Role And Mechanism Of IFN-α In Regulation Of Bone Marrow Mesenchymal Stem Cells

As immunomodulatory cytokines, Type1interferons (IFNs) have a long history of efficacy in treating chronic myeloid leukemia (CML). Recently, many research have reported that the combination of IFN-a and imatinib significantly increased the rates of molecular responses, comparing to single imatinib treatment. Related molecular mechanism may be the direct effect of IFN-alpha on stem cells. Therefore, IFN-a was renewed to be a vital candidate for CML treatment. Bone marrow mesenchymal stem cells (MSCs), which also be defined as mesenchymal stromal cells, are important to the survival of leukemia cells. In this study, we investigate the effect of IFN-a in regulation of MSCs, and the role of IFN-a in the protection function of MSCs. The study is divided into three parts:(1) study the effect of IFN-a in regulation of MSCs,(2) study on the role of PML in the proliferation and differentiation of MSCs,(3) MSCs protect chronic myeloid leukemia cells from imatinib induced apoptosis:the role of IFN-a/PML signaling axis.Part1Role of IFN-a in regulation of mesenchyaml stem cellsAims:In this part, we investigated the effect of IFN-a on human bone marrow mesenchymal stem cells. The role of PML involved in this process was definited.Methods:After approval by institutional review board, MSCs were isolated from the bone marrow of volunteers by Ficoll-paque. Passage3-6MSCs were used for research. Cells were treated with different concentration of IFN-a up to14days. SA-β-gal staining was performed by senescence β-Galactosidase Staining Kit. Cell apotosis was detected by flow cytometry. Expression of target genes was detected by PCR, immunofluorescence or western blot. The recombinant lentiviral vector which encodes shRNA against PML or full-length PML cDNA was constructed. Human embryonic kidney293T cells were used as packaging cells. High-titer lentivirus was produced and was used to transduce MSCs which have reached50%-60%confluence.Empty vector transfected and normal cells were used as controls.Results:MSCs were treated with different concentration of IFN-a up to14days. We found that IFN-a treated cells were growing slowly, and had a dramatically decreased number of colone in a dose dependent manner. However, IFN-a did not induce significant cell apoptosis. Then a variety of senescence-associated detection was measured. MSCs senescence induced by IFN-a had a dose and time dependent manner. Real time PCR analysis proved this process by an increase in production of the senescence marker p53and p21. Consistent with other studies, mRNA expression of PML can be up-regulated by IFN-a in hMSCs. At the same time, both the number and size of PML-NBs were increased markedly and had a concentration dependent manner. Then, PML-overexpressed hMSCs were used to analyse.7days post-transfection, PML overexpressing hMSCs were strongly positive for SA-β-gal activity (47.43±3.8%), as compared with normal and empty vector transfected cells (4.9±0.7%,5.97±0.75%)(P<0,001). mRNA levels of P53and P21were also enhanced in PML-overexpressed hMSCs. To further characterize the effect of PML on cellular senescence in hMSCs, PML expression was inhibited using an RNAi-mediated PML knockdown system. After treated with IFN-a at1000U/ml for7days, hMSCs senescence can be rescued by the knocking down of PML. The percentage of SA-β-gal positive cells in PML knocking down hMSCs has a significant decrease as compared with cell transfected with control-RNAi (4.49±1.27%vs.17.26±1.44%, P<0.05). In the process of IFN-a induced hMSCs senescence, an increasing co-localization of PML and P53was observed in IFN-a treated cells as compared with untreated cells Treated with IFN-a, we did not find significant location of P53in PML-knocking down cells as compared with control.Conclusons:Our results suggested that MSCs incurred senescence upon IFN-a stimulation, while PML levels were observed significant increase. By knocking-down and overexpressing PML, we demonstrated that PML was indispensable to IFN-a mediated MSCs senescence.Part2Role of PML in the proliferation and osteogenic differentiation of human mesenchymal stem cellsAims:In this study, the role of Promyelocytic leukemia gene (PML) on regulating the proliferation and differentiation of human mesenchymal stem cells (hMSCs) was explored.Methods:After approval by institutional review board, MSCs were isolated from the bone marrow of volunteers. PCR and western blot were used to detect mRNA and protein expression. The location of PML protein during cell proliferation and differentiation was detected by immunofluorescence. The lentiviral vector which encodes full-length human PML cDNA or shRNA against PML was transfected into MSCs. Proliferation level was measured by CCK-8assay, while flow cytometry and western blot was used to analyze the cell apoptosis of hMSCs. Osteogenic differentiation of hMSCs was induced by culturing hMSCs with osteo-inductive medium for two weeks, and