CD45 Negatively Regulates Monocytic Cell Differentiation by
Inhibiting Phorbol 12-Myristate 13-Acetate-dependent
Activation and Tyrosine Phosphorylation of Protein Kinase C
*
Eric L.
Deszo
,
Danett K.
Brake
,
Keith A.
Cengel
,
Keith W.
Kelley
, and
Gregory G.
Freund
§¶
From the Departments of
Animal Sciences and
§ Pathology, University of Illinois at Urbana-Champaign,
Urbana, Illinois 61801
Received for publication, November 22, 2000, and in revised form, December 20, 2000
 |
ABSTRACT |
The protein-tyrosine phosphatase CD45 is
expressed on all monocytic cells, but its function in these cells is
not well defined. Here we report that CD45 negatively regulates
monocyte differentiation by inhibiting phorbol 12-myristate 13-acetate
(PMA)-dependent activation of protein kinase C (PKC)
.
We found that antisense reduction of CD45 in U937 monocytic cells
(CD45as cells) increased by 100% the ability of PMA to enlarge cell
size, increase cell cytoplasmic process width and length, and induce
surface expression of CD11b. In addition, reduction in CD45 expression
caused the duration of peak PMA-induced MEK and extracellular
signal-regulated kinase (ERK) 1/2 activity to increase from 5 min to 30 min while leading to a 4-fold increase in PMA-dependent
PKC
activation. Importantly, PMA-dependent tyrosine
phosphorylation of PKC
was also increased 4-fold in CD45as cells.
Finally, inhibitors of MEK (PD98059) and PKC
(rottlerin) completely
blocked PMA-induced monocytic cell differentiation. Taken together,
these data indicate that CD45 inhibits PMA-dependent PKC
activation by impeding PMA-dependent PKC
tyrosine
phosphorylation. Furthermore, this blunting of PKC
activation leads
to an inhibition of PKC
-dependent activation of ERK1/2
and ERK1/2-dependent monocyte differentiation. These findings suggest that CD45 is a critical regulator of monocytic cell development.
 |
INTRODUCTION |
CD45 is the dominant leukocyte plasma membrane phosphatase. It is
a long single-chain type I transmembrane protein with an extracellular
portion that suggests three fibronectin type III domains and an
intercytoplasmic tail that contains two phosphatase domains in direct
repeat (1). There are currently five expressed isoforms of CD45, and
these are generated by differential splicing of exons 4-6, while exons
1-3 and 7-33 remain constant (2). Presently, the functional
variations in these CD45 isoforms appear primarily due to their
extracellular motifs and cell of expression as opposed to differences
in their catalytic domains (3). To date, the best described bioaction
for CD45 is an activator of the Src family kinases Lck and Lyn in T
cell and B cell receptor complexes (4-7). In thymocytes lacking CD45,
maturation from immature double positive CD4/CD8 cells to single
positive CD4 and CD8 cells is blocked (8). In B cells, loss of CD45
inhibits proliferation induced by antigen-dependent
cross-linking of surface IgM and IgD (9). However, in monocytic cells,
the function of CD45 is less clear. Recent studies in monocytes have
shown that cross-linking of CD45 stimulates an increase in the
respiratory burst following tumor necrosis factor-
treatment (10)
and that ligation of CD45 induces both tumor necrosis factor-
and
granulocyte/macrophage colony-stimulating factor production in
monocytes (11).
Phorbol-12-myristate-13-acetate
(PMA)1 can induce monocytic
cells to differentiate by a mechanism dependent on the activation of
protein kinase C (PKC). The PKC family currently comprises 10 related
isozymes that have been divided into three groups based on their
structure and cofactor requirements. The conventional PKC isoforms
,
I,
II, and
require diacylglyerol (DAG), phosphatidylserine, and Ca2+ for activity. The novel PKC isoforms
,
,
, and
do not require Ca2+ as a cofactor but do
require DAG and phosphatidylserine. The atypical PKC isoforms
and
do not require either Ca2+ or DAG but do bind
phosphatidylserine when active (reviewed in Refs. 12-16). PMA
activates the atypical PKC isoforms and conventional PKCs because it is
similar in structure to DAG (17). PMA, like DAG, binds to a
cysteine-rich region contained within the PKC C1 domain producing a
contiguous hydrophobic region that allows PKC to associate with the
cell membrane (18). This facilitates conventional PKC and novel PKC
isoform binding of phosphatidylserine (19). Binding of PKC family
members to relevant cofactors initiates removal of the inhibitory
pseudosubstrate domain from the PKC core inducing autophosphorylation
on COOH-terminal serines and release of activated PKC from the cell
membrane to the cytosol (20).
Protein kinase activation is a critical step in PMA-induced
differentiation of promyelocytic cells (21) and is associated with
activation of the Raf-1/MAP kinase-signaling pathway (22). The MAP
kinase family can be loosely divided into three arms that end in the
activation of Jun amino-terminal kinases, p38/HOG1 kinases, or
extracellular signal-regulated kinases (ERKs) (23-25). PMA-induced ERK
activation is through a kinase cascade that requires Raf-1-dependent phosphorylation of MEK1/2 on serines 217 and 221 and MEK1/2-dependent phosphorylation of ERK1/2 on
threonine 202 and tyrosine 204 (26, 27). PKC-dependent
activation of Raf-1 appears to be by direct phosphorylation of serines
43, 259, and/or 499 (28, 29). Importantly, inhibitors of PKCs like H-7,
GF 109203X, and staurosporine (30-32) and of MEK (PD98059) block
PMA-induced monocytic cell differentiation as measured by a variety of
methods including morphometric analysis and surface marker studies
(33). Here we report that that CD45 negatively regulates PMA-induced monocytic cell differentiation by impeding PMA-dependent
PKC
tyrosine phosphorylation, PKC
activation, and
PKC
-dependent activation of ERK1/2. These findings
indicate that CD45 is a critical regulator of monocytic cell development.
 |
EXPERIMENTAL PROCEDURES |
Materials--
The monocytic cell line U937 and the myeloma cell
line U266 were purchased from American Type Culture Collection
(Manassas, VA). Enhanced chemiluminescence (ECL) detection reagents,
Hybond-ECL nitrocellulose, Protein G-Sepharose were purchased from
Amersham Pharmacia Biotech. Fluorescein isothiocyanate
(FITC)-conjugated CD45 (catalog no. 6603838) and FITC-conjugated CD11b
(catalog no. IM0530) antibodies were purchased from Beckman Coulter
(Fullerton, CA). Phospho-ERK1/2 (catalog no. 91065), phospho-MEK1/2
(catalog no. 91215), MEK1/2 (catalog no. 9122), and the phospho-PKC
,
-
I, -
II, -
, and -
(catalog no. 93715) antibodies were
purchased from Cell Signaling Technology (Beverly, MA). ERK1/2 (catalog no. 06-182), and the PKC
, -
I, -
II, and -
(catalog no.
06-870) antibodies were purchased from Upstate Biotechnology, Inc.
(Lake Placid, NY). PKC
antibody (catalog no. SC-937) was purchased from Santa Cruz Biotechnologies, Inc. (Santa Cruz, CA). FITC-conjugated CD33 (catalog no. F0832) antibody was purchased from Dako (Carpinteria, CA). Trizol reagent was purchased from Life Technologies, Inc. Bio-Rad
protein assay was purchased from Bio-Rad. PD98059 and rottlerin were
purchased from Calbiochem (San Diego, CA). The Lab-Tek chamber slide
system was purchased from Nalge Nunc International (Naperville, IL).
All other cell culture reagents and chemicals were purchased from
Sigma. Oligonucleotide primers were purchased from Operon (Almeda, CA).
The episomal replicating mammalian expression vector pCEP4 was
purchased from Invitrogen (Carlsbad, CA). All other molecular biology
reagents and chemicals were purchased from Promega (Maddison, WI).
Cell Culture--
U937 and U266 cells were grown in growth media
(RPMI 1640 media supplemented with 10% fetal bovine serum, 2.0 g/liter
sodium bicarbonate, 2.5 g/liter glucose, 100,000 units/liter
penicillin, 100 mg/liter streptomycin, 1 mM sodium
pyruvate, and 10 mM HEPES pH 7.4). Cells were passaged 1:1
with fresh media every 3 days. For PMA treatment, cells were washed
twice and resuspended in growth media supplemented with 100 nM PMA.
Vector Construction--
The episomal CD45 antisense (CD45as)
vector was created to target the expressed 5' sequence in exons 1, 2, and 3 that is shared by all known CD45 isoforms (1, 2). In brief, total
cellular RNA from 5 × 106 U266 cells was isolated in
Trizol reagent and the target sequence amplified by polymerase chain
reaction from random hexamer-primed cDNA using the forward primer
(5'-GCGGATCCGGAAATTGTTCCTCGTCTGA-3') and the reverse primer
(5'-GCAAGCTTCAGTGGGGGAAGGTGTTGG-3') by methods we have described (34).
The resultant 196-base pair polymerase chain reaction product was
introduced into the pCEP4 vector using the BamHI and
HindIII restrictions sites included in the forward and
reverse primer sequences and the construct verified by sequence analysis.
Vector Transfection--
The U937 cell lines U937-CD45as and
U937-pCEP4 were created by introducing the vectors CD45as and pCEP4,
respectively, by electroporation. 20 × 106 U937 cells
were washed twice in and resuspended in 800 µl of phosphate-buffered
saline (80 mM Na2HPO4, 20 mM
NaH2PO4·H2O, and 100 mM NaCl, pH 7.4). Cells were added to 4-mm gap
electroporation cuvettes with either 20 µg of CD45as or pCEP4 vector
and electroporated at 400 V in an EC 100 electroporator (Fisher,
Pittsburgh, PA). Cells were recovered in growth media for 24 h and
then removed to growth media supplemented with 250 µg/ml hygromycin
B. After 2 weeks, CD45as cells were labeled with FITC-conjugated CD45
antibody (35) and sorted for low CD45 expression on a MoFlo flow
cytometer (Cytomation, Fort Collins, CO).
Flow Cytometry--
Immunolabeling of cells was performed as
previously described (36). In brief, after indicated treatments, cells
were incubated in growth media supplemented with 5 mM EDTA
for 1 h at 37 °C and then washed once in 0.5% bovine serum
albumin containing phosphate-buffered saline. FITC-conjugated
antibodies at 7 µg/ml/test were added to 1 × 106
cells and incubated on ice for 15 min. Fluorescence was detected at an
excitation of 480 nm on an Epics XL flow cytometer (Beckman Coulter,
Fullerton, CA) quantifying 1 × 104 events using gates
to exclude nonviable cells as determined by propidium iodide staining.
Western Analysis--
Western analysis was performed as
described previously (37). In brief, 15 × 106 cells
were lysed in 1 ml of ice-cold lysis buffer (1% Tritron X-100, 150 mM NaCl, 1 mM NaF, 1 mM EGTA, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 2 µg/ml aprotinin, 2 µg/ml leupeptin, 1 mM sodium
orthovanadate, and 50 mM Tris base, pH 7.4). Proteins were
resolved by SDS-polyacrylamide gel electrophoresis (250 µg/lane)
under reducing conditions in 10% gels and then electrotransferred to
nitrocellulose. Immunoreactive proteins were visualized using the
indicated primary antibodies and enhanced ECL reagents followed by
autoradiography and densitometry.
