Modulation of osteopontin post-translational state by 1, 25-(OH)2-vitamin D3. Dependence on Ca2+ influx

In osteoblastic ROS 17/2.8 cells, 1,25-(OH)2-vitamin D3 stimulates transcription of the extracellular matrix phosphoprotein osteopontin (OPN). We now show post-translational regulation of OPN production by 1,25-(OH)2D3. Prior to transcriptional up-regulation of OPN, 1, 25-(OH)2D3 induces a shift in OPN isoelectric point (pI) from 4.6 to 5.1. Loading equal amounts of OPN recovered from ROS 17/2.8 cells exposed to 1,25-(OH)2D3 or carrier for 3 h reveals that the pI shift represents reduced phosphorylation. Trypsin cleavage patterns of OPN produced after 1,25-(OH)2D3 treatment indicates phosphorylation changes in the resulting peptides. Using structural analogs to 1, 25-(OH)2D3, we found that analog AT (25-(OH)-16-ene-23-yne-D3), which triggers Ca2+ influx but does not bind to the vitamin D receptor, mimicked the OPN pI shift, whereas analog BT (1, 25-(OH)2-22-ene-24-cyclopropyl-D3), which binds to the vitamin D receptor without triggering Ca2+ influx, did not. Likewise, inclusion of the Ca2+ channel blocker nifedipine blocks the charge conversion of OPN. Isolation of OPN from rat femurs and tibiae demonstrates the existence of two OPN charge forms in vivo. We conclude that 1,25-(OH)2D3 regulates OPN not only at the transcriptional level, but also modulates OPN phosphorylation state. The latter involves a short term (<3 h) treatment and is associated with membrane-initiated Ca2+ influx.