Proteolytic action of duodenase is required to induce DNA synthesis in pulmonary artery fibroblasts.
Duodenase is a 29-kDa serine endopeptidase that displays selective trypsin- and chymotrypsin-like substrate specificity. This enzyme has been localized to epitheliocytes of Brunner's glands, and as described here, to mast cells within the intestinal mucosa and lungworm-infected lung, implying an important additional role in inflammation and tissue remodelling. In primary cultures of pulmonary artery fibroblasts, duodenase induced a concentration-dependent increase in [3H]thymidine incorporation with a maximal effect observed at 30 nm. Pretreating duodenase with soybean trypsin inhibitor abolished DNA synthesis, confirming that proteolytic activity was an essential requirement for this response. PAR1, PAR2 and PAR4 activating peptides were unable to induce [3H]thymidine incorporation in pulmonary artery fibroblasts. Likewise, pretreatment of fibroblasts with TNFalpha, known to up-regulate PAR2 expression in other systems, and IL-1beta, did not enhance the potential of duodenase to induce DNA synthesis. Furthermore, duodenase increased GTPgammaS binding to fibroblast membranes indicating that a G-protein-coupled receptor may mediate the effects of duodenase. Duodenase-induced DNA synthesis and GTPgammaS binding were both found to be inhibited by pertussis toxin, implying a role for Gi/o. Selective inhibitors of MEK1 (PD98059) and protein kinase C (GF109203X) only partially inhibited duodenase-induced DNA synthesis, but both wortmannin (100 nm) and LY294002 (10 microm) inhibited this response completely, indicating a key role for PtdIns 3-kinase. Furthermore, duodenase induced a 2.3 plus minus 0.1-fold increase in PtdIns 3-kinase activity in p85 immunoprecipitates, which was sensitive to inhibition by wortmannin. These results suggest that duodenase can induce pulmonary artery fibroblast DNA synthesis in a PtdIns 3-kinase-dependent manner via a G-protein-coupled receptor which is activated by a proteolytic mechanism.
Pemberton
Alan D
AD
Department of Veterinary Studies, University of Edinburgh, Easter Bush Veterinary Centre, Roslin, Edinburgh, UK.
Zamolodchikova
Tatyana S
TS
Scudamore
Cheryl L
CL
Chilvers
Edwin R
ER
Miller
Hugh R P
HR
Walker
Trevor R
TR
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D004247:DNA / Q000096:biosynthesis; D005347:Fibroblasts / Q000166:cytology / Q000201:enzymology*; D007375:Interleukin-1 / Q000494:pharmacology; D007422:Intestines / Q000166:cytology / Q000201:enzymology; D008969:Molecular Sequence Data; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D011651:Pulmonary Artery / Q000166:cytology / Q000201:enzymology*; D044463:Receptor, PAR-1; D044464:Receptor, PAR-2; D018179:Receptors, Thrombin / Q000378:metabolism; D012697:Serine Endopeptidases / Q000378:metabolism*; D015398:Signal Transduction; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D007375:Interleukin-1; D044463:Receptor, PAR-1; D044464:Receptor, PAR-2; D018179:Receptors, Thrombin; D014409:Tumor Necrosis Factor-alpha; D004247:DNA; D019869:Phosphatidylinositol 3-Kinases; D012697:Serine Endopeptidases; C078261:duodenase; C112908:protease-activated receptor 4