br Involvement of p MAPK pathway The NF B
Involvement of p38 MAPK pathway The NF-κB signal transduction cascade is a major stress response signaling pathway for the COX-2 gene expression. In mice and humans, the COX-2 promoter has oicr in for many transcription factors, including NF-κB in the 5′ region of the COX-2 gene , and the requirement of the activation of NF-κB to induce the expression of COX-2 in the lipopolysaccharide-stimulated macrophages has been described . Based on the discovery of HNE as a potential inducer of COX-2, several studies focusing on the HNE-induced signaling mechanisms for the COX-2 expression have been performed , . Initially, it was anticipated that the NF-κB-dependent signaling pathway might mediate the HNE-stimulated COX-2 induction. However, no significant change in the IκB and NF-κB levels after treatment with HNE was observed. Kumagai et al. ,  found that, instead of the NF-κB pathway, HNE elicited a rapid and significant phosphorylation of p38 mitogen-activated protein kinase (MAPK) and activate MAPK kinase (MKK)3/MKK6, a specific MAPKK of p38 MAPK. In addition, the relationship between COX-2 mRNA stability and HNE-activated p38 MAPK pathway was also revealed (Fig. 2). Involvement of a Src-dependent p38, ERK/c-Jun pathway was recently proposed as a major regulator of HNE-induced COX-2 expression in YPEN-1 cells . Thus, the HNE-induced COX-2 gene expression is, at least in part, regulated at post-transcriptional levels via the p38 MAPK pathway.
Involvement of p53 and Sp1 To investigate transcriptional regulation of the COX-2 gene in response to HNE, Kumagai et al.  examined whether the HNE-induced COX-2 expression was mechanistically linked to the expression of p53, a transcription factor that regulates the response to a variety of stimuli, and found that the COX-2 levels were inversely correlated with the p53 levels. In addition, the down-regulation of p53 with the antisense oligonucleotides against p53 significantly enhanced the expression of COX-2 mRNA and protein. These findings and the fact that COX-2 protein is undetectable in normal epithelial cells suggest that mutations of p53 may contribute to the increased expression of COX-2. Of interest, 4-oxo-2-nonenal, an analog of HNE, is unable to induce COX-2 while it activates a p53 signaling pathway . On the other hand, it was hypothesized that Sp1, a general transcription factor that is involved in various inducible and constitutive gene expressions, might also be involved in the induction of COX-2 in response to HNE . This speculation was based on the facts that (i) p53 suppresses various gene expressions through preventing Sp1 activity, (ii) The rat COX-2 promoter region has no putative p53-binding elements, and a minimal promoter region required for the basal transcription of the human COX-2 gene has been demonstrated to contain GC-rich proximal sequences that are specifically bound by Sp1, and (iii) p53 negatively regulates Sp1 through the formation of a p53-Sp1 heterocomplex. The immunoprecipitation experiments indeed showed that p53 bound to Sp1 in intact cells under normal conditions and HNE elicited the dissociation. It was also observed that the dissociation of p53-Sp1 complexes was accompanied by the nuclear translocation of Sp1. In addition, electrophoretic mobility shift assays using the oligonucleotide containing the Sp1 consensus element as a probe showed that HNE treatment resulted in a time-dependent increase in the Sp1 DNA binding activity. Although the regulatory mechanism of dissociation of p53-Sp1 complex remains unclear, the involvement of a phosphatidylinositol 3-kinase pathway, which induces p53 degradation through mdm2 phosphorylation, may not be unlikely. HNE indeed activates phosphatidylinositol 3-kinase/AKT pathway in vascular smooth muscle cells and wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase, significantly inhibited the COX-2 expression . Taken together, it has been hypothesized that down-regulation of p53 followed by the activation of a transcription factor Sp1 might be involved in the HNE-induced COX-2 gene expression (Fig. 2).