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 pheno typic transformation known as "activation" in which they become myofibrobla st-like and express high levels of the tissue inhibitor of metalloproteinas e 1 (TIMP-1). HSC activation is accompanied by transactivation of the TIMP- 1 promoter. Truncation mutagenesis studies delineated a minimal active prom oter consisting of nucleotides -102 to +60 relative to the major start site for transcription, Removal of an AP-I 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 tr ansient expression of proteins capable of forming a low mobility AP-1 DNA b inding complex (LMAP-1), LMAP-1 was maximally induced at 24 hours of cultur e and then fell to undetectable levels at 120 hours. Western blot studies s howed that both c-Fos and c-Jun underwent similar transient inductions. The se temporal changes in c-Fos and c-Jun activities were unexpected because T IMP-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 lineag es has established a key role for Pea3 binding proteins (Ets-l) in AP-1 med iated transactivation of the TIMP-1 promoter. We show that HSCs express rel atively low levels Ets-l and Ets-2 and show that mutagenesis of the Pea3 DN A 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 activitie s in 7- to 14-day culture activated HSCs led to the discovery of high mobil ity AP-1 complexes (HMAP-1). HMAP-1 DNA binding activities were sequence sp ecific with respect to AP-1 and absent from freshly isolated HSCs, Supershi ft EMSA and Western blot studies identified JunD, Fra2, and FosB as potenti al components of the HMAP-1. Mutations of the AP-1 site of the TIMP-1 promo ter that prevented formation of HMAP-1 caused a 70% loss of activity in tra nsfected activated HSCs. Taken together the data indicate that sustained up regulation of TIMP-1 gene expression may be at least partially controlled b y a novel AP-1 dependent regulation of TIMP-1 promoter activity.