Min Le, Leon Silvia, Li Huan, Pinilla Leonor, Carroll Rona S, Tena-Sempere Manuel, Kaiser Ursula B
Division of Endocrinology, Diabetes and Hypertension (L.M., H.L., R.S.C., U.B.K.), Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115; Department of Cell Biology, Physiology and Immunology (S.L., L.P., M.T.-S.), University of Córdoba; CIBER Fisiopatología de la Obesidad y Nutrición, Instituto de Salud Carlos III, and Instituto Maimónides de Investigación Biomédica de Córdoba/Hospital Universitario Reina Sofia, Córdoba, 14004 Spain; and FiDiPro Program, Department of Physiology (M.T.-S.), University of Turku, FIN-20520 Turku, Finland.
Endocrinology. 2015 Dec;156(12):4639-48. doi: 10.1210/en.2015-1635. Epub 2015 Sep 29.
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.
RF9是一种已报道的哺乳动物促性腺激素抑制激素受体拮抗剂，可刺激哺乳动物的促性腺激素分泌。最近的研究表明，RF9对促性腺激素分泌的刺激作用依赖于完整的 kisspeptin 受体（KISS1R）信号传导，但其潜在机制仍有待阐明。我们使用稳定转染了KISS1R的中国仓鼠卵巢细胞，发现RF9以1.6×10(-5)M的解离常数（Kd）特异性结合KISS1R，并以KISS1R依赖的方式刺激细胞内钙增加和肌醇磷酸积累，其半数有效浓度（EC50）值分别为3.0×10(-6)M和1.6×10(-7)M。RF9还刺激细胞外调节蛋白激酶（ERK）磷酸化，其时间进程与kisspeptin-10相似。NPFFR1的假定内源性配体RFRP-3既不刺激肌醇磷酸积累或pERK，也不改变对kisspeptin-10或RF9的反应。与这些体外数据一致，我们发现RF9在Npffr1(-/-)小鼠中刺激促黄体生成素（LH）显著增加，类似于野生型同窝小鼠，而在Kiss1r(-/-)和双敲除Kiss1r(-/-)/Npfrr1(-/-)小鼠中，RF9的刺激作用明显降低。在Kiss1r(-/-T)小鼠中，通过GnRH神经元中Kiss1r表达的选择性拯救，恢复了RF9对LH分泌的刺激作用。综上所述，我们的研究表明，RF9主要作为KISS1R激动剂而非变构调节剂来刺激LH分泌。我们的发现对RF9用于评估NPFF1R功能的效用提出了疑问，并弱化了该信号系统在生殖中枢调节中的主要作用。
