Induction of myofibroblast MMP-9 transcription in three-dimensional collagen I gel cultures: regulation by NF-kappaB, AP-1 and Sp1

Int J Biochem Cell Biol. 2004 Feb;36(2):353-63. doi: 10.1016/s1357-2725(03)00260-7.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Binding Sites
  • Blotting, Northern
  • Cell Culture Techniques / methods
  • Cells, Cultured
  • Collagen / metabolism
  • Collagen Type I / metabolism*
  • Extracellular Matrix / metabolism
  • Gene Expression Regulation*
  • Luciferases / metabolism
  • Male
  • Matrix Metalloproteinase 9 / metabolism*
  • NF-kappa B / metabolism*
  • Phenotype
  • Plasmids / metabolism
  • Promoter Regions, Genetic
  • Protein Structure, Tertiary
  • RNA, Messenger / metabolism
  • Rats
  • Rats, Sprague-Dawley
  • Sp1 Transcription Factor / metabolism*
  • Transcription Factor AP-1 / metabolism*
  • Transcription, Genetic*
  • Transcriptional Activation

Substances

  • Collagen Type I
  • NF-kappa B
  • RNA, Messenger
  • Sp1 Transcription Factor
  • Transcription Factor AP-1
  • Collagen
  • Luciferases
  • Matrix Metalloproteinase 9