then cells were collected on day7for further detection. Differentiated cells were identified with Von kossa staining and ALP activity analysis.Results:PML was positively expressed in the MSCs and both mRNA and protein concentrations were detectable over9days along with cell proliferation. The induction of MSCs to differentiate into osteoblasts increased the expression of PML, which is consistent with the up-regulation of ALP mRNA in a time-dependent manner. PML-NBs in MSCs were not found to significantly change along with cell proliferation as detected by immunofluorescence staining on day4,6and8. However, when cells were induced to differentiate into osteoblast for2weeks and3weeks, the PML-NBs became larger and unequal in size compared to the normal cells. Cell proliferation was inhibited by20.57±4.18%in MSCs overexpressing PML as compared to that of the normal controls (P<0.05) and18.46±3.53%compared to the mock-transfected cells on day3(P<0.05). PML overexpression also significantly increased cell apoptosis. There were no obvious changes to the cell cycle when PML expression was enhanced. However, when the expression of PML was knocked-down for3days, the proportion of hMSCs in S+G2/M phase had clearly increased.Then PML-transfected cells were induced to osteogenic differentiation, while normal and mock-transfected cells served as controls. Mineralized matrix production was strongly enhanced on day7in PML-overexpressed hMSCs under osteogenic differentiation conditions and ALP activity was obviously increased in comparison to mock-transfected and normal MSCs. Next, calcium deposition and ALP activity was detected in PML-transfected cells under non-osteogenic differentiation conditions. We found that PML overexpressed MSCs had an obvious increase in mineralized matrix production on day7compared with normal and mock-transfected cells. Under normal culture conditions for7days, ALP activity was also obviously increased in comparison to mock-transfected and normal MSCs. ALP as an early marker of osteoblastic differentiation, its mRNA expression was dramatically enhanced on day7in PML overexpressed MSCs. Knock-down of PML expression decreased ALP mRNA expression when the cells were cultured under normal conditions for7days.Conclusions:Our data indicates that PML was positively expressed in the MSCs, and PML regulates MSCs as an inhibitor of cell proliferation but a promoter of osteogenic differentiation.Part3MSCs protect chronic myeloid leukemia cells from imatinib induced apoptosis:the role of IFN-α/PML signaling axisAims:In this part, we tried to explore the role of IFN-a/PML signaling axis in process of MSCs protecting chronic myeloid leukemia cells from imatinib induced apoptosis.Methods:Chronic myeloid leukemia cell line K562was cultured alone or co-cultured with MSCs. Then, cells were treated with imatinib at1μM for72h. Apoptosis cells (annexin V staining) were determined by flow cytometry. cDNA microarray was used to analyze gene expression profile and look for meaningful changes.Results:The proliferation rate was significantly reduced in K562cells which were co cultured with MSCs, but there was no significant difference in cell apoptosis. Then MSCs were treated with IFN-α (1000U/ml) or IM (1M) for3days, and co cultured with K562cells. Compared with IM treated group, K562cells in IFN-treated group expanded rapidly, and K562cell count was significantly higher than that in group IM. Next, K562cells were cultured alone or co-cultured with MSCs and received1μM of imatinib. Results showed that the apoptosis rate in K562cultured alone group was62.32±8.44%, and co-cultured group was33.15±7.78%, suggesting that MSCs can protect K562from IM induced apoptosis. Then we used IFN-a treated MSCs for further study and found that the the protective effect of MSCs was decreased in IFN-a treated MSCs. K562was more sensitivly to imatinib cytotoxicity. Apoptosis of K562cells which was co-cultured with IFN-treated MSCs was48.83±2.02%, higher than that of the control group (33.15±7.78%). Then the expression of PML was changed in MSCs. In the PML high expression group, the apoptosis rate of K562cells was49.1±5.03%, significantly higher than control group (36.37±1.63%), while the expression of PML was knocked down in MSCs, the apoptosis rate of K562was35.15±13.03%, lower than that of the control group (41.18±12.94%).Finally, we investigated genome-wide expression profiles of the normal, mock-and PML-transfected hMSCs. After performing t-test analyses, a total of357genes were found to have significantly altered expression levels of>1.5-fold. Except for the PML gene, in the PML-transfected cells, the3most upregulated genes were interleukin (IL)-1β, IL-8and IBSP, while the most downregulated gene was fibroblast growth factor receptor2(FGFR2).Conclusions:MSCs can protect K562cells from imatinib induced apoptosis.The protective function was inhibit when MSCs were treated with IFN-α.Changing the expression of PML can also influence the protective function of MSCs.