Chondrogenic activity of the heparan sulfate proteoglycan perlecan maps to the N-terminal domain I
Chondrogenic activity of the heparan sulfate proteoglycan perlecan maps to the N-terminal domain I
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DOI：
10.1359/jbmr.2002.17.1.48
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发表时间：
2002-01-01
影响因子：
6.2
通讯作者：
Carson, DD
中科院分区：
文献类型：
--
作者：
French, MM;Gomes, RR;Carson, DD
当铺板到细胞外基质 (ECM) 蛋白基底膜蛋白 (Pln) 上时，C3H10T1/2 细胞沿着软骨形成途径分化。为了鉴定大 Pin 分子内提供分化信号的区域，分析了代表不同结构域的基于重组 Pln 序列的多肽促进 C3H10T1/2 细胞中软骨形成的能力。测试了五个不同的域以及结构变化。 N 端结构域 I 以两种形式（IA 和 IB）进行测试，分别仅包含硫酸乙酰肝素 (HS) 链或同时包含 HS 和硫酸软骨素 (CS) 链。还测试了缺乏 HS 和 CS 附着位点的结构域 I 的突变形式 (Pln I-mut)。其他连续指定为 Pln 结构域 II、III(A-C)、IV(A,B) 和 V(A,B) 的构建体用于完成结构功能分析。细胞铺板到 Pin IA 或 Pln IB 上，但没有其他结构域，快速组装成平均 40-120 μm 的细胞聚集体。聚集体的形成取决于糖胺聚糖 (GAG) 链的存在，因为基于 Pin I 的多肽；通过酶去除或 HS/CS 附着位点突变而缺乏 GAG 链的物质是无活性的。在用阿新蓝染色的带有 GAG 的 Pin IA 上形成的聚集体被 II 型胶原蛋白和聚集蛋白聚糖的抗体识别，但不被 X 型胶原蛋白（软骨细胞肥大的标志物）的抗体识别。总的来说，这些研究表明 Pin 的 GAG 结构域 I 提供了足够的信号来触发 C3H10T1/2 细胞进入软骨形成分化途径。因此，在体内软骨形成部位发现的这种基质蛋白多糖（PG）可能会增强可溶性软骨形成因子诱导的早期分化。
C3H10T1/2 cells differentiate along a chondrogenic pathway when plated onto the extracellular matrix (ECM) protein perlecan (Pln). To identify the region(s) within the large Pin molecule that provides a differentiation signal, recombinant Pln-sequence-based polypeptides representing distinct structural domains were assayed for their ability to promote chondrogenesis in C3H10T1/2 cells. Five distinct domains, along with structural variations, were tested. The N-terminal domain I was tested in two forms (IA and IB) that contain only heparan sulfate (HS) chains or both HS and chondroitin sulfate (CS) chains, respectively. A mutant form of domain I lacking attachment sites for both HS and CS (Pln I-mut) was tested also. Other constructs consecutively designated Pln domains II, III(A-C), IV(A,B), and V(A,B) were used to complete the structure-function analysis. Cells plated onto Pin IA or Pln IB but no other domain rapidly assembled into cellular aggregates of 40-120 mum on average. Aggregate formation was dependent on the presence of glycosaminoglycan (GAG) chains, because Pin I-based polypeptides; lacking GAG chains either by enzymatic removal or mutation of HS/CS attachment sites were inactive. Aggregates formed on GAG-bearing Pin IA stained with Alcian Blue and were recognized by antibodies to collagen type II and aggrecan but were not recognized by an antibody to collagen type X, a marker of chondrocyte hypertrophy. Collectively, these studies indicate that the GAG-bearing domain I of Pin provides a sufficient signal to trigger C3H10T1/2 cells to enter a chondrogenic differentiation pathway. Thus, this matrix proteoglycan (PG) found at sites of cartilage formation in vivo is likely to enhance early stage differentiation induced by soluble chondrogenic factors.