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Blood, Vol. 93 No. 4 (February 15), 1999:
pp. 1178-1188
Lineage-Restricted Expression of Protein Kinase C Isoforms in
Hematopoiesis
By
Alessandra Bassini,
Giorgio Zauli,
Giovanni Migliaccio,
Anna Rita Migliaccio,
Massimiliano Pascuccio,
Sabina Pierpaoli,
Lia Guidotti,
Silvano Capitani, and
Marco Vitale
From the Institute of Histology and General Embryology, University of
Bologna, Bologna, Italy; the Human Anatomy Section, Department of
Morphology and Embryology, University of Ferrara, Ferrara, Italy; the
Laboratory of Cell Biology, Istituto Superiore di Sanità, Roma,
Italy; the Human Anatomy Section, Department of Biomedical Sciences and
Biotechnology, University of Brescia, Brescia, Italy; and the Institute
of Cytomorphology NP CNR, Bologna, Italy.
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ABSTRACT |
The pattern of expression of several protein kinase C (PKC) isoforms
( ,  I,  ,  ,  , and  ) during the course of
hematopoietic development was investigated using primary human
CD34+ hematopoietic cells and stable cell lines subcloned
from the growth factor-dependent 32D murine hematopoietic cell line.
Each 32D cell clone shows the phenotype and growth factor dependence characteristics of the corresponding hematopoietic lineage. Clear-cut differences were noticed between erythroid and nonerythroid lineages. (1) The functional inhibition of PKC- in primary human
CD34+ hematopoietic cells resulted in a twofold increase
in the number of erythroid colonies. (2) Erythroid 32D Epo1 cells
showed a lower level of bulk PKC catalytic activity, lacked the
expression of and PKC isoforms, and showed a weak or absent
upregulation of the remaining isoforms, except I, upon readdition
of Epo to growth factor-starved cells. (3) 32D, 32D GM1, and 32D G1
cell lines with mast cell, granulo-macrophagic, and granulocytic
phenotype, respectively, expressed all the PKC isoforms investigated,
but showed distinct responses to growth factor readdition. (4) 32D Epo
1.1, a clone selected for interleukin-3 (IL-3) responsiveness from 32D
Epo1, expressed the isoform only when cultured with IL-3. On the
other hand, when cultured in Epo, 32D Epo1.1 cells lacked the
expression of both and PKC isoforms, similarly to 32D Epo1. (5)
All 32D cell lines expressed the mRNA for PKC-, indicating that the
downmodulation of the isoform occurred at a posttranscriptional
level. In conclusion, the PKC isoform expression during hematopoiesis
appears to be lineage-specific and, at least partially, related to the
growth factor response.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
THE INTERLEUKIN-3 (IL-3)-dependent 32D
cell line1 is multipotent in that factor-dependent
subclones have been obtained under selective culture conditions in
vitro.2 Subclones responsive to and dependent for growth
upon granulocyte-macrophage colony-stimulating factor (GM-CSF) 32D GM1,
granulocyte colony-stimulating factor (G-CSF) 32D G1, and
erythropoietin (Epo) 32D Epo1 have been isolated.2 Each of
these subclones shows a specific phenotype. Parental 32D and the
subclones 32D GM1, 32D G1, and 32D Epo1 have predominantly mast cell,
granulo-macrophagic, granulocytic, and erythroid features, respectively. 32D Epo1.1 is an IL-3-dependent revertant of 32D Epo12,3 that shows the ability to proliferate in the
presence of IL-3 or Epo. Whereas 32D G1 cells are strictly dependent on G-CSF for their growth, as 32D Epo1 from Epo, 32D GM1 can be cultured equally well in GM-CSF or IL-3.
All of these cell lines, which have the same diploid genotype and do
not induce tumors when injected into syngeneic recipients, have the
features of the lineages characteristic of the growth factors they are
supported by.2,3 The most terminally differentiated cell
lines (the G-CSF- and Epo-dependent subclones) cannot be interconverted, ie, Epo-dependent subclones will not give rise to
G-CSF-dependent clones and vice versa. Thus, the lineage commitment of
the cells on which the relevant growth factors work appears univocally
determined, although 32D Epo1 cells can give rise with low frequency to
IL-3-dependent revertants (such as 32D Epo1.1).4 Therefore, these cell lines represent an optimal model to explore the
relationships existing between a given hematopoietic phenotype and the
effect of a specific growth factor.
The intracellular signal transduction pathways elicited by the
different hematopoietic growth factors is now delineated in some
details.5 An important role in mediating at least some of
the pleiotropic effects of hematopoietic growth factors on the
survival, growth, and differentiation of their target cells is thought
to be played by members of the protein kinase C (PKC) family of
serine/threonine kinases.6 PKC enzymes are well-conserved among species, participate in signal transduction in many cells, and mediate a wide number of intracellular functions. At least 12 different isoforms of PKC have been characterized so
far,7,8 and these have been grouped into three categories
based on Ca2+ requirement for activation and phorbol ester
binding activity. Conventional PKCs ( , I, II, and  )
are Ca2+-dependent phorbol ester receptor/kinases;
novel PKCs (,  ,  , and ) are Ca2+-independent
phorbol ester receptor/kinases; and atypical PKCs (,  ,  , and
µ) are kinases independent of both Ca2+ and phorbol
esters. Most PKC isoforms exist in an inactive form in the cytosol of
resting cells, with an amino-terminal pseudosubstrate sequence
occupying the active site. The generation of diacylglycerol (DAG)
causes redistribution of conventional and novel PKC isoforms to the
membrane by binding the regulatory domain of the kinase. It is believed
that ceramide plays a similar role in the activation of the atypical
PKC- isoform.9
The primary aims of this study were to compare the presence and
distribution of various PKC isoforms in hematopoietic cells of
different lineages and to evaluate how their expression is affected by
the lineage-specific growth factors.
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MATERIALS AND METHODS |
Primary human CD34 hematopoietic progenitor cells.
Informed consent to the study was obtained according to the Helsinki
declaration of 1975 from six normal donors. Mononuclear cells were
isolated from leukapheresis units by Ficoll-Paque (d = 1.077 g/mL;
Pharmacia, Uppsala, Sweden) and adherence-depleted overnight. After
removal of adherent cells, CD34+ cells were isolated using
a magnetic cell sorting program Mini-MACS (Miltenyi Biotec, Auburn, CA)
and the CD34 isolation kit in accordance with the manufacturer's
recommendations. The purity of CD34-selected cells was determined for
each isolation by flow cytometry using a monoclonal antibody that
recognizes a separate epitope of the CD34 molecule (HPCA-2; Becton
Dickinson, San Jose, CA) followed by a goat antimouse IgG directly
conjugated to fluorescein (GAM-FITC). CD34+ cells averaged
about 90% to 98%. Freshly isolated CD34+ cells were
either subjected to Western blot analysis or assayed in semisolid
culture for clonogenic progenitors.
Assay for primary human clonogenic cells.
CD34+ cells (105) were seeded in 300 µL of
medium containing 75 µg of an inhibitor peptide
(H-Glu-Ala-Val-Ser-Leu-Lys-Pro-Thr-OH; Calbiochem, La Jolla, CA) that
specifically blocks the translocation of PKC- from the cytosol to
the cell membrane for 2 hours at room temperature. This procedure was
slightly modified with respect to the original description, in which
the peptide inhibitor was used in cardiomyocytes.10 In
particular, the transient permeabilization step with saponin or other
detergent was avoided, because it resulted in being extremely toxic for
primary hematopoietic cells. Moreover, the time of exposure of
CD34+ cells to the peptide was prolonged to 2 hours before
performing semisolid cultures. A myelin basic protein peptide substrate
(H-Ala-Pro-Arg-Thr-Pro-Gly-Gly-Arg-Arg-OH; Calbiochem) was used as a
control peptide. Colony assays for erythroid (burst-forming
unit-erythroid [BFU-E]) and granulocyte-macrophage (colony-forming
unit-granulocyte-macrophage [CFU-GM]) were performed in
serum-free fibrinclot cultures as previously described.11 Briefly, 10,000 CD34+ cells were seeded in Iscove's
modified Dulbecco's medium (IMDM), supplemented with 300 µg/mL
iron-saturated transferrin, 3 mg bovine serum albumin (BSA), 280 µg/mL CaCl2, 10 4 mol/L BSA-adsorbed
cholesterol, 20 µg of L-asparagine, 1.7 × 106 mol/L
insulin, nucleosides (10 µg/mL each), 0.1 mL of 0.2% (wt/vol) purified fibrinogen resuspended in phosphate-buffered saline (PBS), and
0.1 mL of 0.2 U/mL purified human thrombin (95%) in PBS. All reagents,
except fibrinogen, which was provided by Kabi (Stockholm, Sweden), were
purchased from Sigma (St Louis, MO).
Pure erythroid and mixed granulocyte-macrophage colonies were
identified in situ according to standard morphological criteria at the
day of maximal growth (12 to 14 days). Whereas BFU-E were readily
detectable for the presence of hemoglobin, for the identification of
CFU-GM, fibrinclots were fixed and stained with Wright-Giemsa.
Cell lines and cytokines.
The murine 32D cell line and its subclones were maintained at 37°C
in a 5% CO2 atmosphere and split biweekly in Kircade
medium supplemented with 15% horse serum (Defined HS; HyClone, Logan, UT) plus 15% fetal calf serum (Defined FCS; HyClone) plus
1% (vol/vol) conditioned medium of a CHO line transfected with the
murine IL-3 gene (CHO; Genetics Institute, La Jolla, CA;
32D, 32D GM1, and 32D Epo1.1), 100 ng/mL of recombinant human G-CSF
(Genzyme, Cambridge, MA; 32D G1), and 1.5 U/mL recombinant human Epo
(Globuren, Dompè, France; 32D Epo1 and 32D Epo1.1), as previously
described.2,3 Cells were kept at an optimal cell density
comprising between 0.05 and 0.9 × 106/mL.
In the experiments aimed to evaluate the effect of specific growth
factors on PKC isoform expression, cells were first cultured for 4 hours in medium deprived of growth factors. At this time point of
growth factor starvation, the number of apoptotic cells was constantly
less than 10%, whereas longer periods of starvation induced a
significant increase of apoptosis that interfered with the subsequent
preparation of cell homogenates and Western blot analysis.12 After readdition of the growth factors, the
cells were collected at 15, 30, and 60 minutes.
In some experiments, 2 × 105 32D cells were
pretreated with 75 µg of the PKC- inhibitor or control peptide
(Calbiochem) in 300 µL of Kircade for 2 hours at room temperature and
then cultured in the presence of IL-3 or Epo, as described above. The
total number of viable cells was scored at days 4 (IL-3) and 7 (Epo) of culture.
Preparation of cell homogenates and fractions.
Cell homogenates were prepared as previously described.13
Freshly isolated human CD34+ cells and 32D cell lines were
initially washed twice with cold (4°C) medium, resuspended in a
lysis buffer (10 mmol/L Tris-HCl, pH 7.4, 10 mmol/L NaCl, 2 mmol/L
MgCl2, 5 µg/mL leupeptin, 1 µg/mL aprotinin, 10 µg/mL
soybean trypsin inhibitor, 1 mmol/L phenylmethylsulfonyl fluoride, 0.1 mmol/L sodium orthovanadate, and 20 nmol/L okadaic acid, all from
Sigma) for 30 minutes at 4°C and then passed through a 22-G needle
and centrifuged for 10 minutes at 1,000 rpm at 4°C. Pellets
containing predominantly nuclei were discharged. Supernatants containing most membrane and cytoplasmic proteins were subjected to
Western blot analysis.
In some cases, fractionation experiments were performed following a
previously described technique.14 32D cells (8 × 106/experimental point) were incubated for 4 hours in the
absence of IL-3. During the last 2 hours of starvation, cells were
supplemented with 1 mg of the PKC- inhibitor or the control peptide
and then supplemented with 1% IL-3 for 30 minutes. Cells were then
harvested in 100 µL of lysis buffer without Triton X-100, sonicated,
and centrifuged at 100,000g for 60 minutes at 4°C.
The supernatants were collected as the cytosolic or soluble fractions.
The pellets were solubilized in 100 µL of lysis buffer
containing 0.1% Triton X-100 and centrifuged again at 100,000g
for 1 hour at 4°C. These supernatants were harvested as the
membrane fractions.
Anti-PKC isoform-specific antibodies and Western blot analysis.
Rabbit peptide-specific antibodies (IgG fraction) that recognize PKC
, I, , , , and isoforms were from Santa Cruz
Biotechnologies (Santa Cruz, CA). Equal amounts of proteins, determined
by Bradford assay (Bio-Rad Labs, Munchen, Germany) were diluted 1:3
with 4× loading buffer (250 mmol/L Tris-HCl, pH 6.8, 20%
-mercaptoethanol, 40% glycerol, 8% Na deoxycholate, and 0.003%
bromophenol blue) and subjected to sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 7.5% gel.
