Synergistic effects of parathyroid hormone and 1,25-dihydroxyvitamin D3 on proliferation and vitamin D receptor expression of rat growth cartilage cells.
We investigated possible interaction of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] and PTH on: 1) proliferation (monolayer culture) and colony formation (agarose stabilized suspension cultures); 2) expression of 1,25-(OH)2D3 receptor (VDR); and 3) cAMP response to PTH, using primary cultures of chondrocytes from rat tibia proximal epiphysis. 1 alpha,25-(OH)2D3 stereospecifically stimulated DNA synthesis, cell counts, and colony formation at low concentration (10(-12) M). Within 6 h bovine PTH (bPTH)(1-34), human PTH (hPTH)(28-48) (10(-10) M), (Bu)2cAMP (1-2 mM), and 12-O-tetradecanoyl-13-acetate (10(-8) M) increased [3H]thymidine incorporation in the absence and presence of 1,25-(OH)2D3. Both PTH fragments also stimulated chondrocyte growth and colony formation in a Ca-dependent fashion. Prolonged exposure to bPTH(1-34) or hPTH(28-48) did not affect baseline DNA synthesis but increased the stimulatory effect of 1,25-(OH)2D3. This increase was inhibited in the presence of H7 (inhibition of PKC) or the monoclonal hPTH(1-38) antibody A1-70. In subconfluent chondrocyte cultures VDR was up-regulated by bPTH(1-34) and hPTH(28-48) (10(-10) M) or activators of protein kinase C (PKC), but not by (Bu)2cAMP. It was blocked by cycloheximide and actinomycin D and persisted in the presence of Ca-channel blockers. Inhibition of PKC by H7 also blocked the effect of bPTH(1-34) on VDR. The cAMP response to bPTH(1-34) was not affected by 1,25-(OH)2D3. We conclude that: 1) DNA synthesis, cell proliferation, and colony formation in chondrocyte monolayer or suspension cultures is increased by aminoterminal and midregional PTH fragments and by cAMP analogs in a Ca- dependent fashion; 2) bPTH(1-34) and hPTH(28-48) up-regulate VDR by cAMP-independent, PKC-dependent steps requiring transcriptional and translational processes; both PTH fragments also amplify the effect of 1,25-(OH)2D3 on DNA synthesis; and 3) no difference is found between the bPTH(1-34) and hPTH(28-48) fragments with respect to chondrocyte proliferation and VDR up-regulation, although the two differ with respect to stimulation of cAMP production.
Klaus
G
G
Department of Pediatrics, University of Heidelberg, Germany.
von Eichel
B
B
May
T
T
Hügel
U
U
Mayer
H
H
Ritz
E
E
Mehls
O
O
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D000818:Animals; D002117:Calcitriol / Q000494:pharmacology*; D002455:Cell Division / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism; D003513:Cycloheximide / Q000494:pharmacology; D004247:DNA / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D003609:Dactinomycin / Q000494:pharmacology; D004357:Drug Synergism; D006132:Growth Plate / Q000737:chemistry* / Q000166:cytology* / Q000378:metabolism; D007546:Isoquinolines / Q000494:pharmacology; D010281:Parathyroid Hormone / Q000494:pharmacology*; D010446:Peptide Fragments / Q000494:pharmacology; D010879:Piperazines / Q000494:pharmacology; D047428:Protein Kinase Inhibitors; D051381:Rats; D017207:Rats, Sprague-Dawley; D018167:Receptors, Calcitriol / Q000032:analysis* / Q000235:genetics / Q000502:physiology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013936:Thymidine / Q000378:metabolism; D014316:Tritium
D007546:Isoquinolines; D010281:Parathyroid Hormone; D010446:Peptide Fragments; D010879:Piperazines; D047428:Protein Kinase Inhibitors; D018167:Receptors, Calcitriol; C069678:parathyroid hormone (28-48); D014316:Tritium; C025076:parathyroid hormone (1-34), bovine; D003609:Dactinomycin; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D004247:DNA; D003513:Cycloheximide; D000242:Cyclic AMP; D002117:Calcitriol; D013755:Tetradecanoylphorbol Acetate; D013936:Thymidine