Regulation of globin-heme balance in Diamond-Blackfan anemia by HSP70/GATA1

Abstract

Diamond-Blackfan anemia (DBA) is a congenital erythroblastopenia that is characterized by a blockade in erythroid differentiation related to impaired ribosome biogenesis. DBA phenotype and genotype are highly heterogeneous. We have previously identified 2 in vitro erythroid cell growth phenotypes for primary CD34⁺ cells from DBA patients and following short hairpin RNA knockdown of RPS19, RPL5, and RPL11 expression in normal human CD34⁺ cells. The haploinsufficient RPS19 in vitro phenotype is less severe than that of 2 other ribosomal protein (RP) mutant genes. We further documented that proteasomal degradation of HSP70, the chaperone of GATA1, is a major contributor to the defect in erythroid proliferation, delayed erythroid differentiation, increased apoptosis, and decreased globin expression, which are all features of the RPL5 or RPL11 DBA phenotype. In the present study, we explored the hypothesis that an imbalance between globin and heme synthesis may be involved in pure red cell aplasia of DBA. We identified disequilibrium between the globin chain and the heme synthesis in erythroid cells of DBA patients. This imbalance led to accumulation of excess free heme and increased reactive oxygen species production that was more pronounced in cells of the RPL5 or RPL11 phenotype. Strikingly, rescue experiments with wild-type HSP70 restored GATA1 expression levels, increased globin synthesis thereby reducing free heme excess and resulting in decreased apoptosis of DBA erythroid cells. These results demonstrate the involvement of heme in DBA pathophysiology and a major role of HSP70 in the control of balanced heme/globin synthesis.

Graphical abstract

Figure 1.

Heme metabolism pathway analysis during normal human erythroid differentiation. (A) Measurement of total heme content from day 4 to day 14. Quantification based, after iron removal, on the protoporphyrin quantification by direct molecule fluorescence. Mean OD relative to that obtained at day 4 (mean OD = 1). The data are mean ± standard deviation of 3 independent experiments. (B) Expression levels of the major proteins involved in heme synthesis (ALAS1, ALAS2, FECH), excess free heme removal (FLVCR1, BACH1, HMOX1), globin transcription (GATA1), globin translation (HRI, EIF2α, α and β globins, some RP involved as well in DBA, RPS19, RPL5, RPL11), and iron uptake (TfR1). Immunoblots of 50 000 human normal primary erythroid cells during the normal erythroid culture time course from day 0 to day 14 (0, 2, 4, 7, 9, 11, and 14) obtained from healthy CD34⁺ cord blood. Proteins were compared with β-actin expression. (C) Protein expression of ALAS2, FECH, BACH1, FLVCR1, EIF2α, and α and β globins relative to β-actin at days 0, 2, 4, 7, 9, 11, and 14 of normal erythroid differentiation. *P < .05, **P < .010.

Figure 2.

Excess free heme in the shRNA model reproducing DBA (human primary erythroid cells depleted in RPS19, RPL5, or RPL11). (A) Quantification of the total heme in depleted RPS19, RPL5, or RPL11 erythroid cells after CD34⁺ cord blood infection with specific shRNAs at day 9 of primary human erythroid cell culture. The data are mean ± standard deviation of 3 independent experiments, relative to total heme content of shcontrol (value = 1). (B) Excess free heme in depleted RPS19, RPL5, or RPL11 erythroid cells after CD34⁺ cord blood infection with specific shRNAs. Pellets of 100 000 RP-depleted erythroid cells were analyzed, and OD scans were measured from 200 to 800 nm with a spectrophotometer. The day-9 measurement is shown. Free heme was calculated as the ratio between 380 nm ± 2 nm (heme band) and 560 nm ± 2 nm (hemoglobin band) (supplemental Figure 2A). The data are mean ± standard deviation of 3 independent experiments, relative to free heme content of shcontrol (value = 1). (C) Major excess free heme after depletion of RPL5 or RPL11 compared with RPS19 lentivirus–depleted erythroid cells. Immunoblots of 100 000 RPL5 or RPL11 depleted erythroid cells revealed a significant decrease in ALAS2 expression levels, whereas ALAS1 was normal, reinforcing the decreased total heme content and the specificity of the defect in erythroid cells. The iron uptake based on TfR1 expression level was decreased significantly under all conditions. Immunoblots also revealed indirect signs of the large amount of excess free heme in depleted RPL5 and RPL11 erythroid cells on the decreased BACH1 and a large increase in FLVCR1 expression levels. Proteins BACH1, FLVCR1, TfR1, ALAS2, and ALAS1 were compared with the β-actin expression level. A western blot representative of 3 experiments at day 9 of the primary erythroid cell culture is shown (statistics are shown in supplemental Figure 3B). (D) Quantification of ROS production in RPS19-, RPL5-, or RPL11-depleted erythroid cells at day 9 of erythroid culture. We show the second method used for ROS production based on flow cytometry with a CellROX Deep Red Reagent kit (Invitrogen). RPL5- and RPL11-depleted erythroid cells at day 9 produced a higher ROS compared with the control, whereas RPS19 ones exhibited a slight increase in ROS production. Data are representative of 3 experiments. (E) Brief reminder of the HRI/EIF2α pathway (adapted from Chen). (F) HRI/EIF2α protein pathway analysis in DBA erythroid cells after CD34⁺ cord blood infection with specific shRNA-RPS19, -RPL5, -RPL11. Immunoblots of 100 000 RPS19-, RPL5-, or RPL11-depleted primary erythroid cells compared with β-actin expression and shcontrol. A western blot representative of 3 experiments at day 9 of the primary erythroid cell culture is shown (statistics are shown in supplemental Figure 3B). (G) HRI (left panel) and EIF2α (right panel) mRNA expression in DBA erythroid cells after CD34⁺ cord blood infection with specific shRNA-RPS19, -RPL5, -RPL11. We observed a significant decrease in HRI and EIF2α mRNA compared with the reporter gene mRNAs at day 9 of terminal erythroid differentiation. The data are mean ± standard deviation of 3 independent experiments relative to mRNA expression level of shcontrol compared with β-actin (value = 1). *P < .05, **P < .01. NS, nonsignificant.

Figure 3.

DBA-affected patients also exhibit excess free heme. (A) Expression levels of the major proteins involved in heme metabolism and their regulators in a DBA patient who carried a mutation in the RPS19 gene during terminal erythroid differentiation compared with a healthy control. Immunoblots of 50 000 human primary erythroid cells in each lane obtained from purified peripheral blood CD34⁺ cells from the affected DBA patient (UPN#35) during terminal erythroid differentiation from day 7 (D7) to day 12 (D12). Protein expression compared with β-actin or GAPDH, depending on the size of the proteins, to optimize the numbers of proteins analyzed on the same western blot. Due the difficulty in obtaining samples from DBA patients, this patient has been studied once; other DBA-affected patients have been studied. We validated the data because the same protein profile on the western blots in all of the mutated RPS19 DBA patients have been seen (as example another mutated RPS19^Mut/+ patient, supplemental Figure 5). (B) Same data as in (A) for a DBA-affected patient who carried a mutation in the RPL11 gene (UPN#1099). (C) From the immunoblot in panel A, representation of the level of protein expression of FLVCR1 in the erythroid precursors of DBA patient UPN#35 relative to β-actin and in a healthy control at each day of the studied terminal erythroid differentiation. (D) From the immunoblot in panel B, representation of the level of FLVCR1 expression in the erythroid precursors of DBA patient UPN#1099 relative to β-actin and to the healthy control during terminal erythroid differentiation (value = 1 at day 7 in the control erythroid cells). (E) Increased FLVCR1 and decreased globin expression levels in a DBA-affected patient with a mutated RPL5 gene (RPL5^Mut/+) (UPN#845). (F) Relative mRNA expression of GATA1, ALAS2, EIF2α, α and β globins, and TfR1 in a DBA-affected patient (UPN#845) compared with a healthy control (value = 1). (G) Relative ROS production by various DBA-affected patients. These DBA patients carried mutations in the RPS19 or RPL5 gene or even an unknown gene compared with their healthy controls (mean fluorescence intensity = 1). *P < .05 in triplicate experiments.

Figure 4.

HSP70 overexpression rescued GATA1 expression, decreased ROS production, limited free heme content, and rescued the heme/globin balance. (A) Relative protein expression of GATA1, HSP70, β-globin chain, TfR1, and RPL11 after depletion of RPS19 or RPL11 in primary erythroid cells and rescue with overexpression of wild-type HSP70 cDNA. Data at day 9 of terminal erythroid culture, obtained from immunoblots of 100 000 human erythroid primary cells derived from CD34⁺ cells from cord blood and depleted in RPS19 or RPL11 by specific shRNAs. Proteins are compared with β-actin. The data are relative to the protein expression levels in the shcontrol for each protein studied (value = 1). (B) Relative mRNA expression compared with β-actin and shcontrol gene expression (value = 1) after depletion of erythroid cells in RPS19 (left panel) or RPL11 (right panel) mRNA after cord blood CD34⁺ cell lentiviral infection by specific shRNAs and rescue with overexpression of wild-type HSP70 cDNA. The data are mean ± standard deviation of 3 independent experiments and correspond to the relative mRNA expression of each gene (HSP70, GATA1, α and β globin, ALAS2, FECH, TfR1, EIF2α, HRI, RPS19, and RPL11) compared with the reporter genes, such β-actin, and the shcontrol (value = 1). No significant effect of HSP70 overexpression has been noted in depleted RPS19 erythroid cells (left panel), whereas a significant increase in all mRNAs tested, with the exception of RPS19 and RPL11 mRNA, was found after HSP70 overexpression in depleted RPL11 erythroid cells (right panel). (C) HSP70 overexpression rescued the FLVCR1 increase in depleted RPS19 erythroid cells and, to a greater extent, in depleted RPL11 erythroid cells. (D) HSP70 overexpression significantly increased erythroid cell survival in depleted RPL11 erythroid cells. Cell mortality was assessed with 0.2% trypan blue. The data are mean ± standard deviation of 3 independent experiments. *P < .05, ***P ≤ .001.

Figure 5.

Heme induced a decrease in HSP70 expression. (A) Immunoblots performed using lysates of UT7 cells (left panel) or day-2 CD36⁺ human primary erythroid cells (right panel) that were treated for 4 hours with the indicated concentrations of heme arginate. (B) Immunoblot quantification. Values are quantified relative to actin and normalized to untreated cells. The mean ± standard deviation of 4 experiments are shown. *P < .05, **P < .01, unpaired Student t test.

Figure 6.

Take home message of this study. Model integrating all of our findings about how HSP70 accounts for the intrinsic defect in DBA by controlling erythroid differentiation and survival in DBA via GATA1, as well as excess free heme, resulting from the imbalanced heme/globin equilibrium.