The PI3K/Akt-NF-kappa B Signaling Pathway and Hypoxia-induced Mitogenic Factor in Vascular Adhesion Molecule 1 Gene Expression - Adhesion Molecules - page 94

Control HIMF 6h HIMF 24h

110 KD

37 KD

8

* 7

*

6

5

4

3

2

1

0

Control HIMF 6 h HIMF 24 h

Fig. 1 Western blot for VCAM-1 protein at er intratracheal instillation of HIMF in mouse lung.
Intratracheal instillation of recombinant HIMF-induced upregulation of VCAM-1 protein in
mouse lung. Results expressed as mean ± SEM. *Signii cant dif erence from the control group at
both time points (D. Li, unpublished data).

Activation of NF- k B is Essential for HIMF-induced VCAM-1
Production

To search for the TFs involved in HIMF-induced VCAM-1 production, sequentially
deleted 5¢-l anking sequences of mouse VCAM-1 promoter were transfected into
cultured endothelial cells, SVEC 4-10, and HIMF stable expression SVEC-HIMF
(Fig. 4) . h e sequences TGGGTTTCCC at -73 bp and AGGGATTTCCC at -58
bp are identical to the consensus sequence (GGG R[C/A/T]TYYCC) of the NF-
κB binding site. h e results showed that treatment of HIMF induces VCAM-1
promoter-luciferase reporter activity signii cantly in SVEC-HIMF, which stably
overexpresses HIMF. h e highest VCAM-1 luciferase activity was found from a
short construct pGLVCAM-1 (-0.3 luc) that contains two NF-κB binding sites
within 329 bp of VCAM-1 promoter, whereas another deletion construct exhibited
lower luciferase activity. h ese results suggest that a negative regulatory element
might exist between the -0.7 and -0.3 region of the VCAM-1 promoter. It has
been reported that NF-κB regulates VCAM-1 expression; it is unknown, however,