Cellular localization of neprilysin in mouse bone tissue and putative rolein hydrolysis of osteogenic peptides

The regulation of osteoblast and osteoclast metabolism is mediated by both hormones and local bone peptide factors. Peptides and hormones are under co ntrol of membrane peptidases such as Neprilysin (NEP). NEP is a widely dist ributed cell-surface zinc-metallopeptidase that is involved in the regulati on of several important physiological processes by controlling the half-lif e of bioactive peptides, Although NEP is known to be present in skeletal ti ssues, neither its cellular localization nor its function have been establi shed. To address this, question, we examined NEP distribution in bones of p ostnatal mouse. In situ hybridization (ISH) and immunohistochemistry showed that NEP messenger RNA (mRNA) and protein are associated with bone-forming cells including presumptive osteoblast precursors, preosteoblasts, osteobl asts, and osteocytes. NEP levels in newborn and adult mice bones also were compared by immunoblotting. Higher amounts of NEP immunoreactivity were obs erved in newborn as compared with adult bones, suggesting a relationship be tween NEP expression and bone growth. To further explore this hypothesis, w e monitored in vitro NEP proteolytic activity using a series of synthetic o steogenic peptides such as parathyroid hormone-related peptide 1-43 (PTHrP( 1-34)), osteostatin (PTHrP(107-139)), osteogenic growth peptide (OGP), calc itonin, alpha-calcitonin gene-related peptide (alpha-CGRP), and PTH1-34. Ex cept for PTH1-34, all peptides were found to be NEP substrates.