Summary
Megakaryocytic genes such as αIIbβ3 can be expressed by malignant cells as part of
the disturbances in their gene regulation. However, the function of the gene product
greatly depends on the interaction of the ectopic protein with the new environment.
The outside-in signaling of the ectopically expressed αIIbβ3 integrin was studied
in B16a murine melanoma cells using a monoclonal antibody, specifically directed to
the activated conformation of αIIbβ3, PAC-1 and the physiological ligand, fibrinogen.
Ligation of αIIbβ3 induced downregulation of FAK but serine phosphorylation of three
protein bands, 20/21, 85 and 140 kDa within 1-15 min. Flow cytometry indicated that
the ligation of the receptor in B16a cells induces ~50% increase in phosphoserine
positive cells within 5-15 min. 12-lipoxygenase is placed downstream in the signaling
pathway, since ligation of αIIbβ3 induces 12-HETE production within 5 min and pretreatment
of tumor cells with select lipoxygenase inhibitior, Baicalein, prevents the increase
in serine phosphorylation. Confocal microscopy of adherent tumor cells demonstrated
rearrangement of actin filaments upon αIIbβ3 ligation paralleled by downregulation
of p125FAK and phoshotyrosine+ adhesion plaques and translocation of PKCα to stress fibers and cortical actin. PKC
appears to be the major effector serine kinase of the αIIbβ3-coupled signaling pathway,
since pretreatment of tumor cells with a select PKC inhibitor, Calphostin C, prevents
the ligationinduced serine phosphorylation. Previous studies have indicated a role
for the 12-lipoxygenase-PKC signaling pathway in platelet aggregation as well as tumor
invasion, therefore the involvement of this cascade in the signaling of the ectopic
αIIbβ3 integrin may partially explain its role in tumor progression.
Key words
Melanoma - integrin αIIbβ3 - signal transduction - flow cytometry - confocal microscopy
