Bonewald L F, Schwartz Z, Swain L D, Ramirez V, Poser J, Boyan B D
Department of Medicine, University of Texas Health Science Center, San Antonio 78284.
J Cell Physiol. 1990 Nov;145(2):200-6. doi: 10.1002/jcp.1041450203.
Transforming growth factor-beta (TGF beta) serves an important role in extracellular matrix formation by stimulating the production of numerous extracellular matrix proteins by connective tissue cells and by osteoblasts or bone-forming cells. TGF beta has been shown to stimulate alkaline phosphatase (ALPase) activity in the rat osteoblast-like osteosarcoma cell line ROS 17/2.8. Previous studies have shown that this enzyme is elevated during calcification of bone and that it is enriched in matrix vesicles, an extracellular organelle associated with initial hydroxyapatite formation. To test the hypothesis that TGF beta plays a role in regulating mineral deposition in the matrix, the effects of TGF beta on ALPase and phospholipase A2, two enzymes associated with mineralization, were examined. ROS 17/2.8 cells were cultured at high and low density with recombinant human TGF beta (0.1-10 ng/ml) to examine the influence of cell maturation on response to TGF beta. Maximal stimulation of ALPase activity in the low density cultures was seen at 5 ng/ml; in high-density cultures, there was further stimulation at 10 ng/ml. There was a dose-dependent increase in ALPase activity seen in the matrix vesicles and plasma membranes in both types of cultures. Matrix vesicle ALPase exhibited a greater response to factor than did the plasma membrane enzyme. However, in low-density cultures, the two membrane fractions exhibited a parallel response with greatest activity consistently in the matrix vesicles. There was a dose-dependent increase in phospholipase A2-specific activity in the plasma membranes and matrix vesicles of both high- and low-density cultures. In agreement with previous studies, TGF beta inhibited cellular proliferation 50%. The results show that addition of TGF beta stimulates the activity of enzymes associated with calcification. The effect of TGF beta is dependent on the stage of maturation of the cell. This study indicates that TGF beta may play an important role in induced bone formation, calcification, and fracture repair in addition to its role in promoting chondrogenesis.
转化生长因子-β（TGF-β）通过刺激结缔组织细胞和成骨细胞（即骨形成细胞）产生多种细胞外基质蛋白，在细胞外基质形成过程中发挥重要作用。TGF-β已被证明能刺激大鼠成骨样骨肉瘤细胞系ROS 17/2.8中的碱性磷酸酶（ALPase）活性。先前的研究表明，这种酶在骨钙化过程中升高，并且在基质小泡中富集，基质小泡是一种与初始羟基磷灰石形成相关的细胞外细胞器。为了验证TGF-β在调节基质中矿物质沉积方面发挥作用的假设，研究了TGF-β对ALPase和磷脂酶A2（两种与矿化相关的酶）的影响。将ROS 17/2.8细胞以高密度和低密度培养，并加入重组人TGF-β（0.1 - 10 ng/ml），以研究细胞成熟度对TGF-β反应的影响。在低密度培养物中，5 ng/ml时观察到ALPase活性的最大刺激；在高密度培养物中，10 ng/ml时出现进一步刺激。在两种类型的培养物中，基质小泡和质膜中的ALPase活性均呈剂量依赖性增加。基质小泡ALPase对因子的反应比质膜酶更大。然而，在低密度培养物中，两个膜组分表现出平行反应，基质小泡中的活性始终最大。在高密度和低密度培养物的质膜和基质小泡中，磷脂酶A2的特异性活性均呈剂量依赖性增加。与先前的研究一致，TGF-β抑制细胞增殖50%。结果表明，添加TGF-β可刺激与钙化相关的酶的活性。TGF-β的作用取决于细胞的成熟阶段。这项研究表明，TGF-β除了在促进软骨形成中发挥作用外，可能在诱导骨形成、钙化和骨折修复中也发挥重要作用。
