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Endocrinology Vol. 143, No. 3 1026-1035
Copyright © 2002 by The Endocrine Society

REPRODUCTION-DEVELOPMENT

MA-10 Cells Transfected with the Human Lutropin/Choriogonadotropin Receptor (hLHR): A Novel Experimental Paradigm to Study the Functional Properties of the hLHR

Takashi Hirakawa, Colette Galet and Mario Ascoli

Department of Pharmacology, The University of Iowa, Iowa City, Iowa 52242-1109

Address all correspondence and requests for reprints to: Dr. Mario Ascoli, Department of Pharmacology, 2-319B BSB, 51 Newton Road, The University of Iowa, Iowa City, Iowa 52242-1109. E-mail: . mario-ascoli{at}uiowa.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
MA-10 cells are a clonal strain of mouse Leydig tumor cells that retain many of the properties of Leydig cells including expression of the endogenous lutropin/choriogonadotropin receptor (LHR) and the ability to respond to LH/CG with increased steroidogenesis. Recently we noted a dramatic decrease in expression of the endogenous LHR. Although we do not have an explanation for this decline, we took advantage of it to devise a method that allows for the expression of the recombinant human LHR (hLHR) in a Leydig cell model that is now practically devoid of endogenous LHR. We show that the recombinant hLHR can be expressed at variable densities in MA-10 cells and that it can stimulate cAMP and steroid synthesis as well as activate the inositol phosphate and MAPK cascades. We also show that two naturally occurring mutants of the hLHR associated with Leydig cell hyperplasia and one mutant associated with Leydig cell adenomas are constitutively active when assayed for activation of cAMP, inositol phosphate, progesterone, and MAPK.

Our ability to express the hLHR in MA-10 cells (now practically devoid of endogenous LHR) provides a novel paradigm to study the cellular and molecular basis of the functions of the LHR in Leydig cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
DURING THE PAST 10 yr or so, many studies have been done in which the functional properties of mutants (either laboratory designed or naturally occurring) of the lutropin/choriogonadotropin receptor (LHR) are examined following expression of the recombinant products in heterologous cell types such as 293 or COS7 (reviewed in Refs. 1 and 2). The use of heterologous cell lines in these studies offers at least three major advantages. First, they can be readily transfected using inexpensive methods such as calcium phosphate coprecipitations. Second, they can express large amounts of the transfected LHR, thus providing a convenient source of material for biochemical studies. Third, they provide an appropriate null background in which to study the properties of the wild-type and mutated receptors. The latter is also the major disadvantage of using heterologous cell lines, however. Because they normally do not express the endogenous LHR or the differentiated function of cells that normally express the LHR, any studies on the functional properties of the LHR expressed in these cell lines are limited and subject to the criticism that they were performed in a cellular context that is not physiologically relevant. The validity of such criticisms is bound to be highly dependent on the signaling network in question because many signaling networks are ubiquitous and highly conserved, whereas others are cell specific (reviewed in Refs. 3, 4, 5). Ultimately then, the analysis of the signaling networks activated by the LHR and the functional properties of mutants of the LHR would benefit from experimental paradigms that would allow for the expression of the recombinant LHR either in ovarian and/or testicular target cells.

About 20 yr ago, we published a report (6) on the establishment of a mouse Leydig tumor cell line (designated MA-10) that retained many of the properties of normal Leydig cells, including the endogenous expression of a functional LHR that can translate the binding of LH/CG into an increase in cAMP and steroid biosynthesis. In spite of their loss of 17{alpha}-hydroxylase/C17–20 lyase (and resulting shift in androgen to progestin production), MA-10 cells have gained wide acceptance as an appropriate model system to study the actions of LH/CG on steroidogenesis and other aspects of the differentiated functions of Leydig cells (reviewed in Ref. 7). During the last 2 yr or so we noticed that the density of endogenous LHR expressed in MA-10 cells (as measured by 125I-hCG binding) decreased to a point where it became barely detectable. Although we do not have a conclusive explanation for this change (see Discussion), we looked at it pragmatically and ultimately took advantage of it to devise a method in which MA-10 cells (now practically devoid of endogenous LHR) can be used as a suitable host to express the human LHR (hLHR) (or mutants thereof) at variable densities. As illustrated herein, this novel paradigm has allowed us, for the first time, to compare the activation of different signaling pathways and of steroidogenesis by the hLHR-wild-type (wt) and several of its naturally occurring constitutively active mutants in a more relevant cellular context (i.e. the Leydig cell).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Plasmids and cells
The preparation of expression vectors (all in pcDNA3.1) encoding for the hLHR-wt (and mutants thereof) modified with the myc-epitope at the N terminus have been described (8). These inserts were excised from pcDNA3.1 and subcloned into the EcoRV site of the pEF1/V5-His vector (Invitrogen, Carlsbad, CA). The use of this restriction site introduced the stop codon of the hLHR cDNA upstream of the V5 and His epitopes present in the PEF1/V5-His vector. This manipulation thus prevented the addition of these two epitopes to the C terminus of the translated hLHR product.

The origin and handling of MA-10 cells has been described (6). Cells were maintained in Waymouths MB752/1 modified to contain 1.1 g/liter of NaHCO3, 20 mM HEPES, 50 µg/ml of gentamicin, and 15% horse serum (pH 7.4) (growth medium) using plasticware that was coated with gelatin. Gelatin coating was accomplished by incubating all wells or flasks with a 0.1% solution of gelatin (prepared in calcium- and magnesium-free PBS) for 45 min at room temperature. This solution was aspirated before seeding the cells. When needed for transfections cells were plated on 35-mm wells at a density of 4–7 x 105/well and transfected 1 d later using up to 2 µg of plasmid and 12 µl of Lipofectamine (Life Technologies, Inc., Gaithersburg, MD) in a total volume of 1 ml of serum-free OPTI-MEM medium (Life Technologies, Inc.), according to the instructions supplied by the manufacturer. After 3 h at 37 C, each well received 150 µl of horse serum and 1 ml of growth medium and the incubation was continued overnight at 37 C. The medium was then replaced with 3 ml of growth medium and the cells were incubated at 30 C for 48 h before use in any of the assays described below.

293T cells were maintained in DMEM containing 10 mM HEPES, 10% newborn calf serum, and 50 µg/ml gentamicin (pH 7.4). Transient transfections were done using the calcium phosphate method of Chen and Okayama (9). After an overnight incubation with the transfection mixture the cells were washed, placed back in medium, and incubated at 30 C for 48 h before use.

Binding assays
Cells were washed and placed in 1 ml of assay medium A (Waymouths MB752/1 without NaHCO3 but containing, 20 mM HEPES, 50 µg/ml of gentamicin, and 1 mg/ml BSA, pH 7.4). After cooling they were incubated with seven different concentrations of 125I-hCG (3 x 10-11 to 3 x 10-8 M) overnight at 4 C. At the end of the overnight incubation, the cells were scrapped from the wells and they were collected and washed twice (using HBSS supplemented with 1 mg/ml BSA) by centrifugation. Cell pellets were counted directly in a {gamma}-counter. Three wells were used for each concentration of 125I-hCG. Two of them received 125I-hCG only but the third one also received 50 IU/ml crude hCG and was used to correct for nonspecific binding. The apparent Kd and maximal binding capacity were calculated by nonlinear regression of the binding isotherms using Prism (GraphPad Software, Inc.).

Second messenger and progesterone assays
Cells were washed and placed in 1 ml of warm Assay Medium B (Waymouths MB752/1 containing 20 mM HEPES; 50 µg/ml of gentamicin; and 1 mg/ml BSA, pH 7.4) supplemented with 0.5 mM isobutylmethylxanthine. After a 15-min preincubation (at 37 C), duplicate wells were incubated with seven different concentrations of hCG (3 x 10-13 to 3 x 10-8 M) for an additional 30 min at 37 C. Total cAMP (i.e. cells + medium) was extracted and measured by RIA as described elsewhere (10, 11, 12, 13). For the inositol phosphate assays, the cells were placed in medium containing 2 µCi/ml of [2-3H]myo-inositol (NEN Life Science Products, Boston, MA) during the last 24 h of the 30-C posttransfection incubation (see above). Before the assay the cells were washed and placed in 1 ml of warm Assay Medium B containing 20 mM LiCl. After a 15-min preincubation (at 37 C) duplicate wells were incubated with seven different concentrations of hCG (3 x 10-12 to 3 x 10-8 M) for an additional 30 min at 37 C. The medium was then aspirated and the total inositol phosphates present in the cells were extracted and quantitated as described before (13). For the progesterone assays, the transfected cells were washed and placed in 1 ml of warm Assay Medium B. After a 15-min preincubation (at 37 C) duplicate wells were incubated with 7 different concentrations of hCG (3 x 10-13 to 3 x 10-9 M) an additional 4 h at 37 C, and the medium was collected. Steroids were extracted from small aliquots (100 µl) of medium with 1 ml of diethyl ether. The ether extracts were evaporated and the extracts were redissolved and assayed for progesterone using an enzyme linked immunoassay kit purchased from Cayman Chemicals according to their instructions.

The EC50s and maximal responses were calculated from the dose response curves analyzed using Prism (GraphPad Software, Inc.).

MAPK assays
The cells were maintained in medium B for the last 24 h of the 30-C posttransfection incubation (see above). At the end of this incubation duplicate wells were incubated with 7 different concentrations of hCG (3 x 10-12 to 3 x 10-8 M) for an additional 30 min at 37 C. The cells were then placed on ice, the medium was aspirated quickly, the cells were washed once with a cold buffer containing 0.15 M NaCl and 20 mM HEPES (pH 7.4) and lysed with 120–200 µl of lysis buffer (1% NP40; 0.25% sodium deoxycholate; 150 mM NaCl; 1 mM EDTA; 1 mM Na3VO4; 1 mM NaF, 50 mM Tris-Cl, pH 7.4) by gentle rocking for 30 min at 4 C. The cell lysates were clarified by centrifugation assayed for protein content using the BCA protein assay kit from Bio-Rad Laboratories, Inc. diluted 5-fold with 5x concentrated SDS gel sample buffer with reducing agents and boiled for 5 min. Aliquots of the lysates containing identical amounts of protein (~180 µg) were resolved on 12% SDS-PAGE gels and transferred electrophoretically to polyvinylidenedifluoride membranes (14, 15). Phosphorylated ERK-1/2 and total ERK-1/2 were visualized in the blots during an overnight incubation with a phospho-ERK-1/2 antibody [E-4 from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) used at a 1:500 dilution] or a total ERK-1/2 antibody (C-14 from Santa Cruz Biotechnology, Inc. used at a 1:1000 dilution) followed by a 1 h incubation with a secondary antibody covalently coupled to horseradish peroxidase [from Bio-Rad Laboratories, Inc. (Hercules, CA) used at a 1:5000 dilution]. All immune complexes were ultimately visualized and quantitated using the SuperSignal West Femto Maximum Sensitivity system of detection from Pierce Chemical Co. (Madison, WI) and a Kodak (Rochester, NY) digital imaging system. This image capture system is set up to alert us when image saturation occurs and to prevent us from measuring the intensity of such images.

Microscopy
Cells were plated and transfected in two-chamber cover slip culture vessels coated with polylysine (BioCoat from Becton Dickinson and Co., Bedford, MA) using the methods described above. After removing the medium the cells were washed twice with PBS (137 mM NaCl, 2.7 mM KCl, 1.4 mM NaH2PO4, 4.3 mM Na2HPO4, pH 7.4) and fixed during a 30 min incubation at room temperature with 4% paraformaldehyde (dissolved in PBS). The fixed cells were washed twice again and then incubated for 1 h at room temperature with PBS containing 50 mg/ml BSA. This solution was removed and the cells were incubated for another hour at room temperature with a 2 µg/ml solution of a fluorescein-conjugated monoclonal antibody (9E10) to the myc epitope (sc-40 from Santa Cruz Biotechnology, Inc.) dissolved in PBS containing 5 mg/ml BSA. After washing twice with PBS the nuclei were counterstained during a 30-sec incubation with 0.5 µg/ml solution of DAPI (4,6- diamindino-2-phenylindole) at room temperature. The cells were washed again twice with PBS, dried and mounted in Vectashield mounting medium (Vector Laboratories, Inc.) for fluorescence microscopy.

Other methods
The methods used to prepare extracts of the transfected cells for visualization of the myc-tagged hLHR in Western blots have been described (16). Statistical analyses were performed using InStat (GraphPad Software, Inc.).

Hormones and supplies
Purified hCG (CR-127, ~13,000 IU/mg) was kindly provided by the Dr. A. Parlow and the National Hormone and Pituitary Agency of the National Institute of Diabetes and Digestive and Kidney Diseases and purified recombinant hCG 1 was provided by Ares Serono (Randolph, MA). 125I-hCG was prepared as described elsewhere (17). Partially purified hCG (~3,000 IU/mg) was purchased from Sigma (St. Louis, MO), and it was used only for the determination of nonspecific binding (see above). Microbiological grade gelatin was purchased from Difco. 125I-cAMP and cell culture medium were obtained from the Iodination Core and the Media and Cell Production Core, respectively, of the Diabetes and Endocrinology Research Center of the University of Iowa. Other cell culture supplies and reagents were obtained from Corning, Inc. and Life Technologies, Inc., respectively. All other chemicals were obtained from commonly used suppliers.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Our most recent paper using MA-10 cells was published in 1999 (18) and at the time those experiments were being conducted their maximal binding capacity was 5,000–20,000 molecules/cell and the nonspecific binding of 125I-hCG was less than 10% of the total binding. Throughout the course of the experiments reported here, however, the nonspecific binding of 125I-hCG to MA-10 cells has been as high as 80% of the total binding and their maximal binding capacity has decreased to 600–1,000 receptors/cell (see below).

