in the presence or absence of increasing concentrations of hCG. Then the media levels of steroids were measured. The androstenedione, cortisol and DHEA levels in control cultures (37.5 ± 7.2, 48.7 ± 6.5, and 41 ± 4.4 µg/dl, respectively) were considered as 100% for the calculation of changes in the others

Effect of hCG on DHEA-ST mRNA Levels

The finding that hCG treatment increased DHEAS but not DHEA secretion suggested that hCG treatment may primarily increase DHEA-ST. Consistent with this possibility, treatment of H295R cells with hCG significantly increased steady-state levels of DHEA-ST mRNA at 4 h of incubation (Fig. 5). This increase was not due to an enhanced stability of the transcripts (data not shown).



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  		FIG. 5. The effect of hCG on DHEA-ST mRNA levels in H295R cells. The cells were cultured for either 4 or 8 h in the presence or absence of 100 ng/ml hCG, and then total cellular RNA was isolated for Northern blot analysis. Inset shows a representative Northern blot. The DHEA-ST/ GAPDH densitometric ratio in control was considered as 100% for the calculation of changes in the others. Asterisk indicates a significant difference at P < 0.05 as compared with the controls

The increase of DHEA-ST mRNA levels by hCG was hormone specific, as LH mimicked hCG, whereas FSH and TSH had no effect. Isolated {alpha} and ß subunits of hCG also had no effect (Fig. 6).



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  		FIG. 6. The hormone specificity of the hCG effect on DHEA-ST mRNA levels in H295R cells. The cells were cultured for 4 h in the presence or absence of 100 ng/ml of various hormones. Then total cellular RNA was isolated for Northern blot analysis. Inset shows a representative Northern blot. The DHEA-ST/GAPDH densitometric ratio in control was considered as 100% for the calculation of changes in the others. Asterisks indicate significant differences at P < 0.05 as compared with control

Receptor Requirement for the hCG Effects

To verify the requirement of cognate receptors for hCG to increase steady-state DHEA-ST mRNA levels and DHEAS secretion, phosphorothioate ODNs synthesized from human receptor sequence were used. Treatment of cells with an antisense but not sense ODN resulted in a significant reduction in receptor protein levels (Fig. 7). In the antisense ODN-treated cells, hCG was unable to increase either DHEA-ST mRNA levels or DHEAS secretion (Fig. 8). In the sense ODN-treated cells, on the other hand, hCG was just as effective as in untreated cells in increasing both DHEA-ST mRNA levels and DHEAS secretion (Fig. 8).



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  		FIG. 7. Inhibition of LH/hCG receptor synthesis in human H295R cells by treatment with antisense but not with sense LH/hCG receptor ODNs. The cells were cultured for 24 h in the presence or absence of 5 µM LH/ hCG receptor ODNs, and then cell lysates were analyzed for LH/hCG receptors and ß-tubulin by Western blotting. Asterisk indicates a significant difference at P < 0.05 as compared with control



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  		FIG. 8. Inhibition of hCG effect on DHEA-ST mRNA levels and DHEAS secretion by pretreatment with antisense but not with sense LH/hCG receptors ODNs. The H295R cells were pretreated for 24 h in the presence or absence of 5 µM LH/hCG receptor ODNs and then treated for 4 h (DHEA-ST) or 8 h (DHEAS) in the presence or absence of 100 ng/ml hCG. Then the cellular DHEA-ST mRNA and media DHEAS levels were measured. The DHEA-ST/GAPDH densitometric ratio and the DHEAS (54.1 ± 6.3 µg/ml) level in the controls were considered as 100% for the calculation of changes in the others. Asterisks indicate significant differences at P < 0.05 as compared with the corresponding controls

Signaling in the hCG Actions

To determine the signaling involved in the hCG actions, we first investigated the hCG effect on cAMP production. Figure 9 shows that treatment of H295R cells with hCG resulted in a time- and dose-dependent significant increase in media cAMP levels. As cAMP increase is expected to activate PKA, we next examined the hCG effect on PKA activation. The results showed indeed that hCG treatment increased PKA but not PKC activity (Fig. 10).



