Mutations in the Insulin-Like Factor 3 Receptor Are Associated With Osteoporosis

Abstract

ABSTRACT:Introduction: Insulin-like factor 3 (INSL3) is produced primarily by testicular Leydig cells. It acts by binding to its specific G protein-coupled receptor RXFP2 (relaxin family peptide 2) and is involved in testicular descent during fetal development. The physiological role of INSL3 in adults is not known, although substantial INSL3 circulating levels are present. The aim of this study was to verify whether reduced INSL3 activity could cause or contribute to some signs of hypogonadism, such as reduced BMD, currently attributed to testosterone deficiency. Materials and Methods: Extensive clinical, biochemical, and hormonal study, including bone densitometry by DXA, was performed on 25 young men (age, 27-41 yr) with the well-characterized T222P mutation in the RXFP2 gene. Expression analysis of INSL3 and RXFP2 on human bone biopsy and human and mouse osteoblast cell cultures was performed by RT-PCR, quantitative RT-PCR, and immunohistochemistry. Realtime cAMP imaging analysis and proliferation assay under the stimulus of INSL3 was performed on these cells. Lumbar spine and femoral bone of Rxfp2-deficient mice were studied by static and dynamic histomorphometry and CT, respectively. Results: Sixteen of 25 (64%) young men with RXFP2 mutations had significantly reduced BMD. No other apparent cause of osteoporosis was evident in these subjects, whose testosterone levels and gonadal function were normal. Expression analyses showed the presence of RXFP2 in human and mouse osteoblasts. Stimulation of these cells with INSL3 produced a dose-and time-dependent increase in cAMP and cell proliferation, confirming the functionality of the RXFP2/INSL3 receptor-ligand complex. Consistent with the human phenotype, bone histomorphometric and CT analyses of Rxfp2 −/− mice showed decreased bone mass, mineralizing surface, bone formation, and osteoclast surface compared with wildtype littermates. Conclusions: This study suggests for the first time a role for INSL3/RXFP2 signaling in bone metabolism and links RXFP2 gene mutations with human osteoporosis.

International Union of Basic and Clinical Pharmacology. XCV. Recent Advances in the Understanding of the Pharmacology and Biological Roles of Relaxin Family Peptide Receptors 1–4, the Receptors for Relaxin Family Peptides

“…In rat gubernacular cells , in a human osteoblast cell line (MG-63) (Ferlin et al, 2008), and in mouse primary Leydig cells (Pathirana et al, 2012), INSL3 stimulation of RXFP2 leads to increased cAMP accumulation, probably involving Ga s . However, and in contrast, primary cultures of testicular germ cells and oocytes respond to INSL3 activation of RXFP2 with a PTX-sensitive inhibition of cAMP accumulation (Kawamura et al, 2004), consistent with RXFP2 coupling to Ga oB .…”

“…RXFP2 mRNA and protein are present in human osteoblasts, and an osteoblast cell line responded to INSL3 with a concentration-dependent increase in cAMP. Mouse osteoblasts also express RXFP2, but not Insl3, and RXFP2 knockout mice are osteopenic and have functional osteoblast impairment (Ferlin et al, 2008). INSL3 regulates expression of genes required for proliferation and differentiation, matrix deposition, and osteoclastogenesis in cultured human osteoblasts .…”

“…Physiologic functions of INSL3 in other tissues have been postulated based on INSL3 and RXFP2 expression patterns. In young adults with cryptorchid hypogonadism associated with the T222P mutation of RXFP2, 64% had reduced bone density but normal plasma testosterone levels (Ferlin et al, 2008). RXFP2 mRNA and protein are present in human osteoblasts, and an osteoblast cell line responded to INSL3 with a concentration-dependent increase in cAMP.…”

Regulation of the reproductive cycle and early pregnancy by relaxin family peptides

“…In this context it is important to observe that recently INSL3 by acting on osteoclasts has been shown to have a significant role in the context of bone turnover and metabolism (Ferlin et al, 2008). Indeed, where the RXFP2 receptor (human subjects with a homozygous RXFP2 mutation) or its ligand INSL3 (INSL3 knockout mice) are dysfunctional, then there is a markedly increased osteopaenia (Ferlin et al, 2008).…”

“…In this context it is important to observe that recently INSL3 by acting on osteoclasts has been shown to have a significant role in the context of bone turnover and metabolism (Ferlin et al, 2008). Indeed, where the RXFP2 receptor (human subjects with a homozygous RXFP2 mutation) or its ligand INSL3 (INSL3 knockout mice) are dysfunctional, then there is a markedly increased osteopaenia (Ferlin et al, 2008). Thus in addition to estradiol, INSL3 is a further player in women of reproductive age contributing to the maintenance of healthy bone metabolism, and whose loss following the menopause may contribute to the increased prevalence of osteoporosis in older women.…”