Apoptosis signal-regulating kinase 1 (ASK1) and HIF-1? protein are essential factors for nitric oxide-dependent accumulation of p53 in THP-1 human myeloid macrophages

Nitric oxide (NO) is a reactive secondary mediator, which has been found to participate in cell cycle regulation and apoptosis in myeloid macrophages, the key effectors of inflammatory and innate immune responses. However, the molecular mechanisms of nitric oxide-induced death of myeloid macrophages are not well understood. In this study we have found that NO derived from S-nitrosoglutathione (GSNO) activates ASK1 in THP-1 human myeloid macrophages in a concentration and time-dependent manner. It also induces accumulation of HIF-1? protein in a concentration-dependent manner, which peaks at 4 h of exposure to 1 mM GSNO. GSNO does not affect the level of HIF-1? mRNA as detected by the RT-PCR. In addition, GSNO was found to induce accumulation of p53 in normal but not HIF-1? knockdown THP-1 cells, where expression of this protein was silenced by specific siRNA. It has also been found that GSNO-mediated accumulation of p53 depends on activation of ASK1 since no GSNO-induced p53 stabilisation was observed in THP-1 cells transfected with dominant-negative form of this kinase. However, in both HIF-1? knockdown THP-1 cells and those transfected with the dominant-negative form of ASK1, GSNO was able to induce cell death as detected by the MTS cell viability assay leading to an increase in release of LDH.