Molecular determinants of the profibrogenic effects of endothelin-1 in pancreatic stellate cells.

Aim: 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.

Conclusion: 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.