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Originally published online as doi:10.1189/jlb.0905539 on March 24, 2006

Published online before print March 24, 2006
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(Journal of Leukocyte Biology. 2006;79:1306-1313.)
© 2006 by Society for Leukocyte Biology

Wnt signaling regulates transendothelial migration of monocytes

Lara Tickenbrock*, Joachim Schwäble*, Anke Strey{dagger}, Bülent Sargin*, Sina Hehn*, Marion Baas*, Chunaram Choudhary*, Volker Gerke{dagger}, Wolfgang E. Berdel*, Carsten Müller-Tidow*,{ddagger} and Hubert Serve*,{ddagger},1

* Department of Medicine, Hematology and Oncology, Division of Hematology/Oncology,
{dagger} Institute of Medical Biochemistry, ZMBE, and
{ddagger} The Interdisciplinary Centre of Clinical Research Münster (IZKF), University of Münster, Germany

1Correspondence: Department of Medicine, Hematology and Oncology, University of Münster, Domagkstr. 3, 48129 Münster, Germany. E-mail: serve{at}uni-muenster.de


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The Wnt-signaling pathway plays a critical role in directing cell fate during embryogenesis. Several lines of evidence also suggest a role in inflammatory processes. Here, we analyzed whether Wnt signaling plays a role in leukocyte inflammatory responses. Monocytes from healthy donors expressed different Frizzled receptors, which are ligands for the Wnt molecules. Activation of the Wnt/ß-catenin pathway by LiCl or Wnt3a increased ß-catenin protein levels in monocytes but not in granulocytes. It is interesting that the activation of Wnt/ß-catenin signaling via Wnt3a in monocytes resulted in a decrease in migration through an endothelial layer (human dermal microvascular endothelial cell-1). Further experiments revealed that the decrease in transendothelial migration was associated with specific monocyte adherence to endothelial cells after Wnt exposure. The specificity was verified by a lack of Wnt3a-induced adhesion to fibronectin, laminin, or collagen compared with endothelial interaction. Analysis of the distribution of ß-catenin revealed a Wnt3a-induced increase of ß-catenin in the cytoplasm. Wnt3a exposure did not result in any activation of the classical Wnt-target gene c-myc or a Wnt-target gene involved in cell adhesion (Connexin43). Our study implicates for the first time a role of canonical Wnt signaling in inflammatory processes in monocytes.

Key Words: ß-catenin • adhesion • inflammation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The Wnt-signaling pathway plays a central role in embryonic development, cell migration, cell polarity, and cell proliferation [1 ]. Also, it has been implicated in the development of many human cancers [2 ]. Wnts mediate signaling through Frizzled receptors, resulting in the activation of intracellular cascades that have profound biological effects [3 4 5 ]. Tight regulation of cytosolic levels of the oncoprotein ß-catenin determines the activity of this pathway [6 ]. ß-Catenin stability is regulated mainly by phosphorylation through the serine/threonine kinase glycogen synthase kinase 3ß (GSK3ß), which targets the protein for proteasomal degradation [7 ]. Activation of the Wnt-signaling cascade inhibits the activity of GSK3ß and thus increases cytosolic ß-catenin levels. The protein shuttles into the nucleus and acts as a transcriptional coactivator of T cell factor/lymphoid enhancer factor (TCF/LEF) family members [8 ]. These transcription factors induce Wnt-target genes, for example, c-myc or cyclin D1. In addition, ß-catenin plays a major role as a cytoplasmic protein, linking adhesion receptors of the cadherin family to the actin cytoskeleton [9 , 10 ]. Thus, ß-catenin levels in cells represent an important regulatory element mediating effects ranging from transcription regulation to membrane structure and cell shape.

A critical process in inflammatory processes is the migration of leukocytes across vascular endothelium. This transendothelial migration is triggered by cellular adhesion molecules and chemoattractants, which are responsible for signaling cascades in inflammation or tissue injury [11 12 13 14 15 ]. Migration of monocytes or granulocytes is initiated by weak binding of the leukocytes to endothelial surface molecules (selectins), resulting in a rolling movement of the blood cells [16 , 17 ]. Subsequently, integrins mediate stronger adhesion of the leukocytes to the endothelial monolayer, which is finally followed by opening of endothelial cell-cell contacts and junctions that facilitate the passage of the leukocytes [18 19 20 ].

