title: Mutations in the insulin-like factor 3 receptor are associated with osteoporosis.
abstract: BACKGROUND
  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.
  
  
  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.
authors.affiliation: Department of Histology, Microbiology and Medical Biotechnologies, Section of Clinical Pathology and Centre for Male Gamete Cryopreservation, University of Padova, Padova, Italy.
authors.forename: Alberto
authors.initials: A
authors.lastname: Ferlin
authors.forename: Anastasia
authors.initials: A
authors.lastname: Pepe
authors.forename: Lisa
authors.initials: L
authors.lastname: Gianesello
authors.forename: Andrea
authors.initials: A
authors.lastname: Garolla
authors.forename: Shu
authors.initials: S
authors.lastname: Feng
authors.forename: Sandro
authors.initials: S
authors.lastname: Giannini
authors.forename: Manuela
authors.initials: M
authors.lastname: Zaccolo
authors.forename: Arianna
authors.initials: A
authors.lastname: Facciolli
authors.forename: Roy
authors.initials: R
authors.lastname: Morello
authors.forename: Alexander I
authors.initials: AI
authors.lastname: Agoulnik
authors.forename: Carlo
authors.initials: C
authors.lastname: Foresta
mesh_terms: D015502:Absorptiometry, Photon; D000328:Adult; D006801:Humans; D007150:Immunohistochemistry; D007328:Insulin / Q000235:genetics*; D008297:Male; D009154:Mutation; D010024:Osteoporosis / Q000000981:diagnostic imaging / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011506:Proteins / Q000235:genetics*
chemical_list: D007328:Insulin; C084797:Leydig insulin-like protein; D011506:Proteins