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Biology of Reproduction 59, 1116-1123 (1998)
©Copyright 1998 Society for the Study of Reproduction, Inc.

Regulation of Luteinizing Hormone Receptor Gene Expression by Insulin-Like Growth Factor-I in an Immortalized Murine Leydig Tumor Cell Line (BLT-1)1

Fu-Ping Zhanga, Talal El-Hafnawya, , and Ilpo Huhtaniemi2,a

a Department of Physiology, University of Turku, FIN-20520 Turku, Finland


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
It is postulated that insulin-like growth factor-I (IGF-I), a 70-amino acid mitogenic polypeptide, regulates Leydig cell steroidogenesis. In the present study, we assessed the effect of IGF-I on LH receptor (LHR) gene expression in an immortalized murine Leydig tumor cell line (BLT-1). Culture of BLT-1 cells in the presence of IGF-I (0.1–100 ng/ml) for 24 or 48 h increased their [125I]iodo-hCG binding in a dose-dependent manner up to 275% of the control level. Northern hybridization analysis revealed four major transcripts of LHR mRNA in BLT-1 cells (6.9, 2.6, 1.7, and 1.2 kilobases), and treatment at 10–100 ng/ml of IGF-I increased steady-state levels of LHR mRNAs in coordinate fashion up to 2.2-fold. IGF-I (30 ng/ml) induced a time-dependent increase in [125I]hCG binding after a lag period of 2–6 h when studied up to 48 h, with a subsequent decrease. A similar response with steady increase up to 72 h was observed in total LHR mRNA. To elucidate the molecular mechanism of IGF-I action on LHR mRNA expression, we measured the transcription rate of the LHR gene by nuclear run-off assay and assessed transcript stability by the actinomycin D blocking method. The results showed that IGF-I treatment had no effect on the transcription rate of the LHR gene, whereas the half-life (t1/2) of LHR mRNA was significantly prolonged (IGF-I-treated cells, 30 ± 3.8 h; controls, 17 ± 2.5 h). Furthermore, IGF-I at 30 ng/ml and 100 ng/ml increased the expression of LHR promoter-driven luciferase and cytomegalovirus-promoter driven ß-galactosidase activities in BLT-1 cells; however, the former increased only marginally more than the latter. This suggests that the increase of LHR mRNA by IGF-I in Leydig cells is mainly due to increased mRNA stability.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The LH receptor (LHR), along with the FSH and thyroid-stimulating hormone receptors, belongs to the G protein-coupled receptor family, with a unique large extracellular domain. It is mainly expressed in Leydig cells in the testis and in theca, granulosa, interstitial, and luteal cells in the ovary, and it plays a crucial role in reproduction [1]. The cDNA and gene of the LHR have been cloned and extensively studied in several species. The LHR gene contains, depending on species, 10 or 11 exons, and it is expressed as a number of splice variants in gonadal and nongonadal tissues [2–5]. Ligand binding of the LHR activates the adenylate cyclase and phospholipase C signal transduction systems, leading to stimulation of synthesis and secretion of sex steroid hormones. There is increasing evidence that LH action is under modulation by various paracrine/autocrine growth factors and peptides [6].

