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.

Calbindin-D28K and -D9K and 1,25(OH)2 vitamin D3 receptor immunolocalization and mineralization induction in long-term primary cultures of rat epiphyseal chondrocytes.

Rat epiphyseal plat chondrocytes were grown on glass slides, as nonadhering monolayer cultures for up to 6 weeks. Chondrocyte growth, differentiation and maturation, matrix formation and mineralization, and the temporospatial distribution of the vitamin D-dependent calcium-binding proteins, calbindin-D9K and -D28K, and the 1,25(OH)2D3 receptor (VDR), were all monitored. Chondrocytes became confluent in 2.5 weeks, differentiated to acquire a chondrocyte (polygonal) morphology, produced extracellular matrix, and finally formed a true monolayer mineralizing cartilaginous tissue, with all the stages of chondrocyte development within a single culture. beta-Glycerophosphate promoted initial matrix mineralization in 4 weeks and accelerated cell differentiation. High nominal calcium and ascorbic acid were needed for abundant matrix formation. VDR occurred at all differentiation stages, in the nuclei and nucleoli and in the cytoplasm. Calbindin-D28K and -D9K were not coexpressed. Calbindin-D28K was found in prechondroblasts, chondroblasts, and in newly differentiated chondrocytes. It was cytoplasmic in prechondroblasts and subsequently also in the perinuclear region and in nuclei, suggesting migration to the nuclear chromatin. Calbindin-D28K was nuclear only in newly differentiated chondrocytes in vitro and was not found in mature chondrocytes. In contrast, calbindin-D9K was present in the cytoplasm of mature and hypertrophic chondrocytes only. It was first in the cell body and eventually migrated within and to the far end of long cell processes with a decreasing cytoplasmic concentration showed by decreased immunostaining intensity, and ultimately hypertrophy of chondrocytes in culture. These in vitro patterns of calbindins-D and VDR accurately reflect their in vivo distributions. The genomic action of vitamin D, in vitro, resulted in the synthesis of nuclear VDR and calbindins-D.(ABSTRACT TRUNCATED AT 250 WORDS)

Parathyroid hormone prevents 1,25 (OH)2D3 induced down-regulation of the vitamin D receptor in growth plate chondrocytes in vitro.

1,25(OH)2D3 has an antiproliferative effect on growth plate chondrocytes when given in high doses, whereas low doses stimulate chondrocyte proliferation. In the present in vitro study we investigated the effects of parathyroid hormone (PTH) when given concomitantly with 1,25(OH)2D3 on cell proliferation and vitamin D receptor (VDR) regulation. Freshly isolated rat tibial chondrocytes were grown in monolayer cultures or in agarose stabilized suspension cultures (10% charcoal-treated FCS). VDR expression was determined by RT-PCR generating a 297 bp fragment and by binding assays (Scatchard analysis) with [3H]-1,25(OH)2D3. Cell proliferation was measured by [3H]-thymidine incorporation, growth curves in monolayer cultures and by colony formation in agarose-stabilized suspension cultures. Optimal concentration of 1,25(OH)2D3 (10(-12) M) and of PTH fragments [bPTH(1-34) or hPTH(28-48), 10(-10)M] showed additive effects on DNA synthesis of and colony formation by growth plate chondrocytes. This may be explained in part by an up-regulation of VDR by PTH: PTH increased both mRNA expression of VDR and binding capacity. 1,25(OH)2D3 (10(-12) M) induced an up-regulation of the VDR within 24 hours followed by a down-regulation after incubation for more than 24 hours. PTH fragments added concomitantly prevented the down-regulation seen with 1,25(OH)2D3. These findings provide evidence that PTH is a growth promoting hormone that also modulates the effects of 1,25(OH)2D3 by regulating the VDR status of 1,25(OH)2D3 target cells.