The JI
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
Right arrow Citation Map
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 Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Gee, K.
Right arrow Articles by Kumar, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Gee, K.
Right arrow Articles by Kumar, A.
The Journal of Immunology, 2002, 169: 5660-5672.
Copyright © 2002 by The American Association of Immunologists

Differential Regulation of CD44 Expression by Lipopolysaccharide (LPS) and TNF-{alpha} in Human Monocytic Cells: Distinct Involvement of c-Jun N-Terminal Kinase in LPS-Induced CD44 Expression

Katrina Gee{dagger}, Wilfred Lim{dagger}, Wei Ma{ddagger}, Devki Nandan§, Francisco Diaz-Mitoma*,{dagger},{ddagger}, Maya Kozlowski and Ashok Kumar*,{dagger},{ddagger}

Departments of * Pediatrics, and {dagger} Biochemistry, Microbiology and Immunology, University of Ottawa, and {ddagger} Division of Virology and Molecular Immunology, Research Institute, Children’s Hospital of Eastern Ontario, Ottawa, Ontario, Canada; § Division of Infectious Diseases, Department of Medicine, Vancouver Hospital, University of British Columbia, Vancouver, British Columbia, Canada; and Therapeutic Products Program, Research Services Division, Health Canada, Ottawa, Ontario, Canada


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Alterations in the regulation of CD44 expression play a critical role in modulating cell adhesion, migration, and inflammation. LPS, a bacterial cell wall component, regulates CD44 expression and may modulate CD44-mediated biological effects in monocytic cells during inflammation and immune responses. In this study, we show that in normal human monocytes, LPS and LPS-induced cytokines IL-10 and TNF-{alpha} enhance CD44 expression. To delineate the mechanism underlying LPS-induced CD44 expression, we investigated the role of the mitogen-activated protein kinases (MAPKs), p38, p42/44 extracellular signal-regulated kinase, and c-Jun N-terminal kinase (JNK) by using their specific inhibitors. We demonstrate the involvement, at least in part, of p38 MAPK in TNF-{alpha}-induced CD44 expression in both monocytes and promonocytic THP-1 cells. However, neither p38 nor p42/44 MAPKs were involved in IL-10-induced CD44 expression in monocytes. To further dissect the TNF-{alpha} and LPS-induced signaling pathways regulating CD44 expression independent of IL-10-mediated effects, we used IL-10 refractory THP-1 cells as a model system. Herein, we show that CD44 expression induced by the LPS-mediated pathway predominantly involved JNK activation. This conclusion was based on results derived by transfection of THP-1 cells with a dominant-negative mutant of stress-activated protein/extracellular signal-regulated kinase kinase 1, and by exposure of cells to JNK inhibitors dexamethasone and SP600125. All these treatments prevented CD44 induction in LPS-stimulated, but not in TNF-{alpha}-stimulated, THP-1 cells. Furthermore, we show that CD44 induction may involve JNK-dependent early growth response gene activation in LPS-stimulated monocytic cells. Taken together, these results suggest a predominant role of JNK in LPS-induced CD44 expression in monocytic cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Acomponent of bacterial cell walls, LPS, induces inflammatory responses that contribute to the pathogenesis of sepsis, inflammation, and a number of autoimmune diseases. Although the mechanisms that lead to these inflammatory processes remain largely unclear, monocytic cells play a major role in the pathogenesis of LPS-induced syndromes. In response to LPS, monocytes express large quantities of proinflammatory cytokines, as well as several costimulatory/adhesion molecules including B7, ICAM-1, VCAM-1, and CD44 (1, 2, 3). The overexpression of proinflammatory cytokines, costimulatory, and adhesion molecules is a hallmark of inflammatory responses.

Adhesion molecules play a fundamental role in inflammation by mediating leukocyte-endothelial cell adhesion, leukocyte migration, and T cell-APC interactions. The induction of CD44 expression is a key event in the migration of monocytic cells to sites of inflammation or injury (4, 5). CD44 is a surface membrane glycoprotein that binds to hyaluronan (HA),3 a component of the extracellular matrix, and engagement of CD44 on immune cells with HA is a key event in inflammatory responses (4, 5, 6, 7). CD44 has been implicated in a number of immunological phenomena such as lymphocyte homing, cell activation, hemopoiesis, and growth factor presentation, as well as in disease processes such as arthritis, allergy, and tumor metastasis (4, 5, 6, 7, 8, 9). The multiple functions of CD44 have been attributed to its extensive molecular heterogeneity generated by alternate mRNA splicing, as well as by posttranslational modifications (10, 11, 12, 13, 14).

It has been demonstrated that in both primary human monocytes, as well as in monocytic cell lines, LPS induces CD44 expression (3, 14). Recently, TNF-{alpha} was shown to be an important positive regulator of LPS-induced CD44-HA interactions in human monocytes (3, 14). In addition, other cytokines influence CD44 expression and CD44-HA interactions in different cell types (15, 16, 17). For example, in murine B cells, IL-5 enhances CD44-mediated binding to HA (18), whereas IL-3 and GM-CSF mediate this effect on human hemopoietic progenitor cells (19). Alterations in the levels of CD44 expression on monocytes/macrophages by endotoxins and immunoregulatory cytokines may have profound effects on the migration of immune cells to sites of inflammation and in the development of immune responses. Therefore, understanding CD44 regulation and characterizing the signal transduction events involved may lead to the development of strategies for the treatment of autoimmune diseases and cancer. The signal transduction events involved in CD44 up-regulation are not well understood. CD44 expression has been shown to be regulated via calmodulin/Ca2+ signaling pathways in PMA-stimulated T lymphoma cells (20), whereas phosphatidylinositol 3-kinase and protein kinase C were shown to regulate CD44 expression in neuroblastoma cells (21). Recently, early growth response gene (Egr-1) was implicated in the transcription of CD44 in murine B cells (22) and in the endothelial cell line ECV304 (23).

During the past few years, significant progress has been made in elucidating the LPS-induced signaling pathways that mediate cytokine expression in monocytes (24). However, the molecular mechanisms by which LPS and cytokines, especially TNF-{alpha}, regulate CD44 expression are not well understood. In this study, we investigated the regulation of CD44 expression by the family of mitogen-activated protein kinases (MAPKs). MAPKs play a key role in cellular responses such as proliferation, differentiation, and apoptosis (25). The three main families of MAPKs are the extracellular signal-regulated kinase (ERK)1/2, the c-Jun N-terminal kinase (JNK)/stress-activated protein kinases, and p38 (25). ERKs respond to mitogens and growth factors that regulate cell proliferation and differentiation, whereas JNK and p38 MAPKs are primarily activated by stress and inflammatory cytokines (IL-1{beta} and TNF-{alpha}; Ref. 25). LPS and TNF-{alpha} have been shown to activate all three types of MAPKs (25, 26, 27, 28, 29, 30). In this study, we show that CD44 expression is up-regulated in normal human monocytes following stimulation with LPS as well as with IL-10 and TNF-{alpha}, which are produced endogenously following LPS stimulation. We examined the MAPK signaling pathways induced by LPS, IL-10, and TNF-{alpha} in normal human monocytes. Our results revealed a partial role for p38 MAPK in LPS- and TNF-{alpha}-induced expression of CD44 in monocytic cells. In contrast, IL-10-induced CD44 expression did not involve p38 or p42/44 MAPK activation. To further dissect the TNF-{alpha}- and LPS-induced signaling pathways regulating CD44 expression independent of IL-10-mediated effects, we used IL-10 refractory THP-1 cells as a model system. We provide evidence that CD44 expression induced by the LPS pathway is mediated through JNK signaling. Furthermore, our results suggest that CD44 expression consequent to LPS treatment of human monocytes may require JNK-dependent activation of Egr-1.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines, cell culture, and reagents

THP-1, a promonocytic cell line derived from a human acute lymphocytic leukemia patient, was obtained from the American Type Culture Collection (Manassas, VA). Five to 15% of these cells express CD14 on their cell surface, and >50% of these cells express CD14Rs following LPS stimulation (30). Cells were cultured in IMDM (Sigma-Aldrich, St. Louis, MO) supplemented with 10% FBS (Life Technologies, Grand Island, NY), 100 U/ml penicillin, 100 µg/ml gentamicin, 10 mM HEPES, and 2 mM glutamine. PD98059 (Calbiochem, San Diego, CA), an inhibitor of MAP/ERK kinase 1 kinase, selectively blocks the activity of ERK MAPK and has no effect on the activity of other serine threonine protein kinases including Raf-1, p38, and JNK MAPKs, or protein kinase C and protein kinase A (31, 32). The pyridinyl imidazole SB202190 (Calbiochem), a potent inhibitor of p38 MAPK, has no significant effect on the activity of the ERK or JNK MAPK subgroups (31, 32). SB202474, an inactive analog of SB202190, was also purchased from Calbiochem. Dexamethasone (Sabex, Boucherville, Quebec, Canada) was used as an inhibitor of JNK activity. SP600125, a specific JNK inhibitor (Biomol, Plymouth Meeting, PA), is a reversible ATP-competitive inhibitor with >300-fold selectivity vs related MAPKs including ERK1 and p38 as well as protein kinase A and IkB kinase-2 (33) LPS derived from Escherichia coli 0111:B4 (Sigma-Aldrich), human rIL-10 (R&D Systems, Minneapolis, MN), human rTNF-{alpha} (BioSource, Montreal, Quebec, Canada), as well as anti-human IL-10R{alpha} and anti-human TNF-{alpha}R1 (R&D Systems) capable of neutralizing the biological activities of IL-10 and TNF-{alpha}, respectively, were also purchased. All other chemicals used for Western blotting were obtained from Sigma-Aldrich.

