LH and hCG action on the same receptor results in quantitatively and qualitatively different intracellular signalling

Abstract

Human luteinizing hormone (hLH) and chorionic gonadotropin (hCG) act on the same receptor (LHCGR) but it is not known whether they elicit the same cellular and molecular response. This study compares for the first time the activation of cell-signalling pathways and gene expression in response to hLH and hCG. Using recombinant hLH and recombinant hCG we evaluated the kinetics of cAMP production in COS-7 and hGL5 cells permanently expressing LHCGR (COS-7/LHCGR, hGL5/LHCGR), as well as cAMP, ERK1/2, AKT activation and progesterone production in primary human granulosa cells (hGLC). The expression of selected target genes was measured in the presence or absence of ERK- or AKT-pathways inhibitors. In COS-7/LHCGR cells, hCG is 5-fold more potent than hLH (cAMP ED(50): 107.1±14.3 pM and 530.0±51.2 pM, respectively). hLH maximal effect was significantly faster (10 minutes by hLH; 1 hour by hCG). In hGLC continuous exposure to equipotent doses of gonadotropins up to 36 hours revealed that intracellular cAMP production is oscillating and significantly higher by hCG versus hLH. Conversely, phospho-ERK1/2 and -AKT activation was more potent and sustained by hLH versus hCG. ERK1/2 and AKT inhibition removed the inhibitory effect on NRG1 (neuregulin) expression by hLH but not by hCG; ERK1/2 inhibition significantly increased hLH- but not hCG-stimulated CYP19A1 (aromatase) expression. We conclude that: i) hCG is more potent on cAMP production, while hLH is more potent on ERK and AKT activation; ii) hGLC respond to equipotent, constant hLH or hCG stimulation with a fluctuating cAMP production and progressive progesterone secretion; and iii) the expression of hLH and hCG target genes partly involves the activation of different pathways depending on the ligand. Therefore, the LHCGR is able to differentiate the activity of hLH and hCG.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Dose-response and time-course experiments.

a. Dose-response experiment to r-hLH and r-hCG in COS7/LHCGR in the presence of 500 µM IBMX. Total cAMP was measured after 3 hours of incubation and the results of four independent experiments were plotted (Mean±SEM). b. Time-course experiment performed by continuous incubation of COS7/LHCGR for different time-points in the presence of 500 µM IBMX and gonadotropins at ED50 doses (500 pM r-hLH; 100 pM r-hCG). Intracellular cAMP was measured (n = 3; Mean±SEM). Asterisk indicates the significant differences of hLH vs hCG (t-test; p<0.05).

Figure 2. Long-term kinetics of intracellular cAMP production and extracellular progesterone and cAMP release in a primary hGLC sample continuously exposed to r-hLH or r-hCG.

Absolute values of intracellular cAMP (bars) and extracellular progesterone (lines) production by hGLC cultured in the presence IBMX (500 µM) under continuous stimulation by hLH (500 pM) or hCG (100 pM) for up to 36 hours: (a) 0–12 hours, (b) 13–24 hours, (c) 25–36 hours. cAMP basal level of the unstimulated control cells is also shown for each time-point. d. Measurement of extracellular cAMP, released in culture medium. One representative of three independent measurement is shown for each graph. Please notice the different progesterone scale on the Y axis on right side.

Figure 3. Long-term kinetics of intracellular cAMP production in hGLC continuously exposed to r-hLH or r-hCG.

hGLC were cultured in the presence IBMX (500 µM) under continuous stimulation by r-hLH (500 pM) or r-hCG (100 pM) for up to 36 hours: (a) 0–12 hours, (b) 13–24 hours, (c) 25–36 hours. Results are means±SEM (n = 3). Asterisks indicate significant differences between hCG and hLH at the given time point (n = 3; t-test; p<0.05).

Figure 4. Cellular localization of LHCGR and morphological changes of hGLC under continuous exposure to hLH 500 (pM) by confocal microscopy.

a. Unstimulated cells (control) after 1 hour. b. hLH-treated cells after 1 hour. c. Unstimulated cells after 15 hours. d. LHCGR sequestration from cell surface in hLH-treated hGLC, after 15 hours. e. Unstimulated cells after 24 hours. f. Cell-rounding in hLH-treated cells after 24 hours. LHCGR is labeled in red (Tritc), the cytoplasmic marker ERK1/2 in green (Fitch) and cell nuclei marker (DAPI) in blue. The merging of the three images is in the lower right plate of each panel. Images are from one experiment and are representative of three independent experiments with similar results. White arrows indicated LHCGR; Scale bar: 10 µm. Western blot control for the anti-LHCGR antibody and non-permeabilized cells control for immunofluorescence staining were also included (Fig. S5).

Figure 5. Dose-response experiment evaluating the maximal phospho-ERK1/2 and phospho-AKT activation in hGLC by Western blotting.

The cells were stimulated for 15 minutes by different r-hLH or r-hCG doses and the phospho-ERK1/2 and phospho-AKT signals were normalized for total ERK. The image is representative of 3 independent experiments.

Figure 6. Time-course analysis of the phospho-ERK1/2 activation in hGLC under 100 pM hLH or 100 pM hCG stimulation.

a. Comparison of phospho-ERK1/2 activation in hLH-stimulated vs unstimulated (control) at different time-points, by Western blotting (image is representative of 4 independent experiments; total ERK as normalizer). b. Comparison of phospho-ERK1/2 activation in hCG-stimulated vs unstimulated (control) at different time-points, by Western blotting (image is representative of 4 independent experiments; total ERK as normalizer). c, d. Relative semi-quantification of the optical density representing phospho-ERK1/2 activation (shown in figs. 6A,B) stimulated by (c) hLH or (d) hCG, compared to unstimulated (Mean±SEM; n = 4; * = significant vs unstimulated; t-test; p<0.05). e. Normalization of the phospho-ERK1/2 signals measured in hLH-stimulated samples (represented as relative units in Fig. 6C) over each unstimulated (Mean±SEM; n = 4; * = significant vs unstimulated; two-way analysis of variance; p<0.05). f. Normalization of the phospho-ERK1/2 signals measured in hCG-stimulated samples (represented as relative units in Fig. 6D) over each unstimulated (Mean±SEM; n = 4; * = significant vs unstimulated; two-way analysis of variance; p<0.05).

Figure 7. Time-course analysis of the phospho-AKT activation in hGLC under 100 pM hLH or 100 pM hCG stimulation.

a. Comparison of phospho-AKT activation in hLH-stimulated vs unstimulated (control) at different time-points, by Western blotting (image is representative of 4 independent experiments; total ERK as normalizer). b. Comparison of phospho-AKT activation in hCG-stimulated vs unstimulated (control) at different time-points, by Western blotting (image is representative of 4 independent experiments; total ERK as normalizer). c, d. Relative semi-quantification of the optical density representing phospho-AKT activation (shown in figs. 7A,B) stimulated by (C) hLH or (D) hCG, compared to unstimulated (Mean±SEM; n = 4; * = significant vs unstimulated; t-test; p<0.05). e. Normalization of the phospho-AKT signals measured in hLH-stimulated samples (represented as relative units in Fig. 7C) over each unstimulated (Mean±SEM; n = 4; * = significant vs unstimulated; two-way analysis of variance; p<0.05). f. Normalization of the phospho-AKT signals measured in hCG-stimulated samples (represented as relative units in Fig. 7D) over each unstimulated (Mean±SEM; n = 4; * = significant vs unstimulated; two-way analysis of variance; p<0.05).

Figure 8. Evaluation of the gene expression induced in hGLC by hLH or hCG stimulation after 12 hours, performed by real-time PCR.

Specific ERK1/2- or AKT-pathways inhibitor (U0126 and LY294002 respectively) were also used where indicated. a. Increase in the gene expression of the EGF-like factors amphiregulin (AREG) and epiregulin (EREG) induced by LH or hCG. b. Effects of the hLH or hCG stimulation and of the ERK1/2- or AKT pathway early inhibition on neuregulin 1 (NRG1) gene expression. c. Effects of the hLH or hCG stimulation and of the ERK1/2- or AKT pathway early inhibition on NRG1 gene expression. In each treatments, RPS7 gene expression was used as normalizer (mean±SEM; n = 4; * = significant vs control; ° = significant vs hLH-stimulated; t-test; p<0.05).