pubmed.ncbi.nlm.nih.gov

Translational initiation regulated by ATM in dendritic cells development - PubMed

  • ️Wed Jan 01 2014

Translational initiation regulated by ATM in dendritic cells development

E Y So et al. Cell Death Dis. 2014.

Abstract

Ataxia telangiectasia mutated (ATM) protein has been implicated in multiple pathways such as DNA repair, cell cycle checkpoint, cell growth, development, and stem cell renewal. In this study, we demonstrate evidence that ATM is involved in granulocyte macrophage colony-stimulating factor (GM-CSF)-induced dendritic cell (DC) development from bone marrow (BM) cells. Inactivation of ATM protein results in decreased BM proliferation, leading to reduced DC development and their activity for T cell activation. Expression of Jak2, STAT5, and mTOR is suppressed in both wild-type and ATM-null BM prior to GM-CSF stimulation. Activation of those proteins is delayed and prolonged hypophosphorylation of 4EBP1 is observed in ATM-null BM when treated with GM-CSF, although Erk and p38 are similarly expressed and activated in both wild-type and ATM-null BM cell types. Akt is also suppressed in wild-type BM, and transduction of constitutively active Akt or STAT5 in ATM-null BM restores DC development. Together, these results illustrate that ATM deficiency causes impaired initiation of protein translation in BM, leading to immature development of DC.

PubMed Disclaimer

Figures

Figure 1
Figure 1

The phosphorylation and expression of ATM in GM-CSF-induced BM. (a) BM precursors were purified from C57BL/6J male mice and developed into DC and MP using GM-CSF (20 ng/ml) for indicated times, as previously published. Suspended and weakly attached cells were harvested by repeated pipetting and strongly attached cells were detached from plates using scrapper. Total lysates and total RNA were isolated from same cell populations, and then subjected to western blot and RT-PCR analysis, respectively. Lane 1. suspended and attached cells (DC) of D2; lane 2. strongly attached cells (macrophages) of D2; lane 3. suspended and attached cells of D4; lane 4. strongly attached cells of D4; lane 5. suspended and attached cells of D5; lane 6. strongly attached cells of D5. Actin protein and GAPDH transcript were used as internal control to validate ATM expression in these cells. (b) BM were treated with MG132 (DMSO only, 50 and 200 nM) at D2. After 24 h, cells were harvested and then subjected to western blot analysis of ATM and SMC1 expression. Total lysate from D3, D5, and D7 BM were studied. (c) D6 BM were washed and cultured in GM-CSF-free media for 24 h, and then re-treated with GM-CSF (20 ng/ml) or NCS (0.5 μg/ml) for indicated times. (d) Nuclear extract (Nuc) and cytoplasmic extract (Cyt) were isolated from D6 or D8 BM after starvation and re-culture with (+) or without (−) GM-CSF. Antibodies against NUP98 and a Tubulin were used as references of nuclear and cytoplasmic extract, respectively. (e) After starvation in GM-CSF-free media for 24 h, D6 wild-type BM cells were re-cultured with GM-CSF (20 ng/ml) or NCS (0.5 mg/ml). DMSO (−) or KU55933 (+, 5 mM) was added and cultured at indicated times

Figure 2
Figure 2

GM-CSF-induced in vitro development of DC was impaired in ATM-null BM. (a) BM were isolated from ATM-null (ATM-/-) or wild-type (ATM+/+) mice, and developed into DC by GM-CSF treatment. (b) BM from wild-type mice were cultured in GM-CSF with or without KU-55933 (5 μM). Cells were harvested on indicated days and stained with anti-CD11c-FITC and anti-MHCII (I-A/I-E)-APC antibodies. Percentage of DC (CD11c+/MHCIIMed-High) to total cells was analyzed by flow cytometry. Left panel; flow cytometry results. Right panel; a Bar graph showed the average percentage of CD11c/MHCII positive cells. Experiments were performed by three independent experiments. All data are present as mean±S.E. of three replicates. *P<0.05; **P<0.01

Figure 3
Figure 3

The effect of ATM deficiency on cell growth and immune functions of DC. BM from wild-type and ATM-null mice were cultured for indicated days with GM-CSF (20 ng/ml), followed by Annexin V staining to analyze their (a) apoptosis and (b) proliferation. (c) BM from wild-type or ATM-null mice were cultured in RPMI 10 media containing GM-CSF (20 ng/ml) for 9 days. DC were harvested and co-cultured for 4 days with allogeneic lymphocytes obtained from SJL/J mice in indicated ratios of DC/lymphocytes. Data represent the percentage of CD71+ cells / total CD3+ T cells. Experiments were performed in duplicates and repeated at least three times

