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Endocrinology, doi:10.1210/en.2007-1529
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Endocrinology Vol. 149, No. 3 1144-1154
Copyright © 2008 by The Endocrine Society

Non-Gonadotropin-Releasing Hormone-Mediated Transcription and Secretion of Large Human Glycoprotein Hormone {alpha}-Subunit in Human Embryonic Kidney-293 Cells

Ida Casella, Herbert Lindner, Christoph Zenzmaier, Daniela Riitano, Peter Berger and Tommaso Costa

Department of Pharmacology (I.C., D.R., T.C.), Istituto Superiore di Sanità, 00161 Rome, Italy; Department of Clinical Biochemistry (H.L.), Medical University of Innsbruck, and Austrian Academy of Sciences (C.Z., P.B.), Institute for Biomedical Aging Research, A-6020 Innsbruck, Austria

Address all correspondence and requests for reprints to: Tommaso Costa, Dipartimento del Farmaco, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161, Rome, Italy. E-mail: tomcosta{at}iss.it; or Peter Berger, Ph.D., Institute for Biomedical Aging Research, Austrian Academy of Sciences, Rennweg 10, A-6020 Innsbruck, Austria. E-mail: peter.berger{at}oeaw.ac.at.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
To identify genes that are most responsive to a sustained activation of a Gs protein-coupled receptor, HEK293 cells were stably transfected with the β2-adrenergic receptor and stimulated with agonist isoproterenol (1 µM). A microarray study indicated that the gene with the highest stimulation index (500-fold) encoded the common {alpha}-subunit of human glycoprotein hormones (GPH{alpha}). Induction of GPH{alpha} transcription in response to cAMP elevations resulted in a dramatic increase (600-fold) of protein secretion as shown by RT-PCR and a highly specific time-resolved immunofluorometric assay. Cloning and sequencing of the GPH{alpha} cDNA and mass spectrometric analysis of HPLC-purified GPH{alpha} derived from serum-free HEK293-β2-adrenergic receptor-stimulated cells verified the nature of the molecule. Enzymatic deglycosylation with subsequent Western blots revealed that this was a large hyperglycosylated form of GPH{alpha} that had not been associated with a β-subunit previously. This uncombined variant is known to be either cosecreted with GPHs from the pituitary, the placenta, and a variety of tumors or secreted without GPHs from APUD cells and rare tumors. Moreover, it is similar to GPH{alpha} found at high concentrations in seminal plasma. As shown by a panel of endogenous or transfected G protein-coupled receptors in HEK293 cells, the expression of large GPH{alpha} was controlled by Gs- and Gq- but not Gi-dependent receptors and mediated via cAMP and Ca++ release. This suggests that Gq- or Gs-coupled receptors other than the classical GnRH receptor may play a role in the regulation of nonpituitary, nonplacental GPH{alpha} secretion under physiological and pathological conditions.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE {alpha}- AND β-subunits of the heterodimeric glycoprotein hormones that control reproduction (chorionic gonadotropin, FSH, and LH) and thyroid function (TSH) are members of the larger family of cysteine knot growth factors (1). Neither the single common {alpha}- nor four types of β-subunits are considered to be hormonally active in their uncombined free form because the full activation of the corresponding glycoprotein hormone receptors requires heterodimeric hormonal forms (2).

However, biological effects triggered by free subunits, but not likely mediated by glycoprotein hormone receptors, have been reported. The glycoprotein hormone {alpha}-subunit (GPH{alpha}) was found to be biologically active on prolactin releasing cells of pituitary and presumably endometrium (3, 4), and was described as the active human chorionic gonadotropin (hCG) molecular species in stimulating human decidualization. Minor effects observed with heterodimeric hCG were assigned to uncombined GPH{alpha}, generated by dissociation of hormone subunits catalyzed via a yet-unknown mechanism in endometrial cells (5, 6). Recent findings indicate that the local production of GPH{alpha} in prostatic tissue inhibits the growth of stromal cells. Thus, decreased expression of GPH{alpha} with age might be a favoring factor for the development of hyperplasia (7).

Physiologically significant amounts of free GPH{alpha} are eutopically released by the pituitary and trophoblastic placental cells during pregnancy or ectopically by neuroendocrine cells. Unlike in serum in which holo-GPHs are in excess over free GPH{alpha}, in human seminal plasma very high concentrations of GPH{alpha} are found in 1000-fold excess over free hCGβ and 10,000-fold over the holohormone hCG (8). In testicular and other cancers, secretion of GPH{alpha} alone, or in excess over hCGβ subunits and holo-hCG, may occur (9, 10). GPH{alpha} was also observed frequently in transformed cell lines derived from neoplastic tissue, such as bronchogenic tumors (11), or HeLa cell clones (12, 13) in which it might exert autoparacrine growth-promoting effects (14).

Regulation of GPH{alpha} subunit expression and secretion in the pituitary was investigated in cellular model systems from human, rat, and mouse. The major stimuli for GPH{alpha} production and secretion in response to GnRH1 are the activation of the cAMP pathway with consequent phosphorylation of cAMP-responsive element sites and the intracellular release of Ca++ from storage pools with subsequent activation of the MAPK pathway (see Refs. 15, 16 for reviews). However, non-GnRH-regulated ectopic production of GPH{alpha} was not investigated to the same extent of detail. This secretion is of relevance because human seminal plasma contains excessive amounts of GPH{alpha} that originates from the APUD and stromal cells from the prostate. APUD cells are also a source of GPH{alpha} in the gastrointestinal tract and a variety of testicular (10) and gastroenteropathic tumors (17).

Whereas investigating the pattern of gene expression induced by agonist stimulation in a HEK293 cell line stably transfected with β2-adrenergic receptors, we found that human GPH{alpha} was the gene with the highest level of induction. Here we show that HEK293 cells express and release uncombined hyperglycosylated GPH{alpha}. This expression, unlike in many previously described transformed cell lines, is strictly dependent on activation of intracellular signaling pathways. In fact, GPH{alpha} transcription and release in HEK293 cells is barely detectable under basal conditions but reaches high levels only on activation of cAMP and Ca2+-dependent signals. Moreover, using HEK293 cells transiently or stably transfected with receptors selective for each of the three G protein family members, Gs, Gq, and Gi, we show that both Gs and Gq but not Gi proteins mediate increases in GPH{alpha} transcription and secretion.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell cultures and transfections
HEK293 cells and derived lines were grown in DMEM supplemented with 10% (vol/vol) fetal calf serum (FCS) (Invitrogen Ltd., Paisley, UK) in a humidified atmosphere of 5% CO2 at 37 C. Transient transfections of cell monolayers were performed according to the calcium phosphate precipitation method. A stable HEK293 cell line expressing β2-adrenergic receptor (β2AR) was obtained as described previously (18).