So far, only preliminary data about the functional role of Wnt in leukocytes during inflammatory processes are available. A recent publication hints at a function for ß-catenin in the monocytic cell line THP-1, as lipopolysaccharide can induce ß-catenin in these cells, which also resulted in the expression of genes positively regulated by ß-catenin [21 ]. Here, we analyzed the effects of the Wnt/ß-catenin signaling pathway on inflammatory processes of leukocytes. After confirmation of the presence of Frizzled receptors in isolated monocytes by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot, these cells were exposed to Wnt3a-conditioned medium (CM), which specifically activates the canonical Wnt-signaling pathway. Although a strong induction of ß-catenin protein expression was observed in monocytes, no influence on the ß-catenin level could be detected in granulocytes. Wnt3a stimulation markedly decreased the migratory potential of monocytes, whereas the adhesion of monocytes to the endothelial layer was increased following Wnt activation. These events correlate with an enhanced stability of ß-catenin in the cytoplasm of monocytes but are most likely not mediated by activation of Wnt-target genes such as c-myc or Connexin43.

Taken together, activation of the Wnt/ß-catenin pathway in monocytes resulted in a decreased migration rate through an endothelial monolayer concomitant with a stronger adhesion of these cells to the endothelial cells. Knowledge about the precise role of the Wnt-signaling pathway for leukocyte adhesion and migration could help to identify new therapeutic targets for the control of inflammatory processes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cells
Human granulocytes and monocytes were isolated from healthy donors’ buffy coats by Ficoll-Paque and subsequent Percoll density gradient centrifugation [22 ]. Monocytes were then kept overnight in McCoy’s 5a medium containing 15% fetal calf serum (FCS) at 37°C in 5% CO2 using hydrophobic cell culture flasks. Granulocytes were kept overnight in RPMI medium supplemented with 10% FCS at 37°C prior to use in transmigration assays or adhesion assays. Cells were used for transmigration or adhesion assays on the next day.

Human dermal microvascular endothelial cells (HMEC-1), kindly provided by Dr. F. Candal (Centers for Disease Control, Atlanta, GA), were cultured at 37°C in 3% CO2 in MCDB131 supplemented with 10% FCSgold (PAA Laboratories, Cölbe, Germany) and 20 mM L-glutamine, 50 µg/ml gentamicin, 10 ng/ml epidermal growth factor, and 1 µg/ml hydrocortisone.

Wnt3a CM was prepared from confluent cultures of Wnt3a-producing L cells [stably transfected with Wnt3a cDNA, American Type Culture Collection (ATCC), Manassas, VA] or control L cells (ATCC), grown in supplemented Dulbecco’s modified Eagle’s medium (DMEM). Culture supernatants were collected after 3–4 days and 7–8 days.

Transmigration assay
Transmigration assays were performed as described previously [23 ]. Briefly, 2.2 x 105 HMEC-1 were seeded on fibronectin-coated 6.5 mm Transwell filters with a 5-µm pore size and grown to confluency. After 48 h, medium and nonadherent cells were removed, and 600 µl assay medium (DMEM supplemented with 10% FCS, 2 mM L-glutamine, 1% nonessential amino acids, and 25 mM Hepes) was added to the lower compartment of a two-chamber system separated by the Transwell filters. A total of 1–2 x 106 monocytes in 100 µl assay medium was added to the upper chamber, and cells were subsequently incubated at 37°C and 3% CO2 for 4 h. To analyze the effect of Wnt/ß-catenin signaling, activation monocytes were preincubated overnight in medium containing 50% of control CM or Wnt3a CM. Cells, which had transmigrated through the endothelial monolayer, were recovered in the lower tissue-culture chamber and quantified by counting in a flow cytometer (FACSCalibur, BD Biosciences, San Jose, CA). Data are presented as numbers of transmigrated cells across the monolayer for the indicated conditions. To verify the integrity of the endothelial monolayer after the assay, the upper chamber was washed twice with phosphate-buffered saline (PBS), fixed, stained with DiffQuick (Dade Behring, Düdingen, Switzerland), air-dried, and mounted on glass slides for microscopic analysis. Experiments were carried out in triplicates and independently performed at least three times.

Analysis of monocyte-endothelium adhesion
Wnt-stimulated or nonstimulated leukocyte adhesion to nonstimulated endothelium was analyzed by quantifying myeloperoxidase activity of adherent leukocytes as described previously [24 ].

RNA extraction and quantitative real-time PCR
Total RNA was extracted from cells using TRIzol reagent (Invitrogen, Carlsbad, CA), and 1 µg total DNase I-treated RNA was reverse-transcribed into cDNA using oligo-d(T)-primers. Quantification of mRNA levels was carried out using real-time fluorescence detection methods as described previously [25 ]. All samples were analyzed independently at least twice for each gene (Table 1 ). The housekeeping gene GAPDH served as an additional control for the cDNA quality.


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  		Table 1. Primer and Probes for RT-PCR

 
Antibodies and Western blot analysis
Whole cell lysates were prepared using radioimmunoprecipitation assay (RIPA) buffer. Briefly, cells were washed once in ice-cold PBS and lysed for 30 min on ice in RIPA buffer containing 150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 50 mM Tris (pH 8.0) with proteinase inhibitors (Complete, Boehringer Mannheim, Germany), and 1 mM sodium orthovanadate. Debris was removed by centrifugation at 20,000 g for 15 min. For isolating nuclear and cytoplasmic fractions, we used the Nucbuster kit (Novagen, Madison, WI) according to the manufacturer’s recommendations. After adjustment of protein concentrations, the lysates were boiled in SDS sample buffer for 5 min and separated by SDS-polyacrylamide gel electrophoresis. Gels were blotted on a polyvinylidene fluoride membrane (Immobilon P, Millipore, Bedford, MA) and stained with the indicated antibody. Antibodies against ß-catenin were obtained from BD Biosciences. Frizzled-1 and Frizzled-4 detection was performed under nonreducing conditions. Frizzled antibodies were obtained from R&D Systems (Wiesbaden, Germany) and the actin antibody, from Sigma (Taufkirchen, Germany). Antibody-binding was detected with a horseradish peroxidase-coupled secondary antibody followed by enhanced chemiluminescence (ECL) detection (ECL Plus, Amersham Pharmacia, Uppsala, Sweden).

For densitometry analyses, ECL was detected using a high-sensitive charged-coupled device camera (INTAS, Göttingen, Germany), and the bands were quantified using the GelPro Analyzer software (INTAS), according to the manufacturer’s instructions.

Differentiation of HL-60 cells
HL-60 cells were cultured in RPMI with L-glutamine and 10% FCS. For 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced monocytic differentiation or dimethyl sulfoxide (DMSO) induction of granulocytic differentiation, HL-60 cells were plated in RPMI medium (containing 10% FCS) at 3 x 105/ml density. Cells were grown in the presence of 10 ng/ml TPA, 1.3% DMSO, or PBS control and harvested at the indicated time-points. HL-60 differentiation was confirmed by fluorescein-activated cell sorter analysis with (phycoerythrin-labeled) anti-CD11b antibody and anti-CD14 antibody (fluorescein isothiocyanate-labeled, BD Biosciences).

Immunofluorescence
The intracellular distribution of ß-catenin was analyzed in monocytes, which became adherent overnight in chamber slides. Following fixation of these cells for 30 min in 3.7% paraformaldehyde in PBS at room temperature, cells were permeabilized for 2 min at room temperature in 0.2% Triton X-100 in PBS. After washing in PBS, cells were stained with a primary antibody directed against ß-catenin for 1 h at room temperature (purchased from BD Transduction Laboratories, Franklin Lakes, NJ). Following several washing steps, the primary antibody was detected by a goat anti-mouse antibody coupled to Alexa Fluor (60 min room temperature). In addition, cells were labeled with rhodamine-conjugated phalloidin for 30 min at 37°C and for 4', 6-diamidino-2-phenylindole (DAPI) after washing in PBS. Finally, cells were mounted in mowiol with 4% n-propyl-gallate as an antifade agent. Microscopic analyses were performed with a fluorescence microscope connected to a digital imaging system.

Statistics
All data were evaluated by a Man-Whitney-U test, and P values ≤0.05 were considered to be significant. All error bars represent standard errors.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Expression of Frizzled receptors in human leukocytes
Frizzleds act as receptors for Wnt proteins. Most Frizzled receptors are coupled to the ß-catenin canonical signaling pathway, which leads to the activation of disheveled proteins, inhibition of GSK3, nuclear accumulation of ß-catenin, and activation of Wnt target genes. We initially used quantitative real-time RT-PCR to evaluate the presence of the Wnt receptor RNA in granulocytes and monocytes. Analyses using primers for several members of the Frizzled family (Frizzled-1, Frizzled-7, and Frizzled-5) revealed their expression in both types of leukocytes (Fig. 1 ; data for granulocytes not shown). We also could verify a slight expression of Frizzled-4 and Frizzled-8 (data not shown). To verify the real-time RT-PCR data, we performed Western blot analyses for Frizzled-1 and for Frizzled-4. Both types of receptors were expressed on the protein level in monocytes of two different donors (Fig. 1B) . We differentiated HL-60 cells with TPA into the monocytic and with DMSO into the granulocytic lineage. In Figure 1C , the expression of Frizzled receptors Frizzled-1 and Frizzled-4 is shown in Western blot analyses of differentiated samples compared with the control.


Figure 1
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  		Figure 1. Expression of Frizzled receptors in leukocytes. (A) Quantification of Frizzled-1, Frizzled-5, and Frizzled-7 as potential Wnt3a mediators on mRNA levels by real-time RT-PCR. Monocytes were isolated and cultured overnight. The next day, samples were taken for RNA preparation, and real-time RT-PCR analyses were performed. Each bar represents the mean relative expression of a Frizzled receptor, expressed as a Frizzled/GAPDH ratio of two independent experiments ± SD. (B) Total protein lysates of monocytes from two different healthy donors were prepared. Western blot analyses revealed the presence of Frizzled-1 and Frizzled-4. (C) HL-60 cells were differentiated into monocytes (TPA) and granulocytes (DMSO). Lysates were prepared after indicated time-points and subjected to Western blot analyses.

 
Modulation of ß-catenin protein stability in monocytes
Having demonstrated the presence of Frizzled receptors, we determined Wnt effects on the ß-catenin pathway in monocytes. Exposure of cells to LiCl is one method for activating Wnt signaling. LiCl inhibits GSK3ß and thus results in an accumulation of ß-catenin but also of other proteins. We cultured purified monocytes overnight in different concentrations of LiCl (Fig. 2A ). To further elucidate whether there is any response of these cells to specific Wnt-activating signals, purified monocytes and granulocytes were incubated with 50% CM containing Wnt3a or control CM overnight. Wnt signal activation was analyzed by recording ß-catenin protein levels. As shown in Figure 2A and 2B , LiCl treatment resulted in a significant induction of ß-catenin. Wnt3a CM induced a significant increase of approximately twofold of ß-catenin in monocytes. To exclude toxic effects of Wnt3a exposure to monocytes, cells were stained for propidium iodide after an incubation time of 12 h with control CM or Wnt3a CM. The results depict that no changes occurred in the fraction of apoptotic cells (data not shown).


Figure 2
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  		Figure 2. Induction of ß-catenin protein levels by LiCl and Wnt3a. (A) Total protein lysates were prepared from monocytes cultured overnight in indicated concentrations of LiCl, 50% Wnt3a, or control CM. Western blot analysis revealed increasing amounts of detectable ß-catenin protein. (B) Densitometry of ß-catenin. The bar diagram indicates the ratio of the optical density (OD) of the ß-catenin and actin bands shown in A. For densitometry analyses, we used an INTAS camera (Epichem3 Darkroom) and GelPro Analyzer (1D-Gel Toolbar). (C) Total protein lysates were prepared from granulocytes, which had been cultured overnight in the indicated concentrations of LiCl, Wnt3a, or control medium. Western blot analyses revealed increasing amounts ß-catenin protein.

 
We could not detect any ß-catenin induction in granulocytes after LiCl or Wnt3a exposure (Fig. 2C) , indicating that ß-catenin levels in these cells are regulated by other mechanisms than the canonical Wnt signaling pathway. As we were interested in the functional relevance of canonical Wnt signaling in inflammatory cells, we performed all further experiments in monocytes, which responded well to Wnt3a stimulation.

Wnt3a signaling decreased transendothelial migration in monocytes
Having shown that monocytes harbor a functional, canonical Wnt signaling pathway, we analyzed its biological effects during inflammatory processes. We used purified monocytes to analyze the effect of Wnt activation on transendothelial migration. Monocytes were exposed to control CM or Wnt3a CM (50%) and subjected to an in vitro transendothelial migration assay. Figure 3 reveals that preincubation of monocytes with Wnt3a CM decreased their transendothelial migration rate by 60%. These results implicated a specific role of Wnt activation in regulating leukocyte-endothelial interactions.


