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Brief Report
RED CELLS, IRON, AND ERYTHROPOIESIS
Induction of fetal hemoglobin through enhanced translation efficiency
of g-globin mRNA
Cynthia K. Hahn1 and Christopher H. Lowrey1-3
1
Department of Pharmacology and Toxicology, Geisel School of Medicine at Dartmouth, Hanover, NH; and 2Department of Medicine, and 3Norris Cotton
Cancer Center, Dartmouth-Hitchcock Medical Center, Lebanon, NH
Key Points
• HbF induction by salubrinal
is not mediated through
changes in globin mRNA
stability, mRNA cellular
localization, or HbA levels.
• Translation efficiency of
g-globin mRNA is increased
during stress recovery
following salubrinal-enhanced
eIF2a phosphorylation.
Fetal hemoglobin (HbF) induction can ameliorate the clinical severity of sickle cell disease
and b-thalassemia. We previously reported that activation of the eukaryotic initiation
factor 2a (eIF2a) stress pathway increased HbF through a posttranscriptional mechanism. In this study, we explored the underlying means by which salubrinal, an activator
of eIF2a signaling, enhances HbF production in primary human erythroid cells. Initial
experiments eliminated changes in globin messenger RNA (mRNA) stability or cellular
location and reduction of adult hemoglobin as possible salubrinal mechanisms. We
then determined that salubrinal selectively increased the number of actively translating
ribosomes on g-globin mRNA. This enhanced translation efficiency occurred in the recovery
phase of the stress response as phosphorylation of eIF2a and global protein synthesis
returned toward baseline. These findings highlight g-globin mRNA translation as a novel
mechanism for regulating HbF production and as a pharmacologic target for induction of
HbF. (Blood. 2014;124(17):2730-2734)
Introduction
Induction of fetal hemoglobin (HbF) is an effective therapeutic
strategy for b-hemoglobinopathies.1-5 Most studies have focused on
understanding transcriptional regulation of hemoglobin switching to
discover new mechanism-based therapeutic approaches to g-globin
gene activation.6 However, there are data indicating that HbF may
also be posttranscriptionally regulated.7-10
Recently, we identified the eukaryotic initiation factor 2a (eIF2a)
pathway as a critical posttranscriptional regulator of HbF.11 We
found that increasing eIF2a phosphorylation (p-eIF2a) increased
HbF protein without changing g/(g1b) messenger RNA (mRNA)
levels. The most dramatic posttranscriptional HbF induction was
elicited by salubrinal (Sal), a pharmacologic enhancer of p-eIF2a,
which increased HbF up to 4.5-fold without altering globin mRNA
levels, cellular differentiation, or total Hb content. Here, we investigate
the posttranscriptional mechanism of HbF induction when eIF2a is
activated by Sal.
Tocris Bioscience) and actinomycin-D (Sigma-Aldrich) were dissolved in
dimethylsulfoxide, and puromycin (Sigma-Aldrich) was dissolved in phosphatebuffered saline.
mRNA and protein analyses
RNA isolation, complementary DNA synthesis, quantitative polymerase
chain reaction, Hb high-performance liquid chromatography, and western
blotting were performed as previously described (see the supplemental Methods,
available on the Blood Web site).11 Transcript levels were calculated by
the method of Larionov et al13 relative to glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) expression. Polymerase chain reaction primers
are listed in supplemental Table 1.
Lentiviral infection
Vectors and virus were generated as previously described.11 The sequence
targeted by hemoglobin b gene (HBB) short hairpin RNA was 59-TGGCCC
ATCACTTTGGCAAAG-39, and infections were performed on days 8 and 9.
Green fluorescent protein was monitored by flow cytometry for transduction
efficiency (range, 90% to 95%).
Methods
Cell culture and chemicals
Results and discussion
K562 cells were maintained in RPMI medium (Cellgro) with 10% fetal bovine
serum and 1% penicillin-streptomycin. CD341 cells were obtained from the
University of Washington or from Dr Patrick Gallagher (Yale Medical
School) using protocols approved by the institutional review board. Cultures were maintained as described in Sankaran et al.12 Salubrinal (Sal-003;
To investigate Sal’s mechanism, we used an erythroid primary cell
differentiation system12 in which Sal was applied at doses previously
shown to increase p-eIF2a and reduce protein translation.11 First,
we assessed whether Sal changed g- and b-globin mRNA stability.
Submitted March 20, 2014; accepted August 9, 2014. Prepublished online as
Blood First Edition paper, August 28, 2014; DOI 10.1182/blood-2014-03564302.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked “advertisement” in accordance with 18 USC section 1734.
The online version of this article contains a data supplement.
There is an Inside Blood Commentary on this article in this issue.
2730
© 2014 by The American Society of Hematology
BLOOD, 23 OCTOBER 2014 x VOLUME 124, NUMBER 17
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BLOOD, 23 OCTOBER 2014 x VOLUME 124, NUMBER 17
SALUBRINAL REGULATES THE TRANSLATION OF g-GLOBIN
2731
Figure 1. Sal does not change mRNA stability, mRNA cellular localization, or total HbA to induce HbF. (A) Neither g-globin nor b-globin relative mRNA half-life is
changed in cells treated with Sal in combination with 5 mg/mL actinomycin D (Act D) versus actinomycin D alone. Expression is reported as fold change relative to the
untreated control (Untx) on day 15 prior to treatment. Actinomycin D and 6 mM Sal were simultaneously applied on day 15. mRNA expression was quantified by using primers
located at either the 59 or 39 end of each mRNA. Error bars represent 6 standard error of the mean (SEM) of 3 independent experiments. (B) Sal does not change the
cytoplasmic (Cyto):nuclear (Nuc) ratio of g- or b-globin mRNA compared with the untreated control. Cells were treated with 6 mM Sal on days 15 and 18. Cytoplasmic and
nuclear RNA were isolated on days 15, 18, and 20, mRNA expression for g- and b-globin was quantified in each compartment by using primers that spanned at least 1 exonexon junction, and the cytoplasmic mRNA:nuclear mRNA ratio was compared. Error bars represent 6 SEM of 4 independent experiments. (C) Western blotting shows that
short hairpin HBB (shHBB) causes 50% knockdown of b-globin protein compared with short hairpin control (shCTRL). Protein lysates were taken on days 13, 15, and 17
of differentiation after infections on days 8 and 9. glyceraldehyde-3-phosphate dehydrogenase was used as a loading control. (D) High-performance liquid chromatography
(HPLC) was performed on day 20 of culture to assess the proportions of HbF, HbA, and HbA2 in shCTRL- and shHBB-infected cells. These HPLC traces from a representative
experiment reveal that shHBB enhances the percentage of HbF and percentage of HbA2 compared with shCTRL. (E) b-globin knockdown does not increase total HbF, but
it does enhance total HbA2. After analysis by HPLC, the area under the curve for HbF, HbA, and HbA2 was corrected for the total Hb concentration in 2 3 106 cells. Total
amounts of HbF, HbA, and HbA2 are depicted as fold change relative to the shCTRL. Error bars represent 6 SEM of 3 independent experiments. P values were determined by
using an unpaired 2-tailed Student t test. NS, not significant. *P , .05; **P , .001.
Cells were treated with Sal in combination with actinomycin D,
an inhibitor of transcription, and compared with actinomycin D
treatment alone. During a 60-hour time course, Sal did not change the
relative half-life of g- or b-globin mRNA (Figure 1A). Next, we
determined whether changes in g- or b-globin mRNA cellular
localization could explain Sal’s ability to increase HbF. Cytoplasmic
and nuclear RNA fractions were compared as cytoplasmic mRNA:
nuclear mRNA ratios. Sal treatment did not alter the cellular location of g- or b-globin when compared with the control (Figure 1B),
indicating that changes in mRNA transport were not sufficient to
account for the difference in HbF after Sal treatment.
Previously, we observed that Sal treatment not only increased
HbF, but concomitantly reduced adult hemoglobin (HbA).11 We
questioned whether reducing HbA was sufficient to increase HbF.
Decreased b-globin translation or inhibition of b-globin chain
association with a-globin could reduce HbA, thereby allowing
a-globin chains to preferentially complex with g-globin chains. To
test this hypothesis, we used a short-hairpin RNA targeting b-globin
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2732
HAHN and LOWREY
BLOOD, 23 OCTOBER 2014 x VOLUME 124, NUMBER 17
Figure 2. Sal selectively increases the translation efficiency of g-globin mRNA. (A) Representative polysome profile on day 15 after 6 hours of 12 mM Sal treatment
reveals a polysome-to-monosome shift, indicative of halted translation initiation and reduced translation. (B) Western blotting shows that p-eIF2a is increased at 3 and 6 hours
after 12 mM Sal treatment on day 15 relative to an untreated control. At 24 hours after treatment, p-eIF2a levels are similar between the 2 conditions. Total eIF2a and b-actin
are used as loading controls. (C) After 6 hours of treatment with 12 mM Sal, g- and b-globin translation efficiency is unchanged relative to the untreated control. Polysome
profiling fractions were pooled into 7 larger fractions containing ribosome-free 40S/60S or 80S, or polysome-bound RNA ranging from lower to higher ribosome occupancy.
g- and b-globin mRNA were quantified in each pooled fraction and normalized to an exogenous luciferase mRNA control. The percent of total mRNA for each fraction was
calculated. Error bars represent 6 SEM of 3 independent experiments. (D) After 24 hours of 12 mM Sal treatment, the representative polysome profile on day 16 shows
a slight polysome-to-monosome shift. (E) After 24 hours, 12 mM Sal significantly increases the translation efficiency of g- and b-globin mRNA, whereas b-actin and activating
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BLOOD, 23 OCTOBER 2014 x VOLUME 124, NUMBER 17
that reduced protein levels by ;50% (Figure 1C). This was an appropriate level of knockdown because Sal only modestly reduced
HbA in previous experiments. When equal amounts of Hb were
analyzed by HPLC, b-globin knockdown increased the percentage
of HbF by twofold (4% to 8%) and the percentage of HbA 2 by
fivefold (2% to 10%) and decreased HbA from 94% to 82%
(Figure 1D). However, the absolute Hb content per cell was reduced
by ;50% with b-globin knockdown. When total Hb levels per cell
were calculated, HbF was unchanged (Figure 1E). In contrast, total
HbA2 was increased, a finding consistent with elevated HbA2 levels
seen in patients with b-thalassemia intermedia.14 These results
confirm that reducing HbA is not sufficient for increasing HbF but it
does increase HbA2.
We next tested whether Sal affected globin translation. By
using polysome profiling, we confirmed that Sal treatment of 6 hours
reduced protein translation, as indicated by a shift in mRNA from
the polysomes to the 80S peak (Figure 2A). This coincided with
an increase in p-eIF2a, which reduces translation initiation15
(Figure 2B). However, quantification of g- and b-globin mRNA
isolated from pooled polysome fractions revealed that Sal did not
significantly change the translation efficiency of either mRNA after
6 hours (Figure 2C).
We then questioned whether translation was changed during stress
recovery, which has been reported for other mRNAs.16,17 After
24 hours of Sal treatment, p-eIF2a recovered to control levels
(Figure 2B) and the polysome profile from Sal-treated cells was
shifted less to the 80S fraction (Figure 2D). During this recovery
phase, the translation efficiency of g- and b-globin was increased
as indicated by a significant shift to higher ribosome occupancy
(Figure 2E). In contrast, b-actin translation efficiency remained unchanged and activating transcription factor 4 (ATF4) mRNA shifted
slightly, but not significantly, to the polysome fractions. Together,
these results suggest that globin translation efficiency is specifically
increased 24 hours after Sal treatment. When the ratio of heavy-tolight polysomes was compared, Sal increased the g-globin ratio by
2.9-fold (P 5 .019) but increased the b-globin ratio by only 1.6fold (P 5 .025) (Figure 2F), indicating that g-globin mRNA was
preferentially translated. Because our previous results showed
that 2 Sal treatments over 5 days increased HbF up to 4.5-fold,11
smaller changes in translation efficiency would be enhanced by
multiple treatments to account for the observed increase in HbF.
However, the steady-state number of ribosomes on a transcript
is not necessarily a measure of active translation.18 To confirm that
changes in globin translation efficiency were the result of the addition
of functional ribosomes, we cotreated cells with Sal and puromycin.
Puromycin requires peptidyl-transferase activity to be incorporated
into nascent peptides and causes premature ribosome release, which
differentiates stalled from active ribosomes.19 At 24 hours after
Sal treatment, puromycin caused disruption of polyribosomes and
increased the 80S peak in Sal-treated and control cells (Figure 2G).
Puromycin also produced a shift to lower ribosome occupancy of g- and
b-globin transcripts (Figure 2H). Notably, puromycin eliminated the
difference in translation efficiency observed between Sal-treated and
SALUBRINAL REGULATES THE TRANSLATION OF g-GLOBIN
2733
control cells (Figure 2H-I). These results confirm that Sal enhances
globin translation efficiency at 24 hours, as evidenced by a greater
number of actively translating ribosomes occupying the transcripts.
To further implicate enhanced protein translation as a mechanism
responsible for increased HbF after Sal treatment, we used the K562
cell line. We first confirmed that Sal increased g-globin protein levels
without changing g-globin mRNA at 24 hours as seen in primary
cells (supplemental Figure 1A-B). Next, we used the nonradioactive SUnSET method to analyze protein synthesis.20 In this method,
low doses of puromycin are incorporated into neosynthesized
peptides20-22 and are monitored by flow cytometry to deduce protein
translation rates. The puromycin fluorescence was not significantly
changed after 24 hours of Sal treatment, confirming similar levels of
total protein synthesis (supplemental Figure 1C). In control experiments, the fluorescence of cells preincubated with the translation
inhibitor cycloheximide was reduced by 70%. We then analyzed
cells that were dual stained for puromycin and HbF and found that Sal
significantly increased HbF fluorescence by 22% (supplemental
Figure 1D). Additionally, immunocytochemistry indicated a similar
subcellular localization of puromycin and HbF (supplemental
Figure 1E), suggesting that nascent g-globin translation enhances
HbF production by Sal.
In summary, we have determined that posttranscriptional induction
of HbF by Sal is the result of enhanced g-globin mRNA translation
that occurs during the recovery phase of the p-eIF2a stress response.
This provides an explanation for previous observations in which
HbF induction exceeded increases in g-globin mRNA.9,10 Our results
suggest that control of g-globin mRNA translation is an important
mechanism for regulating HbF production that could be used to
benefit patients with b-hemoglobinopathies.
Acknowledgments
The authors thank Dr Patrick Gallagher for kindly supplying CD341
cells, Dr Lionel Lewis and Bernie Beaulieu for assistance with Hb
HPLC, Dr Elizabeth McCoy and Dr Alexei Kisselev for guidance
regarding polysome profiling, and Dr Edwin Hahn, Dr Rodwell
Mabaera, Dr Rachel West, and Dr Emily Schaeffer for valuable
discussions.
This work was supported by the National Institutes of Health
(National Heart, Lung and Blood Institute grant HL73442 [C.H.L.]
and the National Institute of Diabetes and Digestive and Kidney
Diseases grant F30DK094540 [C.K.H.]) and by the Knights of the
York Cross of Honour and the Royal Order of Scotland (Masonic
charitable organizations).
Authorship
Contribution: C.K.H. designed and performed the research, analyzed
and interpreted the data, and wrote the manuscript; and C.H.L.
Figure 2 (continued) transcription factor 4 translation is not significantly changed. mRNA was quantified in each pooled fraction and normalized to an exogenous luciferase
control, and the percentage of total mRNA found in each fraction was calculated. Error bars represent 6 SEM of 3 independent experiments. P values were determined by
using an unpaired 2-tailed Student t test. *P , .05; **P , .01. (F) At 24 hours, Sal increases translation efficiency of g-globin to a greater extent than b-globin. Percentage of
mRNA in the lightest 2 polysome fractions was compared with the percentage of mRNA in the heaviest 2 polysome fractions by using a heavy-to-light polysome ratio. Error
bars represent 6 SEM of 3 independent experiments. P values were determined by using an unpaired 2-tailed Student t test. *P , .05. (G) The representative polysome
profile at 24 hours shows ribosome dissociation and a dramatic shift to the monosome peak when 12 mM Sal and the untreated control are incubated with 1 mM puromycin
(Puro). (H) Puromycin treatment after 24 hours of 12 mM Sal shifts the percentage of g- and b-globin mRNA to lighter polysome fractions to the same extent as in the untreated
control. Error bars represent 6 SEM of 2 independent experiments. (I) Puromycin reduces the heavy:light polysome ratio to the same extent in 12 mM Sal-treated and
untreated cells. Error bars represent 6 SEM of 2 independent experiments.
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2734
BLOOD, 23 OCTOBER 2014 x VOLUME 124, NUMBER 17
HAHN and LOWREY
designed and oversaw the research, analyzed and interpreted the
data, and wrote the manuscript.
Conflict-of-interest disclosure: The authors declare no competing
financial interests.
Correspondence: Christopher H. Lowrey, Section of Hematology/
Oncology, Dartmouth-Hitchcock Medical Center, One Medical
Center Dr, Lebanon, NH 03756, e-mail: christopher.h.lowrey@
dartmouth.edu.
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2014 124: 2730-2734
doi:10.1182/blood-2014-03-564302 originally published
online August 28, 2014
Induction of fetal hemoglobin through enhanced translation efficiency of
γ-globin mRNA
Cynthia K. Hahn and Christopher H. Lowrey
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