The insulin-like growth factor (IGF) system consists of the IGF ligands (IGF-I and IGF-II), IGF receptors, and IGF binding proteins [7]. IGF-I is a well-characterized peptide hormone, consisting of 70 amino acids, that promotes cellular proliferation and protein synthesis in a variety of cell types [8, 9]. The IGF-I gene is expressed in Leydig cells of the rat testis, in the interstitial compartment of the prepubertal mouse testis, and in the seminiferous epithelium of the adult mouse testis [10, 11]. The IGF receptor is located in Leydig cells in the rat and mouse testis [12]. The receptor binding of IGF-I stimulates tyrosine kinase activity of the ß subunit, leading to receptor autophosphorylation and tyrosine phosphorylation of several cellular substrates, including insulin receptor substrate-1 [13]. IGF-I apparently plays an important role in the regulation of Leydig cells. The serum level of IGF-I increases dramatically during puberty in humans, coincident with a rise in gonadotropins [8]. The IGF-I increase begins at Day 14 in the rat, and plasma and interstitial fluid IGF-I levels in the mouse increase significantly during puberty, reaching a peak at Day 24 and declining thereafter [14, 15]. These levels are temporally correlated with the onset of marked developmental changes in the testis, such as organ growth, increase in Leydig cell population, onset of steroidogenesis, and gametogenesis [15, 16]. In vitro studies have indicated that IGF-I enhances Leydig cell steroidogenesis and DNA synthesis in primary culture [17, 18]. Moreover, IGF-I increases Leydig cell steroidogenic responsiveness to hCG due to increased hCG receptor binding [19]. A recent study on IGF-I gene null mutation demonstrated that homozygous mutant mice are infertile dwarfs (about 30% of normal size). Their testis size is reduced and Leydig cell development delayed, with reduced serum testosterone levels [11, 20]. Although these data demonstrate a clear role for IGF-1 in the regulation of Leydig cell steroidogenesis, little is known about the interactions of IGF-I with LH action, the main regulator of Leydig cell function.

Recently we produced transgenic mice bearing a 6-kilobase (kb) fragment of the mouse inhibin {alpha}-subunit promoter fused with the simian virus 40 T-antigen (SV 40 Tag) coding sequences. Gonadal tumors appeared in the mice at the age of 5–6 mo, and those present in the testis originated from Leydig cells [21]. An immortalized cell line, BLT-1, established from one of the testicular tumors was found to express the LHR and P450 side-chain cleavage mRNAs and to display specific [125I]iodo-hCG binding, as well as LH-mediated cAMP and progesterone production [21]. Therefore, these immortalized Leydig cells provide a suitable model for studying the effects of IGF-I on LHR function. In the present study, we provide new insights into the molecular mechanisms involved in regulation of LHR gene expression by IGF-I.


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

BLT-1 cells, an immortalized murine Leydig cell line [21], were maintained in Dulbecco's Modified Eagle's/F12 medium (1:1) (DMEM/F12; Life Technologies, Gibco BRL, Glasgow, UK) supplemented with 10% fetal calf serum (Bio-Clear, Berkinghamshire, UK) and 0.1 mg/ml gentamycin (Biological Industries, Bet-HaEmek, Israel). For the [125I]iodo-binding measurements, the cells (1 x 107) were plated in 60-mm plastic dishes. For LHR mRNA measurements, cells (2 x 107) were plated in 90-mm plastic dishes. After 1 day of culture, the medium was removed and the cells were washed with PBS twice; medium was then replaced with fresh medium containing 0.1% BSA, and incubation was performed with reagents, or identical vehicles for control, for selected times as specified below. Cell passages between 25 and 40 were used for the studies.

IGF-I Dose-Response and Time Course Experiments

For dose-response studies, BLT-1 cells were treated with various doses (0, 0.1, 1, 10, 30, and 100 ng/ml) of IGF-I (R&D Systems, Minneapolis, MN) for 24 h and 48 h. For time course experiments, cells were treated with IGF-I (30 ng/ml) for 0, 2, 6, 12, 24, 48, and 72 h. Control samples were cultured for the respective times with vehicle alone. All groups were harvested at the same time, and RNA was extracted as described below.

