Growth factors such as EGF and TGF-beta1 use multiple signalling pathways in controlling the responses of cells. EGF transduces some of its positive effects on gene expression through AP-1 consensus sequences of affected genes. The potential signalling pathways utilized by EGF include tyrosine kinases, serine/threonine kinases and pathways regulated by increases in intracellular Ca2+. Much less is known about the pathways through which TGF-beta1 regulates expression of affected genes. Elucidation of how TGF-beta1 is able to both stimulate and inhibit gene expression in coordinated physiological processes should be an important goal in understanding the role of TGF-beta1 in development, wound repair, and control over cell proliferation. Progress in the past granting period has focused in part on the role played by TGF-beta1-stimulated second messenger production in controlling transcriptional regulation of stromelysin and other regulated genes, and in part on elucidating the specific TGF-beta1-responsive cis-regulatory sequences involved in both positive and negative control over gene expression. Evidence has accumulated that a specific nucleotide sequence (TIE or beta-RE) from the stromelysin gene (GAGTTGGTGA) is capable of acting in the context of AP-1 sequences to both inhibit EGF-stimulated expression and augment TPA- stimulated expression of the endogenous stromelysin gene and of reporter gene constructs in which the TIE/beta-RE has been placed near an enhancer harboring AP-a sequences. Protein complexes of the fos/jun family bind to the TIE/beta-RE DNA sequence and appear to be involved in regulating its activity as a bifunctional modulator of gene expression. There are several lines of evidence which implicate junB protein as an important component of that complex. The appearance and availability of junB mRNA and protein induced by TGF- beta1, EGF and TPA will be investigated using Western immunoblot analysis and methods to measure mRNA accumulation and transcription. Determination of whether induction of junB by TGF-beta1 is both necessary and sufficient for modulation of transcription will be accomplished by using antisense oligonucleotides to block the induction of junB and by overexpressing junB in both transient and stable expression systems. Characterization of the independent interactions between fos/jun heterodimers and the TIE/beta-RE and AP-1 elements will be performed using specific anti-junB antibodies coupled with mobility shift assays. Interactions will be investigated both in nuclear extracts and in proteins translated in vitro. Experiments will determine whether different fos/jun heterodimers induced by specific agonists are likely to compete for binding to the same nucleotide sequence, either the TIE/beta-RE of AP-1 sites, and whether stimulation of different signalling pathways is capable of altering the affinity of these protein complexes. Finally, the ability of the TIE/beta-RE to serve as an independent enhancer will be investigated. These lines of investigation should delineate the pathways by which TGF-beta1, in concert with EGF or TPA, serves to modulate its positive and negative effects on gene expression through the TIE/beta-RE element.
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