Array analysis
Atlas human cDNA arrays version 1.2 (1176 cDNA tags x 3) were purchased from CLONTECH (Palo Alto, CA). 32P-labeled cDNA was synthesized according to the manufacturer’s instructions from poly-A RNA enriched by streptavidin conjugated magnetic beads using [32P]dATP (6000 Ci/mmol; Amersham, Aylesbury, UK) and the components supplied with the array. The hybridization of the radiolabeled probe to the cDNA array filters and the subsequent washes were performed as outlined by CLONTECH. The dried membranes were measured in a Phosphor imager (Cyclone; PerkinElmer, Palo Alto, CA) and analyzed using CLONTECH software (AtlasImage).

Stimulation of GPH{alpha} expression and secretion in HEK293 cells
Cells were plated in 6- or 24-well plates and cultured until close to confluence before replacing the medium with 1 ml FCS-free DMEM. Twelve hours later, isobutyl-methyl-xanthine (IBMX) 100 µM, rolipram 10 ng/ml, 8-bromoadenosine-cAMP (8-Br-cAMP) 1 µM, cholera toxin 0.5 µg/ml, forskolin 1 µM, calcium ionophore A2318 10 µM, isoproterenol 1 µM, and 5’-(N-ethyl-carboxamido) adenosine (NECA) 100 µM (all from Sigma, St. Louis, MO) were for 24 h, or, in kinetics experiments, for variable time intervals as indicated. Subsequently the medium was collected and stored frozen for the immunofluorometric assay (IFMA) assay of released GPH{alpha}, whereas the cell monolayers were treated with Trizol reagent for RNA extraction and RT-PCR analysis. To determine the relative concentration of secreted and intracellular GPH{alpha} immunoreactivity, the cell monolayers were washed thrice in PBS, scraped into 0.25 ml ice-cold Tris/HCl [50 mM (pH 7.4)], and sonicated. After centrifugation (40,000 x g, 30 min), the supernatants were stored frozen at –80 C until analyzed by IFMA.

Analysis of mRNA expression by RT-PCR
Total RNA was extracted from cells monolayers by directly lysing cells with Trizol reagent (Invitrogen). About 2 µg were reverse transcribed using SuperScript III reverse transcriptase and an oligo(dT)12–18 as primer (Invitrogen). Aliquots, corresponding to one twentieth of total cDNAs were amplified through 25 cycles of PCR, using Platinum Taq DNA polymerase (Invitrogen), according to the manufacturer’s instructions. The pairs of sequence-specific oligonucleotides used for amplifications were: 5' primer, 5'-CCA TGG ATT ACT ACA GAA AAT ATG CAG C-3'; 3' primer, 5'-GCA CGC CGT GTG GTT CTC CAC TTT GGA AA-3' for GPH{alpha} mRNA; 5' primer, 5'-TCC GTG CCT CCA AGA TGA CAA A-3'; and 3' primer, 5'-CAG AGA AGA GCC TGT CTT CAG TC-3' for mRNA amplification of the housekeeping gene coding for the ribosomal protein S26 used for normalize the expression of GPH{alpha}. Synthetic oligonucleotides were from MWG Biotech (Ebersberg, Germany).

Molecular cloning and sequencing of GPH{alpha} cDNA in HEK293 cells
The full-length cDNA encoding the GPH{alpha} protein was amplified from HEK293 reverse transcribed RNA using the following pair of sequence-specific oligonucleotides: 5'primer, 5'-GCC CTG AAC ACA TCC TGC AAA A-3' and 3'primer, 5'-GCA GTC ATC AAG ACA GCA CTT G-3'. The amplified fragment was purified by phenol/chloroform extraction, cloned into pCRII vector (Invitrogen), and sequenced by MWG Biotech.

Time-resolved IFMAs (TR-IFMA) for hCG{alpha} and hCGβ
Coating, blocking, incubation, and washing procedures of the IFMAs for uncombined, i.e. free hCG{alpha} and hCGβ, respectively, were performed as described previously (9). Samples were diluted in IFMA buffer [50 mM Tris-HCl (pH 7.75); 0.9% NaCl, 5 g/liter BSA, 0.1 g/liter Tween 40, and 20 mM diethylenetriaminepenta acid; Sigma-Aldrich, Milwaukee, WI] and run in duplicate. The used monoclonal antibodies (mAbs) against hCG{alpha}, and hCGβ served as reference reagents in the international TD-7 Workshop on antibodies to hCG and hCG-related molecules, and their characteristics have been described elsewhere (19). Coating mAbs were coded INN(sbruck)-hCG-72 recognizing selectively free hCG{alpha} (hCG{alpha} assay) and INN-hCG-68 reacting with hCGβ but not with holo-hCG (hCGβ assay). The detection mAbs INN-hFSH-158, a pan {alpha}-mAb, for the hCG{alpha} assay and INN-hCG-22, a pan β-mAb, for the hCGβ-assay, were labeled with isothiocyanatophenylene triamintetraacetic acid-europium (Wallac, Turku, Finland) according to the manufacturer’s recommendations. Time-resolved fluorescence was measured for a second in a fluorometer (Victor2; Wallac). Hormone standards (hCG{alpha} first I.R.P. 75/569, hCGβ first I.R.P. 75/551) to determine sensitivity and specificity of both IFMAs were kindly provided by the National Institute for Biological Standards and Control (South Mimms, UK). Inter- and intraassay variations of either IFMA were less than 10% over the entire assay ranges.

