PTHrP produced by osteoblastic cells is a paracrine regulator of bone remodelling. Because we found that genetic ablation of PTHrP in osteocytes results in decreased bone formation and strength, we investigated the nature of PTHrP released by osteocytes, using the osteocyte cell line, Ocy454.

Knockdown of PTHrP in Ocy454 cells resulted in increased osteocyte genes Sost, Dmp1 and Mepe as well as reduced cAMP formation in short incubations with phosphodiesterase inhibitor, indicating that autocrine/paracrine actions of PTHrP regulate osteocyte gene expression. A similar result was obtained with PTHR1 knockout using CRISPR/Cas9.  

To investigate how PTHrP is processed and secreted in osteocytes we prepared five PTHrP mutant constructs, to overexpress full length secreted (-36-139), non-secreted (1-139), truncated (-36-67), PTHrP lacking its nuclear localizing sequence (-36-67:95-139), and vector control. All three constructs containing the leader sequence/ secretion signal were associated with substantial activity in conditioned media, assessed by biological assay and radioimmunoassay.  No activity was detected in media from those cells infected with the (1-139) construct, confirming its lack of secretion.

We determined the molecular form of PTHrP secreted by osteocytes and therefore available for autocrine/paracrine action. After collecting biological activity from cation exchange chromatography of media, samples were subjected to polyacrylamide gel electrophoresis, gel slices extracted and assayed for PTHrP by N-terminal directed radioimmunoassay.  PTHrP(-36-139) cells yielded medium activity of approximately 17 kDa with no evidence of any lower molecular weight secreted forms. The constructs for PTHrP (-36-67:95-139) and PTHrP(-36-67) yielded peaks at 14 kDa. As expected, electrophoresis of synthetic PTHrP(1-34) appeared at a much lower molecular weight. Together the findings show that PTHrP is not processed from the Ocy454 osteocytes to any lower molecular weight forms that act through the PTHR1, and identify full length PTHrP as the form acting as a local physiological stimulus of bone formation.