title: Molecular determinants of the profibrogenic effects of endothelin-1 in pancreatic stellate cells.
abstract: OBJECTIVE
  To gain molecular insights into the expression and functions of endothelin-1 (ET-1) in pancreatic stellate cells (PSC).
  
  
  METHODS
  PSCs were isolated from rat pancreas tissue, cultured, and stimulated with ET-1 or other extracellular mediators. Cell proliferation was assessed by measuring the incorporation of 5-bromo-2'-deoxyuridine into DNA and cell migration was studied in a transwell chamber assay. Gene expression at the level of mRNA was quantified by real-time polymerase chain reaction. Expression and phosphorylation of proteins were monitored by immunoblotting, applying an infrared imaging technology. ET-1 levels in cell culture supernatants were determined by an enzyme immunometric assay. To study DNA binding of individual transcription factors, electrophoretic mobility shift assays were performed.
  
  
  RESULTS
  Among several mediators tested, transforming growth factor-beta1 and tumour necrosis factor-alpha displayed the strongest stimulatory effects on ET-1 secretion. The cytokines induced binding of Smad3 and NF-kappaB, respectively, to oligonucleotides derived from the ET-1 promoter, implicating both transcription factors in the induction of ET-1 gene expression. In accordance with previous studies, ET-1 was found to stimulate migration but not proliferation of PSC. Stimulation of ET-1 receptors led to the activation of two distinct mitogen-activated protein kinases, p38 and extracellular signal-regulated kinases (ERK)1/2, as well as the transcription factor activator protein-1. At the mRNA level, enhanced expression of the PSC activation marker, alpha-smooth muscle actin and two proinflammatory cytokines, interleukin (IL)-1beta and IL-6, was observed.
  
  
  CONCLUSIONS
  This study provides novel lines of evidence for profibrogenic and proinflammatory actions of ET-1 in the pancreas, encouraging further studies with ET-1 inhibitors in chronic pancreatitis.
authors.affiliation: Department of Medicine II, Division of Gastroenterology, Medical Faculty, University of Rostock, E.-Heydemann-Str. 6, 18057 Rostock, Germany.
authors.forename: Anika
authors.initials: A
authors.lastname: Jonitz
authors.forename: Brit
authors.initials: B
authors.lastname: Fitzner
authors.forename: Robert
authors.initials: R
authors.lastname: Jaster
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D019332:Endothelin-1 / Q000235:genetics / Q000494:pharmacology*; D005355:Fibrosis; D005786:Gene Expression Regulation; D008297:Male; D010179:Pancreas / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D051381:Rats; D011917:Rats, Inbred Lew; D053773:Transforming Growth Factor beta1 / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
chemical_list: D019332:Endothelin-1; D053773:Transforming Growth Factor beta1; D014409:Tumor Necrosis Factor-alpha