Taking advantage of the low density of endogenous LHR that is currently being expressed in MA-10 cells, we tested a number of conditions for expression of the recombinant hLHR-wt in this cell type. Using 125I-hCG binding to intact cells as a readout, we found that optimal expression of the hLHR-wt was attained by using an expression vector driven by the elongation factor 1{alpha}-subunit promoter instead of the more commonly used cytomegalovirus promoter in combination with the lipofectamine method of transfection (see Materials and Methods). Maintaining the transfected cells at 30 C (instead of 37 C) for 2 d after the transfection also enhanced the cell surface expression of the hLHR as reported earlier for the rLHR expressed in 293 cells (19). Using these conditions about 24% of MA-10 cells expressed the transfected hLHR, a value that compares rather well with the 26% transfection efficiency attained in 293T cells transfected with the same vector but using calcium phosphate as the method of transfection (Fig. 1AGo). Western blots of transfected 293 and MA-10 cells are shown in Fig. 1BGo, and they reveal the presence of a predominant 85- to 95-kDa form of the hLHR, a less prominent 65- to 75-kDa band and several aggregates of at least 165 kDa. These forms of the hLHR have been identified in transfected 293 cells as the mature cell surface hLHR, an immature intracellular precursor and aggregates of the precursor, respectively (reviewed in Ref. 2). These Western blots also show that the total amount of receptor expressed is lower in MA-10 than in 293T cells. Quantitation of receptors by 125I-hCG saturation analysis to intact cells showed that the density of cell surface receptors in transfected MA-10 cells is roughly 70% lower than in transfected 293T cells (Fig. 1CGo). The apparent Kd for 125I-hCG binding to intact cells is comparable in both cell types (Fig. 1CGo), however, and similar to that previously reported by us in hLHR-transfected 293 cells (20, 21, 22).



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  		Figure 1. Expression of the hLHR-wt in transiently transfected MA-10 and 293T cells. MA-10 and 293T cells were transiently transfected with the hLHR-wt (2 µg plasmid/35-mm well) as described in Materials and Methods, and the expression of the receptor was ascertained by fluorescent microscopy, Western blotting, or 125I-hCG binding. The myc-tagged receptor was visualized in intact transfected cells using a fluorescein-coupled monoclonal antibody to the myc epitope (9E10). The nuclei were counterstained with DAPI (see Materials and Methods) and the percentage (±SEM) of cells transfected was calculated by visual inspection of at least 10 fields of approximately 100 cells each in two independent transfections. The myc-tagged receptor was also visualized in Western blots of cell lysates using the 9E10 monoclonal antibody and the equilibrium binding parameters of 125I-hCG to the transfected cells were measured using intact cells incubated at 4 C as described in Materials and Methods.

 
Although the density of endogenous LHR currently being expressed by MA-10 cells is much lower than that reported previously, the endogenous receptors are still functional. In agreement with the data of many investigators (reviewed in Ref. 7), the results summarized in Table 1Go show that the endogenous LHR in MA-10 cells can translate the binding of hCG into an increase in cAMP and progesterone accumulation but cannot translate the binding of hCG into an increase in inositol phosphate accumulation. Because many G protein-coupled receptors (GPCRs) have now been shown to activate the MAPK cascade (23, 24) we also tested the ability of hCG to provoke this response in MA-10 cells. As shown in Table 1Go we found that hCG provokes a 2- to 3-fold increase in the levels of phosphorylated ERK-1/2 in these cells.


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  		Table 1. Human CG-induced responses in MA-10 cells transiently transfected with an empty vector or a vector encoding the hLHR-wt

 
Table 1Go also shows a comparison of the basal and maximal hCG-induced responses detected in MA-10 cells transfected with an empty vector or with the hLHR-wt. These results clearly show that expression of the hLHR-wt enhances the hCG-induced cAMP, progesterone, and MAPK responses of MA-10 cells. It is also interesting to note that the levels cAMP and progesterone measured in hLHR-transfected MA-10 cells incubated without hCG are actually higher than those attained in empty vector-transfected MA-10 cells incubated with a maximally effective concentration of hCG. The same is not true for the MAPK response, however. Lastly, expression of the hLHR-wt also increased the basal levels of inositol phosphates in MA-10 cells and, perhaps more importantly, bestowed onto them an ability to respond to hCG with a robust increase in inositol phosphate accumulation. The increased basal levels of cAMP, progesterone and MAPK activity detected in MA-10 cells transfected with the hLHR-wt and incubated without hCG are to be expected based on the revised ternary model for GPCR activation (see Refs. 2 , 25 , 26).

Representative dose-response curves for the different hCG-induced responses measured in hLHR-wt-transfected MA-10 cells are shown in Figs. 2Go and 3Go and a summary of the results obtained in several experiments is shown in Table 2Go. The sensitivity (i.e. EC50) of these responses to hCG is variable, but the various EC50s are comparable to the EC50s of the same responses previously reported in MA-10 cells (when they were expressing a higher density of endogenous LHR) or in 293 cells transfected with the hLHR and expressing a receptor density that is similar or somewhat higher than that of the transfected MA-10 cells. For example, the EC50 for the cAMP response in hLHR-transfected MA-10 cells is comparable to that previously reported by us in 293 cells expressing the hLHR-wt (0.1 nM, see Ref. 21). For obvious reasons the EC50 for the hCG-induced progesterone response in hLHR-transfected MA-10 cells cannot be compared with hLHR-transfected 293 cells. The EC50s for the hCG-induced cAMP and progesterone accumulation in hLHR-transfected MA-10 cells are similar to those previously measured using similar conditions in MA-10 cells when they expressed a higher density of endogenous LHR (0.2 and 0.04 nM, respectively, see Ref. 27). The robust inositol phosphate response to hCG induced by expression of the hLHR in MA-10 cells displays the highest EC50 of all responses, but this EC50 is approximately 5-fold lower than the EC50 obtained in 293T cells expressing a comparable or higher density of the hLHR-wt (i.e. ~3 nM, data not shown). The hCG-induced MAPK response, which is also greatly augmented by expression of the hLHR-wt was characterized by an EC50 that is comparable to that of the inositol phosphate response. Lastly, MA-10 cells transfected with the hLHR-wt display a lower EC50 for the hCG-induced steroid response relative to that of the hCG-induced cAMP response (Table 2Go) This is characteristic of the steroidogenic response of normal rat Leydig cells (28) and untransfected MA-10 cells (27).



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  		Figure 2. Dose-response curves for four hCG-induced responses in MA-10 cells transiently transfected with the hLHR-wt. MA-10 cells were transiently transfected with hLHR-wt (2 µg of plasmid/35-mm well) and incubated with buffer only or with the indicated concentrations of hCG. cAMP (A), progesterone (B), inositol phosphates (C), or phospho-ERK-1/2 (D) were measured as described in Materials and Methods. The results of a representative experiment for each response are shown. Each point is the average of duplicate determinations. No error bars are shown because they are small and obscured by the symbols.

 


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  		Figure 3. hCG-induced phosphorylation of ERK-1/2 in MA-10 cells expressing the hLHR-wt. MA-10 cells were transiently transfected with hLHR-wt (2 µg of plasmid/35-mm well) and incubated with buffer only or with the indicated concentrations of hCG for 30 min. Electrophoretic blots of cell lysates resolved on SDS gels were incubated with an antibody to total ERK-1/2 (top panel) or phospho-ERK-1/2 (bottom panel) followed by a secondary antibody covalently labeled with horseradish peroxidase. All immune complexes were ultimately visualized and quantitated using the SuperSignal West Femto Maximum Sensitivity system of detection from Pierce Chemical Co. and a Kodak digital imaging system as described in Materials and Methods. Only the relevant portions of a representative blot are shown.

 

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  		Table 2. Human CG-induced responses in MA-10 cells transiently transfected with the hLHR-wt

 
The functional properties of the hLHR-wt were next measured in MA-10 cells transfected with increasing amounts of the hLHR-wt expression vector chosen to vary the levels of receptor expression (as measured by 125I-hCG binding) from approximately 600–125,000 receptors/cell (Fig. 4Go). As shown in Fig. 5Go the magnitude of all hCG-induced responses increased as a function of the amount of receptor expressed but there are some interesting differences among the four responses measured. The hCG-induced cAMP response (Fig. 5AGo) showed a clear biphasic dependence on receptor density with a steep slope as the receptor density increased from approximately 600–18,000 receptors/cell and a shallower slope as the density increased from approximately 18,000–126,000 receptors/cell. This response did not appear to saturate even at the highest density of receptor expressed (~126,000 receptors/cell). The progesterone response (Fig. 5BGo) increased in a linear fashion as the receptor density increased from approximately 600–90,000 receptors/cell and then it saturated as the receptor density increased to approximately 126,000 receptors/cell. The hCG-induced inositol phosphate response was also biphasic, but the shallow slope of this response occurred at low receptor densities and the steeper slope occurred at higher receptor densities (Fig. 5CGo). There was little or no change in the magnitude of the inositol phosphate response as the density of cell surface hLHR increased from approximately 600–8,000 receptors/cell. The magnitude of this response increased in a fairly linear fashion as the density of cell surface hLHR increased from approximately 8,000–90,000 receptors/cell, and then it saturated as the receptor density increased to approximately 126,000 receptors/cell. Lastly, the hCG-induced MAPK kinase response (Fig. 5DGo) increased in a linear fashion as the receptor density increased from approximately 600–65,000 receptors/cell. This response saturated (or declined slightly) as the receptor density increased from approximately 65,000–126,000 receptors/cell.



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  		Figure 4. 125I-hCG binding to MA-10 cells transiently transfected with different amounts of the hLHR-wt expression vector. MA-10 cells were transiently transfected with the indicated amounts of hLHR-wt plasmid. Their hCG binding capacity was determined during an overnight incubation with 30 nM 125I-hCG (±crude hCG to correct for nonspecific binding) as described in Materials and Methods. Each point is the mean of two to five independent transfections. When the results of two transfections are shown, the error bars extend to the range of the values obtained in the two transfections. When the results of three to five transfections are shown, the error bars represent the SEM.

