A synthetic double-stranded RNA, poly I:C, induces a rapid apoptosis of human CD34+ cells
2011; Elsevier BV; Volume: 40; Issue: 4 Linguagem: Inglês
10.1016/j.exphem.2011.12.002
ISSN1873-2399
AutoresJiajia Liu, Yong‐Mei Guo, Makoto Hirokawa, Keiko Iwamoto, Kumi Ubukawa, Yoshihiro Michishita, Naohito Fujishima, Hiroyuki Tagawa, Naoto Takahashi, Weiguo Xiao, Junsuke Yamashita, Toshiaki Ohteki, Kenichi Sawada,
Tópico(s)Immune Cell Function and Interaction
ResumoToll-like receptor 3 (TLR3), retinoic acid-inducible gene I, and melanoma differentiation-associated antigen 5 (RIG-I/MDA-5) helicases are known to sense double-stranded RNA (dsRNA) virus and initiate antiviral responses, such as production of type-I interferons (IFNs). Recognition of dsRNA by TLR3 or RIG-I/MDA-5 is cell-type–dependent and recent studies have shown a direct link between TLRs and hematopoiesis. We hypothesized that viral dsRNA recognized by either TLR3 or RIG-I/MDA-5, affects the growth of human hematopoietic stem/progenitor cells. Here we show that polyinosinic polycytidylic acid (poly I:C)–mediated very rapid apoptosis occurs within 1 hour in CD34+ cells in a dose-dependent manner. Polyadenylic-polyuridylic acid, another synthetic dsRNA that signals only through TLR3, had no effect. Poly I:C-LMW/LyoVec, a complex between low molecular-weight poly I:C and the transfection reagent LyoVec, which signals only through RIG-I/MDA-5, induces apoptosis of CD34+ cells. A strong and sustained upregulation of messenger RNA and protein levels of Noxa, a proapoptotic BH3-only protein that can be induced by RIG-I/MDA-5 pathway, is found in CD34+ cells treated by poly I:C. Although poly I:C upregulates type-I IFNs in CD34+ cells, neither exogenous IFN-α nor IFN-β induces rapid apoptosis in CD34+ cells and neutralization or blocking of type-I IFN receptor does not rescue CD34+ cells, whereas Z-VAD, a pan-caspase inhibitor, rescues the cells from apoptosis. These results suggest that RIG-I/MDA-5, but not TRL3, signaling triggers poly I:C–induced rapid apoptosis of human CD34+ cells, which will provide an insight into the mechanisms of dsRNA virus-mediated hematopoietic disorders. Toll-like receptor 3 (TLR3), retinoic acid-inducible gene I, and melanoma differentiation-associated antigen 5 (RIG-I/MDA-5) helicases are known to sense double-stranded RNA (dsRNA) virus and initiate antiviral responses, such as production of type-I interferons (IFNs). Recognition of dsRNA by TLR3 or RIG-I/MDA-5 is cell-type–dependent and recent studies have shown a direct link between TLRs and hematopoiesis. We hypothesized that viral dsRNA recognized by either TLR3 or RIG-I/MDA-5, affects the growth of human hematopoietic stem/progenitor cells. Here we show that polyinosinic polycytidylic acid (poly I:C)–mediated very rapid apoptosis occurs within 1 hour in CD34+ cells in a dose-dependent manner. Polyadenylic-polyuridylic acid, another synthetic dsRNA that signals only through TLR3, had no effect. Poly I:C-LMW/LyoVec, a complex between low molecular-weight poly I:C and the transfection reagent LyoVec, which signals only through RIG-I/MDA-5, induces apoptosis of CD34+ cells. A strong and sustained upregulation of messenger RNA and protein levels of Noxa, a proapoptotic BH3-only protein that can be induced by RIG-I/MDA-5 pathway, is found in CD34+ cells treated by poly I:C. Although poly I:C upregulates type-I IFNs in CD34+ cells, neither exogenous IFN-α nor IFN-β induces rapid apoptosis in CD34+ cells and neutralization or blocking of type-I IFN receptor does not rescue CD34+ cells, whereas Z-VAD, a pan-caspase inhibitor, rescues the cells from apoptosis. These results suggest that RIG-I/MDA-5, but not TRL3, signaling triggers poly I:C–induced rapid apoptosis of human CD34+ cells, which will provide an