GPH{alpha} purification via reversed phase-HPLC (RP-HPLC)
GPH{alpha} was purified from 80 ml of supernatant from HEK293 stably transfected with β2AR and stimulated with isoproterenol (1 µM). The supernatant was 4-fold concentrated by centrifugation with Ultrafree (Biomax 5 kDa cutoff, 15 ml; UFV2BCC10; Millipore, Anderson, MA) according to the manufacturer’s recommendations, dialyzed against 0.01 M NaHCO3 overnight at 4 C (Slide-A-Lyzer, 3.5-kDa cutoff; Pierce, Rockford, IL), frozen (–70 C), lyophilized, resolved in 5 ml aqua bidest, dialyzed against 10 mM Na-phosphate buffer (pH 3.5; 4 C, overnight; Slide-A-Lyzer, 7-kDa cut-off; Pierce), and purified by RP-HPLC.

The RP-HPLC equipment used consisted of a 127 Solvent Module and a model 166 UV-visible-region detector (Beckman Instruments, Fullerton, CA). The effluent was monitored at 214 nm. Separation was performed on a Nucleosil C4 column (250 mm x 8 mm inner diameter; 5 µm particle pore size; 30 nm pore size; end capped; Seibersdorf, Austria). Samples of 1 ml were injected onto the column and chromatographed within 60 min at a constant flow of 1.5 ml/min with a two-step acetonitrile gradient starting at solvent A-solvent B (70:20) (solvent A: water containing 0.1% trifluoroacetic acid; solvent B: 70% acetonitrile and 0.1% trifluoroacetic acid). Next, the concentration of solvent B was increased from 20 to 70% in 50 min and from 70 to 100% in 10 min. Fractions were collected and, after adding 100 µl 1% NH3, lyophilized and stored at –20 C. Recovery of GPH{alpha} at each purification step was monitored by IFMA. The 80-ml starting material contained 188 µg; this was concentrated to 66.6 µg per 0.4 ml after RP-HPLC purification.

Mass-spectrometric (MS) analysis
RP-HPLC purified GPH{alpha} (25 µl equivalent to 3.8 µg) from HEK293 stably transfected with β2AR stimulated with isoproterenol (1 µM) (see above) was reduced by 10 mM dithiothreitol in 100 mM NH4HCO3 (pH 8.3) (30 min at 56 C) and alkylated by addition of 25 of 55 mM iodoacetamide in 100 mM NH4HCO3. After 20 min incubation at room temperature in the dark, the sample was digested with {alpha}-chymotrypsin [EC 3.4.21.1; Sigma type I-S, 1:100 (wt/wt)] for 4 h at 37 C. The digest was subjected to nanospray-MS. Protein digest was analyzed using a LCQ ion trap instrument (ThermoFinnigan, San Jose, CA) equipped with a nanospray interface. The nanospray voltage was set at 1.6 kV, and the heated capillary was held at 200 C. MS/MS spectra were searched against a human database using SEQUEST (LCQ BioWorks; ThermoFinnigan).

Digestion with glycosidases
GPH{alpha} purified by HPLC as described above from supernatant of HEK293 stably transfected with β2AR and specifically stimulated with isoproterenol (10 µM), and for comparative purposes the frozen carrier-free concentrate (FC 862) of the new World Health Organization (WHO)-adopted first International Reference Preparation for Immunoassay of hCG{alpha} (first IRR hCG{alpha} 99/720) were deglycosylated according to the manufacturer’s recommendations (New England BioLabs, Frankfurt/Main, Germany). N-linked carbohydrate antennae were digested either with peptide N-glycanase PNGase F [peptide-N4-(N-acetyl-β-glucosaminyl) asparagine amidase] (EC no. 3.5.1.52) or with endoglycosidase H when the extent of trimming of the carbohydrate moiety was determined.

In brief, 22 µl sample equivalent to 1 µg GPH{alpha} or hCG{alpha} were incubated with 2 µl 10 x denaturing buffer (5% sodium dodecyl sulfate, 10% β-mercaptoethanol) for 10 min (100 C) and put on ice. Then 2.2 µl 0.5 M sodium phosphate buffer (pH 7.5), 2.2 µl Nonidet P-40, and 1 µl enzyme (PNGase F, 500 U) were added and incubated for 2 h at 37 C.

For digestion with endoglycosidase H, 1 µg GPH{alpha} or hCG{alpha} was denatured as described above, 2.2 µl sodium citrate (0.5 M, pH 5.5), and 500 U (1 µl) endoglycosidase H added. Deglycosylation was carried out for 2 h at 37 C.

Western blot
Approximately 500 µg each of PNGase-F- or endoglycosidase H-deglycosylated, and native GPH{alpha} and hCG{alpha}, were diluted with sample buffer to 25 µl and loaded on precast polyacrylamide gradient gels (4–20% Tris-glycine gels; Cambrex BioScience Rockland, ME) and separated by SDS-PAGE (SDS-PAGE; Mighty Small II; Hofer Scientific Instruments, San Francisco, CA; 150 V, 75 min). Proteins were electrophoretically transferred (3.5 h, 400 mA) to polyvinyl difluoride-membranes (Immunoblot 0.2 µm; Bio-Rad, Hercules, CA) and blocked with 5% (wt/vol) skimmed milk powder in PBS (45 min, room temperature). A mixture of three mAbs directed against three distinct epitopes on hCG{alpha} (codes INN-hCG-72, -hFSH-132, and -158) (20) were diluted in 5% (wt/vol) milk powder in PBS/0.1% Tween 20 to approximately 5 µg/ml each and incubated overnight at 4 C. After extensive washing with 5% milk powder/PBS/0.1% Tween 20, membranes were incubated for 1 h with goat antimouse IgG horseradish peroxidase (W 4021; Promega, Mannheim, Germany) diluted 1:2500 in 5% (wt/vol) milk powder in PBS. Chemiluminescent substrate conversion (Super Signal West Dura; Pierce) was detected by Hyperfilm ECL (Amersham) using exposure times between 10 sec and 45 min.

Signaling pathways involved in GPH{alpha} transcription and secretion
The cDNA clones for the receptors indicated below were obtained from the UMR cDNA Resource Centre (www.cdna.org).