 


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  		Figure 5. hCG-induced responses in MA-10 cells expressing increasing densities of the hLHR-wt. MA-10 cells were transiently transfected with the increasing amounts of the hLHR-wt plasmid as shown in Fig. 3Go and their hCG-responses were measured using saturating concentrations of hCG as described in the legend to Table 1Go and in Materials and Methods. The maximal hCG-induced responses are plotted against the maximal hCG binding capacity determined from the data shown in Fig. 3Go. Each point is the mean of two to five independent transfections. When the results of two transfections are shown, the error bars extend to the range of the values obtained in the two transfections. When the results of three to five transfections are shown, the error bars represent the SEM.

 
Having characterized this system, we next set up to examine the functional properties of selected hLHR mutants that have been found in boys with gonadotropin-independent precocious puberty associated with Leydig cell hyperplasia or Leydig cell adenomas (2, 29, 30). When expressed in heterologous cell types these mutants consistently induce an increase in basal cAMP levels, but data on their ability to activate other signaling pathways in an agonist-independent fashion are controversial (reviewed in Ref. 2). In the experiments described below, we expressed three of these mutants in MA-10 cells and compared their signaling properties with those of the hLHR-wt. Two of the mutants chosen, L457R and D578Y, are germ line mutations found in boys with precocious puberty and Leydig cell hyperplasia (31, 32, 33, 34), whereas the third one, D578H is a somatic mutation found in boys with precocious puberty and Leydig cell adenomas (35).

MA-10 cells were transfected with equivalent amounts of each of these plasmids chosen to give maximal and comparable levels of receptor expression at the cell surface as documented in Table 3Go. The different cellular responses characterized above were then measured in cells incubated without or with a maximally effective concentration of hCG as shown in Fig. 6Go. In agreement with the data obtained in heterologous cell types (8, 31, 32, 33, 34, 35, 36) MA-10 cells expressing the L457R, D578Y, and D578H mutants display a dramatically enhanced basal level of cAMP and are refractory to further hCG stimulation (Fig. 6AGo). Expression of the L457R, D578Y, and D578H mutants in MA-10 cells also resulted in elevated levels of basal progesterone synthesis that are comparable to those detected in cells transfected with the hLHR-wt and incubated with hCG (Fig. 6BGo). Cells expressing the mutants did not respond with an additional increase in progesterone synthesis when stimulated with hCG (Fig. 6BGo). The inositol phosphate response also followed the same pattern. MA-10 cells expressing the L457R, D578Y, and D578H mutants displayed an obvious elevation of basal inositol phosphate accumulation and these cells were refractory to further hCG stimulation (Fig. 6CGo). The increase in agonist-independent levels of inositol phosphates seemed higher in cells expressing the D578H mutant than in cells expressing either of the other two mutants, however (Fig. 6CGo). Lastly, expression of the L457R, D578Y, and D578H mutants in MA-10 cells resulted in a small but measurable increase in basal ERK-1/2 phosphorylation (2- to 3-fold over MA-10 cells expressing hLHR-wt incubated without hCG). In contrast to the other responses measured above, however, MA-10 cells expressing any of these mutants still responded to hCG with a further increase in ERK-1/2 phosphorylation (Fig. 6DGo).


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  		Table 3. 125I-hCG binding to MA-10 cells transiently transfected with the hLHR-wt and mutants thereof

 


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  		Figure 6. hCG-induced responses in MA-10 cells expressing the hLHR-wt or mutants thereof. MA-10 cells were transiently transfected with the hLHR-wt, L457R, D578Y, or D578H mutants (2 µg of plasmid/35-mm well) and incubated with buffer only or with a saturating concentration of hCG as described in the legend to Table 1Go and in Materials and Methods. cAMP (A), progesterone (B), inositol phosphates (C), or phospho-ERK-1/2 (D) were measured as described in Materials and Methods. Each bar shows the mean ± SEM of three independent transfections.

 
We conclude that the L457R, D578Y, and D578H mutants are constitutively active toward all responses measured.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The overwhelming majority of experiments currently being pursued on the structure and functions of the LHR are being done using heterologous cell lines expressing the recombinant LHR or mutants thereof. Although these experiments have provided important and useful information (reviewed in Refs. 1 and 2), there is an obvious need for experimental paradigms that allow for the expression of the recombinant hLHR in target cells. The experiments presented were designed to address this issue, and they accomplish three important goals. First, we describe a method that can be used to express the hLHR in a previously established and well characterized Leydig tumor cell line (MA-10 cells, see Ref. 6). Second, we characterize the signaling properties of the hLHR-wt expressed in MA-10 cells. Third, we use this novel paradigm to document the pleiotropic nature of the constitutive activity of several naturally occurring gain-of-function mutations of the hLHR in Leydig cells.

