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Previous Article | Table of Contents | Next Article 
Blood, 1 November 2000, Vol. 96, No. 9, pp. 3241-3248
RED CELLS
Regulation of hemoglobin synthesis and proliferation of
differentiating erythroid cells by heme-regulated
eIF-2 kinase
John S. Crosby,
Peter J. Chefalo,
Irene Yeh,
Shong Ying,
Irving M. London,
Philippe Leboulch, and
Jane-Jane Chen
From the Harvard-Massachusetts Institute of Technology
Division of Health Sciences and Technology and the Department of
Biology, Massachusetts Institute of Technology, Cambridge MA;
Harvard Medical School, Hematology Division, Department of Medicine,
Brigham and Women's Hospital, Boston, MA.
 |
Abstract |
Protein synthesis in reticulocytes depends on the
availability of heme. In heme deficiency, inhibition of protein
synthesis correlates with the activation of heme-regulated eIF-2
kinase (HRI), which blocks the initiation of protein synthesis by
phosphorylating eIF-2 . HRI is a hemoprotein with 2 distinct
heme-binding domains. Heme negatively regulates HRI activity by binding
directly to HRI. To further study the physiological function of HRI,
the wild-type (Wt) HRI and dominant-negative inactive mutants of HRI
were expressed by retrovirus-mediated transfer in both non-erythroid
NIH 3T3 and mouse erythroleukemic (MEL) cells.
Expression of Wt HRI in 3T3 cells resulted in the inhibition of protein
synthesis, a loss of proliferation, and eventually cell death.
Expression of the inactive HRI mutants had no apparent effect on the
growth characteristics or morphology of NIH 3T3 cells. In contrast,
expression of 3 dominant-negative inactive mutants of HRI in MEL cells
resulted in increased hemoglobin production and increased proliferative
capacity of these cells upon dimethyl-sulfoxide
induction of erythroid differentiation. These results directly
demonstrate the importance of HRI in the regulation of protein
synthesis in immature erythroid cells and suggest a role of HRI in
the regulation of the numbers of matured erythroid cells.
(Blood. 2000;96:3241-3248)
© 2000 by The American Society of Hematology.
 |
Introduction |
Phosphorylation of the -subunit of translational
initiation factor 2 (eIF-2 ) is important in the regulation of
protein synthesis in mammalian cells under various conditions of stress
such as heme-deficiency,1 viral infection (reviewed in
Proud2), nutrient starvation,3-5 heat
shock,3,5,6 and stress in endoplasmic reticulum (ER)
(reviewed in Brostrom and Brostrom7). Phosphorylation of
the -subunit of eIF-2 at the serine 51 residue by activated eIF-2
kinases results in the formation of an eIF-2( P)/eIF-2B complex that
renders eIF-2B nonfunctional. eIF-2B is required for the exchange of
guanosine triphosphate for guanosine diphosphate bound to eIF-2
in the recycling of eIF-2 for another round of initiation. Since eIF-2B
is limiting, the phosphorylation of only a fraction of the total
eIF-2 present in the cell is sufficient to shut off protein
synthesis (reviewed in Jackson8 and Clemens9). Overexpression of either nonphosphorylatable
eIF-2 Ala51,10 inactive mutant forms of double-stranded
RNA (dsRNA)-dependent eIF-2 kinase (PKR),11-13 or the
p58 inhibitor of PKR14 has been shown to result in
malignant transformation of NIH 3T3 cells. On the other hand,
overexpression of PKR resulted in apoptosis.15-18 These
studies underscore the importance of eIF-2 phosphorylation by
eIF-2 kinases with respect to regulation of protein synthesis and
cell growth.
