Synergistic effects of parathyroid hormone and 1,25-dihydroxyvitamin D3 on proliferation and vitamin D receptor expression of rat growth cartilage cells.

Abstract

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.

Dexamethasone Impairs Growth Hormone (GH)-Stimulated Growth by Suppression of Local Insulin-Like Growth Factor (IGF)-I Production and Expression of GH- and IGF-I-Receptor in Cultured Rat Chondrocytes*

“…Epiphyseal chondrocytes from 60-to 80-g Sprague Dawley rats (Charles River, Kieslegg, Germany) were isolated and cultured as described previously (15). Epiphyseal chondrocytes from 60-to 80-g Sprague Dawley rats (Charles River, Kieslegg, Germany) were isolated and cultured as described previously (15).…”

“…Cells were cultured as described earlier (15)(16)(17) in 35-mm plastic dishes (Nunc, Wiesbaden, Germany) for growth curves and determination of local IGF-I synthesis, in 96-well plates for proliferation assays and in 24-well plates (Nunc, Wiesbaden, Germany) for binding studies. Cells were cultured as described earlier (15)(16)(17) in 35-mm plastic dishes (Nunc, Wiesbaden, Germany) for growth curves and determination of local IGF-I synthesis, in 96-well plates for proliferation assays and in 24-well plates (Nunc, Wiesbaden, Germany) for binding studies.…”

“…Cells were cultured in agarose according to Benya and Schaffer (16) as described earlier (15)(16)(17) in 35-mm dishes (Falcon Plastics). Cells were cultured in agarose according to Benya and Schaffer (16) as described earlier (15)(16)(17) in 35-mm dishes (Falcon Plastics).…”

Thyroid hormone actions on cartilage and bone: interactions with other hormones at the epiphyseal plate and effects on linear growth

“…Thus 1,25(OH) 2 -vitamin D 3 inhibits precocious chondrocyte hypertrophy and its deficiency destroys the balance of inhibition and stimulation of chondrocyte maturation, leading to widening of the hypertrophic zone in rachitic growth plates. Nuclear receptors for 1,25(OH) 2 -vitamin D 3 have been demonstrated in murine, rat and rabbit chondrocytes in vitro ( Jikko et al 1993, Klaus et al 1994, Balmain et al 1995, Akiyama et al 1996 and in sections of chicken and rat growth plates (Klaus et al 1991, Berry et al 1996. They are expressed throughout the growth plate region, but are most concentrated in proliferative and hypertrophic cells (Klaus et al 1991, Berry et al 1996.…”

PTH Stimulated Growth and Decreased Col‐X Deposition are Phosphotidylinositol‐3,4,5 Triphosphate Kinase and Mitogen Activating Protein Kinase Dependent in Avian Sterna

“…PPR may regulate Col-X, Bcl-2, and vitamin D receptor expression via the PKC pathway (Klaus et al, 1994;Zhen et al, 2001). The concerted participation of many proteins control chondrogenesis including Ihh, TGFb2, and cyclin D1 are within pathways downstream and upstream of the PPR.…”