Figure 3
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  		Figure 3. Wnt-signaling activation affects transendothelial migration of monocytes. Transendothelial migration assays were performed with monocytes cultured in Wnt3a CM. In each assay, results obtained for Wnt3a-treated cells were compared with those of the control cells kept for the same time in Wnt3a-free CM. Experiments were carried out in at least triplicates in three independent experiments. Each bar represents the mean of three independent experiments ± SEM.

 
Wnt signaling modulates adhesion in monocytes
Next, we analyzed whether alterations in endothelial cell adhesion of monocytes could offer an explanation for their decreased propensity to transmigrate. As shown in Figure 4A , Wnt3a-treated monocytes adhered to endothelial cells significantly stronger than control-treated cells. The increased adhesion appears not to be a result of integrin-mediated interactions between the extracellular matrix proteins, laminin, fibronectin, or collagen, and monocytes: Preincubation with Wnt3a CM did not change their interaction in adhesion assays (Fig. 4B) . Taken together, these findings indicate that the activated Wnt-signaling pathway triggers leukocyte-endothelium interaction under the conditions chosen.


Figure 4
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  		Figure 4. Wnt signaling results in higher adhesion properties in monocytes, which were exposed to Wnt3a CM overnight before subjected to adhesion assays to endothelial cells (A) or fibronectin, laminin, or collagen (B). Experiments were carried out in at least quadruplicates in four independent experiments. Each bar represents the mean of four independent experiments ± SEM. (C and D) Adherent monocytes on an endothelial layer. Monocytes were preincubated with control (C) or Wnt3a (D) CM. The same number of monocytes (1x106 per chamber) was seeded upon HMEC-1 cells, allowed to migrate for 4 h, and subsequently stained for microscopic analyses (original, 200x).

 
Influence of Wnt activation or adhesion on the cellular ß-catenin distribution
The results so far implicated a specific interaction of monocytes and endothelial cells induced by preincubation of monocytes with Wnt3a. To identify whether cytoplasmic or nuclear functions of ß-catenin predominate in this effect, we analyzed the subcellular distribution of ß-catenin in nonadherent and plastic-adherent monocytes with and without Wnt3a stimulation. As shown in Figure 5A , nonadherent monocytes express high amounts of nuclear and only moderate amounts of cytoplasmic ß-catenin. As has been reported earlier, adherence of monocytes to a plastic surface induced cellular ß-catenin [21 ]. However, we show here for the first time that this increase occurs predominantly in the cytoplasm and not in the nucleus. Similarly, incubation of nonadherent monocytes with Wnt3a CM results in an increase of cytoplasmic but not of nuclear ß-catenin levels in these cells, which already express considerable nuclear ß-catenin in unstimulated conditions. Taken together, these results indicate that the observed effects of Wnt3a on the migratory and adhesive properties of monocytes are a result of increased levels of cytoplasmic and not nuclear ß-catenin. These results were supported by immunofluorescence studies shown in Figure 5C 5D 5E 5F . The merge picture (Fig. 5F) verifies nicely that a fraction of ß-catenin was localized in the nucleus. In addition, we observed detectable amounts of ß-catenin in the cytoplasm.


Figure 5
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  		Figure 5. Distribution of cellular ß-catenin in Wnt-activated and adherent monocytes. (A) After preparation, monocytes were exposed to control or Wnt3a CM overnight. Cells then were separated in cytoplasmic (c) or nuclear (n) fractions with the Nucbuster kit (Novagen). Western blot analyses revealed that adherence and Wnt3a activation resulted in higher amounts of ß-catenin in the cytoplasm. (B) Densitometry of ß-catenin. The bar diagram indicates the ratio of the OD of the ß-catenin and actin bands shown in A. For densitometry analyses, we used an INTAS camera (Epichem3 Darkroom) and GelPro Analyzer (1D-Gel Toolbar). (C–F) Monocytes were incubated overnight without Wnt3a in chamber slides to induce adherence. Cells were fixed and stained for ß-catenin, rhodamine-phalloidin, or DAPI (original, 1000x). (C) DAPI; (D) ß-catenin; (E) rhodamine-phalloidin; (F) overlay DAPI/ß-catenin/rhodamine-phalloidin.

