Schwartz Z, Sylvia V L, Dean D D, Boyan B D
Department of Orthopaedics, The University of Texas Health Science Center at San Antonio, 78284-7774, USA.
Endocrinology. 1998 Feb;139(2):534-45. doi: 10.1210/endo.139.2.5753.
Transforming growth factor-beta (TGFbeta), as well as the vitamin D3 metabolites 1,25-dihydroxyvitamin D3 (1,25) and 24,25-dihydroxyvitamin D3 (24,25), regulate chondrocyte differentiation and maturation during endochondral bone formation. Both the growth factor and secosteroids also affect protein kinase C (PKC) activity, although each has its own unique time course of enzyme activation. Vitamin D3 metabolite effects are detected soon after addition to the media, whereas TGFbeta effects occur over a longer term. The present study examines the interrelation between the effects of 1,25, 24,25, and TGFbeta on chondrocyte differentiation, matrix production, and proliferation. We also examined whether the effect is hormone-specific and maturation-dependent and whether the effect of combining hormone and growth factor is mediated by PKC. This study used a chondrocyte culture model developed in our laboratory that allows comparison of chondrocytes at two stages of differentiation: the more mature growth zone (GC) cells and the less mature resting zone chondrocyte (RC) cells. Only the addition of 24,25 with TGFbeta showed synergistic effects on RC alkaline phosphatase-specific activity (ALPase). No similar effect was found when 24,25 plus TGFbeta was added to GC cells or when 1,25 plus TGFbeta were added to GC or RC cells. The addition of 1,25 plus TGFbeta and 24,25 plus TGFbeta to GC and RC cells, respectively, produced a synergistic increase in [35S]sulfate incorporation and had an additive effect on [3H]thymidine incorporation. To examine the signal transduction pathway involved in producing the synergistic effect of 24,25 and TGFbeta on RC cells, the level of PKC activity was examined. Addition of 24,25 and TGFbeta for 12 h produced a synergistic increase in PKC activity. Moreover, a similar effect was found when 24,25 was added for only the last 90 min of a 12-h incubation. However, a synergistic effect could not be found when 24,25 was added for the last 9 min or the first 90 min of incubation. To further understand how 24,25 and TGFbeta may mediate the observed synergistic increase in PKC activity, the pathways potentially leading to activation of PKC were examined. It was found that 24,25 affects PKC activity through production of diacylglycerol, not through activation of G protein, whereas TGFbeta only affected PKC activity through G protein. The results of the present study indicate that vitamin D metabolites and TGFbeta produced a synergistic effect that is maturation-dependent and hormone-specific. Moreover, the synergistic effect between 24,25 and TGFbeta was mediated by activation of PKC through two parallel pathways: 24,25 through diacylglycerol production and TGFbeta through G protein activation.
转化生长因子-β（TGFβ）以及维生素D3代谢产物1,25-二羟基维生素D3（1,25）和24,25-二羟基维生素D3（24,25）在软骨内骨形成过程中调节软骨细胞的分化和成熟。生长因子和甾醇类激素均会影响蛋白激酶C（PKC）的活性，不过它们各自具有独特的酶激活时间进程。维生素D3代谢产物的作用在添加到培养基后很快就能检测到，而TGFβ的作用则需要更长时间才会出现。本研究探讨了1,25、24,25和TGFβ对软骨细胞分化、基质产生和增殖的影响之间的相互关系。我们还研究了这种作用是否具有激素特异性和成熟依赖性，以及激素与生长因子联合作用的效果是否由PKC介导。本研究使用了我们实验室开发的软骨细胞培养模型，该模型能够比较处于两个分化阶段的软骨细胞：更成熟的生长区（GC）细胞和不太成熟的静止区软骨细胞（RC）细胞。仅24,25与TGFβ联合添加时，对RC碱性磷酸酶特异性活性（ALPase）显示出协同作用。当将24,25加TGFβ添加到GC细胞中，或把1,25加TGFβ添加到GC或RC细胞中时，未发现类似效果。分别向GC和RC细胞中添加1,25加TGFβ和24,25加TGFβ，会使[35S]硫酸盐掺入产生协同增加，并对[3H]胸腺嘧啶核苷掺入产生相加作用。为了研究参与产生24,25和TGFβ对RC细胞协同作用的信号转导途径，检测了PKC活性水平。添加24,25和TGFβ 12小时会使PKC活性协同增加。此外，当在12小时孵育的最后90分钟仅添加24,25时，也发现了类似效果。然而，在孵育的最后9分钟或最初90分钟添加24,25时，未发现协同作用。为了进一步了解24,25和TGFβ如何介导观察到的PKC活性协同增加，研究了可能导致PKC激活的途径。发现24,25通过二酰基甘油的产生来影响PKC活性，而非通过G蛋白的激活，而TGFβ仅通过G蛋白来影响PKC活性。本研究结果表明，维生素D代谢产物和TGFβ产生了一种成熟依赖性和激素特异性的协同作用。此外，24,25与TGFβ之间的协同作用是通过两条平行途径激活PKC介导的：24,25通过二酰基甘油的产生，TGFβ通过G蛋白的激活。