[125I]Iodo-hCG Binding

Human CG (CR-127, NICHD, NIH, Bethesda, MD) was radioiodinated with sodium [125I]iodide (Amersham, Aylesbury, UK) to a specific activity of 35 000 cpm/ng and 37% specific binding to an excess of LHRs, as determined according to Catt et al. [22]. To measure binding, the cells were washed twice with ice-cold PBS and scraped into Dulbecco's (D)-PBS using a rubber policeman. Cells were pelleted by centrifugation at 1500 rpm and washed twice with D-PBS. The final pellet was resuspended at a concentration equivalent to 1 x 107 cells/ml and frozen (-80°C) until measurement of binding. Triplicate aliquots of cells in D-PBS/0.1% BSA (0.2 x 106) were incubated with 150 000 cpm of [125I]iodo-hCG in the presence or absence of 50 IU of unlabeled hCG (Pregnyl; Organon, Oss, The Netherlands) in a total volume of 250 µl. After overnight incubation at room temperature, the cells were washed with 4 ml of ice-cold D-PBS/0.1% BSA, and after centrifugation, the radioactivity of the cell pellets was counted in a gamma spectrometer. All data were corrected for nonspecific binding, which was assessed in the presence of 50 IU of Pregnyl per assay tube.

RNA Preparation

The RNA from cells was extracted by the single-step method as described previously, with minor modifications [23]. Briefly, after removal of the culture medium, 1 ml of denaturing solution (4 M guanidine thiocyanate, 25 mM sodium citrate, pH 7.0, 0.5% Sarkosyl [Sigma Chemical Co., St. Louis, MO], 0.1% ß-mercaptoethanol) was added; the cells were scraped from plates using a rubber policeman and passing the lysate through a pipette several times. Subsequently, 100 µl of 2 M sodium acetate (pH 4.0), 1 ml of water-saturated phenol, and 200 µl of chloroform-isoamyl alcohol (49:1) were added to the lysate. After centrifugation, RNA in the supernatant was precipitated with isopropanol and extracted twice using phenol/chloroform. After precipitation with ethanol, the RNA pellets were dissolved in water treated with diethyl pyrocarbonate for the Northern hybridization analysis.

Northern Hybridization Analysis

Northern hybridization was performed as described previously [23]. Briefly, 15 µg of total RNA was resolved in a 1.2% denaturing agarose gel and blotted onto a nylon membrane (Hybond-N; Amersham) by capillary transfer. The filter was prehybridized for at least 4 h at 65°C in a solution containing 50% formamide, 3-strength SSC (single-strength SSC is 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0), 5-strength Denhardt's solution, 1% SDS, 0.1 µg/ml heat-denatured calf thymus DNA, and 100 µg/ml yeast tRNA (Sigma). Hybridization was carried out at 66°C overnight in the same solution after addition of the 32P-labeled cRNA probe. After hybridization, the membrane was washed with double-strength SSC and 0.1% SDS at room temperature for 30 min twice, and with 0.1-strength SSC and 0.1% SDS at 66°C for 2 h twice; then the membrane was exposed to x-ray film (Kodak XAR-5; Eastman Kodak, Rochester, NY) at -70°C for 1–3 days. The molecular sizes of the mRNA species were determined by comparison with the RNA molecular weight marker (Promega, Madison, WI). The 32P-labeled cRNA was synthesized using a riboprobe synthesis II kit (Promega) and [32P]UTP (Amersham); LHR cDNA subcloned into pGEM-4Z was used as the template. The cDNA corresponded to bases 441–849 of the extracellular domain of the rat LHR cDNA as reported by McFarland et al. [2]. All values for the LHR mRNA levels were normalized relative to the intensity of the 18S rRNA to correct for potential differences in the amounts of loaded RNA.

Measurement of Stability of the Transcripts

BLT-1 cells were incubated with and without 30 ng/ml of IGF-I for 36 h. Thereafter transcription was arrested in the presence of 10 mg/ml actinomycin D (Sigma). Control groups were removed at Time 0, and the remaining groups were incubated for an additional 4–20 h. Incubations could not be continued beyond 20 h because of poor cell survival. Total RNA was then isolated from the samples as described above for the Northern hybridization analysis.

Nuclear Run-Off Assays

BLT-1 cells were plated on 9-cm2 plates (15 x 106 cells per plate) in 8 ml of 10% fetal calf serum-DMEM. One day after plating, cells were treated with IGF-I (30 ng/ml) or vehicle in 8 ml of DMEM/F12 with 0.1% BSA for 36 h in triplicate. The isolation of nuclei was performed according to the protocol described by Bender [24]. The in vitro elongation reaction was carried out according to the protocol described by Fei and Drake [25], with minor modifications. Briefly, for each reaction, 2.5 x 106 nuclei in 93 µl of glycerol storage buffer (50 mM Tris-Cl, pH 8.1, 40% [v:v] glycerol, 5 mM MgCl2, 0.1 mM EDTA, 0.1 mM dithiothreitol) were incubated in a 2.0-ml Eppendorf tube with 100 µl of double-strength reaction buffer, 6 µl of [{alpha}-32P]UTP (~800 Ci/mmol; Amersham), and 1 µl RNasin (39 U/µl; Promega) at room temperature for 30 min. DNA was digested by adding 20 µl of RNase-free DNase (1 U/µl; Promega) at room temperature for 10 min. Yeast tRNA (50 µg) was then added as a carrier. Nuclear RNA was isolated by adding 500 µl of RNA isolation solution (4 M guanidine thiocyanate, 25 mM sodium citrate, pH 7.0, 0.5% Sarkosyl, 0.1% ß-mercaptoethanol), 70 µl of 2.0 M sodium acetate (pH 4.0), 700 µl water-saturated phenol, and 140 µl chloroform/isoamyl alcohol (49:1). After incubation on ice for 15 min, the samples were centrifuged in a microcentrifuge (13000 rpm, 20 min, at 4°C), the supernatant was transferred to another Eppendorf tube, one volume of isopropanol was added, and the samples were incubated at -20°C for 1 h. RNA was pelleted by centrifugation (13000 rpm, 20 min), then washed with 70% ethanol and resuspended in 400 µl of TES buffer (10 mM Tris, pH 8.0, 10 mM EDTA, 0.2% SDS).

Five micrograms of linearized and denatured plasmids containing cDNAs (rat LHR and glyceraldehyde phosphate dehydrogenase) and empty pBluescript-SK was slot-blotted and UV cross-linked onto nylon membrane. Strips were prehybridized at 42°C for 12 h in prehybridization buffer (50% formamide, 5-strength SSC, 0.5-strength Denhardt's, 0.5% SDS, and 10 µg/ml heat-denatured calf thymus DNA). Hybridization was performed at 42°C for 60 h in the same solution by adding equal amounts of radioactivity (1 x 106 cpm/ml hybridization buffer) of the corresponding experimental groups. After hybridization, the strips were washed with 30 ml of double-strength SSC and 0.1% SDS twice at room temperature for 30 min, then with double-strength SSC and 0.1% SDS twice at 65°C for 30 min. Strips were exposed to x-ray film at -70°C for 10–25 days.

Transfection and Luciferase Assay

BLT-1 cells were transiently transfected using electroporation as previously described [26]. Briefly, 8 x 106 cells were electroshocked using 350 V and 960 µF. Cells in each cuvette were transfected with 25 µg of a plasmid (pBS) carrying 1.6 kb of the 5'-flanking region of the LHR gene (p1.6LHR-Luc) in front of the luciferase reporter gene + poly (A) and control plasmid, 2 µg of cytomegalovirus (CMV) promoter-driven ß-galactosidase. After electroporation, the cells were left at room temperature for 10 min; they were then plated onto 9-cm culture dishes in complete growth medium. Eighteen hours later, the medium was changed to one without serum but containing IGF-I (30 or 100 ng/ml). The cells were harvested after 24-h stimulation and lysed through three cycles of freezing and thawing. The lysate was separated from the cell debris by centrifugation at 13 000 rpm, at 4°C for 15 min, and was used to measure the luciferase and ß-galactosidase activities and total protein contents.

