Relaxin stimulates osteoclast differentiation and activation Alberto Ferlin, Anastasia Pepe, Arianna Facciolli, Lisa Gianesello, Carlo Foresta ⁎ Section of Clinical Pathology and Center for Male Gamete Cryopreservation, Department of Histology, Microbiology and Medical Biotechnologies, University of Padova, Via Gabelli 63, 35121 Padova, Italy abstract article info Article history: Received 11 April 2009 Revised 5 October 2009 Accepted 6 October 2009 Available online 13 October 2009 Edited by: R. Baron Keywords: Osteoclast Relaxin RXFP1 RANK Osteoclastogenesis 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. © 2009 Elsevier Inc. All rights reserved. Introduction The peptide hormone relaxin was originally described as an insulin-like hormone produced in the ovaries during pregnancy, but it was later recognized as a multi-functional factor in a broad range of target tissues including several non-reproductive organs also in males, where it is produced mainly by the prostate [1]. Major effects of relaxin include extracellular matrix remodelling, collagen degra- dation and up-regulation of matrix metalloproteinases [2], and it has a role in fibrosis, wound healing, angiogenesis and response to infarction [2,3]. Relaxin is also a cardiotropic hormone [4], is a potent endothelium-dependent vasodilator of human systemic arteries [5], and regulates platelet and mast cell function [6]. Furthermore, recently relaxin has been implicated as autocrine/ paracrine factor important for tumour biology [7]. It is implicated in various mechanisms associated with tumour cell growth, differenti- ation, local invasion and metastasis of different tumours, especially those that give bone metastases, such as breast, prostate, thyroid, and myeloma [7]. Importantly, relaxin expression has been shown to correlate with cancer progression. For example, high levels of relaxin expression are associated with aggressive features of endometrial and prostate carcinoma [8,9], serum relaxin concentrations are signifi- cantly elevated in advanced breast cancer patients [10,11], and serum relaxin levels are higher in patients with metastatic prostate cancers with respect to those without metastases [12,13]. However, the possible role of relaxin in bone metastases has not been investigated. In osteolytic metastases, the destruction of bone is primarily mediated by osteoclasts, the cells that resorb the bone. Osteoclasts are giant, multinucleated cells that derive from the differentiation of hematopoietic precursors of the monocyte–macrophage lineage [14]. The bone micro-environment plays a critical role in the differentiation and activation of osteoclasts. Osteoblasts, the cells that form the bone, express key factors that modulate the differentiation of osteoclast precursors in mature osteoclasts (osteoclastogenesis): macrophage colony-stimulating factor (M-CSF), which acts on osteoclasts through its receptor c-FMS, and receptor activator of nuclear factor-κB (RANK) ligand (RANKL), which acts on osteoclasts through its receptor RANK and stimulates osteoclast differentiation and activity. On the contrary, the decoy receptor for RANKL, osteoprotegerin (OPG, a member of the superfamily of the tumour necrosis factor receptors), prevents the activation of RANK and thus it inhibits osteoclasts recruitment [15]. Previous studies demonstrated that peripheral blood monocyte cells (PBMC) express the mRNA for the relaxin receptor and respond Bone 46 (2010) 504–513 ⁎ Corresponding author. Fax: +39 049 8218520. E-mail address: carlo.foresta@unipd.it (C. Foresta). 8756-3282/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.bone.2009.10.007 Contents lists available at ScienceDirect Bone journal homepage: www.elsevier.com/locate/bone