Control of the tissue inhibitor of metalloproteinases-1 promoter in culture-activated rat hepatic stellate cells: regulation by activator protein-1 DNA binding proteins.
In the injured liver hepatic stellate cells (HSCs) undergo a dramatic phenotypic transformation known as "activation" in which they become myofibroblast-like and express high levels of the tissue inhibitor of metalloproteinase 1 (TIMP-1). HSC activation is accompanied by transactivation of the TIMP-1 promoter. Truncation mutagenesis studies delineated a minimal active promoter consisting of nucleotides -102 to +60 relative to the major start site for transcription. Removal of an AP-1 site located at nucleotides -93 to -87 caused almost a complete loss of promoter activity. Analysis of AP-1 DNA binding activities during culture activation of HSCs initially indicated transient expression of proteins capable of forming a low mobility AP-1 DNA binding complex (LMAP-1). LMAP-1 was maximally induced at 24 hours of culture and then fell to undetectable levels at 120 hours. Western blot studies showed that both c-Fos and c-Jun underwent similar transient inductions. These temporal changes in c-Fos and c-Jun activities were unexpected because TIMP-1 mRNA expression is not detected in HSCs until culture day 3 to 5 and is thereafter sustained at a high level. Previous work in other cell lineages has established a key role for Pea3 binding proteins (Ets-1) in AP-1 mediated transactivation of the TIMP-1 promoter. We show that HSCs express relatively low levels Ets-1 and Ets-2 and show that mutagenesis of the Pea3 DNA binding site in the TIMP-1 promoter has less than a twofold effect on its activity in activated HSCs. Further analysis of AP-1 DNA binding activities in 7- to 14-day culture activated HSCs led to the discovery of high mobility AP-1 complexes (HMAP-1). HMAP-1 DNA binding activities were sequence specific with respect to AP-1 and absent from freshly isolated HSCs. Supershift EMSA and Western blot studies identified JunD, Fra2, and FosB as potential components of the HMAP-1. Mutations of the AP-1 site of the TIMP-1 promoter that prevented formation of HMAP-1 caused a 70% loss of activity in transfected activated HSCs. Taken together the data indicate that sustained upregulation of TIMP-1 gene expression may be at least partially controlled by a novel AP-1 dependent regulation of TIMP-1 promoter activity.
University Medicine, Southampton General Hospital, Southampton, UK.
M J
MJ
Bahr
K J
KJ
Vincent
M J
MJ
Arthur
A V
AV
Fowler
D E
DE
Smart
M C
MC
Wright
I M
IM
Clark
R C
RC
Benyon
J P
JP
Iredale
D A
DA
Mann
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000502:physiology; D002478:Cells, Cultured; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism / Q000502:physiology*; D008099:Liver / Q000166:cytology / Q000378:metabolism / Q000502:physiology*; D008969:Molecular Sequence Data; D009154:Mutation / Q000502:physiology; D011401:Promoter Regions, Genetic / Q000502:physiology*; D011499:Protein Processing, Post-Translational / Q000502:physiology; D016760:Proto-Oncogene Proteins c-fos / Q000378:metabolism; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000235:genetics*; D018808:Transcription Factor AP-1 / Q000502:physiology*; D014157:Transcription Factors / Q000378:metabolism; D015533:Transcriptional Activation / Q000502:physiology
D004268:DNA-Binding Proteins; C513243:Etv5 protein, rat; D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D019715:Tissue Inhibitor of Metalloproteinase-1; D018808:Transcription Factor AP-1; D014157:Transcription Factors; C065788:transcription factor PEA3; D004247:DNA