A DNA element in the alpha 1 type III collagen promoter mediates a stimulatory response by angiotensin II

Background. Angiotensin II (Ang II) plays an important role in extracellula r matrix deposition and tissue scarring in the kidney and the heart. The me chanism for extracellular matrix stimulation by Ang II is currently hypothe tical, with one possibility pointing to a direct effect on cell synthesis o f specific collagens. Methods. We studied the molecular mechanism for activation of type III coll agen synthesis by Ang II in an in vitro cell model of myofibroblasts by eva luating (I) al(III) collagen mRNA expression; (2) alpha 1(III) collagen pro moter activity; (3) DNA/protein binding with characterization of binding si tes; (4) expression of transcription factors; and (5) the role of a short D NA segment as Ang II responsive element. Results. We found a specific dose-dependent stimulation of alpha 1(III) col lagen mRNA expression and a parallel effect on alpha 1(III) collagen promot er activity. Transfection of constructs containing arl(III) collagen promot er fragments of different lengths localized the site of activation within t he shortest 178 bp construct. By gel-retardation experiments, we observed t he formation of a DNA-protein complex with crude extracts from Ang II-stimu lated cells and an oligonucleotide spanning the 3 to 20 sequence. This comp lex was due to a sequence-specific interaction and was abolished by a 3 bp substitution mutation. The introduction of this mutation into the 178 bp co nstruct abolished the stimulatory effect of Ang II. Conclusions. These results demonstrate that Ang II stimulates the expressio n of alpha 1(III) collagen mRNA in myofibroblasts in vitro by activating th e alpha 1(III) collagen promoter at the level of a factor recognition site localized immediately downstream of the transcription start site. This mech anism could be involved in Ang II-induced renal and heart fibrosis.