Figure 4
Figure 4

LPS-induced DC maturation in both wild-type and ATM-null BM cultures. Wild-type and ATM-null BM at D9 were harvested, followed by treatment with LPS (1 and 2 μg/ml) for additional 24 h. The percentage of maturated BMDC was quantified by ratio of mature (CD11c+MHCIIHigh) to immature+mature DCs (CD11c+MHCIIMed-High). A representative of two independent experiments was shown

Figure 5
Figure 5

The effect of ATM deficiency and KU-55933 on GM-CSF-induced kinase signaling in BM culture. (a) Wild-type BM were cultured in GM-CSF-containing media with or without KU (1 μM) for 9 days. (b) Wild-type and ATM-null BM were cultured and harvested at D0, D3, D6, and D9. Cells were then lysed and total cell extracts were subjected to SDS-PAGE and western blotting assay. Phospho-protein (Akt at Ser473; Erk at Tr202/Tyr204; p38 at Thr180/Try182) and total proteins were detected. Actin protein was used as loading control. (c) Wild-type BM were cultured in the presence of GM-CSF with or without Akt inhibitor (Akt IN) or KU55933 (KU) at indicated concentration. Cells were harvested at D9 and subjected to flow cytometric analysis after staining with anti-CD11c/MHCII. (d) BM were infected with retrovirus expressing constitutively active Akt-IRES-GFP at D3, and BM were harvested at D6. GFP-negative (Akt not-expressing) and GFP-positive (Akt expressing) cells were sorted and MHCII expression were analyzed. Data were expressed by a percentage of MHCII+ cells and representative of two independent experiments

Figure 6
Figure 6

Essential roles of STAT5 in DC development. (a) BM were isolated from wild-type (+/+) or ATM-null mice (−/−), then cultured in GM-CSF-containing media for indicated days. Cells were then lysed and total cell extracts were subjected to SDS-PAGE and western blotting assay with antibodies against phospho-proteins (Jak2 at Tyr1007/1008; STAT5 at Tyr649; Akt at Ser473) and total proteins. Actin protein was used as loading control. (b) Total RNA was isolated from wild-type and ATM-null BM at indicated days, followed by RT-PCR to examine indicated gene expression. The level of GAPDH mRNA was used to validate control gene expression. (c) BM at D3 were infected with retrovirus expressing GFP or STAT5-GFP (STAT5), and cultured until D8. Percentage of MHCIIMed-High of GFP-positive cells was analyzed and shown. Data were representative of two different experiments

Figure 7
Figure 7

ATM is required for the phosphorylation of 4EBP1 and expression of mTOR. (a) BM were harvested at D6 and D8, and then cultured in GM-CSF-free media. After 24 h, cells were re-treated with (GM+) or without GM-CSF (GM−) for 6 h. Total lysate was mixed with m7-GTP-sepharose and then subjected to pull-down assay, followed by immunoblot analysis of eIF4E and 4EBP1. (b) Total lysates of wild-type (ATM+/+) or ATM-null (ATM−/−) were prepared at D3, D6, and D8, followed by western blot assay of protein levels of mTOR, Akt, and phosphorylated mTOR (Ser2481, S2481P) and Akt (Ser487, S487P). Actin serves as a loading control. Three experiments were performed independently

Figure 8
Figure 8

Model of the roles of ATM in BM differentiation to DC induced by GM-CSF. At early stage (∼ D6), ATM transduces signaling from GM-CSF to Akt, leading to efficient translation of proteins essential in development of DC. At late stage, ATM-dependent expression of Jak2/STAT5 is essential for maturation of DC

Similar articles

Cited by

References

    1. Inaba K, Inaba M, Romani N, Aya H, Deguchi M, Ikehara S, et al. Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colony-stimulating factor. J Exp Med. 1992;176:1693–1702. - PMC - PubMed
    1. Gilliet M, Boonstra A, Paturel C, Antonenko S, Xu XL, Trinchieri G, et al. The development of murine plasmacytoid dendritic cell precursors is differentially regulated by FLT3-ligand and granulocyte/macrophage colony-stimulating factor. J Exp Med. 2002;195:953–958. - PMC - PubMed
    1. Banchereau J, Briere F, Caux C, Davoust J, Lebecque S, Liu YJ, et al. Immunobiology of dendritic cells. Annu Rev Immunol. 2000;18:767–811. - PubMed
    1. Derheimer FA, Kastan MB. Multiple roles of ATM in monitoring and maintaining DNA integrity. FEBS Lett. 2010;584:3675–3681. - PMC - PubMed
    1. Lavin MF, Kozlov S. ATM activation and DNA damage response. Cell Cycle. 2007;6:931–942. - PubMed

Publication types

MeSH terms

Substances