Certain lung injuries induce large increases in connective tissue content, particularly collagen, resulting in fibrosis. During injury, cells are exposed to effector substances that regulate production of matrix components such as transforming growth factor-beta (TGF-beta). this proposal focuses on establishing the mechanisms involved during the stimulation of collagen transcription by TGF-beta. Our preliminary work demonstrated that TGF-beta stimulated lung fibroblasts to accumulate type I collagen and alpha1(I) mRNA. TGF-beta increased alpha1(I) transcription involving a cis-regulatory region, referred to as a TGF- beta activating element (TAE), 1.6 Kb from the transcriptional start site. Although this site resembled a nuclear factor I (NF-1) consensus site, TAE, but not NF-1, abrogated the TGF-beta response of a collagen promoter driving chloramphenicol acetyl transferase (CAT) co-transfected into lung fibroblasts. The TAE site had an activator protein-2 (AP-2) binding site within a NF-1-like binding site. When cells were grown with increasing doses of TGF-beta, the nuclear TAE, NF-1 and AP-2 DNA-binding protein increased with a corresponding decrease in cytoplasmic DNA binding protein. We hypothesize that treatment with TGF-beta results in the phosphorylation of a specific nuclear binding protein which translocates from the cytoplasm to the nucleus. This nuclear protein binds to the TAE sequence in the alpha1(I) collagen promoter to activate transcription. This proposal will characterize the TAE binding proteins and examine the mechanism whereby TGF-beta alters its activity. In addition, preliminary data using transgenic mice with a full length collagen promoter CAT construct had 8 fold increased CAT activity in lungs of bleomycin treated animals suggesting that these mice will provide an excellent model for examining cis-activating elements directing collagen transcription in fibrosis. This proposal will further investigate TGF-beta responsive cis and trans-activating elements necessary for stimulated collagen transcription using cell cultures and transgenic animals.
The specific aims of this proposal are to characterize the proteins that bind to TAE. Both nuclear and cytoplasmic binding proteins derived from cells stimulated with and without TGF-beta will be examined, to analyze the mechanism of the TGF-beta response. We will determine if the phosphorylation state and/or the localization of transcription factors change with TGF-beta treatment, to characterize further the inhibition of TGF-beta response by TAE. We will determine if the double stranded (ds) TAE blocks the increased nuclear binding activity of proteins or endogenous collagen transcription. We will determine if dsTAE enters the nucleus or acts through a cytoplasmic mechanism to inhibit TGF-beta action, to perform site directed mutagenesis of rat alpha1(I) promoter to establish critical bases for TGF-beta response and to determine if other regions are involved in TGF-beta action. This will continue our efforts to map cis-acting response elements using transfection into human lung fibroblasts. When site directed deletions are established that obliterate the promoter TGF-beta response in transfected cells, then transgenic animals will be made to test constructs in vivo.
