Induction of matrix metalloproteinase-1 gene transcription by tumour necrosis factor alpha via the p50/p50 homodimer of nuclear factor-kappa B in activated human hepatic stellate cells.
OBJECTIVE
Liver injury results in the activation of hepatic stellate cells (HSCs), which in turn produce matrix metalloproteinase (MMP) in response to pro-inflammatory cytokines for tissue remodelling. This study explored the transcriptional induction of the MMP-1 gene by tumour necrosis factor-alpha (TNF-alpha) in HSCs.


METHODS
The LI90 human HSC line was used in the present study. Gelatin zymography, enzyme-linked immunosorbent assay, Northern blotting and gene promoter-reporter assays were used to analyse the induction of MMP-1 protein, mRNA expression and gene transcription respectively. Deletional or site-directed mutations were introduced into the promoter region and transiently transfected into LI90 cells to determine the cis-acting elements necessary for TNF-alpha inducibility. Gel shift mobility assays were used to determine the transcriptional factors involved in the TNF-alpha responsiveness.


RESULTS
TNF-alpha upregulated MMP-1 protein and mRNA expression in a dose-dependent manner. A time-course experiment revealed a rapid induction of MMP-1 mRNA expression after TNF-alpha treatment. Mutation in a putative nuclear factor (NF)-kappaB-binding site at -2541 bp almost completely abolished the TNF-alpha response to MMP-1 gene-promoter activity, suggesting transcriptional regulation of MMP-1 expression by TNF-alpha via this site. Electrophoretic mobility shift assay and supershift assays indicated that this transcriptional regulation was regulated via the p50/p50 homodimer of NF-kappaB.


CONCLUSIONS
MMP-1 gene expression might be induced by TNF-alpha via the p50/p50 homodimer of NF-kappaB in activated human HSCs.
Hozawa
Shigenari
S
Division of Gastroenterology, Keio University School of Medicine, Tokyo, Japan. hozawas@sc.itc.keio.ac.jp
Nakamura
Tetsuya
T
Nakano
Masaru
M
Adachi
Masayuki
M
Tanaka
Hirotoshi
H
Takahashi
Yoko
Y
Tetsuya
Mine
M
Miyata
Naoteru
N
Soma
Hiromitsu
H
Hibi
Toshifumi
T
D015152:Blotting, Northern; D002460:Cell Line; D024202:Electrophoretic Mobility Shift Assay; D004790:Enzyme Induction / Q000502:physiology*; D004797:Enzyme-Linked Immunosorbent Assay; D055166:Hepatic Stellate Cells / Q000378:metabolism*; D006801:Humans; D020781:Matrix Metalloproteinase 1 / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D052002:NF-kappa B p50 Subunit / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*
D052002:NF-kappa B p50 Subunit; D014409:Tumor Necrosis Factor-alpha; D020781:Matrix Metalloproteinase 1