B-Myb represses trans-activation of the Col5A2 collagen promoter indirectly via inhibition of binding of factors interacting with positive elements within the first exon

B-myb, a member of the myb gene family, was originally isolated based on it s high homology with c-myb in the DNA-binding domain. Previously we showed that B-myb is expressed in bovine vascular smooth muscle cells (SMCs) in a cell cycle-dependent fashion, and inhibits type I collagen gene promoter ac tivity. Here, we have explored its role in regulation of another fibrillar collagen gene, Col5A2, encoding the alpha 2 chain of type V collagen. Ectop ic expression of B-Myb decreased alpha 2(V) promoter activity and endogenou s alpha 2(V) collagen mRNA levels. The responsive region of the alpha 2(V) collagen gene was localized to a fragment including 100 bp of basal promote r and 150 bp of exon 1 sequences, which contained two CRE-like elements. Bi nding to these elements increased upon deprivation of serum-growth factors, when expression of the Col5A2 gene is elevated, leading us to test their r ole despite the failure of excess unlabelled CRE oligonucleotide from the s omatostatin gene to successfully compete for binding. Mutation of the eleme nts significantly decreased the basal level of alpha 2(V) collagen promoter activity and ablated inhibition by B-Myb. Furthermore, addition of B-Myb-g lutathionine S-transferase fusion protein inhibited complex formation. Thus , these results confirm a major role for B-Myb in mediating intracellular s ignals controlling collagen gene expression in vascular SMCs. A model of in direct repression of the Col5A2 gene by B-Myb, via interaction with a posit ively-acting matrix regulatory factor, termed MRF-V, is discussed. (C) 1999 Elsevier Science B.V./International Society of Matrix Biology. AU rights r eserved.