Developmental regulation of proenkephalin gene expression in osteoblasts.

Proenkephalin (PENK), a classically defined opioid gene, was originally thought to be expressed almost exclusively in the mature nervous and neuroendocrine systems. In the last few years, it was demonstrated, however, that high levels of PENK messenger RNA and PENK-derived peptides are expressed in embryonic mesenchymal tissues during differentiation into mature tissues and organs. Shortly after birth, as development progresses, PENK expression drops in those tissues to undetectable levels. Very little is known about the molecular mechanisms regulating this transient expression. To investigate those mechanisms, we used primary cell cultures of calvaria-derived osteoblasts. These cultures express PENK and exhibit a normal pattern of osteoblastic differentiation. In the present study we demonstrate that 1) a reciprocal interrelationship exists between PENK expression and osteoblastic differentiation in vivo, ex vivo, and in vitro; namely, PENK expression is down-regulated upon cellular differentiation; 2) PENK promoter usage and messenger RNA splicing function similarly in osteoblasts and in neural cells; 3) osteoblastic PENK expression is modulated by bone-targeting hormones; and 4) this down-regulation is inhibited by the serine/threonine kinase inhibitor H-8. The link between osteoblastic differentiation and down-regulation of PENK expression together with our preliminary findings indicating the existence of an osteoblastic opioid receptor suggest that opioids act in an autocrine/paracrine mechanism on undifferentiated osteoblasts and play a significant role in bone development.