Expression, function and cooperating partners of protease-activated receptor type 3 in vascular endothelial cells and B lymphocytes studied with specific monoclonal antibody.
Receptor-specific antibodies can both prevent ligand-receptor interaction and initiate receptor signaling. Previously we generated monoclonal antibody 8E8 (mAb 8E8) against protease-activated receptor type 3 (PAR3) which inhibited proliferation of B cell hybridoma. Here we used mAb 8E8 and PAR1-specific polyclonal antibody to reveal the functions and cooperating partners of PAR3 in endothelial cells and in B lymphocytes. MAb 8E8 or PAR1 agonist peptide stimulated IL-6 and IL-8 production and VCAM-1 expression in HPMEC-ST1.6R cells. PAR1 antibody stimulated only VCAM-1 expression, while ICAM-1 expression was stimulated with mAB 8E8 or PAR3 peptide. MAb 8E8 stimulated weak mitogenic response, while PAR1 antibody inhibited it in normal but not in malignant B lymphocytes. Sandwich ELISA assay demonstrated the interaction of PAR3 with PAR1 in malignant cell lines and with IgM in normal B lymphocytes. It is concluded that PAR3 cooperates with PAR1 to mediate the effect of thrombin on cytokine production and VCAM-1 expression in endothelial cells and on cell proliferation in malignant B cells. ICAM-1 expression in endothelial cells requires PAR3 without PAR1. The inhibitory effect of thrombin in normal B lymphocytes is mediated by PAR1 alone, while mitogenic and pro-survival signaling in B lymphocytes is provided through PAR3 in cooperation with BCR.
Palladin Institute of Biochemistry, Kyiv, Ukraine.
Olena
O
Kalashnyk
Yuliya
Y
Petrova
Olena
O
Lykhmus
Lyubov
L
Mikhalovska
Sergiy
S
Mikhalovsky
Anastasiya
A
Zhukova
Dmitri
D
Gnatenko
Wadie
W
Bahou
Sergiy
S
Komisarenko
Maryna
M
Skok
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D001402:B-Lymphocytes / Q000276:immunology* / Q000378:metabolism*; D002460:Cell Line; D049109:Cell Proliferation; D042783:Endothelial Cells / Q000276:immunology* / Q000378:metabolism*; D006801:Humans; D015850:Interleukin-6 / Q000276:immunology / Q000378:metabolism; D016209:Interleukin-8 / Q000276:immunology / Q000378:metabolism; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D044463:Receptor, PAR-1 / Q000276:immunology / Q000378:metabolism; D018179:Receptors, Thrombin / Q000096:biosynthesis / Q000276:immunology* / Q000378:metabolism*; D013917:Thrombin / Q000276:immunology / Q000378:metabolism; D019010:Vascular Cell Adhesion Molecule-1 / Q000276:immunology / Q000378:metabolism
D000911:Antibodies, Monoclonal; D015850:Interleukin-6; D016209:Interleukin-8; D044463:Receptor, PAR-1; D018179:Receptors, Thrombin; D019010:Vascular Cell Adhesion Molecule-1; C105295:protease-activated receptor 3; D013917:Thrombin