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 cells synthesize PTHrP, but its role in bone turnover is unclear. Intermittent treatment with N-terminal PTHrP or PTH stimulates bone growth in vivo, possibly by increasing local bone factors. In addition, C-terminal PTHrP (107-139), which does not bind to the PTH/PTHrP receptor, appears to affect bone resorption in vivo and in vitro, although its effect on bone formation in vivo remains controversial. Bone angiogenesis is an often overlooked 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 found that PTHrP (107-139), between 10 nM and 1 pM, increased VEGF mRNA in human osteoblastic (hOB) cells from trabecular bone. This effect of this agonist, at 10 nM, was maximal (fivefold for VEGF(165), and twofold for VEGF(121), compared to control) within 1 to 4 h. This effect was similar to that induced 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 peptides, added together at 100 pM, was not higher than that observed with each peptide 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 protein kinase C inhibitor staurosporine, but not the protein kinase A inhibitor H89, inhibited the increase in VEGF mRNA induced by 10 nM PTHrP (107-139). PTHrP (107-139), at 10 nM, also stimulated cytosolic VEGF immunostaining in hOB cells, and VEGF secretion into the medium conditioned by hOB or MG-63 cells for 24 h, which was (ng/mg protein): 10 +/- 1 or 5 +/- 3 (control), respectively, and 21 +/- 1 or 11 +/- 2 (PTHrP [107-139]-stimulated), respectively. 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), increased 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 incubation medium. These findings demonstrate that the C-terminal domain of PTHrP induces expression and secretion of VEGF, a main angiogenic factor, in hOB cells and MG-63 cells. This relationship between PTHrP and VEGF has potential implications for both bone vascularization and bone formation, and neoangiogenesis in PTHrP-producing tumors.
Esbrit
Pedro
P
Bone and Mineral Metabolism Laboratory, Fundación Jiménez Díaz, Madrid, Spain.
Alvarez-Arroyo
Maria Victoria
MV
Nephrology Laboratory, Fundación Jiménez Díaz, Madrid, Spain.
DE Miguel
Fernando
F
Bone and Mineral Metabolism Laboratory, Fundación Jiménez Díaz, Madrid, Spain.
Martin
Olga
O
Nephrology Laboratory, Fundación Jiménez Díaz, Madrid, Spain.
Martinez
Maria Eugenia
ME
Biochemistry Division, Hospital La Paz, Madrid, Spain.
Caramelo
Carlos
C
Nephrology Laboratory, Fundación Jiménez Díaz, Madrid, Spain.
D000704:Analysis of Variance; D015152:Blotting, Northern; D002478:Cells, Cultured; D016228:Endothelial Growth Factors / Q000378:metabolism* / Q000494:pharmacology*; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007124:Immunoenzyme Techniques; D008222:Lymphokines / Q000378:metabolism* / Q000494:pharmacology*; D010006:Osteoblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D010281:Parathyroid Hormone / Q000494:pharmacology*; D044162:Parathyroid Hormone-Related Protein; D010446:Peptide Fragments / Q000494:pharmacology; D011506:Proteins / Q000494:pharmacology*; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
D016228:Endothelial Growth Factors; D008222:Lymphokines; C470121:PTHLH protein, human; D010281:Parathyroid Hormone; D044162:Parathyroid Hormone-Related Protein; D010446:Peptide Fragments; D011506:Proteins; D012333:RNA, Messenger; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors