Latent transforming growth factor-beta 1 and its binding protein are components of extracellular matrix microfibrils.
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.
Taipale
J
J
Departments of Virology, University of Helsinki, Finland.
Saharinen
J
J
Hedman
K
K
Keski-Oja
J
J
D002352:Carrier Proteins / Q000302:isolation & purification*; D002472:Cell Transformation, Viral; D002478:Cells, Cultured; D003094:Collagen / Q000302:isolation & purification; D017077:Culture Media, Conditioned / Q000737:chemistry; D005109:Extracellular Matrix / Q000737:chemistry* / Q000648:ultrastructure; D005347:Fibroblasts / Q000166:cytology; D005353:Fibronectins / Q000302:isolation & purification; D005455:Fluorescent Antibody Technique; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D053779:Latent TGF-beta Binding Proteins; D016253:Microscopy, Immunoelectron; D010446:Peptide Fragments; D011485:Protein Binding; D011498:Protein Precursors; D011506:Proteins / Q000302:isolation & purification; D013539:Simian virus 40; D016212:Transforming Growth Factor beta / Q000302:isolation & purification*; D053773:Transforming Growth Factor beta1
D002352:Carrier Proteins; D017077:Culture Media, Conditioned; D005353:Fibronectins; D047908:Intracellular Signaling Peptides and Proteins; C509032:LTBP1 protein, human; D053779:Latent TGF-beta Binding Proteins; D010446:Peptide Fragments; D011498:Protein Precursors; D011506:Proteins; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D003094:Collagen