Macrophage-associated nitric oxide (NO) production plays a crucial role in the pathogenesis of tissue damage. However, negative factors that regulate NO production remains poorly understood despite its significance of NO homeostasis. Here, we show that activating transcription factor 3 (ATF3), a transcriptional regulator of cellular stress responses, was strongly induced in activated macrophages and its depletion resulted in pronounced enhancement of inducible nitric oxide synthase (iNOS) gene expression and subsequently the induction of high levels of NO production. In response to lipopolysaccharide (LPS) and IFN-γ, ATF3 inhibited transcriptional activity of NF-κB by interacting with the N-terminal (1-200 amino acids) of p65 and was bound to the NF-κB promoter, leading to suppression of iNOS gene expression. In addition, inhibitory effects of ATF3 on iNOS and NO secretion were suppressed by inhibitor of casein kinase II (CK2) activity or its knockdown. Moreover, the levels of ATF3 were highly elevated in established cecal ligation and puncture or LPS-injected mice, a model of endotoxemia. ATF3 is also elevated in peritoneal macrophages. Collectively, our findings suggest that ATF3 regulates NO homeostasis by associating with NF-κB component, leading to the repression of its transcriptional activity upon inflammatory signals and points to its potential relevance for the control of cell injuries mediated by NO during macrophage activation.

Allergies are immediate hypersensitive responses to antigens and IL‐4 is involved in the initiation and development of allergic responses. Diosgenin, a steroidal sapogenin, is extracted from the root of wild yam and has been shown to have a variety of biological activities including anti‐inflammatory activity. The present study was undertaken to examine whether diosgenin has an inhibitory effect on allergic response in mouse splenocytes and mouse model. Our results showed that diosgenin suppressed both T and B cells proliferation induced by mitogens. RT‐PCR analysis also revealed that mRNA levels of both IL‐4 and NFAT1 were in vitro suppressed in the presence of diosgenin. In allergic mouse model, application of diosgenin on atopic dermatitis like skin lesions improved skin condition and inhibited starching behaviors. In addition, diosgenin application suppressed IL‐4 mRNA levels and its secretion. Levels of NFAT1 mRNA were also attenuated by diosgenin in mouse. Taken together, these results suggest that diosgenin has a potent inhibitory role in T cell activation and may be a candidate for therapeutic agent in allergy.

Activating transcription factor 3(ATF3), known as a stress inducible gene, is a member of the CREB/ATF family of transcription factors which is expressed only under conditions such as inflammation, cell injury or oxidative stress. LPS‐induced ATF3 has been known to suppress productions of Nitric oxide (NO) and cytokines such as IL‐6, and IL‐12 in macrophages. However, the mechanism of action of ATF3 on the macrophage function has not been characterized in detail. In the present study, we examined the regulatory role of ATF3 on inducible nitric oxide synthase (iNOS) expression in LPS/IFN‐γ‐treated macrophages. ATF3 knockdown enhanced NO in macrophages. Western blot and RT‐PCR analysis revealed that protein and mRNA levels of iNOS were also attenuated in ATF3‐knockdown cells. In addition, activities of NF‐κB and AP‐1 were increased by ATF3‐knockdown. These data indicate that ATF3 acts as s negative regulator on iNOS expression via suppression of NF‐κB and AP‐1 activities. Treatment with casein kinase ¥±(CK¥±) inhibitor decreased iNOS expression. LPS/IFN‐γ induced‐phosphorylation and nuclear translocation of ATF3 were also attenuated by CK¥± inhibitor. Overall, these results suggest that ATF3 regulates NO production through modulation of CK¥±, NF‐κB and AP‐1 signaling pathway.

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