Preproenkephalin (Penk) is expressed in differentiated osteoblasts, and its deletion in Hyp mice partially rescues their bone mineralization defect.

Although our understanding of the molecular mechanisms controlling osteoblast differentiation and function is steadily increasing, there are still many open questions, especially regarding the regulation of bone matrix mineralization. For instance, while there is hallmark evidence for the importance of the endopeptidase Phex, whose inactivation in Hyp mice or human patients causes X-linked hypophosphatemic rickets, it is still largely unknown how Phex controls bone mineralization since a physiological substrate for its endopeptidase activity has not been identified yet. Using a genome-wide expression analysis comparing primary calvarial osteoblasts, we have identified preproenkephalin (Penk) as a gene that is selectively expressed in mineralized cultures. Since a role of enkephalin in the regulation of bone remodeling has been suggested previously and since Leu-enkephalin is known to be cleaved by Phex, we analyzed whether Penk expression in osteoblasts is physiologically relevant. Through skeletal analysis of a Penk-deficient mouse model, we found that Penk expression is dispensable for bone development and remodeling since we could not detect any defect following nondecalcified bone histology and histomorphometry compared to wild-type littermates. When Penk was deleted in Phex-deficient Hyp mice, however, we observed a significant reduction of the osteoid enrichment at 24 weeks of age, whereas their disturbance of mineral homeostasis was not affected by the additional absence of the Penk gene. Taken together, our data provide the first in vivo analysis concerning the role of Penk in osteoblasts.