Isolation of monocytes from PBMCs

PBMCs were isolated from the blood of healthy adult volunteers following approval of the protocol by the Ethics Review Committee of the Ottawa General Hospital (Ottawa, Ontario, Canada). PBMCs were isolated by density-gradient centrifugation over Ficoll-Hypaque (Amersham Pharmacia Biotech, Piscataway, NJ) as previously described (15, 34). Briefly, the cell layer containing mononuclear cells was collected and washed three times in PBS. Purified, nonactivated monocytes were isolated by negative selection by depletion of T and B cells using magnetic polystyrene M-450 Dynabeads (Dynal Biotech, Oslo, Norway) coated with Abs specific for CD2 (T cells) and CD19 (B cells), as described earlier (15, 34). Briefly, PBMCs (10 x 106/ml) were resuspended with CD2 and CD19 Dynabeads and were incubated for 30 min on ice with constant rocking. Cells were incubated at 37°C for 2 h following which nonadherent cells were removed. The adherent mononuclear cells obtained contained <1% CD2+ T cells and CD19+ B cells as determined by flow cytometric analysis.

Cell stimulation and collection of culture supernatants

To determine the effects of p38 and p42/44 MAPK inhibitors on CD44 expression and IL-10 and TNF-{alpha} production, purified human monocytes (1 x 106/ml) and THP-1 cells (0.5 x 106/ml) were incubated in 24-well culture plates (BD Labware, Franklin Lakes, NJ). Cells were left untreated or treated for 24 h in the presence or absence of MAPK inhibitors and with either LPS (1 µg/ml), IL-10 (10 ng/ml), or TNF-{alpha} (10 ng/ml). Cell supernatants were frozen at -80°C and thawed at the time of analysis. Cells were analyzed for CD44 expression by flow cytometry, and the supernatants analyzed for IL-10 and TNF-{alpha} production by ELISA.

Measurement of IL-10 and TNF-{alpha} in the culture supernatants by ELISA

IL-10 and TNF-{alpha} were measured by ELISA using two different mAbs recognizing distinct epitopes, as described earlier (30, 34). Briefly, the plates (Nunc, Roskilde, Denmark) were coated overnight at 4°C with the primary Ab (either anti-IL-10 mAb no. 18551D from BD PharMingen, San Diego, CA; final concentration 5 µg/ml; or anti-TNF-{alpha} mAb no. AHC3712 from BioSource; final concentration 5 µg/ml) in coating buffer (0.04 M Na2CO3, 0.06 M NaHCO, pH 9.6). The plates were washed with PBS-Tween 20 and blocked with PBS/10% FBS. IL-10 or TNF-{alpha} were detected using a second biotinylated mAb in PBS/10% FBS (anti-IL-10 Ab no. 18562D from BD PharMingen; final concentration 4 µg/ml; anti-TNF-{alpha} Ab no. AHC3419 from BioSource; final concentration 4 µg/ml). Streptavidin-peroxidase was used at a final concentration of 1:1000 (Jackson ImmunoResearch Laboratories, West Grove, PA). The color reaction was developed by o-phenylenediamine (Sigma-Aldrich) and hydrogen peroxide, and read at 450 nm. rIL-10 (R&D Systems) and rTNF-{alpha} (BioSource) were used as standards. The level of sensitivity for both IL-10 and TNF-{alpha} production by ELISA was 16 pg/ml.

Flow cytometric analysis

CD44 expression on CD14+ monocytes and THP-1 cells was determined by flow cytometry as described earlier (16, 35). Briefly, cells were washed once at the time of harvesting with PBS/0.1% sodium azide and aliquoted into polystyrene tubes (Falcon, Lincoln Park, NJ). Cells were double-stained with PE-labeled anti-CD14 mAbs (BD Biosciences, Mountain View, CA) and FITC-labeled anti-CD44 mAbs (BD PharMingen). Autofluorescence and isotype (IgG2b)-matched control Abs (BD Biosciences) were also included. The gates were set in accordance with gates obtained with the isotype-matched control Abs and mean channel fluorescence (MCF) was determined. Data were acquired on a BD FACScan Flow Cytometer (BD Biosciences) and analyzed using the WinMDI version 2.8 software package (provided by J. Trotter, Scripps Institute, San Diego, CA). Validity of comparisons in the expression levels of CD14 and CD44 between different samples was ensured through the use of Calibrite Beads (BD Biosciences).

Western blot analysis

Phosphorylation of p38, p42/44, and JNK MAPKs was determined by Western blot analysis using MAPK-specific Abs as previously described (30, 35, 36). Cells were stimulated at 37°C for 0–15 min with either LPS, IL-10, or TNF-{alpha}. Cells were then placed in ice and washed with cold PBS. Cell pellets were lysed for 30 min with lysis buffer (50 mM HEPES (pH 7.5), 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl2, 100 mM NaF, 100 mM sodium orthovanadate, and 1 mM EGTA (pH 7.7)) followed by centrifugation for 20 min at 14,000 x g, 4°C. The protein concentration of the supernatants was determined using the Bio-Rad protein determination assay (Bio-Rad, Hercules, CA). Total cell proteins were subjected to electrophoresis on 8% polyacrylamide SDS gels and the proteins were transferred onto polyvinylidene difluoride membranes (Pall Gelman Laboratory, Ann Arbor, MI). The membranes were probed with either rabbit anti-phospho-human-p38 Ab (NEB, Mississauga, Ontario, Canada), mouse anti-phospho-human-p42/44 Ab, or rabbit anti-phospho-human JNK Ab (Santa Cruz Biotechnology, Santa Cruz, CA), followed by either goat anti-mouse or goat anti-rabbit polyclonal Abs conjugated to HRP (Bio-Rad). The membranes were stripped and reprobed with rabbit polyclonal Abs specific for each of the unphosphorylated p38, p42, and JNK MAPKs (Santa Cruz Biotechnology), as described earlier (30). All immunoblots were visualized by ECL (Amersham Pharmacia Biotech).

Transient transfection

THP-1 cells were transfected with either a pcDNA-3 plasmid expressing a dominant-negative (DN) mutant of MAPK kinase (MKK) 4/stress-activated protein/ERK kinase 1 (SEK1; provided by Dr. J. R. Woodgett, Princess Margaret Hospital, Toronto, Ontario, Canada) or a control pcDNA-3 plasmid, as described earlier (30, 36). Before transfection, 10 µg of plasmid was incubated with 10 µl of LIPOFECTAMINE reagent (Invitrogen, Burlington, Ontario, Canada) in 200 µl of OPTI-MEM I Reduced Serum medium (Invitrogen) for 45 min to allow formation of DNA-liposome complexes. These complexes were added to the cell suspension (1.5 x 106 cells/ml) in each well and cultured for 24 h. Cells were stimulated with 1 µg/ml of LPS for another 24 h followed by analysis for CD44 expression. Two positive control vectors, pcDNA3.1/His/lacZ (Invitrogen) and pSV-{beta}-galactosidase (Promega, Madison, WI) were used for determination of transfection efficiency by using a commercially available {beta}-Gal Staining kit (Invitrogen) as per the manufacturer’s instructions. An average of 18% of cells were found to be transfected.

Gel mobility shift assays

Gel mobility assays were performed as per the standard technique and as described earlier (30, 36). Cells (1 x 107) were harvested in Tris-EDTA-saline buffer (pH 7.8) and centrifuged at 200 x g for 5 min at 4°C. The cells were lysed for 10 min at 4°C with buffer A (10 mM HEPES, 10 mM KCl, 1.5 mM MgCl2, 0.5 mM DTT, 0.5 mM PMSF, pH 7.9) containing 0.1% Nonidet P-40. The lysates were centrifuged at 20,000 x g for 10 min at 4°C. The pellets containing the nuclei were suspended in buffer B (20 mM HEPES, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 25% glycerol) at 4°C for 15 min. Both buffers A and B contained the proteolytic inhibitors at 0.5 mM each of DTT, PMSF, and spermidine, as well as 0.15 mM spermine, and 5 µg/ml each of aprotinin, leupeptin, and pepstatin. The supernatants containing the nuclear proteins were collected and frozen at -80°C. Nuclear proteins (5 µg) were mixed for 20 min at room temperature with 32P-labeled Egr-1 oligonucleotide probes, and the complexes were subjected to nondenaturing 17% PAGE for 90 min. The gels were dried and exposed to x-ray film. The Egr-1 oligonucleotide sequence used was: 5'-GGA TCC AGG GGG GGC GAG CGG GGG CGA-3' (Santa Cruz Biotechnology).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
LPS, IL-10, and TNF-{alpha} up-regulate CD44 expression on the surface of normal human monocytes

Stimulation of normal human monocytes with LPS results in the release of pro- and anti-inflammatory cytokines (IL-1, IL-6, TNF-{alpha}, and IL-10), and induces the expression of various cell surface receptors including CD44 (1, 2, 3). We confirmed that stimulation of normal human monocytes with LPS resulted in CD44 up-regulation as determined by flow cytometric analysis (Fig. 1Go). The up-regulation occurred in a dose-dependent manner (data not shown). It has been suggested that TNF-{alpha} and IL-10 are the major cytokines that regulate interaction of CD44 with its ligand HA in human monocytes (3, 14). Because TNF-{alpha} and IL-10 are produced in response to LPS stimulation in these cells, we investigated whether these endogenously produced cytokines also modulate CD44 expression. We show that in the presence of exogenous TNF-{alpha} and IL-10, CD44 expression is increased on normal human monocytes (Fig. 1GoA). To determine that endogenously produced IL-10 and TNF-{alpha} regulate LPS-induced CD44 expression, we used Abs specific for human IL-10R{alpha} (0.1 µg/ml) and human TNF-{alpha}R1 (5 µg/ml) capable of neutralizing the biological activity of 1 ng/ml of IL-10 and TNF-{alpha}, respectively, as recommended by the supplier (R&D Systems). The results show that anti-IL-10R{alpha} and anti-TNF-{alpha}R1 Abs inhibited the LPS-induced CD44 expression but not by the control Abs (Fig. 1GoB).



