The roles of Akt and NOSs in regulation of VLA-4-mediated melanoma cell adhesion to endothelial VCAM-1 after UVB-irradiation

Abstract

UVB-reduced avidity between M624 melanoma and HUVEC cells is dependent on the interaction of VLA-4 with its endothelial ligand VCAM-1. Our previous studies suggested that a spatial organization of α4 integrin, one of the two subunits of VLA-4, on the melanoma cell surface contributed to the changes in avidity for VCAM-1 upon UVB-irradiation. In this study, we demonstrate that Akt plays an important role in regulation of the expression and surface level of α4 integrin on melanoma cells upon UVB-irradiation. While the cell surface level of α4 integrin is not significantly affected by UVB-irradiation or Akt inhibitor alone, it is dynamically altered after UVB-irradiation when Akt is inhibited. Inhibition of Akt also reverses the reduction of avidity of cells after the irradiation. Our data also shows that UVB reduces the level of Akt. The inhibition of Akt activity correlates with a reduced amount of coupled cNOS and reduced amount of iNOS after UVB-irradiation. However, the effect of NOSs on melanoma cell adhesion appears due to their roles in regulation of apoptosis after UVB-irradiation. Base on these results, we propose that the UVB-induced reduction of avidity of melanoma cells is coordinatively regulated by NOSs and Akt through two differential mechanisms.

Fig. 1. The expression of α4 integrin

M624 melanoma cells were treated with L-NAC, L-NAME, 1400w or Akt I in the presence or absence of UVB (50 mJ/cm²) as indicated. The total cellular levels of Akt and β-actin were determined by western blot analysis using corresponding antibodies.

Fig. 2. The expression of iNOS, eNOS and Akt, and coupling of eNOS

M624 melanoma cells were treated with L-NAC, L-NAME, 1400w or Akt I in the presence or absence of UVB (50 mJ/cm²) as indicated. Panel A: The expression of iNOS and Akt was determined by western blot analysis using corresponding antibodies. Panel B: The monomers and dimers of eNOSs were first separated on a low-temperature SDS-resistant SDS-PAGE and then detected by western blot analysis using anti-eNOS antibody. The levels of β-actin were also probed as a loading control.

Fig. 3. The cell surface level of α4 integrin

M624 melanoma cells were treated with L-NAC, L-NAME, 1400w or Akt I in the presence or absence of UVB (50 mJ/cm²) as indicated. FITC-conjugated mouse anti-human α4 integrin antibody was added in each sample and the intensity of cell surface α4 integrin was determined by flow cytometry. 2×10⁴ cells were counted for each sample. The data represents three independent experiments.

Fig. 4. The adhesive affinity of melanoma cells

M624 melanoma cells were treated with L-NAC, L-NAME, 1400w or Akt I and then untreated or UVB irradiated. The cells were harvested for use in the flow adhesion assay at 18 h post-UVB. The cells were perfused over immobilized soluble VCAM-1 in the parallel plate flow chamber, at a physiologic shear stress of 0.45 dyn/cm². The number of adhesive interactions in a single field of view was counted over a 5 min perfusion period. The experiments were repeated three times. The bars represented as mean±SD. The student t-test was used to compare the control with UVB irradiation with the inhibitor treatment after UVB irradiation. *: p<0.05.