Cellular Localization of Neprilysin in Mouse Bone Tissue and Putative Role in Hydrolysis of Osteogenic Peptides

Abstract The regulation of osteoblast and osteoclast metabolism is mediated by both hormones and local bone peptide factors. Peptides and hormones are under control of membrane peptidases such as Neprilysin (NEP). NEP is a widely distributed cell‐surface zinc‐metallopeptidase that is involved in the regulation of several important physiological processes by controlling the half‐life of bioactive peptides. Although NEP is known to be present in skeletal tissues, neither its cellular localization nor its function have been established. To address this question, we examined NEP distribution in bones of postnatal 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, osteoblasts, and osteocytes. NEP levels in newborn and adult mice bones also were compared by immunoblotting. Higher amounts of NEP immunoreactivity were observed in newborn as compared with adult bones, suggesting a relationship between NEP expression and bone growth. To further explore this hypothesis, we monitored in vitro NEP proteolytic activity using a series of synthetic osteogenic peptides such as parathyroid hormone‐related peptide 1–43 (PTHrP 1–34 ), osteostatin (PTHrP 107–139 ), osteogenic growth peptide (OGP), calcitonin, α‐calcitonin gene‐related peptide (α‐CGRP), and PTH 1–34 . Except for PTH 1–34 , all peptides were found to be NEP substrates.