insight into the mechanisms of dsRNA virus-mediated hematopoietic disorders. The initial sensing of infection is mediated by innate pattern recognition receptors, which include Toll-like receptors (TLRs), retinoic acid–inducible gene I (RIG-I)-like receptors, nucleotide binding oligomerization domain–like receptors, and C-type lectin receptors (for review see [1Barral P.M. Sarkar D. Su Z.Z. et al.Functions of the cytoplasmic RNA sensors RIG-I and MDA-5: key regulators of innate immunity.Pharmacol Ther. 2009; 124: 219-234Crossref PubMed Scopus (139) Google Scholar, 2Takeuchi O. Akira S. Pattern recognition receptors and inflammation.Cell. 2010; 140: 805-820Abstract Full Text Full Text PDF PubMed Scopus (5197) Google Scholar, 3Yoneyama M. Fujita T. Recognition of viral nucleic acids in innate immunity.Rev Med Virol. 2010; 20: 4-22Crossref PubMed Scopus (231) Google Scholar]). Each pattern recognition receptor, which recognizes microbe-specific pathogen-associated molecular patterns, activates specific signaling cascades to induce gene expression of targets such as proinflammatory cytokines and type-I interferons (IFNs) that coordinate the elimination of pathogens and infected cells [4Akira S. Uematsu S. Takeuchi O. Pathogen recognition and innate immunity.Cell. 2006; 124: 783-801Abstract Full Text Full Text PDF PubMed Scopus (8299) Google Scholar]. A set of TLRs including TLR3, TLR7, TLR8, and TLR9 recognizes nucleic acids derived from viruses and bacteria, as well as endogenous nucleic acids in pathogens [4Akira S. Uematsu S. Takeuchi O. Pathogen recognition and innate immunity.Cell. 2006; 124: 783-801Abstract Full Text Full Text PDF PubMed Scopus (8299) Google Scholar]. Recently, several lines of evidence have demonstrated a direct or indirect link between TLRs and hematopoiesis [5Guo Y.M. Ishii K. Hirokawa M. et al.CpG-ODN 2006 and human parvovirus B19 genome consensus sequences selectively inhibit growth and development of erythroid progenitor cells.Blood. 2010; 115: 4569-4579Crossref PubMed Scopus (30) Google Scholar, 6Nagai Y. Garrett K.P. Ohta S. et al.Toll-like receptors on hematopoietic progenitor cells stimulate innate immune system replenishment.Immunity. 2006; 24: 801-812Abstract Full Text Full Text PDF PubMed Scopus (598) Google Scholar, 7Sioud M. Floisand Y. Forfang L. Lund-Johansen F. Signaling through toll-like receptor 7/8 induces the differentiation of human bone marrow CD34+ progenitor cells along the myeloid lineage.J Mol Biol. 2006; 364: 945-954Crossref PubMed Scopus (122) Google Scholar]. Nagai et al. [6Nagai Y. Garrett K.P. Ohta S. et al.Toll-like receptors on hematopoietic progenitor cells stimulate innate immune system replenishment.Immunity. 2006; 24: 801-812Abstract Full Text Full Text PDF PubMed Scopus (598) Google Scholar] showed that TLRs and their coreceptors were expressed by multipotential hematopoietic stem cells, whose cell cycle entry was triggered by TLR ligation. They also showed that TLR signaling via the Myd88 adaptor protein drove differentiation of myeloid progenitors and also drove lymphoid progenitors to become dendritic cells. Sioud et al. [7Sioud M. Floisand Y. Forfang L. Lund-Johansen F. Signaling through toll-like receptor 7/8 induces the differentiation of human bone marrow CD34+ progenitor cells along the myeloid lineage.J Mol Biol. 2006; 364: 945-954Crossref PubMed Scopus (122) Google Scholar] showed that human BM CD34+ progenitor cells constitutively express functional TLR7/8, a receptor that recognizes single-stranded RNAs from RNA viruses, and ligation of which can induce differentiation along the myeloid lineage without the addition of any exogenous cytokines. More recently, we demonstrated selective inhibition of erythroid growth and downregulation of the expression of erythropoietin receptor messenger RNA (mRNA) in human CD34+ cells by CpG (cytosine linked to guanine by a phosphate bond) oligodeoxynucleotide-2006 (CpG-ODN2006), a TLR9 ligand that shares a consensus sequence with the parvovirus B19 genome [5Guo Y.M. Ishii K. Hirokawa M. et al.CpG-ODN 2006 and human parvovirus B19 genome consensus sequences selectively inhibit growth and development of erythroid progenitor cells.Blood. 2010; 115: 4569-4579Crossref PubMed Scopus (30) Google Scholar]. Thus, nucleic acids derived from viruses and bacteria directly or indirectly affect hematopoiesis. TLR3 detects viral double-stranded RNAs (dsRNAs) in the endolysosome and is involved in the recognition of polyinosinic polycytidylic acid (poly I:C), a synthetic dsRNA analog. A second family of pattern recognition receptors comprises the cytoplasmic sensors of viral nucleic acids, including RIG-I, melanoma differentiation-associated gene 5 (MDA-5), and laboratory of genetics and physiology 2. RIG-I and MDA-5 are DExD/H RNA helicases, possessing two caspase activation and recruitment domains at their amino terminus. Together with laboratory of genetics and physiology 2 they form the RIG-I-like receptors family. Laboratory of genetics and physiology 2 possesses only the helicase domain and lacks the caspase activation and recruitment domain [2Takeuchi O. Akira S. Pattern recognition receptors and inflammation.Cell. 2010; 140: 805-820Abstract Full Text Full Text PDF PubMed Scopus (5197) Google Scholar, 3Yoneyama M. Fujita T. Recognition of viral nucleic acids in innate immunity.Rev Med Virol. 2010; 20: 4-22Crossref PubMed Scopus (231) Google Scholar]. RIG-I and MDA-5 share ∼25% homology within the caspase activation and recruitment domain regions and 40% within the helicase domain. Poly I:C, which has been used frequently to study RNA sensing is at least 3 kb in length. By producing poly I:C of various lengths, it was determined that short segments of the polymer (∼300 bp) do not activate MDA-5, but are potent ligands for RIG-I. In contrast, longer segments of poly I:C preferentially activate MDA-5 [8Kato H. Takeuchi O. Mikamo-Satoh E. et al.Length-dependent recognition of double-stranded ribonucleic acids by retinoic acid-inducible gene-I and melanoma differentiation-associated gene 5.J Exp Med. 2008; 205: 1601-1610Crossref PubMed Scopus (1086) Google Scholar]. The mechanism underlying the discrimination between long and short dsRNA of MDA-5 and RIG-I remains to be elucidated. We hypothesized that triggering TLR3, RIG-I, and/or MDA-5 with their cognate dsRNA ligands affects hematopoiesis through production of type-I IFNs and proinflammatory cytokines, and at the same time may trigger endogenous apoptosis as part of an antiviral host response. The results presented here partly support this hypothesis and even extend our understanding of dsRNAs in association with hematopoiesis. We identify a very rapid induction of apoptosis of human CD34+ cells by poly I:C, and suggest that RIG-I and MDA-5 signaling triggers apoptosis of human CD34+ cells. Bovine serum albumin (BSA), Iscove's modified Dulbecco's medium (IMDM), and propidium iodide (PI) were purchased from Sigma (St Louis, MO, USA). Fetal bovine serum was from HyClone (Logan, UT, USA). Penicillin and streptomycin were from Invitrogen (Carlsbad, CA, USA). Insulin (porcine sodium, activity 28.9 U/mg) was obtained from Wako Pure Chemical Industries (Osaka, Japan). Interleukin-3 (IL-3), stem cell factor, and thrombopoietin were kind gifts from the Kirin Brewery Co. Ltd. (Tokyo, Japan), and erythropoietin, and granulocyte colony-stimulating factor were from Chugai Pharmaceutical Co. (Tokyo, Japan). Vitamin B-12 was from Eisai Co. Ltd. (Tokyo, Japan) and folic acid was from Takeda Pharmaceutical Co. Ltd. (Osaka, Japan). RNase (Type III-A) was from Sigma. Carboxy-fluorescein diacetate succinimidyl ester was from Invitrogen. Poly I:C was from InvivoGen (San Diego, CA, USA) and Alexis Biochemicals (Lausen, Switzerland). Lipopolysaccharide (LPS) and bafilomycin A1 were from Sigma. R848 was purchased from Alexis Biochemicals. CpG-oligodeoxynucleotide (ODN) 2006 with a modified nuclease-resistant backbone, phosphorothioate (PS) (ODN2006-PS), and CpG-ODN with the natural phosphodiester (PO) nuclease-sensitive backbone (ODN2006-PO) were commercially synthesized by Hokkaido System Science Co. Ltd. (Sapporo, Japan). Poly I:C/LMW/LeoVec, a complex between low molecular-weight poly I:C and the transfection reagent LyoVec, which signals only through RIG-I/MDA-5 [9Gitlin L. Barchet W. Gilfillan S. et al.Essential role of mda-5 in type I IFN responses to polyriboinosinic:polyribocytidylic acid and encephalomyocarditis picornavirus.Proc Natl Acad Sci U S A. 2006; 103: 8459-8464Crossref PubMed Scopus (884) Google Scholar], Poly I:C-rhodamine, BX795, and polyadenylic-polyuridylic acid, another synthetic dsRNA that signals only through TLR3 [10Conforti R. Ma Y. Morel Y. et al.Opposing effects of toll-like receptor (TLR3) signaling in tumors can be therapeutically uncoupled to optimize the anticancer efficacy of TLR3 ligands.Cancer Res. 2010; 70: 490-500Crossref PubMed Scopus (82) Google Scholar], were purchased from InvivoGen. Interferon (IFN)-α, IFN-β, mouse monoclonal antibody (Ab) against human IFN-α (anti–IFN-α Ab), and mouse monoclonal antibody against human IFN-α/β receptor (R) 1 (anti-IFNR Ab) were from PBL Biomedical Laboratories (Piscataway, NJ, USA). Goat monoclonal antibody against human IFN-β (anti–IFN-β Ab) and Z-VAD were from R&D Systems (Minneapolis, MN, USA). Fluorescein isothiocyanate (FITC)–labeled monoclonal antibodies (mAb) specific for CD15 (H198) and phycoerythrin (PE)-labeled mAb specific for CD123 (IL-3Rα; 9F5) were purchased from Becton Dickinson (Mountain View, CA, USA). FITC or PE-labeled mAbs for glycophorin A (JC159), PE-CD34 (BIRMA-k3), and FITC-CD61 (Y2/51), FITC-CD11c (KB90) were purchased from Dako Japan Co. (Kyoto, Japan). Anti-Noxa mouse mAb (114C307) was purchased from Calbiochem (Tokyo, Japan). Anti-Puma (p53 upregulated modulator of apoptosis) rabbit polyclonal Ab, horse anti-mouse IgG/horseradish peroxidase–linked Ab and goat anti-rabbit IgG/horseradish peroxidase–linked Ab were purchased from Cell Signaling (Tokyo, Japan). FITC-Annexin V apoptosis detection kit was from Sigma. PE-Annexin V was from R&D Systems. PE-anti–IFN-α was from Miltenyi Biotec (Auburn, CA, USA). Granulocyte colony-stimulating factor–mobilized human peripheral blood CD34+ cells were purified from healthy volunteers and stored in liquid nitrogen until use as described previously [11Saito K. Hirokawa M. Inaba K. et al.Phagocytosis of codeveloping megakaryocytic progenitors by dendritic cells in culture with thrombopoietin and tumor necrosis factor-alpha and its possible role in hemophagocytic syndrome.Blood. 2006; 107: 1366-1374Crossref PubMed Scopus (12) Google Scholar]. Informed consent was obtained from each subject before entry into this study, and the Akita University Graduate School of Medicine Committee preapproved the study for the Protection of Human Subjects. For the generation of progenitor cells toward various lineages, CD34+ cells were thawed and prepared for the culture, as described previously. Cells were cultured in multilineage medium (IMDM containing 20% fetal bovine serum, 10% heat-inactivated pooled human AB serum, 1% BSA, 10 μg/mL insulin, 0.5 μg/mL vitamin B-12, 15 μg/mL folic acid, 50 nM β-mercaptoethanol, 50 U/mL penicillin, and 50 μg/mL streptomycin, in the presence of 50 ng/mL IL-3, 50 ng/mL stem cell factor, 2 IU/mL erythropoietin, 100 ng/mL granulocyte colony-stimulating factor, and 100 ng/mL thrombopoietin) at a cell density of 2 × 104 cells/mL. For the short-term culture within 4 hours, CD34+ cells were cultured at cell densities ranging from 5 to 10 × 104 cells/mL. Cells were maintained at 37°C in a 5% CO2 incubator as described previously. The yield was measured by dye exclusion using 0.2% trypan blue dye and a hemocytometer. The cells collected from culture were washed twice with IMDM containing 0.3% BSA. The cells were then incubated with FITC- and PE-labeled mAb, washed twice with MACS buffer (10 mM phosphate-buffered saline [pH 7.4], 0.5% BSA and 2 mM EDTA), and analyzed using a FACS Calibur (Becton Dickinson), as reported elsewhere [11Saito K. Hirokawa M. Inaba K. et al.Phagocytosis of codeveloping megakaryocytic progenitors by dendritic cells in culture with thrombopoietin and tumor necrosis factor-alpha and its possible role in hemophagocytic syndrome.Blood. 2006; 107: 1366-1374Crossref PubMed Scopus (12) Google Scholar]. Cells collected from culture were washed twice with IMDM containing 0.3% BSA. Cells were then incubated with 5 μg/mL ethidium monoazide bromide for 15 minutes in the dark, and washed twice with MACS buffer. After a 10-minute light exposure on ice, the cells were fixed and stained for intracellular PE-anti–IFN-α with BD Cytofix/Cytoperm kit (BD Biosciences, Franklin Lakes, NJ, USA) according to the manufacturer's instructions. Annexin V-PE or Annexin V-FITC and PI (Sigma) were used to assess the incidence of apoptosis. A cell pellet was suspended in Annexin V binding buffer (10 mM HEPES/NaOH [pH 7.4] + 140 mM NaCl + 2.5 mM CaCl2) with 0.2 ng/μL Annexin V-PE or FITC and 20 ng/μL PI. After incubation on ice in the dark for 15 minutes, cells were analyzed using a FACSCalibur (BD Biosciences). Cells were harvested, washed with cold phosphate-buffered saline, and fixed in 70% ethanol. Cells were then stored at −20°C until analysis. Fixed cells were centrifuged at 1200 rpm, washed with cold phosphate-buffered saline twice and RNase A added at a final concentration of 0.5 mg/mL. Cells were then incubated for 10 minutes at 37°C. Next, 25 μg/mL PI was added and the cells were incubated for 30 minutes at room temperature in the dark. Cells were analyzed using a FACSCalibur. FlowJo software (Tree Star, Inc, Ashland, OR, USA) was used to determine the percentage of cells in the different cell cycle phases. Fluorescence staining was imaged using a Confocal Laser Scanning Microscope 510 (LSM510; Carl Zeiss Microscope Systems, Germany) equipped with a 100× objective lens and a 10× camera lens (Carl Zeiss Microscope Systems). Rhodamine was excited using a Hene laser at 543 nm. Detector slits were configured to minimize cross talk between channels and processed using a software package (LSM510, version 3.2) and Adobe Photoshop (Adobe Systems, San Jose, CA, USA). Total RNA was extracted from 1 × 105 cells per sample using TRizol reagent (Invitrogen). The extracted RNA was then reverse-transcribed using the SuperScript III First-Strand Synthesis System for RT-PCR (Invitrogen) in a 20-μL reaction volume. Complementary DNA was then subjected to real-time RT-PCR using LightCycler 480 SYBR Green I Master (Roche Applied Science, Basel, Switzerland). The relative gene expression levels were normalized with glyceraldehyde 3-phosphate dehydrogenase. Primer sequences are presented in Table 1 and were purchased from Nippon Gene Research Laboratories (Sendai, Japan).Table 1Primers used in this studyPrimerDirectionSequenceTLR3Forward5′- AAC AGC ATC AAA AGA AGC AG -3′Reverse5′- ACA GAG TGC ATG GTT CAG TT -3′RIG-IForward5′- GGG ACG AAG CAG TAT TTA G -3′Reverse5′- CAT CTC CAA GCA CAG TGT A -3′MDA-5Forward5′- CCA AAG CTG AAG AAC ACA T -3′Reverse5′- ATC TTC TCT GGT TGC