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Control of the tissue inhibitor of metalloproteinases‐1 promoter in culture‐activated rat hepatic stellate cells: Regulation by activator protein‐1 DNA binding proteins
Author(s) -
Bahr Matthias J.,
Vincent Karen J.,
Arthur Michael J. P.,
Fowler Andrew V.,
Smart David E.,
Wright Matthew C.,
Clark Ian M.,
Benyon R. Christopher,
Iredale John P.,
Mann Derek A.
Publication year - 1999
Publication title -
hepatology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.488
H-Index - 361
eISSN - 1527-3350
pISSN - 0270-9139
DOI - 10.1002/hep.510290333
Subject(s) - transactivation , microbiology and biotechnology , hepatic stellate cell , biology , electrophoretic mobility shift assay , transcription factor , promoter , transcription (linguistics) , gene expression , biochemistry , gene , endocrinology , linguistics , philosophy
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 ( lm AP‐1). lm AP‐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 ( hm AP‐1). hm AP‐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 HM AP‐1. Mutations of the AP‐1 site of the TIMP‐1 promoter that prevented formation of hm AP‐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.