Latent transforming growth factor-β1 and its binding protein are components of extracellular matrix microfibrils

Abstract

We studied the localization of latent transforming growth factor-β1 (TGF-β1) 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-β1 propeptide (β1-LAP). Both LTBP-1 and β1-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-β1 complexes are integral components of the extracellular matrix. SV-40-transformed human fibroblasts lacked extracellular LTBP-1 fibers. EM analysis revealed ≃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 fibronection matrix could be reconstituted in vitro. When isolated matrix components were immobilized on nitrocellulose and incubated with fibroblasts conditioned medium, LTBP-1 from the medium associated with cellular fibronection 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-β1 complexes are components of the extracellular matrix and suggest that alterations of the pericellular matrix could result in aberrant TGF-β signaling.