ATC T -3′PKRForward5′- TGG CTA TTC ATC ATG GCT GG-3′Reverse5′- CTC AGC AGC ATT CCT TTT GG-3′IFN-βForward5′- TCA TGA GCA GTC TGC ACC T-3′Reverse5′- AGA GGC ACA GGC TAG GAG AT-3′p21Forward5′- ACT CTC AGG GTC GAA AAC G-3′Reverse5′- CAC ACA AAC TGA GAC TAA GGC-3′BimForward5′- TGG CAA AGC AAC CTT CTG-3′Reverse5′- TGG CTC TGT CTG TAG GGA GGT A-3′p53Forward5′- AGA TGT TCC GAG AGC TGA-3′Reverse5′- CAG TGG GGA ACA AGA AGT-3′Bcl-2Forward5′- TGT GGC CTT CTT TGA GTT C-3′Reverse5′- CGG TTC AGG TAC TCA GTC ATC-3′Bcl-xlForward5′- GGT ATT GGT GAG TCG GAT CG -3′Reverse5′- TCG GCT GCT GCA TTG TTC -3′NoxaForward5′- TGA TAT CCA AAC TCT TCT GC -3′Reverse5′- ACC TTC ACA TTC CTC TCA A -3′PumaForward5′- GAC CTC AAC GCA CAG TA -3′Reverse5′- CTA ATT GGG CTC CAT CT -3′GAPDHForward5′- GAA GGT GAA GGT CGG AGT C-3′Reverse5′- GAA GAT GGT GAT GGG ATT TC-3′GAPDH = Glyceraldehyde 3-phosphate dehydrogenase; Puma = p53 upregulated modulator of apoptosis. Open table in a new tab GAPDH = Glyceraldehyde 3-phosphate dehydrogenase; Puma = p53 upregulated modulator of apoptosis. CD34+ cells were incubated in multilineage medium for 4 hours with or without 50 μg/mL poly I:C. Western blot analysis was carried out according to manufacturer's protocol (Invitrogen). Significant differences between groups were calculated using the unpaired Student's t-test. Tests were undertaken using Stat View 4.0 and significant differences were defined as p < 0.05. To examine the effects of poly I:C on the growth of hematopoietic progenitors, human CD34+ cells were cultured for 7 days in multilineage medium. Under these conditions, simultaneous differentiation of erythroid (glycophorin A+), neutrophilic (CD15+), and megakaryocytic (CD61+) progenitors occurred [5Guo Y.M. Ishii K. Hirokawa M. et al.CpG-ODN 2006 and human parvovirus B19 genome consensus sequences selectively inhibit growth and development of erythroid progenitor cells.Blood. 2010; 115: 4569-4579Crossref PubMed Scopus (30) Google Scholar]. During 7 days of culture, poly I:C significantly reduced the number of progenitor cells generated from CD34+ cells in a dose-dependent manner (Fig. 1A ). We also found that the decrease of the number of cells by poly I:C was due to decreases of erythroid, neutrophilic, and megakaryocytic progenitors (Fig. 1B). These data indicate that poly I:C inhibits growth of CD34+ cells independent of the cell lineage and suggest that poly I:C inhibits multipotential progenitor cells. To further understand the kinetics of the growth inhibition of CD34+ cells by poly I:C, purified CD34+ cells were cultured in multilineage medium in the presence or absence of poly I:C. The cell yield substantially increased in the cultures that did not contain poly I:C, and decreased in the cultures that did contain poly I:C after 4 days in culture (Fig. 1C), which indicated that poly I:C affected the growth of CD34+ cells in the early stage of development. When purified CD34+ cells were cultured for 7 days in multilineage medium and poly I:C was added at the indicated time points (Fig. 1D), only the addition of poly I:C to the medium from the beginning of the culture resulted in significant inhibitory effects on the generation of progenitors (Fig. 1D). Additions of poly I:C to the medium later than 1 day after the initiation of culture resulted in no inhibitory effects on the generation of progenitors, which suggests that poly I:C inhibits growth of CD34+ cells in the very early stage of development. To further investigate the developmental stage of CD34+ cells susceptible to poly I:C, CD34+ cells were transiently exposed to poly I:C and cultured in multilineage medium for 7 days. Surprisingly, only 30 minutes of exposure of CD34+ cells to poly I:C significantly inhibited the growth of CD34+ cells (Fig. 1E). These findings