title: Lipopolysaccharide stimulates the tyrosine phosphorylation of mitogen-activated protein kinases p44, p42, and p41 in vascular endothelial cells in a soluble CD14-dependent manner. Role of protein tyrosine phosphorylation in lipopolysaccharide-induced stimulation of endothelial cells.
abstract: Vascular endothelial cell (EC) injury or activation by LPS plays a critical role in the pathogenesis of Gram-negative meningitis and endotoxic shock. EC do not express membrane CD14, but respond to LPS in a soluble CD14-dependent manner. The signal transduction mechanisms involved in LPS-induced EC responses are largely unknown. We used bovine and human brain microvessel EC (BBMEC, and HBMEC) to study LPS-induced protein tyrosine phosphorylation. LPS rapidly induced the tyrosine phosphorylation of several proteins in BBMEC and HBMEC, which was detectable by 5 to 15 min, reached a maximum by 30 min, and declined by 60 to 90 min. The increase in tyrosine phosphorylation was apparent following stimulation with LPS at 0.1 ng/ml and was dose dependent up to 100 ng/ml. Similar changes in tyrosine phosphorylation were induced by smooth and rough LPS as well as lipid A, but not by the inactive lipid A analogue, Rhodopseudomonas sphaeroides diphosphoryl lipid A. Pretreatment of EC with the tyrosine kinase inhibitor, herbimycin A, inhibited LPS-stimulated protein tyrosine phosphorylation and LPS-mediated lactic dehydrogenase release from BBMEC and IL-6 release from HBMEC in a dose-dependent manner. Three proteins with apparent m.w. of 44, 42, and 41 kDa were predominant among the LPS-induced tyrosine phosphoproteins, and they were identified as mitogen-activated protein kinase isoforms ERK1, ERK2, and p38, respectively. LPS-induced protein tyrosine phosphorylation in HBMEC and BBMEC was soluble CD14 dependent, since pretreatment of these cells with anti-hCD14 mAb inhibited the LPS-induced tyrosine phosphorylation of p44, p42, and p41. Additionally, LPS induced a mobility shift in p44 and p42 mitogen-activated protein kinase isozymes, which was inhibited by herbimycin A pretreatment of the EC. These findings demonstrate for the first time that increased protein tyrosine phosphorylation and activation of mitogen-activated protein kinases occur rapidly after LPS stimulation of EC in the presence of soluble CD14. Our data also suggest that a herbimycin-sensitive step, presumably a tyrosine kinase, is involved in mediating LPS-induced human EC activation and IL-6 secretion.
authors.affiliation: Division of Infectious Diseases, Childrens Hospital Los Angeles, University of Southern California School of Medicine 90027, USA.
authors.forename: M
authors.initials: M
authors.lastname: Arditi
authors.forename: J
authors.initials: J
authors.lastname: Zhou
authors.forename: M
authors.initials: M
authors.lastname: Torres
authors.forename: D L
authors.initials: DL
authors.lastname: Durden
authors.forename: M
authors.initials: M
authors.lastname: Stins
authors.forename: K S
authors.initials: KS
authors.lastname: Kim
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016227:Benzoquinones; D001921:Brain / Q000098:blood supply; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000187:drug effects / Q000276:immunology / Q000378:metabolism*; D002417:Cattle; D002478:Cells, Cultured; D002648:Child; D002675:Child, Preschool; D004306:Dose-Response Relationship, Immunologic; D004730:Endothelium, Vascular / Q000187:drug effects / Q000201:enzymology* / Q000276:immunology; D006801:Humans; D047029:Lactams, Macrocyclic; D018950:Lipopolysaccharide Receptors / Q000502:physiology*; D008070:Lipopolysaccharides / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D051379:Mice; D008833:Microcirculation / Q000201:enzymology / Q000276:immunology; D008969:Molecular Sequence Data; D010766:Phosphorylation / Q000187:drug effects; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000187:drug effects / Q000378:metabolism*; D011809:Quinones / Q000494:pharmacology; D017828:Rifabutin / Q000031:analogs & derivatives; D014443:Tyrosine / Q000378:metabolism*
chemical_list: D016227:Benzoquinones; D047029:Lactams, Macrocyclic; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D011809:Quinones; D017828:Rifabutin; D014443:Tyrosine; C020754:herbimycin; D011505:Protein-Tyrosine Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases