Abstract
Close talk between inflammatory mediators and immunological cytokines has been discovered and reported. In this study, the role of nuclear factor of activated T cell-2 (NFAT2) in regulation of high mobility group box-1 (HMGB1) release was investigated. THP-1 cell and HEK293T cell were incubated and stimulated by lipopolysaccharide (LPS). Firstly, binding site between HMGB1 and NFAT2 was identified by co-immunoprecipitation (IP). Box A, Box B and CT domain of HMGB1 were constructed, as well as Rel-homology-domain (RHD), pre-RHD and pro-RHD of NFAT2. THP-1 cell was harvested, cell lysate and culture medium were collected at appointed times. Binding between HMGB1 and NFAT2 was measured, HMGB1 protein level in culture medium was analyzed at the same time. Secondly, the role of NFAT2 in regulating HMGB1 release was investigated. When THP-1 cell was cultured for 24 h, HMGB1 protein level was measured at appointed times with or without siRNA to inhibit NFAT2 expression. Our data show that HMGB1 bound to NFAT2 in THP-1 cell cytoplasm. Further experiments showed that box B domain of HMGB1 could bind to pre-RHD of NFAT2. After stimulation by LPS, interaction between HMGB1 and NFAT2 was discovered decreasing gradually. However, HMGB1 protein level increased in culture medium at the same time. Furthermore, HMGB1 release could be enhanced by NFAT2 inhibition. Taken together, release of HMGB1 could be regulated by NFAT2 in human monocytes.