Statistical Analysis--
Where indicated, experimental data was
analyzed by Student's t test for comparison of medians
using Excel (Microsoft, Redmond, WA).
 |
RESULTS |
CD45 Negatively Regulates PMA-induced Monocytic Cell
Differentiation--
PMA is a potent activator of monocytic cells
increasing surface expression of CD45 and CD11b and inducing phenotypic
maturation (21). To determine the role of CD45 in
PMA-dependent monocytic cell activation, CD45, CD11b, and
cell morphology were examined in U937 cells transfected with the CD45
antisense vector (U937-CD45as cells) and U937 cells transfected with
the vector backbone (U937-pCEP4 cells) treated with 100 nM
PMA for 2 days. Fig. 1A shows
that PMA increased CD45 surface expression in U937-pCEP4 cells by 80% at 2 days from a median fluorescence of 22.8 ± 2.3 to 41.7 ± 7.2. In contrast, CD45 surface expression in U937-CD45as cells was 50% that of U937-pCEP4 cells basally (day 0) at a median fluorescence of 11.9 ± 0.9 and after 2 days of PMA treatment at a median
fluorescence of 20.9 ± 2.1. Fig. 1B demonstrates that
in U937-pCEP4 cells PMA induced a 3-fold elevation in surface CD11b
expression at 2 days as median fluorescence increased from 0.31 ± 0.13 to 1.03 ± 0.06. However, in U937-CD45as cells, PMA exposure
increased CD11b expression by 14-fold as median fluorescence increased
from 0.28 ± 0.01 to 4.05 ± 0.79. To confirm that the CD45as
vector did not down-regulate all surface makers, CD33 expression was
examined. As Fig. 1C shows, CD33 surface expression in
U937-pCEP4 and U937-CD45as cells was similar both basally (median
fluorescence, 1.72 ± 0.11 versus 1.60 ± 0.07)
and after PMA treatment (median fluorescence, 1.93 ± 0.03 versus 1.91 ± 0.07). Finally, Fig. 1D
demonstrates that PMA increased U937-pCEP4 cell nuclear size, cell
size, and cell process length by 2-fold after 2 days. In U937-CD45as
cells, PMA induced a 2-fold increase in nuclear size but a 4-fold
increase in cell size and cell process length. Additionally, when cells were pre-treated with the MEK inhibitor PD98059 (25 µM)
for 15 min prior to PMA treatment, PMA-induced morphologic
differentiation was blocked. Taken together, these findings demonstrate
that transfection of the episomal CD45as vector in U937 cells reduces
by 50% the surface expression of CD45. Importantly, reduction in CD45
expression induces at least a doubling of PMA-dependent
CD11b expression, cell size, and cell process length when compared with
control (U937-pCEP4) cells and that these phenotypic changes are
sensitive to MEK inhibition.

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Fig. 1.
CD45 negatively regulates PMA-induced
monocytic cell differentiation. A, U937-pCEP4 and
U937-CD45as cells were treated with 100 nM PMA for
0, 1. or 2 days. Surface expression of CD45 was quantified by flow
cytometry using a FITC-conjugated CD45 antibody. Results significantly
different from U937-CD45as, day 0 at = 0.05 are indicated by
an asterisk (*). Results represent an average of four
independent experiments ± S.E. B, cells were treated
as in A and surface CD11b quantified by flow cytometry using
a FITC-conjugated CD11b antibody. Results significantly different from
U937-pCEP4, day 0 at = 0.05 are indicated by an
asterisk (*). Results represent an average of four
independent experiments ± S.E. C, cells were treated
as in A, and CD33 was quantified by flow cytometry using a
FITC-conjugated CD33 antibody. Results represent an average of four
independent experiments ± S.E. D, cells were treated
as in A and grown in Lab-Tek chamber slides with or without
a 15-min pretreatment with 25 µM PD98059. After 2 days,
slides were air-dried and cells stained with Wright-Giemsa. Results are
representative of three independent experiments (original
magnification, ×200).
|
|
PMA-dependent Activation of ERK1/2 Is Regulated by
CD45--
PMA mediates its effect on monocytic cell differentiation
through activation of the MAP kinases (22). To determine whether CD45
affected PMA-dependent MAP kinase activation, MEK and
ERK1/2 activation were examined by Western analysis. Fig.
2A shows that in U937-pCEP4
cells PMA induced a 5-fold activation of MEK at 5 min and that by 30 min MEK activation had decreased to 2-fold over basal. In U937-CD45as
cells, PMA treatment also caused a 5-fold activation of MEK at 5 min
but at 30 min MEK activation still remained 5-fold over basal.
Likewise, in Fig. 2B, PMA induced a 5-fold increase in
ERK1/2 activation at 5 min and by 30 min this increase had returned to
basal levels. In U937-CD45as cells, however, ERK1/2 activation was
5-fold over basal at 5 min and remained elevated at 4-fold over basal
at 30 min. Importantly, in split samples, mass of MEK and ERK1/2 was
unchanged by antisense expression (Fig. 2, A and
B, lower panels). Taken together,
these findings demonstrate that antisense reduction of CD45 leads to an
extension of peak MEK and MAP kinase activation from 5 to 30 min,
indicating that CD45 attenuates MAP kinase activation in PMA activated
monocytic cells.

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Fig. 2.