In addition to the 2 extensively studied mammalian heme-regulated
eIF-2 kinases (HRIs) and the PKR, there are also the GCN2 protein
kinase and the recently identified mammalian ER resident eIF-2
kinase (PERK),19 which is identical to the
pancreatic eIF-2 kinase (PEK) highly expressed in the
pancreas.20 These eIF-2 kinases share extensive
homology in the kinase catalytic domains19-25 and
phosphorylate eIF-2 at the serine 51 residue.19,20,26,27 However, the regulatory domains and
the mechanism of each of these eIF-2 kinases are very different. HRI
is regulated by heme, the prosthetic group of
hemoglobin.1,28,29 PKR is regulated by dsRNA through the 2 N-terminal dsRNA-binding domains (reviewed in Proud2). GCN2
is activated under condition of amino acid starvation through the
C-terminal domain, which contains a His-transfer-RNA-synthase-like sequence (reviewed in Hinnebusch30). PERK is activated by
ER stress and contains a lumenal domain that is similar to the sensor domain of the ER-stress kinase, Irel.19
It is well documented that protein synthesis in intact reticulocytes
and their lysates is dependent upon the availability of heme. In heme
deficiency, inhibition of protein synthesis correlates with the
activation of HRI (reviewed in Chen and London,1
Jackson,8 Clemens,9 and
Hershey31). Hemin has been shown to inhibit both the
autokinase and eIF-2 kinase of HRI by inhibiting adenosine triphosphate binding to HRI.32,33 We have shown previously that expression of Wt HRI in insect Sf9 cells causes global inhibition of protein synthesis and that baculovirus-expressed Wt HRI is a
hemoprotein and is regulated by micromolar concentrations of hemin both
in vitro and in vivo.28,29 Furthermore, there are 2 distinct heme-binding sites in HRI. The heme binding to the N-terminal
domain of HRI is stable and copurified with HRI to homogeneity. The
heme binding to the kinase-insertion domain is reversible and is
responsible for sensing the heme and regulates HRI
activity.34
We have reported earlier that the protein and messenger RNA (mRNA) of
HRI are expressed predominantly in erythroid cells of adult rabbits. In
addition, the level of HRI mRNA is increased during erythroid
differentiation of mouse erythroleukemic (MEL) cells, and this increase
in HRI mRNA is dependent on the presence of heme.35 Since
then, 2 reports have described the presence of small amounts of HRI
mRNA in non-erythroid rat and mouse tissues and the presence of
HRI-like activity in mouse liver and NIH 3T3 cells.25,36
These reports suggested a possible role of HRI in translational
regulation in non-erythroid cells in addition to its role in erythroid
cells. It is to be noted that no HRI protein was reported in either of
these 2 studies.
To directly examine the physiological function of HRI, we have
expressed Wt and inactive mutants of HRI in both non-erythroid 3T3
cells and erythroid MEL cells. We report here that expression of Wt HRI
in NIH 3T3 cells results in severe inhibition of cell growth and
ultimately cell death, while cells that express inactive HRI mutants
appear to grow normally. The expression of inactive HRI mutants in 3T3
cells resulted in no detectable change in morphology and did not cause
oncogenic transformation. In contrast, the expression of these inactive
HRI mutants in MEL cells significantly increased the hemoglobin content
and proliferative capacity of the differentiating MEL cells. These
results demonstrate a functional role of HRI in erythroid MEL cells,
but not in non-erythroid 3T3 cells.
 |
Materials and methods |
Plasmid constructions
All HRI constructs contained the 5' untranslated leader of
tobacco mosaic virus and the entire 3' untranslated region as described previously.21 K199R (single-letter amino acid codes) HRI
complementary DNA (cDNA) was prepared and reported
previously.28 The internal deletion of amino acids 375 through 384 of HRI ( 10 HRI) and of amino acids 375 through 394 of
HRI ( 20 HRI) was prepared by recombinant polymerase chain reaction
(PCR) as described.37 Briefly, the primers used for
deletions were 30 nucleotides long, flanking the desired deleted
sequence with 15 nucleotides each on both the 5' and 3' ends. The
fragments of Dra III to Hpa I sites (nucleotides 805 through 1875) with
the desired deletions were amplified as 2 separate fragments by 2 separate PCR reactions. The first half was from the Dra III site to the
start site of the deletion. The second half was from 3' terminal to the
site of deletion to Hpa I site. These PCR reactions and the
following recombinant PCR for the production of the Dra III to Hpa I
fragment were as described previously.28 The Dra III to
Hpa I fragment with 10 or 20 deletion was digested with Dra III
and Hpa I and then subcloned to TMV-HRI vector.21 The
deletions were confirmed by DNA sequencing. In vitro transcription and
translation of 10 and 20 HRI in the TMV-HRI plasmid were also
performed to ensure that protein products with the expected sizes were
made. The 10 and 20 HRI cDNAs were then excised from the TMV-HRI
plasmid and subcloned into 1392 baculovirus-transferring vector as
described previously.28 Production of recombinant HRI
baculoviruses and the expression of HRI were as
described.28
Wt, K199R, 10, and 20 HRI retroviral constructs were prepared by
the use of pLXSN retroviral vector38 (provided by D. Miller, Fred Hutchinson Cancer Research Center, Seattle, WA) with neomycin- or puromycin-resistant gene. The HRI cDNA inserts of Wt,
K199R, 10, and 20 were excised by digestion with Bgl II and Eco
RI. These inserts were blunt-ended by Klenow fragment and ligated to
the pLXSN vector, which was digested with Eco RI and blunt-ended.