indicate that the inhibition of CD34+ cells by poly I:C occurs within a very short time period. To investigate whether the inhibitory effects of poly I:C mediate the inhibition of cell division, CD34+ cells labeled with carboxy-fluorescein diacetate succinimidyl ester were cultured in multilineage medium for 7 days with or without poly I:C. The progenitor cells generated with or without poly I:C exhibited the same carboxy-fluorescein diacetate succinimidyl ester intensity (Fig. 2A ), which indicates that poly I:C-mediated inhibition of progenitor development does not depend on the decrease of cell division. On the other hand, after 4 hours of exposure to poly I:C, CD34+ cells showed a marked heterogeneity in size and contained many shrunken cells (Fig. 2B), and an increase of Annexin V+/PI− apoptotic cells from 8.3% ± 1.8% to 46.4% ± 7.6% (p < 0.01, Fig. 2C), in a dose-dependent manner (Fig. 2E). These data indicate that poly I:C induces apoptosis of CD34+ cells within a very short time period. In addition, when CD34+ cells, cultured for 2 days in multilineage medium (day 2 cells), were treated with poly I:C for 4 hours and apoptosis was measured, an increase of Annexin V+/PI− apoptotic cells from 3.2% ± 0.2% to 7.0% ± 0.5% (p < 0.01, Fig. 2D) was observed, but to a lesser extent compared to that seen in day 0 CD34+ cells (Fig. 2C). Consistent with the result shown in Figure 1D, this result also suggests that poly I:C inhibits the growth of CD34+ cells in the very early stage of development. To examine the specificity of poly I:C in an immediate induction of apoptosis of CD34+ cells, purified CD34+ cells were cultured in multilineage medium for 4 hours in the presence or absence of various TLR ligands, such as LPS (a TLR4 ligand), R848 (a TLR7/8 ligand), nuclease-resistant 2006-PS, and nuclease-sensitive 2006-PO (TLR9 ligands). As illustrated in Figure 3A , poly I:C but not LPS, R848, 2006-PS, and 2006-PO, exhibited an increase of Annexin V+/PI− apoptotic cells. These data indicate that the effect of inducing immediate apoptosis of CD34+ cells is specific in poly I:C among various TLR ligands. In order to examine the intracellular localization of poly I:C, purified CD34+ cells were cultured in multilineage medium for 4 hours with rhodamine-labeled poly I:C (Fig. 3B). Poly I:C-rhodamine was found in cells with a feature of flat and blurry cytoplasm (Fig. 3B, arrows in the middle panel), which suggests that the internalization of poly I:C in CD34+ cells rapidly induces apoptosis of these cells. When bafilomycin A, a potent inhibitor of autophagosome-lysosome fusion [12Yoshimori T. Yamamoto A. Moriyama Y. Futai M. Tashiro Y. Bafilomycin A1, a specific inhibitor of vacuolar-type H(+)-ATPase, inhibits acidification and protein degradation in lysosomes of cultured cells.J Biol Chem. 1991; 266: 17707-17712Abstract Full Text PDF PubMed Google Scholar], was tested against the effect of poly I:C, bafilomycin A completely blocked poly I:C–induced apoptosis (Fig. 3C), suggesting that CD34+ cells internalize poly I:C through the autophagosome-lysosome pathway. Recognition of dsRNA by TLR3 or RIG-I/MDA-5 is cell-type–dependent. To identify the downstream molecules of poly I:C signaling, purified CD34+ cells were cultured in multilineage medium in the presence or absence of poly I:C and expressions of TLR3, RIG-I, MDA-5, and dsRNA-dependent protein kinase R (PKR) mRNA were monitored at the indicated time points. PKR is a serine–threonine kinase that binds dsRNA [13Saunders L.R. Barber G.N. The dsRNA binding protein family: critical roles, diverse cellular functions.FASEB J. 2003; 17: 961-983Crossref PubMed Scopus (298) Google Scholar]. PKR-deficient mouse embryonic fibroblasts have defective type-I IFN responses to poly I:C and some RNA viruses [14Yang