C-terminal parathyroid hormone-related protein increases vascular endothelial growth factor in human osteoblastic cells

The N-terminal region of parathyroid hormone (PTH) and PTH-related protein (PTHrP) interacts with a common PTH/PTHrP receptor in osteoblasts. These ce lls synthesize PTHrP, but its role in bone turnover is unclear. Intermitten t treatment with N-terminal PTHrP or PTH stimulates bone growth in vivo, po ssibly by increasing local bone factors. In addition, C-terminal PTHrP (107 -139), which does not bind to the PTH/PTHrP receptor, appears to affect bon e resorption in vivo and in vitro, although its effect on bone formation in vivo remains controversial. Bone angiogenesis is an often over-looked but critical event in the process of bone remodeling. Recently, PTH (1-34) has been shown to induce gene expression of vascular endothelial growth factor (VEGF), a potent angiogenic factor, by osteoblastic cells. However, no data are available on the effect of PTHrP (107-139) on VEGF expression in these cells. Using semiquantitative reverse transcription followed by PCR, we fo und that PTHrP (107-139), between 10 nM and 1 pM, increased VEGF mRNA in hu man osteoblastic (hOB) cells from trabecular bone. This effect of this agon ist, at 10 nM, was maximal (fivefold for VEGF(165), and twofold for VEGF(12 1), compared to control) within 1 to 4 h. This effect was similar to that i nduced by PTHrP (1-34) in these cells, as well as in human osteosarcoma MG- 63 cells, using Northern blot analysis. Moreover, the effect of both peptid es, added together at 100 pM, was not higher than that observed with each p eptide alone in hOB cells. The effects of PTHrP (107-139) and that of PTHrP (1-34) were abolished by actinomycin D in hOB cells. In these cells, the p rotein kinase C inhibitor staurosporine, but not the protein kinase A inhib itor H89, inhibited the increase in VEGF mRNA induced by 10 nM PTHrP (107-1 39). PTHrP (107-139), at 10 nM, also stimulated cytosolic VEGF immunostaini ng in hOB cells, and VEGF secretion into the medium conditioned by hOB or M G-63 cells for 24 h, which was (ng/mg protein): 10 +/- 1 or 5 +/- 3 (contro l), respectively, and 21 +/-. 1 or 11 +/- 2 (PTHrP [107-139]-stimulated), r espectively. Furthermore, medium conditioned by these cells for 24 h in the presence of 10 nM PTHrP (107-139), with or without 10 nM PTHrP (1-34), inc reased about 30% bovine aortic endothelial cell (BAEC) growth at 48 h. This effect was inhibited by adding a specific anti-VEGF antibody to the BAEC i ncubation medium. These findings demonstrate that the C-terminal domain of PTHrP induces expression and secretion of VEGF, a main angiogenic factor, i n hOB cells and MG-63 cells. This relationship between PTHrP and VEGF has p otential implications for both bone vascularization and bone formation, and neoangiogenesis in PTHrP-producing tumors.