Ribosomal Protein S19 Deficiency Leads to ... - Wiley Online Library

TISSUE-SPECIFIC STEM CELLS Ribosomal Protein S19 Deficiency Leads to Reduced Proliferation and Increased Apoptosis but Does Not Affect Terminal Erythroid Differentiation in a Cell Line Model of Diamond-Blackfan Anemia KOICH MIYAKE,a,b TAIJU UTSUGISAWA,a JOHAN FLYGARE,a THOMAS KIEFER,a,c ISAO HAMAGUCHI,d JOHAN RICHTER,a STEFAN KARLSSONa a

Molecular Medicine and Gene Therapy, Lund University, Lund, Sweden; bDepartment of Biochemistry and Molecular Biology, Nippon Medical School, Tokyo, Japan; cClinic for Internal Medicine C, Department of Hematology and Oncology, Ernst-Moritz-Arndt University of Greifswald, Greifswald, Germany; dDepartment of Safety Research on Blood and Biologics, National Institute of Infectious Diseases, Tokyo, Japan

Key Words. Anemia • Apoptosis • Erythropoiesis • Lentiviral vector

ABSTRACT Diamond-Blackfan anemia (DBA) is a congenital red-cell aplasia in which 25% of the patients have a mutation in the ribosomal protein (RP) S19 gene. It is not known how the RPS19 deficiency impairs erythropoiesis and proliferation of hematopoietic progenitors. To elucidate molecular mechanisms in RPS19-deficient DBA, we analyzed the effects of RPS19 deficiency on erythropoietin (EPO)-induced signal transduction, cell cycle, and apoptosis in RPS19-deficient TF-1 cells. We did not find any abnormality in EPO-induced signal transduction. However, RPS19-deficient TF-1 cells showed G0/G1 arrest (82% vs. 58%; p < .05) together with accumulation of p21 and p27. The fraction of apoptotic cells detected by Annexin V analysis also increased compared with control cells (13% vs. 3.1%; p < .05). Western blot

analysis of apoptosis-related proteins showed that the level of bcl-2 and Bad was decreased and Bax was increased in RPS19-deficient TF-1 cells. Moreover, primary CD34-positive cells from DBA patients detected by Annexin V analysis also generated a higher number of apoptotic cells compared with normal CD34-positive cells during in vitro culture (38% vs. 8.9%; n ⴝ 5; p < .001). Finally, we show that although RPS19 silencing reduces EPO-induced development of erythroid progenitors expressing glycophorin A (GPA), RPS19 silencing in cells already expressing GPA does not affect GPA expression. These findings indicate that RPS19 deficiency causes apoptosis and accelerated loss of erythroid progenitors in RPS19-deficient DBA. STEM CELLS 2008;26:323–329

Disclosure of potential conflicts of interest is found at the end of this article.

INTRODUCTION Diamond-Blackfan anemia (DBA; MIM: 205900) is a rare congenital pure red-cell hypoplasia characterized by a normocytic or macrocytic anemia, reticulocytopenia, and paucity of bone marrow erythroid precursors [1, 2]. Approximately 30%– 45% of DBA patients have malformations, usually involving the upper limbs, the head, the urogenital or cardiovascular system, and short stature [3–5]. Most cases are sporadic, although in 10%–25% of patients, a positive family history has been described [5, 6]. In vitro progenitor culture studies indicate that DBA results from an intrinsic defect in progenitors predominantly affecting erythroid differentiation [7]. Approximately 25% and 2% of the patients have mutations in the genes encoding ribosomal proteins S19 (RPS19) and S24 (RPS24), respectively [8 –10]. Both RPS19 and RPS24 are components of the small 40S ribosomal subunit, suggesting that the erythroid failure in DBA is somehow linked to ribosomal biogenesis in the nucleolus or translational regulation [11]. We have previously shown that transduction of the RPS19 gene into CD34⫹ cells from several RPS19-deficient DBA

patients improved the number of erythroid colonies [12]. RPS19 transgene overexpression also improved the erythroid progenitor proliferation defect in RPS19-deficient DBA patients [13]. We further demonstrated that downregulation of RPS19 mRNA by lentivirus-mediated expression of small interfering RNA (siRNA) against RPS19 in primary human CD34⫹ hematopoietic cells significantly impaired erythroid development and mimicked defects in erythroid differentiation seen in cells from DBA patients [14]. These studies suggested that RPS19 plays an important role for erythroid differentiation and proliferation. However, the exact mechanism by which RPS19 mutations affect erythropoiesis remains unclear. Since DBA is a rare disease, it is difficult to obtain sufficient hematopoietic cell samples from DBA patients to allow detailed biochemical studies of the mechanisms causing DBA. Therefore, we developed cellular models for RPS19-deficient DBA using inducible expression of siRNA against RPS19 in TF-1 and UT-7 cells [15]. Upon RPS19 silencing, these cell lines present a phenotype similar to that of progenitor cells from RPS19deficient DBA patients, including reduced erythroid differentiation and a reduction in proliferative capacity. Recently, we used these TF-1 cell lines to demonstrate that RPS19-deficient

Correspondence: Stefan Karlsson, M.D., Ph.D., Molecular Medicine and Gene Therapy, Lund University, BMC A12, 221 84, Lund, Sweden. Telephone: 46-222-05-77; Fax: 46-222-05-78; e-mail: [email protected] Received July 18, 2007; accepted for publication October 22, 2007; first published online in STEM CELLS EXPRESS October 25, 2007. ©AlphaMed Press 1066-5099/2008/$30.00/0 doi: 10.1634/ stemcells.2007-0569

STEM CELLS 2008;26:323–329 www.StemCells.com

Molecular Mechanism of Diamond-Blackfan Anemia

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cells exhibit a specific defect in processing of the 18S rRNA and maturation of the 40S ribosomal subunit [16]. In the present study, we again used the RPS19-deficient TF-1 cell lines to study how a deficiency of RPS19 affects erythropoietin (EPO) signaling, cell cycle regulation, and apoptosis. We found that RPS19 silencing in TF-1 cells does not affect EPO signaling but instead induces a cell cycle block in G1 phase and increased apoptosis. Furthermore, we found that the early EPO-dependent stage of erythroid differentiation colony forming unit-erythmoid (CFU-E) is more affected by the RPS19 deficiency than the later proerythroblast stage and stages beyond it.

MATERIALS

AND

METHODS

Cell Lines, Culture Conditions, and Cytokines The cytokine-dependent TF-1 cells, TF-1-B cells, (an inducible RPS19-deficient DBA model cell line) and TF-1-S control cells were maintained in RPMI 1640 supplemented with 10% fetal bovine serum (FBS) and antibiotics in the presence of 5 ng/ml GMCSF (a gift from Novartis International, Basel, Switzerland, http:// www.novartis.com) as described previously [15]. To differentiate into the erythroid lineage, these cells were cultured with 5 U/ml recombinant EPO (Janssen-Cilag, Sollentuna, Sweden, http://www. janssen-cilag.com). After informed consent was obtained, bone marrow (BM) samples were collected from RPS19-deficient DBA patients (all DBA samples had abnormalities in the RPS19 gene) and healthy volunteers using a protocol approved by Lund University Hospital Ethics Committee. After isolation of mononuclear cells using a Lymphoprep density gradient (Nycomed, Oslo, Norway, http://www.nycomed.com), CD34⫹ cell enrichment was performed using Midi MACS LS separation columns and isolation kit (Miltenyi Biotec, Auburn, CA, http://www.miltenyibiotec.com).

Bad, phosphorylated Stat1, phosphorylated Stat2, phosphorylated Stat5, phosphorylated Erk1/2 (Cell Signaling Technology, Beverly, MA, http://www.cellsignal.com), p21, p27, ␤-actin (Santa Cruz Biotechnology Inc., Santa Cruz, CA, http://www.scbt.com), and RPS19 [15]. Proteins were visualized using chemiluminescence reagents (Western Lightning; PerkinElmer Life and Analytical Sciences, Boston, http://www.perkinelmer.com) according to the manufacturer’s protocol. Densitometry was performed on developed film by using ImageJ software, version 1.30, to quantify the data. For immunoprecipitation analysis, cells were solubilized with cell lysis buffer containing 50 mM HEPES, pH 7.5, 0.5% Nonidet P-40, 150 mM NaCl, 1 mM EDTA, 1 mM sodium orthovanadate, 1 mM dithiothreitol, 1 mM NaF, and protease inhibitors (Calbiochem, Darmstadt, Germany, http://www.emdbiosciences.com). After centrifugation, soluble proteins were incubated with antibody specific for p85 (Santa Cruz Biotechnology) for 1 hour at 4°C and precipitated with protein A/G-agarose (Santa Cruz Biotechnology) for an additional 1 hour. The immune complexes were washed with the cell lysis buffer, separated by SDS-PAGE, and analyzed with antibody for phosphotyrosine (PY-99; Santa Cruz Biotechnology) as previously described [19].

Apoptosis Analysis The relative number of apoptotic cells in culture was assessed by Annexin V binding. Briefly, cells were incubated with APC-conjugated Annexin V according to the recommendations of the manufacturer (Boehringer Mannheim, Mannheim, Germany, http://www. boehringer.com) in the presence of 1 mmol/l CaCl2. Nuclei of cells were counterstained with 7-aminoactinomycin D, and cells were analyzed by flow cytometry. To confirm the apoptotic change, we also determined the fraction of apoptotic cells by the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) method [20] using the DeadEnd Colorimetric TUNEL system (Promega, Madison, WI, http://www.promega.com) according to the manufacturer’s instructions.

Production of Lentiviral Vectors

Statistical Analysis

Construction of lentiviral packaging (pCMV-⌬R8.91 and pMD.G) and vector (pLV-TH-B, pLV-TH-S, and pLV-mRIY) plasmids has been described previously [14, 15]. All recombinant lentivirus vectors were produced by transient transfection of 293T cells as previously described [14, 15].

Data were analyzed using the two-tailed Student t test as appropriate for the data set. p ⬍ .05 was considered statistically significant.

Analysis of Expression of EPO Receptor and GlycophorinA Expression of EPO receptor (EPO-R) was analyzed by staining with allophycocyanin (APC)-conjugated anti-human EPO-R monoclonal antibody (mAb), and expression of glycophorin A (GPA) was measured by staining with APC-conjugated anti-GPA mAb (Becton, Dickinson and Company, San Jose, CA, http://www.bd.com). In brief, cells were pelleted, washed once with phosphate-buffered saline (PBS), and thereafter incubated for 30 minutes at 4°C with the mAb. After washing twice in PBS containing 2% FBS, the cells were analyzed using flow cytometry (FACSort; Becton Dickinson). Background fluorescence was assessed using isotype-matched irrelevant APC-conjugated mAb.

Cell Cycle Analysis Cell cycle analysis was performed as previously described [17, 18]. Briefly, the cells were fixed for 30 minutes with 50% methanol, stained with 100 ␮g/ml propidium iodide after treatment with RNase A, and subjected to flow cytometry analysis using a FACScan instrument (Becton Dickinson).

RESULTS Expression of EPO-R Is Intact in RPS19-Deficient Cells DBA patients display an impaired in vivo and in vitro response to pharmacologic doses of EPO. Serum EPO concentration is also high in proportion to the severity of anemia. Therefore, abnormal EPO-R function has been speculatively advanced as a cause of DBA. However, it has been reported that the structure of EPO is normal and anti-EPO antibodies are absent in DBA patients [21]. In this study, we asked whether RPS19 deficiency may cause abnormalities in EPO-R expression, EPO binding, or EPO signal transduction. First, we analyzed the EPO-R expression in RPS19-deficient TF-1-B cells by fluorescence-activated cell sorting analysis. As shown in Figure 1A, no abnormal EPO-R expression was detected in RPS19-deficient TF-1-B cells expressing siRNA targeting RPS19 compared with control TF-1-S cells expressing a scrambled unspecific siRNA sequence.

Western Blot Analysis

EPO Signal Transduction in RPS19-Deficient Cells Is Normal

Western blot analysis was performed as previously described [15]. Briefly, soluble proteins were separated by 15% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membrane (Hybond-P; Amersham Biosciences, Uppsala, Sweden, http://www.amersham.com). The membranes were incubated with antibodies specific for Bcl-XL, Bcl-2, Bax,

Next, we examined the EPO signal transduction in TF-1 cells. EPO induces the stimulation of Jak2 tyrosine kinase that leads to the tyrosine phosphorylation of several proteins, including the EPO-R itself [22]. As a result, different intracellular pathways are activated, such as STAT transcription factors [23], mitogen-

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Figure 2. Cell cycle analysis of ribosomal protein (RP) S19-deficient cells. (A): TF-1 cells were transduced with LV expressing small interfering RNA against RPS19 (LV-TH-B) or control vector (LV-TH-S). Four days after transduction, the cells were stained with propidium iodide, and cell cycle analysis was performed by flow cytometry. (B): After TB and TS were cultured with erythropoietin and Dox for 4 days, Western blot analysis was performed using p21 or p27 antibody. Abbreviations: LV, lentiviral vector; TB, TF-1-B cells; TS, TF-1-S cells.

Increased Apoptosis in RPS19-Deficient Cells

Figure 1. Expression of EPO-R on TB and EPO-induced activation of Stat, Erk, and PI3K. (A): After TB and TS were cultured with EPO and Dox for 4 days, expression of EPO-R was analyzed by flow cytometry. (B): Four days after Dox induction, TB and TS were starved for 8 hours and subsequently stimulated with EPO (10 U/ml) for 15 minutes. Total cell lysates were prepared for WB with the indicated anti-pStat, pErk1,2, and RPS19 antibodies. (C): First, lysates were incubated with antibody specific for PI3K (p85) and precipitated. In this step, all proteins that combined with PI3K were precipitated (IP anti-PI3K). After separating by SDS-polyacrylamide gel electrophoresis, we analyzed with antibody for PY-99 (WB anti-PY). Therefore, we could detect several phosphorylated bands [19]. Abbreviations: EPO, erythropoietin; EPO-R, erythropoietin receptor; GAB1, Grb2-associated binder-1; IP, immunoprecipitation; IRS2, insulin receptor substrate-2; PI3K, phosphatidylinositol 3 kinase; PY, phosphotyrosine; RP, ribosomal protein; SHIP, SH2containing inositol 5-phosphatase; TB, TF-1-B cells; TS, TF-1-S cells; WB, Western blot.

activated protein kinase [24], and phosphatidylinositol 3 (PI-3) kinase [19, 25]. When we analyzed phosphorylation of STAT transcription in RPS19-deficient TF-1-B cells, we did not find any abnormalities in pSTAT-1, -3, and/or pErk 1,2 (Fig. 1B). Moreover, immunoprecipitation of PI-3 kinase showed normal signal transduction in RPS19-deficient TF-1-B cells (Fig. 1C).

Increased Proportion of Cells in G0/G1 and Accumulation of p21 and p27 in RPS19-Deficient Cells Although there was no alteration of expression of EPO receptor or signal transduction, RPS19-deficient cell lines exhibited reduced proliferative capacity and impaired erythroid differentiation [15]. We therefore examined the cell cycle status in RPS19deficient cell lines by propidium iodide staining. As shown in Figure 2A, a strong increase in the proportion of G0/G1 cells was detected in RPS19-deficient TF-1-B cells compared with TF-1-S cells (mean, 82% vs. 58%), together with a severe reduction in S-phase cells (mean, 13% vs. 32%; n ⫽ 4; p ⬍ .05). Western blot analysis of cyclin-dependent kinase inhibitors showed accumulation of p21 and p27 in RPS19-deficient TF1-B cells (Fig. 2B). www.StemCells.com

We also analyzed apoptotic change in RPS19-deficient TF-1 cells by Annexin V staining. Annexin V-positive cells were increased in RPS19-deficient cells compared with control TF-1 cells (mean, 13% vs. 3.1%; n ⫽ 4; p ⬍ .05) (Fig. 3A). The increased proportion of apoptotic cells among RPS19-deficient cells was confirmed by TUNEL assay (Fig. 3B). These results indicate that suppression of RPS19 induces apoptosis. Because EPO can promote erythroid progenitor survival by repressing apoptosis through Bcl-XL and Bcl-2, we analyzed Bcl-XL and Bcl-2 antiapoptotic proteins together with proapoptotic proteins Bax and Bad. Western blot analysis of apoptosis-related proteins showed that the level of Bcl-2 and Bad was decreased and that the level of Bax was increased in RPS19-deficient TF-1 cells compared with control cells (Fig. 3C). These alterations were corrected by transduction with a lentiviral vector expressing modified RPS19, which is not affected by siRNA and which rescues the DBA phenotype [14, 15]. Quantitative data on apoptosis-related protein expression is shown in Figure 3D. These results suggest that downregulation of Bcl-2 and Bad, together with upregulation of Bax protein, is specifically caused by deficient RPS19 expression.

