CD45 Negatively Regulates Monocytic Cell Differentiation by Inhibiting Phorbol 12-Myristate 13-Acetate-dependent Activation and Tyrosine Phosphorylation of Protein Kinase Cdelta *

Eric L. DeszoDagger , Danett K. BrakeDagger , Keith A. CengelDagger , Keith W. KelleyDagger , and Gregory G. FreundDagger §

From the Departments of Dagger  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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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) delta . 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 PKCdelta activation. Importantly, PMA-dependent tyrosine phosphorylation of PKCdelta was also increased 4-fold in CD45as cells. Finally, inhibitors of MEK (PD98059) and PKCdelta (rottlerin) completely blocked PMA-induced monocytic cell differentiation. Taken together, these data indicate that CD45 inhibits PMA-dependent PKCdelta activation by impeding PMA-dependent PKCdelta tyrosine phosphorylation. Furthermore, this blunting of PKCdelta activation leads to an inhibition of PKCdelta -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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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-alpha treatment (10) and that ligation of CD45 induces both tumor necrosis factor-alpha 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 alpha , beta I, beta II, and gamma  require diacylglyerol (DAG), phosphatidylserine, and Ca2+ for activity. The novel PKC isoforms delta , epsilon , eta , and theta  do not require Ca2+ as a cofactor but do require DAG and phosphatidylserine. The atypical PKC isoforms zeta  and iota  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 PKCdelta tyrosine phosphorylation, PKCdelta activation, and PKCdelta -dependent activation of ERK1/2. These findings indicate that CD45 is a critical regulator of monocytic cell development.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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-PKCalpha , -beta I, -beta II, -gamma , and -delta (catalog no. 93715) antibodies were purchased from Cell Signaling Technology (Beverly, MA). ERK1/2 (catalog no. 06-182), and the PKCalpha , -beta I, -beta II, and -gamma (catalog no. 06-870) antibodies were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). PKCdelta 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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha  = 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 alpha  = 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 alpha  = 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.

PMA-dependent Activation and Tyrosine Phosphorylation of PKCdelta 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 alpha , beta I, beta II, gamma , and delta . 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 PKCalpha , beta I, beta II, gamma , and delta , measured in split samples, were not altered by antisense expression (Fig. 4A, lower panels). Since PKCdelta 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 PKCdelta 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 PKCdelta and that PKCdelta autophosphorylation was regulated by CD45, Western analysis was performed on using a PKCdelta -specific immunoprecipitating antibody. Fig. 4C (top panel) shows that, in U937-pCEP4 cells, 100 nM PMA induced a 3-fold autophosphorylation of PKCdelta at 5 min but that, in U937-CD45as cells, PMA caused a 12-fold autophosphorylation of PKCdelta . To determine the potential mechanism by which CD45 modulates PMA-dependent PKCdelta activity, phosphorylation of PKCdelta was examined in split immunoprecipitates. Fig. 4C (upper panel) demonstrates that PMA induced a 3-fold increase in PKCdelta 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 PKCdelta 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 PKCdelta was unaffected by CD45 expression or PMA treatment. Finally, to examine PKCalpha , -beta I, -beta II, and -gamma activation and tyrosine phosphorylation, Western analysis was again performed on split immunoprecipitates. Fig. 4D demonstrates that the PKC isoforms alpha , beta I, beta II and gamma  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 PKCalpha , -beta I, -beta II, and -gamma were present in the immunoprecipitates from PMA-treated cells and that PKCalpha , -beta I, -beta II and -gamma mass were unaffected by CD45 expression. These findings show that PKCdelta is required for PMA-induced CD11b surface expression and that CD45 negatively regulates the autophosphorylation and tyrosine phosphorylation of PKCdelta . Overall, these data indicate that control of PKCdelta tyrosine phosphorylation by CD45 regulates PMA-induced PKCdelta activity and subsequent monocyte differentiation.


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Fig. 4.   PMA-dependent activation and tyrosine phosphorylation of PKCdelta is negatively regulated by CD45. A, U937-pCEP4 and U937-CD45as cells were treated with 100 nM PMA for the times indicated and autophosphorylated PKCalpha , -beta I, -beta II, -gamma , and -delta (PKCalpha ,beta I,beta II,gamma ,delta -P) were measured by Western analysis from whole cell lysates using a phospho-PKCalpha ,beta I,beta II,gamma ,delta antibody (upper panel). PKCalpha ,beta I,beta II,gamma ,delta mass were measured by Western analysis from whole cell lysates from U937-pCEP4 and U937-CD45as cells using a PKCalpha ,beta I,beta II,gamma antibody (lower panel, left) and a PKCdelta 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 alpha  = 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 PKCdelta (PKCdelta -P) and mass of PKCdelta were measured in PKCdelta 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) PKCalpha , -beta I, -beta II, and -gamma (PKCalpha ,beta I,beta II,gamma -) and mass of PKCalpha , -beta I, -beta II, and -gamma (lower panel) were measured in PKCalpha , -beta I, -beta II, and -gamma immunoprecipitates (IP) by Western analysis. PKCdelta immunoprecipitates from U937-CD45as cells serve as positive controls. Results are representative of three independent experiments.


    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

These data establish that CD45 negatively regulates PMA-dependent monocytic cell differentiation by impairing PKCdelta 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 PKCdelta inhibitor rottlerin reduced by 66% PMA-dependent CD11b expression (Fig. 4B) while CD45 antisense expression quadrupled PMA-dependent PKCdelta activation and tyrosine phosphorylation (Fig. 4C). Notably, PKC isoforms alpha , beta I, beta II, and gamma  were neither activated nor tyrosine-phosphorylated by PMA (Fig. 4D). Taken together, these findings indicate that CD45 blunts PKCdelta activation by reducing PMA-dependent PKCdelta tyrosine phosphorylation and that this inhibition in PKCdelta 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 PKCalpha and PKCdelta were essential for PMA-induced macrophage differentiation while PKCbeta II, -epsilon , -zeta , and -eta were not required (40). Subsequent studies in the same cell system demonstrated that only PKCdelta appeared to be required for phorbol ester-induced macrophage differentiation (41). Our data support these findings in that we found that PKCdelta but not PKCalpha , -beta I, -beta II, and -gamma 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 delta  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 PKCdelta 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 PKCdelta 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, PKCdelta 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 PKCdelta 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 PKCdelta (46-48). Currently, the role of PKCdelta tyrosine phosphorylation is not clear. Studies have demonstrated that PKCdelta tyrosine phosphorylation can both increase (38) and reduce (49) PCKdelta activity. Here we show that PKCdelta is tyrosine-phosphorylated in response to PMA and that antisense reduction in CD45 expression increases PMA-dependent PKCdelta tyrosine phosphorylation. Importantly, the increase in PKCdelta tyrosine phosphorylation seen with loss of CD45 expression is associated with increased PKCdelta autophosphorylation, ERK1/2 activation, and differentiation, indicating that CD45-regulated tyrosine phosphorylation is likely responsible for reducing PKCdelta serine 660 phosphorylation and, hence, its autophosphorylation and activation. It is then this reduction in PKCdelta activity that results in reduced ERK1/2 activation and reduced CD11b expression.

Unclear, however, is how CD45 might regulate PKCdelta 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 PKCdelta 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 PKCdelta tyrosine phosphorylation in CD45-deficient monocytic cells. Finally, CD45 may directly dephosphorylate PKCdelta controlling PKCdelta activity at the inner membrane surface. Preliminary data from our laboratory show that CD45 can dephosphorylate in vitro tyrosine-phosphorylated PKCdelta , but, as indicated above, tyrosine phosphatases tend to have little in vitro substrate specificity. In summary, we found that CD45 negatively regulates PMA-induced PKCdelta autophosphorylation, PKCdelta tyrosine phosphorylation, and monocytic cell differentiation. We conclude that CD45 inhibits PMA-dependent PKCdelta tyrosine phosphorylation, thereby blunting PKCdelta activation and PKCdelta -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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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