High Mobility Group Box-1 Protein Acts As A Co-activator Of Nuclear Factor Of Activated T Cells -2 In Promoting Interleukin-2 Transcription

High mobility group box protein 1 (HMGB1), a nuclear and Cytosolic protein, was originally identified as an intranuclear factor with an important structural function in chromatin organization. Recently, HMGB1 has been identified as a proinflammatory cytokine that mediates endotoxin lethality, local inflammation, and macrophage activation. HMGB1 induces arthritis when it is injected into murine joints and also acute lung injury. HMGB1 is released by activated macrophages and monocytes after exposure to LPS, TNF-α, or IL-1β and as a result of tissue damage. It is a also a potent stimulating signal to monocytes and induces the delayed synthesis of proinflammatory cytokines. The intracellular abundance of HMGB1 and its proinflammatory activities suggest that its release at sites of cell injury or damage plays a role in the initiation and/or perpetuation of an immune response. Furthermore, as HMGB1 is found in the serum of patients with acute (sepsis) and chronic (rheumatoid arthritis) inflammatory conditions, and it may be involved in maladaptive or autoimmune responses. HMGB1 protein can trigger the release of IL-2 from T cells and IL-12 from dendric cells (DC). Our present study showed that addition of recombinant HMGB1 protein into culture induced apoptosis of a large number of T cells, and could promote the translocation of nuclear factor of activating T cells(NFAT) from cytoplasm to nucleus. However, the underlying mechanism remained to be determined and required further research.It is well known that NFAT family transduces major immunological signals in T cells. NFAT translocates into nucleus and regulates gene transcription. Fivemembers of NFAT are identified now, NFATl(NFATp,NFATc2), NFAT2(NFATcl,NFATc), NFAT3(NFATc4), NFAT4(NFATc3, NFATx) and NFAT5. NFAT5 is the only NFAT-related protein represented in the Drosophila genome. NFAT5 is identical to TonEBP (tonicity element binding protein), a transcription factor crucial for cellular reponses to hypertonic tress. NFAT1 and NFAT2 are highly expressed in peripheral T cells and strongly activate the IL-2 promoter in transfection assays. Recombinant plasmids of HMGB1 and NFAT2 were constructed for the examination of their coordination in promoting activity of IL-2 reporter gene, for the determination of the role of their binding, and for the exploration of their binding site. This may be helpful for identifying the target for interventional strategy. Main results:1. HMGB1 coordinates with NFAT2 in promoting IL-2 reporter gene transcription. Dramatic increase of IL-2 reporter gene was only obtained when both of HMGB1 and NFAT2 were transfected.2. Either of HMGB1 and NFAT2 was indispensable in promoting activity of IL-2 reporter gene, and it was verified by sRNAi analysis.3. HMGB1 could bind to NFAT2 in cells or in vitro.4. B box of HMGB1 was identified to be the binding site for NFAT2.