Gerstenfeld L C, Kelly C M, Von Deck M, Lian J B
Department of Orthopedic Surgery, Harvard Medical School, Boston, Massachusetts 02115.
Endocrinology. 1990 Mar;126(3):1599-609. doi: 10.1210/endo-126-3-1599.
Chondrocytes, derived from a tissue that remains as permanent hyaline cartilage in vivo (embryonic chicken caudal sterna) were treated with 10(-8) to 10(-8) M 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. These nonadherent rounded chondrocytes acquired an adherent, polygonal morphology in a dose-dependent fashion with 1,25(OH)2D3 treatment. During the first 4 days of 1,25(OH)2D3 treatment cell flattening was associated with a 10-fold increase in beta-actin and fibronectin and their corresponding messenger RNAs (mRNAs). After adherence over the 12 days of continuous hormone treatment, a 2- to 4-fold increase in DNA synthesis and DNA accumulation were observed for the highest hormone dose (10(-8) M). Over the same time course total collagen synthesis decreased 35-50% primarily due to decreased type II collagen synthesis, which accompanied comparable decreases in its mRNA. In contrast, both alpha 1(I) and alpha 2(I) showed a continuous 5- to 10-fold increase; however, type I collagen protein synthesis remained undetectable, indicating translational control of the type I collagen synthesis. alpha 1(X) mRNAs showed a 2- 3-fold increase after 12 days of hormone treatment, and its polypeptide was clearly detected by sodium dodecyl sulfate polyacrylamide gel analysis. Type IX collagen synthesis showed a 2-fold increase in synthesis and its mRNA levels during the first 4 days of 1,25(OH)2D3 treatment but thereafter had levels comparable to control cultures. Analysis of proteoglycan synthesis and core protein mRNA levels showed there was a 2-fold increase in core protein mRNAs while proteoglycan synthesis, as assessed by 35S incorporation, showed only a 10-20% increase. Direct hormone effects vs. those secondary to altered cellular morphology were determined by blocking cell adherence by growth of the 1,25(OH)2D3-treated cultures on bacteriological petri dishes. All of the observed effects on cytoskeletal and collagen mRNAs were blocked except the elevations observed in proteoglycan core protein and alpha 1(IX) mRNAs. DNA contents in hormone-treated cultures also remained elevated. These results suggest that 1,25(OH)2D3 both activates and suppresses specific genes, promoting chondrocyte maturation toward a more hypertrophic phenotype. However, prevention of the initial morphological alterations that are induced by 1,25(OH)2D3 blocks many of the subsequent changes in connective tissue expression.
从在体内保持为永久性透明软骨的组织（胚胎鸡尾胸骨）中分离出软骨细胞，用10⁻⁸至10⁻⁸ M的1,25 - 二羟维生素D₃ [1,25(OH)₂D₃] 对其进行处理。这些非贴壁的圆形软骨细胞在1,25(OH)₂D₃处理下以剂量依赖的方式获得了贴壁的多边形形态。在1,25(OH)₂D₃处理的前4天，细胞扁平化与β - 肌动蛋白和纤连蛋白及其相应信使核糖核酸（mRNA）增加10倍有关。在持续激素处理的12天内细胞贴壁后，观察到最高激素剂量（10⁻⁸ M）下DNA合成和DNA积累增加2至4倍。在相同的时间进程中，总胶原蛋白合成下降了35 - 50%，主要是由于II型胶原蛋白合成减少，其mRNA也有相应程度的下降。相比之下，α1(I)和α2(I)均持续增加5至10倍；然而，I型胶原蛋白的蛋白质合成仍未检测到，表明I型胶原蛋白合成存在翻译控制。激素处理12天后，α1(X) mRNA增加了2至3倍，通过十二烷基硫酸钠聚丙烯酰胺凝胶分析可清楚检测到其多肽。在1,25(OH)₂D₃处理的前4天，IX型胶原蛋白合成及其mRNA水平增加了2倍，但此后与对照培养物水平相当。对蛋白聚糖合成和核心蛋白mRNA水平的分析表明，核心蛋白mRNA增加了2倍，而通过³⁵S掺入评估的蛋白聚糖合成仅增加了10 - 20%。通过将1,25(OH)₂D₃处理的培养物在细菌培养皿上生长来阻断细胞贴壁，以确定激素的直接作用与细胞形态改变继发的作用。除了在蛋白聚糖核心蛋白和α1(IX) mRNA中观察到的升高外，所有对细胞骨架和胶原蛋白mRNA的观察到的作用均被阻断。激素处理的培养物中的DNA含量也保持升高。这些结果表明，1,25(OH)₂D₃既激活又抑制特定基因，促进软骨细胞向更肥大的表型成熟。然而，防止由1,25(OH)₂D₃诱导的初始形态改变会阻断结缔组织表达中的许多后续变化。