View larger version (15K):
[in this window]
[in a new window]
  		FIGURE 1. A, LPS, IL-10, and TNF-{alpha} enhance CD44 expression in normal human monocytes. Purified normal human monocytes (0.5 x 106/ml) were stimulated with LPS (1 µg/ml), IL-10 (10 ng/ml), or TNF-{alpha} (10 ng/ml) for 24 h. B, LPS-treated normal human moncytes were treated with either anti-IL-10R{alpha} (0.1 µg/ml, upper panel), anti-TNF-{alpha}R1 (5 µg/ml, middle panel), or by anti-IL-10R{alpha} (0.1 µg/ml) plus anti-TNF-{alpha}R1 (5 µg/ml) (lower panel) or with isotype matches control Abs. Shaded histograms represent untreated cells. Bold lines indicate LPS-stimulated cells in the presence of control Abs, whereas broken lines represent LPS-stimulated cells treated with anti-IL-10R and anti-TNF-{alpha}R Abs. The cells were double-stained with PE-labeled mouse anti-human CD14 Abs and with FITC-labeled mouse anti-human CD44 Abs. CD44 expression was analyzed on CD14+ monocytes by flow cytometry. The experiments shown are representative of five different experiments.

 
Role of p38 MAPKs in LPS-mediated CD44 expression

The MAPKs play a major role in the LPS- and cytokine-mediated induction of several cell surface molecules (30, 37, 38, 39, 40). Therefore, it was of interest to identify the members of the MAPK family that are involved in the LPS, IL-10, and TNF-{alpha}-induced regulation of CD44 expression in monocytic cells. We first investigated the involvement of p38 and p42/44 ERK MAPKs by examining their activation following stimulation of monocytes with LPS. LPS induced the phosphorylation of both p38 and p42/44 ERKs (Fig. 2GoA). The role of p38 and p42/44 ERKs has been studied through the use of specific kinase inhibitors: SB202190 for p38-mediated signaling, and PD98059 for p42/44-mediated signaling (31, 32). To confirm that SB202190 and PD98059 inhibited the phosphorylation of p38 and ERKs, respectively, in our system, freshly isolated monocytes were pretreated with these inhibitors for 2 h followed by stimulation with LPS for 10 min. The results show that prior incubation of monocytes with SB202190 or PD98059 resulted in the inhibition of LPS-induced phosphorylation of p38 and p42/44 ERKs, respectively, (Fig. 2GoA).



View larger version (31K):
[in this window]
[in a new window]
  		FIGURE 2. A, LPS stimulation induces p38 and p42/44 MAPK phosphorylation in purified human monocytes. Purified normal human monocytes (1.0 x 106/ml) were pretreated with either SB202190 or PD98059 at varying concentrations ranging from 0–50 µM for 2 h before LPS (1 µg/ml) stimulation for 10 min. Crude protein extracts (50 µg) were subjected to SDS-PAGE followed by Western blot analysis. The membranes were blotted with either anti-phospho-p38 (pp38) Ab or anti-phospho-p42 (pp42) Ab, and to control for loading, the membranes were stripped and reprobed with either anti-p38 (p38) or anti-p42 (p42), respectively. The p38 MAPK inhibitor modulates LPS-induced CD44 expression (B) and TNF-{alpha} and IL-10 production (C) in normal human monocytes. Purified normal human monocytes (0.5 x 106/ml) were pretreated with either SB202190 or PD98059 at varying concentrations ranging from 0–50 µM for 2 h before LPS (1 µg/ml) stimulation for 24 h. CD44 expression was then analyzed by flow cytometry. Supernatants were harvested and analyzed by ELISA for TNF-{alpha} and IL-10 production. The histogram shown is a representative of five different experiments performed. ELISA results are represented as mean ± SD of five experiments.

 
To determine the role of p38 and p42/44 MAPKs in the LPS-mediated induction of CD44, monocytes were treated with SB202190 or PD98059 for 2 h before stimulation with LPS and analyzed for CD44 expression. SB202190 partially inhibited CD44 expression at doses as low as 10 µM (Fig. 2GoB). Complete inhibition was not observed even if 25 and 50 µM of SB202190 were used (Fig. 2GoB). Because of large variations in CD44 expression levels on monocytic cells among individuals, the data in Table IGo do not show a significant inhibition at 10 µM; however, at doses of 25 and 50 µM, the partial effect of SB202190 is significant (p < 0.05). Treatment of cells with SB202474, an inactive analog of SB202190, for 2 h before LPS stimulation did not influence CD44 expression (data not shown), thus confirming the specificity of SB202190. In contrast, PD98059 did not inhibit CD44 expression even at doses of 50 µM (Fig. 2GoB and Table IGo), indicating that the p42/44 pathway is not directly involved in the regulation of LPS-induced CD44 expression. Doses higher than 50 µM were not used, as these concentrations were cytotoxic as determined by the trypan blue exclusion test (data not shown).


View this table:
[in this window]
[in a new window]
  		Table I. Effect of p38 and p42/44 MAPK inhibitors on LPS- and TNF-{alpha} induced CD44 expression in normal human monocytesa

 
Because LPS stimulation of primary monocytes also results in the production of cytokines such as IL-10 and TNF-{alpha}, and because these cytokines affect CD44 expression, it is possible that the down-regulation of CD44 expression observed following treatment with SB202190 of LPS-stimulated monocytes may be due to the inhibition of endogenous IL-10 and/or TNF-{alpha} production. Therefore, we evaluated the effects of MAPK inhibitors on IL-10 and TNF-{alpha} production in LPS-stimulated monocytes. SB202190, but not PD98059, inhibited LPS-induced IL-10 as well as TNF-{alpha} production in a dose-dependent manner (Fig. 2GoC). The same cells were used for CD44 expression as well as IL-10 and TNF-{alpha} production analyses in the experiments shown in Fig. 2Go, B and C. These results suggest that the partial inhibition of LPS-induced CD44 expression by SB202190 may be, at least in part, due to the decreased endogenous production of TNF-{alpha} and/or IL-10.

Role of p38 MAPK in TNF-{alpha}-induced CD44 expression

It is likely that IL-10 and/or TNF-{alpha} induced by stimulation of monocytes with LPS may up-regulate CD44 expression by activating either p38 or p42/44 MAPKs. To clarify the role of p38 and p42/44 MAPKs in TNF-{alpha}-induced CD44 expression, we examined the effects of the MAPK inhibitors on TNF-{alpha}-induced CD44 up-regulation. Similar to the LPS-induced activation of p38 and p42/44 (Fig. 2GoA), SB202190 and PD98059 significantly reduced TNF-{alpha}-induced phosphorylation of p38 and p42/44 ERK MAPKs, respectively, in normal monocytes (Fig. 3GoA). Similar to the results obtained for LPS-induced CD44 expression (Fig. 2GoA), neither SB202474 (data not shown) nor PD98059 influenced CD44 expression. SB202190 partially inhibited CD44 expression at doses as low as 10 µM (Fig. 3GoB). Complete inhibition was not observed even if 25 and 50 µM of SB202190 were used (Fig. 3GoB). Because of large variations in the level of CD44 expression on monocytic cells among individuals, the data in Table IGo do not show a significant inhibition at 10 µM; however, at doses of 25 and 50 µM, the partial effect of SB202190 is significant (p < 0.05). These results suggest that LPS-induced CD44 expression may be partially regulated by the enhanced production of TNF-{alpha} which is p38 MAPK-dependent. Furthermore, although the interaction of TNF-{alpha} with its receptor activated both the p38 and p42/44 MAPK pathways, p38 may play a role, at least in part, in TNF-{alpha}-induced CD44 expression.



View larger version (39K):
[in this window]
[in a new window]
  		FIGURE 3. A, TNF-{alpha} induces p38 and p42/44 MAPK phosphorylation in purified human monocytes. Purified normal human monocytes (1.0 x 106/ml) were pretreated with either SB202190 or PD98059 at varying concentrations ranging from 0–50 µM for 2 h before TNF-{alpha} (10 ng/ml) stimulation for 10 min. Crude protein extracts (50 µg) were subjected to SDS-PAGE followed by Western blot analysis. The membranes were blotted with either anti-phospho-p38 (pp38) Ab or anti-phospho-p42 (pp42) Ab. To control for loading, the membranes were stripped and reprobed with anti-p38 (p38) or anti-p42 (p42) Abs. B, Effect of p38 and p42/44 MAPK inhibitors on TNF-{alpha}-induced CD44 expression in normal human monocytes. Purified normal human monocytes (0.5 x 106/ml) were pretreated with either SB202190 or PD98059 at varying concentrations ranging from 0–50 µM for 2 h before TNF-{alpha} (10 ng/ml) stimulation for 24 h. CD44 expression was then analyzed by flow cytometry. The experiment shown is representative of five different experiments.

 
IL-10-induced CD44 expression does not involve p38 or p42/44 MAPK activation

To determine the role of p38 and p42/44 MAPKs in IL-10-mediated CD44 expression, we first determined whether IL-10 could activate p38 or p42/44 ERK MAPKs. Stimulation of normal monocytes with IL-10 induced the phosphorylation of p42/44, but not of p38 MAPK. Furthermore, IL-10-induced activation of p42/44 ERK was inhibited by PD98059 in a dose-dependent manner (Fig. 4GoA). However, p42/44 ERK was not found to be involved in IL-10-induced CD44 expression, since incubation of monocytes with PD98059 did not affect IL-10-induced CD44 expression (Fig. 4GoB).



View larger version (37K):
[in this window]
[in a new window]
  		FIGURE 4. A, Effect of IL-10 on p38 and p42/44 MAPK phosphorylation in purified human monocytes. Purified normal human monocytes (1.0 x 106/ml) were pretreated with either SB202190 or PD98059 at varying concentrations ranging from 0–50 µM for 2 h before IL-10 (10 ng/ml) stimulation for 10 min. Crude protein extracts (50 µg) were subjected to SDS-PAGE followed by Western blot analysis. The membranes were blotted with either anti-phospho-p38 (pp38) Ab or anti-phospho-p42 (pp42) Ab. To control for loading, the membranes were stripped and reprobed with anti-p38 (p38) or anti-p42 (p42) Abs. B, The p42/44 ERK MAPK inhibitor does not prevent IL-10-mediated down-regulation of CD44 expression in normal human monocytes. Monocytes (1.0 x 106/ml) were treated with 50 µM PD98059 for 2 h before stimulation with IL-10. After 48 h, cells were analyzed by flow cytometry for the expression of CD44. The experiment shown is representative of five different experiments.

 
Role of p38 MAPK in LPS- and TNF-{alpha}-induced CD44 expression in THP-1 cells

LPS-induced CD44 expression in normal human monocytes is complex and regulated by interaction of LPS with its CD14/Toll-like receptor (TLR) complex as well as by the interaction between IL-10 and TNF-{alpha} and with their corresponding receptor complexes. Because of the inherent endogenous production of IL-10 in LPS-stimulated monocytic cells, the role of p38 and p42/44 MAPK in LPS- and TNF-{alpha}-induced CD44 expression could not be studied. To investigate the role of MAPKs in LPS-induced CD44 expression independent of IL-10, we used IL-10 refractory THP-1 cells as a model system. THP-1 cells responded in a similar manner as monocytes with respect to CD44 induction following stimulation with LPS and TNF-{alpha}, but did not respond to IL-10 (Fig. 5Go). We show that LPS induced the activation of p38 and p42/44 ERK MAPKs; this activation was inhibited by their respective specific kinase inhibitors (Fig. 6GoA). As for primary monocytes, treatment of THP-1 cells with low doses of SB202190 (10 µM) before LPS stimulation resulted in only a partial inhibition of CD44 expression (Fig. 6GoB and Table IIGo, p < 0.05). Complete inhibition was not observed even if 25 and 50 µM of SB202190 were used (Fig. 6GoB and Table IIGo). In contrast, LPS-induced CD44 expression in THP-1 cells was not inhibited by either SB202474, an inactive analog of SB202190 (data not shown), nor by the p42/44 inhibitor PD98059 (Fig. 6GoB and Table IIGo), thereby eliminating the role of p42/44 signaling in LPS-induced CD44 expression and suggesting the involvement of p38, albeit in a partial manner.



View larger version (33K):
[in this window]
[in a new window]
  		FIGURE 5. LPS and TNF-{alpha} enhance CD44 expression in THP-1 cells. THP-1 cells (0.5 x 106/ml) were treated with LPS (1 µg/ml), IL-10 (10 ng/ml), or TNF-{alpha} (10 ng/ml) for 24 h. Following the stimulation, cells were examined for CD44 surface expression by flow cytometry. The experiment shown is representative of five different experiments.

 


View larger version (33K):
[in this window]
[in a new window]
  		FIGURE 6. A, LPS stimulation induces p38 and p42/44 MAPK phosphorylation in THP-1 cells. Cells (1.0 x 106/ml) were pretreated with either SB202190 or PD98059 at varying concentrations ranging from 0–50 µM for 2 h before LPS (1 µg/ml) stimulation for 10 min. Crude protein extracts (50 µg) were subjected to SDS-PAGE followed by Western blot analysis. The membranes were blotted with either anti-phospho-p38 (pp38) Ab or anti-phospho-p42 (pp42) Ab. To control for loading, the membranes were stripped and reprobed with anti-p38 (p38) or anti-p42 (p42) Abs. The Western blot shown is a representative of five different experiments. Effect of p38 and p42/44 MAPK inhibitors on LPS-induced CD44 expression (B) and TNF-{alpha} production (C) in THP-1 cells. Cells (0.5 x 106/ml) were treated with various concentrations of either SB202190 or PD98059 for 2 h before stimulation with LPS. After 24 h, cells were analyzed by flow cytometry for the expression of CD44. Supernatants were harvested and analyzed for TNF-{alpha} production by ELISA. ELISA data are represented as a mean ± SD of five different experiments.

 

View this table:
[in this window]
[in a new window]
  		Table II. Effect of p38 and p42/44 MAPK inhibitors on LPS- and TNF-{alpha}-induced CD44 expression in THP-1 cellsa

 
Similar to normal human monocytes, LPS may induce CD44 expression in THP-1 cells via activation through either the CD14-mediated pathway or by endogenously produced TNF-{alpha}. LPS-induced TNF-{alpha} production has been shown to be mediated, at least in part, via the activation of p38 MAPK in normal human monocytes (Ref. 39 and Fig. 2Go). Therefore, it was of interest to determine whether the inhibition of p38 MAPK by SB202190 would result in the loss of TNF-{alpha} production in THP-1 cells concomitant with CD44 inhibition. Our results show that SB202190 did not inhibit LPS-induced TNF-{alpha} production in THP-1 cells (Fig. 6GoC).

It is likely that LPS-induced CD44 expression in THP-1 cells may be regulated via the activation of the p38 and p42/44 MAPK following interaction of endogenously produced TNF-{alpha} with the TNF-{alpha} receptor. To determine the role of p38 and p42/44 MAPKs in TNF-{alpha}-induced CD44 expression, we first demonstrated that TNF-{alpha} induced the phosphorylation of both p38 and p42/44 in THP-1 cells, and that this phosphorylation was inhibited by their respective inhibitors SB202190 and PD98059 in a dose-dependent manner (Fig. 7GoA). Treatment of THP-1 cells with 25 and 50 µM of SB202190 before stimulation with TNF-{alpha} resulted in a partial inhibition of CD44 expression (Fig. 7GoB and Table IIGo, p < 0.05). In contrast, TNF-{alpha}-induced CD44 expression in THP-1 cells was not affected by either SB202474 (data not shown) or PD98059 (Fig. 7GoB and Table IIGo). Taken together, these results suggest that both LPS and TNF-{alpha} enhance CD44 expression, at least in part, through the activation of p38 MAPK in both primary monocytes and THP-1 cells.



View larger version (40K):
[in this window]
[in a new window]
  		FIGURE 7. A, TNF-{alpha} induces p38 and p42/44 MAPK phosphorylation in THP-1 cells. Cells (1.0 x 106/ml) were pretreated with either SB202190 or PD98059 at varying concentrations ranging from 0–50 µM for 2 h before TNF-{alpha} (10 ng/ml) stimulation for 10 min. Crude protein extracts (50 µg) were subjected to SDS-PAGE followed by Western blot analysis. The membranes were blotted with either anti-phospho-p38 (pp38) or anti-phospho-p42 (pp42) anitbodies. To control for loading, the membranes were stripped and reprobed with anti-p38 (p38) or anti-p42 (p42) Abs, respectively. B, Effect of p38 and p42/44 MAPK inhibitors on TNF-{alpha}-induced CD44 expression in THP-1 cells. Cells (0.5 x 106/ml) were pretreated with either SB202190 or PD98059 at varying concentrations ranging from 0–50 µM for 2 h before TNF-{alpha} (10 ng/ml) stimulation for 24 h. CD44 expression was then analyzed by flow cytometry. The experiment shown is representative of five different experiments.

 
JNK MAPK plays a distinct role in LPS-induced but not in TNF-a-induced CD44 expression in THP-1 cells

The partial involvement of p38 and the lack of involvement of p42/44 MAPKs in LPS- and TNF-{alpha}-induced CD44 expression prompted us to examine the role of JNK, the third major member of the MAPK family. To assess the role of JNK, we performed experiments using the glucocorticoid dexamethasone, an inhibitor of JNK MAPK activation (41). We examined whether LPS or TNF-{alpha} could induce JNK phosphorylation in THP-1 cells, and whether dexamethasone could inhibit its phosphorylation. Both LPS and TNF-{alpha} induced the phosphorylation of JNK in THP-1 cells, and this activation was inhibited in a dose-dependent manner by dexamethasone (Fig. 8GoA). Furthermore, treatment of THP-1 cells with dexamethasone before stimulation with LPS resulted in complete abrogation of CD44 expression in a dose-dependent manner (Fig. 8GoB). In contrast, dexamethasone did not influence TNF-{alpha}-induced CD44 expression in THP-1 cells at any concentration (Fig. 8GoB). These results show that JNK signaling may be necessary for LPS-induced but not for TNF-{alpha}-induced CD44 expression. Recently, a specific JNK inhibitor, SP600125, has become commercially available (33). To confirm the involvement of JNK, THP-1 cells were pretreated with varying concentrations of SP600125 before stimulation with LPS or TNF-{alpha}. SP600125 inhibited JNK phosphorylation induced by LPS and TNF-{alpha} in a dose-dependent manner (Fig. 9GoA). Consistent with the results obtained with dexamethasone, SP600125 inhibited LPS-induced CD44 expression in a dose-dependent manner, whereas TNF-{alpha}-induced CD44 expression remained unaffected (Fig. 9GoB).



View larger version (41K):
[in this window]
[in a new window]
  		FIGURE 8. A, Dexamethasone inhibits LPS-induced and TNF-{alpha}-induced phosphorylation of JNK MAPK in THP-1 cells. THP-1 cells (1.0 x 106/ml) were treated with dexamethasone at varying concentrations ranging from 0–50 nM for 2 h before stimulation with LPS (1 µg/ml) or TNF-{alpha} (10 ng/ml) for 10 min. Total cell proteins were analyzed for phosphorylation of JNK MAPK using an anti-phospho-JNK rabbit polyclonal Ab. To control for protein loading, the membranes were stripped and reprobed with anti-JNK rabbit polyclonal Abs. B, Dexamethasone inhibits LPS-induced but not TNF-{alpha}-induced CD44 expression in THP-1 cells. Cells (0.5 x 106/ml) were treated with various concentrations of dexamethasone ranging from 0–50 nM for 2 h before stimulation with LPS followed by flow cytometric analysis of CD44 expression. The experiment shown is representative of five different experiments.

 


View larger version (49K):
[in this window]
[in a new window]
  		FIGURE 9. A, SP600125 inhibits LPS-induced and TNF-{alpha}-induced phosphorylation of JNK MAPK in THP-1 cells. THP-1 cells (1.0 x 106/ml) were treated with SP600125 at varying concentrations ranging from 0–50 µM for 2 h before stimulation with LPS (1 µg/ml) or TNF-{alpha} (10 ng/ml) for 10 min. Total cell proteins were analyzed for phosphorylation of JNK MAPK using an anti-phospho-JNK rabbit polyclonal Ab. To control for protein loading, the membranes were stripped and reprobed with anti-JNK rabbit polyclonal Abs. B, SP600125 inhibits LPS-induced but not TNF-{alpha}-induced CD44 expression in THP-1 cells. Cells (0.5 x 106/ml) were treated with various concentrations of SP600125 ranging from 0–50 µM for 2 h before stimulation with LPS followed by flow cytometric analysis of CD44 expression. The experiment shown is representative of two different experiments.

