O'Keefe R J, Loveys L S, Hicks D G, Reynolds P R, Crabb I D, Puzas J E, Rosier R N
Department of Orthopaedics, University of Rochester, NY 14642, USA.
J Orthop Res. 1997 Mar;15(2):162-74. doi: 10.1002/jor.1100150203.
Parathyroid hormone-related protein is a critical autocrine regulator of endochondral ossification in the growth plate, as demonstrated by the severe disruption of growth-plate structure and function in parathyroid hormone-related protein-deficient transgenic mice. In the present study, the effects of parathyroid hormone-related protein on the synthesis of collagen mRNA and protein were studied in short-term cultures of isolated chick growth-plate chondrocytes. Parathyroid hormone-related protein selectively inhibits type-X collagen protein synthesis with no significant effect on type-II collagen protein synthesis. These effects were present in all maturationally distinct populations of chondrocytes separated by countercurrent centrifugal elutriation. In cultures of resting chondrocytes, the onset of type-X collagen expression was inhibited, while the synthesis of type-X collagen was decreased in cultures of hypertrophic chondrocytes. Synthesis of type-II and type-X collagen mRNA was examined by nonradioactive in situ hybridization with synthetic oligonucleotide cDNA probes, and the level of expression was quantified using digital image analysis. Dose-dependent suppression of type-X collagen gene expression by parathyroid hormone-related protein was observed, with no significant effect on type-II collagen mRNA detected. The results were confirmed by analysis of Northern blots of total chondrocyte mRNA. These experiments demonstrated differential transcriptional regulation of type-II and type-X collagen, with selective suppression of type-X collagen expression, by parathyroid hormone-related protein in growth-plate chondrocytes. In addition, excellent agreement was found between traditional protein and mRNA analyses and microscopic digital image analysis techniques, supporting the use of this convenient and sensitive assay method. Parathyroid hormone-related protein inhibits chondrocyte maturation and is known to stimulate proliferation, suggesting that this autocrine factor may function to regulate premature hypertrophy in the growth plate.
甲状旁腺激素相关蛋白是生长板软骨内成骨的关键自分泌调节因子，甲状旁腺激素相关蛋白缺陷转基因小鼠生长板结构和功能的严重破坏就证明了这一点。在本研究中，在分离的鸡生长板软骨细胞的短期培养中研究了甲状旁腺激素相关蛋白对胶原蛋白mRNA和蛋白质合成的影响。甲状旁腺激素相关蛋白选择性抑制X型胶原蛋白的合成，对II型胶原蛋白的合成无显著影响。这些作用在通过逆流离心淘析分离的所有成熟不同的软骨细胞群体中均存在。在静止软骨细胞培养物中，X型胶原蛋白表达的起始受到抑制，而在肥大软骨细胞培养物中X型胶原蛋白的合成减少。使用合成寡核苷酸cDNA探针通过非放射性原位杂交检测II型和X型胶原蛋白mRNA的合成，并使用数字图像分析对表达水平进行定量。观察到甲状旁腺激素相关蛋白对X型胶原蛋白基因表达的剂量依赖性抑制，未检测到对II型胶原蛋白mRNA有显著影响。通过对总软骨细胞mRNA的Northern印迹分析证实了结果。这些实验表明，甲状旁腺激素相关蛋白在生长板软骨细胞中对II型和X型胶原蛋白具有差异转录调节作用，选择性抑制X型胶原蛋白的表达。此外，传统蛋白质和mRNA分析与显微数字图像分析技术之间具有极好的一致性，支持使用这种方便且灵敏的检测方法。甲状旁腺激素相关蛋白抑制软骨细胞成熟，并且已知会刺激增殖，这表明这种自分泌因子可能起到调节生长板中过早肥大的作用。
