Klaus G, von Eichel B, May T, Hügel U, Mayer H, Ritz E, Mehls O
Department of Pediatrics, University of Heidelberg, Germany.
Endocrinology. 1994 Oct;135(4):1307-15. doi: 10.1210/endo.135.4.7523093.
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.
我们使用大鼠胫骨近端骨骺软骨细胞原代培养物，研究了1,25 - 二羟基维生素D3 [1,25-(OH)2D3] 与甲状旁腺激素（PTH）在以下方面可能的相互作用：1）增殖（单层培养）和集落形成（琼脂糖稳定的悬浮培养）；2）1,25-(OH)2D3受体（VDR）的表达；3）对PTH的cAMP反应。低浓度（10(-12) M）的1α,25-(OH)2D3特异性刺激DNA合成、细胞计数和集落形成。在6小时内，牛甲状旁腺激素（bPTH）(1 - 34)、人甲状旁腺激素（hPTH）(28 - 48)（10(-10) M）、(Bu)2cAMP（1 - 2 mM）和12 - O - 十四烷酰 - 13 - 乙酸酯（10(-8) M）在有无1,25-(OH)2D3的情况下均增加了[3H]胸腺嘧啶核苷掺入。两种PTH片段也以钙依赖的方式刺激软骨细胞生长和集落形成。长时间暴露于bPTH(1 - 34)或hPTH(28 - 48)不影响基线DNA合成，但增加了1,25-(OH)2D3的刺激作用。在存在H7（抑制蛋白激酶C（PKC））或单克隆hPTH(1 - 38)抗体A1 - 70的情况下，这种增加受到抑制。在亚汇合软骨细胞培养物中，VDR被bPTH(1 - 34)和hPTH(28 - 48)（10(-10) M）或蛋白激酶C（PKC）激活剂上调，但不受(Bu)2cAMP上调。它被放线菌酮和放线菌素D阻断，并在存在钙通道阻滞剂的情况下持续存在。H7对PKC的抑制也阻断了bPTH(1 - 34)对VDR的作用。对bPTH(1 - 34)的cAMP反应不受1,25-(OH)2D3影响。我们得出结论：1）氨基末端和中间区域的PTH片段以及cAMP类似物以钙依赖的方式增加软骨细胞单层或悬浮培养中的DNA合成、细胞增殖和集落形成；2）bPTH(1 - 34)和hPTH(28 - 48)通过不依赖cAMP、依赖PKC的步骤上调VDR，这需要转录和翻译过程；两种PTH片段也放大了1,25-(OH)2D3对DNA合成的作用；3）尽管bPTH(1 - 34)和hPTH(28 - 48)片段在刺激cAMP产生方面有所不同，但在软骨细胞增殖和VDR上调方面未发现差异。
