The Effect Of Scaffold Protein RACK1 On The Expression Of Myeloid Cells And Inflammatory Cytokines

BackgroundReceptor for Activated C Kinase 1?RACK1?,a scaffold protein with molecular weight36 kDa,contains seven Trp-Asp 40?WD40?repeats.Numerous studies have shown that RACK1 plays a pivotal role in cancer development through modulating the apoptosis and proliferation of tumor cells.Our research group has also revealed that overexpressed RACK1promotes the oncogenic growth of human hepatocellular carcinoma through enhancing anchorage-independent growth and resistance to TRAIL-or Fas-mediated apoptosis.Recent studies suggest that RACK1 may regulate the expression of inflammatory cytokines by suppressing the NF-?B signaling pathway.Therefore,we presume that RACK1may play an important role in the inflammatory response,because the expression of inflammatory cytokines relies on the NF-?B signaling pathway and macrophages are the major source of inflammatory cytokines.However,all reports in the literature make use of RNA interference in cell lines to explore how RACK1 might regulate the expression of inflammatory cytokines.Off-target effects might occur and its in vivo role remains elusive.Acute promyelocytic leukemia?APL?is a special type of acute myeloid leukemia?AML?.It characteristically shows the expansion and accumulation of hematopoietic precursor cells due to the impaired differentiation and augmented proliferation of APL cells.As a classic differentiation agent,all-trans retinoic acid?ATRA?has been widely used in the treatment of APL with success.However,the clinical application of ATRA has strict limitations,due to its severe side effects.To overcome the limitations,efforts have been made by investigators worldwide to search for effective alternative regimens.Therefore,combination therapies may be promising for relapsed or refractory patients.Our previous work has revealed that RACK1plays a key role in the proliferation of THP1 cells,but the function of RACK1 in APL cells remains unknown.In this scenario,we generated a myeloid cell-specific conditional knockout mouse model for RACK1(Rack1^?mye)and made use of lentivirus carrying RACK1 short hairpin RNA?shRNA?to explore the function of RACK1 in myeloid cells.Our work provides insight into the treatment of acute or chronic inflammatory diseases as well as APL.ObjectivesWith Rack1^?mye mice as the main model,we set out to explore how myeloid RACK1might impact on septic shock and the secretion of inflammatory cytokines.With APL cell lines as the main model,we set out to explore the effect of RACK1 knockdown on cell survival and the secretion of inflammatory cytokines.The underlying mechanisms also explored.Methods1?DNA was extracted from the mouse tails,and then the genotypes were identified.2?Mononuclear cells were isolated respectively from the bone marrow,peripheral blood,and spleen of 1 week-old mice.Then the surface markers and the cell survival were analyzed by flow cytometry.3?APL cells were infected with control lentivirus or lentivirus carrying RACK1 shRNA,followed by ATRA treatment.Then the cell death and differentiation analyzed by flow cytometry and the secretion of inflammatory cytokines analyzed by ELISA.4?RACK1 knockdown in APL cells was achieved as above,followed by treatment with ATRA and lysosomal protease inhibitors.Then analyzed the cell death by flow cytometry.In addition,whole cell lysates harvested and subjected to western blot to detect membrane-bound LC3B-II,an indicator of autophagy.5?4 week-old mice were intraperitoneally injected with LPS at a dose of 25 mg/kg,and the mortality was monitored in 72 h.6?Bone marrow mononuclear cells were isolated from 1 week-old mice and bone marrow-derived macrophages?BMDM?were induced with 50 ng/ml M-CSF for 7 days.Then BMDM were stimulated with 100 ng/ml LPS for various time.Supernatants harvested and subjected to ELISA to detect the secretion of inflammatory cytokines.Moreover,cellular survival monitored with ATPlite.7?BMDM were induced as above.Then BMDM were stimulated with 100 ng/ml LPS for various time.Whole cell lysates harvested and subjected to Western Blot to detect the activation of inflammatory signaling pathways.8?RAW264.7 cells were infected with control lentivirus or lentivirus carrying RACK1shRNA.Then cells were transfected with IL-10 promoter activity reporting plasmids,followed by luciferase assay.Results1.Effects of RACK1 on the differentiation and survival of myeloid cellsFirstly,with APL cell lines as the main model,the results indicate that knockdown of RACK1and then combined with ATRA induces apoptosis,while does not affect the cell differentiation induced by ATRA.As our previous work reveals RACK1 promotes autophagy,we presume that the apoptosis of APL cells is affected by RACK1 through autophagy.And the result of treatment with lysosomal protease inhibitors shows that RACK1 protect cells from apoptosis by enhancing autophagy.On the other hand,with Rack1^?mye mice as the main model,we set out to explore the impact of RACK1 deficiency on the differentiation and survival of myeloid cells.The results show that the knock-out of RACK1 in myeloid cell does not affect cell differentiation and survival in the resting state.However,cell survival is affected significantly after treatment with the LPS.2.Effects of RACK1 on cytokine expressionWith APL cell lines as the main model,the data show that RACK1 knockdown does not affect differentiation induced by ATRA and the secretion of inflammatory cytokines as revealed by ELISA.So RACK1 may be a new target for the treatment of APL.In addition,with Rack1^?mye mice as the main model to detect the impact of myeloid RACK1 deficiency on septic shock,and the data indicate that myeloid RACK1 limits the inflammatory response in the mouse.Further exploration revealed that the expression of inflammatory cytokine IL-10significantly decreased.3?Mechanism by which RACK1 regulates the inflammatory cytokine expressionWith Rack1^?mye mice as the main model,the underlying mechanism by which RACK1regulates the inflammatory cytokine expression explored.Western blot about the NF-?B signaling pathway indicates that the re-synthesis of I?B?significantly decreased.In this scenario,knockdown of RACK1 in RAW264.7 cells exploited to explore whether the activity of IL-10 promoter is affected.And the results indicate that the activity of IL-10 promotor significantly decreased upon RACK1 knockdown.The site of IL-10 promoter affected by RACK1 is from 158bp upstream to 64b downstream of the transcription initiation site.The NF-?B p50/p50 cis element is located at the position-55/-46 of IL-10 promoter.Therefore,we hypothesized whether RACK1 regulates the transcription of IL-10 though NF-?B p50/p50complex.To prove this,we generated a mutant p50/p50 cis element in IL-10 promoter,and further exploration shows that RACK1 knockdown does not affect the activity of IL-10promoter without a functional p50/p50 cis element.Then we purchased p50 knockout mice,ELISA assay revealed that the secretion of inflammatory cytokine IL-10 decreased significantly upon p50 deficiency.Taken together,our work suggests that myeloid RACK1plays a pivotal role to prevent septic shock through,at least partially,promoting p50/p50 cis element-mediated transcription of IL-10.ConclusionsRACK1 knockdown in APL cells could induce cell apoptosis without enhancing differentiation and the secretion of inflammatory cytokines.Further exploration revealed that the lysosome-autophagy pathway is likely to be responsible for the anti-apoptotic ability of RACK1.So RACK1 may be a new target for the treatment of APL.With Rack1^?mye mice as the main model,our data indicate that the knock-out of RACK1 in myeloid cells does not affect cell differentiation and survival in the resting state,however,myeloid RACK1 plays a pivotal role to prevent septic shock through,at least partially,promoting p50/p50 cis element-mediated transcription of IL-10.