title: Thrombin modulates vectorial secretion of extracellular matrix proteins in cultured endothelial cells.
abstract: We have identified a novel cellular action of thrombin on cultured rat adrenal medullary endothelial cells (RAMEC). Five-minute incubation of RAMEC with physiological concentrations of thrombin (<1 U/ml) caused within 3 h an increase in the basolateral deposition of the extracellular matrix (ECM) proteins fibronectin, laminin, and collagens IV and I, concomitant with a corresponding decrease in the apical release of these proteins into the medium. This shift in vectorial secretion of ECM proteins, quantitated with enzyme-linked immunoassays, was time dependent. Maximal stimulation of ECM protein deposition was observed after incubation of cells with thrombin for 5-15 min. Prolonged exposure (>1 h) to thrombin resulted in loss of proteins from the ECM. Thrombin-stimulated ECM protein deposition exhibited a bell-shaped dose dependence, peaking for all proteins at 0.25 U/ml of thrombin, and was independent of de novo mRNA or protein synthesis. Maximal amounts of deposited proteins increased between 2.5-fold (fibronectin) and 4-fold (collagen I) over baseline values. Similar results were obtained with thrombin receptor agonist peptide (TRAP), proteolytically active gamma-thrombin, and, to a lesser extent, other serine proteases such as trypsin and plasmin. A scrambled TRAP, proteolytically inactive PPACK-thrombin, DIP-thrombin, and type IV collagenase were ineffective. Together, these results suggest that the thrombin effects are mediated by proteolytic activation of the thrombin receptor. Possible involvement of the phospholipase C-signaling pathway in thrombin-mediated ECM protein deposition was also investigated. Inhibition or downregulation of protein kinase C (PKC) and chelation of intracellular or extracellular Ca2+ did not suppress, but rather enhanced, basal and thrombin-stimulated ECM protein deposition. Quantitative differences in augmentation of basolateral deposition by these treatments suggest differential regulatory pathways for individual ECM proteins. Our data indicate that, in cultured RAMEC, short-term activation of the thrombin receptor causes an increase in amounts of deposited ECM protein by a cellular signaling pathway that is independent of PKC activation and/or elevation of intracellular Ca2+.
authors.affiliation: Department of Biology, Marquette University, Milwaukee, Wisconsin 53201-1881, USA.
authors.forename: E
authors.initials: E
authors.lastname: Papadimitriou
authors.forename: V G
authors.initials: VG
authors.lastname: Manolopoulos
authors.forename: G T
authors.initials: GT
authors.lastname: Hayman
authors.forename: M E
authors.initials: ME
authors.lastname: Maragoudakis
authors.forename: B R
authors.initials: BR
authors.lastname: Unsworth
authors.forename: J W
authors.initials: JW
authors.lastname: Fenton
authors.forename: P I
authors.initials: PI
authors.lastname: Lelkes
mesh_terms: D000313:Adrenal Medulla / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000502:physiology; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004727:Endothelium / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D016326:Extracellular Matrix Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D010446:Peptide Fragments / Q000494:pharmacology; D010447:Peptide Hydrolases / Q000494:pharmacology; D011493:Protein Kinase C / Q000502:physiology; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D018179:Receptors, Thrombin / Q000819:agonists; D012697:Serine Endopeptidases / Q000494:pharmacology; D013917:Thrombin / Q000494:pharmacology*; D013997:Time Factors
chemical_list: D016326:Extracellular Matrix Proteins; D010446:Peptide Fragments; D012333:RNA, Messenger; D018179:Receptors, Thrombin; D011493:Protein Kinase C; D010447:Peptide Hydrolases; D012697:Serine Endopeptidases; D013917:Thrombin; D002118:Calcium