Journal of Leukocyte Biology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Junttila, I.
Right arrow Articles by Silvennoinen, O.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Junttila, I.
Right arrow Articles by Silvennoinen, O.
(Journal of Leukocyte Biology. 2003;73:281-288.)
© 2003 by Society for Leukocyte Biology

M-CSF induced differentiation of myeloid precursor cells involves activation of PKC-{delta} and expression of Pkare

Ilkka Junttila*, Roland P. Bourette{dagger}, Larry R. Rohrschneider{ddagger} and Olli Silvennoinen*,§

* Laboratory of Molecular Immunology, Institute of Medical Technology, and
§ Department of Clinical Microbiology, Tampere University Hospital, University of Tampere, Finland;
{dagger} Centre de Génetique Moléculaire et Cellulaire, Université Claude Bernard Lyon 1, Villeurbanne Cedex, France; and
{ddagger} Fred Hutchinson Cancer Research Center, Division of Basic Sciences, Seattle, Washington

Correspondence: Dr. Olli Silvennoinen, Institute of Medical Technology, University of Tampere Lenkkeilijänkatu 8, 33014 Tampere, Finland. E-mail: olli.silvennoinen{at}uta.fi


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Macrophage-colony stimulating factor (M-CSF) regulates proliferation and differentiation of cells belonging to the monocytic lineage. We investigated the mechanisms of M-CSF differentiation signaling in follicular dendritic cell-P1 cells and analyzed the catalytic activation of different protein kinase C (PKC) isoforms. M-CSF induced rapid catalytic activation of PKC-{delta} and membrane translocation of the tyrosine phosphorylated form of PKC-{delta}. Mutation of tyrosine 807 in the M-CSF receptor (Fms) abrogates cell differentiation but not a proliferative response to M-CSF, and FmsY807F failed to activate PKC-{delta}. We also investigated the downstream signaling pathways from PKC-{delta}. A cyclic adenosine monophosphate-regulated Ser/Thr kinase gene, protein kinase X (PRKX), has been associated with macrophage differentiation in human cells. We found that M-CSF and PKC-{delta} induced the expression of the PRKX murine homologue: PKA-related gene. Taken together, our results indicate that PKC-{delta} functions as a critical mediator of M-CSF-induced differentiation signaling.

Key Words: signal transduction • tyrosine phosphorylation • ser/thr kinase


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Monocytes and their terminally differentiated counterparts, macrophages, have a central role in immune and inflammatory responses [1 ]. Differentiation of macrophages from bone marrow stem cells is precisely regulated by hematopoietic cytokines. Interleukin-3 (IL-3) is a growth and survival factor for immature and mature myeloid cells. For monocytes, macrophage-colony stimulating factor (M-CSF; also termed colony stimulating factor-1, CSF-1) is the principal regulator of proliferation and terminal differentiation. The receptor for M-CSF (Fms) is a receptor tyrosine kinase. Ligand binding induces dimerization and activation of kinase domains of Fms receptor chains resulting in phosphorylation of specific tyrosine residues such as Y559, Y697, Y706, Y721, Y807, and Y973 in the cytoplasmic domain of the receptor. Phosphorylated tyrosine residues then serve as docking sites for Src homology-2 domain-containing signaling molecules including Src family members, suppressor of cytokine signaling-1, signal transducer and activator of transcription 1, phosphatidylinositol 3-kinase (PI-3K), phospholipase C-{gamma}2 (PLC-{gamma}2), and c-Cbl [2 3 4 5 6 7 8 9 10 11 ].

Functional characterization of Fms mutants has indicated that Y697, Y706, and Y721 are facilitating M-CSF-induced differentiation, but they are not required for the process. In contrast, mutation of Y807, which controls PLC-{gamma}2 binding to phosphorylated Y721, completely abrogates the differentiation response but also simultaneously enhances M-CSF-dependent proliferation [12 ]. Signaling mechanisms related to Y721 and Y807 in Fms have been analyzed in detail, and the differentiation signaling is dependent on the balance between PLC-{gamma}2 and PI-3K activation. However, the downstream events of Fms-mediated differentiation signaling have remained uncharacterized but the end product of PLC-{gamma}2 catalysis; diacylglycerol (DAG) is a critical activator of protein kinase C (PKC).

Several lines of evidence have implicated PKC in regulation of macrophage differentiation. Activation of PKC by pharmacological agents such as 12-O-tetradecanoyl-phorbol-13-acetate (TPA) induces macrophage differentiation, and also M-CSF stimulation results in increased PKC activity [13 ]. PKC is a Ser/Thr kinase family consisting of at least 12 isoforms. The activation and expression patterns of different isoforms vary in tissues, and increasing evidence is accumulating about specific cellular functions for various PKC isoforms [14 15 16 ]. Recent evidence from genetically modified mice lacking functional PKC genes also confirms this concept by showing distinct phenotypes and clear differences in the nonredundant functions of various PKC isoforms [17 18 19 ]. PKC isoforms {alpha}, ß, and {delta} have been implicated in induction of macrophage differentiation in normal progenitor cells as well as in human and murine myeloid cell lines [20 , 21 ].

PKC isoforms are divided into three classes: conventional, novel, and atypical, according to the requirements for cofactors Ca2+ and DAG for their activation [14 ]. In addition, tyrosine phosphorylation has been shown to regulate PKC {alpha}, {delta}, and {theta} isoforms [22 , 23 ]. Certain growth factors, such as platelet-derived growth factor-ß and epidermal growth factor (EGF), induce tyrosine phosphorylation of PKC-{delta} [24 , 25 ]. Several tyrosine phosphorylation sites of PKC-{delta} have been identified [26 27 28 ]. Phosphorylation of these different tyrosine residues has diverse and even cell type-specific effects on PKC-{delta} activity, such as increased resistance to proteolytic degradation and modulation of downstream signaling [27 , 28 ].

Identification of downstream targets for PKC is essential for understanding the signaling mechanisms underlying the various PKC-regulated cellular responses. One putative differentiation-related target for PKC signaling is a novel Ser/Thr kinase, protein kinase X (PRKX), which is expressed in human myeloid cells, and its expression is induced by PKC-ß during terminal myeloid differentiation. [29 ]. PRKX belongs to a family of cyclic adenosine monophosphate (cAMP)-dependent protein kinases [30 ]. A homologous mouse gene, PKA-related gene (Pkare), was recently identified, but its function remains unknown [31 ].

Early findings that PKC is activated during M-CSF-induced differentiation [13 ] led us to study the catalytic activation of different PKC isoforms that are expressed in myeloid progenitor cells. PKC-{delta} was rapidly activated and tyrosine-phosphorylated upon M-CSF stimulation, and this activation directly correlated with macrophage differentiation signaling. Finally, we show that M-CSF induces expression of Pkare and that PKC-{delta} regulates Pkare expression in differentiating myeloid progenitor cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture and growth factor stimulation
Myeloid follicular dendritic cells (FDC)-P1 (clone 19) expressing wild-type (wt) or mutant Fms have been described previously [12 ]. 32D and 32D PKC-{delta} cells were kind gift from Dr. Weigun Li (Georgetown University, Washingon, D.C.) and have been described previously [20 ]. For growth factor stimulation, cells were starved in 1% medium as indicated in figure legends and stimulated with specified growth factors.

Subcellular fractionation
Cells were lysed by vortexing without sonication on ice for 10 min in 600 µl hypotonic buffer (10 mM HEPES, 1 mM MgCl2, 1 mM EDTA, 4 mM di-isopropylfluorophosphate, 0.5% aprotinin, 10 µg/ml leupeptin, pH 7.5) containing 1 mM phenylmethylsulfonyl fluoride and 200 µM orthovanadate. Particulate fraction-containing membranes and nuclei and soluble fraction were separated by a 30-min centrifugation (4°C, 100,000 g). To obtain the soluble fraction, the supernatant was centrifuged once again (30 min, 4°C, 100,000 g). To purify particulate fraction, the pellet was washed by 1 ml hypotonic buffer to eliminate soluble contaminations and then lysed in 600 µl radio immunoprecipitation assay (RIPA) lysis buffer [1% deoxycholic acid, 1% Nonidet P-40 (NP-40), 0.1% sodium dodecyl sulfate (SDS), 10 mM Tris-base, 150 mM NaCl, 0.5% aprotinin, pH 7.4] supplemented with protease inhibitors. Finally, to obtain the purified particulate fraction, insoluble particles were removed by 30-min centrifugation (4°C, 100,000 g).

Immunoprecipitation and Western blotting
Cells were lysed in NP-40 lysis buffer (0.5% NP-40, 10 mM Tris-base, 50 mM NaCl, 30 mM Na4P2O7, 50 mM sodium fluoride, 20 mM iodoacetimide, 5 µM ZnCl2, pH 7.3), RIPA lysis buffer (described above), or Triton lysis buffer (50 mM Tris-HCL, 10% glycerol, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 50 mM NaF, 1 mM Na3VO4, pH 7.5) supplemented with protease inhibitors. Immunoprecipitations were performed as described previously [32 ]. Immunocomplexes were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to nitrocellulose membrane (Micron Separation, Westborough, MA). Immunodetection was performed using specific primary antibodies, biotinylated anti-mouse or anti-rabbit secondary antibodies (Dako A/S, Glustrup, Denmark), and streptavidin-biotin horseradish peroxidase conjugate and electrochemiluminescence detection (Amersham Pharmacia Biotech, Piscataway, NJ). When mentioned, the filter was stripped for 1 h at 56°C in a solution containing 100 mM 2-mercaptoethanol, 2% SDS, 62.5 mM Tris-HCL, pH 6.8, washed twice for 15 min in rinse solution (0.05% Tween-Tris-buffered saline), and subjected to a new immunodetection. Antibodies used were antiphosphotyrosine antibody (clone 4G10; Upstate Biotechnology, Lake Placid, NY), monoclonal anti-PKC-{delta} N-terminal antibody (Transduction Laboratories, Lexington, KY), polyclonal anti-PKC-{delta} C-terminal antibody (Gibco-BRL Life Technologies, Gaithersburg, MD), polyclonal anti-PKC-{alpha} and -ß antibodies (Gibco-BRL Life Technologies), monoclonal anti-PKC-{varepsilon} antibody (Transduction Laboratories), and polyclonal anti-PKC-{zeta} antibody (Chemicon International Inc., Temecula, CA). The polyclonal antimurine-Fms antibody was made against the cytoplasmic domain of Fms [10 ].

PKC in vitro kinase assay
The PKC in vitro kinase assay has been described previously [33 ]. Briefly, PKC was immunoprecipitated, suspended in kinase-reaction buffer (0.04 mg/ml phosphatidyl-L-serine, 10% glycerol, 0.1 mM CaCl2, 0,02% Triton X-100, 10 mM MgCl2, 20 mM HEPES, 0.25 µCi/ml {gamma}-adenosine 5'-triphosphate, pH 7.4) for 30 min at 30°C. The immunocomplexes were resolved in SDS-PAGE, and the gels were vacuum-dried and exposed for autoradiography.

Reverse transcriptase-polymerase chain reaction (RT-PCR)
For 32D and 32D PKC-{delta} cells, RT-PCR was performed as described previously [31 ]. Briefly, cells were stimulated as indicated in figure legends, followed by total RNA isolation using Trizol reagent (Gibco-BRL Life Technologies). Total RNA was then used for the first-strand cDNA synthesis with monkey murine leukemia virus transcriptase (Gibco-BRL Life Technologies) and random hexamers (Amersham Pharmacia Biotech), according to the manufacturers’ protocol. PCR was performed with primers for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) [34 ] and Pkare [31 ] with PCR conditions as follows: 94°C, 1 min; 55°C, 1 min; 72°C, 3 min. Reaction was repeated 25 times, and 25 µl of the product was loaded on a 1.2% ethidiumbromide-containing agarose gel. For FDC-P1 myeloid precursor cells that stably express Fms wt (FD-Fms-wt) cells, RNA was extracted from cells maintained in the presence of IL-3 or M-CSF for 1, 2, or 3 days, using the Rneasy kit (Qiagen, Valencia, CA). Total RNA (2 µg) was reverse-transcribed using random hexamers and the Omniscript RT kit (Qiagen). One-tenth of the first-strand cDNA synthesis reaction was used as template for the PCR reactions using Pkare and GAPDH primers (described above). PCR conditions were 95°C, 30 s; 60°C, 30 s; and 72°C, 1 min; repeated 35 times. Products were size-fractionated on a 1.5% ethidiumbromide-containing agarose gel.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
M-CSF induces PKC-{delta} kinase activity
We wanted to characterize the roles of different PKC isoforms in M-CSF signaling by analyzing their activation in FD-Fms-wt. FDC-P1 cells are self-renewing in the presence of IL-3, but they lack endogenous Fms. Expression of Fms renders the cells responsive to M-CSF-induced differentiation.

Previously, activation of PKC by physiological ligands has been measured mainly by protein translocation rather than by kinase activity. The very rapid kinetics of PLC-{gamma}2 activation (5 s) in M-CSF signaling [10 ] led us to consider the possibility that the catalytic activation of PKC may be very rapid. Starved FD-Fms-wt cells were treated with M-CSF for 45 s or left untreated. We focused our analysis to the five PKC isoforms that are mainly expressed in hematopoietic cells, namely the conventional PKC isoforms {alpha} and ß, the novel isoforms {delta} and {varepsilon}, and the atypical isoform {zeta}. The different PKC isoforms were immunoprecipitated and subjected to PKC in vitro kinase assay followed by separation in SDS-PAGE. The expression of various PKC isoforms in FDC-P1 cells was confirmed by Western blotting (data not shown). The 80-kDa PKC-{alpha} was constitutively active in FD-Fms-wt cells and M-CSF stimulation for 45 s or 3, 10, or 30 min and had no effect on its kinase activity (Fig. 1A and data not shown). No catalytic activity was detected corresponding to the molecular weight of full-length PKC-ß (80 kDa). Nonetheless, M-CSF increased the phosphorylation of an unknown 50-kDa protein in PKC-ß immunoprecipitates. PKC-ß is proteolytically cleaved into C- and N-terminal fragments upon activation with TPA [35 ]. However, the observed phosphoprotein is not likely to represent the C-terminal, catalytic domain of PKC-ß, as the antibody is targeted against the N terminus of PKC-ß. It remains possible that this phosphoprotein is a coprecipitating substrate protein that is efficiently phosphorylated by PKC-ß. PKC-{varepsilon} or -{zeta} showed no marked differences in autokinase activity in response to M-CSF treatment upon 45-s or 3-, 10-, or 30-min stimulation (Fig. 1A and data not shown). M-CSF did not stimulate the autokinase activity of full-length (78 kDa) PKC-{delta}, but increased phosphorylation of a 35-kDa phosphoprotein was observed. The 35-kDa phosphoprotein is likely to represent the catalytic fragment of PKC-{delta}, as the antibody used in this experiment was against the C terminus of PKC-{delta}, and PKC-{delta} is degraded upon activation into a 30- to 40-kDa catalytically active C-terminal fragment and a 40-kDa regulatory fragment [36 ]. By using an antibody against the N terminus of PKC-{delta}, we observed a modest increase by M-CSF in full-length PKC-{delta} activity, but we did not detect the 35-kDa fragment (data not shown).



View larger version (33K):
[in this window]
[in a new window]
  		Figure 1. M-CSF induces PKC-{delta} kinase activity. (A) FD-Fms-wt cells were starved for 12 h [1% fetal bovine serum (FBS)] and were stimulated with murine recombinant (mr)M-CSF (5000 U/ml) as indicated. Total cellular protein (1000 µg) was immunoprecipitated (ip) with indicated PKC antibody. The immunocomplexes were subjected to PKC in vitro kinase assay and resolved by SDS-PAGE before autoradiography. PKC-{delta} catalytic fragment, immunoglobulin (Ig) complexes, and molecular weight marker are indicated. (B) Experiment was performed as in A but with the indicated times of mrM-CSF stimulation, and only PKC-{delta} was immunoprecipitated.

 
A kinetic experiment was performed to verify the activation of PKC-{delta} (Fig. 1B) . FD-Fms-wt cells were both left untreated or were stimulated with M-CSF for 45 s or 3 min, and the kinase activity of PKC-{delta} was analyzed. The autokinase activity of full-length PKC-{delta} and the C-terminal catalytic fragment were markedly increased upon 45-s M-CSF stimulation, and the activity returned to basal level within 3 min after stimulation. The PKC-{delta} protein levels were equal, as shown by the Western blot from the total cell lysates.

Taken together, our results demonstrate that the PKC-{delta} isoform was rapidly and transiently activated by M-CSF. PKC isoforms {alpha} and {zeta} were found to be constitutively active in FD-Fms-wt cells, but their activities were not regulated by M-CSF. It remains possible that M-CSF induced the catalytic activity of PKC-ß or the activity of a PKC-ß-associated, unknown protein.

M-CSF induces PKC-{delta} tyrosine phosphorylation
The rapid catalytic activation of PKC-{delta} led us to investigate more closely the regulation of PKC-{delta} in myeloid growth factor signaling, and we wanted to analyze the effects of IL-3, granulocyte M-CSF (GM-CSF), and M-CSF on tyrosine phosphorylation of PKC-{delta}. First, induction of cellular tyrosine phosphorylation in FD-Fms-wt cells was investigated. M-CSF induced very prominent cellular tyrosine phosphorylation when compared with IL-3 or GM-CSF-treated cells (Fig. 2A ). Tyrosine phosphorylation of PKC-{delta} was detected after M-CSF but not after IL-3 or GM-CSF stimulations, and the phosphorylated form of PKC-{delta} exhibited slower migration when compared with the nonphosphorylated form (Fig. 2A) . To determine if PKC-{delta} tyrosine phosphorylation is an early event in Fms signaling, starved FD-Fms-wt cells were stimulated with M-CSF for different times (Fig. 2B) . Tyrosine-phosphorylated PKC-{delta} was detected as early as 5 s after M-CSF stimulation, with a maximum between 30 s and 1 min. Tyrosine phosphorylation then decreased rapidly and was not detectable after 5 min. Reprobing the filter with anti-PKC-{delta} antibody showed that the pattern of the slowly migrating PKC-{delta} paralleled with the tyrosine-phosphorylated fraction of PKC-{delta}.



View larger version (53K):
[in this window]
[in a new window]
  		Figure 2. PKC-{delta} tyrosine phosphorylation is induced by M-CSF. (A) FD-Fms-wt cells were starved for 3 h (1% FBS) and were stimulated as indicated on ice for 1 h. mrIL-3 (500 ng/ml), 5000 U/ml mrGM-CSF, and 5000 U/ml mrM-CSF were used. Total cellular protein (25 µg) was loaded on each lane and resolved by SDS-PAGE, and tyrosine phosphorylation (PY) was detected by immunoblotting. For PKC-{delta} immunoprecipitation (IP), 1000 µg total cellular protein was immunoprecipitated with N-terminal antibody. After immunoblotting, the filter was stripped and reprobed with N-terminal anti-PKC-{delta} antibody. PKC-{delta}, Ig complexes, and molecular weight marker are indicated. (B) FD-Fms-wt cells were starved for 3 h (1% FBS) and were stimulated with mrM-CSF (5000 U/ml) at 37°C as indicated. Total cellular protein (1000 µg) was immunoprecipitated with N-terminal anti-PKC-{delta} antibody, and immunocomplexes were separated by SDS-PAGE. Tyrosine phosphorylation was detected by immunoblotting. After immunoblotting, the filter was stripped and reprobed with the N-terminal anti-PKC-{delta} antibody.

 
M-CSF causes cellular translocation of PKC-{delta}
One hallmark of PKC activation is translocation of PKC from cytosol to cell membranes, where different anchoring proteins facilitate interaction of PKC with substrates [37 ]. A comparison of M-CSF-induced tyrosine phosphorylation revealed clear differences in the pattern of phosphoproteins between soluble and particulate fractions. The intensity of phosphorylation was more prominent in a particulate fraction, where the mature and immature forms of Fms also localized (Fig. 3A , upper and lower panels). The decrease in Fms levels upon longer stimulation (>5 min) is likely to be a result of the internalization and degradation of Fms [38 , 39 ].



View larger version (72K):
[in this window]
[in a new window]
  		Figure 3. PKC-{delta} is rapidly translocated to cell membranes upon M-CSF stimulation. (A) FD-Fms-wt cells were starved for 3 h (1% FBS) and were stimulated with mrM-CSF (5000 U/ml) at 37°C as indicated. Soluble and particulate cell fractions were separated as described in Materials and Methods. Total cellular protein (25 µg) from different fractions was loaded on each lane and resolved by SDS-PAGE. Tyrosine phosphorylation was detected by immunoblotting. After immunoblotting, the filter was stripped and reprobed with antimurine-Fms-antibody. (B) FD-Fms-wt cells were starved for 3 h (1% FBS) and were stimulated with mrM-CSF (5000 U/ml) at 37°C for indicated times. Soluble and particulate cell fractions were separated as described in Materials and Methods. Equal amounts of protein from both fractions were immunoprecipitated with N-terminal anti-PKC-{delta} antibody and resolved by SDS-PAGE. Tyrosine phosphorylation was detected by immunoblotting. After immunoblotting, the filter was stripped and reprobed with N-terminal anti-PKC-{delta} antibody.

 
PKC activity has been shown to translocate to cellular membranes in human monocytes after M-CSF stimulation [13 ], but the involvement of different PKC isoforms was not analyzed. We wanted to study if M-CSF stimulation causes PKC-{delta} translocation to a membrane fraction. In untreated, starved FD-Fms-wt cells, PKC-{delta} was not tyrosine-phosphorylated and was localized in a soluble fraction (Fig. 3B) . Tyrosine phosphorylation of PKC-{delta} was observed after 5 s of M-CSF stimulation in soluble and particulate fractions. After 30-s and 1-min stimulations, all tyrosine-phosphorylated PKC-{delta}, as well as most of the protein, was in the particulate fraction. Tyrosine phosphorylation was not observed any more at 5 min, and gradually, the PKC-{delta} protein returned to a soluble fraction. These results indicate that tyrosine phosphorylation of PKC-{delta} is an immediate event in M-CSF-induced signal transduction.

Mutation of Fms intracellular tyrosine residues decreases PKC-{delta} tyrosine phosphorylation
Upon activation, Fms has been shown to induce several cellular-signaling pathways [2 ]. To investigate the signal requirements for PKC-{delta} activation, the effects of the previously characterized Fms tyrosine mutations on PKC-{delta} activation were analyzed. Mutation of Y721 has no significant effect on M-CSF-induced differentiation, and Y807 is absolutely required for differentiation [12 ]. M-CSF-triggered PKC-{delta} activation was investigated in FDC-P1 cells stably expressing tyrosine-to-phenylalanine mutation at Y721 or Y807 of Fms (FD-Fms-721 and FD-Fms-807). FDC-P1 parental cells, which lack expression of Fms, were used as a control. Total cellular phosphorylation was slightly decreased in mutant Y807F (Fig. 4A ). The M-CSF-induced tyrosine phosphorylation of PKC-{delta} was reduced in FD-Fms-721, and in FD-Fms-807 cells, PKC-{delta} tyrosine phosphorylation was strongly decreased (Fig. 4B) . PKC-{delta} protein levels were similar in different cell lines (Fig. 4B , lower panel). We also investigated the effects of the mutations on PKC-{delta} translocation. The cellular translocation of PKC-{delta} in response to M-CSF was decreased in the FD-Fms-721 mutant, but some tyrosine-phosphorylated PKC-{delta} was detected in the membrane fraction (Fig. 4C) . In contrast, FD-Fms-807 was completely unable to induce membrane translocation of PKC-{delta}. Taken together, these results indicate that mutation of Y807 disrupts the ability of Fms to activate PKC-{delta}, which is consistent with the role of PKC-{delta} in Fms-induced differentiation.



View larger version (36K):
[in this window]
[in a new window]
  		Figure 4. Mutation of Fms tyrosines 721 and 807 decreases PKC-{delta} tyrosine phosphorylation and translocation to cell membranes. (A) FDC-P1 clone 19, FD-Fms-wt, FD-Fms-721, and FD-Fms-807 cells were starved for 12 h (1% FBS) and were stimulated with mrM-CSF (5000 U/ml) as indicated. Total cellular protein (20 µg) was loaded on each lane and resolved by SDS-PAGE. Tyrosine phosphorylation was detected by immunoblotting. (B) FDC-P1 clone 19, FD-Fms-wt, FD-Fms-721, and FD-Fms-807 cells were starved for 12 h (1% FBS) and were stimulated with mrM-CSF (5000 U/ml) as indicated. Total cellular protein (1000 µg) was immunoprecipitated with N-terminal anti-PKC-{delta} antibody, and immunocomplexes were resolved by SDS-PAGE. Tyrosine phosphorylation was detected by immunoblotting. After immunoblotting, the filter was stripped and reprobed with N-terminal anti-PKC-{delta} antibody. (C) FD-Fms-721 and FD-Fms-807 cells were starved for 12 h (1% FBS) and were stimulated with mrM-CSF (5000 U/ml) as indicated. Soluble and particulate cell fractions were separated as described in Materials and Methods. Equal amounts of protein from both fractions were immunoprecipitated with N-terminal anti-PKC-{delta} antibody and resolved by SDS-PAGE. Tyrosine phosphorylation was detected by immunoblotting. After immunoblotting, the filter was stripped and reprobed with N-terminal anti-PKC-{delta} antibody. To detect the weak tyrosine phosphorylation signal of Fms mutants, the X-ray film exposure time in the phosphotyrosine blot was 10 min compared with 1 min used in B.

 
M-CSF and PKC-{delta} induce Pkare expression
We then wanted to study the downstream signaling events from PKC-{delta} activation. During monocytic differentiation of human HL-60 cells, PKC-ß has been shown to induce expression of PRKX, which appears to be required for macrophage differentiation [29 ]. The murine counterpart gene for PRKX has been cloned and termed Pkare, but the function of this kinase is currently unknown [31 ]. To investigate whether Pkare expression associates with monocytic differentiation in murine myeloid cells, the role of Pkare was first analyzed in FDC-P1 cells during M-CSF-induced differentiation. The FD-Fms-wt cells were stimulated with M-CSF for 24, 48, or 72 h or were cultured in the presence of IL-3. Total RNA was extracted from differentiated and nondifferentiated cells (Fig. 5A , lane 1) transcribed into cDNA, and PCR was performed with Pkare and GAPDH primers. Pkare expression was significantly increased after the M-CSF treatment, and the highest expression was after 3 days; i.e., when the cells were fully differentiated into macrophage-like cells (Fig. 5A) . To directly assess the role of PKC-{delta} in Pkare expression, we used another IL-3-dependent murine myeloid progenitor cell, 32D. 32D cells express low levels of PKC-{delta} and fail to differentiate in response to TPA treatment, but ectopic expression of PKC-{delta} renders the cells responsive to TPA-induced macrophage differentiation [20 ]. It is interesting that TPA induced Pkare expression in 32D-PKC-{delta} cells but not in 32D cells (Fig. 5B) . Taken together, both M-CSF and PKC-{delta} induced differentiation results in increased Pkare expression.



View larger version (23K):
[in this window]
[in a new window]
  		Figure 5. M-CSF and PKC-{delta} induce Pkare expression. (A) FD-Fms-wt cells were cultivated in the presence of mrIL-3 or mrM-CSF (2500 U/ml) for 1, 2, or 3 days. Total RNA was isolated, and PCR reactions were performed with equal amounts of reverse-transcribed cDNA with Pkare and GAPDH primers as described in Materials and Methods. (B) 32D and 32D PKC-{delta} cells were stimulated with TPA (100 ng/ml) for 16 h as indicated. Experiment was performed as in A. PCR products were separated in a 1.2% ethidiumbromide-containing agarose gel and were photographed under UV light. (C) 32D and 32D PKC-{delta} cells, maintained in IL-3, were lysed, and 20 µg total cellular protein was loaded on each lane and resolved by SDS-PAGE. PKC-{delta} expression was detected by immunoblotting.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Hematopoietic growth factors regulate gene expression during differentiation of multipotent stem cells into terminally differentiated macrophages. Here we show that M-CSF-induced differentiation of macrophages involves rapid activation of PKC-{delta}, which in turn stimulates the expression of Pkare.

PKC isoforms show clear differences in terms of expression pattern, activation mechanisms, and functions [17 18 19 20 21 , 40 ]. In monocytes, both the total PKC activity and PKC-{alpha} translocation have been shown to be increased by M-CSF [13 , 41 ]. Our results now demonstrate rapid and transient activation of PKC-{delta} in M-CSF-induced signaling. The rapid PKC-{delta} activation correlates well and probably reflects the rapid activation kinetics of the upstream activator of PKC, namely PLC-{gamma}2. It is possible that the transient activation reflects the translocation of PKC-{delta}, first into the cell membranes and after proteolytic cleavage, into the nucleus.

Membrane translocation of PKC-{delta} after M-CSF stimulation was rapid and transient. Most of the tyrosine-phosphorylated PKC-{delta} was detected in the particulate fraction. The precise mechanism of PKC-{delta} tyrosine phosphorylation in M-CSF signaling is currently unknown but decreased tyrosine phosphorylation associated with decreased cellular translocation. This suggests that Fms, either directly or by activating other tyrosine kinases, increases PKC-{delta} tyrosine phosphorylation, which results in translocation. In the case of EGF stimulation, tyrosine phosphorylation of PKC-{delta} required a downstream kinase from the EGF receptor [25 ]. However, M-CSF induced PKC-{delta} tyrosine phosphorylation at 4°C (Fig. 2A) , which favors the possibility that the receptor itself is mediating the phosphorylation.

In the FDC-P1 myeloid differentiation model, the magnitude of PKC-{delta} activation and tyrosine phosphorylation correlated with the differentiation responses induced by the different receptors (Y721F, Y807F). As mutation of Y807 of Fms abrogates differentiation but simultaneously enhances proliferation of FDC-P1 cells [12 ], it is probable that this mutation alleviates a negative signal for M-CSF- and IL-3-mediated proliferation. We have previously shown that IL-3-induced mitogenic signaling is inhibited by PKC-{delta} [33 ]. These results support the concept that PKC-{delta} could mediate the M-CSF-induced growth-inhibitory signal at least partially. In support of this, PKC-{delta} has been implicated in the negative regulation of cell division, also in other cell types such as fibroblasts and B cells [19 , 42 ].

PKC activation is associated with regulation of several cellular-signaling cascades leading to cell growth, differentiation, and apoptosis. However, the critical target genes of PKC in differentiation signaling are still poorly characterized. One potentially important target gene is PRKX, which is regulated by PKC-ß during myeloid differentiation of human HL-60 cells. Of human tissues, PRKX is abundantly expressed only in freshly isolated granulocytes and macrophages. Inhibition of PRKX by antisense oligonucleotides blocked M-CSF-induced differentiation of human peripheral blood monocytes [29 ]. We found that the mouse counterpart of PRKX, Pkare, is regulated by M-CSF in mouse macrophage differentiation models, and its expression was induced by PKC-{delta} (Fig. 5) . The precise function of Pkare in myeloid differentiation is still unknown and an important question for future studies. Pkare is related to PKA, and human homologue PRKX is regulated by cAMP. Increased intracellular cAMP is associated with monocytic differentiation, and cAMP inhibits IL-3- and M-CSF-induced proliferation in FD-Fms-wt cells [12 ]. cAMP-dependent kinases have been shown to play an important role in the regulation of transcription through phosphorylation of the cAMP response element binding protein (CREB), and also PRKX has been shown to participate in CREB-mediated transcription [29 , 43 , 44 ]. Based on these findings and the data presented here, Pkare could be an effector for M-CSF-induced differentiation. Our results indicate that Fms-induced PLC-{gamma}2 activation results in PKC-{delta} activation, which in turn induces the expression of the Pkare gene. Increased Pkare expression might then regulate the transcription of other differentiation-associated genes. The mechanism of Pkare regulation remains unknown, but PKC-{delta} is likely to regulate the promoter and transcription of Pkare. However, it is also possible that PKC-{delta} could directly regulate Pkare activity.

Macrophage differentiation is associated with growth inhibition and induction of a specific set of genes. We hypothesize that PKC-{delta} mediates at least two signals in regulation of myeloid differentiation. Our results strongly suggest that PKC-{delta} is mediating M-CSF-induced differentiation signaling, and a potential target gene for PKC-{delta}-induced signaling is Pkare. Second, the growth-inhibitory signal of Fms appears to be, at least partially, mediated by PKC-{delta}. These findings place PKC-{delta} in a critical position to coordinate cellular homeostasis during macrophage differentiation.


   ACKNOWLEDGEMENTS
 
This work has been supported by the Academy of Finland, the Finnish Cultural Foundation, the Finnish Cancer Foundation, the Sigrid Juselius Foundation, and the Medical Research Fund of Tampere University Hospital. We thank Paula Kosonen for technical assistance, Dr. O. Jaakkola for help with subcellular fractionation, and Dr. W. Li for 32D cells.

Received July 15, 2002; revised November 1, 2002; accepted November 4, 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Unanue, E. R., Allen, P. M. (1987) The basis for the immunoregulatory role of macrophages and other accessory cells Science 236,551-557[Abstract/Free Full Text]
  2. Hamilton, J. A. (1997) CSF-1 signal transduction J. Leukoc. Biol. 62,145-155[Abstract]
  3. Bourette, R. P., Rohrschneider, L. R. (2000) Early events in M-CSF receptor signaling Growth Factors 17,155-166[Medline]
  4. Sengupta, A., Liu, W. K., Yeung, Y. G., Yeung, D. C., Frackelton, A. R., Jr, Stanley, E. R. (1988) Identification and subcellular localization of proteins that are rapidly phosphorylated in tyrosine in response to colony-stimulating factor 1 Proc. Natl. Acad. Sci. USA 85,8062-8066[Abstract/Free Full Text]
  5. Marks, D. C., Csar, X. F., Wilson, N. J., Novak, U., Ward, A. C., Kanagasundarum, V., Hoffmann, B. W., Hamilton, J. A. (1999) Expression of a Y559F mutant CSF-1 receptor in M1 myeloid cells: a role for Src kinases in CSF-1 receptor-mediated differentiation Mol. Cell. Biol. Res. Commun. 1,144-152[CrossRef][Medline]
  6. van der Geer, P., Hunter, T. (1993) Mutation of Tyr697, a GRB2-binding site, and Tyr721, a PI 3-kinase binding site, abrogates signal transduction by the murine CSF-1 receptor expressed in Rat-2 fibroblasts EMBO J. 12,5161-5172[Medline]
  7. Bourette, R. P., De Sepulveda, P., Arnaud, S., Dubreuil, P., Rottapel, R., Mouchiroud, G. (2001) Suppressor of cytokine signaling 1 interacts with the macrophage colony-stimulating factor receptor and negatively regulates its proliferation signal J. Biol. Chem. 276,22133-22139[Abstract/Free Full Text]
  8. Novak, U., Nice, E., Hamilton, J. A., Paradiso, L. (1996) Requirement for Y706 of the murine (or Y708 of the human) CSF-1 receptor for STAT1 activation in response to CSF-1 Oncogene 13,2607-2613[Medline]
  9. Reedijk, M., Liu, X., van der Geer, P., Letwin, K., Waterfield, M. D., Hunter, T., Pawson, T. (1992) Tyr721 regulates specific binding of the CSF-1 receptor kinase insert to PI 3'-kinase SH2 domains: a model for SH2-mediated receptor-target interactions EMBO J. 11,1365-1372[Medline]
  10. Bourette, R. P., Myles, G. M., Choi, J. L., Rohrschneider, L. R. (1997) Sequential activation of phoshatidylinositol 3-kinase and phospholipase C-{gamma}2 by the M-CSF receptor is necessary for differentiation signaling EMBO J. 16,5880-5893[CrossRef][Medline]
  11. Wilhelmsen, K., Burkhalter, S., van der Geer, P. (2002) C-Cbl binds the CSF-1 receptor at tyrosine 973, a novel phosphorylation site in the receptor’s carboxy-terminus Oncogene 21,1079-1089[CrossRef][Medline]
  12. Bourette, R. P., Myles, G. M., Carlberg, K., Chen, A. R., Rohrschneider, L. R. (1995) Uncoupling of the proliferation and differentiation signals mediated by the murine macrophage colony-stimulating factor receptor expressed in myeloid FDC-P1 cells Cell Growth Differ. 6,631-645[Abstract]
  13. Imamura, K., Dianoux, A., Nakamura, T., Kufe, D. (1990) Colony-stimulating factor 1 activates protein kinase C in human monocytes EMBO J. 9,2389-2423–2428
  14. Liu, W. S., Heckman, C. A. (1998) The sevenfold way of PKC regulation Cell. Signal. 10,529-542[CrossRef][Medline]
  15. Newton, A. C. (1997) Regulation of protein kinase C Curr. Opin. Cell Biol. 9,161-167[CrossRef][Medline]
  16. Mellor, H., Parker, P. J. (1998) The extended protein kinase C superfamily Biochem. J. 332,281-292
  17. Malmberg, A. B., Chen, C., Tonegawa, S., Basbaum, A. I. (1997) Preserved acute pain and reduced neuropathic pain in mice lacking PKC{gamma} Science 278,279-283[Abstract/Free Full Text]
  18. Hodge, C. W., Mehmert, K. K., Kelley, S. P., McMahon, T., Haywood, A., Olive, M. F., Wang, D., Sanchez-Perez, A. M., Messing, R. O. (1999) Supersensitivity to allosteric GABA(A) receptor modulators and alcohol in mice lacking PKC{epsilon} Nat. Neurosci. 2,997-1002[CrossRef][Medline]
  19. Miyamoto, A., Nakayama, K., Imaki, H., Hirose, S., Jiang, Y., Abe, M., Tsukiyama, T., Nagahama, H., Ohno, S., Hatakeyama, S., Nakayama, K. I. (2002) Increased proliferation of B cells and auto-immunity in mice lacking protein kinase C{delta} Nature 416,865-869[CrossRef][Medline]
  20. Mischak, H., Pierce, J. H., Goodnight, J., Kazanietz, M. G., Blumberg, P. M., Mushinski, J. F. (1993) Phorbol ester-induced myeloid differentiation is mediated by protein kinase C-{alpha} and -{delta} and not by protein kinase C-ß II, -{epsilon}, -{zeta}, and -{eta} J. Biol. Chem. 268,20110-20115[Abstract/Free Full Text]
  21. Macfarlane, D. E., Manzel, L. (1994) Activation of beta-isozyme of protein kinase C (PKC ß) is necessary and sufficient for phorbol ester-induced differentiation of HL-60 promyelocytes. Studies with PKC beta-defective PET mutant J. Biol. Chem. 269,4327-4331[Abstract/Free Full Text]
  22. Liu, F., Roth, R. A. (1994) Insulin-stimulated tyrosine phosphorylation of protein kinase C {alpha}: evidence for direct interaction of the insulin receptor and protein kinase C in cells Biochem. Biophys. Res. Commun. 200,1570-1577[CrossRef][Medline]
  23. Crosby, D., Poole, A. W. (2002) Interaction of Bruton’s tyrosine kinase and protein kinase C{theta} in platelets. Cross-talk between tyrosine and serine/threonine kinases J. Biol. Chem. 277,9958-9965[Abstract/Free Full Text]
  24. Li, W., Yu, J. C., Michieli, P., Beeler, J. F., Ellmore, N., Heidaran, M. A., Pierce, J. H. (1994) Stimulation of the platelet-derived growth factor beta receptor signaling pathway activates protein kinase C-{delta} Mol. Cell. Biol. 14,6727-6735[Abstract/Free Full Text]
  25. Denning, M. F., Dlugosz, A. A., Threadgill, D. W., Magnuson, T., Yuspa, S. H. (1996) Activation of the epidermal growth factor receptor signal transduction pathway stimulates tyrosine phosphorylation of protein kinase C {delta} J. Biol. Chem. 271,5325-5331[Abstract/Free Full Text]
  26. Szallasi, Z., Denning, M. F., Chang, E. Y., Rivera, J., Yuspa, S. H., Lehel, C., Olah, Z., Anderson, W. B., Blumberg, P. M. (1995) Development of a rapid approach to identification of tyrosine phosphorylation sites: application to PKC {delta} phosphorylated upon activation of the high affinity receptor for IgE in rat basophilic leukemia cells Biochem. Biophys. Res. Commun. 214,888-894[CrossRef][Medline]
  27. Blake, R. A., Garcia-Paramio, P., Parker, P. J., Courtneidge, S. A. (1999) Src promotes PKC{delta} degradation Cell Growth Differ. 10,231-241[Abstract/Free Full Text]
  28. Kronfeld, I., Kazimirsky, G., Lorenzo, P. S., Garfield, S. H., Blumberg, P. M., Brodie, C. (2000) Phosphorylation of protein kinase C{delta} on distinct tyrosine residues regulates specific cellular functions J. Biol. Chem. 275,35491-35498[Abstract/Free Full Text]
  29. Semizarov, D., Glesne, D., Laouar, A., Schiebel, K., Huberman, E. (1998) A lineage-specific protein kinase crucial for myeloid maturation Proc. Natl. Acad. Sci. USA 95,15412-15417[Abstract/Free Full Text]
  30. Klink, A., Schiebel, K., Winkelmann, M., Rao, E., Horsthemke, B., Ludecke, H. J., Claussen, U., Scherer, G., Rappold, G. (1995) The human protein kinase gene PKX1 on Xp22.3 displays Xp/Yp homology and is a site of chromosomal instability Hum. Mol. Genet. 4,869-878[Abstract/Free Full Text]
  31. Blaschke, R. J., Monaghan, A. P., Bock, D., Rappold, G. A. (2000) A novel murine PKA-related protein kinase involved in neuronal differentiation Genomics 64,187-194[CrossRef][Medline]
  32. Saharinen, P., Ekman, N., Sarvas, K., Parker, P., Alitalo, K., Silvennoinen, O. (1997) The Bmx tyrosine kinase induces activation of the Stat signaling pathway, which is specifically inhibited by protein kinase C{delta} Blood 90,4341-4353[Abstract/Free Full Text]
  33. Kovanen, P. E., Junttila, I., Takaluoma, K., Saharinen, P., Valmu, L., Li, W., Silvennoinen, O. (2000) Regulation of Jak2 tyrosine kinase by protein kinase C during macrophage differentiation of IL-3-dependent myeloid progenitor cells Blood 95,1626-1632[Abstract/Free Full Text]
  34. Worm, M., Krah, J. M., Manz, R. A., Henz, B. M. (1998) Retinoic acid inhibits CD40 + interleukin-4-mediated IgE production in vitro Blood 92,1713-1720[Abstract/Free Full Text]
  35. Bastiaens, P. I., Jovin, T. M. (1996) Microspectroscopic imaging tracks the intracellular processing of a signal transduction protein: fluorescent-labeled protein kinase C ß I Proc. Natl. Acad. Sci. USA 93,8407-8412[Abstract/Free Full Text]
  36. Emoto, Y., Manome, Y., Meinhardt, G., Kisaki, H., Kharbanda, S., Robertson, M., Ghayur, T., Wong, W. W., Kamen, R., Weichselbaum, R., et al (1995) Proteolytic activation of protein kinase C {delta} by an ICE-like protease in apoptotic cells EMBO J. 14,6148-6156[Medline]
  37. Mochly-Rosen, D., Gordon, A. S. (1998) Anchoring proteins for protein kinase C: a means for isozyme selectivity FASEB J. 12,35-42[Abstract/Free Full Text]
  38. Manger, R., Najita, L., Nichols, E. J., Hakomori, S., Rohrschneider, L. (1984) Cell surface expression of the McDonough strain of feline sarcoma virus fms gene product (gp 140fms) Cell 39,327-337[CrossRef][Medline]
  39. Guilbert, L. J., Stanley, E. R. (1986) The interaction of 125I-colony-stimulating factor-1 with bone marrow-derived macrophages J. Biol. Chem. 261,4024-4032[Abstract/Free Full Text]
  40. Wang, Q. J., Acs, P., Goodnight, J., Giese, T., Blumberg, P. M., Mischak, H., Mushinski, J. F. (1997) The catalytic domain of protein kinase C-delta in reciprocal {delta} and {epsilon} chimeras mediates phorbol ester-induced macrophage differentiation of mouse promyelocytes J. Biol. Chem. 272,76-82[Abstract/Free Full Text]
  41. Whetton, A. D., Heyworth, C. M., Nicholls, S. E., Evans, C. A., Lord, J. M., Dexter, T. M., Owen-Lynch, P. J. (1994) Cytokine-mediated protein kinase C activation is a signal for lineage determination in bipotential granulocyte macrophage colony-forming cells J. Cell Biol. 125,651-659[Abstract/Free Full Text]
  42. Kang, Y., Park, J. S., Kim, S. H., Shin, Y. J., Kim, W., Joo, H. J., Chun, J. S., Kim, H. J., Ha, M. J. (2000) Overexpression of protein kinase C {delta} represses expression of proliferin in NIH3T3 cells that regulates cell proliferation Mol. Cell. Biol. Res. Commun. 4,181-187[CrossRef][Medline]
  43. Di Pasquale, G., Stacey, S. N. (1998) Adeno-associated virus Rep78 protein interacts with protein kinase A and its homolog PRKX and inhibits CREB-dependent transcriptional activation J. Virol. 72,7916-7925[Abstract/Free Full Text]
  44. Chiorini, J. A., Zimmermann, B., Yang, L., Smith, R. H., Ahearn, A., Herberg, F., Kotin, R. M. (1998) Inhibition of PrKX, a novel protein kinase, and the cyclic AMP-dependent protein kinase PKA by the regulatory proteins of adeno-associated virus type 2 Mol. Cell. Biol. 18,5921-5929[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
B. Pakuts, C. Debonneville, L. M. Liontos, M. P. Loreto, and C. J. McGlade
The Src-like Adaptor Protein 2 Regulates Colony-stimulating Factor-1 Receptor Signaling and Down-regulation
J. Biol. Chem., June 22, 2007; 282(25): 17953 - 17963.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
L. Pearn, J. Fisher, A. K. Burnett, and R. L. Darley
The role of PKC and PDK1 in monocyte lineage specification by Ras
Blood, May 15, 2007; 109(10): 4461 - 4469.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
O. Saijonmaa, T. Nyman, and F. Fyhrquist
Atorvastatin inhibits angiotensin-converting enzyme induction in differentiating human macrophages
Am J Physiol Heart Circ Physiol, April 1, 2007; 292(4): H1917 - H1921.
[Abstract] [Full Text] [PDF]

Home page
 J. Histochem. Cytochem.Home page
W. Li, Z.-X. Yu, and R. M. Kotin
Profiles of PrKX Expression in Developmental Mouse Embryo and Human Tissues
J. Histochem. Cytochem., August 1, 2005; 53(8): 1003 - 1009.
[Abstract] [Full Text] [PDF]

Home page
 Hum Reprod UpdateHome page
R. Wu, K. H. Van der Hoek, N. K. Ryan, R. J. Norman, and R. L. Robker
Macrophage contributions to ovarian function
Hum. Reprod. Update, March 1, 2004; 10(2): 119 - 133.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
G. Li, Y.-J. Kim, C. Mantel, and H. E. Broxmeyer
P-Selectin Enhances Generation of CD14+CD16+ Dendritic-Like Cells and Inhibits Macrophage Maturation from Human Peripheral Blood Monocytes
J. Immunol., July 15, 2003; 171(2): 669 - 677.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Junttila, I.
Right arrow Articles by Silvennoinen, O.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Junttila, I.
Right arrow Articles by Silvennoinen, O.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS