RF9 Acts as a KISS1R Agonist In Vivo and In Vitro - PubMed (original) (raw)

RF9 Acts as a KISS1R Agonist In Vivo and In Vitro

Le Min et al. Endocrinology. 2015 Dec.

Abstract

RF9, a reported antagonist of the mammalian gonadotropin-inhibitory hormone receptor, stimulates gonadotropin secretion in mammals. Recent studies have suggested that the stimulatory effect of RF9 on gonadotropin secretion relies on intact kisspeptin receptor (KISS1R) signaling, but the underlying mechanisms remain to be elucidated. Using Chinese Hamster Ovary cells stably transfected with KISS1R, we show that RF9 binds specifically to KISS1R, with a Kd of 1.6 × 10(-5)M, and stimulates an increase in intracellular calcium and inositol phosphate accumulation in a KISS1R-dependent manner, with EC50 values of 3.0 × 10(-6)M and 1.6 × 10(-7)M, respectively. RF9 also stimulated ERK phosphorylation, with a time course similar to that of kisspeptin-10. RFRP-3, the putative endogenous ligand for NPFFR1, did not stimulate inositol phosphate accumulation or pERK, nor did it alter responses to of kisspeptin-10 or RF9. In agreement with these in vitro data, we found that RF9 stimulated a robust LH increase in Npffr1(-/-) mice, similar to that in wild-type littermates, whereas the stimulatory effect of RF9 was markedly reduced in Kiss1r(-/-) and double Kiss1r(-/-)/Npfrr1(-/-) mice. The stimulatory effect of RF9 on LH secretion was restored by the selective rescue of Kiss1r expression in GnRH neurons, in Kiss1r(-/-T) mice. Taken together, our study demonstrates that RF9 acts primarily as a KISS1R agonist, but not as an allosteric modulator, to stimulate LH secretion. Our findings raise questions regarding the utility of RF9 for assessing NPFF1R function and de-emphasize a predominant role of this signaling system in central regulation of reproduction.

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Figures

Figure 1.

Figure 1.

RF9 binds specifically to KISS1R. Competitive displacement binding assays were performed in CHO-KISS1R cells incubated with 0.05nM 125I-KP10 and increasing concentrations of either unlabeled KP10 or RF9. Data represent mean ± SEM from three independent experiments.

Figure 2.

Figure 2.

RF9 stimulates KISS1R-mediated increases in [Ca2+]i.[Ca2+]i responses were measured in CHO-KISS1R cells using a fluo-4 [Ca2+]i assay. CHO-KISS1R cells were treated with increasing concentrations of RF9 or KP10 as indicated. Data are shown as the percent of the maximum response and represent mean ± SEM from three independent experiments.

Figure 3.

Figure 3.

RF9 stimulates KISS1R-mediated increases in IP accumulation. Intracellular IP accumulation was measured as described in the Methods. CHO-KISS1R cells were treated with increasing concentrations of KP10 or RF9 in the presence or absence of 10−5M RFRP-3, or with increasing concentrations of RFRP-3 alone. In addition, cells were treated with increasing concentrations of KP10 in the presence of 10−6M RF9. Intracellular IP levels are expressed as counts per minute. Data are shown as the percent of the KP10-induced maximal response and represent the mean ± SEM from at least three independent experiments.

Figure 4.

Figure 4.

RF9 stimulates ERK phosphorylation. A, CHO-KISS1R (left panel) and CHO K1 (right panel) cells were treated with 10−7M KP10 or 10−6M RF9 in the presence or absence of 10−5M RFRP-3, or with 10−5 M RFRP-3 alone, as indicated. Cell lysates were collected for Western blot analysis after 2 or 30 minutes of treatment. The image shown is a representative blot from at least three independent experiments performed with similar results. B, The Western blot data were quantified and the results of three independent experiments were pooled and are depicted as a bar graph, as pERK1/2 normalized for total extracellular-signal-regulated kinase 1/2 levels. Statistical analysis of the quantitative pERK results was performed by one-way ANOVA followed by multiple comparisons to compare the mean of each group with the mean of the control group (Fisher's LSD test). *, P < .05; **, P < .01; ***, P < .001.

Figure 5.

Figure 5.

RF9 stimulates robust increases in LH in both WT and _Npffr1_−/− male (A) and female (B) mice. LH levels were measured in WT and _Npffr1_−/− mice 15 min after ICV injection of RF9 (5 nmol/5 μL) or vehicle. Each experimental group was composed of 7–10 mice. Statistical significance was analyzed by ANOVA followed by post-hoc Student-Newman-Keuls tests. **, P < .01 vs corresponding unstimulated groups of the same genotype; a, P < .05 vs corresponding WT groups.

Figure 6.

Figure 6.

Stimulation of LH secretion by RF9 is blunted in _Kiss1r_−/− and Kiss1r_−/−/Npffr1_−/− mice but restored in Kiss1r_−/−_T mice with selective reactivation of Kiss1r expression in GnRH neurons. LH levels were measured in WT, _Kiss1r_−/−, Kiss1r_−/−_T, and Kiss1r_−/−/Npffr1_−/− mice. Blood samples were collected 15 min after ICV injection of RF9 (5 nmol/5 μL). Each experimental group was composed of 4–10 mice. All tests were performed in both male (A) and female (B) mice, except for Kiss1r_−/−/Npffr1_−/− mice, which were only studied in females. Statistical significance was analyzed by ANOVA followed by post-hoc Student-Newman-Keuls tests. *, P < .05; **, P < .01 vs corresponding unstimulated groups of the same genotype; a, P < .01 vs corresponding WT values.

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