 
To further determine the role of JNK MAPK in LPS-induced CD44 expression, we transiently transfected THP-1 cells with either a DN SEK1 plasmid (DN SEK1) or with a control plasmid pcDNA3. Activation of SEK1 is responsible for the induction of the JNK signaling cascade, and the presence of DN SEK1 effectively blocks JNK activation (42, 43, 44). THP-1 cells transfected for 12 h with either the DN SEK1 or the control plasmid were stimulated with LPS and analyzed for JNK phosphorylation and CD44 expression. The optimal time period of 12 h of posttransfection before LPS-stimulation was chosen based on the results of a series of experiments in which cells were stimulated with LPS for 2, 4, 8, 12, or 24 h posttransfection (data not shown). LPS stimulation of THP-1 cells transfected with the DN SEK1 revealed reduced JNK phosphorylation compared with that observed in cells transfected with the control plasmid (Fig. 10A). LPS stimulation of THP-1 cells transfected with the DN SEK1 revealed significantly lower expression of CD44 compared with that of the LPS-stimulated cells transfected with the control vector (Fig. 10B). In contrast, transfection of THP-1 cells with DN SEK1 did not impact TNF-{alpha}-induced CD44 expression (Fig. 10GoB). The effect of DN SEK1 on CD44 expression was specific since the expression of surface CD14 and IL-10 production remained unaffected following LPS stimulation (data not shown). These results suggest a distinct role for JNK-mediated signals in LPS-induced CD44 expression in THP-1 cells. In contrast, TNF-{alpha}-induced regulation of CD44 expression did not involve JNK activation in these cells.



View larger version (18K):
[in this window]
[in a new window]
  		FIGURE 10. A, JNK phosphorylation is inhibited by LPS stimulation in DN SEK1-transfected THP-1 cells. Cells were transfected with either a DN SEK1 kinase or with control vector followed by stimulation with LPS for 10 min. Crude protein extracts (50 µg) were subjected to SDS-PAGE analysis followed by Western blot analysis. The membranes were blotted with an anti-phospho JNK rabbit polyclonal Ab, and to control for equal loading of proteins, the membranes were stripped and reprobed with anti-JNK rabbit polyclonal Ab. B, A DN mutant of MKK-4 selectively inhibits LPS-induced, but not TNF-{alpha}-induced, CD44 expression in THP-1 cells. THP-1 cells were transfected with either a DN SEK1 kinase, or with a control vector. Transfected cells were stimulated with either LPS (1 µg/ml) or TNF-{alpha} (10 ng/ml) for 24 h. Following the incubations, cells were analyzed by flow cytometry for CD44 surface expression. The experiment shown is representative of three different experiments.

 
Egr-1 binding to the CD44 promoter in LPS-stimulated cells is regulated by JNK MAPK

Egr-1 has been shown to play a role in CD44 expression in human B cells and endothelial cells (22, 23). Although the role of Egr-1 in the regulation of CD44 expression in human monocytic cells has not been demonstrated, it was of interest to investigate whether LPS and TNF-{alpha} induce Egr-1 activation in THP-1 cells, and whether LPS-induced Egr-1 activation can be inhibited by dexamethasone and a specific inhibitor of JNK. Cells were stimulated with LPS or TNF-{alpha} over a period of time ranging from 0–240 min, and the nuclear extracts were analyzed for binding to the Egr-1 oligonucleotide probe by a gel shift assay. Maximum binding of Egr-1 to the Egr-1 oligonucleotide sequence occurred at 60–240 min following stimulation with LPS and TNF-{alpha} (Fig. 11Go). We observed a major band corresponding to the Egr-1 DNA-protein complex that was blocked by competition with cold Egr-1 oligonucleotides, indicating its specificity. Incubation of THP-1 cells with dexamethasone or SP600125 for 2 h before stimulation with LPS inhibited Egr-1 binding to the oligonucleotide containing the Egr-1 sequence (Fig. 11GoA). We also show that LPS stimulation of THP-1 cells transfected with the DN SEK1, in contrast to cells transfected with the control plasmid, showed decreased binding of Egr-1 to the Egr-1 oligonucleotide sequence (Fig. 11GoA). Because treatment of THP-1 cells with dexamethasone and SP600125 as well as transfection with DN SEK1 did not affect TNF-{alpha}-induced CD44 expression in our studies, we did not investigate the effect of dexamethasone or SP600125 on TNF-{alpha}-induced Egr-1 binding activity. These results show that dexamethasone and SP600125 regulate LPS-induced Egr-1 activation, suggesting a role for JNK in Egr-1 activation and possibly for CD44 induction in monocytic cells following LPS stimulation.



View larger version (36K):
[in this window]
[in a new window]
  		FIGURE 11. Dexamethasone, DN SEK 1, and SP600125 inhibit LPS-induced activation of the Egr-1 transcription factor in THP-1 cells. THP-1 cells were stimulated with LPS (1 µg/ml) (A) or TNF-{alpha} (10 ng/ml) (B) for various times ranging from 15–240 min followed by centrifugation and collection of cell pellets. To determine the effects of dexamethasone and SP600125 on LPS-induced activation of Egr-1, cells were treated with dexamethasone (50 nM) or SP600125 (10–50 µM) for 2 h before stimulation with LPS (1 µg/ml). To determine the effect of JNK on Egr-1 activation, THP-1 cells were transfected with either a vector containing a DN SEK1 or control vector, followed by treatment with LPS for 60 min. To perform the gel shift assay, nuclear extracts were harvested from the cell pellets obtained at each time point. Nuclear extracts containing 5 µg of protein were incubated for 1 h with 32P-labeled oligonucleotides corresponding to the consensus sequence for Egr-1. To determine the specificity of Egr-1 transcription factor binding, the nuclear extracts were incubated with 100-fold unlabeled oligonucleotides (cold competitor) corresponding to the consensus sequence of Egr-1. The complexes were subjected to electrophoresis followed by autoradiography. The experiment shown is representative of three experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
It is well established that CD44-mediated HA interactions play a vital role at multiple steps in the generation of immune responses including inflammation (4, 5). LPS is a major inducer of CD44 expression and may modulate CD44-mediated biological effects in monocytes during inflammation (3). In this study, we have investigated the role of MAPKs in the regulation of CD44 expression in human monocytic cells stimulated with either LPS, IL-10, or TNF-{alpha}, the cytokines produced by LPS-stimulated monocytic cells. We provide evidence for the involvement of p38 MAPK, at least in part, in TNF-{alpha}-induced CD44 expression in both normal monocytes and THP-1 cells. In contrast, neither p38 nor p42/44 MAPKs were found to be involved in IL-10-induced CD44 expression in monocytes. To dissect the TNF-{alpha}-/LPS-induced signaling pathway regulating CD44 expression independent of IL-10, we used IL-10 refractory THP-1 cells as a model system. Our results reveal the distinct involvement of JNK MAPK in the LPS-mediated but not in the TNF-{alpha}-mediated pathway resulting in CD44 induction.

LPS signaling through CD14 has been shown to involve TLR-4 which associate with CD14 (45, 46). LPS interaction with CD14 promotes dimerization of TLR and subsequent recruitment of MyD88, a myeloid differentiation marker which functions as an adaptor molecule (45, 47). MyD88 associates via its C-terminal toll homology domain with TLR, and via its N-terminal death domain with a serine-threonine protein kinase, IL-1R-associated kinase (47). Upon interaction with MyD88, IL-1R-associated kinase is autophosphorylated and binds to TNFR-associated factor-6. TNFR-associated factor-6 subsequently activates TGF{beta}-activated kinase-1 and MKK kinase, members of the MAPK signaling cascade (47, 48, 49). This interaction activates the NF-{kappa}B complex in addition to the p38 MAPK (48, 49).

Regulation of CD44 induction in monocytic cells following LPS stimulation may involve a combination of signals delivered by the interaction of LPS with the CD14/TLR complex as well as by the interaction of endogenously produced cytokines with their cognate receptors. TNF-{alpha} and IL-10 have been shown to be the primary cytokines that regulate CD44 expression in LPS-stimulated normal human monocytes (3, 14). In this study, we attempted to elucidate the molecular mechanism, specifically the role of MAPKs underlying CD44 induction in monocytic cells stimulated with LPS, TNF-{alpha}, or IL-10. Our results show that the p42/44 ERK MAPK was not involved in the regulation of CD44 expression induced either by LPS, IL-10, or TNF-{alpha}. However, SB202190, a p38 inhibitor, partially prevented LPS-induced CD44 expression. Because SB202190 inhibited LPS-induced IL-10 and TNF-{alpha} production, down-regulation of CD44 expression may be attributed to a block in the production of these cytokines.

TNF-{alpha} is a pleiotropic cytokine that has growth modulatory, cytotoxic, and inflammatory activities (50). Interaction of TNF-{alpha} with its receptor activates complex intracellular signaling pathways, including protein tyrosine kinase-dependent cascades, leading to the activation of transcription factors such as NF-{kappa}B, AP-1, etc. (28, 51). TNF-{alpha} has been shown to activate all three types of MAPKs in different cell types (28, 29). However, the role of intracellular signaling molecules, especially the MAPK, in TNF-{alpha}-induced CD44 expression remains largely unknown. Our results consistently show partial inhibition of CD44 expression by the p38 inhibitor in both monocytes and THP-1 cells. The partial inhibition of CD44 induction by SB202190 in both cell types was observed at low doses of 10 µM, and a complete inhibition was not observed even when concentrations of 25 and 50 µM of SB202190 were used. Considering the complex mechanism underlying CD44 regulation, partial inhibition of CD44 expression by SB202190 may be attributed to other signaling molecules that may cooperate with the members of p38 MAPK pathway in TNF-{alpha}-induced CD44 expression. Because high concentrations of SB202190 may nonspecifically inhibit some kinases (52), the possible role of these kinases, however minor, in the observed partial inhibition of CD44 expression cannot be ruled out.

The signaling molecules that mediate the biological effects of IL-10, especially those involved in IL-10-mediated CD44 up-regulation, are not well understood. IL-10 has been shown to mediate its inhibitory effects on murine macrophages through the activation of STAT3 (53, 54). Recently, IL-10 was shown to increase the expression of the cell cycle inhibitor, cyclin-dependent kinase inhibitor p19INK4D in macrophages. IL-10-induced expression of p19INK4D was later shown to be dependent on STAT3 activation (55). Herein, we show that IL-10 is capable of inducing p42/44 but not p38 activation. However, p42/44 was not found to be involved in IL-10-induced CD44 expression. Whether IL-10-induced CD44 expression is also mediated by STAT-3-dependent activation of cyclin-dependent kinase inhibitors p19INK4D needs to be investigated.

The role of MAPKs in LPS- and TNF-{alpha}-induced CD44 expression could not be studied in normal human monocytes because endogenously produced IL-10 up-regulates CD44 expression independent of p38 and ERK kinases. IL-10 refractory THP-1 cells provide a useful model to study the LPS-mediated signaling events in CD44 expression without the additional effects of endogenously produced IL-10. Our results show the distinct involvement of JNK in LPS-induced CD44 expression. This conclusion is based on results derived by using the specific JNK inhibitor, SP600125, as well as following transfection of THP-1 cells with a DN SEK1 kinase. Two MKKs, MKK4 and MKK7, have been found to be the primary activators of JNK (56, 57). MKK4 is an essential component of the JNK signal transduction pathway, and disruption of the MKK4 gene blocks JNK activity (58). DN SEK1 has also been shown to act as a specific inhibitor of the JNK signal transduction pathway (42, 43, 44). However, it was recently shown that MKK4 can also activate p38 MAPK (57, 59). Although p38 MAPK can be modulated by MKK4, the p38 MAPK inhibitor had only a partial effect on CD44 expression. This is in contrast to the significant reduction of LPS-induced CD44 expression observed following transfection of DN SEK1. Furthermore, transfection of THP-1 cells with DN SEK1 did not influence TNF-{alpha}-induced CD44 expression. These results suggest a key role for JNK and/or its specific upstream kinases and regulators in LPS-induced CD44 induction.

To determine the role of JNK, we initially used dexamethasone, an anti-inflammatory glucocorticoid and inhibitor of JNK/stress-activated protein kinase activation (41) which mediates its biological effects on cytokine production primarily by complexing with AP-1, thus down-regulating AP-1 activity (60, 61). Furthermore, dexamethasone has been shown to inhibit AP-1 activity by interfering with JNK phosphorylation (41, 62). In this study, dexamethasone inhibited LPS- and TNF-{alpha}-induced phosphorylation of JNK. However, dexamethasone treatment reduced CD44 expression to basal levels in LPS-stimulated, but not in TNF-{alpha}-stimulated cells. Similar results were obtained by using SP600125, a specific JNK inhibitor. These results may also suggest a role for JNK in LPS-, but not in TNF-{alpha}-, mediated CD44 up-regulation.

The JNK MAPK pathway includes JNK1, JNK2, and JNK3 (63). JNK1 and JNK2 are widely expressed in several tissues, whereas JNK3 is more selectively expressed in the brain, testis, and heart. The JNK3 gene has been shown to be involved in neuronal cell death (64), whereas JNK1 and JNK2 have been implicated in Th1/Th2 cell differentiation (65, 66). JNK1 has also been shown to regulate the development of T cell-mediated immunity against Leishmania major infections in an experimental mouse model (67). Whether JNK1 or JNK2 regulate LPS-induced CD44 expression needs to be investigated.

The findings of this study raise the question of how activation of JNK and its upstream regulators may serve to induce CD44 expression. To understand the signaling events downstream of JNK MAPK activation responsible for CD44 gene transcription, we attempted to identify the transcription factors involved. Recently, Egr-1 was shown to be involved in PMA-induced CD44 expression in murine B cells (22) and also in IL-1{alpha}-induced CD44 expression in human endothelial cells (23). The Egr-1 gene is a prototypic member of a gene family encoding transcription factors which share a conserved zinc finger DNA-binding motif (68). This gene is induced rapidly and transiently in response to B cell receptor cross-linking or treatment with PMA (69). It has been shown that both p38 and JNK MAPKs are involved in Egr-1 activation (70). Although the role of Egr-1 in the regulation of CD44 expression in human monocytic cells has not been clearly demonstrated, our results suggest that both LPS and TNF-{alpha} induce Egr-1 activation in THP-1 cells. Furthermore, treatment of cells with dexamethasone, SP600125, or transfection of cells with DN SEK1 reduced Egr-1 activation in LPS-stimulated THP-1 cells, suggesting a role for JNK in LPS-induced Egr-1 activation. However, the molecular mechanism by which TNF-{alpha} induces Egr-1 activation, as well as the role of Egr-1 in CD44 expression in human monocytic cells, needs to be investigated.

In summary, regulation of CD44 expression in LPS-stimulated monocytic cells involves a complex sequence of intracellular signaling events. Our results reveal that the regulation of CD44 expression in monocytic cells involves cross-talk between the signals delivered by the interaction of LPS with the CD14/TLR complex and by the interaction of IL-10 and TNF-{alpha} with their respective receptor complexes. In this study, we show for the first time a role for JNK in the LPS-mediated, but not in the TNF-{alpha}-mediated, signaling pathway resulting in CD44 induction. In contrast, p38 MAPK was found to partially regulate TNF-{alpha}-induced CD44 expression in both normal monocytes and in THP-1 cells. We also show that activation of JNK MAPK may result in the activation of Egr-1, the transcription factor implicated in CD44 transcription. Therefore, JNK MAPK represents an important and novel target for anti-inflammatory JNK inhibitors capable of inhibiting CD44 expression. Further understanding of the mechanism leading to CD44 induction may help in devising strategies for the manipulation of immune responses such as inflammation.


   Acknowledgments
 
We thank Dr. G. Graziani-Bowering for critically reading the manuscript. We also thank Dr. J. R. Woodgett for providing us with pcDNA plasmid expressing the DN mutant SEK1.


   Footnotes
 
1 This work was supported by grants from the Cancer Research Society, Canada, Natural Sciences and Engineering Research Council of Canada, and the Research Institute, Children’s Hospital of Eastern Ontario (to A.K.). W.M. and W.L. were supported by scholarships from the Ontario HIV Treatment Network. K.G. was supported by fellowships from the Medical Research Council of Canada and from the Strategic Areas of Development from the University of Ottawa. Back

2 Address correspondence and reprint requests to Dr. Ashok Kumar, Division of Virology, Research Institute, Children’s Hospital of Eastern Ontario, University of Ottawa, 401 Smyth Road, Ottawa, Ontario, Canada, K1H 8L1. E-mail address: akumar{at}med.uottawa.ca Back

3 Abbreviations used in this paper: HA, hyaluronan; DN, dominant negative; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; MKK, MAPK kinase; SEK1, stress-activated protein/ERK kinase 1; TLR, Toll-like receptor; Egr-1, early growth response gene; DN SEK1, DN SEK1 plasmid; MCF, mean channel fluorescence. Back

Received for publication January 31, 2002. Accepted for publication September 17, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Heinzelmann, M., H. C. J. Polk, A. Chernobelsky, T. P. Stites, L. E. Gordon. 2000. Endotoxin and muramyl dipeptide modulate surface receptor expression on human mononuclear cells. Immunopharmacology 48:117.[Medline]
  2. Verhasselt, V., C. Buelens, F. Willems, D. De Groote, N. Haeffner-Cavaillon, M. Goldman. 1997. Bacterial lipopolysaccharide stimulates the production of cytokines and the expression of costimulatory molecules by human peripheral blood dendritic cells: evidence for a soluble CD14-dependent pathway. J. Immunol. 158:2919.[Abstract]
  3. Levesque, M. C., B. F. Haynes. 1997. Cytokine induction of the ability of human monocyte CD44 to bind hyaluronan is mediated primarily by TNF-{alpha} and is inhibited by IL-4 and IL-13. J. Immunol. 159:6184.[Abstract]
  4. Pure, E., C. A. Cuff. 2001. A crucial role for CD44 in inflammation. Trends Mol. Med. 7:213.[Medline]
  5. Siegelman, M. H., H. C. DeGrendele, P. Estess. 1999. Activation and interaction of CD44 and hyaluronan in immunological systems. J. Leukocyte Biol. 66:315.[Abstract]
  6. Lesley, J., R. Hyman, P. W. Kincade. 1993. CD44 and its interaction with extracellular matrix. Adv. Immunol. 54:271.[Medline]
  7. Naor, D., R. V. Sionov, D. Ish-Shalom. 1997. CD44: structure, function, and association with the malignant process. Adv. Cancer Res. 71:241.[Medline]
  8. Shimizu, Y., G. A. Van Seventer, R. Siraganian, L. Wahl, S. Shaw. 1989. Dual role of the CD44 molecule in T cell adhesion and activation. J. Immunol. 143:2457.[Abstract]
  9. Miyake, K., K. L. Medina, S. Hayashi, S. Ono, T. Hamaoka, P. W. Kincade. 1990. Monoclonal antibodies to Pgp-1/CD44 block lympho-hemopoiesis in long-term bone marrow cultures. J. Exp. Med. 171:477.[Abstract/Free Full Text]
  10. Stamenkovic, I., A. Aruffo, M. Amiot, B. Seed. 1991. The hematopoietic and epithelial forms of CD44 are distinct polypeptides with different adhesion potentials for hyaluronate-bearing cells. EMBO J. 10:343.[Medline]
  11. Gunthert, U., M. Hofmann, W. Rudy, S. Reber, M. Zoller, I. Haussmann, S. Matzku, A. Wenzel, H. Ponta, P. Herrlich. 1991. A new variant of glycoprotein CD44 confers metastatic potential to rat carcinoma cells. Cell 65:13.[Medline]
  12. Dougherty, G. J., D. L. Cooper, J. F. Memory, R. K. Chiu. 1994. Ligand binding specificity of alternatively spliced CD44 isoforms: recognition and binding of hyaluronan by CD44R1. J. Biol. Chem. 269:9074.[Abstract/Free Full Text]
  13. Katoh, S., Z. Zheng, K. Oritani, T. Shimozato, P. W. Kincade. 1995. Glycosylation of CD44 negatively regulates its recognition of hyaluronan. J. Exp. Med. 182:419.[Abstract/Free Full Text]
  14. Levesque, M. C., B. F. Haynes. 1999. TNF-{alpha} and IL-4 regulation of hyaluronan binding to monocyte CD44 involves posttranslational modification of CD44. Cell. Immunol. 193:209.[Medline]
  15. Kryworuchko, M., F. Diaz-Mitoma, A. Kumar. 1999. Interferon-{gamma} inhibits CD44-hyaluronan interactions in normal human B lymphocytes. Exp. Cell Res. 250:241.[Medline]
  16. Kryworuchko, M., K. Gee, F. Diaz-Mitoma, A. Kumar. 1999. Regulation of CD44-hyaluronan interactions in Burkitt’s lymphoma and Epstein-Barr virus-transformed lymphoblastoid B cells by PMA and interleukin-4. Cell. Immunol. 194:54.[Medline]
  17. Mackay, C. R., H. J. Terpe, R. Stauder, W. L. Marston, H. Stark, U. Gunthert. 1994. Expression and modulation of CD44 variant isoforms in humans. J. Cell Biol. 124:71.[Abstract/Free Full Text]
  18. Murakami, S., K. Miyake, C. H. June, P. W. Kincade, R. J. Hodes. 1990. IL-5 induces a Pgp-1 (CD44) bright B cell subpopulation that is highly enriched in proliferative and Ig secretory activity and binds to hyaluronate. J. Immunol. 145:3618.[Abstract]
  19. Legras, S., J. P. Levesque, R. Charrad, K. Morimoto, C. Le Bousse, D. Clay, C. Jasmin, F. Smadja-Joffe. 1997. CD44-mediated adhesiveness of human hematopoietic progenitors to hyaluronan is modulated by cytokines. Blood 89:1905.[Abstract/Free Full Text]
  20. Sionov, R. V., D. Naor. 1998. Calcium- and calmodulin-dependent PMA-activation of the CD44 adhesion molecule. Cell Adhes. Commun. 6:503.[Medline]
  21. Fichter, M., R. Hinrichs, G. Eissner, B. Scheffer, S. Classen, M. Ueffing. 1997. Expression of CD44 isoforms in neuroblastoma cells is regulated by PI 3-kinase and protein kinase C. Oncogene 14:2817.[Medline]
  22. Maltzman, J. S., J. A. Carman, J. G. Monroe. 1996. Role of EGR1 in regulation of stimulus-dependent CD44 transcription in B lymphocytes. Mol. Cell. Biol. 16:2283.[Abstract]
  23. Fitzgerald, K. A., L. A. O’Neill. 1999. Characterization of CD44 induction by IL-1: a critical role for Egr-1. J. Immunol. 162:4920.[Abstract/Free Full Text]
  24. DeFranco, A. L., M. T. Crowley, A. Finn, J. Hambleton, S. L. Weinstein. 1998. The role of tyrosine kinases and MAP kinases in LPS-induced signaling. Prog. Clin. Biol. Res. 397:119.[Medline]
  25. Lewis, T. S., P. S. Shapiro, N. G. Ahn. 1998. Signal transduction through MAP kinase cascades. Adv. Cancer Res. 74:49.[Medline]
  26. Liu, M. K., P. Herrera-Velit, R. W. Brownsey, N. E. Reiner. 1994. CD14-dependent activation of protein kinase C and mitogen-activated protein kinases (p42 and p44) in human monocytes treated with bacterial lipopolysaccharide. J. Immunol. 153:2642.[Abstract]
  27. Hambleton, J., S. L. Weinstein, L. Lem, A. L. DeFranco. 1996. Activation of c-Jun N-terminal kinase in bacterial lipopolysaccharide-stimulated macrophages. Proc. Natl. Acad. Sci. USA 93:2774.[Abstract/Free Full Text]
  28. Hatzoglou, A., J. Roussel, M. F. Bourgeade, E. Rogier, C. Madry, J. Inoue, O. Devergne, A. Tsapis. 2000. TNF receptor family member BCMA (B cell maturation) associates with TNF receptor-associated factor (TRAF) 1, TRAF2, and TRAF3 and activates NF-{kappa}B, elk-1, c-Jun N-terminal kinase, and p38 mitogen-activated protein kinase. J. Immunol. 165:1322.[Abstract/Free Full Text]
  29. Darnay, B. G., B. B. Aggarwal. 1997. Early events in TNF signaling: a story of associations and dissociations. J. Leukocyte Biol. 61:559.[Abstract]
  30. Ma, W., W. Lim, K. Gee, S. Aucoin, D. Nandan, M. Kozlowski, F. Diaz-Mitoma, A. Kumar. 2001. The p38 mitogen activated kinase pathway regulates the human IL-10 promoter via the activation of Sp1 transcription factor in lipopolysaccharide-stimulated human macrophages. J. Biol. Chem. 17:13664.
  31. Dudley, D. T., L. Pang, S. J. Decker, A. J. Bridges, A. R. Saltiel. 1995. A synthetic inhibitor of the mitogen-activated protein kinase cascade. Proc. Natl. Acad. Sci. USA 92:7686.[Abstract/Free Full Text]
  32. Lee, J. C., P. R. Young. 1996. Role of CSB/p38/RK stress response kinase in LPS and cytokine signaling mechanisms. J. Leukocyte Biol. 59:152.[Abstract]
  33. Bennett, B. L., D. T. Sasaki, B. W. Murray, E. C. O’Leary, S. T. Sakata, W. Xu, J. C. Leisten, A. Motiwala, S. Pierce, Y. Satoh, et al 2001. SP600125, an anthrapyrazolone inhibitor of Jun N-terminal kinase. Proc. Natl. Acad. Sci. USA 98:13681.[Abstract/Free Full Text]
  34. Daftarian, P. M., A. Kumar, M. Kryworuchko, F. Diaz-Mitoma. 1996. IL-10 production is enhanced in human T cells by IL-12 and IL-6 and in monocytes by tumor necrosis factor-{alpha}. J. Immunol. 157:12.[Abstract]
  35. Gee, K., M. Kozlowski, M. Kryworuchko, F. Diaz-Mitoma, A. Kumar. 2001. Differential effect of IL-4 and IL-13 on CD44 expression in the Burkitt’s lymphoma B cell line bl30/b95–8 and in Epstein-Barr virus (EBV) transformed human B cells: loss of IL-13 receptors on Burkitt’s lymphoma B cells. Cell. Immunol. 211:131.[Medline]
  36. Lim, W., W. Ma, K. Gee, S. Aucoin, D. Nandan, F. Diaz-Mitoma, M. Kozlowski, A. Kumar. 2002. Distinct role of p38 and c-jun N-terminal kinases in IL-10-dependent and IL-10-independent regulation of the co-stimulatory molecule B7.2 in lipopolysaccharide-stimulated human monocytic cells. J. Immunol. 168:1759.[Abstract/Free Full Text]
  37. Arrighi, J. F., M. Rebsamen, F. Rousset, V. Kindler, C. Hauser. 2001. A critical role for p38 mitogen-activated protein kinase in the maturation of human blood-derived dendritic cells induced by lipopolysaccharide, TNF-{alpha}, and contact sensitizers. J. Immunol. 166:3837.[Abstract/Free Full Text]
  38. De Cesaris, P., D. Starace, G. Starace, A. Filippini, M. Stefanini, E. Ziparo. 1999. Activation of Jun N-terminal kinase/stress-activated protein kinase pathway by tumor necrosis factor {alpha} leads to intercellular adhesion molecule-1 expression. J. Biol. Chem. 274:28978.[Abstract/Free Full Text]
  39. Foey, A. D., S. L. Parry, L. M. Williams, M. Feldmann, B. M. Foxwell, F. M. Brennan. 1998. Regulation of monocyte IL-10 synthesis by endogenous IL-1 and TNF-{alpha}: role of the p38 and p42/44 mitogen-activated protein kinases. J. Immunol. 160:920.[Abstract/Free Full Text]
  40. Lee, J. C., J. T. Laydon, P. C. McDonnell, T. F. Gallagher, S. Kumar, D. Green, D. McNulty, M. J. Blumenthal, J. R. Heys, S. W. Landvatter. 1994. A protein kinase involved in the regulation of inflammatory cytokine biosynthesis. Nature 372:739.[Medline]
  41. Hirasawa, N., Y. Sato, Y. Fujita, S. Mue, K. Ohuchi. 1998. Inhibition by dexamethasone of antigen-induced c-Jun N-terminal kinase activation in rat basophilic leukemia cells. J. Immunol. 161:4939.[Abstract/Free Full Text]
  42. Whitmarsh, A. J., P. Shore, A. D. Sharrocks, R. J. Davis. 1995. Integration of MAP kinase signal transduction pathways at the serum response element. Science 269:403.[Abstract/Free Full Text]
  43. Zanke, B. W., K. Boudreau, E. Rubie, E. Winnett, L. A. Tibbles, L. Zon, J. Kyriakis, F. F. Liu, J. R. Woodgett. 1996. The stress-activated protein kinase pathway mediates cell death following injury induced by cis-platinum, UV irradiation or heat. Curr. Biol. 6:606.[Medline]
  44. Verheij, M., R. Bose, X. H. Lin, B. Yao, W. D. Jarvis, S. Grant, M. J. Birrer, E. Szabo, L. I. Zon, J. M. Kyriakis, et al 1996. Requirement for ceramide-initiated SAPK/JNK signalling in stress-induced apoptosis. Nature 380:75.[Medline]
  45. Takeuchi, O., K. Hoshino, S. Akira. 2000. Cutting edge: TLR2-deficient and MyD88-deficient mice are highly susceptible to Staphylococcus aureus infection. J. Immunol. 165:5392.[Abstract/Free Full Text]
  46. Takeuchi, O., K. Hoshino, T. Kawai, H. Sanjo, H. Takada, T. Ogawa, K. Takeda, S. Akira. 1999. Differential roles of TLR2 and TLR4 in recognition of Gram-negative and Gram-positive bacterial cell wall components. Immunity 11:443.[Medline]
  47. Muzio, M., G. Natoli, S. Saccani, M. Levrero, A. Mantovani. 1998. The human toll signaling pathway: divergence of nuclear factor {kappa}B and JNK/SAPK activation upstream of tumor necrosis factor receptor-associated factor 6 (TRAF6). J. Exp. Med. 187:2097.[Abstract/Free Full Text]
  48. Ninomiya-Tsuji, J., K. Kishimoto, A. Hiyama, J. Inoue, Z. Cao, K. Matsumoto. 1999. The kinase TAK1 can activate the NIK-I{kappa}B as well as the MAP kinase cascade in the IL-1 signalling pathway. Nature 398:252.[Medline]
  49. Yang, R. B., M. R. Mark, A. L. Gurney, P. J. Godowski. 1999. Signaling events induced by lipopolysaccharide-activated toll-like receptor 2. J. Immunol. 163:639.[Abstract/Free Full Text]
  50. Smith, C. A., T. Farrah, R. G. Goodwin. 1994. The TNF receptor superfamily of cellular and viral proteins: activation, costimulation, and death. Cell 76:959.[Medline]
  51. Rothe, M., V. Sarma, V. M. Dixit, D. V. Goeddel. 1995. TRAF2-mediated activation of NF-{kappa}B by TNF receptor 2 and CD40. Science 269:1424.[Abstract/Free Full Text]
  52. Davies, S. P., H. Reddy, M. Caivano, P. Cohen. 2000. Specificity and mechanism of action of some commonly used protein kinase inhibitors. Biochem. J. 351:95.[Medline]
  53. O’Farrell, A. M., Y. Liu, K. W. Moore, A. L. Mui. 1998. IL-10 inhibits macrophage activation and proliferation by distinct signaling mechanisms: evidence for Stat3-dependent and -independent pathways. EMBO J. 17:1006.[Medline]
  54. Takeda, K., B. E. Clausen, T. Kaisho, T. Tsujimura, N. Terada, I. Forster, S. Akira. 1999. Enhanced Th1 activity and development of chronic enterocolitis in mice devoid of Stat3 in macrophages and neutrophils. Immunity 10:39.[Medline]
  55. O’Farrell, A. M., D. A. Parry, F. Zindy, M. F. Roussel, E. Lees, K. W. Moore, A. L. Mui. 2000. Stat3-dependent induction of p19INK4D by IL-10 contributes to inhibition of macrophage proliferation. J. Immunol. 164:4607.[Abstract/Free Full Text]
  56. Tournier, C., A. J. Whitmarsh, J. Cavanagh, T. Barrett, R. J. Davis. 1997. Mitogen-activated protein kinase kinase 7 is an activator of the c-Jun NH2-terminal kinase. Proc. Natl. Acad. Sci. USA 94:7337.[Abstract/Free Full Text]
  57. Derijard, B., J. Raingeaud, T. Barrett, I. H. Wu, J. Han, R. J. Ulevitch, R. J. Davis. 1995. Independent human MAP-kinase signal transduction pathways defined by MEK and MKK isoforms. Science 267:682.[Abstract/Free Full Text]
  58. Yang, D., C. Tournier, M. Wysk, H. T. Lu, J. Xu, R. J. Davis, R. A. Flavell. 1997. Targeted disruption of the MKK4 gene causes embryonic death, inhibition of c-Jun NH2-terminal kinase activation, and defects in AP-1 transcriptional activity. Proc. Natl. Acad. Sci. USA 94:3004.[Abstract/Free Full Text]
  59. Guan, Z., S. Y. Buckman, A. P. Pentland, D. J. Templeton, A. R. Morrison. 1998. Induction of cyclooxygenase-2 by the activated MEKK1->SEK1/MKK4->p38 mitogen-activated protein kinase pathway. J. Biol. Chem. 273:12901.[Abstract/Free Full Text]
  60. Jonat, C., H. J. Rahmsdorf, K. K. Park, A. C. Cato, S. Gebel, H. Ponta, P. Herrlich. 1990. Antitumor promotion and antiinflammation: down-modulation of AP-1 (Fos/Jun) activity by glucocorticoid hormone. Cell 62:1189.[Medline]
  61. Schule, R., P. Rangarajan, S. Kliewer, L. J. Ransone, J. Bolado, N. Yang, I. M. Verma, R. M. Evans. 1990. Functional antagonism between oncoprotein c-Jun and the glucocorticoid receptor. Cell 62:1217.[Medline]
  62. Shalom-Barak, T., J. Quach, M. Lotz. 1998. Interleukin-17-induced gene expression in articular chondrocytes is associated with activation of mitogen-activated protein kinases and NF-{kappa}B. J. Biol. Chem. 273:27467.[Abstract/Free Full Text]
  63. Gupta, S., T. Barrett, A. J. Whitmarsh, J. Cavanagh, H. K. Sluss, B. Derijard, R. J. Davis. 1996. Selective interaction of JNK protein kinase isoforms with transcription factors. EMBO J. 15:2760.[Medline]
  64. Yang, D. D., C. Y. Kuan, A. J. Whitmarsh, M. Rincon, T. S. Zheng, R. J. Davis, P. Rakic, R. A. Flavell. 1997. Absence of excitotoxicity-induced apoptosis in the hippocampus of mice lacking the Jnk3 gene. Nature 389:865.[Medline]
  65. Yang, D. D., D. Conze, A. J. Whitmarsh, T. Barrett, R. J. Davis, M. Rincon, R. A. Flavell. 1998. Differentiation of CD4+ T cells to Th1 cells requires MAP kinase JNK2. Immunity 9:575.[Medline]
  66. Dong, C., D. D. Yang, C. Tournier, A. J. Whitmarsh, J. Xu, R. J. Davis, R. A. Flavell. 2000. JNK is required for effector T-cell function but not for T-cell activation. Nature 405:91.[Medline]
  67. Constant, S. L., C. Dong, D. D. Yang, M. Wysk, R. J. Davis, R. A. Flavell. 2000. JNK1 is required for T cell-mediated immunity against Leishmania major infection. J. Immunol. 165:2671.[Abstract/Free Full Text]
  68. Cao, X. M., R. A. Koski, A. Gashler, M. McKiernan, C. F. Morris, R. Gaffney, R. V. Hay, V. P. Sukhatme. 1990. Identification and characterization of the Egr-1 gene product, a DNA-binding zinc finger protein induced by differentiation and growth signals. Mol. Cell. Biol. 10:1931.[Abstract/Free Full Text]
  69. Seyfert, V. L., S. McMahon, W. Glenn, X. M. Cao, V. P. Sukhatme, J. G. Monroe. 1990. Egr-1 expression in surface Ig-mediated B cell activation: kinetics and association with protein kinase C activation. J. Immunol. 145:3647.[Abstract]
  70. Lim, C. P., N. Jain, X. Cao. 1998. Stress-induced immediate-early gene, egr-1, involves activation of p38/JNK1. Oncogene 16:2915.[Medline]



