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Prepublished online as a Blood First Edition Paper on October 10, 2002; DOI 10.1182/blood-2002-05-1517.
HEMATOPOIESIS
From the Herman B Wells Center for Pediatric Research,
Indiana University School of Medicine, Indianapolis; the Department of
Pediatrics, Indiana University School of Medicine, Indianapolis; the
Department of Biochemistry and Molecular Biology, Indiana University
School of Medicine, Indianapolis; and the Department of Microbiology
and Immunology, Indiana University School of Medicine, Indianapolis.
The cullin family of proteins is involved in the ubiquitin-mediated
degradation of cell cycle regulators. Relatively little is known about
the function of the CUL-4A cullin, but its overexpression in breast cancer suggests CUL-4A might also regulate the
cell cycle. In addition, since other cullins are required for normal development, we hypothesized that CUL-4A is involved in
regulating cell cycle progression during differentiation. We observed
that CUL-4A mRNA and protein levels decline 2.5-fold during
the differentiation of PLB-985 myeloid cells into granulocytes. To
examine the significance of this observation, we overexpressed
CUL-4A in these cells and found that modest (< 2-fold),
enforced expression of CUL-4A attenuates terminal
granulocytic differentiation and instead promotes proliferation. This
overexpression similarly affects the differentiation of
these cells into macrophages. We recently reported that nearly one half of CUL-4A+/ Ubiquitin-mediated degradation plays a critical
role in controlling the turnover of cell cycle
regulators.1,2 The adenosine triphosphate
(ATP)-dependent attachment of ubiquitin to a ubiquitin-activating enzyme (E1) activates ubiquitin for transfer to a ubiquitin-conjugating enzyme (E2) and then to a ubiquitin ligase (E3), which transfers ubiquitin to a substrate protein.3 Repetition of this
ubiquitin transferase reaction results in the attachment of a
polyubiquitin chain to the substrate, which is then recognized by the
26S proteasome and degraded. Much of the ubiquitin pathway's substrate
specificity derives from E3 ligases, so regulating E3 activity is a
likely mechanism for controlling ubiquitin-mediated degradation.
Cullins are a core component of a subset of E3 ligases (described
below). In this report, we examine the function of the
CUL-4A cullin and find it plays a role in regulating
granulocytic differentiation.
Multisubunit RING (Really Interesting New Gene) E3 ligases contain at
least 5 subunits: a RING finger protein, an E2, an adaptor protein, a
substrate recognition subunit, and a cullin.3 An extensively studied type of multisubunit RING E3 is SCF
(Skp1, Cdc53/cullin, F-box
protein), which was initially characterized in Saccharomyces
cerevisiae.1,2 Skp1 (the adaptor protein), Cdc53 (the
cullin), and Rbx1 (the RING finger protein) make up the SCF core. This
core binds an E2 and various F-box proteins (such as Cdc4, Grr1, and
Met30), which are responsible for substrate recognition. The resulting
SCF complexes (SCFCdc4, SCFGrr1, and
SCFMet30) target for degradation a variety of cell cycle
regulators, including Sic1, Far1, Cdc6, Cln1, Cln2, Gic2, and Swe1.
Mammals encode 6 cullins, Cul-1, Cul-2, Cul-3, Cul-4A, Cul-4B, and
Cul-5,4 and these appear to be components of SCFs or related complexes.2 Cul-1 is a component of
SCFSkp2, which is implicated in the ubiquitination of p21,
p27, and E2F-1, and is also a subunit of SCF In vivo studies demonstrate that cullins are required for normal
development. Inactivation of a Caenorhabditis
elegans cullin, cul-1, causes hyperplasia in all
tissues of the developing embryo.4 Mice that are deficient
for CUL-1 fail to develop past 5.5 days after coitus
(dpc),12,13 and CUL-3-null mice fail
to develop past 7.5 dpc.9
Thus, cullins and their E3s are involved in the ubiquitin-mediated
degradation of cell cycle regulators and are required for embryonic
development. Recent findings suggest a similar function for
CUL-4A. CUL-4A mRNA is cell cycle-regulated and
is high during the transition from G1 to
S-phase,14 and it is amplified and/or overexpressed
in breast cancer and in hepatocellular carcinomas, suggesting a role in
regulating cell cycle progression.15,16 DDB2 is
mutated in some xeroderma pigmentosum group-E individuals and is
required for the repair of ultraviolet radiation-damaged DNA,17,18 and the encoded protein is a substrate of a
Cul-4A-containing E3.19-21 Also, CUL-4A was
overexpressed in normal mammary epithelial cells, and after exposure to
ionizing radiation, these cells failed to accumulate with
G2/M DNA content and did not accumulate p53, suggesting
that overexpression of CUL-4A interferes with a
G2/M checkpoint and indicating another function for this
gene.22 However, since the ultraviolet
irradiation-induced arrest at S-phase was not affected by
CUL-4A overexpression, the connection between CUL-4A and DDB2 in these cells remains unclear.
In addition, we recently determined that
CUL-4A Cell culture and differentiation
Generation of anti-Cul-4A antiserum and immunoblots
For immunoblots, cells were lysed for 30 minutes on ice in 50 mM Tris (tris(hydroxymethyl)aminomethane)-hydrochloric acid (HCl), pH 7.5; 10 mM EDTA (ethylenediaminetetraacetic acid); 1% sodium dodecyl sulfate (SDS); 1 mM dithiothreitol (DTT); 2 mM phenylmethylsulfonyl fluoride (PMSF); 15 µg/mL aprotinin; 2µg/mL pepstatin; and 5 µg/mL leupeptin, followed by probe sonication for 10 seconds at 4°C. Protein lysates were cleared by centrifugation at 16 000g at 4°C for 20 minutes, protein concentration was measured by Bradford assay (Bio-Rad, Hercules, CA), 10 or 20 µg total cell lysate was fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and immunoblotting was performed as previously described.27 Antibodies were visualized by enhanced chemiluminescence (NEN Life Science Products, Boston, MA), densitometry was performed with an Eagle Eye II still video system and software (Stratagene, La Jolla, CA), and loading was normalized with respect to actin. Cul-4A was detected with 1:3000 diluted anti-Cul-4A antiserum (described above) followed by 1:10 000 diluted anti-rabbit immunoglobulin G (IgG) antibodies (A-6154; Sigma, St Louis, MO). Monoclonal antibodies for gp91phox diluted 1:5000 (a generous gift from M. Dinauer, Indiana University School of Medicine) were followed by 1:10 000 diluted anti-rabbit IgG antibodies, anti-actin monoclonal antibodies (H5441; Sigma) diluted 1:4000 were followed by anti-mouse IgG antibodies (NA931; Amersham Life Science, Buckinghamshire, United Kingdom) diluted 1:10 000, and the hemagglutinin antigen (HA) epitope tag was detected with horseradish peroxidase (HRP)-conjugated anti-HA antibodies (Roche Biochemicals, Indianapolis, IN). Polyclonal antibodies for c-myc (06-303; Upstate Biotechnology, Lake Placid, NY) or p130 (sc-317; Santa Cruz Biotechnology, Santa Cruz, CA) were diluted to 2 µg/mL and followed by 1:10 000 diluted anti-rabbit IgG antibodies. Northern blot analysis of mRNA Northern blotting was performed using standard methods, except that MagnaGraph nylon membranes were used under conditions suggested by the manufacturer (Osmonics, Westborough, MA). Full-length cDNAs were used to make probes for CUL-4A, gp91phox, -actin mRNA, and 18S rRNA, and all
probes were radiolabeled with random oligonucleotide primers (NEBlot
Kit; New England Biolabs, Beverly, MA) and  -32P
deoxycytidine triphosphate (dCTP). Blots were washed twice with 2 ×
sodium chloride/sodium citrate (SSC), 0.5% SDS at room
temperature for 10 minutes each and twice with 0.1 × SSC, 0.1% SDS
at 55°C for 20 minutes each. Blots were quantified with a
phosphorimager (Storm; Molecular Dynamics, Sunnyvale, CA) or by
autoradiography and an Eagle Eye II still video system (Stratagene),
and loading was normalized with respect to -actin or 18S rRNA. Two
species of CUL-4A mRNA (3.8 and 3.5 kilobase [kb]) were
detected, as previously reported.15
Plasmid constructions and transfections To construct an HA epitope-tagged CUL-4A cDNA, a NotI fragment containing full-length mouse CUL-4A cDNA was inserted into the NotI site of pRc/CMV (Invitrogen, Carlsbad, CA) to generate pRc/CMV-CUL-4A. Using oligonucleotides (mCUL4A 26 [5'-tcagcgacaggatggtgc-3'] and 3'Cul4A-XbaI [5'-gctctagatcagcggccgcttgccacgtagtggtactgatttgg-3']), a polymerase chain reaction (PCR)-amplified fragment was synthesized with a NotI site upstream of the CUL-4A stop codon and subcloned into pRc/CMV-CUL-4A. Into this NotI site, a 111-base pair (bp) NotI fragment that encodes 3 tandem copies of the HA epitope28 was then inserted in frame with the CUL-4A coding sequence to generate pRc/CMV-CUL-4A-HA. The DNA sequence of the PCR-amplified fragment included in the final construct was confirmed by dideoxy sequencing. An XbaI fragment containing this CUL-4A-HA cDNA was then inserted into the XbaI site of pEF-PGKpac,29 which contains a puromycin N-acetyltransferase selectable marker, to generate pEFpac-CUL-4A-HA.PLB-985 myeloid cells were transfected by electroporation as previously described30 with KpnI-linearized pEF-PGKpac-CUL-4A-HA or pEF-PGKpac. Individual clones were selected as previously described,25 except that transfected clones were selected with 2 µg/mL of puromycin. After 2 to 3 weeks of selection, individual clones were screened by probing immunoblots of total lysate with anti-HA antibodies. Morphologic analysis Cytocentrifuge (105 cells per slide, 340 rpm, 5 minutes; Cytospin3, Shandon, Pittsburgh, PA) slide preparations of cells were stained with Diff-Quik Stain Set (Dade Behring, Newark, DE) and viewed with oil immersion optics (original magnification × 40 and × 100). Each morphologic subtype of neutrophil lineage cells was identified on the basis of conventional criteria (cell size, ratio of nucleus to cytoplasm, and characteristics of nuclear chromatin31), and at least 200 cells per slide were scored.Flow cytometric analysis of cell cycle and apoptosis For cell cycle analyses, cells were washed 2 times in phosphate-buffered saline (PBS) and incubated in PBS containing 0.1% TritonX-100 and RNAse A (50 µg/mL) for 30 minutes at 4°C. Propidium iodide (PI; 50 µg/mL) was added, and the cells were incubated for another 30 minutes at 4°C.32 Following staining, samples were analyzed using a FACScan flow cytometer (Becton Dickinson, San Jose, CA) and ModFit LT software. Apoptosis assays were performed essentially as previously described.33 Briefly, cells were harvested, washed once with PBS, and resuspended in 100 µL binding buffer (10 mM HEPES [N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid]/sodium hydroxide [NaOH], pH 7.4; 140 mM sodium chloride [NaCl]; 2.5 mM calcium chloride [CaCl2]). Cells were then stained with 5µL Annexin V-fluorescein isothiocyanate (FITC; Pharmingen, San Diego, CA) and 500 ng PI, and incubated at room temperature for 15 minutes in the dark. Binding buffer (400 µL) was then added prior to flow cytometric analysis using a FACScan flow cytometer. Apoptotic cells were defined as the fraction of Annexin V-positive per PI-negative cells.Hematopoietic progenitor cell assay Bone marrow cells were harvested from 6-week-old CUL-4A+/ mice and wild-type littermate
controls by flushing bone marrow from femurs into 10 mL RPMI 1640 supplemented with 10% fetal bovine serum (FBS; Hyclone Laboratories,
Logan, UT). The cells (25 000 per plate) were plated in progenitor
cell methylcellulose colony assays as previously
described.34 Briefly, the cells were suspended in
triplicate into 10 × 35-mm grid culture dishes (no. 171099; Nunc,
Naperville, IL) with 1.2% methylcellulose (MethoCult M3134; StemCell
Technologies, Vancouver, BC, Canada), 30% FBS, 1% deionized fraction
V bovine serum albumin (Sigma), 104 M -mercaptoethanol
(Sigma), and cytokines (100 ng/mL recombinant murine interleukin 3 [IL-3; Peprotech, Rocky Hill, NJ], 100 ng/mL recombinant human
erythropoietin [Amgen, Thousand Oaks, CA], and 4 U/mL recombinant
murine stem cell factor [Peprotech, Rocky Hill, NJ]), followed
by incubation at 37°C, 5% carbon dioxide [CO2] in a
humidified incubator. After 7 days of incubation colony types were
determined by in situ observation using an inverted microscope and
according to documented criteria.34
CUL-4A expression declines during granulocytic and macrophage differentiation In an attempt to gain insight into CUL-4A function, we examined its expression during the differentiation of PLB-985 cells, a myelomonoblastic human cell line capable of differentiating into granulocytes on induction with 0.5% DMF24 or into macrophages on induction with 100 nM PMA.26 These cells were differentiated into granulocytes for 6 days, and CUL-4A mRNA and protein were measured. CUL-4A mRNA declines approximately 2.6-fold (± 0.60) and the protein decreases about 2.4-fold (± 0.2) (Figure 1A). Two different molecular-weight species were detected by our anti-Cul-4A polyclonal antiserum. The mobility of the lower-molecular weight protein is consistent with the predicted 84 kD for a CUL-4A protein. The higher-molecular weight form is Cul-4A posttranslationally modified by the attachment of a ubiquitin-related peptide, Nedd8, a previously reported modification of Cul-4A and cullins in general.35,36 To show that these cells differentiated into granulocytes, the expression of gp91phox, whose transcription is induced during terminal myeloid differentiation into either granulocytes or macrophages and which is a component of an NADPH oxidase complex,37 was also measured and confirmed to have increased during this time course (Figure 1A).
Similar results were obtained when these cells were differentiated for 3 days into macrophages (Figure 1B), although greater changes in CUL-4A expression were observed. Both CUL-4A mRNA and protein levels declined about 10-fold. Enforced expression of CUL-4A inhibits granulocytic and macrophage differentiation If the down-regulation of CUL-4A expression is required for differentiation into either granulocytes or macrophages, overexpressing this gene might interfere with these differentiation pathways. Cell lines that stably overexpress a CUL-4A cDNA from a constitutive promoter were generated ("Materials and Methods"). To facilitate the distinction between the endogenous and exogenous Cul-4A, the coding sequence for a triple HA epitope was inserted at the 3' end of the CUL-4A coding sequence. By immunoblot analysis with anti-HA antibodies, more Cul-4A-HA is expressed in the CUL-4A-HA2 cell line than in CUL-4A-HA1 cells (Figure 2, top panel). Two immunoreactive bands were detected in each transfected cell line. The lower band corresponds to Cul-4A-HA and the upper band corresponds to HA-tagged Cul-4A with Nedd8. Using anti-Cul-4A antisera to measure total Cul-4A levels (both endogenous and HA-tagged), we found that CUL-4A-HA1 expressed 1.6-fold more Cul-4A than PLB-985 cells transfected with plasmid alone, and CUL-4A-HA2 expressed 1.9-fold more (Figure 2, middle panel). In each CUL-4A-HA cell line, 3 immunoreactive bands were detected. The 2 lower bands exhibit the same mobilities as the 2 detected in untransfected cells. The middle and top bands have the same mobilities as the proteins detected by anti-HA antibodies (B.L. and K.T.C., unpublished result, January 2002). Therefore, it follows that the lowest band corresponds to unmodified Cul-4A, the middle band corresponds to both Cul-4A-HA and Cul-4A modified by Nedd8, and the top band corresponds to Cul-4A-HA modified by Nedd8. Of note, the relative amounts of Cul-4A associated and not associated with Nedd8 appear to have been altered in CUL-4A-HA1 and CUL-4A-HA2 cells compared with control cells, and this altered ratio may have some impact on either endogenous or exogenous Cul-4A activity.38-44
These 3 cell lines were treated with 0.5% DMF, and during a 6-day time
course gp91phox expression was measured to
assess the extent of granulocytic differentiation. As shown in Figure
3A, after 6 days the
gp91phox mRNA level in CUL-4A-HA1
cells reached only 66% (± 7%) of the control cell (transfected with
empty vector only) level, and the level in CUL-4A-HA2 cells
was induced to only 40% (± 4%). Correspondingly, the extent of
gp91phox protein induction is less when
CUL-4A is overexpressed (Figure 3B). The p65 form of
gp91phox is high mannose glycosylated and the
smear at roughly 91 kD is the mature, glycosylated species. Overall,
gp91phox induction is less when more
CUL-4A-HA is expressed. The activity of the NADPH oxidase
complex, of which gp91phox is an essential
component, was also measured in individual cells by colorimetric assay.
Following 4 days of DMF induction, 94.5% (± 0.9%) of the control
cells had detectable levels of oxidase activity (Figure 3C). However,
significantly fewer CUL-4A-HA1 and CUL-4A-HA2
cells (only 74.0% [± 2.6%] and 68.5% [± 5.3%], respectively) exhibited activity. Therefore, enforced expression of
CUL-4A inhibits the gp91phox
induction that occurs during terminal granulocytic differentiation.
To directly measure the extent of granulocytic differentiation in these cell lines, the degree of nuclear segmentation during DMF-induced differentiation was examined. Following 4 days of DMF induction, 91.7% (± 3.0%) of the control cells terminally differentiated into granulocytes (percentage of the total exhibiting band or polymorphonuclear morphology; Figure 3D,Eii). In contrast, only 39.3% (± 2.7%) of the CUL-4A-HA1 cells and 30% (± 3.6%) of the CUL-4A-HA2 cells exhibit these morphologies (Figure 3D,Eiii-iv). Correspondingly, more of the cells overexpressing CUL-4A remain undifferentiated. Only 2% of the control cells exhibit myeloblast or promyelocyte morphology, while 30% of the CUL-4A-HA1 cells and 34% of the CUL-4A-HA2 cells exhibit these morphologies. Hence, enforced CUL-4A expression inhibits granulocytic differentiation. Similar results were observed when CUL-4A-HA2 and control
cells were induced to differentiate into macrophages. These cell lines
were treated with 100 nM PMA, and during a 3-day time course gp91phox mRNA was measured to determine the
extent of macrophage differentiation. After 3 days, the
gp91phox mRNA level in CUL-4A-HA2
cells reached only about 10% of the level observed in control cells,
an even greater attenuation than was observed for granulocytic
differentiation (Figure 4).
Enforced expression of CUL-4A promotes proliferation in cells induced to differentiate into granulocytes As PLB-985 cells terminally differentiate, they exit the cell cycle and cease proliferating. If cells overexpressing CUL-4A differentiate less than controls, perhaps they proliferate more. Focusing on the effect of enforced CUL-4A expression on granulocytic differentiation, we treated the 3 cell lines described above with DMF, and during the following 6 days, cell number in each culture was quantified. As shown in Figure 5A, while control cells increase in number only 4-fold (± 0.2) during this time course, CUL-4A-HA1 cells increase 8-fold (± 0.4) and CUL-4A-HA2 cells increase 10.7-fold (± 0.8). In the absence of DMF, all 3 cell lines increased in number about 25-fold after 6 days in culture, and their growth rates were not significantly different from one another. Therefore, enforced expression of CUL-4A promotes proliferation after induction of differentiation, and the cell line that expresses more CUL-4A exhibits greater proliferation.
