Sullivan T A, Uschmann B, Hough R, Leboy P S
Department of Biochemistry, School of Dental Medicine, University of Pennsylvania, Philadelphia 19104-6003.
J Biol Chem. 1994 Sep 9;269(36):22500-6.
During development and fracture repair, endochondral bone formation is preceded by an orderly process of chondrocyte hypertrophy and cartilage matrix calcification. Analysis of calcifying versus noncalcifying cartilage has identified several differences in matrix proteins; among these are appearance of a novel collagen, type X, and decreased synthesis of type II collagen, the major component of cartilage matrix. In addition, there is a marked increase in alkaline phosphatase, an enzyme expressed at high levels in all mineralizing tissues. Cultured chondrocytes can be induced to undergo these changes in gene expression and to produce calcified matrix by exposure to ascorbic acid. The mechanism by which ascorbate produces these changes has been examined by analyzing the effect of the vitamin on prehypertrophic chick embryo sternal chondrocytes. Nuclear run-on assays demonstrated that ascorbate alters mRNA levels in chondrocytes by changing the transcription rates. The fact that marked changes in mRNA levels require 1-2 days of ascorbate exposure suggested that the effect of this vitamin on gene transcription may be secondary to other, earlier ascorbate-induced effects. Since cells cultured with ascorbate produce a collagen-enriched matrix, we examined the hypothesis that transcriptional changes were secondary to altered cell-matrix interactions. Chondrocytes were cultured after attachment to tissue culture plastic, in suspension, or on plates coated with collagen type I. Comparison of alkaline phosphatase activity with and without ascorbate addition demonstrated that under all of these conditions, induction of enzyme was dependent on the presence of ascorbate. When plates containing ascorbate-conditioned chondrocyte matrix were used as substrate for naive chondrocytes, the cells continued to require ascorbate for induction of high levels of alkaline phosphatase and type X collagen mRNA. Addition of the hydroxylation inhibitor, 3,4-dehydroproline, caused marked inhibition of collagen secretion as well as accumulation of underhydroxylated collagens within the cells. However, even in the presence of this inhibitor ascorbate was effective in inducing elevated alkaline phosphatase and type X collagen. These results indicate that the ability of ascorbate to induce chondrocyte hypertrophy does not depend on production of a collagen-rich matrix.
在发育和骨折修复过程中，软骨内成骨之前会经历软骨细胞肥大和软骨基质钙化的有序过程。对钙化软骨与非钙化软骨的分析已确定基质蛋白存在若干差异；其中包括一种新型胶原蛋白X型的出现以及软骨基质主要成分II型胶原蛋白合成的减少。此外，碱性磷酸酶显著增加，该酶在所有矿化组织中均高水平表达。培养的软骨细胞通过暴露于抗坏血酸可被诱导发生这些基因表达变化并产生钙化基质。通过分析维生素对肥大前鸡胚胸骨软骨细胞的影响，研究了抗坏血酸产生这些变化的机制。核转录分析表明，抗坏血酸通过改变转录速率来改变软骨细胞中的mRNA水平。mRNA水平的显著变化需要1至2天的抗坏血酸暴露这一事实表明，这种维生素对基因转录的影响可能继发于其他更早的抗坏血酸诱导效应。由于用抗坏血酸培养的细胞产生富含胶原蛋白的基质，我们检验了转录变化继发于细胞 - 基质相互作用改变这一假设。将软骨细胞接种到组织培养塑料板上、悬浮培养或接种到包被有I型胶原蛋白的平板上进行培养。比较添加和不添加抗坏血酸时的碱性磷酸酶活性表明，在所有这些条件下，酶的诱导均依赖于抗坏血酸的存在。当含有经抗坏血酸处理的软骨细胞基质的平板用作未处理软骨细胞的底物时，细胞仍需要抗坏血酸来诱导高水平的碱性磷酸酶和X型胶原蛋白mRNA。添加羟基化抑制剂3,4 - 脱氢脯氨酸会导致胶原蛋白分泌显著抑制以及细胞内羟基化不足的胶原蛋白积累。然而，即使在存在这种抑制剂的情况下，抗坏血酸仍能有效诱导碱性磷酸酶和X型胶原蛋白升高。这些结果表明，抗坏血酸诱导软骨细胞肥大的能力不依赖于富含胶原蛋白基质的产生。