Densitometry

The relative optical densities of autoradiographic bands of LHR and 18S rRNAs and hybridization signals from nuclear run-off experiments were quantified using the TINA 2.0 program (Raytest, Straubenhardt, Germany).

Data Analysis

A Macintosh version of the SuperANOVA program (Abacus Concepts, Berkeley, CA) was used for one-factor ANOVA, followed by Duncan's new multiple range test; p values less than 0.05 were regarded as statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of IGF-I on LHR Number and mRNA Levels

BLT-1 cells were cultured for 24 h at 37°C. After change of medium, the cells were incubated at increasing concentrations of IGF-I (0.1, 1, 10, 30, and 100 ng/ml) in DMEM medium with 0.1% BSA for 24 h and for 48 h. As shown in Figures 1A and 2A, using [125I]iodo-hCG-binding assay, IGF-I increased the number of LHR-binding sites in a dose-dependent manner. During the 24-h incubation, IGF-I at concentrations of 30 ng/ml and 100 ng/ml significantly increased hCG binding to 225% and 275% of the control value, respectively (p < 0.05 and p < 0.01). Similarly, during the 48-h incubation, IGF-I at concentrations of 30 ng/ml and 100 ng/ml increased hCG binding to 230% and 180% of the control level, respectively (p < 0.001 and p < 0.01).



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  		FIG. 1. Effect of various doses of IGF-I on hCG binding and LHR mRNA levels of BLT-1 cells during 24-h incubation. The cells were incubated in DMEM/0.1% BSA at the concentrations of IGF-I, as shown, for 24 h. At the end of the incubation period, hCG binding and LHR mRNA levels were analyzed by radioreceptor assay and Northern hybridization. ADU, Arbitrary densitometric units. A) Specific [125I]iodo-hCG binding to cells. B) Densitometric quantitation of the LHR mRNA splice variants. The values were normalized to the intensity of the 18S RNA to correct for loading differences and are expressed as percentages of the levels measured in control cells. The results are mean ± SEM of three independent experiments; the mean of controls was assigned 100%. C) A representative Northern hybridization blot of LHR mRNA. 18S and 28S, migration of the 18S and 28S rRNAs, respectively; kb, sizes and migration of the LHR mRNA splice variants in kilobase pairs; * p < 0.05, ** p < 0.01, *** p < 0.001, compared with controls.



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  		FIG. 2. Effect of various doses of IGF-I on hCG binding and LHR mRNA levels of BLT-1 cells during 48-h incubation. With the exception of the longer 48-h incubation time, all details of this experiment were identical to those described for Figure 1.

Figures 1B, 1C, 2B, and 2C show the effect of various doses of IGF-I on the expression of LHR mRNA levels after 24-h and 48-h treatments. Northern hybridization analysis of cultured Leydig cells revealed four major transcripts of LHR mRNA with sizes of 6.9, 2.6, 1.7, and 1.2 kb, as previously reported by us [17, 19]. IGF-I stimulated a dose-dependent increase in the LHR mRNA levels. Treatment of BLT-1 cells with 30 and 100 ng/ml of IGF-I for 24 h resulted in increases, respectively, to 1.4-fold and 1.6-fold of control, of the total LHR mRNA (p < 0.05 and p < 0.01). Treatment of BLT-1 cells with 30 and 100 ng/ml of IGF-I for 48 h resulted in increases, respectively, to 1.8-fold and 2.2-fold of the control, of total LHR mRNA (p < 0.05 and p < 0.01). IGF-I coordinately increased the four major transcripts of LHR mRNA at both time points.

We next evaluated the time course effects of IGF-I on both LHR number and mRNA levels. Cells were treated with 30 ng/ml of IGF-I, and their hCG binding and the level of LHR mRNA were measured at various times after the addition of IGF-I. The results shown in Figure 3A indicate that IGF-I caused an increase in the number of LHR after a lag period of 2–6 h. Thereafter the receptor number progressively increased to a maximum (300% of control) at 48 h (p < 0.001), followed by a decrease at 72 h (p < 0.01). Similarly, IGF-I increased total LHR mRNA levels after a lag period of 2–6 h. Thereafter, the levels of LHR total mRNA progressively increased, reaching a plateau between 48 and 72 h (p < 0.001) (Fig. 3, A and B).



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  		FIG. 3. Time course effects of IGF-I on hCG binding and LHR mRNA levels of cultured BLT-I cells. The cells were incubated for increasing lengths of time in the absence and presence of 30 ng/ml IGF-I, and the hCG binding and LHR mRNA levels were measured by radioreceptor assay and Northern hybridization. A) Specific [125I]iodo-hCG binding into cells (circles) and densitometric quantitation of total LHR (sum of the 6.9-, 2.6-, 1.7-, and 1.2-kb transcripts) (squares). The values of total LHR mRNA were normalized to the intensity of 18S RNA to correct for loading differences and are expressed as percentage of control. Results are mean ± SEM of three independent experiments; the mean of controls was assigned 100%. ADU, Arbitrary densitometric units. B) Representative Northern hybridization. 18S and 28S, migration of the 18S and 28S rRNAs, respectively; kb, sizes and migration of the LHR mRNA splice variants in kilobase pairs; * p < 0.05, ** p < 0.01, *** p < 0.001, compared with controls.

Effects of IGF-I on Stability of the LHR mRNA Levels

We determined whether the increased level of LHR mRNA in IGF-I-treated BLT-1 cells was due to increased mRNA stability using the actinomycin D blocking method. In these experiments, cells were treated either with vehicle or with 30 ng/ml of IGF-I for 36 h, after which transcription was arrested by addition of actinomycin D. As shown in Figure 4, after inhibition of transcription, the decrease of LHR mRNA was slower when the BLT-1 cells were pretreated with IGF-I for 36 h, as compared with the control. The mRNA half-lives, as calculated by regression analysis of the slopes, were 30.5 ± 3.8 h for the IGF-I-treated samples and 17 ± 2.5 h for the controls (n = 3, p < 0.01).



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  		FIG. 4. Effect of IGF-I on LHR mRNA stability. The cells were incubated in DMEM/0.1% BSA with or without 30 ng/ml of IGF-I for 36 h. Total RNA was extracted from cells at zero time (as control) and at 4, 8, 12, and 20 h after addition of actinomycin D (10 µg/ml). The LHR mRNA was determined by Northern hybridization. A) Densitometric values of total LHR mRNA (i.e., the sum of the 6.9-, 2.6-, 1.7-, and 1.2-kb transcripts). The values shown are normalized to the intensity of 18S RNA to correct for loading differences and are presented by regression slopes of the average of three independent experiments. Half-life of controls, 17 ± 2.5 h; half-life of IGF-I-treated cells, 30.5 ± 3.8 h. B) A representative Northern hybridization blot of LHR mRNA. 18S and 28S, migration of the 18S and 28S rRNAs, respectively; kb, sizes and migration of the LHR mRNA splice variants in kilobase pairs.

Effects of IGF-I on the Rate of Transcription of LHR mRNA

To determine whether IGF-I increases LHR mRNA levels by stimulating the transcription rate of the LHR gene in BLT-1 cells, we performed nuclear run-off experiments. The results (Fig. 5) demonstrated that IGF-I treatment had no significant effect on the transcription rate of the LHR gene as compared with control (p > 0.05). To define further the mechanism of IGF-I stimulation of the LHR gene expression, BLT-1 cells were transiently transfected with a 1.6-kb fragment of the LHR promoter subcloned into the luciferase reporter plasmid. IGF-I at concentrations of 30 ng/ml and 100 ng/ml induced a clear, significant increase in the luciferase activity after 24-h incubation. However, IGF-I also displayed a similar increase in the ß-galactosidase activity (Table 1). The luciferase/ß-galactosidase ratio was slightly but significantly elevated (1.37, p < 0.05) after stimulation with 100 ng/ml IGF-I.



