HMBOX1 was a novel transcription factor possibly involving in function of pancreas and cytotoxicity of NK cells. For function determination, recombinant human HMBOX1 protein was obtained and purified, and the monoclonal antibodies against HMBOX1 were prepared. The full-length cDNA fragment encoding HMBOX1 was amplified from NK-92 cells and inserted into prokaryotic expression vector pET22b. The pET22b-HMBOX1-6his vector was then transformed into E. coli Rosetta （DE3） and induced by 1 mM IPTG for 4 h at 37℃. The fusion HMBOX1 protein was mainly expressed in inclusion bodies, which was purified and refolded using Ni^2＋-affinity chromatography. With the purified fusion HMBOX1 protein as antigen, monoclonal antibodies against HMBOX1 were generated, providing a potentially useful tool for further study in HMBOX1 functions. Using these anti-HMBOX1 mAbs, we identified that HMBOX1 is located in both cytoplasm and nucleus and could be detected in 10 human normal tissues, including cerebrum, pancreas, kidney and liver tissues. Moreover, the expression in hepatic carcinoma was significantly lower than that in adjacent tissues.
Jun DaiLongyan WuCai ZhangXiaodong ZhengZhigang TianJian Zhang
HMBOX1 is a new member of the homeobox family.Homeobox members have been reported to participate in embryonic development and systemic metabolism,but the function of HMBOX1 remains unclear,especially in the hematopoietic system.Here,we show that HMBOX1is expressed at a high level in primary humanNKcells but is expressed at much lower levels inNKcell lines.Overexpression of HMBOX1 significantly inhibited NK cell activities,including natural cytotoxicity against tumor cells,the level of CD107a(a marker protein for degranulation)and the production of cytolytic proteins(perforin and granzymes).More interestingly,HMBOX1 negatively regulated the expression of NKG2D and the activation of the NKG2D/DAP10 signaling pathway in NK cells.This effect was reversed by knocking down HMBOX1.Taken together,these findings demonstrate that HMBOX1 may act as a negative regulator of NK cell functions via suppressing the NKG2D/DAP10 signaling pathway.