This article has been cited by other articles:


Home page
 BloodHome page
E. Vachon, R. Martin, V. Kwok, V. Cherepanov, C.-W. Chow, C. M. Doerschuk, J. Plumb, S. Grinstein, and G. P. Downey
CD44-mediated phagocytosis induces inside-out activation of complement receptor-3 in murine macrophages
Blood, December 15, 2007; 110(13): 4492 - 4502.
[Abstract] [Full Text] [PDF]

Home page
 J. Leukoc. Biol.Home page
M. Zoller, P. Gupta, R. Marhaba, M. Vitacolonna, and P. Freyschmidt-Paul
Anti-CD44-mediated blockade of leukocyte migration in skin-associated immune diseases
J. Leukoc. Biol., July 1, 2007; 82(1): 57 - 71.
[Abstract] [Full Text] [PDF]

Home page
 Mol Cancer ResHome page
N. Muthukumaran, K. E. Miletti-Gonzalez, A. K. Ravindranath, and L. Rodriguez-Rodriguez
Tumor Necrosis Factor-{alpha} Differentially Modulates CD44 Expression in Ovarian Cancer Cells
Mol. Cancer Res., August 1, 2006; 4(8): 511 - 520.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
J. P. Mishra, S. Mishra, K. Gee, and A. Kumar
Differential Involvement of Calmodulin-dependent Protein Kinase II-activated AP-1 and c-Jun N-terminal Kinase-activated EGR-1 Signaling Pathways in Tumor Necrosis Factor-{alpha} and Lipopolysaccharide-induced CD44 Expression in Human Monocytic Cells
J. Biol. Chem., July 22, 2005; 280(29): 26825 - 26837.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
A. A. R. Rahimi, K. Gee, S. Mishra, W. Lim, and A. Kumar
STAT-1 Mediates the Stimulatory Effect of IL-10 on CD14 Expression in Human Monocytic Cells
J. Immunol., June 15, 2005; 174(12): 7823 - 7832.
[Abstract] [Full Text] [PDF]

Home page
 FASEB J.Home page
Y. CHEN, Q. KE, Y. YANG, J. S. RANA, J. TANG, J. P. MORGAN, and Y.-F. XIAO
Cardiomyocytes overexpressing TNF-{alpha} attract migration of embryonic stem cells via activation of p38 and c-Jun amino-terminal kinase
FASEB J, December 1, 2003; 17(15): 2231 - 2239.
[Abstract] [Full Text] [PDF]

Home page
 Drug Metab. Dispos.Home page
A. Joiakim, P. A. Mathieu, C. Palermo, T. A. Gasiewicz, and J. J. Reiners Jr.
THE JUN N-TERMINAL KINASE INHIBITOR SP600125 IS A LIGAND AND ANTAGONIST OF THE ARYL HYDROCARBON RECEPTOR
Drug Metab. Dispos., November 1, 2003; 31(11): 1279 - 1282.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
K. Gee, M. Kozlowski, and A. Kumar
Tumor Necrosis Factor-{alpha} Induces Functionally Active Hyaluronan-adhesive CD44 by Activating Sialidase through p38 Mitogen-activated Protein Kinase in Lipopolysaccharide-stimulated Human Monocytic Cells
J. Biol. Chem., September 26, 2003; 278(39): 37275 - 37287.
[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
Right arrow Citation Map
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 Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Gee, K.
Right arrow Articles by Kumar, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Gee, K.
Right arrow Articles by Kumar, A.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS