A DNA element in the alpha1 type III collagen promoter mediates a stimulatory response by angiotensin II.
BACKGROUND
Angiotensin II (Ang II) plays an important role in extracellular matrix deposition and tissue scarring in the kidney and the heart. The mechanism for extracellular matrix stimulation by Ang II is currently hypothetical, with one possibility pointing to a direct effect on cell synthesis of specific collagens.


METHODS
We studied the molecular mechanism for activation of type III collagen synthesis by Ang II in an in vitro cell model of myofibroblasts by evaluating (1) alpha1(III) collagen mRNA expression; (2) alpha1(III) collagen promoter activity; (3) DNA/protein binding with characterization of binding sites; (4) expression of transcription factors; and (5) the role of a short DNA segment as Ang II responsive element.


RESULTS
We found a specific dose-dependent stimulation of alpha1(III) collagen mRNA expression and a parallel effect on alpha1(III) collagen promoter activity. Transfection of constructs containing alpha1(III) collagen promoter fragments of different lengths localized the site of activation within the shortest 178 bp construct. By gel-retardation experiments, we observed the formation of a DNA-protein complex with crude extracts from Ang II-stimulated cells and an oligonucleotide spanning the 3 to 20 sequence. This complex 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 construct abolished the stimulatory effect of Ang II.


CONCLUSIONS
These results demonstrate that Ang II stimulates the expression of alpha1(III) collagen mRNA in myofibroblasts in vitro by activating the alpha1(III) collagen promoter at the level of a factor recognition site localized immediately downstream of the transcription start site. This mechanism could be involved in Ang II-induced renal and heart fibrosis.
Department of Nephrology, and Laboratory of Molecular Genetics, G. Gaslini Institute, University of Genova, Italy. labnefro@tin.it
G M
GM
Ghiggeri
R
R
Oleggini
L
L
Musante
G
G
Caridi
R
R
Gusmano
R
R
Ravazzolo
D000804:Angiotensin II / Q000378:metabolism* / Q000494:pharmacology; D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D003094:Collagen / Q000235:genetics* / Q000378:metabolism*; D016326:Extracellular Matrix Proteins / Q000235:genetics / Q000378:metabolism; D005355:Fibrosis; D015870:Gene Expression / Q000187:drug effects / Q000502:physiology; D005816:Genetic Complementation Test; D006331:Heart Diseases / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D007674:Kidney Diseases / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D008969:Molecular Sequence Data; D018485:Muscle Fibers, Skeletal / Q000166:cytology; D016297:Mutagenesis, Site-Directed / Q000502:physiology; D009206:Myocardium / Q000166:cytology; D010957:Plasmids; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011507:Proteinuria / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D012333:RNA, Messenger / Q000494:pharmacology; D051381:Rats; D014157:Transcription Factors / Q000378:metabolism; D014162:Transfection; D014662:Vasoconstrictor Agents / Q000378:metabolism* / Q000494:pharmacology
D016326:Extracellular Matrix Proteins; D012333:RNA, Messenger; D014157:Transcription Factors; D014662:Vasoconstrictor Agents; D000804:Angiotensin II; D003094:Collagen