| Abstract: | Transforming growth factor beta (TGF-β) stimulates protein complex formation on a TGF-β response element (TAE) found in the distal portion (−1624) of the collagen alpha 1(I) promoter. To identify the fibroblast proteins in this complex, an expression library constructed from human embryonic lung fibroblasts mRNA was screened using a tetramer of TAE. Y-box binding protein (YB-1), was identified as a protein in the TAE–protein complex. The protein expressed by phage clones formed a specific complex with labeled TAE but not mutated TAE (mTAE) similar to the complex formed with nuclear protein. Nuclear protein–TAE complexes isolated from native gels contained YB-1 by Western analysis. TGF-β treatment increased the amount of YB-1 protein in nuclear extracts, decreased its amount in cytoplasm, but did not alter the steady state levels of YB-1 mRNA. A full-length YB-1 protein expressed in human lung fibroblasts was primarily located in the nucleus with punctate staining in cytoplasmic regions. The expression of YB-1 decreased in the cytoplasm after 2 h of TGF-β treatment. Therefore, the increased binding activity seen in TGF-β-stimulated nuclear extracts was due primarily to relocalization of YB-1 from the cytoplasm to the nuclear compartment. Co-transfection of YB-1 cDNA with a collagen promoter–reporter construct caused a dose-dependent activation of collagen promoter activity in rat fibroblasts whereas the promoter with a mutation in the TAE element was not sensitive to YB-1 co-expression. In conclusion, we have identified YB-1 as a protein that interacts with a TGF-β response element in the distal region of the collagen alpha 1(I) gene. YB-1 protein activates the collagen promoter and translocates into the nucleus during TGF-β addition to fibroblasts, suggesting a role for this protein in TGF-β signaling. |