CD34ⴙ BM Cells from DBA Patients Generate a High Number of Apoptotic Cells During In Vitro Culture To analyze whether primary cells from RPS19-deficient DBA patients exhibit increased apoptosis, we analyzed CD34⫹ BM cells from RPS19-deficient DBA patients by Annexin V staining. To maintain the hematopoietic progenitor cells, we cultured CD34⫹ cells in the presence of interleukin (IL)-3 (20 ng/ml), IL-6 (50 ng/ml), stem cell factor (SCF) (100 ng/ml), and EPO (5 U/ml) for 4 days. During in vitro culture, a high number of apoptotic cells were observed in CD34⫹ BM cells from RPS19deficient DBA patients compared with normal CD34⫹ cells (Fig. 4) (38% vs. 8.9%; n ⫽ 5; p ⬍ .001).

Terminal Erythroid Differentiation Is Not Affected by Suppression of RPS19 in TF-1 Cells That Are Stimulated to Differentiate into Erythroid Cells Since erythropoiesis is extensively regulated by apoptosis at the CFU-E to erythroblast stage, we investigated how an induced

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Figure 3. Increased apoptosis in ribosomal protein (RP) S19-deficient cells. After culture of TB and TS with erythropoietin (EPO) and Dox for 4 days, cells were analyzed by Annexin V staining (A) and TUNEL assay (B). (C): TS, TB, and TB⫹mRPS19 were cultured with EPO and Dox for 4 days. Western blot analysis was performed using antibodies for apoptosis-related proteins. (D): Quantitative data on apoptosis-related protein expression by densitometry analysis of Western blots (means of two independent experiments). The graph shows the ratio of RPS19 protein expression (Dox(⫹)/Dox(⫺)) normalized to ␤-actin protein expression. Abbreviations: TB, TF-1-B cells; TB⫹mRPS19, TF-1-B cells transduced with LV-mRIY; TS, TF-1-S cells; TUNEL, terminal deoxynucleotidyl transferase dUTP nick-end labeling.

induces apoptosis and G0/G1 cell cycle arrest in cells progressing from GPAlow (CFU-E) to GPAhigh (proerythroblasts) cells during erythroid development.

DISCUSSION

Figure 4. An increase in apoptosis in CD34⫹ cells from DiamondBlackfan anemia (DBA) Pt. CD34-positive bone marrow cells from normal donors or DBA Pt were cultured with SCF (100 ng/ml), interleukin (IL)-3 (20 ng/ml), IL-6 (50 ng/ml), and EPO (5 U/ml), and apoptotic cells were analyzed for 4 days by Annexin V staining. Abbreviation: Pt, patients.

RPS19 deficiency affects erythroid differentiation in TF-1 cells before and after EPO induction. Figure 5A shows expression of GPA during erythroid differentiation induced by EPO in TF-1-B cells and RPS19 expression level after induction by Dox. The cell surface expression of GPA was gradually increased during culture with EPO and reached a plateau at day 10. To analyze the effect of RPS19 downregulation at different stages of erythroid differentiation, we performed two experimental plans (Fig. 5B). First, Dox was added at day 0 and cultured with EPO for 10 days. Since it takes at least 4 days to suppress the protein level of RPS19 by expression of this siRNA construct (Fig. 5A) [15], the effect of suppression of RPS19 appeared at stage a (Fig. 5Ba). In the second experimental setup, TF-1-B cells were first cultured with EPO for 4 days, and then Dox was added and cultured for 10 days. In this experimental design, the effect of suppression of RPS19 appeared at stage b (Fig. 5Bb). Ten days after Dox induction, expression of GPA was analyzed by flow cytometry. Down-modulation of GPA was detected only when RPS19 was suppressed prior to the increase in GPA (Fig. 5B). The results from three independent experiments are summarized in Table 1. These results suggest that the deficiency of RPS19

We previously generated models for RPS19-deficient DBA by using lentivirus-mediated siRNA against RPS19 in primary human CD34⫹ hematopoietic cells [14] and in TF-1 and UT-7 multipotent progenitor cell lines [15]. A similar decrease in cell proliferation and erythroid differentiation was observed in these RPS19-deficient cells as in primary cells from RPS19-deficient DBA patients. However, it is not known how the RPS19 deficiency impairs erythropoiesis and proliferation of hematopoietic progenitors. DBA patients display an impaired in vivo and in vitro response to pharmacologic doses of EPO. Hence, abnormalities in EPO-R expression, EPO binding, or EPO signal transduction may be present. Ohene-Abuakwa et al. speculate that the defect lies downstream of the EPO-R, influencing survival and/or proliferation of erythroid progenitors in DBA cells [26]. Our findings presented here demonstrate that the level of EPO-R expression and EPO signal transduction is normal in an RPS19-deficient DBA model cell line. We have previously demonstrated down-modulation of GPA and a reduction of 2,7-diaminofluorene (DAF)-positive cells in RPS19-deficient TF-1 cells [15]. However, when we analyzed GPA expression in erythroid-differentiated TF-1 cells, down-modulation of GPA was not detected in EPO-induced, erythroid-differentiated GPAhigh TF-1 cells. These results strongly suggest that the early EPO-dependent stage of erythroid differentiation (CFU-E) is more affected by a deficiency of RPS19 than later stages (proerythroblast and later). Suppression of proliferation was detected in RPS19-deficient TF-1 cells as well as primary CD34⫹ cells treated with siRNA against RPS19 [14, 15]. This phenomenon was detected in myeloid cultures as well. Moreover, suppressed proliferation was observed in hematopoietic and nonhematopoietic (293-T, HeLa, and 3T3 cells) (data not shown). Therefore, we believe


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Figure 5. Suppression of RPS19 does not affect terminal erythroid differentiation. (A): Expression of GPA in TF-1-B cells during EPO-induced erythroid differentiation. TF1-B cells were cultured with EPO, and GPA expression was analyzed by flow cytometry every 2 days. Fluorescence-activated cell sorting profiles with the mean fluorescent intensity of GPA expression are presented. The lower panel shows the time course of RPS19 protein expression in TF-1-B cells cultured with Dox for 7 days, determined by Western blot using RPS19 or ␤-actin-specific antibodies. The band density ratio of RPS19/␤-actin was defined as 1.0 in TF-1-B cells at day 0 (without Dox), and relative ratios were calculated. (B): Experimental design for analysis of the effect of suppression of RPS19 on EPO-differentiated TF-1-B cells. TF-1-B cells were cultured with EPO and Dox from day 0 (Ba) or with EPO for 4 days before addition of Dox (Bb). Ten days after Dox induction, expression of GPA was analyzed by flow cytometry. Abbreviations: EPO, erythropoietin; GPA, glycophorin A; RP, ribosomal protein. Table 1. Mean fluorescent intensity of GPA in RPS19 deficient cells Experiment

a) b)

Dox(ⴚ)

Dox(ⴙ)

1507 ⫾ 286 1526 ⫾ 168

478 ⫾ 82* 1506 ⫾ 213

*p ⬍ .05 compared to Dox(⫺). Mean fluorescent intensity of GPA expression in Dox induced TF-1-B cells differentiated by EPO according to experimental protocol a) or b) (Fig. 5B). Each result represents the average ⫾ standard deviation (SD) from three independent experiments.

that suppression of RPS19 affects cell proliferation in a general fashion. In fact, malformation and short stature detected in DBA patients could be the outcome of increased apoptosis or prolonged G1 phase during embryogenesis. In addition, the primary pharmacological treatment for DBA patients is dexamethasone, which acts by stimulating proliferation of early erythroid progenitors [27], suggesting that the proposed proliferative defect in DBA can be restored in patients responding to steroid treatment. Drosophila Minute phenotypes, characterized by delayed larval development, diminished viability, reduced body size, diminished fertility, and thin bristles, are due to mutations in RP genes [28, 29]. Thus, it is possible that ribosomal proteins play important roles in control of cell proliferation in humans, as well as in Drosophila. We observed increased apoptosis in RPS19-deficient cells detected by Annexin V staining and TUNEL assay. Western blot analysis showed downregulation of Bcl-2 and Bad proteins in RPS19-deficient cells. Bcl-2 exerts a survival function in response to a wide range of apoptotic stimuli through inhibition of mitochondrial cytochrome c release [30], whereas Bad is a www.StemCells.com

proapoptotic member of the Bcl-2 family that connects the growth factor signaling pathway with the cell death pathway by displacing Bax from binding to Bcl-2 and Bcl-XL [31, 32]. Therefore, Bcl-2 downregulation is an expected finding in apoptotic cells, whereas the decrease of Bad protein seems to contradict the increased level of apoptosis in RPS19-deficient TF-1-B cells. However, Seo et al. reported that Bad has a function as a prosurvival factor, prior to its role in promoting cell death [33]. Downregulation of Bad protein may act as a death factor in RPS19-deficient cells. We also detected increased Bax protein levels in RPS19-deficient TF-1 cells. Recently, Gazda et al. demonstrated a significant increase of Bax and other proapoptotic agents in RPS19-mutated DBA patients by global gene expression analysis [34]. Our findings suggest that apoptosis of erythroid progenitors contributes to development of anemia in RPS19-deficient DBA patients. In normal erythropoiesis, the numbers of CFU-E and proerythroblasts are tightly regulated by and are very sensitive to apoptosis [35]. The important role of apoptosis as a regulator of erythropoiesis may explain why erythroid cells are specifically reduced in RPS19-deficient DBA patients with a generally high level of apoptosis. We also detected increased apoptosis in primary cells from RPS19-deficient DBA patients. We have previously demonstrated a severe reduction in erythroid progenitors (CFU-E, Burst forming unit (BFU-E)) but not myeloid progenitors (CFUM/GM) [12] and described a proliferation deficiency of CD34⫹ DBA samples compared with normal CD34⫹ cells [13] using the same CD34⫹ RPS19-deficient DBA patient samples used in this study. Since only the erythroid progenitors were negatively affected in this cell population where increased apoptosis was detected, we propose that it is mainly the erythroid progenitors in DBA that show increased apoptosis.


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It has previously been demonstrated that ribosome biogenesis and rRNA processing are impaired in RPS19-deficient DBA cells [16, 36]. We speculate that these defects in ribosome biogenesis may induce a state of “nucleolar stress” that indirectly activates the p53 pathway that leads to transcriptional activation of p21 and p27 and prolonged G1 cell cycle phase [37, 38]. Induction of this nucleolar stress signal could explain the prolonged G1 cell cycle phenotypes seen in RPS6- and RPL24-deficient mice [39, 40], as well as the increased proportion of cells in G0/G1 and accumulation of p21 and p27 in RPS19-deficient cells.

CONCLUSIONS We found that suppression of RPS19 inhibits cell proliferation and early erythroid differentiation but not late erythroid maturation in RPS19-deficient DBA model cell lines. We also detected apoptotic change in RPS19-deficient cells, as well as primary CD34⫹ cells, from DBA patients. These results suggest that suppression of proliferation and increased apoptosis in early erythroid progenitors are major factors leading to suppressed erythroid development in RPS19-deficient DBA.

ACKNOWLEDGMENTS We thank Karin Olsson, Eva Nilsson, Ann Brun, and Noriko Miyake for expert technical assistance. This work was supported by the European Commission (INHERINET), the Swedish Medical Research Council, the Swedish Gene Therapy Program, the Swedish Cancer Society, the Swedish Children’s Cancer Society, and the Diamond-Blackfan Anemia Foundation, Inc. (U.S.A.) and by clinical research support from Lund University Hospital (to S.K.). This work was also supported by grants from the Wennergren Foundation, Sweden (to K.M.); Kungliga Fysiografiska Sa¨llskapet, Sweden (to K.M. and J.F.); the Ronald McDonald Foundation, Sweden (to J.F.); and Deutsche Forschungsgemeinschaft (to T.K.). The Lund Stem Cell Center is supported by a Center of Excellence grant in life sciences from the Swedish Foundation for Strategic Research.

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CONFLICTS

The authors indicate no potential conflicts of interest.

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