Effect Of Neddylation On Hematopoietic Stem Cells

Objective Conditional knockout of NEDD8 specifically in type I interferon-reactive cells was constructed to investigate the role of neddylation in hematopoietic stem cells and its potential mechanism,which provide a theoretical basis for drug research related to this pathway.Methods Firstly,we confirmed the localization of NEDD8 by IHC and then we constructed mice model of conditional knockout of NEDD8 specifically in type I interferon-reactive cells.Gel electrophoresis and Western blot were used to confirm the construction of the knockout mouse model.We counted the death of Nedd8^F/F mice and Nedd8^F/F,Mx1-Cre/F,Mx1-Cre mice.Bone marrow pathology of Nedd8^F/F mice and Nedd8^F/F,Mx1-Cre mice was observed.The proportion and absolute number of hematopoietic stem cells in Nedd8^F/F,Mx1-Cre/F,Mx1-Cre mice and their littermates were detected.Apoptosis and proliferation of hematopoietic stem cells in Nedd8^F/FMx1-Cre mice and their littermates were measured.In order to exclude the influence of environmental factors in Nedd8^F/F mice and Nedd8^F/F,Mx1-Cre/F,Mx1-Cre mice,the same experiments were conducted in a chimeric mice model.In addition,we measured the proportion of various hematopoietic precursor cells of chimeric mice.We isolated the lineage-negative cells from the bone marrow of Nedd8^F/F mice and Nedd8^F/F,Mx1-Cre mice and measured the protein levels of C-myc,N-myc and Cullin1.To further verify this hypothesis,we intraperitoneally injected Nedd8^F/F,Mx1-Cre with a myc inhibitor.In addition,we intraperitoneally injected Nedd8^F/F with MLN and DMSO and then measured the protein levels of Cullin4A of Lin^-cells.Result 1.The effects of Mx1-Cre-mediated NEDD8 conditional knockout on hematopoiesis in mice In this study,Western blot,surface marker staining,BrdU staining,flow cytometry and other methods were used to study the effect of Mx1-Cre-mediated NEDD8conditional knockout on hematopoiesis in mice.The results showed that NEDD8 and CD117 co-localized in bone marrow cavity.NEDD8 had been knocked out in Nedd8^F/F,Mx1-cre/F,Mx1-cre mice at gene and protein level.The survival rate of Nedd8^F/F,Mx1-cre/F,Mx1-cre mice was lower than Nedd8^F/F mice.The number of bone marrow cavity cell of Nedd8^F/F,Mx1-cre/F,Mx1-cre mice was lower than Nedd8^F/F mice.The absolute number of Bone marrow leukocyte,LSK and hematopoietic stem cells from Nedd8^F/F,Mx1-cre/F,Mx1-cre mice was lower than Nedd8^F/F mice.In chimeric mice,the absolute number of LSK,hematopoietic stem cells from Nedd8^F/F,Mx1-cre/F,Mx1-cre mice was lower than those from Nedd8^F/F mice.The percentages of CLP,CMP and MEP from Nedd8^F/F,Mx1-cre/F,Mx1-cre mice was lower than those from Nedd8^F/F mice.However,the percentages of GMP from Nedd8^F/F,Mx1-cre/F,Mx1-cre mice was higher than those from Nedd8^F/F mice.The percentages of myeloid cells in bone marrow,spleen and peripheral blood of chimeric mice and of B lymphocyte in spleen and peripheral blood of chimeric mice from Nedd8^F/F,Mx1-cre/F,Mx1-cre mice was lower than those from Nedd8^F/F mice.There was no significant difference between the two groups of B lymphocyte in bone marrow.In the bone marrow and peripheral blood of chimeric mice,the percentages of T lymphocyte from Nedd8^F/F,Mx1-cre mice was higher than that from Nedd8^F/F mice,but there was no significant difference between them in spleen.The apoptosis and proliferation of hematopoietic stem cells in Nedd8^F/F,Mx1-cre mice were significantly higher than those in Nedd8^F/F mice.In chimeric mice,the apoptosis and proliferation of hematopoietic stem cells from Nedd8^F/F,Mx1-cre mice were also significantly higher than those from Nedd8^F/F mice.2.The mechanism of hematopoietic exhaustion caused by Mx1-Cre-mediated NEDD8 conditional knockout The possible mechanism of hematopoietic exhaustion caused by Mx1-Cre-mediated NEDD8 conditional knockout was studied by magnetic bead sorting,Western blot and flow cytometry.It showed that the Mx1-Cre-mediated NEDD8 conditional knockout inhibited the neddylation of Cullin1 and then resulted in the accumulation of C-myc and N-myc in lineage negative bone marrow cells.Injection with a myc inhibitor partially rescued the hematopoietic exhaustion caused by Mx1-Cre-mediated NEDD8conditional knockout.The protein content of NEDD8-Cul4A of Lin-negative cells from Nedd8^F/F mice and those injected with DMSO had significantly higher than Cul4A.The protein content of Cul4A of Lin-negative cells from Nedd8^F/F,Mx1-cre mice was significantly higher than that from Nedd8^F/F mice.The protein content of NEDD8-Cul4A of Lin-negative cells from mice injected with DMSO was slightly higher than that from mice injected with MLN4924.It suggests that NEDD8 deficiency may affect the ubiquitination of Fbw7 to C-myc and N-myc by inhibiting the neddylation of Cullin1 and eventually result in hematopoietic failure in mice.Conclusion Neddylation can affect the differentiation,proliferation and apoptosis of hematopoietic stem cells.The mechanism is that the deletion of NEDD8 may affect the ubiquitination of Fbw7 to C-myc and N-myc by inhibiting the neddylation of Cullin1 and eventually result in hematopoietic failure in mice.