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  		FIG. 9. The effect of hCG on media cAMP levels in H295R cells. In time-dependency experiments, the cells were cultured for increasing lengths of time in the presence or absence of 100 ng/ml hCG. In dose-dependency experiments, the cells were cultured for 2 h in the presence or absence of increasing concentrations of hCG. Then the media cAMP levels were measured, which in the controls (34.4 ± 4.7 pmol/ml) were considered as 100% for the calculation of changes in the others. Asterisks indicate significant differences at P < 0.05 as compared with the corresponding controls



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  		FIG. 10. The effect of hCG on PKA and PKC activity in H295R cells. The cells were cultured for 2 h in the presence or absence of 100 ng/ml hCG, and then cell lysates were assayed for the enzymes' activity. The kinase activities in controls were considered as 100% for the calculation of changes in the others. Insets show representative profiles of phosphorylated and nonphosphorylated synthetic peptide substrates. Asterisk indicates a significant difference at P < 0.05 as compared with the control

The involvement of PKA but not PKC in the hCG actions was further investigated by using corresponding inhibitors. The results showed that while H-89 alone, a selective PKA inhibitor, had no effect either on DHEA-ST mRNA levels or on DHEAS secretion, it blocked the hCG ability to increase both of them (Fig. 11). The Bis, a PKC inhibitor, on the other hand, had no effect on its own, nor could it block the hCG effects (Fig. 11). The use of inhibitors alone suggests that basal DHEAS secretion is not controlled by either signaling pathway.



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  		FIG. 11. The effect of kinase inhibitors on hCG-induced increase in DHEA-ST mRNA levels and DHEAS secretion. The H295R cells were cultured for 4 h (DHEA-ST) or 8 h (DHEAS) in the presence or absence of hCG, bisindolylmaleimide (Bis), or isoquinolinesulfonamide (H-89) alone or their indicated combinations, and then cellular DHEA-ST mRNA and media DHEAS levels were measured. Inset shows a representative Northern blot. The DHEA-ST/GAPDH densitometric ratio and DHEAS level (42.6 ± 5.1 µg/dl) in controls were considered as 100% for the calculation of changes in the others. Asterisks indicate significant differences at P < 0.05 as compared with the corresponding controls

Finally, the cAMP mediation of the hCG effects was further tested by determining whether 8-bromo-cAMP, a stable cAMP analog, could mimic hCG and whether 8-Cl-cAMP, a site selective cAMP analogue that inhibits type 1 PKA by enhancing the degradation of the RI{alpha} regulatory subunit [31], could block the effects of hCG and 8-bromo-cAMP.

Figure 12 shows that indeed 8-bromo-cAMP mimics hCG and that 8-Cl-cAMP blocks the effects of both 8-bromo-cAMP and hCG in increasing DHEA-ST mRNA levels and DHEAS secretion.



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  		FIG. 12. The effects of 8-bromo-cAMP, 8-Cl-cAMP, and hCG on DHEA-ST mRNA levels and DHEAS secretion. The H295R cells were cultured for 4 h (DHEA-ST) or 8 h (DHEAS) in the presence or absence of hCG, 8-bromo-cAMP, and 8-Cl-cAMP alone or in their indicated combinations, and then the cellular DHEA-ST mRNA and media DHEAS levels were measured. Inset shows a representative Northern blot. The DHEA-ST/ GAPDH densitometric ratio and the media DHEAS level in controls were considered as 100% for calculation of change in the others. Asterisks indicate significant differences at P < 0.05 as compared with the corresponding controls


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Adrenals from human [4–7], mouse [32–34], guinea pig [35], ferret [36], turkey [37], and catfish [38] contain LH/ hCG receptors, which suggests that the adrenal receptors are well conserved during evolution. As adrenals and ovaries share embryonic origin, it may not be too surprising to find that adrenals contain LH/hCG receptors, as do ovaries. The knockout of LH/hCG receptors predicts an ovarian as well as adrenal phenotype, which was found to be the case [39–41].

Human adrenal cortical carcinoma H295R cells contain LH/hCG receptor mRNA transcripts and receptor protein, which could bind both hCG and LH but not the other members of the glycoprotein hormone family or dissociated hCG subunits. Activation of these receptors with hCG resulted in a time- and dose-dependent increase in DHEAS secretion. The increases were seen between 4 and 12 h of treatment and with 50–100 ng/ml hCG. The lack of response at longer treatment time and at higher hCG concentration could be due to receptor downregulation. The lack of response at lower hCG concentrations (i.e., low sensitivity) may be reflecting a mechanism to prevent inappropriate stimulation. Alternatively, it may be an indication of local control by endogenously synthesized hormones or an endocrine control only when serum gonadotropin levels are elevated. It could also be an intrinsic property of H295R cells, which are not very sensitive even to ACTH in increasing cortisol, androstenedione, and DHEA [23].

The secretory response of DHEAS was hormone specific, as other members of the glycoprotein hormone family had no effect. A previous study has shown that ACTH also had no effect, which is the reason for not repeating the ACTH stimulation experiments in the present study [23]. The hCG effect on DHEAS secretion was steroid specific, as hCG treatment could not increase the secretion of androstenedione, cortisol, or DHEA, which ACTH treatment could in H295R cells [23]. The lack of the hCG effect on DHEA secretion suggests that hCG does not increase de novo steroid biosynthesis. Instead, it may increase its sulfation. Consistent with this possibility, the hCG treatment increased steady-state mRNA levels of DHEA-ST at 4 h, which were not sustained at 8 h, even though DHEAS secretion remained elevated. This paradox could perhaps be due to an elevation of DHEA-ST protein and/or its catalytic activity, neither of which was measured in the present study.

As adrenals are nontraditional targets of LH/hCG action, some verification of receptor involvement in their actions was needed. This verification was obtained by inhibiting LH/hCG receptor synthesis in H295R cells by using phosphorothioate ODNs synthesized from human receptor sequence. The use of antisense but not sense LH/hCG receptor ODN, which inhibited receptor synthesis, prevented hCG from increasing DHEA-ST mRNA levels and DHEAS secretion. This reaffirms the concept that receptor activation is a prerequisite for hCG to increase DHEA-ST mRNA levels and DHEAS secretion.

Depending on the tissue and physiological context, hCG uses many signaling pathways. However cAMP/PKA is the most common among them [3]. The signaling studies revealed that treatment of H295R cells with hCG resulted in a time- and dose-dependent increase in cAMP secretion. These increases were not fully coincidental with the changes in DHEAS secretion, which led to further testing on the importance of cAMP/PKA signaling in the LH and hCG actions. These studies have demonstrated an activation of PKA but not PKC by hCG treatment and inhibition of PKA activation by H-89 but not PKC by bis prevented hCG from increasing DHEA-ST mRNA levels and DHEAS secretion.

In further support of the possibility that hCG uses cAMP/PKA signaling, 8-bromo-cAMP was found to be just as effective as hCG in increasing both DHEA-ST mRNA levels and DHEAS secretion. A site-selective inhibitor of type I PKA [31], 8-Cl-cAMP, prevented not only the effects of hCG but also those of 8-bromo-cAMP, suggesting that this enzyme plays an essential role in the hCG action. Thus, multiple studies on signaling pathways provided strong support for the essential role of cAMP/PKA in the action of hCG and LH to increase DHEAS secretion by H295 cells. The use of cAMP signaling in the hCG action was also observed in transgenic mice adrenals [32–34].

The hCG and LH responses found in the present study were rather modest and occurred at higher concentrations, which is a typical feature of nongonadal tissues in general [3]. Nevertheless, they were genuine as demonstrated by hormone and steroid specificity, the ability of receptor antisense ODN to block the hCG effects, and the specificity in the signaling pathway that hCG used in adrenal cells.

A number of previous studies demonstrating higher serum adrenal androgen levels in the face of normal serum ACTH levels and elevated LH or hCG levels, as well as the present findings, support the possibility that LH drives the secretion of adrenal androgens [13–17]. This concept may be valid despite the contradictory evidence on whether hCG can increase adrenal androgen secretion in castrated or intact individuals. For example, while some studies demonstrated that hCG can increase adrenal androgens [42–45], others failed to find an effect [46–50]. This discrepancy could be due to downregulation of adrenal receptors by chronically elevated serum LH levels in long-term castrated individuals and the use of nonoptimal conditions with respect to the hCG dose and time allowed to measure response in intact individuals. The previous findings on homologous LH/hCG receptor downregulation [51] and the present finding on dose and time dependency of the hCG effect on DHEAS secretion support these possibilities.

Cushing's syndrome due to spontaneously formed LH- and hCG-sensitive adrenal tumors during pregnancy and menopause causes secretion of higher levels of glucocorticoids and androgens [13–17, 52–61]. It is possible that these tumors may arise in part from the changes in sensitivity and/or zonal distribution of normal adrenal LH/hCG receptors. Serum DHEAS levels decrease and LH levels increase with age [62, 63]. This seemingly paradoxical relationship now appears to have been resolved by studies demonstrating an age-dependent regression of zona reticularis and loss of cells that express DHEA-ST [64, 65]. Thus, even though serum LH levels may have been elevated, they will not be able to stimulate DHEAS secretion. In addition, chronically elevated LH levels may downregulate adrenal receptors, resulting in a loss of response.

In summary, our results demonstrate the presence of LH/ hCG receptors in human adrenal cortical H295R cells, which are functionally coupled to increasing DHEAS secretion in a hormone- and steroid-specific manner and mediated by cAMP/PKA signaling pathway. These findings support the possibility that LH and hCG could influence adrenal function in normal and pathological conditions.


   FOOTNOTES
 
1 Correspondence: Ch. V. Rao, Division of Research, Department of Obstetrics, Gynecology, and Women's Health, 438 MDR Building, 511 South Floyd St., University of Louisville Health Sciences Center, Louisville, KY 40292. FAX: 502 852 0881; cvrao001{at}gwise.louisville.edu Back

2 Current address: Department of Psychiatry, Emory University School of Medicine, Atlanta, GA 30322 Back

Received: 10 January 2004.

First decision: 30 January 2004.

Accepted: 1 April 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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