Enforced expression of CUL-4A reduces exit from the cell cycle after induction of granulocytic differentiation To measure proliferation by a different method, the fraction of cells in each phase of the cell cycle was measured before and after DMF induction. As shown in Figure 5B, the cell cycle profiles of the control, CUL-4A-HA1, and CUL-4A-HA2 cells are essentially the same before DMF treatment. Four days after induction, the proportion of control cells in S-phase decreased from 55% to only 2%, and the proportion of cells in G0/G1 increased from 34% to 86%. However, of CUL-4A-HA2 cells, 26% were in S-phase and only 49% were in G0/G1 after induction. The CUL-4A-HA1 cells exhibit an intermediate shift, with 13% in S-phase and 77% in G0/G1. Therefore, while a 28-fold decrease occurs in the proportion of control cells in S-phase, only a 2- to 4-fold decrease occurs in cells with enforced CUL-4A expression.Enforced expression of CUL-4A has little effect on apoptosis after induction of granulocytic differentiation Enforced CUL-4A expression might also reduce apoptosis to promote the expansion of PLB-985 cell cultures induced to differentiate. Therefore, apoptosis was measured before and after DMF induction. The 3 PLB-985 cell lines were each induced with DMF, and Annexin V was used to detect the presence of phosphatidylserine on the cell surface of cells undergoing apoptosis. Before induction, there was little or no difference in the proportion of apoptotic control, CUL-4A-HA1, and CUL-4A-HA2 cells (3.6% ± 0.7%, 4.3% ± 0.7%, and 3.4% ± 0.5%, respectively). After 4 days of DMF induction, there was only a small difference between the proportion of apoptotic control and CUL-4A-HA2 cells (4.1% ± 0.2% and 7.6% ± 0.3%, respectively; P = .01) and no significant difference between the control and CUL-4A-HA1 cells (4.3% ± 1.1%). Since after induction to differentiate, the CUL-4A-HA2 cells underwent slightly more apoptosis than control cells, their greater proliferation is not due to a reduction in apoptosis.Enforced expression of CUL-4A alters the expression of p130 and c-myc after induction of granulocytic differentiation Cul-4A was found to promote the ubiquitin-mediated degradation of DDB2.19-21 Therefore, it is likely that Cul-4A is a component of an E3 ligase and that a Cul-4A-containing E3 ligase targets for degradation one or more regulators that promote differentiation and/or inhibit proliferation in PLB-985 cells induced to differentiate into granulocytes. However, there is no direct evidence that DDB2 is required for granulocytic differentiation. Since cullin-containing E3s often target multiple substrates for degradation, and each cullin participates in different E3 complexes, each with distinct substrates,2 it is likely that Cul-4A-containing E3s have additional substrates and that the enforced expression of CUL-4A and inappropriate degradation of one or more of these substrates results in attenuated differentiation and greater proliferation in cells induced to differentiate into granulocytes. These substrates might be one or more positive regulators of granulocytic differentiation45,46 and/or negative regulators of proliferation during granulocytic differentiation, including the p130 pocket protein, p27, STAT-3, and C/EBP.47-53 To begin to test this hypothesis, we
measured the expression of p130 before and after CUL-4A-HA2
and control cells were induced to differentiate into granulocytes. The
expression of c-myc, whose expression must be down-regulated for the
differentiation of myeloid cell lineages, including granulocytes, was
also measured in these cells. As shown in Figure
6, in cells with enforced
CUL-4A expression, the expression pattern of each of these
regulators is altered from that of the control. While p130 expression
was induced in the control cells, this protein was undetectable in CUL-4A-HA2 cells, both before and after induction. Although
c-myc expression declined during the induction of both cell lines, this decline was attenuated in the CUL-4A-HA2 cells.
These results further corroborate the finding that CUL-4A-HA2 cells exhibit attenuated differentiation, continued proliferation, and reduced cell cycle exit after induction with DMF. They are also consistent with CUL-4A functioning upstream of c-myc and p130. However, it remains to be determined whether these effects are direct or indirect. Reduced CUL-4A expression in
CUL-4A+/ mice express about half as much
Cul-4A protein as wild-type mice.23 To investigate whether
this altered CUL-4A expression affects hematopoietic
differentiation in vivo, we analyzed the peripheral blood counts in
these animals but found no significant difference from wild-type
littermates (data not shown). However, culture of bone marrow
hematopoietic progenitors in methylcellulose with recombinant
hematopoietic growth factors revealed that the number of multipotent
progenitor (colony-forming unit-mixed lineage
[granulocyte-erythrocyte-macrophage-megakaryocyte], CFU-GEMM) colonies cultured from
CUL-4A+/ bone marrow was dramatically reduced
(about 3-fold) compared with cultures initiated with bone marrow from
wild-type littermates (Table 1). In
addition, primitive erythroid progenitors (BFU-Es) were reduced to an
even greater extent, more than 5-fold. Still, the numbers of
granulocyte-macrophage progenitor colonies (CFU-GMs) and the total
numbers of progenitor colonies observed were not significantly
different from controls. However, it is important to note that these
measurements were made under basal, unstressed conditions and that
additional differences in hematopoiesis might become apparent between
heterozygous and wild-type mice that have been stressed (eg, by
microbial infection or treatment with a chemotherapeutic
agent).
These results clearly demonstrate that down-regulation of CUL-4A expression is required for granulocytic differentiation and that in cells induced to differentiate into granulocytes, enforced CUL-4A expression instead promotes proliferation. The proportion of cells undergoing apoptosis after induction to differentiate is the same or only slightly greater in cells with enforced CUL-4A expression, so their greater cell numbers (Figure 5A) are not a consequence of a reduction in apoptosis. Therefore, following induction to differentiate, the reduced cell-cycle exit of these CUL-4A-overexpressing cells is consistent with CUL-4A's acting to promote proliferation and/or inhibit differentiation. The molecular mechanism for how CUL-4A overexpression promotes proliferation and reduces granulocytic differentiation remains to be determined. However, our findings that the expression patterns of p130 and c-myc are altered in cells overexpressing CUL-4A are consistent with Cul-4A's acting upstream of these regulators required for granulocytic differentiation. The observation that the amount of c-myc in uninduced CUL-4A-HA2 cells is roughly the same as the amount in control cells (Figure 6) suggests that CUL-4A affects c-myc indirectly. Whether CUL-4A affects p130 expression directly or indirectly and whether this or additional regulators are substrates of a Cul-4A-containing E3 remain to be determined. The finding that CUL-4A plays a role in the regulation of
the cell division cycle in cells induced to differentiate into
granulocytes may be related to our report that CUL-4A is
required for normal mammalian embryonic development.23 We
observed that CUL-4A Although the numbers of multipotential and erythroid progenitors in
CUL-4A+/ We also found that 44% of the expected
CUL-4A+/ We observed a modest reduction (about 2.5-fold) in CUL-4A expression during granulocytic differentiation and found that appropriate CUL-4A expression is essential for regulating granulocytic and macrophage differentiation and for regulating proliferation during differentiation. Since modestly increased CUL-4A expression does not alter the cell cycle distribution in uninduced cells but dramatically alters the cell cycle distribution in cells induced to differentiate, our results show that this CUL-4A cell cycle regulatory function is interconnected with differentiation, a novel finding for mammalian cullins.
The authors wish to thank Merv Yoder, Loren Field, Mary Dinauer, and Dave Skalnik for insightful advice and discussions; Nan Pazdernik for generously helping to characterize the anti-Cul-4A antiserum; and Scott Johnson, Chris Shelley, and Prianto Moeljadi for important technical assistance.
Submitted May 23, 2002; accepted October 3, 2002.
Prepublished online as Blood First Edition Paper, October 10, 2002; DOI 10.1182/blood-2002-05-1517.
Supported by the American Heart Association Midwest Affiliate (9930341Z), the Indiana University Cancer Center (NIH P30CA82709), the Showalter Research Trust, the American Cancer Society (IRG-84-002-14), the Indiana University School of Medicine Core Centers of Excellence in Molecular Hematology (NIH P30DK49218), and the Riley Memorial Association (K.T.C.). A grant from the National Institutes of Health (HL63219) also provided support (D.W.C.).
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: Kristin T. Chun, Wells Research Center, Cancer Research Bldg Room 474, 1044 W Walnut St, Indianapolis, IN 46202; e-mail: kchun{at}iupui.edu.
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© 2003 by The American Society of Hematology.
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  D. L. Waning, B. Li, N. Jia, Y. Naaldijk, W. S. Goebel, H. HogenEsch, and K. T. Chun Cul4A is required for hematopoietic cell viability and its deficiency leads to apoptosis Blood, July 15, 2008; 112(2): 320 - 329. [Abstract] [Full Text] [PDF]
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B. Li, N. Jia, D. L. Waning, F.-C. Yang, L. S. Haneline, and K. T. Chun Cul4A is required for hematopoietic stem-cell engraftment and self-renewal Blood, October 1, 2007; 110(7): 2704 - 2707. [Abstract] [Full Text] [PDF]
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B. Li, N. Jia, R. Kapur, and K. T. Chun Cul4A targets p27 for degradation and regulates proliferation, cell cycle exit, and differentiation during erythropoiesis Blood, June 1, 2006; 107(11): 4291 - 4299. [Abstract] [Full Text] [PDF]
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J. A.F. Marteijn, L. van Emst, C. A.J. Erpelinck-Verschueren, G. Nikoloski, A. Menke, T. de Witte, B. Lowenberg, J. H. Jansen, and B. A. van der Reijden The E3 ubiquitin-protein ligase Triad1 inhibits clonogenic growth of primary myeloid progenitor cells Blood, December 15, 2005; 106(13): 4114 - 4123. [Abstract] [Full Text] [PDF]
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 N. Minegishi, N. Suzuki, Y. Kawatani, R. Shimizu, and M. Yamamoto Rapid turnover of GATA-2 via ubiquitin-proteasome protein degradation pathway Genes Cells, July 1, 2005; 10(7): 693 - 704. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
-TRCP, which
targets for degradation I
B
N306 (encoding
Cul-4A's 453 carboxy-terminal amino acids, approximately 60% of its
full length) in Escherichia coli BL21(DE3) (Novagen;
Madison, WI), as previously described.
; CUL-4A
protein, 
; gp91phox mRNA, 
).
Representative results from 4 independent experiments are shown; SEM is
reported in "Results." (B) PLB-985 cells were induced to
differentiate into macrophages with 100 nm PMA. Northern blot analysis
was used to measure CUL-4A mRNA at each time point.
CUL-4A protein level was measured as described for panel A. The same controls were measured, and the relative amounts of mRNA or
protein were quantified and graphed as described for panel
A.
). 100%
corresponds to the amount of gp91phox mRNA
measured in control cells 6 days after induction. Error bars denote
SEM. For data points with no visible error bars, the error bars are
smaller than the symbol. (B) The 3 cell lines were induced with DMF as
described for panel A, and total protein was isolated from a sample
taken at each time point. Protein (10 µg) from each sample
was electrophoresed, and the amount of gp91phox
protein was determined by immunoblot and normalized with respect to the
amount of actin. The results from a representative experiment are
shown. The symbols are as described for panel A. 100% corresponds to
the amount of gp91phox protein measured in
control cells 6 days after induction. (C) The 3 cell lines were induced
with DMF for 4 days, and cells with oxidase activity were detected by
their ability to reduce nitro blue tetrazolium to generate violet
formazan granules. Cells with oxidase activity were counted and the
results from 3 independent experiments were graphed. Error bars
represent SEM, which is reported in the text. Comparing either
CUL-4A-HA1 or CUL-4A-HA2 and the control,
P < .01 by Student t test. (D) The 3 cell
lines were induced with DMF as described above and slide preparations
were stained with Diff-Quick Stain. Cells with band or
polymorphonuclear nuclear morphology were counted, and the results of 3 independent experiments were graphed. Error bars represent SEM,
which is reported in the text. Comparing either CUL-4A-HA1
or CUL-4A-HA2 and the control, P = .0002 by
Student t test. (E) Fields representative of 3 separate
experiments graphed in panel D were photographed (magnification × 40
and × 100 for inset images) and appear as follows: (i) empty vector
control cells before DMF addition, (ii) empty vector control cells 4 days after induction, (iii) CUL-4A-HA1 cells 4 days after
induction, and (iv) CUL-4A-HA2 cells 4 days after induction.
Arrows in panel i point to examples of cells with undifferentiated cell
morphology, while those in panel ii show differentiated
cells.
mice results in reduced erythroid and multipotential
progenitors