Gels were then transferred to nitrocellulose membrane by Trans-Blot
(Bio-Rad). SDS-PAGE and transfer were performed according to the
manufacturer's recommendations. Equal loading of each fraction was
verified by staining duplicate gels with Coomassie Brilliant Blue R-250
(Sigma). The membranes were incubated in PBS, supplemented with 3%
(wt/vol) BSA (Sigma) for 30 minutes at room temperature, and then
incubated overnight at 4°C with anti- (dilution,
1:500), -I (dilution, 1:500), - (dilution, 1:50), -
(dilution, 1:250), - (dilution, 1:125), and - (dilution, 1:500)
isoform-specific antibodies. After four washings with PBS/0.1% Tween
20/0.1% BSA, peroxidase-conjugated goat antirabbit IgG antibody (1:1,500; Cappel, Durham, NC) was applied to the membrane for 60 minutes at room temperature. Bound antibody was visualized by the ECL
Western blotting detection reagents and Hyperfilm-ECL luminescence
detection film (Amersham Corp, Arlington Heights, IL). Semiquantitative
densitometric analysis was performed with an imaging densitometer
(Model GS 670; Bio-Rad) using the Molecular Analyst software (Bio-Rad).
The following criteria were used to confirm that the immunostaining was
specific for the monitored enzymes.13 No immunostaining was
obtained by antibodies preadsorbed with 1 mg of the corresponding immunizing peptide. When the blots were stripped, reblocked, and reprobed with antibodies, which had been previously incubated 1:1 for
10 minutes with the corresponding immunizing peptide (1 mg/mL), the
specific peptide blocked immunoreactivity.
Assay of PKC activity.
Cells were washed twice with cold (4°C) PBS and directly
resuspended in a lysis buffer (20 mmol/L Tris-HCl, pH 7.4, 137 mmol/L NaCl, 10% glycerol, 0.1% SDS, 0.5% Na deoxycholate, 1% Triton X-100, 2 mmol/L EDTA, 1 mmol/L phenylmethylsulfonyl fluoride, plus
inhibitors of proteases and phosphatases) for 20 minutes. One hundred
micrograms of proteins were incubated at 37°C for 10 minutes in a
reaction mixture containing 50 mmol/L Tris-HCl, pH 7.4, 5 mmol/L
MgCl2, 0.3 mmol/L dithiothreitol, 100 µmol/L sodium
orthovanadate, 100 µmol/L CaCl2, 5 µg/mL DAG, 100 µg/mL phosphatidylserine, 13 µmol/L ATP, and 1 µCi of
(32P)ATP in a final volume of 50 µL. PKC activity was
assayed by measuring the incorporation of 32Pi
into a serine substituted peptide corresponding to amino acids 19 through 36 of PKC (UBI, Lake Placid, NY). The reactions were stopped
with an appropriate volume of 4× loading buffer, boiled 5 minutes, and electrophoresed on 18% PAGE. The gels were stained with
Coomassie R-250, destained, and autoradiographed on Kodak X-OMATS S
films (Eastman Kodak, Rochester, NY). Under these
conditions, the peptide was clearly separated and migrated to the
bottom of the gel according to the calculated molecular weight (2.342 kD, dephosphorylated form). The peptide spots were excised and
radioactivity was counted in a liquid scintillation counter.
RNA isolation, reverse transcription, and polymerase chain reaction
(PCR) amplification.
Total RNA was prepared with the commercial (Trizol; GIBCO, Paisley, UK)
guanidine thiocyanate/phenol protocol15 and reverse transcribed (1 µg RNA/reaction) at 42°C for 30 minutes in 20 µL of 10 mmol/L Tris-HCl, pH 8.3, containing 5 mmol/L MgCl2, 1 U RNAse inhibitor, dNTPs (200 µmol/L each), 2.5 U cloned Moloney murine leukemia virus reverse transcriptase, and 2.5 µmol/L random hexamers (all from Perkin-Elmer, Norwalk, CT). An aliquot of the cDNA
(2.5 µL corresponding to 125 ng of total RNA) was dissolved in 10 mmol/L Tris-HCl, pH 8.3, containing 2 mmol/L MgCl2, dNTPs (200 µmol/L each), 2 U AmpliTaq DNA polymerase (final volume, 100 µL), and the sense and antisense primers (100 nmol/L each) specific
for each isoform of human PKC.16 As an internal control of
the PCR amplification, the primers specific for the -actin cDNA (50 nmol/L each) were added to each tube after the first 20 cycles of
amplification as described.17 The amplification reactions were performed in a PTC-100 Thermocycler (MJ Research Inc,
Watertown, MA) with the following parameters: denaturing for 60 seconds
at 95°C, annealing for 60 seconds at 60°C, and primer extension
for 60 seconds at 72°C. Two positive controls (whole human cord
blood and murine bone marrow cDNA) and a negative control
(H2O) were included in each amplification. The
amplification products were separated on standard 1.5% agarose gel and
evidentiated by ethidium staining. All the procedures were performed
according to standard protocols.18
Statistical analysis.
The results were expressed as means ± standard deviations (SD) of
three or more experiments performed in duplicate. Statistical analysis
was performed using the two-tailed Student's t-test for unpaired data.
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RESULTS |
Enhancing effect of a PKC- peptide inhibitor on the
development of human erythroid colonies and Epo-dependent 32D cells.
The pattern of PKC isoform expression was evaluated by Western blot
analysis in primary human CD34+ hematopoietic progenitor
cells (Fig 1). Among the PKC isoforms analyzed,
CD34+ cells showed a specific expression of isoforms
belonging to classical ( and I), novel (, , and ), and
atypical () groups. Because previous studies performed on both human
and murine erythroleukemic cells have shown that modulation of PKC-
may play a prominent role in erythroid maturation,19-21
primary CD34+ cells were pretreated with a recently
described inhibitory peptide, which specifically blocks PKC-
activation,10 for 2 hours at room temperature and then
seeded in semisolid culture. At the end of the incubation period, a
twofold increase (P < .01) in the yield of erythroid colonies
was observed in the presence of PKC- inhibitory peptide, but not in
the presence of a chemically similar control peptide
(Fig 2A). It is noteworthy that the PKC- inhibitory
peptide was unable to induce any modification in the number of
nonerythroid colonies, independently of the cytokine combination added
in culture (Fig 2A).
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| Fig 1.
Western blot analysis of PKC-, I , , , ,
and isoforms in homogenates obtained from freshly isolated
CD34+ cells. Isoform-specific antibodies were added to
the membranes in the absence (left of each couple lanes) or presence of
the corresponding immunizing peptide (right of each couple of lanes). A
representative of three separate experiments is reported.
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| Fig 2.
Effect of PKC- inhibitory peptide on the growth of
erythroid (BFU-E) and granulo-macrophagic (CFU-GM) colonies (A) and the
translocation of PKC- to the membrane fraction (B). In (A), data are
expressed as the means ± SD of three separate experiments performed
in triplicate. A statistically significant (*P < .01)
increase in the number of BFU-E was noticed in the presence of the
PKC- inhibitory peptide. In (B), Western blot analysis of PKC- in
the fractionated lysates of untreated and IL-3-treated 32D cells
cultured in the presence or absence of PKC- inhibitory peptide or
control peptide. S, soluble fractions. M, membrane fractions.
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Parallel experiments were performed on the multipotent 32D cell line,
which shows the ability to give rise to factor-dependent subclones in
the presence of specific cytokines.1 When 2 × 105 32D cells were exposed to the PKC- inhibitory
peptide for 2 hours and then seeded in liquid culture in the presence
of IL-3 or Epo, the total number of viable cells scored in
IL-3-containing cultures at various days of culture was similar in the
presence or absence of the PKC- inhibitory peptide (27.6 × 105 v 28 × 105, respectively, at
day 4). On the other hand, the total number of viable cells scored in
Epo-containing cultures was significantly (P < .01) higher in
the presence than in the absence of the PKC- inhibitory peptide (190 × 104 v 6.4 × 104,
respectively, at day 7). Furthermore, the specificity of the PKC-
inhibitory peptide was formally demonstrated in experiments performed
on parental 32D cells, in which the amount of PKC- translocated to
the membrane in response to 30 minutes of treatment with IL-3 was
significantly reduced in the presence of the PKC- peptide inhibitor
but not in the presence of the control peptide (Fig
2B).
Selective expression of PKC isoforms in various hematopoietic
lineages.
Because the previous data strongly suggest that the modulation of
PKC- activity is an important step for erythroid development, the
next groups of experiments were performed using, as a model system, the
32D, 32D GM1, 32D G1, and 32D Epo1 murine cell clones (Fig 3A through D). A specific expression pattern of PKC
isoforms was observed in the lineage-restricted 32D cell lines. PKC
isoforms , I, , , , and were all expressed in the
mast cell (Fig 3A), granulo-monocytic (Fig 3B), and granulocytic (Fig
3C) lineages. The erythroid cell lines only expressed , I, ,
and , but not and (Fig 3D).
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| Fig 3.
Western blot analysis of PKC-, I, , , ,
and isoforms in homogenates obtained from 32D (A), 32D GM1 (B), 32D
G (C), and 32D Epo1 (D) cell lines. Cells were examined before (lane 1)
and after 4 hours of serum-starvation (lanes 2 through 5) in the
absence (lane 2) or presence of the specific growth factor that was
readded for 15 (lane 3), 30 (lane 4), and 60 (lane 5) minutes. The
positive control was represented by a rat brain homogenate (lane 6). A
representative of five separate experiments is reported.
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Because most PKC isoforms show similar substrate specificities,
reflecting the conservation of the substrate binding site,6 we next performed an assay using a serine substituted peptide of the
amino-terminal region of PKC to investigate the whole PKC catalytic
activity in cell homogenates obtained from the various 32D subclones
(Fig 4). Although some variability in the
basal PKC catalytic activity was noticed among 32D cell lines, 32D Epo1 showed markedly lower levels with respect to all the nonerythroid cell
lines investigated. The absence of and isoforms likely accounts
for the lower levels of PKC catalytic activity observed in erythroid
cell lines in comparison with the other cell clones (Fig 3). In this
respect, we have previously demonstrated the existence of a strict
positive correlation between the amount of a given isoform observed at
Western blot analysis and the catalytic activity immunoprecipitated by
the same isoform-specific antibody.13
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| Fig 4.
Assay of PKC catalytic activity in homogenates obtained
from various 32D subclones. Data are reported as the means ± SD of
three separate experiments performed in duplicate.
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Effects of growth factor starvation and readdition on PKC isoform
expression.
The various 32D cell clones are dependent for proliferation and
survival on the continuous presence of the specific growth factor.2 In preliminary experiments, we found that, after 4 hours of incubation at 37°C in medium without growth factor, about 90% of the cells were still Tripan Blue negative and were able to
respond to the readdition of the specific growth factor. The cell lines
were next incubated for 4 hours in the absence of growth factors, after
which each sample was divided into aliquots and the growth factors were
readded. The amount of the PKC isoforms was quantified at time 0 (after
4 hours of growth factor deprivation) and at 15, 30, and 60 minutes
from the readdition of the growth factor.
In the 32D parental cell line, all the PKC isoforms were decreased
after the starvation and slowly recovered to the initial values after
60 minutes of incubation with the growth factors, with the fastest
isoform to return to normal levels being the PKC I at 15 minutes
(Figs 3A and 5A).
In the 32D GM1 cell line, all but the isoform were reduced after
the starvation. The isoform was only marginally reduced and
returned to the normal level in about 15 minutes of culture with the
growth factor. The isoform , I, , and were strongly decreased, and only the I recovered to the initial level after 60 minutes of incubation with the growth factor (Figs 3B and 5B).
In the 32D G1, the amounts of the isoforms , , and were not
modified after the starvation. The concentration of isoform was
decreased and did not recover after 60 minutes, whereas the
concentrations of isoform I and both returned to normal values
after 15 minutes of incubation (Figs 3C and 5C).
In the 32D Epo1, all of the isoforms present (, I, , and
) were decreased after the starvation and did not return to the normal level after the reincubation with the growth factor, at least
for the time investigated (Figs 3D and 5D).
Growth factor-dependent modulation of PKC- in 32D Epo1.1 cells.
To ascertain whether the pattern of PKC isoform expression was
modulated by growth factors, besides being dependent on the specific
phenotype, we used as a model system the 32D Epo1.1 revertant cell
clone (Fig 6A and B). This cell line was
derived from the 32D Epo1 by selection with IL-3 in serum-free medium
and can be grown alternatively in medium containing IL-3 or
Epo.4 The pattern of isoform expression in the steady-state
culture of 32D Epo1.1 cells supplemented with Epo was not modified with
respect to the parental 32D Epo1. When the 32D Epo1.1 cells were grown in the presence of IL-3, the only major modification regarded the isoform, which was absent in Epo- but present in IL-3-supplemented cultures (Figs 6 and 7).
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| Fig 6.
Western blot analysis of PKC-, I, , , ,
and isoforms in homogenates obtained from 32D Epo1.1 cells cultured
in Epo (A) or IL-3 (B). Cells were examined before (lane 1) and after 4 hours of serum-starvation (lanes 2 through 5) in the absence (lane 2)
or presence of the specific growth factor that was readded for 15 (lane
3), 30 (lane 4), and 60 (lane 5) minutes. The positive control was
represented by a rat brain homogenate (lane 6). A representative of
four separate experiments is reported.
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| Fig 7.
Time-course analysis of the effect of growth factor
readdition on PKC isoform expression in 32D Epo1.1 cells cultured in
Epo (A) or IL-3 (B). Semiquantitative densitometric analysis on Western
blot experiments shown in the legend of Fig 6 is reported. The values
represent the means ± SD of five separate experiments. ( ) ;
( ) I; ( ) ; ( ) ; ( ) ; ( ) .
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To further evaluate the regulation of PKC- during erythroid
differentiation, we have also analyzed the expression of PKC- and
- mRNA in all 32D cell lines (Fig 8).
Both 32D Epo1 and 32D Epo1.1 cells, either cultured in IL-3 or Epo,
expressed detectable levels of PKC- and - mRNA by reverse
transcriptase-PCR (RT-PCR). Although not quantitative,
these data suggest that PKC- downregulation in erythroid cell lines
occurs at the posttranscriptional level.
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| Fig 8.
RT-PCR analysis of the expression of PKC- and -
mRNA in the subclones of the 32D cell lines. The ethidium bromide
staining of products amplified by 30 cycles of PCR with primers
specific for the PKC- and - or for murine actin and separated by
electrophoresis on agarose gel are presented. Comparable amounts of
products of the expected molecular weights were amplified not only from
cDNA obtained from human cord blood (last lane) and mouse bone marrow
(first lane) mononuclear cells, as controls, but also from cDNA
obtained from all subclones of the 32D cell lines. The specificity of
the PKC- band amplified from the 32D Epo cell line was proved by
direct sequencing of the band eluted from the gel.
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| |
DISCUSSION |
The complexity of hematopoiesis arises from the requirement for highly
regulated progression through successive stages involving commitment of
pluripotent hematopoietic stem cells to specific cell lineages,
terminal differentiation of lineage-restricted progenitors, and growth
arrest with production of functional end cells with a defined life
span, which must be constantly replenished.22 Hematopoiesis
is under control of soluble hematopoietic cytokines, which are capable
of stimulating the proliferation and enhancing the survival of the
appropriate hematopoietic progenitor cells.5 However, the
mechanisms by which cytokines promote hematopoietic differentiation
remain controversial. Two general models for the role of cytokines in
controlling hematopoietic differentiation have been proposed. In the
instructive model,23 cytokines transmit specific signals to
multipotent hematopoietic cells directing lineage commitment. In the
stochastic model,24 cytokines only support the
proliferation and survival of lineage-committed cells. A major
distinction between these two models is that in the instructive model
the cytokine receptors are transmitting specific lineage commitment
signals. Recently, an hybrid model has been proposed,25 using the 32Dcl3 mouse hematopoietic cell line that is capable of
differentiating into granulocytes in response to G-CSF. When 32Dcl3
cells were transfected with the antiapoptotic genes bcl-2 or
bcl-Xl, some features of granulocytic differentiation, namely nuclear segmentation, appear to be preprogrammed and did not require G-CSF, whereas the complete maturation of 32D cells to granulocytes, involving the induction of myeloperoxidase activity, was dependent on
G-CSF. Thus, the regulation of lineage commitment is, at least in part,
extrinsic: the growth factors in which the cells are cultured can
influence the type of mature cells formed.
Most hematopoietic growth factors are able to activate common signaling
pathways: increased intracellular pH, which appears to be required for
the survival of hematopoietic progenitors with suppression of
programmed cell death26,27 and activation of the
Ras/Raf/MAPK pathway.28 However, what is still not clear is
how growth factors elicit varied developmental responses in hematopoietic progenitors. Candidate intracellular regulatory molecules, which may be involved in the development of hematopoietic cells, are members of the serine- and threonine-specific PKC
family.13,19-21,29-33 Because most of previous studies used
transformed or leukemic cell lines that were induced to differentiate
by pharmacological agents, their physiological implications for normal
hematopoiesis remain to be determined. To further address the role of
the different PKC isoforms in hematopoiesis, we used primary human
CD34+ cells and cell lines subcloned from the 32D cell
line, a pluripotent murine myeloid cell line, that has a diploid
karyotype. 32D cells were originated by a bone marrow long-term culture
in the presence of murine IL-31-3 and are widely used as a
model for myeloid progenitor cells also to study signal transduction
pathways.34
A potentially important role for PKC- downmodulation in the process
of erythroid maturation was suggested in experiments performed on
primary human CD34+ cells that gave rise to a significant
higher number of erythroid colonies in the presence of a specific
PKC- peptide inhibitor. Similarly, the total number of parental 32D
cells cultured in Epo for 7 days, showing the potentiality to give rise
to Epo-dependent subclones, was significantly higher in the presence of
the PKC- inhibitory peptide. Moreover, analysis of the cytoplasmatic
levels of the PKC isoforms in murine 32D cell lines showed that the and isoforms were absent in the erythroid lineage (32D Epo1) and
present in the granulomonocytic (32D, 32D GM1, and 32D G1).
The PKC isoforms were variably upregulated in response to growth factor
readdition, with each cell line showing a distinctive expression
pattern. However, the time necessary to recover from growth factor
starvation changed from less than 15 minutes to more than 60 minutes,
indicating that translation of the PKC isoform mRNAs is differently
regulated both inside the same cell line and between the different
phenotypes. In fact, at 15 minutes, PKC I returned to the levels
observed in unstarved cells in the 32D cell clone. In the 32D GM1, PKC
recovered to the basal values, albeit from a small decrease, in the
15-minute time frame. In the 32D G1, both PKC isoforms I and recovered at 15 minutes. The erythroid 32D Epo1 did not show any
isoform recovering in the short time after starvation. Interestingly,
some of the isoforms investigated were not affected by the growth
factor starvation at all, as the in the 32D GM1 and , , and
in the 32D G1.
The linkage between stimulation of specific growth factor receptor and
PKC isoform composition was evident in the 32D Epo1.1, an
IL-3-dependent revertant of 32D Epo1 that displays an hybrid behavior.
In fact, when Epo1.1 cells were cultured with IL-3, they only lacked
the expression of isoform and showed an upregulated expression of
I and isoforms in response to IL-3. On the other hand, when
Epo 1.1 cells were supplemented with Epo, they did not express both and isoforms and did not upregulate the I isoform, similarly
to 32D Epo. In this respect, it is noteworthy that the 32D Epo1.1 are
as differentiated as the 32D Epo1 when grown in Epo (high levels of and globin expression and erythroid-specific splicing of the
spectrin-binding domain of band 4.1) but are less differentiated when
grown in IL-3 (only high levels of expression of and globin).4
To further evaluate the role of PKC- in erythroid differentiation,
we have analyzed the expression of PKC- mRNA in erythroid cell
lines. 32D Epo1 and 32D Epo1.1 cells, cultured in either IL-3 or Epo,
expressed apparently normal levels of PKC- mRNA by RT-PCR. This
result was expected because posttranscriptional regulation of PKC-
has been found alreadly in other hematopoietic cell
lines35,36 and indicates that experiments of forced PKC- expression will probably require the isolation of stable transfectants and high levels of expression.36 In this respect, we have
obtained a recently described PKC- construct37 that will
allow us to develop stable transfectants to identify both the
posttranscriptional mechanisms that downmodulate the activity of
PKC- in 32D Epo cells and how downmodulation of PKC- favors the
erythroid differentiation program (run-off experiments of several genes
after forced expression, etc).
Thus, each hematopoietic phenotype shows a cell type-specific pattern
of expression of the different PKC isoenzymes, fully consistent with
the hypothesis that the different isoenzymes might fulfill different
biological functions in hematopoietic development. In particular, the
erythroid commitment induced by Epo does not seem to involve PKC
activation and is rather accompanied by the downregulation of specific
isoforms. The only isoform upregulated by growth factor readdition in
all myeloid lineages was PKC-I, which has been previously
implicated in mediating survival/proliferation signals rather than
differentiation.30 Our findings support the notion that
both qualitative and quantitative differences in the levels of PKC
isoform expression may play a role in mediating differentiation
decisions along distinct hematopoietic lineages.
| |
FOOTNOTES |
Submitted March 16, 1998; accepted October 6, 1998.
Supported by "Blood Project" of the Istituto Superiore di
Sanità and "Sant'Anna Hospital of Ferrara Project."
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Giorgio Zauli, MD, PhD, Human Anatomy
Section, Department of Morphology and Embryology, Via Fossato di
Mortara 66, 44100 Ferrara, Italy.
| |
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Abstract
Introduction