Transfection, infection, and selection of cells
NIH 3T3 cells were obtained from American Type Culture
Collection (Manassas, VA); BOSC23 and Bing
cells39 were provided by W. Pear and D. Baltimore
(Massachusetts Institute of Technology, Cambridge, MA); and crip and
cre cells40 were provided by R. Mulligan (Harvard
Medical School, Boston, MA). NIH 3T3/eIF-4E and PKR 6-12 cells were
provided by N. Sonenberg (McGill University, Montreal, Quebec, Canada).
NIH 3T3 cells, crip, and cre cells were grown at 37°C in
Dulbecco modified Eagle medium (DMEM) supplemented with 10%
heat-inactivated calf serum, 4.5 mg/mL glucose, 2 mmol/L glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin.
For BOSC23, Bing, and MEL cells, 10% heat-inactivated fetal calf serum was used.
The packaging cell lines BOSC23, Bing, crip, and cre were grown
and transfected as described.39,40 Plasmid DNAs (20 µg) prepared by the Qiagen, Valencia, CA procedure were
transfected into these cells by means of a calcium phosphate
precipitation method (Ependorf-5 Prime Inc, Boulder, CO). The
recombinant retroviruses produced were harvested from the culture
supernatants by filtering through the 0.22 µm filter units
as described.39,40 All infections were carried out in the
presence of 8 µg/mL polybrene (Sigma; St Louis, MO). The
recombinant retroviruses were used to infect NIH 3T3 or MEL cells. For
cells infected with Wt HRI, 5 µmol/L hemin was added to the culture
media to maintain cell viability. Retrovirally transduced cells were
selected 2 days after infection with 500 µg/mL G418 (Gibco/BRL,
Rockville, MD) or 1.5 µg/mL puromycin (Sigma). For
experiments, these transduced cells were cultured in the absence of
selection agents.
Lysate preparations and Western blot analysis
Preparations of cell lysates and Western blot analysis of lysate
proteins were performed as previously described.35 Mouse antirabbit HRI monoclonal antibody was used to detect HRI protein by
means of either ECL (Amersham, Piscataway, NJ) or NBT-BCIP color development system (Promega, Madison, WI).
 |
Results |
Inhibition of the Wt HRI activity by inactive mutant
HRI
Since we planned to use the inactive HRI mutants to diminish
endogenous HRI activity, we examined the abilities of inactive HRI
mutants to inhibit the activity of Wt HRI by coexpression of Wt HRI
with 3 HRI mutants in Sf9 cells using recombinant baculoviruses. The
expression of large quantities of HRI in this system readily permits
the biochemical analysis of HRI kinase activity.28 The K199R mutant HRI has a mutation of K199 in catalytic domain II to R. K199R HRI is inactive when expressed in insect cells28 and
in yeast cells.27 The 10 and 20 HRIs are the
internal deletions of amino acids 375 through 384 and amino acids 375 through 394, respectively, which are located in kinase domain VI and
highly conserved among all eIF-2 kinase. These 2 deletion mutants
are similar to PKR 6 mutation.
As shown in Figure 1A, in contrast to Wt
HRI (lane 1), K199R, 10, and 20 HRI lacked eIF-2 kinase
activity (lanes 2, 3, and 4). In addition, co-expressions of these
inactive mutants with Wt HRI decreased the rate of eIF-2
phosphorylation by 40% to 50% (Figure 1B-C). We did the
co-expressions 4 times with similar results. We have shown previously
that co-expression of Wt HRI with interleukin 1- does not affect HRI
activity.28 Thus, these observations indicate that
inhibition of theWt HRI activity by these 3 inactive mutant HRIs is
specific and is not due to the general effect of co-expression.
Similarly, co-expression of the mutant HRI with Wt HRI in NIH 3T3 cells
relieved the inhibitory effect of Wt HRI (data not shown). Previous
work by others and by us has demonstrated that HRI forms
homodimers,29,41-43 It is most likely that inactive HRI
mutants inhibit the activity of Wt HRI by forming heterodimers with Wt
HRI. Consistent with this interpretation, it is important to note that
during co-expression there is always a fraction of active Wt HRI dimer
present, at least 25%. Therefore, one would not expect complete
inhibition of the HRI activity by co-expression.

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| Figure 1.
Inhibition of the protein kinase activity of Wt HRI by
co-expression with the HRI mutants.
Wt and mutant HRI (K199R, 10, and 20) were expressed individually
or co-expressed in Sf9 cells as described previously.28
Protein kinase assays were performed with the use of cytoplasmic cell
extracts and purified eIF-2 as described previously.28 (A)
eIF-2 kinase activity of Wt, K199R, 10, or 20 HRI. (B)
eIF-2 kinase activity of co-expressed HRI. Wt HRI alone (lanes 1, 5, and 9); co-expression of Wt HRI with K199R (lanes 2, 6, and 10), with
10 (lanes 3, 7, and 11), or with 20 (lanes, 4, 8, and 12). The
time intervals of protein kinase assays are 2, 5, and 10 minutes as indicated. (C) The extent of eIF-2
phosphorylation was quantified by scintilation counting of the gel
slices containing eIF-2 . , wt;
, 10/wt; , 20/wt;
, K199R/wt.
|
|
Expression of Wt HRI in NIH 3T3 cells
inhibits their growth
We produced the Wt and inactive K199R HRI retroviruses and used
them to infect NIH 3T3 cells as described in "Materials and methods." Western blot analyses of the retrovirally transduced cells
with the anti-HRI monoclonal antibody showed that full-length Wt and
K199R HRI protein were synthesized in these cells (Figure 2A, lanes 2 and 3). The level of Wt HRI
protein expression in these cells was much lower than that of the
inactive K199R HRI (lanes 2 and 3). Since Wt HRI expressed in 3T3 cells
was an active eIF-2 kinase when analyzed by in vitro protein kinase
assays (data not shown), it is likely that Wt HRI inhibited its own
synthesis as well as protein synthesis generally in NIH 3T3 cells.
Similar observations were made when HRI was expressed in Sf9 cells by recombinant baculoviruses.28

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| Figure 2.
Growth characteristics of NIH 3T3 cells overexpressing
Wt and K199R HRI.
(A) Levels of HRI protein expression. NIH 3T3 cells overexpressing
vector alone (lane 1), Wt HRI (lane 3), or K199R HRI (lane 2) were
generated by infections with retroviruses as described in "Materials
and methods." Cytoplasmic extracts (20 µg) from these cells, as
indicated in the Figure, were separated by 7.5% sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed for
HRI by Western blot analysis. Purified rabbit reticulocyte HRI (lane 4)
and reticulocyte lysates (lane 5) were used as positive controls. (B)
Growth curve using total cell numbers. NIH 3T3 cells expressing either
the pLXSN retroviral vector alone, Wt HRI, or K199R HRI were seeded at
equal densities of (2.5 × 105) per 100 mm. Cells
expressing Wt HRI were washed 3 times with DMEM to remove the hemin
supplemented in the medium and maintained in the culture medium without
hemin supplement for 24 hours prior to seeding. Total numbers
of cells at different times of culture were plotted. (C) Growth curve
of viable cells. Cells were grown as described in panel B, and the cell
viability was determined by staining with trypan blue. The number of
trypan blue-negative cells was plotted relative to time in culture.
(D) Phase contrast photographs. NIH 3T3 cells expressing vector, Wt, or
K199R HRI at day 3 of growth as shown in panels B and C were
photographed at 200 × magnification.
|
|
Forced expression of Wt HRI in NIH 3T3 cells resulted in a severe
inhibition of cell growth and ultimately in cell death (Figure 2B-D).
The addition of a low concentration (5 µmol/L) of hemin to
the culture medium enabled us to maintain Wt HRI-expressing 3T3 cells.
All the experiments described here used pooled cells and were,
therefore, not subject to clonal variation. For characterizing the
growth curve of cells expressing Wt HRI, these cells were cultured for
24 hours in the absence of hemin and plated in the absence of hemin. As
shown in Figures 2B and 1C, the initial growth of Wt HRI was much
slower than that of cells expressing vector alone or K199R HRI, and
after 4 days in culture most of the cells were detached. These cells
appeared condensed and round and displayed an increased refractivity
similar to that of apoptotic cells. There were few trypan
blue-negative cells remaining by day 4 and none by day 5 (Figure 2C).
In contrast, expression of the K199R HRI had no apparent effect on the
morphology or growth rate of 3T3 cells (Figure 2B-D). Similar results
were obtained with overexpression of 10 or 20 HRI (data not shown).
Overexpression of inactive K199R, 10, or 20
HRI does not cause malignant transformation of
NIH 3T3 cells
It has been shown that overexpression of the K296R or 6
inactive mutants of PKR in 3T3 cells results in malignant
transformation.11,13,44 We examined the growth in
monolayer, cloning efficiency, and tumorigenicity of 3 K199R HRI
clones. Each of our K199R HRI-expressing clones exhibited a normal
morphology and had doubling times and saturation densities unchanged
from those of uninfected or vector-only control cells (Table
1). We observed similar results using
pooled K199R, 10, or 20 HRI-expressing 3T3 cells (data not
shown). Cells expressing K199R, 10, or 20 HRI failed to grow on
soft agar (Tables 1 and 2), in contrast
to NIH 3T3 cells expressing the inactive mutant PKR 6, which
exhibited cloning efficiencies of 19.8% ± 0.9% (Table 1). None of
our K199R HRI 3T3 clones developed tumors in athymic
(nu/nu) nude mice within the 10-week
observation period, while all 3 nude mice injected with PKR
6-12 cells developed tumors within 28 to 34 days. The
inability of inactive HRI to transform 3T3 cells is consistent with our
earlier finding that HRI is expressed predominantly in eythroid
cells in contrast to the ubiquitous expression of PKR.
It has been reported that 3T3 cells that overexpressed eIF-4E exhibit a
morphology indicative of a transformed phenotype and grew in soft
agar.45-47 We examined the ability of Wt HRI to inhibit the transformation by eIF-4E and PKR 6. Interestingly, we found that
the cloning efficiency of eIF-4E cells was dramatically reduced by
co-expression with Wt HRI, but not by co-expression with K199R or 20
HRI. Similarly, the cloning efficiency of PKR 6 was also reduced
significantly by co-expression of Wt HRI. These results indicated that
HRI could inhibit protein synthesis in cells transformed by eIF-4E or
PKR 6. The ability of HRI to inhibit protein synthesis in
eIF-4E-overexpressing cells is consistent with the current knowledge
of independent regulation of protein synthesis by eIF-4E and eIF-2. The
ability of Wt HRI to inhibit PKR 6 transformation suggests that PKR
and HRI do not form a heterodimer and that both PKR and HRI function as homodimers.
Expression of inactive mutant HRI increases
hemoglobin production and proliferative capacity of differentiating
MEL cells
Next, we examined the effects of expression of these mutants in
MEL cells in which endogenous HRI resides.35 MEL cells
were infected with amphotropic retroviruses containing mutant HRI cDNA and/or puromycin selection marker. Pooled selected cells were plated in
the absence of puromycin, and erythroid differentiation was induced by
dimethyl sulfoxide (DMSO). The levels of HRI expression before and
after DMSO induction were determined by Western blot analysis. As shown
in Figure 3A, the levels of K199R and
10 HRI expression were increased substantially upon DMSO induction
of MEL cells (lanes 4 and 8 vs lanes 3 and 7). It has been shown previously that the promoter activity of Moloney retrovirus LTR is
enhanced by DMSO induction.48

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| Figure 3.
Expression of inactive mutant HRI increases the and
globin contents of differentiating MEL cells.
(A) Expression of retrovirally transduced HRI in MEL cells. MEL cells
expressing vector alone, K199R HRI, or 10 HRI were plated at
1 × 107 cells per 100-mm plate the night before
treatment with 2% DMSO as indicated. Cells were counted and harvested
on day 5. Cells were lysed at 1 × 108 cells/mL.
Cytoplasmic extracts, equivalent to 2 × 106 cells, were
used for anti-HRI Western blot analysis. Lanes 1 and 2 are uninfected
MEL cell controls; lanes 3 and 4 are K199R-infected cell extracts;
lanes 5 and 6 are 10 HRI-infected cell extracts; lane 7, vector-infected cell extracts. (B) and globin contents of MEL
cells expressing mutant HRI. Lanes 2, 4, and 6 are induced with DMSO.
Cytoplasmic extracts, equivalent to 5 × 105 cells, were
separated by 15% SDS-PAGE and stained with Coomassie blue. The
positions of and globin were marked. Lane 1, rabbit
reticulocyte lysates. Lanes 2 and 3, cell extracts; lanes 4 and 5, K199R HRI-expressing cell extracts; lanes 6 and 7, 10 HRI-expressing
cell extracts; lanes 3, 5, and 7 are treated with DMSO.
|
|
Since hemoglobin is the major protein in erythroid cells, the effects
of forced expression of the dominant-negative HRI mutants on the levels
of and globin chains in differentiating MEL cells were
examined. The contents of and globin were determined by
SDS-PAGE and Coomassie blue staining of the cell extracts from equal
numbers of cells. As shown in Figure 3B, DMSO induction of MEL cells
induced the globin expression in the cells expressing puromycin-resistant gene alone as expected (lane 3 vs 2). Upon DMSO
induction, MEL cells overexpressing K199R or 10 HRI had significantly higher levels of globin chains (lanes 5 and 7 vs lane 3).
Before DMSO induction, there were no apparent changes in the profiles
of protein expressed in MEL cells expressing inactive mutants. This is
perhaps to be expected since the levels of the expression of the mutant
HRI were low before induction.
Since there was an increase in the and globin chains upon
expression of K199R and 10 HRI, the amounts of hemoglobin in differentiating MEL cells were also determined by scanning the visible
spectra of the cell extracts for the characteristic Soret band of
hemoglobin. There was no detectable signal of Soret band without DMSO
induction (Figure 4). Upon DMSO
induction, the peak of Soret band was visible. Overexpression of the
K199R or 10 HRI in MEL cells resulted in a 2- to 3-fold increase in
the hemoglobin content of these cells upon DMSO induction as compared
with uninfected MEL cells or MEL cells expressing the
puromycin-resistant vector control (Figure 4) or Bcl-2 (data not
shown). This increase in hemoglobin synthesis is most likely the result
of the inhibition of the endogenous HRI activity by these inactive HRI
mutants. Thus, the increase of hemoglobin synthesis is the consequence of increase of general protein synthesis.

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| Figure 4.
Hemoglobin contents of differentiating MEL cells
expressing inactive mutant HRI.
The same cytoplasmic extracts described in Figure 3 were diluted 1:5
and then analyzed for the presence of the Soret band of hemoglobin at
414 nm by scanning from 300 to 600 nm as described
previously.29
|
|
It is well established that upon terminal differentiation, cells lose
their ability to divide (proliferative capacity). We have shown above
that expression of Wt HRI in 3T3 cells resulted in the inhibition of
the proliferation of these cells. We therefore asked whether HRI was
involved in the loss of the proliferative capacity of differentiating
MEL cells. The cell numbers of differentiating MEL cells expressing
mutant HRI at days 4 and 5 after DMSO induction were determined and are
shown in Table 3. The cell pellets of K199R and 10 HRI-expressing cells were visibly larger and redder than those of uninfected cells, cells expressing vector alone, or cells
expressing Bcl-2. At both 4 and 5 days after DMSO induction, there was
a significant increase in the number of K199R or 10 HRI-expressing
cells as compared with the number of control uninfected cells or cells
expressing the puromycin-resistant gene only or Bcl-2 (Table 3). In
addition, cell numbers of K199R and 10 HRI-expressing cells
continued to increase substantially from day 4 to day 5 while the cell
number of vector control cells, Bcl-2-expressing cells, or uninfected
cells did not increase much. These observations suggested that K199R
and 10 HRI cells continued to proliferate from day 4 to day 5 after
DMSO induction. Therefore, it appears that HRI may play a role in the
loss of proliferative capacity of differentiating erythroid cells. In
the absence of DMSO, there was no significant change in cell number of
K199R or 10 HRI-expressing cells. This may be due to the low-level
expression of this mutant HRI without DMSO induction as shown
in Figure 3A. Thus, these results demonstrate that overexpression of
K199R or 10 HRI in DMSO-induced MEL cells affects not only the
production of hemoglobin, but also the proliferative capacity of these
cells.
