The PI3K/Akt-NF-kappa B Signaling Pathway and Hypoxia-induced Mitogenic Factor in Vascular Adhesion Molecule 1 Gene Expression - Adhesion Molecules - page 95

Control
HIMF IT
Fig. 2 Immunohistochemical staining for VCAM-1 in mouse lung tissue at er intratracheal
instillation of HIMF. h ere is marked increase of VCAM-1 protein expression in HIMF-treated
mouse lungs. h e upregulated VCAM-1 is mainly localized to airway epithelium and alveolar type
II cells and vascular endothelium (arrows). Scale bars = 100 μm. Reprint from Tong et al. (2006),
with permission.
whether HIMF enhances VCAM-1 expression through NF-κB pathway. To assess
this possibility, transfection of several dominant-negative mutants in NF-κB
pathway, IKKα (K44A), IKKβ (K44A) and IκBα (S32A/S36A), was performed in
SVEC 4-10 and MLE-12 cells. In response to HIMF, IKK was phosphorylated,
which in turn phosphorylates IκBα leading to NF-κB activation in both SVEC
4-10 and MLE-12 cells (Fig. 4) .
PI-3K/Akt Pathway is Involved in HIMF-Induced NF- k B
Activation and VCAM-1 Production
To test whether PI-3K/Akt participates in HIMF-mediated NF-κB activation,
SVEC 4-10 and MLE-12 cells were treated with HIMF and western blot was
performed. h e results showed that HIMF strongly induced Akt phosphorylation
at Ser473 and h r308. Furthermore, only the PI-3K inhibitor LY294002 (10
μmol/L) inhibited HIMF-activated Akt phosphorylation (Fig. 5) . However,
incubation of cells with SB203580 (5 μmol/L), PD098059 (5 μmol/L) or U0126 (5
μmol/L), inhibitors against p38 and ERK1/2 MAPK pathways respectively, had no
ef ect on HIMF-induced Akt phosphorylation. In addition, transfection of Δp85, a