Src-family kinases mediate an outside-in signal necessary for beta2 integrins to achieve full activation and sustain firm adhesion of polymorphonuclear leucocytes tethered on E-selectin.
In cell suspensions subjected to high-shear rotatory motion, human PMN (polymorphonuclear cells) adhered to E-selectin-expressing CHO (Chinese-hamster ovary) cells (CHO-E), and formed homotypic aggregates when challenged by E-selectin-IgG fusion protein, by a mechanism that involved beta2 integrins. Both heterotypic and homotypic PMN adhesion was accompanied by tyrosine phosphorylation of a 110 kDa protein (P110). This event was prevented by blocking anti-(beta2 integrin) antibodies and by inhibitors of Src-family kinases, suggesting that it was part of an 'outside-in' signalling that was initiated by integrin engagement. Interestingly, Src-family kinase inhibitors prevented beta2-integrin-mediated (i) homotypic PMN adhesion triggered by E-selectin-IgG, (ii) heterotypic CHO-E/PMN adhesion in mixed-cell suspensions, and (iii) firm adhesion of PMN to CHO-E monolayers under physiological flow. Similarly to PMN treated with Src-family kinase inhibitors, PMN from hck-/-fgr-/- and hck-/-fgr-/-lyn-/- mice showed significant impairment of beta2-integrin-mediated adhesion to CHO-E. Moreover, the expression of beta2 integrin activation epitopes at the sites of cell-cell contact in CHO-E/PMN conjugates was abolished by Src-family kinase inhibitors. One component of P110 was identified as the FAK (focal adhesion kinase) Pyk2 (proline-rich tyrosine kinase 2), which was phosphorylated in a beta2 integrin- and Src-family-kinase-dependent manner. Thus, Src-family kinases, and perhaps Pyk2, mediate a signal necessary for beta2 integrin function in PMN tethered by E-selectin.
Totani
Licia
L
Laboratory of Vascular Biology and Pharmacology, Consorzio Mario Negri Sud, 66030 Santa Maria Imbaro, Italy. totani@negrisud.it
Piccoli
Antonio
A
Manarini
Stefano
S
Federico
Lorenzo
L
Pecce
Romina
R
Martelli
Nicola
N
Cerletti
Chiara
C
Piccardoni
Paola
P
Lowell
Clifford A
CA
Smyth
Susan S
SS
Berton
Giorgio
G
Evangelista
Virgilio
V
D000818:Animals; D018821:CD18 Antigens / Q000502:physiology*; D016466:CHO Cells; D002448:Cell Adhesion / Q000502:physiology*; D006224:Cricetinae; D003412:Cricetulus; D019040:E-Selectin / Q000502:physiology*; D004533:Egtazic Acid / Q000494:pharmacology; D005434:Flow Cytometry; D051418:Focal Adhesion Kinase 2 / Q000502:physiology; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009504:Neutrophils / Q000166:cytology*; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D011720:Pyrazoles / Q000494:pharmacology; D011743:Pyrimidines / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000502:physiology; D012212:Rheology; D015398:Signal Transduction / Q000502:physiology*; D013314:Stress, Mechanical; D014162:Transfection; D019061:src-Family Kinases / Q000037:antagonists & inhibitors / Q000502:physiology*
C108123:4-amino-5-(4-methylphenyl)-7-(tert-butyl)pyrazolo(3,4-d)pyrimidine; D018821:CD18 Antigens; D019040:E-Selectin; D011720:Pyrazoles; D011743:Pyrimidines; D011993:Recombinant Fusion Proteins; D004533:Egtazic Acid; D051418:Focal Adhesion Kinase 2; D019061:src-Family Kinases