Cells plated in 24 wells were transiently transfected with cDNAs coding for different G protein-coupled receptors (GPCRs) [muscarinic M3, histamine H1, histamine H2, opioid type-µ (MOP), β2-adrenergic, and neurokinin-1]. To assess the signaling activity of transiently transfected GPCRs, cells were switched 24 h after transfection to serum-free medium containing [3H]myo-inositol (1 µCi/ml) (NEN Life Science Products, PerkinElmer, Shelton, CT) and incubated for an additional 24 h. After removal of the culture media and washing, monolayers were incubated for 30 min at 20 C in PBS containing LiCl (1 mM) and the agonists active on the transfected receptors. After agonist removal, reactions were arrested by adding 0.5 ml ice-cold HCl 0.1 N. Inositol phosphate accumulation and cAMP levels were measured in the same extracts as described previously (21).


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression and release of GPH{alpha} in HEK cells
β2AR-mediated GPH{alpha} expression.
To identify genes that are most responsive to a sustained activation of a Gs-coupled receptors, we used a HEK293 cell clone stably transfected with the human β2AR. RNA isolated from control cells and cells exposed to the agonist isoproterenol for 24 h were used to synthesize cDNA probes, which were subsequently hybridized with a set of three human cDNA arrays (Atlas v.1.2; CLONTECH) covering a total of approximately 3600 genes. The results of the screening showed that only a few dozen genes underwent up- or downmodulation in response to the agonist. Most of them exhibited modest changes (<10-fold), compared with the basal level. However, there was one outstanding exception. The cDNA encoding for the common {alpha}-subunit of the heterodimeric glycoprotein hormones family was up-regulated 500-fold as a result of agonist treatment (Fig. 1AGo). The expression of GPH{alpha} in this cell line had not been previously reported in the literature.


Figure 1
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  		FIG. 1. Macroarray analysis of HEK293 mRNA expression on activation of β2AR and PCR analysis of GPH{alpha} expressing in different cell lines A, HEK293 cells expressing β2AR were incubated in the absence (basal) or presence of isoproterenol (1 µM) for 24 h. Radiolabeled cDNA probes synthesized from the extracted RNA were hybridized to CLONTECH Atlas 1.2 membranes as described in Materials and Methods. Left side, Autoradiogram of the membranes with the spots corresponding to the GPH{alpha} transcripts marked by arrows (only the membrane set containing the GPH{alpha} gene is shown). Right side, Quantification of the data. Only the transcripts exhibiting changes greater than 2-fold are shown. The letter codes correspond to the following genes: a, G2/mitotic-specific cyclin B1; b, protein kinase B; c, ataxia telangiectasia mutated protein; d, zinc finger protein 9; e, BRG1 (brahma-related gene 1); f, RNA polymerase II elongation factor SII; g, guanine nucleotide-binding protein-{alpha} stimulating activity polypeptide 1; h, early growth response protein 1; i, glycoprotein hormone {alpha}-subunit; l, methionine aminopeptidase 2. B, Parental HEK293 cells and HeLa, A431, and SH-EP cells were grown under conditions previously described for such cell lines (22 23 ). The cells were plated into 6-well flasks and, once confluent, were incubated for 6 h with the indicated stimulators at the following concentrations: isoproterenol (Iso), 1 µM; forskolin (Frk), 10 µM; {alpha}-thrombin (Thr), 1 U/ml; epidermal growth factor (EGF), 20 ng/ml. Bas, Basal level. Steady-state mRNA levels of GPH{alpha} and the housekeeping gene S26 were detected by RT-PCR. The data are representative of two independent experiments.

 
To verify that the overexpression of GPH{alpha} was not the result of the fortuitous selection of a particular HEK293 phenotype during the isolation of the β2AR-expressing clone, total RNA was prepared from parental untransfected HEK293 cells and analyzed by RT-PCR, using primers specific for the human GPH{alpha} gene sequence. Basal GPH{alpha} expression was extremely low under nonstimulated conditions but strongly induced in the presence of forskolin (which directly activates adenylyl cyclase bypassing receptors). In addition, despite the much lower level of endogenous β2AR in the wild-type cell line (≤25 fmol/mg), compared with the transfected clone (32 pmol/mg), a smaller extent of isoproterenol-stimulated GPH{alpha} expression was also detectable in the parental cell line (Fig. 1BGo). Thus, the expression of the GPH{alpha} gene is an intrinsic property of HEK293 cells in general.

To investigate whether signaling-dependent activation of GPH{alpha} gene expression might be a widespread phenomenon, we examined three additional human transformed cell lines, including HeLa cells and A341 epidermoid carcinoma, both of which express β-adrenergic receptors (22, 23), and the SH-EP line, a transdifferetiated epithelial phenotype from human neuroblastoma, known to express protease-activated receptor 1 (24). Hela cells expressed high levels of GPH{alpha} mRNA, but unlike in HEK293, the transcription was constitutive, as the levels were virtually identical in basal and isoproterenol or forskolin stimulated conditions (Fig. 1BGo). No detectable mRNA was observed in A341 cells, whereas a very low extent of expression slightly stimulated by thrombin was observed in SH-EP cells (Fig. 1BGo). Thus, the peculiar characteristic of HEK293 cells is their ability to express high levels of GPH{alpha} only in response to activation of intracellular signals.

cAMP-mediated synthesis and secretion of GPH{alpha}
To verify that the remarkable induction of GPH{alpha} transcription in HEK293 cells was matched by a corresponding enhancement of protein synthesis and release, we compared mRNA induction and protein production of GPH{alpha} in both HEK293-β2AR and parental untransfected HEK293 cells. As shown in Fig. 2AGo, not only stimulated levels (lanes 2 and 4), but also basal mRNA expression (lanes 1 and 3) were greater in HEK293-β2AR than in the parental cells. This indicates that the higher degree of receptor expression in the transfected clone can generate enough constitutive signaling to activate the expression of the gene. Using TR-IFMA, we found that protein synthesis and release followed a quite similar pattern. Immunoreactive GPH{alpha} was stimulated 113-fold over basal in wild-type cells exposed to forskolin and 457-fold in the β2AR-expressing clone exposed to isoproterenol. Basal protein levels were also 4-fold greater in β2AR-expressing cells than in wild type (Fig. 2BGo). Thus, the stimulation of GPH{alpha} mRNA in HEK293 cells is paralleled by a very similar enhanced production of {alpha}-subunit protein.


Figure 2
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  		FIG. 2. Stimulation of GPH{alpha} expression in HEK293 cells by modulators of cAMP signaling. Parental cells (HEK) or the monoclonal line expressing β2AR (HEK-β2AR) were incubated with 1 µM forskolin (Frk) or 1 µM isoproterenol (Iso), as indicated. A, Steady-state mRNA levels of GPH{alpha} and the housekeeping gene S26 detected by RT-PCR. Bas, Basal level. B, GPH{alpha} protein levels measured by IFMA in cell extracts (intracellular) and culture media (secreted). Data are presented as cumulative histograms and the percentage of secreted GPH{alpha} protein is indicated within or on top of the bars. Data are averages (±SEM) of triplicate determinations.

 
A TR-IFMA selective for hCGβ was used to assess the possible coproduction of hCGβ-subunit. Levels of 100 pg/ml or less of immunoreactive β-subunit were detected in nonstimulated cells and remained unchanged after addition of either forskolin or isoproterenol (data not shown). Thus, HEK293 express primarily uncombined GPH{alpha} and only traces of hCGβ.

Immunoreactive GPH{alpha} was found in both culture medium and cell extracts. In the parental untransfected cells, the fraction of total {alpha}-subunit in the medium was only 16% under basal conditions but increased to 87% after stimulation. In the receptor-expressing cells, 61% of the protein was released in the medium even in the absence of agonist, in line with the greater level of constitutive signaling noted by PCR analysis, but in the presence of agonist, the fraction of released GPH{alpha} was similarly enhanced to 83% (Fig. 2BGo). Such shifts in released vs. intracellular ratios induced by stimulation suggest that the protein is actively secreted from the cells after the enhanced transcription triggered by the elevation of cAMP levels.

Coupling of GPH{alpha} expression and secretion
To contrast the temporal evolutions of agonist-induced mRNA transcription with that of protein release, we performed RT-PCR assays and TR-IFMA determinations on cell extracts and media of HEK-β2AR cells collected at different time intervals after stimulation by 1 µM isoproterenol. As expected, the induction of mRNA transcription was detectable earlier than release. The transcription of mRNA started within the first 30 min of agonist stimulation, reached high levels after about 1 h, and continued to increase more slowly during the remaining period (Fig. 3AGo). The release of protein in the medium took a longer time lag to start (2.5 h) but then displayed a time course consistent with the kinetics of mRNA accumulation. The rate was nearly log linear with time during the first 5 h and then slowed down without reaching any apparent plateau (Fig. 3BGo).


Figure 3
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  		FIG. 3. Time course of GPH{alpha} transcription and protein release. HEK-β2AR cells were stimulated with 1 µM isoproterenol for the indicated times. GPH{alpha} peptide in cell media was measured by IFMA, and the mRNA levels of the genes (inset) were revealed by RT-PCR. Representative experiment repeated a second time with similar results.

 
We also studied the concentration dependency for isoproterenol-mediated enhancement of GPH{alpha} production in receptor-expressing cells. Both cellular and released {alpha}-subunit accumulation were enhanced with very similar concentration-response relationships by the agonist (Fig. 4AGo). However, the EC50s measured in such curves (1.9–2.1 µM) were 2 orders of magnitude greater than the EC50 for stimulation of cAMP accumulation (14 nM) determined in the same cells (Fig. 4BGo). The ratio between released and cellular GPH{alpha} was also increased by isoproterenol in concentration-dependent fashion, but the apparent EC50 for the maximal enhancement of this ratio (17 nM) was in very good agreement with that for cAMP stimulation (Fig. 4AGo, inset).


Figure 4
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  		FIG. 4. Concentration-response curves for isoproterenol-mediated induction of GPH{alpha} release and intracellular cAMP. HEK293-β2AR cells were treated with the indicated concentrations of isoproterenol (Iso). A, The levels of immunoassayable GPH{alpha} determined by IFMA in the cell extracts (intracell), in the media (secreted), and their sum (total) are plotted as function of the logarithm of isoproterenol concentration. The inset shows the same data, but the release of GPH{alpha} is expressed as ratio between extracellular and intracellular amounts. B, Concentration-response curve for isoproterenol-induced enhancement of intracellular cAMP measured in cells exposed to agonist for 30 min in a parallel experiment. Data are means of triplicate determinations. The experiment was repeated twice with similar results.

 
Genuine large free GPH{alpha}: identification by cDNA cloning, MS/MS, and glycosylation pattern
GPH{alpha} was purified by RP-HPLC from a serum-free supernatant of cells in which maximal accumulation of the released protein was achieved by exposure to 10 µM isoproterenol for 48 h. From 80 ml of starting material approximately 188 µg of purified GPH{alpha} were obtained. An aliquot (3.8 µg) was reduced, alkylated, digested with {alpha}-chymotrypsin, and analyzed by MS/MS. Verification that this specifically induced protein in fact is GPH{alpha} was achieved by coverage of nearly half of the amino acid sequence of the hCG{alpha} apoprotein by the analyzed peptides (Table 1Go).


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  		TABLE 1. MS/MS analysis of purified GPH{alpha}

 
To verify whether the sequence of the GPH{alpha} expressed by HEK293 cells may bear spontaneous mutations or polymorphisms, the full-length cDNA encoding the secreted protein was cloned and sequenced. As shown in Table 2Go, the predicted amino acid sequence of the protein including the 24 residues signal peptide is identical with that originally published for the human gene (25).


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  		TABLE 2. Cloned GPH{alpha} sequence

 
Free GPH{alpha} that has been secreted as uncombined protein differs in glycosylation pattern from hCG{alpha} dissociated from pregnancy-derived holo-hCG. It contains additional oligosaccharide branching and core fucosylation, which result in a slightly elevated molecular mass (change in apparent molecular size = ~2 kDa). The comparison of GPH{alpha} purified from HEK293 with dissociated hCG{alpha} (WHO first International Reference Preparation, 99/720, FC 862) (Fig. 5Go, lanes 1 and 2) shows a similar upshift of apparent molecular weight for HEK293-β2AR-derived material, suggesting that it is large hyperglycosylated GPH{alpha}. Accordingly, purified GPH{alpha} from HEK293 cells was resistant to endoglycosidase H deglycosylation (Fig. 5Go, lane 4), unlike dissociated hCG{alpha}, which was partially digested under the same conditions (Fig. 5Go, lane 3). Both preparations were completely deglycosylated by PNGase F resulting in core molecules of identical sizes (Fig. 5Go, lanes 5 and 6). Thus, HEK293-β2AR cells produce large hyperglycosylated GPH{alpha} on agonist stimulation.


Figure 5
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  		FIG. 5. HEK293 large GPH{alpha} differs in glycosylation from hCG{alpha} dissociated from pregnancy-derived hCG. Western blot analysis was conducted under reducing conditions of hCG{alpha} (lane 1), RP-HPLC purified GPH{alpha} from supernatant of HEK293 cells stably transfected with β2AR and stimulated with isoproterenol (lane 2). Endoglycosidase H (Endo H)-treated hCG{alpha} (lane 3) and -purified GPH{alpha} (lane 4) or PNGase F-deglycosylated hCG{alpha} (lanes 5) and purified GPH{alpha} (lane 6) are shown. hCG{alpha} dissociated from pregnancy-derived hCG is smaller in size (lane 1), sensitive to partial endoglycosidase H digestion, resulting in monoglycosyl hCG{alpha} (Mr app ~18 kDa) (lane 3) and fully deglycosylated by PNGase-F (lane 5). Deglycosylated large GPH{alpha} (lane 6) and hCG{alpha} (lane 5) are identical in size.

 
Signal specificity for the induction of GPH{alpha} expression
We performed a series of experiments directed to further investigate second-messenger regulation of GPH{alpha} expression in HEK293 cells. Steady-state mRNA levels and protein release were measured simultaneously in cells treated with a variety of cAMP raising agents or a calcium ionophore (summarized in Table 3Go). Expression and release of GPH{alpha} was strictly regulated by changes of intracellular cAMP concentrations (Fig. 6Go). In fact, agonist stimulation of the endogenous Gs-coupled adenosine A2B receptor (26), irreversible activation of Gs by cholera toxin modification, and direct activation of protein kinase A (PKA) by 2Br-cAMP were all capable of producing large induction of GPH{alpha} expression and release. The cAMP phosphodiesterase inhibitor IBMX also stimulated GPH{alpha} accumulation but to a smaller extent and only significantly in the β2AR-expressing clone. This may reflect tonic stimulation of cAMP signaling exerted by the overexpressed receptor in the transfected line. The calcium ionophore A-23187 produced a marked stimulation of GPH{alpha} accumulation (Fig. 6Go), indicating the involvement of Ca2+ signaling in the control of GPH{alpha} expression.


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  		TABLE 3. Receptor cDNAs for transfection and stimulatory/inhibitory agents used in this study

 

Figure 6
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  		FIG. 6. Dual second-messenger control of GPH{alpha} transcription and release in HEK293 cells. Effects of various cAMP stimulators and of the Ca2+ ionophore A23187 on GPH{alpha} transcription (A), and release (B), in parental and receptor-expressing HEK293 cells incubated for 24 h in the absence (Ctr; control) or presence of the indicated agents used at the following concentrations: 100 µM IBMX; 1 µM isoproterenol (Iso); 1 µM Neca; 0.5 mM 8-Br-cAMP; 10 ng/ml cholera toxin (CTX); 100 µM Rolipram (Rol); 1 µM forskolin (Frk); and 10 µM A23187. GPH{alpha} and S26 mRNA levels are revealed by RT-PCR. GPH{alpha} protein concentrations in the culture media are measured by IFMA (note: y-axes of the plots are log scaled). Data were averaged from three experiments.

 
To further investigate the dual signaling control of GPH{alpha} transcription, we used a strategy based on receptor-mediated activation of the endogenous G proteins in HEK293 cells. A number of diverse GPCRs capable of interacting with Gs, Gq, or both or Gi (Table 3Go) were transfected in HEK293 and stimulated via their specific agonists to evaluate the relative role of the signaling pathways triggered by the two G proteins in the control of GPH{alpha} expression. In addition to Gs-coupled receptors, GPCRs that primarily interact with Gq (such as the muscarinic M3, the histamine H1 receptors) or both G protein types (neurokinin-1 receptors), also strongly induced GPH{alpha} expression (Fig. 7Go). Only the Gi/o-coupled µ-opioid receptor (MOP), which neither enhances cAMP levels nor phosphatidyl inositols turnover in HEK293 cells (Fig. 7BGo), was unable to up-regulate GPH{alpha} mRNA. Thus, both Gs and Gq, through cAMP and Ca2+, respectively, regulate the expression of the common gonadotropin {alpha}-subunit in HEK293 cells.


Figure 7
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  		FIG. 7. Effect of transfected GPCRs displaying different G protein specificity on the transcription of GPH{alpha} in HEK293 cells. A, HEK293 cells plated in 24 wells were transfected with the following receptors: muscarinic M3 (M3), histamine H1 (H1) and H2 (H2), β2AR, neurokinin 1 (NK1), MOP, or nontransfected (A2B). PI turnover and cAMP levels were measured as described in Materials and Methods. The following agonists were used to stimulate the corresponding receptor: 100 µM carbachol (Carb) for M3; 100 µM histamine (His) for H1 and H2; 1 µM isoproterenol (Iso) for β2AR; 1 µM substance P (SP) for NK1; 10 µM [D-Ala2, Gly5-ol]enkephalin (DG) for MOP; 1 µM NECA was added to nontransfected cells to activate the endogenous adenosine A2B receptor. Inositol-1 phosphate (IP1) accumulation computed as a fraction of total cellular [3H]inositol (21 ) is expressed as net stimulation by subtracting the values in the absence of agonists. Enhancement of cAMP is given as ratio of stimulated vs. basal levels (means ± SEM; three independent experiments). Data were averaged from three experiments B, Cell monolayers plated in 6-well flasks were transfected with noncoding plasmid (minus signs) or cDNAs coding for the indicated receptors (plus signs). On the next day, cells were treated for an additional 18 h with serum-free medium containing the corresponding agonists named on the top of the pictures (concentrations are given in the legend of A). GPH{alpha} and S26 mRNA levels were measured by RT-PCR. Note that a detectable GPH{alpha} transcript is also present in cells transfected with noncoding cDNA in the presence of Iso because HEK293 express low levels of endogenous β-adrenoceptors. Data are representative of two independent experiments.

 
Finally, we investigated whether the two signaling pathways that control GPH{alpha} expression in HEK 293 cells may exhibit functional antagonism or rather exert their effects in an additive/synergic fashion. To this end, we transfected HEK293 cells with the Gq-coupled H1-histamine receptor and examined the consequence of the concurrent stimulation of GPH{alpha} transcription via Ca2+ and cAMP signaling. As shown in Fig. 8Go, the transcription induced via Gs/cAMP by NECA acting at the endogenous adenosine A2B receptor was further enhanced by the H1 agonist histamine acting via Gq/Ca2+. Thus, there is a substantial degree of additivity between the two signaling pathways.


Figure 8
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  		FIG. 8. Additivity of cAMP- and Ca2+-mediated signaling in stimulating GPH{alpha} transcription. HEK wild-type cells, transiently transfected with histamine H1 receptor (H1R) cDNA and further cultured for 18 h in serum-free medium (as in Fig. 7BGo), were incubated for an additional 6 h in the absence (Bas) or presence of the adenosine agonist (NECA, 1 µM), histamine (His, 100 µM), and both (NECA + His). GPH{alpha} and S26 mRNA levels were measured by RT-PCR. The density of the mRNA bands (right-hand histogram) was quantified from a high-resolution digital scan of the gel film using the software Optiquant (PerkinElmer). Values are given as arbitrary density units after subtraction of the basal. The data are representative of two independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have shown in this study that HEK293 cells can express and release large amounts of the common {alpha}-chain for glycoprotein hormones in response to the activation of the cAMP and Ca2+ signaling pathways via stimulation of endogenous and transfected GPCRs other than classical GnRH receptors.

Purification of secreted GPH{alpha} from stimulated HEK293-β2AR cells followed by tryptic digestion and MS/MS identification of the peptide fragments showed that the sequence of the released protein unequivocally corresponded to GPH{alpha} with minor impurities present in the final preparation, mainly consisting of residual FCS proteins. Molecular cloning of the expressed mRNA confirmed the sequence of the entire GPH{alpha} precursor. It is worth noting that the HEK293 {alpha}-subunit does not carry the single amino acid substitution (A56E) previously described in a GPH{alpha} ectopically secreted by a human carcinoma. This mutation was deemed responsible for the abnormal glycosylation and the consequent high molecular size of the protein (27).

Earlier work suggested that uncombined GPH{alpha} contains an additional oligosaccharide O linked to T39 (T43 in bovine GPH{alpha}), which might be responsible for the shift in molecular weight and the poor ability to dimerize with free β-subunits (28, 29). However, subsequent studies reported little or no extent of O-glycosylation in several free {alpha}-producing systems (30, 31, 32) and show that the N-linked oligosaccharides are primarily responsible for the increase in molecular size of the free {alpha}-subunit and its reduced ability to form {alpha}β-heterodimers (33, 34). Thus, it is conceivable that the GPH{alpha} secreted by HEK293 cells is similar the N-hyperglycosylated form that is frequently produced in many eu- and ectopic sources (30, 31, 32, 33, 35, 36, 37, 38).

Western blot analysis indicated that the apparent molecular weight of the released protein is larger than that of the first WHO reference preparation of hCG{alpha}, which was dissociated from pregnancy hCG (39). Large uncombined GPH{alpha} has been described to be resistant to the digestion by endoglycosidase H (40), which cleaves the chitobiose core of hybrid and high mannose N-linked carbohydrate antennae. Purified HEK293 protein is resistant to deglycosylation with endoglycosidase H, but after PNGaseF digestion, its apparent molecular weight is identical with dissociated hCG{alpha}.

Thus, by gene array screening, RT-PCR, molecular cloning, nanospray MS analysis, SDS-PAGE molecular size, and sensitivity to deglycosylation, we unambiguously show that the major molecule induced by intracellular messengers in HEK293 cells is a genuine large uncombined form of GPH{alpha} that is heavily glycosylated.

The ectopic overproduction of GPH{alpha} in a transformed cell line as we observed in HEK293 is not a new finding. Early reports showed that bronchogenic tumor cell lines can produce unbalanced amounts of hCG subunits, with some clones expressing virtually only uncombined hCG{alpha} (11). Later, selected clones of HeLa cells (a continuous line derived from a cervical carcinoma) were found to prevalently express {alpha}-subunits in cell culture (12, 41).

However, the second-messenger control of GPH{alpha} transcription and release in neoplastic cells has not been investigated in detail. Modest increases of hCG subunits transcription induced by PKA and protein kinase C (PKC) activators were described in choriocarcinoma cells (42), and more recently release of free CG{alpha} subunit in response to PKA and PKC activation was reported in a hepatoma cell line (43). In HeLa cell lines variable levels of constitutive {alpha}-subunit expression were reported, but in most cases the expression could be enhanced only by treatment with nonspecific activators of gene expression, such as sodium butyrate or inhibitors of DNA synthesis (44, 45). As shown here in a side-to-side comparison, GPH{alpha} transcription in HeLa cells is constitutive and cannot be regulated by cAMP signaling.

Thus, unlike other transformed cell lines, the peculiar feature of HEK293 cells GPH{alpha} expression is the tight regulation in response to physiological signals. The expression was very low or undetectable under basal conditions but rapidly induced by stimulation of cAMP and Ca2+ signaling pathways, leading to the accumulation of microgram per milliliter concentrations of GPH{alpha} in the serum-free medium of the treated cells.

To investigate the signal specificity of GPH{alpha} induction in HEK293 cells, we used two different approaches. In the first, we used a panel of stimulators or inhibitors involved in cAMP signaling and a calcium ionophore. The results clearly indicated that GPH{alpha} mRNA expression and protein secretion of GPH{alpha} can be stimulated by intracellular elevation of cAMP and Ca2+ ions.

It is known that the GnRH receptor, the major physiological regulator of gonadotropin expression and release, can interact in various cell types with at least three different types of G proteins, Gq/11, Gs, and Gi (46, 47, 48). Therefore, in the second approach, we used a number of GPCRs that show a more restricted G protein specificity than the GnRH receptor as transfectable tools to investigate the G protein subtypes capable of mediating transcriptional control of GPH{alpha} in HEK293 cells. Our data clearly show that both Gq/11 and Gs, but not Gi, can regulate the expression of large GPH{alpha}.

This dual Ca2+ and cAMP responsiveness of GPH{alpha} expression and release in HEK293 is in accord with the mechanisms of gene regulation that occur in cells in which GPH{alpha} is physiologically secreted, such as pituitary and placental cells. Distinct cAMP and PKC response sequences have been identified in the promoter region of the human GPH{alpha} gene and explain the dual control by these two signal transduction pathways (49).

Consistent with that previous work on promoter regulation, we found that there is a significant level of additivity between Ca2+- and cAMP-induced transcription, which agrees with the observation that promoter activity was enhanced in synergistic fashion by Ca2+ and cAMP stimuli (42, 49). This further confirms the physiological nature of the regulation of GPH{alpha} expression in HEK293 cells and indicates that this cell line may provide a better model for investigations on the mechanism of signal regulated secretion of free glycoprotein hormone {alpha}-subunit in an ectopic system.

APUD cells of several organs secrete uncombined GPH{alpha}. Neuroendocrine cells of the prostate might be responsible for the highly concentrated levels of large free GPH{alpha} found in the seminal plasma (8), but very little is known about the signaling mechanisms that control secretion of this hyperglycosylated form of GPH{alpha} Therefore, HEK293 cells may constitute a useful experimental model for unraveling the contribution of different signaling pathways to the transcriptional and secretory regulation of this protein in such tissues.

Taking advantage of the monoclonal line expressing large amounts of recombinant β2AR, we also performed a series of experiments on cAMP-mediated induction of GPH{alpha} production. They showed a close relationship between enhancement of cAMP levels and extent of {alpha}-subunit induction, particularly when comparing the effect of isoproterenol in the receptor-expressing clone and the parental line. In wild-type HEK293 cells with very low levels of endogenous β-adrenoceptors, the enhancement of cAMP levels induced by isoproterenol is barely detectable (1.5- to 2-fold). In contrast, in the β2AR-transfected clone isoproterenol produces a 500- to 1000-fold enhancement of intracellular cAMP. The release of GPH{alpha} at 24 h induced by isoproterenol in the two cell lines was consistent with such differences in second-messenger responses but also appeared to magnify minor changes in intracellular nucleotide concentrations because the stimulation of {alpha}-subunit release via endogenous β-AR in the nontransfected cells was significant (10-fold). There was also a 3-fold difference in basal GPH{alpha} release between the receptor clone and the parental line, indicating that such readout is capable of detecting small changes in steady-state basal nucleotide levels caused by the constitutive signaling activity of the overexpressed receptors. GPH{alpha} release in HEK293 may thus be used as a highly sensitive assay for cAMP-mediated signaling, particularly when combined with the ultrasensitive TR-IFMA procedure used here. Such an assay might also be one of the most sensitive available to date for the detection of mutational-induced changes in the constitutive activity of Gs-coupled GPCRs.

Despite the agreement between cAMP mobilization and GPH{alpha} release, the EC50 for isoproterenol-mediated enhancement of GPH{alpha} production was approximately 2 orders of magnitude higher than the EC50 for stimulation of intracellular cAMP. However, such discrepancy disappeared when release was expressed as change of ratios between extracellular and intracellular amounts of GPH{alpha}. This indicates that the initial agonist-induced elevation of cAMP levels is more directly linked to the shift between intra- and extracellular pools of GPH{alpha}, rather than to the overall accumulation of protein over a 24-h period.

It may be speculated that the discrepancy in agonist EC50 between second-messenger response and GPH{alpha} synthesis might be related to the biphasic kinetics of expression noted in this study. GPH{alpha} mRNA increased rapidly within the first hour after agonist addition and then more slowly during the remaining 24 h. GPH{alpha} production displayed a similar biphasic course, although delayed in onset. We suspect that only the rapid phase may be directly triggered by the initial receptor-induced elevation of cAMP, whereas the slower and more sustained phase of accumulation might be the result of secondary changes that are independent of receptor activation. If so, the discrepancy in concentration-response relationships between signaling and release may be explained, particularly if release is measured as protein accumulation at later end points when the slower phase is predominant.

In conclusion, we have shown that HEK293 cells express and secrete large amounts of a hyperglycosylated form of human GPH{alpha} on stimulation of endogenous and transfected β2AR and other GPCRs in a manner that is stringently regulated at the transcriptional level by the cAMP and calcium signaling pathways. GPH{alpha} expression in this readily available and highly transfectable cell line can be an experimental model for the investigation of glycoprotein hormone biosynthesis and regulation and could also be exploited as an endogenous reporter gene assay for studies on orphan GPCRs activity.

Moreover, the finding that several receptors coupled with either Gs and Gq, but not Gi proteins, can specifically control the transcription and secretion of large GPH{alpha} is important. It suggests that yet-to-be-identified Gq- and Gs-coupled GPCR types potentially expressed in APUD cells or ectopically producing tumors might have a role in orchestrating the control of nonpituitary, nonplacental regulation of free GPH{alpha}, both under physiological and pathophysiological conditions.


   Acknowledgments
 
We thank Dr. W. Merz (Biochemistry Center, Heidelberg University, Heidelberg, Germany) for critical reading of the manuscript and Regine Gerth for performing the TR-IFMAs. Dr. M. Crescenzi (Environment and Primary Prevention, Istituto Superiore di Sanità, Rome, Italy) provided the A341 cell line. The International Federation of Clinical Chemistry Working Group on hCG kindly provided the frozen carrier-free concentrate of starting material (FC 862) of the WHO first IRP hCG{alpha} 99/720.


   Footnotes
 
First Published Online December 13, 2007

Abbreviations: β2AR, β2-Adrenergic receptor; 8-Br-cAMP, 8-bromoadenosine-cAMP; FC, carrier-free concentrate; FCS, fetal calf serum; GPCR, G protein-coupled receptor; GPH{alpha}, {alpha}-subunit of human glycoprotein hormone; hCG, human chorionic gonadotropin; IBMX, isobutyl-methyl-xanthine; IFMA, immunofluorometric assay; mAb, monoclonal antibody; MOP, opioid type-µ; MS, mass spectrometric; PKA, protein kinase A; PKC, protein kinase C; RP-HPLC, reversed phase-HPLC; TR-IFMA, time-resolved IFMA.

Disclosure Statement: The authors of this paper have nothing to declare.

Received June 11, 2007.

Accepted for publication November 27, 2007.


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