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RINF maintenance of SMAD7 sustains human erythropoiesis
proposed to contribute to the development or progression of (pre)leukemia, such as myelodysplastic syndrome (MDS).1,3-5 We and others have demonstrated that RINF mRNA expression is an unfavorable6,7 and independent3 prognostic factor in acute myeloid leukemia (AML) as well as in solid tumors.8,9 However, its role in normal hematopoiesis has hitherto been poorly investigated and its contribution to the erythroid lineage and red blood cell (RBC) expansion is unknown.
RINF contains a nuclear localization signal that has been functionally validated10 and, in most studies, its subcellular localization is reported to be mainly or exclusively nuclear and it acts as a transcriptional cofactor.1,3,8,10-17 RINF associ- ates strongly with chromatin1 through its conserved zinc- finger domain (CXXC) which plays an essential role in pro- viding the capacity to bind CpG islands.18,19 Interestingly, this domain is almost identical to the one harbored by TET1 and TET3, two epigenetic modulators involved in the erasure of DNA-methylation marks,20 pointing to the possi- bility that RINF might interfere with TET activities, hydroxymethylation, and gene transcription, as recently demonstrated in mice.12,15 RINF has also been reported to bind ATM, mediate DNA-damage-induced activation of TP533,10 and inhibit the WNT-b-catenin signaling path- way3,21-24 through a cytoplasmic interaction with disheveled proteins DVL and DVL2.21
Transforming growth factor b (TGFb) is a powerful and widespread cell growth inhibitor in numerous mammalian tissues.25,26 In the hematopoietic system, TGFb is known to regulate hematopoietic stem and progenitor cells (HSPC) and is also described as a potent inducer of erythroid differ- entiation and inhibitor of cell proliferation.27-30 TGFb signals through cell surface serine/threonine kinase receptors, mainly TGFbRI and TGFbRII. Activated TGFbRI phospho- rylates SMAD2 and SMAD3 which translocate into the nucleus and form complexes that regulate transcription of target genes. TGFb can also elicit its biological effects by activation of SMAD-independent pathways.31,32 Inhibitory SMAD (SMAD6 and SMAD7) inhibit TGFb signaling. Importantly, a reduced SMAD7 expression sensitizes cells to the antiproliferative effects of TGFb and contributes to anemia in patients suffering from MDS, suggesting, firstly, that identifying transcriptional regulators of SMAD7 could enlighten our understanding of erythropoiesis and, second- ly, that inhibiting TGFb signaling could be a therapeutic strategy that would mitigate ineffective hematopoiesis in disease states.33-35
In the present work, we used primary human CD34+ cells to demonstrate that RINF knockdown affects human ery- thropoiesis and mitigates RBC production through a mech- anism that is mediated by SMAD7, the main inhibitor of TGFb signaling.
Methods
The methods for flow cytometric cell sorting of megakary- ocyte-erythroid progenitor (MEP) cells, immunofluorescence stud- ies, chromatin Immunoprecipitation (ChIP) experiments, and the primer sequences used for quantitative reverse transcriptase poly- merase chain reaction (qRT-PCR) and ChIP-qPCR are described in the Online Supplementary Methods.
Primary culture of hematopoietic cells
Mononuclear cells were isolated from cord blood (CRB Saint-
Louis Hospital, Paris, France) or adult bone marrow donors with written informed consent for research use, in accordance with the Declaration of Helsinki. The ethics evaluation com- mittee of INSERM, the Institutional Review Board (IRB00003888), approved our research project (n. 16-319). Mononucleated cells were separated by Ficoll-Paque (Life Technologies) and purified with a human CD34 MicroBead Kit (cat. n. 130100453, Miltenyi Biotec). A two-step culture method for cell expansion and erythroid differentiation was used to obtain highly stage-enriched erythroblast populations.36,37 Briefly, CD34+ cells (purity 94-98%) were cultured for 7 days with interleukin (IL)6, IL3 (10 ng/mL), and stem cell factor (SCF, 100 ng/mL) to expand hematopoietic progenitors.37 CD36+ ery- throid progenitors were purified using magnetic microbeads (CD36 FA6.152 from Beckman Coulter and anti-mouse IgG1 MicroBeads from Miltenyi Biotec). Then, erythropoietin (EPO) was added for 10-18 days to allow erythroid differentiation. For assays of colony-forming cells (CFC, counted at 11-14 days), CD34+ hematopoietic stem cells were seeded in methylcellulose (H4034, StemCell Technologies). TGFbRI inhibitor SB431542 was from Selleckchem (cat. n. S1067)38 and TGFb was from Peprotech (cat. n. 100-21).
Flow cytometry and differentiation analysis
To monitor end-stage erythroid differentiation of primary cells, cells were labeled with anti-human PE/Cy7-conjugated glycophorin A (GPA/CD235a) (cat n. A71564), PE-conjugated CD71 (IM2001U), and APC-conjugated CD49d (cat. n. B01682) from Beckman Coulter, PE-conjugated CD233/Band3 from IBGRL (cat. n. 9439-PE). Fluorescence-activated cell sorting (FACS) analysis was performed on an AccuriTM C6 Flow Cytometer (Becton-Dickinson). For morphological characteriza- tion cells were cytospun and stained with May-Grünwald- Giemsa. For the benzidine assay, 2x105 cells were incubated for 5 min with 0.01% benzidine and H2O2 at 0.3% (Sigma-Aldrich).
Culture of cell lines and treatments
K562 (ATCC#CCL243) and UT7 39 cells were cultured in 5.3
RPMI 1640 medium and minimum essential medium-a supple- mented with 2.5 ng/mL of granulocyte-macrophage colony- stimulating factor (GM-CSF; Miltenyi Biotech), respectively. Both media were supplemented with 10% inactivated fetal bovine serum, 2 mM L-glutamine, 50 U/mL penicillin G and 50 mg/mL streptomycin (Life Technologies). To induce hemoglobin production, K562 cells were treated with hemin at 40 mM (Sigma-Aldrich). Erythroid maturation of UT75.3 was triggered by replacing GM-CSF with EPO 5 U/mL.
Lentiviral and retroviral transduction of hematopoietic cells
For shRNA-mediated RINF knockdown, we used the pTRIPDU3/GFP lentiviral vector40 which drives the same shRNA sequences downstream of the H1 promoter as our pre- viously described pLKO.1/puroR vector.1 For RINF overexpres- sion, the retroviral vector MigR/IRES-GFP was used with the previously described experimental conditions.1 Green fluores- cent protein (GFP) sorting was performed on a FACSAriaIII (BD Biosciences). For SMAD7 overexpression, the retroviral vector pBABE-puro-SMAD7-HA (Addgene plasmid#37044) was used as previously described.1,41 For doxycycline-inducible lentiviral expression, SMAD7-HA cDNA was inserted into the pINDUCER21 vector by gateway technology (Addgene plasmid #46948) and shRNA sequences were inserted in the Tet-pLKO- GFP vector (modified from Tet-pLKO-puro, Addgene plas- mid#21915, deposited by Dmitri Wiederschain).
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