Naondo Sohara1, Maria Trojanowska, Adrian Reuben. 1. Division of Gastroenterology and Hepatology, Department of Medicine, Medical University of South Carolina, 96 Jonathan Lucas Street, Suite 210, Charleston, SC 29425, USA.
Abstract
BACKGROUND/AIMS: We previously showed that in cultured human myofibroblasts (hMFBs), Oncostatin M (OSM)-stimulated collagen accumulation is associated with increased tissue inhibitor of metalloproteinase (TIMP)1 message. However, the mechanism is unknown. METHODS: hMFBs were isolated by outgrowth from cirrhotic liver explants and cultured. Using OSM (10 ng/ml) stimulation, with and without PD98059 (PD, a specific mitogen-activated protein kinase/extracellular signal-related kinase (MEK) inhibitor), we measured: TIMP-1 protein in culture medium by Western blot, TIMP-1 mRNA levels and stability by Northern analysis, TIMP-1 promoter activity (including transcription site mutation analysis), DNA binding activity to nuclear proteins by electrophoretic mobility shift assay (EMSA), and total and phosphorylated MAP kinase in hMFB extracts by Western blot. RESULTS: OSM stimulation of hMFBs increased TIMP-1 protein production 1.69-fold, TIMP-1 mRNA levels 2.36-fold, promoter activity 2.22-fold, TIMP-1 message stability, and phosphorylation of mitogen-activated protein kinase (MAPK). PD inhibited OSM-mediated stimulation of TIMP-1 protein, mRNA, promoter activity, phosphorylation of MAPK, and TIMP-1 message stability. An SP-1 transcription site of the TIMP-1 promoter is essential for OSM induction of TIMP-1 promoter activity. EMSA demonstrates that this site binds to transcriptional factors SP-1 and SP-3. CONCLUSIONS: OSM stimulates the TIMP-1 axis in hMFBs in vitro via a MEK-MAP kinase cascade.
BACKGROUND/AIMS: We previously showed that in cultured human myofibroblasts (hMFBs), Oncostatin M (OSM)-stimulated collagen accumulation is associated with increased tissue inhibitor of metalloproteinase (TIMP)1 message. However, the mechanism is unknown. METHODS: hMFBs were isolated by outgrowth from cirrhotic liver explants and cultured. Using OSM (10 ng/ml) stimulation, with and without PD98059 (PD, a specific mitogen-activated protein kinase/extracellular signal-related kinase (MEK) inhibitor), we measured: TIMP-1 protein in culture medium by Western blot, TIMP-1 mRNA levels and stability by Northern analysis, TIMP-1 promoter activity (including transcription site mutation analysis), DNA binding activity to nuclear proteins by electrophoretic mobility shift assay (EMSA), and total and phosphorylated MAP kinase in hMFB extracts by Western blot. RESULTS: OSM stimulation of hMFBs increased TIMP-1 protein production 1.69-fold, TIMP-1 mRNA levels 2.36-fold, promoter activity 2.22-fold, TIMP-1 message stability, and phosphorylation of mitogen-activated protein kinase (MAPK). PD inhibited OSM-mediated stimulation of TIMP-1 protein, mRNA, promoter activity, phosphorylation of MAPK, and TIMP-1 message stability. An SP-1 transcription site of the TIMP-1 promoter is essential for OSM induction of TIMP-1 promoter activity. EMSA demonstrates that this site binds to transcriptional factors SP-1 and SP-3. CONCLUSIONS: OSM stimulates the TIMP-1 axis in hMFBs in vitro via a MEK-MAP kinase cascade.