Y.L. Reis L.F. Pavlovic J. et al.Deficient signaling in mice devoid of double-stranded RNA-dependent protein kinase.EMBO J. 1995; 14: 6095-6106Crossref PubMed Scopus (559) Google Scholar]. As illustrated in Figure 4A , TLR3, RIG-I, and MDA-5 mRNA, but not PKR mRNA, were found to be induced by poly I:C. Inductions of RIG-I and MDA-5 mRNA were evident as early as 2 hours into incubation with poly I:C. Polyadenylic-polyuridylic acid, another synthetic dsRNA, which signals only through TLR3 [10Conforti R. Ma Y. Morel Y. et al.Opposing effects of toll-like receptor (TLR3) signaling in tumors can be therapeutically uncoupled to optimize the anticancer efficacy of TLR3 ligands.Cancer Res. 2010; 70: 490-500Crossref PubMed Scopus (82) Google Scholar], had no effect on the proliferation (Fig. 4B) and apoptosis (Fig. 4C) of CD34+ cells. On the other hand, poly I:C-LMW/LyoVec, a complex between low molecular-weight poly I:C and the transfection reagent LyoVec, which signals only through RIG-I/MDA-5 [9Gitlin L. Barchet W. Gilfillan S. et al.Essential role of mda-5 in type I IFN responses to polyriboinosinic:polyribocytidylic acid and encephalomyocarditis picornavirus.Proc Natl Acad Sci U S A. 2006; 103: 8459-8464Crossref PubMed Scopus (884) Google Scholar], induced apoptosis of CD34+ cells accompanied with an increase of PI+/Annexin V− late apoptotic fraction (Fig. 4D). Collectively, RIG-I/MDA-5, but not TLR3, signaling induces poly I:C–mediated apoptosis of CD34+ cells. Apoptosis is regulated by various factors, such as transcription factors (e.g., c-Jun, p53, and c-Myc), cell cycle regulators (e.g., p21 and cyclin D1), Bcl-2 subfamily (e.g., Bcl-2 and Bcl-xl) and Bcl-2 homology 3 (BH3)-only proteins (e.g., Bim, Puma and Noxa) (for review see [15Adams J.M. Cory S. The Bcl-2 apoptotic switch in cancer development and therapy.Oncogene. 2007; 26: 1324-1337Crossref PubMed Scopus (1632) Google Scholar, 16Kurokawa M. Kornbluth S. Caspases and kinases in a death grip.Cell. 2009; 138: 838-854Abstract Full Text Full Text PDF PubMed Scopus (338) Google Scholar]). These alterations promote the sensitivity of CD34+ cells toward cell death. Analyses of p53, p21, Bcl-2, and Bcl-xl during poly I:C–mediated apoptosis of CD34+ cells showed strong transcriptional induction of p21 with RNA levels increasing 45.2- ± 10.1-fold within 2 hours of exposure to poly I:C. Among proapoptotic BH3-only proteins, critical initiators of mitochondrial apoptosis [17Huang D.C. Strasser A. BH3-Only proteins-essential initiators of apoptotic cell death.Cell. 2000; 103: 839-842Abstract Full Text Full Text PDF PubMed Scopus (889) Google Scholar], a strong and sustained upregulation (up to 22-fold) of Noxa mRNA and protein levels was found (Fig. 5A and B ), and Bim mRNA to a lesser extent (Fig. 5A), whereas the changes of p53, Bcl-2, Bcl-xl, and Puma mRNA levels were minimal (Fig. 5A). There is evidence that RIG-I and MDA-5 trigger a p53-independent alternative pathway for the induction of Noxa [18Besch R. Poeck H. Hohenauer T. et al.Proapoptotic signaling induced by RIG-I and MDA-5 results in type I interferon-independent apoptosis in human melanoma cells.J Clin Invest. 2009; 119: 2399-2411Crossref PubMed Google Scholar]. Therefore, these results suggest functional links between dsRNA sensors, RIG-I and MDA-5, and the apoptosis program via Noxa. The stress pathway initiated by BH3-only proteins can permeabilize the mitochondrial outer membrane, releasing cytochrome c, which provokes apoptotic protease-activating factor 1 to activate caspase-9 [15Adams J.M. Cory S. The Bcl-2 apoptotic switch in cancer development and therapy.Oncogene. 2007; 26: 1324-1337Crossref PubMed Scopus (1632) Google Scholar]. To analyze the relationships between poly I:C–mediated apoptosis and ca
Referência(s)