Anabolic effects of 1,25-dihydroxyvitamin D-3 on osteoblasts are enhanced by vascular endothelial growth factor produced by osteoblasts and by growth factors produced by endothelial cells

Human osteoblast-like cells (HOB) produce vascular endothelial growth factor (VEGF), the steady state level of which is stimulated by 1,25-dihydroxyvitamin D-3 [1,25-(OH)(2)D-3]. As osteoblasts and endothelial cells are proximally located in skeletal tissue, we investigated the anabolic effects of 1,25-(OH)(2)D-3 and VEGF on HOB cocultured with endothelial cells. When KOB with high alkaline phosphatase (AI-P) activity and human umbilical vein endothelial cells (HUVEC) with little activity were cultured together, AI-P activity increased, accompanied by an increase in cell number. When KOB and HUVEC were cultured separately, 1,25-(OH)(2)D-3 did not directly stimulate [H-3]thymidine incorporation into HUVEC, but stimulated it in the presence of HOB. VEGF did not directly stimulate the AI-P activity of HOB but stimulated it in the presence of HUVEC. The conditioned medium of HOB stimulated the proliferation of HUVEC, and this was partially blocked by anti-VEGF antibody. Conversely, the conditioned medium of HUVEC increased Al-P activity and [H-3]thymidine incorporation into HOB, and this was partially blocked by antiinsulin-like growth factor I antibody and BQ-123, a specific antagonist of the endothelin-l (ET-1) receptor. 1,25-(OH)(2)D-3 stimulated the release of VEGF and ET-1 from HOB and HUVEC, respectively. Furthermore, the 1,25-(OH)(2)D-3-induced release of VEGF was enhanced in KOB cocultured with HUVEC. A quantitative reverse transcription-PCR study revealed that genes for VEGF receptors (Flt-1 and KDR) were expressed in HUVEC, but not in HOB, and that 1,25-(OH)(2)D-3 increased the levels of expression of VEGF receptor genes in endothelial cells only when cocultured with HOB. In summary, we demonstrated that 1,25-(OH)(2)D-3 exerts an anabolic effect on osteoblasts by enhancing their production of VEGF, which stimulates its receptors on endothelial cells, followed by increased production of osteotropic growth factors, such as insulin-like growth factor I and ET-1. These in vitro findings suggest that the VEGF/VEGF receptor system may be involved in both bone formation and bone remodeling in vivo.