Induction of myofibroblast MMP-9 transcription in three-dimensional collagen I gel cultures: regulation by NF-kappaB, AP-1 and Sp1.
Chronic liver injury leads to a progressive wound healing response that eventually results in hepatic fibrosis characterised by net deposition of fibrillar extracellular matrix (ECM) and a qualitative shift from type IV to type I/III collagen. The pivotal cellular event underlying this response is hepatic stellate cell (HSC) activation towards a myofibroblast-like phenotype. Activated HSC contribute to ECM remodelling via secretion of type I/III collagens and matrix metalloproteinases (MMPs). Previous studies showed that three-dimensional (3D) contact of activated HSC with type I collagen further stimulates the ECM remodelling properties of HSC by inducing the type IV gelatinase, MMP-9. The aim of the current study was to confirm transcriptional activation of the MMP-9 gene and identify transcription factors regulating this response. Gelatin zymography and Northern blotting were used to confirm induction of MMP-9 protein and mRNA expression in primary rat HSC cultured in a three-dimensional collagen I gel lattice. MMP-9 promoter studies in transfected HSC and electrophoretic mobility shift assay (EMSA) were employed to study transcriptional events. Both NF-kappaB and AP-1 DNA were induced in HSC cultured in 3D collagen I gels and binding sites for these factors in the MMP-9 promoter were crucial for induction of transcription. By contrast removal of an Sp1 site in the promoter enhanced transcription, while over-expression of either Sp1 or Sp3 repressed transcription. It is concluded that 3D contact of activated HSC with collagen I stimulates MMP-9 expression by elevating NF-kappaB and AP-1 activities which are able to overcome the repressive influence of Sp1/Sp3 on MMP-9 gene transcription.
Takahra
Terumi
T
Third Department of Internal Medicine, Toyama Medical and Pharmaceutical University, 2630 Sugitani, 930-0194, Toyama, Japan.
Smart
David E
DE
Oakley
Fiona
F
Mann
Derek A
DA
D000818:Animals; D001665:Binding Sites; D015152:Blotting, Northern; D018929:Cell Culture Techniques / Q000379:methods; D002478:Cells, Cultured; D003094:Collagen / Q000378:metabolism; D024042:Collagen Type I / Q000378:metabolism*; D005109:Extracellular Matrix / Q000378:metabolism; D005786:Gene Expression Regulation; D008156:Luciferases / Q000378:metabolism; D008297:Male; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism*; D016328:NF-kappa B / Q000378:metabolism*; D010641:Phenotype; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D018808:Transcription Factor AP-1 / Q000378:metabolism*; D014158:Transcription, Genetic; D015533:Transcriptional Activation
D024042:Collagen Type I; D016328:NF-kappa B; D012333:RNA, Messenger; D016329:Sp1 Transcription Factor; D018808:Transcription Factor AP-1; D003094:Collagen; D008156:Luciferases; D020780:Matrix Metalloproteinase 9