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  		FIG. 5. Effect of IGF-I on the rate of LHR gene transcription. The cells were incubated in DMEM/0.1% BSA with or without 30 ng/ml of IGF-I for 36 h, and nuclei were isolated. The relative transcription rates were determined by elongation of preinitiated transcripts in the presence of [{alpha}-32P]UTP, followed by purification and hybridization with linear plasmid DNAs. A) Representative autoradiograph of hybridization of a run-off assay. B) The intensities of the bands were quantitated using the TINA 2.0 program. The values were corrected for nonspecific binding by subtracting the hybridization signal of the vector, and the results were normalized to the glyceraldehyde phosphate dehydrogenase values. Each bar represents the mean ± SEM of triplicates from two independent experiments, with the mean of controls taken as 100%.


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  		TABLE 1. Stimulation of BLT-1 cells transfected with murine LHR promoter-driven luciferase and CMV promoter-driven ß-galactosidase cDNAs by IGF-I.a


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, we demonstrated that culturing mouse tumor Leydig cells (BLT-1) with recombinant human IGF-I resulted in a dose-dependent increase in the steady-state levels of LHR mRNA and [125I]iodo-hCG binding. These increases paralleled each other, though after a lag period of 2–6 h, and thereafter with different kinetics. Thus, the increase in receptor binding of about 300% of the control value was attained after 48-h treatment with IGF-I, whereas the 2.2-fold maximum increase of the LHR mRNA was reached at 72 h. In contrast, the binding activity was decreased to about 150% of the control level between 48 and 72 h; this did not occur in the cognate mRNA levels. Furthermore, IGF-I coordinately increased the four major transcripts of the LHR mRNA in BLT-1 cells, in agreement with previous reports [1, 27]. Although the size, number, and relative abundance of the various LHR mRNA transcripts differ from one tissue to another and between species, Northern hybridization analyses have demonstrated that all transcripts are coordinately regulated by LH, and by 8-bromo-cAMP in mouse Leydig cells and porcine Leydig cells [1, 27]. In contrast, studies by LaPolt et al. [28] and us [23, 29] indicated that during the hCG-mediated reduction of testicular LHR mRNA transcripts in intact and ethylene dimethane sulfonate-treated rats, the 1.6- to 1.8-kb transcript did not change as much as the others.

IGF-I is a mitogenic polypeptide, and it regulates the proliferation and differentiation of multiple cell types and also has insulin-like metabolic effects [8, 9]. Recently, increasing evidence has emerged indicating that IGF-I plays an important autocrine or paracrine role in the regulation of Leydig cell function [12]. Both IGF-I and its receptor are expressed in the rat and mouse testis, and they are regulated by hCG in different ways [30–32]. In primary cultures, IGF-I can enhance Leydig cell steroidogenesis [17–19, 33]. The role of IGF-I and growth hormone (GH) in testicular function has also been studied in Snell dwarf mice. These mice have an anterior pituitary defect and secrete little GH and prolactin, which is associated with delayed puberty and a low serum testosterone level. Treatment of these dwarf mice with GH and IGF-I increased testicular LH receptors and steroidogenic response [34]. Most recently, a study demonstrated that mice carrying a null mutation of the IGF-I gene exhibit a dramatic reduction in the sizes of reproductive organs and are infertile. The volume and number of Leydig cells were reduced (50% and 33% of normal, respectively), and the Leydig cells were delayed in their development [11, 20]. Our present data provide direct evidence that IGF-I up-regulates LHR mRNA and binding sites in Leydig cells. Taken together, these results suggest that IGF-I has an important stimulatory role in the regulation of Leydig cells. Whether it is locally produced in the testis or is of circulatory origin remains to be explored.

The LHR mRNA level is regulated in a complex fashion. The major regulator of LHR expression is the ligand, LH/hCG [1]. In mouse Leydig tumor cells, hCG and 8-bromo-cAMP down-regulate the LHR mRNA level, mainly through inhibition of transcription, without change or with a small increase in the mRNA stability [35, 36]. This has been further confirmed by transfection experiments, using expression plasmids containing the 5'-flanking region of rat or murine LHR promoter and a luciferase reporter gene, in which 8-bromo-cAMP significantly decreased the relative luciferase activity [26, 37]. In contrast, Chuzel et al. [27] reported that in porcine Leydig cells, the main mechanism by which LH/hCG down-regulates its receptor mRNA is by decreasing receptor mRNA stability. Likewise, in the pseudopregnant rat, the hCG-induced down-regulation of LHR mRNA mainly occurs through decreased stability of the receptor mRNA [38]. A recent study indicated that homologous down-regulation of nongonadal LHR in human uterine endometrial stromal cells was not due to decreased transcription rate of the LHR gene. It was rather due to a significant decrease in the half-life of receptor transcripts [39]. Therefore, there appear to be species- and tissue-specific differences in the response of LHR to LH/hCG.

In the present study, the molecular mechanism of IGF-I action responsible for the increased steady-state levels of LHR mRNA was elucidated. We examined the stability of the LHR transcripts, as done by others, using the actinomycin D blocking method. The result demonstrated that IGF-I significantly increases the half-life of the receptor transcripts from 17 ± 2.5 h in controls to 30 ± 3.8 h after IGF-I treatment. It is known that actinomycin D increases the stability of receptor transcripts [40]. However, since in the present experiment actinomycin D was used in both control and IGF-I-treated cells, the difference between the two experimental groups was a specific IGF-I effect. We then determined the transcription rates of isolated nuclei from control and IGF-I-pretreated Leydig cells in order to determine possible effects on transcription rate of the LHR message. Our results indicate that the increase in the steady-state levels of LHR mRNA during the IGF-I treatment was not due to increased transcription. Furthermore, a transfection study with an LHR promoter/luciferase gene construct showed that IGF-I increased both the LHR promoter-driven luciferase and the viral promoter-driven ß-galactosidase activities, the former only marginally more than the latter. These results suggest that IGF-I increases the LHR mRNA levels mainly by increasing the stability of mRNA; if there are changes in the LHR promoter activity, as reflected by an increased transcription rate, these changes are marginal and of only limited importance in the increased LHR mRNA steady-state levels.

The regulation of mRNA stability is an important process in controlling the steady-state levels of mRNAs in various systems [41]. The fact that IGF-I can increase the stability of LHR transcripts suggests that this growth factor may increase the synthesis of protein(s) that stabilize the transcripts or may decrease the synthesis of those that destabilize transcripts. These proteins function by binding to the 3'- or 5'-untranslated regions and the open reading frame of mRNA and to trans-acting factors such as RNA-binding proteins and RNase [41–43]. The exact nature of these proteins remains to be investigated.

In summary, the present study provides direct evidence that IGF-I up-regulates the LHR binding and mRNA levels of murine Leydig tumor cells in a dose- and time-dependent manner. The increase in the steady-state level of LHR mRNA was attributable mainly to increased stability of the LHR transcripts. The present results further elucidate the molecular mechanism of the up-regulation of Leydig cell function by IGF-I.


   FOOTNOTES
 
1 This study was supported by grants from the Academy of Finland and the Sigrid Jusélius Foundation. Back

2 Correspondence: Ilpo Huhtaniemi, Department of Physiology, University of Turku, Kiinamyllynkatu 10, FIN-20520 Turku, Finland. FAX: 358 2 2502610; ilpo.huhtaniemi{at}utu.fi Back

Accepted: June 25, 1998.

Received: April 13, 1998.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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