We repeated the experiments described above in Figures 3 and 4 and
Table 3 with a separate batch of MEL cells infected with another batch
of HRI retroviruses. In this experiment, MEL cells were plated at one
fifth of the cell density employed in the previous experiment presented
in Table 3. Under this condition, the loss of the proliferative
capacity of DMSO-treated differentiating MEL cells was observed more
clearly; ie, the cell number of DMSO-treated MEL cells expressing
vector is 55.5% of that of the untreated cells (Figure
5). In contrast to the loss of
proliferative capacity of the control vector-differentiating cells, MEL
cells expressing K199R, 10, or 20 HRI had increased proliferative
capacity, with higher cell numbers upon DMSO-induction as compared with
the untreated cells. The cell-cycle status of these cells was also
analyzed by flow cytometry and is presented in Table
4. At 4.5 days after subculture and in
the absence of DMSO, approximately 20% to 30% of cells were in
S+G2/M phase. Upon DMSO induction for 4.5 days, the
percentage of cells in S+G2/M decreased substantially to
6% to 8%, a 3-fold drop as compared with untreated cells. This
decrease was indicative of loss of proliferative capacity of
differentiating cells. Significantly, the cells expressing K199R or
10 HRI had a higher percentage of cells in S+G2/M. The
20 HRI-expressing cells had only a slightly higher percentage of
cells in S+G2/M, perhaps because these cells had already
reached the saturation density since the number of 20 HRI-expressing
cells was considerably higher than the number of K199R or 10
HRI-expressing cells. Collectively, these results from Tables 3 and 4
and Figure 5 indicate that HRI also regulates the proliferation of
differentiating erythroid cells.

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| Figure 5.
Effects of expression of inactive mutant HRI on the
proliferative capacities of differentiating MEL cells.
Cells expressing vector or mutant HRI as indicated were plated at
2 × 106 cells per 100-mm plate and induced as described
in the Figure 4 legend. Cell numbers were counted on day 4.5 after
induction.
|
|
Recently, it has been reported that there is HRI-like activity in NIH
3T3 cells.25 As shown in Figure 2, a very low-level expression of Wt HRI in NIH 3T3 cells resulted in cell death. However,
expression of inactive mutant HRI alone in nonerythroid 3T3 cells did
not result in any enhancement in cell growth or changes in cell
morphology (Figure 2; Tables 1 and 2). These results strongly suggest
that if there is HRI protein present in 3T3 cells, it is not active
under our experimental conditions. Most important, we observed that
expression of inactive mutant HRI in erythroid MEL cells resulted in
phenotypical changes in these cells, ie, increased hemoglobin content
and proliferative capacity. The levels of expression of these
dominant-negative mutants were comparable in 3T3 and MEL cells (data
not shown). Thus, the most plausible explanation for the observed
phenotypic changes upon expression of dominant-negative mutants in
erythroid cells, but not in non-erythroid cells, is that active HRI is
present in MEL cells but not in 3T3 cells.
 |
Discussion |
In this study, we have examined the consequence of overexpression
of Wt and inactive mutant HRI in nonerythroid and erythroid cells. We
have shown here that forced expression of Wt HRI in 3T3 cells caused an
inhibition of cell growth and ultimately cell death (Figure 2). We were
able to maintain the growth of Wt HRI-expressing 3T3 cells by the
addition of hemin to the culture medium. Upon removal of hemin, Wt HRI
became active, shut off protein synthesis by phosphorylating eIF-2
(data not shown), and killed cells. This lethal effect of
overexpression of Wt HRI in 3T3 cells is similar to that of the
overexpression of PKR in these cells.16 Interestingly, we
found that expression of Wt HRI can inhibit the transformation
phenotype of PKR 6-overexpressing 3T3 cells (Table 2). This finding
indicates that HRI and PKR do not form heterodimers in vivo. Thus, HRI
and PKR act independently of each other and represent different targets
of regulation of protein synthesis in response to different kinds
of stress.
