Profiling insulin like factor 3 (INSL3) signaling in human osteoblasts.
BACKGROUND
Young men with mutations in the gene for the INSL3 receptor (Relaxin family peptide 2, RXFP2) are at risk of reduced bone mass and osteoporosis. Consistent with the human phenotype, bone analyses of Rxfp2(-/-) mice showed decreased bone volume, alterations of the trabecular bone, reduced mineralizing surface, bone formation, and osteoclast surface. The aim of this study was to elucidate the INSL3/RXFP2 signaling pathways and targets in human osteoblasts.


RESULTS
Alkaline phosphatase (ALP) production, protein phosphorylation, intracellular calcium, gene expression, and mineralization studies have been performed. INSL3 induced a significant increase in ALP production, and Western blot and ELISA analyses of multiple intracellular signaling pathway molecules and their phosphorylation status revealed that the MAPK was the major pathway influenced by INSL3, whereas it does not modify intracellular calcium concentration. Quantitative Real Time PCR and Western blotting showed that INSL3 regulates the expression of different osteoblast markers. Alizarin red-S staining confirmed that INSL3-stimulated osteoblasts are fully differentiated and able to mineralize the extracellular matrix.


CONCLUSIONS
Together with previous findings, this study demonstrates that the INSL3/RXFP2 system is involved in bone metabolism by acting on the MAPK cascade and stimulating transcription of important genes of osteoblast maturation/differentiation and osteoclastogenesis.
Section of Clinical Pathology, Department of Histology, Microbiology and Medical Biotechnologies, University of Padova, Padova, Italy.
Alberto
A
Ferlin
Lisa
L
Perilli
Lisa
L
Gianesello
Giuseppe
G
Taglialavoro
Carlo
C
Foresta
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D015870:Gene Expression; D006801:Humans; D007328:Insulin / Q000378:metabolism*; D051379:Mice; D010006:Osteoblasts / Q000378:metabolism*; D010766:Phosphorylation; D011506:Proteins / Q000378:metabolism*; D015398:Signal Transduction
D007328:Insulin; C084797:Leydig insulin-like protein; D011506:Proteins; D000469:Alkaline Phosphatase; D002118:Calcium