Relaxin stimulates osteoclast differentiation and activation.

Relaxin is a pleiotropic hormone with actions in reproductive and non-reproductive tissues, and has a role in tumor biology. It can promote growth, differentiation and invasiveness of different tumors, especially those that give bone metastases, and relaxin serum concentrations are increased in patients with bone metastasis. In osteolytic metastasis the destruction of bone is mediated by osteoclasts that are multinucleated cells derived from hematopoietic progenitors. We found that human hematopoietic precursors and mature osteoclasts express the relaxin receptor RXFP1. Then, we investigated the effects of relaxin on the differentiation, activation and gene expression of osteoclasts during in vitro osteoclastogenesis from human hematopoietic progenitor cells. Relaxin alone was able to induce the multistep differentiation process of human osteoclastogenesis with timing similar to that obtained with the classical stimulators of osteoclastogenesis RANKL, M-CSF and PTH. The expression profile of several osteoclast genes was studied with quantitative RT-PCR during the entire process of osteoclastogenesis. This analysis showed that relaxin induced genes that are implicated in the differentiation, survival and activation of osteoclasts. Relaxin-induced osteoclasts were fully differentiated, positive for tartrate resistant acid phosphatase and vitronectin receptor, expressing a typical F-actin ring and able to resorb the bone. Furthermore, relaxin induced the expression of its specific receptor RXFP1 in osteoclasts. This study demonstrates for the first time that relaxin is a potent stimulator of osteoclastogenesis from hematopoietic precursors and regulates the activity of mature osteoclasts, opening new perspectives on the role of this hormone in bone physiology, diseases and metastasis.

RXFP1 is expressed on the sperm acrosome, and relaxin stimulates the acrosomal reaction of human spermatozoa.

The function of relaxin in human reproductive processes remains poorly understood. Nevertheless, relaxin is produced by the prostate in men and by the corpora lutea of ovaries and endometrium in women. Previous studies with contrasting results have suggested a possible role of relaxin on sperm function and fertilization. Here we show the expression of the relaxin receptor RXFP1 on the acrosome of human spermatozoa. Furthermore, relaxin induced the acrosome reaction and, interestingly, the percentage of viable sperm with the acrosome reaction induced by relaxin was similar to that obtained with progesterone. These data suggest a physiological role on sperm for relaxin produced by the prostate and/or the female genital tract.

New roles for INSL3 in adults.

Insulin-like factor 3 (INSL3) is produced primarily by testicular Leydig cells and acts by binding to its specific G-protein-coupled receptor, RXFP2 (relaxin family peptide 2). INSL3 is involved in testicular descent during fetal development, and mutations in the INSL3 and RXFP2 genes cause cryptorchidism. The physiological role of INSL3 in adults is not known, although substantial INSL3 circulating levels are present. After extensive clinical, biochemical, and hormonal investigations, including bone densitometry with dual energy X-ray absorptiometry, on 25 young men (age, 27-41 years) who have the well-characterized T222P mutation in the RXFP2 gene, we found that 16 of them (64%) had significantly reduced bone density. No other cause of osteoporosis was evident in these subjects, whose testosterone and gonadal function were normal. Expression analysis of INSL3 and RXFP2 on human bone biopsy and human and mouse osteoblast cell cultures performed by reverse transcription-PCR and immunohistochemistry showed the presence of RXFP2 in these cells. Real-time cAMP imaging analysis and proliferation assays under the stimulus of INSL3 showed a dose- and time-dependent increase in cAMP and cell proliferation, and specific osteoblast gene activation was observed by real-time PCR after INSL3 stimulation. Lumbar spine and femoral bone of Rxfp2-deficient mice were studied by static and dynamic histomorphometry and micro-computed tomography, respectively, and showed decreased bone mass, mineralizing surface, bone formation, and osteoclast surface compared to wild-type littermates, compatible with a functional osteoblast impairment. This study identified for the first time a role for INSL3 in adults, demonstrating a modulating effect on bone metabolism and linking RXFP2 gene mutations with human osteoporosis.

INSL3 plays a role in the balance between bone formation and resorption.

We have recently demonstrated that the disruption of the INSL3 hormonal system, as observed in humans with mutations in the RXFP2 gene and in Rxfp2-deficient mice, might affect the equilibrium of the osteoblast-osteoclast system, resulting in an imbalance between bone formation and bone resorption that may result in reduced bone mass. In the present study we have better characterized the in vitro effects of INSL3 on human osteoblasts. Stimulation of human primary osteoblasts with INSL3 at serial concentrations (1 pM, 1 nM, and 1 microM) induced a dose-dependent increase in osteoblast proliferation and expression of specific osteoblast genes.

Role of relaxin in human osteoclastogenesis.

Recently, we have demonstrated that INSL3 is important for bone metabolism, and in this study we analyzed the possible role of the cognate hormone relaxin on human osteoclasts. In fact, previous studies showed an effect of relaxin on peripheral blood mononuclear cells (PBMCs), the precursors of osteoclasts. Analysis of the expression of the relaxin receptor RXFP1 and RLN-2 mRNA in primary cell cultures of human osteoclasts obtained from PBMCs showed by reverse transcriptase PCR and immunofluorescence only the presence of RXFP1 transcripts. Analysis of the in vitro effects of relaxin on osteoclastogenesis showed that relaxin is able alone to induce the differentiation of PBMCs into mature osteoclasts. This study shows, for the first time, that relaxin has an effect on bone metabolism, facilitating the differentiation of osteoclasts. A possible role for relaxin in osteolytic bone metastasis is also proposed.

Mutations in the insulin-like factor 3 receptor are associated with osteoporosis.

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. Real-time 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 muCT, 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 muCT 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.