It has been demonstrated that overexpression of inactive mutants of PKR
in NIH 3T3 cells results in the malignant transformation of those
cells.11,13,44 Because PKR is a dimer, it is believed that
inactive PKR protein acts in a transdominant-negative manner to form an
inactive heterodimer with the endogenous PKR.11 However, we found that overexpression of the inactive K199R, 10, or 20 HRI
did not result in any change in the morphology that would be indicative
of malignant transformation. These cells did not grow in soft agar or
form tumors in nude mice. We have previously shown that co-expression
of Wt HRI with K199R HRI in baculovirus-infected insect cells leads to
diminished kinase activity of the Wt HRI.28 This reduced
activity is thought to be the result of the formation of an inactive
HRI heterodimer. We showed similar results here using the inactive HRI
deletion mutants 10 and 20 (Figure 1). Our observation that
forced expression of K199R, 10, or 20 HRI fails to confer upon
NIH 3T3 cells a transformed phenotype is consistent with there being no
active HRI in these cells. These results are in agreement with our
earlier finding that HRI is expressed predominantly in erythroid
cells.35
Recently, it has been reported that HRI mRNA is expressed in NIH 3T3
cells and in nonerythroid tissues.25 They have also partially purified HRI-like activity from young mouse liver (6 to 8 weeks old) and NIH 3T3 cells. It is important to note that fetal liver
is a site of erythropoiesis during development and that erythropoiesis
persists in the liver of the mouse after birth. In addition, NIH 3T3
cells are embryonic in origin. It is to be noted that in these 2 reports no Western blot analysis of HRI in tissues or 3T3 cells has
been performed to establish that HRI protein is indeed present
in nonerythroid cells. We show here that a very low-level expression of
Wt HRI in NIH 3T3 cells (1/100 to 1/500 of rabbit reticulocytes) is
sufficient to inhibit cell growth (Figure 2). These results indicate
that if there is HRI protein present in NIH 3T3 cells, it is probably
not active under our experimental conditions. Recently, 2 more
mammalian eIF-2 kinases have been cloned, the PEK
(PERK)19,20 and the GCN2.49,50 Both of these
eIF-2 kinases are, like PKR, universally expressed.
In contrast to no phenotypical change in 3T3 cells, forced expression
of the inactive mutant HRI in differentiating MEL cells results in
increased hemoglobin synthesis (Figures 3 and 4) and proliferative
capacity of these cells (Tables 3 and 4; Figure 5). These phenotypical
changes not only provide the direct in vivo evidence for the function
of HRI in the regulation of hemoglobin synthesis in erythroid cells,
but also suggest a role of HRI in the regulation of the proliferation
of the differentiating erythroid cells through, most likely, the
inhibition of protein synthesis. However, it is possible that HRI may
have an as yet unidentified substrate other than eIF-2 , and the
phosphorylation of this substrate might be involved in the regulation
of cell proliferation. The regulation of hemoglobin synthesis by HRI in
erythroid cells is the result of its regulation of general protein
synthesis. We have shown recently that HRI is a hemoprotein with 2 distinct heme-binding sites.29, 34 Thus, HRI serves as a
sensor of heme and provides a feedback mechanism to coordinate the
synthesis of globins according to heme concentration in erythroid cells.
In addition to regulating HRI activity, heme also regulates its own
synthesis in reticulocytes at one or more steps prior to the formation
of -aminolevulinic acid, the first intermediate in the heme
biosynthetic pathway.51 Furthermore, inhibition of protein
synthesis in reticulocytes by the addition of cycloheximide or
puromycin also results in the inhibition of heme biosynthesis at the
formation of -aminolevulinic acid.52 These earlier
studies indicate that in the absence of globin synthesis, the
accumulation of heme inhibits heme biosynthesis. Thus, heme coordinates
the synthesis of heme and globins in erythroid cells by regulating HRI
and its own synthesis.
 |
Acknowledgments |
We thank all investigators who provided materials as indicated in
"Materials and methods" and Dr Karen Westerman for providing expertise in the production of retroviruses.
 |
Footnotes |
Submitted January 17, 2000; accepted June 26, 2000.
Supported by National Institutes of Health grants DK-16272 (J.-J.C.)
and HL-55435 (P.L.B).
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 U.S.C.
section 1734.
Reprints: Jane-Jane Chen, E25-545, Massachusetts Institute
of Technology, Cambridge, MA 02139; e-mail: j-jchen{at}mit.edu.
 |
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