MA-10 cells are a clonal strain of mouse Leydig tumor cells that were adapted to culture in this laboratory over 20 yr ago (6) and retained many of the properties of its normal counterparts including the expression of the endogenous LHR and the ability to respond to LH/CG with an increase in steroid biosynthesis (reviewed in Ref. 7). In the past 2 yr or so, we noted that MA-10 cells changed from being firmly attached and having an epithelial-like morphology to being loosely attached with a more rounded morphology. Their ability to proliferate in culture also diminished and the density of cell surface LHR declined dramatically from 5,000–20,000 receptors/cell (reviewed in Ref. 7) to 600–1,000 receptors/cell (Fig. 4Go and Table 3Go). Despite the low density of endogenous LHR currently being expressed by MA-10 cells, these receptors remain functional as judged by the ability of these cells to respond to hCG with increased cAMP and progesterone accumulation (Table 1Go).

Although the reasons behind these phenotypic alterations are not known with certainty, we suspect that they are related to recent changes in the formulations used to manufacture and/or treat the surfaces of the plasticware used for cell culture because 1) the changes described above can be readily detected upon thawing MA-10 cells that were frozen in 1981 and maintained in liquid nitrogen since then; and 2) two of these phenotypic alterations (i.e. the change in cell morphology and proliferation capacity) can be readily reversed by coating the plasticware used to culture the cells with gelatin (see Materials and Methods). The decrease in LHR density cannot be reversed by this manipulation, however. The batch of horse serum used to culture MA-10 cells is also known to have a dramatic negative effect on LHR density in MA-10 cells but current batches of horse serum obtained from different vendors also failed to reverse the decline in LHR density. Because MA-10 cells cultured under current conditions can express the LHR when transfected with an expression vector driven by a strong heterologous promoter (Fig. 1Go), we speculate that the decrease in the expression of the endogenous LHR in MA-10 cells is being caused by contaminants (present in the preparations of gelatin used to coat the plasticware) or by endocrine disruptors (released from the cell culture plasticware) that induced a decrease the transcription of the endogenous LHR gene. For example, we know that some hormones such as epidermal growth factor, as well as second messenger analogs such as 8Br-cAMP and phorbol esters can decrease the density of endogenous LHR in MA-10 cells by decreasing the transcription of the LHR gene (37, 38) and that cell culture plasticware has steroid-like compounds (39, 40) that could have a similar effect. Lastly, it should be pointed out that MA-10 cells have been made available by us to many investigators throughout the world and it is not known whether the changes described above are restricted to our laboratory or if they have also been observed in other laboratories.

The availability of a Leydig cell line that retains many of the differentiated functions of its normal counterparts (but is practically devoid of endogenous LHR) provided us with a unique opportunity to willfully manipulate the expression of the LHR and mutants thereof in Leydig cells. This is an important step because it will allow us to investigate the signaling pathways activated by the LHR in the appropriate cellular context. For example, there is a growing body of evidence suggesting that GPCRs can affect the proliferation and differentiation of endocrine cells and that they may do so by using signaling mechanisms that are much more complex than previously recognized. For example, it is now generally accepted that most GPCRs can independently activate more than one subfamily of heterotrimeric G proteins (reviewed in Ref. 41); that heterotrimeric G proteins may in fact not be the only mediators of GPCR signaling (reviewed in Refs. 42 and 43) and that the agonist-stimulated trafficking of GPCRs among subcellular compartments may be an important event in GPCR signaling (reviewed in Refs. 44, 45, 46, 47). Whereas some of these events may be ubiquitous and highly conserved others may be cell specific, and thus an analysis of LHR-activated signaling networks, will ultimately have to be done in target cells.

Some of the results presented here already shed light on an important issue, the ability of the LHR to activate the inositol phosphate pathway. The ability of hCG to stimulate this signaling pathway has been clearly demonstrated in heterologous cell lines transfected with either the mouse (48, 49), the rat (50, 51), or the human LHR (35, 36, 52, 53, 54), but it could not be demonstrated in Leydig tumor cells by us (Table 1Go herein and Ref. 13) or others (55). Because the inositol phosphate response of MA-10 cells can be stimulated with arginine vasopressin (13) 2 and the hCG-induced inositol phosphate response of transfected cells requires high levels of receptors and high concentrations of hCG (see references cited above), we hypothesized that our inability to measure this response in MA-10 cells was due to their low density of endogenous LHR (2, 7). The data presented here supports that hypothesis. An hCG-induced inositol phosphate response is undetectable in untransfected MA-10 cells (expressing 600–1,000 receptors/cell) or in transfected MA-10 cells expressing approximately 8,000 receptors/cell (Table 1Go and Fig. 5CGo). This response becomes readily detectable in MA-10 cells expressing approximately 15,000 copies of the hLHR-wt and it continues to increase as the density of receptors increases up to approximately 90,000 receptors/cell (Fig. 5CGo). Clearly then, we can conclude that the density of LHR is the main determinant of this response and that our previous inability to detect an hCG-induced stimulation of the inositol phosphate response in MA-10 cells is due to the low density of endogenous LHR rather than to the absence of the appropriate signaling molecules. The hCG-induced activation of the inositol phosphate response in transfected heterologous cell lines is believed to be mediated by the Giß’2f{gamma}-mediated activation of a phospholipase Cß (49, 56). We now have the tools to determine if the same pathway is operative in MA-10 cells.

The importance of the LHR in the proliferation of Leydig cells has been highlighted recently by the phenotype of humans who harbor mutations of the LHR gene and by the phenotype of LHR null mice. Thus, some germ-line loss-of-function mutations of the LHR in humans result in Leydig cell hypoplasia, whereas all familial or sporadic gain-of-function mutations of the LHR result in Leydig cell hyperplasia (reviewed in Refs. 29 , 30). Likewise, targeted deletion of the LHR in mice results in Leydig cell hypoplasia (57, 58). Given these findings, it is not unreasonable to expect a role for the LHR in the proliferation and/or neoplastic transformation of Leydig cells. Such expectation has been recently fulfilled by the discovery of a somatic gain-of-function mutation of the LHR gene in several Leydig cell tumors (35). We show here that there are at least two GPCR-dependent signaling pathways that participate in cell proliferation (the inositol phosphate cascade and the phospho-ERK-1/2 cascade) that can be activated by the LHR and could be responsible for this effect. Thus, as an additional step in testing the applicability of this new experimental paradigm to the study of the hLHR, we compared the functional properties of two previously characterized gain-of-function mutants of the hLHR found in boys with Leydig cell hyperplasia (hLHR-L457R and hLHR-D578Y) with one gain-of-function somatic mutation of the hLHR (D578H) found in boys with Leydig cell tumors. This is an important issue for two reasons. First, because these hLHR mutants have not been previously expressed in Leydig cells their effects on steroidogenesis have not been evaluated. The data presented here thus represent the first demonstration that steroidogenesis is constitutively activated by each of these mutants (Fig. 6BGo). Second, it has been suggested (35) that hLHR mutations associated with Leydig cell tumors (such as D578H) may activate the inositol phosphate and the cAMP pathway constitutively, whereas mutations associated with Leydig cell hyperplasia (such as D578Y and L457R) may be constitutively active only toward the cAMP pathway but not the inositol phosphate pathway. As shown herein, such differences are not apparent in MA-10 cells. The L457R, D578Y, and D578H mutants were found to be constitutively active on cAMP and inositol phosphate accumulation as well as on the phosphorylation of ERK-1/2, another pathway that is involved in cell proliferation (Fig. 6Go). In fact, the data presented here (Fig. 6Go) show that the constitutive activity of these three mutants is pleiotropic in nature. They are constitutively active when measured for cAMP, progesterone, inositol phosphate, and ERK-1/2 phosphorylation. The phospho-ERK-1/2 response can be clearly distinguished from the other three responses, however. The extent of constitutive activation of this response is relatively minor and, in contrast to the other three responses, which are insensitive to further hCG stimulation, the phospho-ERK-1/2 response of cells expressing the L457R, D578Y, or D578H mutants is still sensitive to hCG (compare Fig. 6DGo with 6AGo, 6BGo, and 6CGo). This finding implies that the phospho-ERK-1/2 response is mediated by G proteins that are distinct from those that mediate the cAMP and inositol phosphate responses. Alternatively, this response could be activated in a G protein-independent fashion. More studies will be needed to address this question.

In summary, the results presented herein serve to establish and characterize a novel experimental paradigm in which the hLHR or mutants thereof can be expressed and analyzed in a Leydig cell line (MA-10). Our ability to transiently express the recombinant hLHR in a Leydig cell line that retains many of the differentiated functions of Leydig cells but is practically devoid of endogenous LHR provides a novel and flexible experimental paradigm that can be used to characterize the multiple signaling pathways that are activated by the LHR and mediate the effects of this receptor on the proliferation and differentiation of Leydig cells. We are also currently trying to select clonal MA-10 cells stably transfected the recombinant hLHR-wt. When available such cell lines may provide a more stable model for the study of LH actions.


   Acknowledgments
 
We thank Dr. Deborah L. Segaloff for reading this manuscript and Ares Serono (Randolph, MA) for the original human LHR construct and for recombinant hCG.


   Footnotes
 
This work was supported by NIH Grant CA-40629 (to M.A.) and a fellowship from the Lalor Foundation (to T.H.). The services and facilities provided by the Diabetes and Endocrinology Research Center of the University of Iowa (supported by NIH Grant DK-25295) are also gratefully acknowledged.

Abbreviations: GPCR, G protein-coupled receptor; hLHR, human lutropin/choriogonadotropin receptor; hLHR-wt, hLHR wild-type.

1 Both preparations were used in this study and were found to be indistinguishable. Back

2 This observation was reproduced during the course of the experiments described herein. Back

Received October 10, 2001.

Accepted for publication November 21, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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