Efects Of RACK1 Knockout On Hematopoietic Stem Cells In IFN-1-responsive Cells

Objective:Conditional knockout of RACK1?gene name Gnb2l1?,an adaptor containing 7 Trp-Asp?WD?repeats,specifically in type I interferon-reactive cells was achived by crossing Gnb2l1^F/F mice with Mx1-Cre-transgenic mice.Whether the mouse model is resistant to VSV infection was then analyzed.Flow cytometry was used to analyze the effects of RACK1 deficiency in type I interferon-reactive cells on hematopoietic stem cells and hematopoietic progenitor cells of each lineage.The apoptosis and proliferation of RACK1-sufficient and-deficent hematopoietic stem cells were also examined.Molecular mechanism of RACK1 regulating hematopoietic stem cells by protein-protein interaction analysis.Methods:1.The genotype was identified with the mouse tails to distinguish Gnb2l1^F/F,MX-Cre/F,MX-Cre mice and Gnb2l1^F/F mice.After Gnb2l1^F/F,MX-Cre mice and Gnb2l1^F/F mice were intraperitoneally injected with Poly?I:C?.Western blot was performed to conform the efficient depletion of RACK1.2.Gnb2l1^F/F,MX-Cre mice and Gnb2l1^F/F mice were injected with VSV via the tail vein,and then the level of IFN?in the serum was detected with ELISA.Liver or lung weight relative to body weight was calculated,respectively.Flow cytometry analysis was performed to explore the percentages of myeloid cells and pDC,the major type 1interferon-producing cells in vivo,in the liver and spleen.3.Hematopoieticstemcells,megakaryocyte/erythroidprogenitorcells,granulocyte/macrophage progenitor cells,myeloid progenitor cells,lymphocyte progenitor cells,B lymphocytes,myeloid cells,and erythroid cells are sorted by surface marker staining.The gene expression levels of RACK1,c-Myc and N-Myc in each stage were detected by Real-Time PCR.Multi-color fluorescence histochemistry was used to analyze whether RACK1 is co-localized with CD117 in hematopoietic cells.HE staining was used to analyze the bone marrow of Gnb2l1^F/F,MX-Cre mice and Gnb2l1^F/F/F mice.Flow cytometry was used to analyze the effects of RACK1 deficiency in type I interferon-reactive cells on hematopoietic stem cells and hematopoietic progenitor cells of each lineage.The apoptosis and proliferation of RACK1-sufficient and-deficient hematopoietic stem cells were also examined with Annexin V staining.Chimeric mice were prepared to verify whether the above changes were due to cell intrinsic defects.Flow cytometry was used to analyze the expression levels of c-Myc and N-Myc in RACK1-sufficient and-deficient HSC,Myc inhibitor was injected intraperitoneally into mice for reversal,which provided a clue for the mechanism.4.CO-IP was performed to detect whether c-Myc,N-Myc and RACK1 interact with each other.GST pull down was performed using purified GST and GST-RACK1 to analyze whether it is a direct interaction.Gnb2l1^F/F,MX-Cre mice and Gnb2l1^F/F mice HSC were purified,and c-Myc or N-Myc was precipitated to analyze the effect of E3ubiquitin ligase Fbxw7.Results:1.The mouse genotype was successfully identified.Western blot revealed RACK1 knockout in type 1 interferon-reactive cells was also achieved at the protein level after Poly?I:C?injection.2.It was found that IFN?in Gnb2l1^F/F,MX-Cre mice was significantly lower than that in Gnb2l1^F/F mice after VSV infection.On the other hand,compared with Gnb2l1^F/F mice,the ratio of wet weight to body weight of liver and lung tended to increase.Flow cytometry showed significantly reduced myeloid cells and type?interferon mainly produces cells pDC in Gnb2l1^F/F,MX-Cre/F,MX-Cre mice.3.Real-Time PCR results showed that at the mRNA levels of Gnb2l1,c-Myc and N-Myc were higher in hematopoietic stem cells and hematopoietic progenitor cells than in mature cells.Fluorescent multiplex immunohistochemistry?mIHC?with tyramide signal amplification showed that RACK1 was co-localized with CD117 in hematopoietic cells.HE staining revealed that specific knockout of RACK1 in type I interferon-reactive cells resulted in much fewer cells in the medullary cavity.Gnb2l1^F/F,MX-Cre mice have significantly reduced proportion and total number of hematopoietic stem cells,CMP and CLP.The proportion of GMP and MEP increased slightly,but their absolute numbers significantly reduced.The apoptosis and proliferation of HSC increased significantly.Chimeric mouse experiments have demonstrated that the above changes are due to cell intrinsic defects.Specific knockout of RACK1 in type I interferon-reactive cells led to increased proportion of c-Myc⁺and N-Myc⁺HSC.Injection of Myc inhibitor can partially reverse HSC.4.CO-IP results showed that RACK1 interacted with c-Myc and N-Myc.GST pull down proved that it was a direct interaction.The interaction of c-Myc or N-Myc with Fbxw7in HSC was reduced in the absence of RACK1.Conclusion:1.The mouse model was successfully identified,and Gnb2l1^F/F,MX-Cre mice and Gnb2l1^F/F mice were distinguished.2.Specific knockout of RACK1 in type I interferon-reactive cells reduces the anti-infective ability of mice.3.Specific knockout of RACK1 in type I interferon-reactive cells significantly reduced HSC in the bone marrow and hematopoietic precursors in each lineage.Gnb2l1^F/F,MX-Cre/F,MX-Cre mice showed a significant increase in the proportion of HSC apoptosis and a significant increase in proliferation.Preparation of chimeric mice confirmed that this phenomenon is caused by intracellular defects.Reverse experiments suggest that our specific knockout of RACK1 in IFN-1-responsive cells causes a series of effects on HSC in bone marrow due to the accumulation of c-Myc and N-Myc.4.RACK1 has direct interaction with c-Myc and N-Myc.When c-Myc or N-Myc was precipitated,the amount of Fbxw7 protein interacting with c-Myc or N-Myc was decreased after specifically knocking out RACK1 in type I interferon-reactive cells.The binding of c-Myc or N-Myc to Fbxw7 is weakened,resulting in a large accumulation of c-Myc or N-Myc.It provides a favorable reference for further research on the mechanism of RACK1 affecting HSC.