PMA-dependent activation of
ERK1/2 is regulated by CD45. A, U937-pCEP4 and
U937-CD45as cells were treated with 100 nM PMA for the
times indicated and MEK activation (MEK ) was measured by
Western analysis from whole cell lysates using a phospho-MEK1/2
antibody (P-MEK, upper panel). MEK1/2
mass was measured by Western analysis from whole cell lysates using a
MEK1/2 antibody (lower panel). Results are
representative of three independent experiments. B, cells
were treated as in A and activated ERK1/2
(ERK1/2 ) was measured by Western analysis from whole cell
lysates using a phospho-ERK1/2 antibody (ERK1/2-P;
upper panel). ERK1/2 mass was measured by Western
analysis from whole cell lysates using an ERK1/2 antibody. Results are
representative of three independent experiments.
|
|
Equalization of ERK1/2 Activation in U937-pCEP4 and U937-CD45as
Cells Results in Equivalent PMA-induced CD11b Expression--
As
demonstrated in Fig. 1D, the MEK kinase inhibitor PD98059
blocks PMA-induced phenotypic maturation. To demonstrate that loss of
CD45-dependent regulation of MAP kinase activation was the
cause for PMA-dependent increased CD11b expression in
U937-CD45as cells when compared with U937-pCEP4 cells, MAP kinase
normalization studies were performed. Fig.
3A shows that, as in Fig.
1B, PMA at 2 days leads to more than a doubling of CD11b
expression in U937-CD45as cells when compared with U937-pCEP4 cells
(median fluorescence, 5.34 ± 0.32 versus 1.91 ± 0.31). Importantly, treating of U937-CD45as cells with 3 µM PD98059 15 min prior to PMA exposure resulted in
U937-CD45as cells and U937-pCEP4 cells having similar CD11b expression
at 2 days (median fluorescence, 2.20 ± 0.03 versus 1.91 ± 0.31). Fig. 3B demonstrates the impact of 3 µM PD98059 on PMA-dependent ERK1/2 activation
in U937-CD45as cells. As with Fig. 2B, PMA-induced ERK1/2
activation is reduced at 15 min by 50% in U937-pCEP4 cells when
compared with U937-CD45as cells. However, when U937-CD45as cells are
treated with 3 µM PD98059 for 15 min prior to PMA
exposure, U937-CD45as cell ERK1/2 activation at 5 and 15 min is
equivalent to that of U937-pCEP4 cells. Taken together, these findings
demonstrate that activation of ERK1/2 is required for
PMA-dependent CD11b expression and indicate that regulation
of ERK1/2 activation by CD45 controls CD11b surface levels.

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Fig. 3.
Equalization of ERK1/2 activation in
U937-pCEP4 and U937-CD45as cells results in equivalent PMA-induced
CD11b expression. A, U937-pCEP4 and U937-CD45as cells
were pretreated for 15 min with (1, 3, or 16 µM) or
without (Control, PMA) the indicated
concentrations of PD98059 and all cells except control were treated
with 100 nM PMA for 2 days. Surface expression of CD11b was
quantified by flow cytometry using a FITC-conjugated CD11b antibody.
Results significantly different from U937-pCEP4, PMA at = 0.05 are indicated by an asterisk (*). Results represent an
average of four independent experiments ± S.E. B,
U937-pCEP4 and U937-CD45as cells were pretreated for 15 min with (+) or
without ( ) 3 µM PD98059 and then treated with 100 nM PMA for the times indicated. Activated ERK1/2
(ERK1/2-P) was measured by Western analysis using a
phospho-ERK1/2 antibody. Results are representative of three
independent experiments.
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|
PMA-dependent Activation and Tyrosine Phosphorylation
of PKC
Is Negatively Regulated by
CD45--
PMA-dependent initiation of the MAP kinases
cascade requires activation of PKCs (21, 22). Therefore, to determine
whether CD45 impacted PKC activation Western analysis was performed
using a phospho-PKC antibody to isoforms
,
I,
II,
, and
. Fig. 4A shows that in
U937-pCEP4 cells 100 nM PMA induced a maximal 3-fold PKC
autophosphorylation on serine 660 at 5 min. In U937-CD45as cells,
however, PMA led to a 12-fold PKC autophosphorylation at the same time
point. In addition, masses of PKC
,
I,
II,
, and
,
measured in split samples, were not altered by antisense expression
(Fig. 4A, lower panels). Since PKC
has been shown to be tyrosine-phosphorylated in the myeloid progenitor
line 32D in response to PMA (38), the next step was to determine its role in monocytic cell differentiation and its regulation by CD45. Fig.
4B demonstrates that the PKC
inhibitor, rottlerin (39), at 2.5 µM blocked by 66% PMA induced surface expression
of CD11b in U937 cells at 2 days reducing median fluorescence from
7.20 ± 0.30 in PMA-treated cells to 2.4 ± 0.32 in
rottlerin/PMA-treated cells. Importantly, rottlerin did not markedly
alter cell viability, as measured by propidium iodide staining in that
control cells were 94% viable, rottlerin-treated cells 88% viable,
PMA-treated cells 88% viable, and rottlerin/PMA-treated cells 73%
viable after 2 days of treatment. To confirm that PMA activated PKC
and that PKC
autophosphorylation was regulated by CD45, Western
analysis was performed on using a PKC
-specific immunoprecipitating
antibody. Fig. 4C (top panel) shows
that, in U937-pCEP4 cells, 100 nM PMA induced a 3-fold
autophosphorylation of PKC
at 5 min but that, in U937-CD45as cells,
PMA caused a 12-fold autophosphorylation of PKC
. To determine the
potential mechanism by which CD45 modulates PMA-dependent
PKC
activity, phosphorylation of PKC
was examined in split
immunoprecipitates. Fig. 4C (upper
panel) demonstrates that PMA induced a 3-fold increase in
PKC
serine 660 phosphorylation in U937-pCEP4 cells and that CD45
antisense expression quadrupled this effect. The middle
panel demonstrates that PMA also induced a 3-fold increase
in PKC
tyrosine phosphorylation in U937-pCEP4 cells and that CD45
antisense expression quadrupled this effect. The lower
panel of Fig. 4C shows that in split
immunoprecipitates the mass of PKC
was unaffected by CD45 expression
or PMA treatment. Finally, to examine PKC
, -
I, -
II, and -
activation and tyrosine phosphorylation, Western analysis was again
performed on split immunoprecipitates. Fig. 4D demonstrates
that the PKC isoforms
,
I,
II and
are neither
phosphorylated on serine 660 (top panel) nor
phosphorylated on tyrosine (middle panel) in
response to PMA. The lower panel of Fig.
