Page 278 - 2022_01-Haematologica-web

A. Astori et al.
Quantitative reverse transcriptase polymerase chain reaction
Cells were collected and stored directly at -80°C for RNA preparation with the TRIzol (Life Technologies) extraction pro- tocol as previously described.1 First-strand cDNA synthesis (reverse trasncription) was carried out using a Transcriptor First Strand cDNA Synthesis Kit (cat. n. 489703000, Roche). RINF mRNA expression was detected using a Lightcycler® 480 ProbesMaster kit (cat. n. 4707494001, Roche). Relative mRNA expression was normalized to RLPL2, PPIA or ACTB gene expression in a two-color duplex reaction. For SMAD7, PU.1 and c-KIT mRNA detection, qRT-PCR was performed using SYBRGreen on a Light Cycler 480 machine (Roche) and gene expression was calculated by the 2-ΔΔCT method. Primer sequences are available in an Online Supplementary File.
Western blot analysis
Total cell extracts were prepared and western blotting was carried out as previously described.1 Blots were incubated with a primary polyclonal antibody and then with an appropriate per- oxidase-conjugated secondary antibody (anti-rabbit and anti- mouse IgG horseradish peroxidase [HRP] antibodies from Cell Signaling, cat. n. 7074S, and 7076S, respectively) and anti-goat IgG HRP antibody from Southern Biotech (cat. n. 6160-05). Proteins were detected using a chemiluminescent system (Clarity Western ECL, cat. n. 170-5060, Biorad). Rabbit polyclon- al antibodies were previously described for RINF,1 P85/PI3KR1,42 or commercially purchased for anti-RINF, anti-SMAD7 (cat. n. 16513-1-AP, cat. n. 25840-1-AP, ProteinTech), anti-b-actin (ACTB, cat. n. A1978, Sigma-Aldrich), anti-phospho-SMAD2/3 (cat. n. 8828, Cell Signaling), and anti-HSC70 mouse monoclonal antibody (SC-7298, Santa Cruz Biotechnology).
Statistical analyses
All analyses were performed using GraphPad Prism software. When the distribution of data was normal, the two-tailed Student t test was used for group comparisons. Contingency tables were established using the Fisher exact test, and the Pearson correlation coefficient was used to determine the corre- lation between the normally distributed RINF mRNA and SMAD7 mRNA expression values. Statistics were carried out on a minimum of three independent experiments. The statistical significance of P values is indicated in the Figures: not significant (ns): P>0.05; *P<0.05; **P<0.01; ***P<0.001. Error bars represent confidence intervals at 95% or standard deviations (SD) for the qRT-PCR analyses.
Results
RINF is expressed during early human erythropoiesis
We employed cytokine-induced erythroid differentiation of CD34+ progenitor cells isolated from human cord blood or adult bone marrow to model human erythropoiesis in vitro. To obtain highly enriched populations of differentiat- ing erythroblasts, cells were grown in a two-phase liquid culture (experimental design in Figure 1A) as previously described.36,37 We found that RINF protein was present at the earliest stages of erythroid differentiation and reached its highest level in progenitors and proerythroblasts (ProE) (Figure 1B). After that, the level of RINF protein decreased in the basophilic erythroblast stage and was barely detectable in late erythroblasts (i.e., polychromatic and orthochromatic erythroblast stages). Similarly, RINF mRNA levels were high in erythroid progenitors but decreased at
the basophilic stage and onwards (Figure 1C). This tempo- ral expression pattern mirrors RINF expression data extract- ed from three independent transcriptome datasets (microar- ray or RNA-sequencing) reported by Merryweather-Clarke et al.,43 An et al.,44 and Keller et al.45 with adult or cord-blood CD34+ cells (Online Supplementary Figure S2). Taken togeth- er, our data show that RINF expression peaks in erythroid progenitors and proerythroblasts during cytokine-induced erythropoiesis.
RINF silencing results in diminished red blood cell production without affecting cell viability
We next investigated the consequences of RINF knock- downonerythropoiesisandcellviability(seeexperimental design in Figure 2A). Knockdown experiments were per- formed with two previously validated short-hairpin RNA (shRINF#4 and shRINF#3) sequences1 driven by the lentivi- ral pTRIPDU3/GFP vector (Online Supplementary Figure S3A, B). We found that RINF was extinguished as early as 2 days after transduction (i.e., at day 0 of EPO) with shRINF#4 at both mRNA (Figure 2B) and protein (Figure 2C) levels, without affecting cell viability, assessed by trypan blue cell counting (Figure 2D) or cell growth up to day 10 of EPO (Figure 2E, left panel). However, in the last days of ex vivo culture (day 11-17), we noticed a reduction in total number of RBC produced (average decrease of ~45%) at 17 days with EPO (Figure 2E, right panel), which was particularly marked for four donors out of six studied.
To investigate how RINF knockdown affects the clono- genic capacity of HSPC, we next performed comparative colony-forming assays for the development and maturation of erythroid (burst-forming unit erythroid progenitors; BFU-E), myeloid (CFU-G, CFU-M, or CFU-GM) and mixed (CFU-GEMM) colonies. No statistically significant changes were noted in the total number of colonies or the number of CFU-M, CFU-GM, or CFU-GEMM (Online Supplementary Figure S3C). However, the proportions of granulocytic colonies (CFU-G) and BFU-E, were slightly reduced or increased, respectively (P<0.01, Fisher exact test). The number of MEP-sorted cells was also increased under RINF knockdown conditions (Online Supplementary Figure S3E), and this increase corresponded with an increase in small (more mature) BFU-E, findings that suggest an accelerated maturation. A more careful analysis of colony size revealed that the median size of BFU-E derived from CD34+ cells dropped by about 30% (i.e., from 0.101 to 0.071 mm2, unpaired t-test, P=0.007, n=3 donors) after RINF knockdown (Figure 2F). Concomitantly with the size reduc- tion of large BFU-E (the most immature ones), we noted a slight but statistically significant increase in small BFU-E (the most mature ones), in agreement with accelerated mat- uration (Figure 2G).
Erythroid maturation is affected by RINF
We next investigated whether the reduced expansion observed from day 10 of EPO could be the consequence of accelerated maturation. Indeed, we observed that ery- throid differentiation was accelerated under conditions of RINF knockdown using quantification by benzidine staining (Figure 3A), morphological analysis of cells stained with May-Grünwald-Giemsa (Figure 3B), and flow cytometry for the erythroid markers GPA and CD71 (Figure 3C, upper panel), and CD49d and Band3 (Figure 3C, lower panel). These differences were statistically sig- nificant (P<0.001) for CD34+ cells derived from both cord
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