Taipale J, Saharinen J, Hedman K, Keski-Oja J
Departments of Virology, University of Helsinki, Finland.
J Histochem Cytochem. 1996 Aug;44(8):875-89. doi: 10.1177/44.8.8756760.
We studied the localization of latent transforming growth factor-beta 1 (TGF-beta 1) and its binding protein (LTBP-1) in the extracellular matrix of cultured human fibroblasts by immunofluorescence and immunoelectron microscopy. Immunofluorescence of confluent fibroblast cultures indicated that LTBP-1 localizes to extracellular fibrillar structures resembling fibronectin-collagen matrix. Similar fibrillar structures were detected in cells stained with antibodies specific for TGF-beta 1 propeptide (beta 1-LAP). Both LTBP-1 and beta 1-LAP colocalized with fibronectin in double immunofluorescence analysis. These fibrillar structures were resistant to extraction with sodium deoxycholate, which is further evidence that LTBP-1 and large latent TGF-beta 1 complexes are integral components of the extracellular matrix. SV-40-transformed human fibroblasts lacked extracellular LTBP-1 fibers. EM analysis revealed approximately 10-nm-thick microfibrils that were labeled by anti-LTBP at 90-140-nm intervals. In addition, LTBP-1 was found in structures that were heavily labeled for fibronectin. The accumulation of LTBP-1 in the fibronectin matrix could be reconstituted in vitro. When isolated matrix components were immobilized on nitrocellulose and incubated with fibroblast conditioned medium, LTBP-1 from the medium associated with cellular fibronectin but not with heparan or chondroitin sulfate, vitronectin, tenascin, laminin, or collagen I or IV. The association of LTBP-1 with cellular fibronectin was abolished by treatment of the medium with plasmin, which cleaves LTBP-1 and inhibits its assembly to matrix. The present results indicate that latent TGF-beta 1 complexes are components of the extracellular matrix and suggest that alterations of the pericellular matrix could result in aberrant TGF-beta signaling.
我们通过免疫荧光和免疫电子显微镜研究了潜伏转化生长因子-β1（TGF-β1）及其结合蛋白（LTBP-1）在培养的人成纤维细胞外基质中的定位。汇合的成纤维细胞培养物的免疫荧光表明，LTBP-1定位于类似于纤连蛋白-胶原基质的细胞外纤维状结构。在用TGF-β1前肽（β1-LAP）特异性抗体染色的细胞中也检测到了类似的纤维状结构。在双重免疫荧光分析中，LTBP-1和β1-LAP均与纤连蛋白共定位。这些纤维状结构对脱氧胆酸钠提取具有抗性，这进一步证明LTBP-1和大的潜伏TGF-β1复合物是细胞外基质的组成成分。SV-40转化的人成纤维细胞缺乏细胞外LTBP-1纤维。电子显微镜分析显示，抗LTBP标记的约10纳米厚的微纤维以90-140纳米的间隔排列。此外，在纤连蛋白标记强烈的结构中发现了LTBP-1。LTBP-1在纤连蛋白基质中的积累可以在体外重建。当将分离的基质成分固定在硝酸纤维素上并与成纤维细胞条件培养基孵育时，培养基中的LTBP-1与细胞纤连蛋白结合，但不与硫酸乙酰肝素或硫酸软骨素、玻连蛋白、腱生蛋白、层粘连蛋白或I型或IV型胶原结合。用纤溶酶处理培养基可消除LTBP-1与细胞纤连蛋白的结合，纤溶酶可切割LTBP-1并抑制其组装到基质中。目前的结果表明，潜伏TGF-β1复合物是细胞外基质的组成成分，并提示细胞周围基质的改变可能导致异常的TGF-β信号传导。