 
No effect of Wnt3a on transcription of classical target genes
ß-Catenin serves a dual function in cells. Although its function in the cytoplasm is defined mainly by its binding to cadherins to enhance cell-cell contacts, its function in the nucleus is thought to be mediated by the formation of ß-catenin-TCF/LEF complexes, which support transcriptional activation of several well-described target genes. As it was previously reported that transfection of ß-catenin in the human monocytic leukemia cell line THP-1 did not alter TCF-dependent promoter activity [21 ], we wanted to elucidate the effect of Wnt3a on monocytes, which were exposed to Wnt3a overnight before RNA and subsequently cDNA were prepared. Quantitative real-time RT analysis of known Wnt target genes revealed no changes in the expression pattern of c-myc, cyclin D1, or Connexin43 (Fig. 6 , and data not shown). These results suggest a different role of ß-catenin in monocytes than the transcriptional activation of Wnt-target genes.


Figure 6
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  		Figure 6. Wnt3a activation in monocytes does not induce conventional Wnt-target genes. Quantification of c-myc and Connexin43 mRNA levels by real-time RT-PCR. Monocytes were cultured overnight in control or Wnt3a CM. Samples were taken for RNA preparation, and real-time RT-PCR analyses were performed. The mean of the c-myc or Connexin43 mRNA level of the control CM level was set as 1. Each bar represents the mean of two independent experiments ± SD.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Wnt signaling has been demonstrated to have profound effects on embryogenesis, cell growth, and certain kinds of cancer [1 ]. Recent studies revealed a role for Wnt signaling in hematopoiesis and stem cell renewal [26 27 28 29 30 ]. However, little is known about Wnt signaling in inflammatory processes. Our results now show that the Wnt/ß-catenin signaling activation has an influence on migration and adhesion in leukocytes. This finding, for the first time, indicates that the Wnt/ß-catenin signaling pathway can modulate inflammatory processes. Also, it suggests that cytoplasmic ß-catenin may play a more important role in leukocyte adhesion than anticipated.

In our study, we demonstrated that Wnt3a activated the Wnt/ß-catenin signaling pathway in isolated monocytes. LiCl and Wnt3a exposure resulted in an increase of total ß-catenin protein. In contrast, no response of the ß-catenin level could be observed in granulocytes. Our data also revealed that activation of the Wnt/ß-catenin pathway led to a decreased migratory potential of monocytes. These data are in line with experiments that showed that Wnt3a caused stronger adhesion of monocytes to the endothelial monolayer. It is interesting that we found a strong, negative correlation of the cytoplasmic but not the nuclear amounts of ß-catenin with the monocyte migratory potential.

As classical adherence through cadherins is absent in monocytes, unknown adhesion molecules might regulate this interaction. Further evidence for the relevance of cytoplasmic in contrast to nuclear ß-catenin levels is provided by the real-time RT-PCR experiments, which revealed no influence of ß-catenin activation on the expression of classical Wnt target genes such as c-myc or cyclin D1. These data fit well to the published results from Thiele and colleagues [21 ]. They could not observe any activation of TCF-promoter activity after ß-catenin transfection into the monocytic cell line THP-1. In addition, Chung et al. [31 ] observed that ß-catenin contributes to homotypic cell aggregation of phytohemagglutinin-activated Jurkat cells, also indicating a role in adhesion. Taken together, these results implicate that the well-described Wnt-target genes, which are thought to be functionally related to cellular proliferation and malignant transformation such as c-myc or cyclin D1, are not included in this setting. One explanation might be that primary monocytes are fully differentiated and have low if any proliferative capacity. It is conceivable that under these circumstances, regulatory regions of Wnt target genes are not accessible for TCF/ß-catenin complexes or that necessary cofactors are not expressed.

Further knowledge about the precise role of the Wnt/ß-catenin signaling pathway in inflammation may lead to new, therapeutic developments of anti-inflammatory drugs.


   ACKNOWLEDGEMENTS
 
This work is supported by grants from the Deutsche Forschungsgemeinschaft (SFB293, Se 600/3-1), the José-Carreras Leukemia Foundation (R03/19f), the Interdisciplinary Centre of Clinical Research Münster (IZKF Project No. Ser 2/041/04, Mül 2/096/04), and the Innovative Medizinische Forschung (IMF Project Nos. TI 110506 and TI 620301) at the University of Münster and the Heisenberg Grant from the Deutsche Forschungsgemeinschaft (MU 1328/3-1).

Received September 29, 2005; revised January 25, 2006; accepted January 31, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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