4D shows that PKC
, -
I, -
II, and -
were present
in the immunoprecipitates from PMA-treated cells and that PKC
,
-
I, -
II and -
mass were unaffected by CD45 expression. These
findings show that PKC
is required for PMA-induced CD11b surface
expression and that CD45 negatively regulates the autophosphorylation
and tyrosine phosphorylation of PKC
. Overall, these data indicate
that control of PKC
tyrosine phosphorylation by CD45 regulates
PMA-induced PKC
activity and subsequent monocyte
differentiation.

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Fig. 4.
PMA-dependent activation and
tyrosine phosphorylation of PKC is negatively
regulated by CD45. A, U937-pCEP4 and U937-CD45as cells
were treated with 100 nM PMA for the times indicated and
autophosphorylated PKC , - I, - II, - , and -
(PKC , I, II, , -P)
were measured by Western analysis from whole cell lysates using a
phospho-PKC , I, II, , antibody (upper
panel). PKC , I, II, , mass were measured by
Western analysis from whole cell lysates from U937-pCEP4 and
U937-CD45as cells using a PKC , I, II, antibody
(lower panel, left) and a PKC
antibody (lower panel, right). Results
are representative of three independent experiments. B, U937
cells were pretreated for 15 min with or without 2.5 µM
rottlerin as indicated then treated with or without 100 nM
PMA for 2 days. Surface expression of CD11b was quantified by flow
cytometry using a FITC-conjugated CD11b antibody. Results significantly
different from control at = 0.05 are indicated by an
asterisk (*). Results represent an average of four
independent experiments ± S.E. C, U937-pCEP4 and
U937-CD45as cells were treated with 100 nM PMA for 5 min as
indicated and serine (Ser660) (upper
panel) and tyrosine (pY) (middle
panel) phosphorylated PKC
(PKC -P) and mass of PKC were measured in
PKC immunoprecipitates (IP) by Western analysis. Results
are representative of three independent experiments. D,
U937-pCEP4 and U937-CD45as cells were treated as in C and
serine-phosphorylated (Ser660) (upper
panel) and tyrosine-phosphorylated (pY)
(middle panel) PKC , - I, - II, and -
(PKC , I, II, -)
and mass of PKC , - I, - II, and - (lower
panel) were measured in PKC , - I, - II, and -
immunoprecipitates (IP) by Western analysis. PKC
immunoprecipitates from U937-CD45as cells serve as positive controls.
Results are representative of three independent experiments.
|
|
 |
DISCUSSION |
These data establish that CD45 negatively regulates
PMA-dependent monocytic cell differentiation by impairing
PKC
activation and tyrosine phosphorylation. Flow cytometry
demonstrated that antisense reduction of CD45 reduces
PMA-dependent expression of CD45 while quadrupling that of
CD11b and not affecting that of CD33 (Fig. 1, A-C).
Morphologic studies showed that phenotypic characteristics of monocyte
differentiation such as increased cell size and cell process length
were doubled in U937-CD45as cells when compared with that of U937-pCEP4
(Fig. 1D). In addition, the MEK inhibitor PD98059 blocked
all morphologic changes induced by PMA in both cell lines (Fig.
1D). Antisense reduction of CD45 increased the duration of
peak PMA-induced MEK and ERK1/2 activation from 5 min to nearly 30 min
(Fig. 2, A and B). Furthermore, normalization of
PMA-induced ERK1/2 activity in U937-CD45as cells to that in U937-pCEP4
cells resulted in equivalent PMA-dependent CD11B surface expression in these two cell lines (Fig. 3, A and
B). Finally, PMA activation in U937-CD45as cells was 4-fold
greater than that in U937-pCEP4 cells (Fig. 4A).
Importantly, the PKC
inhibitor rottlerin reduced by 66%
PMA-dependent CD11b expression (Fig. 4B) while
CD45 antisense expression quadrupled PMA-dependent PKC
activation and tyrosine phosphorylation (Fig. 4C). Notably,
PKC isoforms
,
I,
II, and
were neither activated nor
tyrosine-phosphorylated by PMA (Fig. 4D). Taken together,
these findings indicate that CD45 blunts PKC
activation by reducing
PMA-dependent PKC
tyrosine phosphorylation and that this
inhibition in PKC
autophosphorylation results in reduced activation
of ERK1/2 and ERK1/2-dependent monocyte differentiation.
PMA-dependent monocytic cell differentiation requires PKC
activation and ERK1/2 activation (21, 22), but the PKC isoforms involved in this process have only recently become better defined. In
the interleukin-3-dependent myeloid cell line 32D,
transfection studies initially demonstrated that PKC
and PKC
were
essential for PMA-induced macrophage differentiation while PKC
II,
-
, -
, and -
were not required (40). Subsequent studies in the
same cell system demonstrated that only PKC
appeared to be required for phorbol ester-induced macrophage differentiation (41). Our data
support these findings in that we found that PKC
but not PKC
,
-
I, -
II, and -
were autophosphorylated on serine 660 after PMA
treatment and autophosphorylation of serine 660 has been shown to be
required for PKC activation (42). In addition, we found that rottlerin
a competitive inhibitor of the PKC ATP-binding site, which
preferentially inhibits the
isoform at concentrations between 3 and
6 µM (39), blocked by 66% PMA-induced monocyte differentiation. Since phorbol esters lead to PKC autophosphorylation (42) and activated PKC initiates the MAP kinase cascade (21, 22), the
increased serine 660 phosphorylation of PKC
we observed in CD45
antisense cells is the likely cause of the increased MEK and ERK1/2
activity we also observed in these cells. In addition, since ERK1/2
activation is required for PMA-dependent monocyte differentiation as shown by us here with MEK inhibition studies and by
others (33), we conclude that PKC
is the critical PKC responsible
for PMA-induced monocyte differentiation in U937 cells and that its
autophosphorylation on serine 660 probably controls this process.
Furthermore, from the 32D cell studies sited above, PKC
would be
expected to play an important role, generally, in the phorbol ester
activation pathway (43) in monocytes.
CD45 is expressed in nearly all cells of hematopoietic origin (44), but
its function in cells of the myeloid lineage is especially ill defined.
In vitro, the tyrosine phosphatase activity of CD45 is
relatively substrate nonspecific consistent with what is found with
many of the transmembrane and intracytoplasmic tyrosine phosphatases
(45). This has made knockout and antisense approaches critical to
defining the role of CD45 in hematopoietic cells. Our stable episomal
antisense vector to the conserved exons 1, 2, and 3 led to a 50%
reduction in CD45 expression in unstimulated and PMA-stimulated U937
cells. Surprisingly, this led to an increase in differentiation as
measured by CD11b expression and morphologic examination. Expected
results were that, as in the T and B cells systems, loss of CD45 would
be associated with decreased maturation and reduced ability to respond
to activating stimuli. The reason for this incongruity may be due to
the role of the Scr family kinases in PMA-induced differentiation.
Unique to PKC
is that it is phosphorylated on tyrosine residues
after cells are PMA-stimulated (38). Although the kinases involved in
this process are not fully delineated, v-Src, Lyn, and Fyn have been
shown to associate and/or phosphorylate PKC
(46-48). Currently, the
role of PKC
tyrosine phosphorylation is not clear. Studies have
demonstrated that PKC
tyrosine phosphorylation can both increase
(38) and reduce (49) PCK
activity. Here we show that PKC
is
tyrosine-phosphorylated in response to PMA and that antisense reduction
in CD45 expression increases PMA-dependent PKC
tyrosine
phosphorylation. Importantly, the increase in PKC
tyrosine
phosphorylation seen with loss of CD45 expression is associated with
increased PKC
autophosphorylation, ERK1/2 activation, and
differentiation, indicating that CD45-regulated tyrosine
phosphorylation is likely responsible for reducing PKC
serine 660 phosphorylation and, hence, its autophosphorylation and activation. It
is then this reduction in PKC
activity that results in reduced
ERK1/2 activation and reduced CD11b expression.
Unclear, however, is how CD45 might regulate PKC
tyrosine
phosphorylation. In terms of the Src family kinases, CD45 is generally thought to dephosphorylate an inhibitory tyrosine thereby activating the kinase (50). In our system, loss of CD45 expression would be
expected to lead to reduced Src family kinase activity and decreased
PKC
tyrosine phosphorylation contrary to what we observed. Interestingly, CD45 has also been implicated in dephosphorylating the
activating tyrosine 394 on Lyn and reducing Lyn kinase activity (6,
50). In CD45 knockout macrophage precursors, Lyn activity is increased
nearly 5-fold (51), implicating Lyn as potentially important to
increased PKC
tyrosine phosphorylation in CD45-deficient monocytic
cells. Finally, CD45 may directly dephosphorylate PKC
controlling
PKC
activity at the inner membrane surface. Preliminary data from
our laboratory show that CD45 can dephosphorylate in vitro
tyrosine-phosphorylated PKC
, but, as indicated above, tyrosine phosphatases tend to have little in vitro substrate
specificity. In summary, we found that CD45 negatively regulates
PMA-induced PKC
autophosphorylation, PKC
tyrosine
phosphorylation, and monocytic cell differentiation. We conclude that
CD45 inhibits PMA-dependent PKC
tyrosine
phosphorylation, thereby blunting PKC
activation and
PKC
-dependent activation of ERK1/2. Reduced ERK1/2
activity then limits monocyte differentiation.
 |
FOOTNOTES |
*
This work was supported by grants from the National
Institutes of Health Grant (CA-61931 to G.G.F. and AG06246 to K.W.K.), The Macula Foundation (to G.G.F), the American Diabetes Association (to
G.G.F.) and USDA/CREES (to G.G.F.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence and reprint requests should be
addressed: Dept. of Pathology, College of Medicine, 506 S. Mathews
Ave., University of Illinois at Urbana-Champaign, Urbana, IL 61801. Tel.: 217-244-8839; Fax: 217-244-5617; E-mail:
freun@ux1.cso.uiuc.edu.
Published, JBC Papers in Press, December 21, 2000, DOI 10.1074/jbc.M010589200
 |
ABBREVIATIONS |
The abbreviations used are:
PMA, phorbol
12-myristate 13-acetate;
ERK, extracellular signal-regulated kinase;
MEK, mitogen or extracellular kinase;
pY, phosphotyrosine;
PKC, protein
kinase C;
FITC, fluorescein isothiocyanate;
MAP, mitogen-activated
protein;
DAG, diacylglyerol.
 |
REFERENCES |
1.
|
Trowbridge, I. S.
(1994)
Annu. Rev. Immunol.
12,
85-116[CrossRef][Medline]
[Order article via Infotrieve]
|
2.
|
Hall, L. R.,
Streuli, M.,
Schlossman, S. F.,
and Saito, H.
(1988)
J. Immunol.
141,
2781-2787[Abstract/Free Full Text]
|
3.
|
Donovan, J. A.,
and Koretzky, G. A.
(1993)
J. Am. Soc. Nephrol.
4,
976-985[Abstract]
|
4.
|
Katagiri, T.,
Ogimoto, M.,
Hasegawa, K.,
Mizuno, K.,
and Yakura, H.
(1995)
J. Biol. Chem.
270,
27987-27990[Abstract/Free Full Text]
|
5.
|
Justment, L. B.,
Campbell, K. S.,
Chien, N. C.,
and Cambier, J. C.
(1991)
Science
252,
1839-1842[Medline]
[Order article via Infotrieve]
|
6.
|
Yanagi, S.,
Sugawara, H.,
Kurosaki, M.,
Sabe, H.,
Yamamura, H.,
and Kurosaki, T.
(1996)
J. Bio. Chem.
271,
30487-30492[Abstract/Free Full Text]
|
7.
|
D'Oro, U.,
and Ashwell, J. D.
(1999)
J. Immunol.
162,
1879-1883[Abstract/Free Full Text]
|
8.
|
Wallace, V. A.,
Penninger, J. M.,
Kishihara, K.,
Timms, E.,
Shahinian, A.,
Pircher, H.,
Kündig, T. M.,
Ohashi, P. S.,
and Mak, T. W.
(1997)
J. Immunol.
158,
3205-3214[Abstract]
|
9.
|
Byth, K. F.,
Conroy, L. A.,
Howlett, S.,
Smith, A. J. H.,
May, J.,
Alexander, D. R.,
and Holmes, N.
(1996)
J. Exp. Med.
183,
1707-1718[Abstract]
|
10.
|
Liles, W. C.,
Ledbetter, J. A.,
Waltersdorph, A. W.,
and Klebanoff, S. J.
(1995)
J. Immunol.
155,
2175-2184[Abstract]
|
11.
|
Hayes, A. L.,
Smith, C.,
Foxwell, B. M. J.,
and Brennan, F. M.
(1999)
J. Biol. Chem.
274,
33455-33461[Abstract/Free Full Text]
|
12.
|
Nishizuka, Y.
(1984)
Nature
308,
693-698[Medline]
[Order article via Infotrieve]
|
13.
|
Newton, A. C.
(1995)
J. Biol. Chem.
270,
28495-28498[Free Full Text]
|
14.
|
Mellor, H.,
and Parker, P. J.
(1998)
Biochem. J.
332,
281-292[Medline]
[Order article via Infotrieve]
|
15.
|
Ron, D.,
and Kazanietz, M. G.
(1999)
FASEB J.
13,
1658-1676[Abstract/Free Full Text]
|
16.
|
Parekh, D. B.,
Ziegler, W.,
and Parker, P. J.
(2000)
EMBO J.
19,
496-503[Free Full Text]
|
17.
|
Sharkey, N. A.,
Leach, K. L.,
and Blumberg, P. M.
(1984)
Proc. Natl. Acad. Sci. U. S. A.
81,
607-610[Abstract]
|
18.
|
Zhang, G.,
Kazanietz, M. G.,
Blumberg, P. M.,
and Hurley, J. H.
(1995)
Cell
81,
917-924[Medline]
[Order article via Infotrieve]
|
19.
|
Newton, A. C.,
and Keranen, L. M.,.
(1994)
Biochemistry
33,
6651-6658[Medline]
[Order article via Infotrieve]
|
20.
|
Orr, J. W.,
Keranen, L. M.,
and Newton, A. C.
(1992)
J. Biol. Chem.
267,
15263-15266[Abstract/Free Full Text]
|
21.
|
Pedrinaci, S.,
Ruiz-Cabello, F.,
Gomez, O.,
Collado, A.,
and Garrido, F.
(1990)
Int. J. Cancer
45,
294-298[Medline]
[Order article via Infotrieve]
|
22.
|
Kharbanda, S.,
Saleem, A.,
Emoto, Y.,
Stone, R.,
Rapp, U.,
and Kufe, D.
(1994)
J. Biol. Chem.
269,
872-878[Abstract/Free Full Text]
|
23.
|
Boulton, T. G.,
Yancopoulos, G. D.,
Gregory, J. S.,
Slaughter, J. S.,
Moomaw, C.,
Hsu, J.,
and Cobb, M. H.
(1990)
Science
249,
64-67[Medline]
[Order article via Infotrieve]
|
24.
|
Kyriakis, J. M.,
Banerjee, P.,
Nikolakaki, E.,
Dai, T.,
Rubie, E. A.,
Ahmad, M. F.,
Avruch, J.,
and Woodgett, J. R.
(1994)
Nature
369,
156-160[CrossRef][Medline]
[Order article via Infotrieve]
|
25.
|
Han, J.,
Lee, J.-D.,
Bibbs, L.,
and Ulevitch, R. J.
(1994)
Science
265,
808-811[Medline]
[Order article via Infotrieve]
|
26.
|
Payne, D. M.,
Rossomando, A. J.,
Martino, P.,
Erickson, A. K.,
Her, J. H.,
Shabanowitz, J.,
Hunt, D. F.,
Weber, M. J.,
and Sturgill, T. W.
(1991)
EMBO J.
10,
885-892[Abstract]
|
27.
|
Alessi, D. R.,
Saito, Y.,
Campbell, D. G.,
Cohen, P.,
Sithanandam, G.,
Rapp, U.,
Ashworth, A.,
Marshall, C. J.,
and Cowley, S.
(1994)
EMBO J.
13,
1610-1619[Abstract]
|
28.
|
Kolch, W.,
Heldecker, G.,
Kochs, G.,
Hummel, R.,
Vahidl, H.,
Mishcak, H.,
Finkenzeller, G.,
Marmé, D.,
and Rapp, U. R.
(1993)
Nature
364,
248-252
|
29.
|
Sözeri, O.,
Vollmer, K.,
Liyanage, M.,
Frith, D.,
Kour, G.,
Mark, I. I. I., G. E.,
and Stabel, S.
(1992)
Oncogene
7,
2259-2262[Medline]
[Order article via Infotrieve]
|
30.
|
Barendsen, N.,
Mueller, M.,
and Chen, B.
(1990)
Leuk. Res.
14,
467-474[Medline]
[Order article via Infotrieve]
|
31.
|
Taoka, T.,
Tasaka, T.,
Tanoka, T.,
Irino, S.,
and Norman, A. W.
(1992)
Blood
80,
46-52[Abstract]
|
32.
|
Bhatia, M.,
Kirkland, J. B.,
and Meckling-Gill, K. A.
(1996)
Exp. Cell Res.
222,
61-69[CrossRef][Medline]
[Order article via Infotrieve]
|
33.
|
He, H.,
Wang, X.,
Gorospe, M.,
Holbrook, N. J.,
and Trush, M. A.
(1999)
Cell Growth Diff.
10,
307-315[Abstract/Free Full Text]
|
34.
|
Cengel, K. A.,
Kason, R. E.,
and Freund, G. G.
(1998)
Biochem. J.
335,
397-404[Medline]
[Order article via Infotrieve]
|
35.
|
Kulas, D. T.,
Freund, G. G.,
and Mooney, R. A.
(1996)
J. Biol. Chem.
271,
755-760[Abstract/Free Full Text]
|
36.
|
Schacher, D. H.,
VanHoy, R. W.,
Liu, Q.,
Arkins, S.,
Dantzer, R.,
Freund, G. G.,
and Kelley, K. W.
(2000)
J. Immunol.
164,
113-120[Abstract/Free Full Text]
|
37.
|
Cengel, K. A.,
and Freund, G. G.
(1999)
J. Biol. Chem.
274,
27969-27974[Abstract/Free Full Text]
|
38.
|
Li, W.,
Mischak, H., Yu, J.-C.,
Wang, L.-M.,
Mushinski, J. F.,
Heidaran, M. A.,
and Pierce, J. H.
(1994)
J. Biol. Chem.
269,
2349-2352[Abstract/Free Full Text]
|
39.
|
Gschwendt, M.,
Müller, H. J.,
Kielbassa, K.,
Zang, R.,
Kittstein, W.,
Rincke, G.,
and Marks, F.
(1994)
Biochem. Biophys. Res. Commun.
199,
93-98[CrossRef][Medline]
[Order article via Infotrieve]
|
40.
|
Mischak, H.,
Pierce, J. H.,
Goodnight, J.,
Kazanietz, M. G.,
Blumberg, P. M.,
and Mushinski, J. F.
(1993)
J. Biol. Chem.
268,
20110-20115[Abstract/Free Full Text]
|
41.
|
Wang, Q. J.,
Acs, P.,
Goodnight, J.,
Giese, T.,
Blumberg, P. M.,
Mischak, H.,
and Mushinski, J. F.
(1997)
J. Biol. Chem.
272,
76-82[Abstract/Free Full Text]
|
42.
|
Edwards, A. S.,
and Newton, A. C.
(1997)
J. Biol. Chem.
272,
18382-18390[Abstract/Free Full Text]
|
43.
|
Kontny, E.,
Kurowska, M.,
Szczepañska, K.,
and Ma liñski, W.
(2000)
J. Leukocyte Biol.
67,
249-258[Abstract]
|
44.
|
Woodford-Thomas, T.,
and Thomas, M. L.
(1993)
Semin. Cell Biol.
4,
409-418[CrossRef][Medline]
[Order article via Infotrieve]
|
45.
|
Tonks, N. K.,
Charbonneau, H.,
Diltz, C. D.,
Fischer, E. H.,
and Walsh, K. A.
(1988)
Biochemistry
27,
8695-8701[Medline]
[Order article via Infotrieve]
|
46.
|
Zang, Q.,
Lu, Z.,
Curto, M.,
Barile, N.,
Shalloway, D.,
and Foster, D. A.
(1997)
J. Biol. Chem.
272,
13275-13280[Abstract/Free Full Text]
|
47.
|
Song, J. S.,
Swann, P. G.,
Szallasi, Z.,
Blank, U.,
Blumberg, P. M.,
and Rivera, J.
(1998)
Oncogene
16,
3357-3368[CrossRef][Medline]
[Order article via Infotrieve]
|
48.
|
Kronfeld, I.,
Kazimirsky, G.,
Lorenzo, P. S.,
Garfield, S. H,
Blumberg, P. M.,
and Brodie, C.
(2000)
J. Biol. Chem.
275,
35491-35498[Abstract/Free Full Text]
|
49.
|
Li, W.,
Li, W.,
Chen, X. -H,.,
Kelley, C. A.,
Alimandi, M.,
Zhang, J.,
Chen, Q.,
Bottaro, D. P.,
and Pierce, J. H.
(1996)
J. Biol. Chem.
271,
26404-26409[Abstract/Free Full Text]
|
50.
|
Katagiri, T.,
Ogimoto, M.,
Hasegawa, K.,
Arimura, Y.,
Mitomo, K.,
Okada, M.,
Clark, M. R.,
Mizuno, K.,
and Yakura, H.
(1999)
J. Immunol.
163,
1321-1326[Abstract/Free Full Text]
|
51.
|
Roach, T.,
Slater, S.,
Koval, M.,
White, L.,
McFarland, E. C.,
Okumura, M.,
Thomas, M.,
and Brown, E.
(1997)
Curr. Biol.
7,
408-417[Medline]
[Order article via Infotrieve]
|
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