Oxidant stress enhances protein binding to an AP-1 like site in the 5′ flanking sequence of murine ICAM-1 ♦ 1622

Intercellular adhesion molecule-1 (ICAM-1), a ligand for neutrophilβ₂ integrins, has been found to be important in the development of hyperoxic lung injury. We have reported that ICAM-1 is upregulated in hyperoxic mouse lungs independent of cytokine upregulation. To explore mechanisms of ICAM-1 upregulation we found that ICAM-1 mRNA was increased in H441 cells exposed to H₂O₂ and TNF-α. We then sequenced 2.5 kb of the 5′ flanking sequence of the murine ICAM-1 gene and constructed serial deletions ligated to chloamphenicol actyl transferase (CAT) transfected into H441 cells. We found CAT expression was consistently increased -398 to -309 bp from the transcription start site, and decreased from -309 to -280. We then performed gel-mobility shift assays and found evidence for protein binding between -309 and -280, with no binding when a cis-element contained in this region similar to an AP-1 binding site was mutated. The binding site sequence (TGACTCC) in the region is similar to a prototypic AP-1 site (TGACTCA), but the signals were not supershifted by antibodies detecting members of the fos/jun superfamily. In addition, protein-DNA binding to the AP-1-like site was increased in H441 cells exposed to TNF-α, and H₂O₂ as well as lung tissue from mice exposed to hyperoxia. Southwestern blot analysis with a radiolabeled oligomer from -309 to -280 found that a 90 kd and a 110 kd protein bound to the sequence. We conclude that the two proteins binding to the ICAM-1 between -309 to -280 may be responsible for ICAM-1 upregulation under oxidant stress. Supported by NIH grant 5 P30 HD27823-04