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The Journal of Immunology, 2001, 167: 6786-6793.
Copyright © 2001 by The American Association of Immunologists

Regulation and Possible Function of {beta}-Catenin in Human Monocytes1

Andrea Thiele2,*, Mark Wasner{dagger}, Claudia Müller*, Kurt Engeland{dagger} and Sunna Hauschildt3,*

* Institute of Zoology, Department of Immunobiology, University of Leipzig, Leipzig, Germany; and {dagger} Medizinische Klinik II, Department of Internal Medicine, University of Leipzig, Max Bürger Research Center, Leipzig, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In this study, we demonstrate that adherence factors, serum constituents, LPS, and zymosan are capable of inducing a cellular accumulation of {beta}-catenin in human monocytes. Whereas adherence-dependent accumulation of {beta}-catenin can be blocked by wortmannin, an inhibitor of phosphatidylinositol 3-kinase, accumulation induced by the remaining stimuli cannot be prevented by inhibition of phosphatidylinositol 3-kinase, implying the involvement of {beta}-catenin in other not yet described signal transduction pathways. A role of {beta}-catenin in adherence-dependent processes by interacting with classical cadherins can be excluded as we could not detect cadherins in monocytes. To test whether it is possible that {beta}-catenin interacts with LEF/TCF (lymphoid enhancer factor/T cell factor) transcription factors, we studied the expression of this protein family. TCF-4 was identified as the LEF/TCF transcription factor present in human monocytes. However, neither cellular induction of {beta}-catenin nor cotransfection experiments with {beta}-catenin conducted in the monocytic cell line THP-1 resulted in the activation of a LEF/TCF-dependent promoter, suggesting the requirement of additional signals. Concurrent with this suggestion, we found that LPS and zymosan, two physiological inducers of {beta}-catenin, caused an increase in the expression of genes that are positively regulated by {beta}-catenin.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Originally {beta}-catenin was isolated as a cytoplasmic protein associated with the cell adhesion molecule E-cadherin (1), a member of the growing family of classical cadherins. The function of this complex was shown to be in the linking of E-cadherin to the cytoskeleton (2). When {beta}-catenin was identified as the mammalian homolog of arm (the product of the segment polarity gene armadillo), which is involved in the Wingless signaling pathway in Drosophila (3), it became clear that {beta}-catenin also has a signaling role. Signaling was shown to be mediated through its interaction with the familiy of lymphoid enhancer factor (LEF)4/T cell factor (TCF) transcription factors and the subsequent activation of target genes. Factors of the LEF/TCF family are high mobility group proteins and were discovered as proteins binding to specific sequences in lymphoid enhancers (4, 5). Their function as ultimate targets for the Wnt/Wingless signal transduction pathway (6, 7) came as a complete surprise. Proteins of the Wnt family (which are the mammalian homologs of Wingless) are growth factors that modulate numerous developmental processes in a variety of organisms. The current prevailing view is that LEF/TCF becomes a transcriptional activator of Wnt-inducible genes upon binding to the transcriptional coactivator {beta}-catenin (8). Alternatively, LEF/TCF may be a transcriptional repressor whose activity is antagonized by {beta}-catenin, a possibility for which there is growing evidence (9). In any case, cytoplasmic stabilization of {beta}-catenin is required before it can act as a transcriptional activator.

If {beta}-catenin-stabilizing signals are absent, the protein is rapidly degraded by the ubiquitin proteasome pathway (10). Ubiquitination requires phosphorylation by the serine-threonine kinase glycogen synthase kinase-3{beta} (GSK-3{beta}) (10), and phosphorylation is facilitated by a large multiprotein machinery that includes APC (the tumor suppressor protein encoded by the adenomatous polyposis coli gene) (11) and axin (12)/conductin (13). Mutations that prevent the degradation of {beta}-catenin are often detected in colon carcinoma (14), skin cancer (15), and hepatoblastoma (16). It has been suggested that aberrant amounts of cytoplasmic {beta}-catenin result in the constitutive activation of LEF/TCF-dependent promoters in colorectal cancers (14, 17). Further implications of this pathway in cancer development have been provided by the finding of cyclin D1 (18), c-myc (19), and genes encoding for metalloproteinases (20, 21) as target genes of the complex.

Recent publications report on novel inducers of {beta}-catenin-LEF/TCF-dependent signaling, including integrin-linked kinase (ILK) (22, 23) and NO (24). Acetyltransferases (25, 26, 27) and pontin52 (28) have been discussed as coactivators of the {beta}-catenin-LEF/TCF complex. Conversely, besides corepressors (29, 30), there are inhibitory proteins such as cadherins (31), caveolin-1 (32), and a novel {beta}-catenin binding protein inhibitor of {beta}-catenin-TCF-4 (33). The latter compete for the binding of {beta}-catenin and/or recruit the protein to distinct cellular compartments.

Among the cell types susceptible to the induction of {beta}-catenin-LEF/TCF-dependent transcriptional activation identified to date are epithelial (14) and endothelial cells (34), fibroblasts (35), and Jurkat T cells (35, 36). There are first reports on a role of {beta}-catenin in signal transduction pathways of primary immune cells (37).

In this study, we provide evidence for the presence of {beta}-catenin and its respective protein partner TCF-4 in human monocytes. Furthermore, we show that {beta}-catenin is modulated by stimuli that trigger monocytes to various biological responses, indicating a central role of {beta}-catenin in cellular processes induced by these stimuli.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents

Latex beads (1 µm) were purchased from Serva (Heidelberg, Germany). PMA was obtained from ICN Biomedicals (Costa Mesa, CA), and human epidermal growth factor (EGF) from PromoCell (Heidelberg, Germany). All other chemicals were purchased from Sigma (St. Louis, MO), unless indicated otherwise.

Cell separation and monocyte purification

PBMCs from healthy donors were isolated by centrifugation over a Ficoll-Paque (Pharmacia, Uppsala, Sweden) density gradient. After repeated washing in PBS containing 0.3 mM EDTA, the monocytes were isolated by counterflow centrifugation using the J6-MC elutriator system (Beckman Instruments, Palo Alto, CA), as described previously (38). For PCR analyses, monocytes were further purified applying the MACS monocyte isolation kit (Miltenyi Biotech, Auburn, CA), resulting in 99% CD14-positive cells.

Cell culture

Monocytes (106/ml) were incubated in RPMI 1640 containing 2 mM glutamine (Seromed Biochrom KG, Berlin, Germany), 100 U/ml penicillin (Seromed Biochrom KG), and 100 µg/ml streptomycin (Seromed Biochrom KG) in the presence or absence of 10% FCS in 24-well cell culture plates (TPP, Trasadingen, Switzerland). To prevent plastic adherence, the wells were coated with 1% agarose (FMC Bioproducts, Rockland, MA) in PBS.

The human colon adenocarcinoma cell line DLD-1 was cultured in RPMI 1640 supplemented with 10% FCS, the human acute monocytic leukemia cell line THP-1 in RPMI 1640 supplemented with 10% FCS and 50 µM 2-ME, and the human acute myeloid leukemia cell line KG-1a in IMDM supplemented with 20% FCS. All media contained 2 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin.

Cell lysis, SDS-PAGE, and Western blotting

Monocytes (2 x 106), THP-1 cells (5 x 105), KG-1a cells (5 x 105), and DLD-1 cells (5 x 105) were washed with PBS and resuspended in 50 µl lysis buffer containing 0.5% Triton X-100, 10 mM Tris-HCl, pH 8, 0.1 mM EDTA, 0.32 M sucrose, 3 mM MgCl2, 1 mM PMSF, and 10 µg/ml leupeptin. After 10 min on ice, cells were sonicated (10 strokes, output 70%, duty cycle 60%, Bandelin Sonoplus GM70; Bandelin, Berlin, Germany) and centrifuged at 12,000 x g for 10 min. Protein concentrations of the supernatants were determined by using Bradford reagent. Aliquots of the supernatants were boiled in Laemmli sample buffer (39) for 5 min and separated on 9 or 10% SDS-polyacrylamide gels (MiniProtean II; Bio-Rad GmbH, Hercules, CA) at 200 V for 45–60 min. Following electrophoresis, the gels were equilibrated in transfer buffer (25 mM Tris, 192 mM glycine, 10% methanol) for 10 min before the proteins were electrotransferred to polyvinylidene difluoride membranes (Amresco, Salon, OH) at 0.7 A and 10°C for 3 h. After blocking in PBS containing 5% dry milk powder (Glücksklee, Nestlé Deutschland, Frankfurt, Germany) and 0.2% Tween 20 (Serva, Heidelberg, Germany) at 4–8°C overnight, membranes were incubated with anti-{beta}-catenin (1/2,000, clone 15B8; Sigma), anti-pan-cadherin (1/3,000, polyclonal antiserum; Sigma), or anti-TCF-4 (1/50; Zymed Laboratories, South San Francisco, CA) Abs at 37°C for 2 h. The membranes were washed five times with PBS at room temperature for 5 min and then incubated with the peroxidase-conjugated goat anti-mouse (1/20,000; Sigma) or goat anti-rabbit (1/30,000; Sigma) secondary Ab at 37°C for 1 h. After washing five more times with PBS at room temperature for 5 min, the proteins were visualized by chemiluminescence (Renaissance Western Blot Chemiluminescence; NEN Life Science Products, Boston, MA), according to the manufacturer’s instructions.

Oligonucleotides

All oligonucleotides were synthesized by MWG Biotech AG (Ebersberg, Germany) or Microsynth (Balgach, Switzerland).

LEF/TCF mRNA was detected by a nested PCR approach using three intron-spanning degenerate primers, which were partly described previously (40): sense primer, 5'-AATGC(AGCT)TT(CT)ATG(CT)T(AGCT)TA(CT)ATGAA; outer antisense primer, 5'-AC(AG)TA(AG)TT(AG)TC(ACT)CG(AGCT)GC(AGCT)G(AT)CCA; inner antisense primer, 5'-C(AG)(AT)(AG)TG(AGCT)AGCTG(AGCT)C(GT)CTCCTT.

Cadherin mRNA was detected by a nested PCR approach using four degenerate primers that were partly described previously (41): outer sense primer, 5'-TA(CT)(AG)(AG)(AGCT)GA(AGCT)GA(AG)GG(AGCT)GG(AGCT); outer antisense primer, 5'-(AGCT)CC(AGCT)CC(AGCT)CC(AG)TACATGTC; inner sense primer, 5'-GA(AG)GG(AGCT)GG(AGCT)GG(AGCT)GA(AG)GA(AG)GAC; inner antisense primer, 5'-(AGCT)A(AG)(AG)TA(AG)T(CT)(AG)TA(AG)T(CT)(CT)TG(AG)TC.

To amplifiy GAPDH, matrilysin, and urokinase-type plasminogen activator (uPAR) cDNA, the following intron-spanning primers were used: GAPDH-sense, 5'-AAC AGC GAC ACC CAC TCC TC; GAPDH-antisense, 5'-GGA GGG GAG ATT CAG TGT GGT; matrilysin-sense, GTG GAG TGC CAG ATG TTG CAG; matrilysin-antisense, AGA CTG CTA CCA TCC GTC CAG; uPAR-sense, GCT GTC ACC TAT TCC CGA AGC; and uPAR-antisense, GTA ACA CTG GCG GCC ATT CTG.

RT-PCR

Total RNA was isolated from 2 x 106 cells using the RNeasy mini kit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions. RNA that was further used for the detection of classical cadherins was treated with DNase I to digest any contaminating genomic DNA (42) before cDNA preparation. cDNA was prepared by annealing RNA with oligo(dT20) for 10 min at 70°C and reverse transcription using Superscript II reverse transcriptase (Life Technologies, Gaithersburg, MD) at 37°C for 60 min, followed by an inactivation phase of 4 min at 94°C.

To detect LEF/TCF transcription factors and classical cadherins, nested PCR approaches were used. For the first PCR amplification step, an aliquot of each reverse-transcribed sample was added to the reaction mixture containing 1.5 mM MgCl2, 200 µM dNTP, 4 µM of both the outer sense and outer antisense primer, and 75 U/ml Taq-DNA polymerase (Life Technologies, Gaithersburg, MD). Reactions were performed in a DNA thermal cycler Crocodile III (Oncor Appligene, Heidelberg, Germany) under the following conditions: An initial denaturation step for 3 min at 95°C was followed by 35 cycles of 60 s at 95°C, 45 s at 45°C, and 45 s at 72°C, with a prolongation of 10 s per 8 cycles. The final extension phase was 5 min at 72°C. An aliquot of the PCR product was used for the nested amplification, employing the inner sense and the inner antisense primer for 25 cycles under the same conditions as used for the first amplification.

For the specific amplification of GAPDH, matrilysin, and uPAR cDNA, an aliquot of each reverse-transcribed sample was added to the reaction mixture containing 1.5 mM MgCl2, 200 µM dNTP, 1 µM of both the sense and antisense primer, and 30 U/ml Taq-DNA polymerase (Life Technologies).

Reactions were performed in a DNA thermal cycler Crocodile III (Oncor Appligene) under the following conditions: An initial denaturation step for 2 min at 95°C was followed by cycles of 60 s at 95°C, 60 s at 60°C, and 90 s at 72°C. The final extension phase was 15 min at 72°C. The numbers of cycles were 24 (GAPDH), 28 (matrilysin), and 25 (uPAR).

The PCR products were separated by electrophoresis on 1.8% agarose gels (FMC Bioproducts) containing 1 µg/ml ethidium bromide and visualized under UV light. The 100-bp ladder (Life Technologies) served as a m.w. standard.

Cloning and sequencing of PCR products

After the amplification of LEF/TCF or cadherin cDNA using degenerate primers, PCR products of the corresponding size were excised from a low-melting agarose gel (FMC Bioproducts). The fragments obtained were cloned using the TOPO TA cloning kit (Invitrogen, Groningen, The Netherlands) according to the manufacturer’s instructions. After DNA preparation using a QIAprep plasmid preparation kit (Qiagen), the clones were sequenced (MWG Biotech, Ebersberg, Germany). BLAST GenBank search was used to find corresponding sequences (43).

Plasmids, transient DNA transfections, and luciferase assays

The reporter plasmid TOPFLASH and FOPFLASH and the expression plasmids for {beta}-catenin and TCF-4 were a kind gift from B. Vogelstein (Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, Baltimore, MD) and have been described previously (14). The Renilla luciferase vector pRL-CMV was purchased from Promega (Madison, WI). The Firefly luciferase construct containing the cyclin B2 promoter has been described (44, 45). Plasmids were purified using a Plasmid Midi Kit (Qiagen) according to the manufacturer’s instructions. The THP-1 cells were transfected using DEAE-dextran, as described previously (46). Briefly, 5 x 105 THP-1 cells/ml were seeded into tissue culture flasks the day before transfection. The next day, 3 ml cell suspension was washed twice with suspension TBS (STBS) (47) and pelleted. A total of 0.1 µg Firefly luciferase reporter plasmid, 0.1 µg expression plasmid, and 0.01 µg Renilla luciferase control vector was mixed with DEAE-dextran (400 µg/ml) in 70 µl STBS and immediately added to the pelleted THP-1 cells. The cells were incubated at 37°C for 20 min, washed twice with STBS, and further cultured in complete cell culture medium for 48 h. Aliquots of the same sample were lysed and either analyzed for luciferase activity using the Dual Luciferase Reporter Assay System (Promega) according to the manufacturer’s instructions or assayed by Western blotting (as described above).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Modulation of {beta}-catenin by adherence factors and serum constituents

Monocytes were incubated in 24-well cell culture plates in RPMI 1640, supplemented with 10% FCS. After different times, cells were lysed and {beta}-catenin was detected by Western blotting. As shown in Fig. 1GoA, the protein was not detectable in freshly isolated monocytes, but was slightly visible after 30 min of cultivation. It was strongly expressed after 5 h, and after cultivation overnight (16 h) {beta}-catenin was hardly detectable anymore.



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  		FIGURE 1. Modulation of {beta}-catenin in human monocytes by adherence factors, serum constituents, and human EGF. Human monocytes (106/ml) were incubated in 24-well cell culture plates in the presence of 10% FCS (A), in the absence of FCS (B), in agarose-coated wells in the presence of 10% FCS (C), or in agarose-coated wells in the presence of 10% FCS or 10 ng/ml human EGF (D). After the indicated times, cells were lysed and {beta}-catenin was detected by Western blotting. No signal was seen after incubation in agarose-coated wells in the absence of FCS.

 
To test whether adherence-induced factors and/or serum constituents caused this effect, we incubated the cells under nonadherent conditions in the absence of serum, under adherent conditions in the absence of serum (adherence induction, shown in Fig. 1GoB), and under nonadherent conditions in the presence of serum (serum induction, shown in Fig. 1GoC). Surface adherence of monocytes was prevented by cultivation in agarose-coated wells. In the absence of serum under nonadherent conditions, {beta}-catenin was not detectable (data not shown). However, incubation in the absence of serum under adherent conditions resulted in a marked induction of the protein (Fig. 1GoB). The kinetics was similar to those observed in the presence of serum (Fig. 1GoA), with the exception that the protein was still detectable after cultivation overnight (16 h). The prolonged expression of {beta}-catenin might be related to a stronger adherence to plastic upon incubation overnight in the absence of serum as compared with the presence of serum. When incubating monocytes in medium containing 10% FCS under nonadherent conditions, a transient accumulation of {beta}-catenin could also be observed (Fig. 1GoC). In contrast to the kinetics presented above, the protein was not detectable until 2 h of incubation, indicating the involvement of a long-term signaling cascade. Since EGF, a growth factor present in the serum, was identified as a potential costimulator of {beta}-catenin-induced transcriptional activation (48), we substituted the serum by EGF. Fig. 1GoD shows that EGF also induced {beta}-catenin, suggesting that the serum induction of the protein might be mediated through growth factor signaling.

Modulation of {beta}-catenin by LPS and zymosan, but not by latex beads

Monocytes play a major part in host defense. In response to bacterial invasion, they ingest and kill bacteria and also secrete a variety of mediators that increase the defense capacity of the host. To test whether stimuli that initiate these events might regulate the expression of {beta}-catenin, cells were incubated either with LPS, a potent monocyte activator, or with particles that are phagocytosed by monocytes such as latex beads or zymosan, a yeast cell wall extract. Since an appropriate response to LPS requires serum and phagocytosis is facilitated by adherence, we cultivated monocytes in 24-well cell culture plates in medium containing 10% FCS before adding the stimuli. To prevent an interference with the cultivation-dependent expression of {beta}-catenin, this precultivation was conducted for 16 h. As shown in Fig. 2GoA, LPS caused a rapid induction of {beta}-catenin (15 min), which reached a maximum after 2 h and decreased again after 5 h of stimulation. A rapid induction of {beta}-catenin was also observed after incubation with zymosan (Fig. 2GoB), whereas latex beads had no effect (Fig. 2GoC). Although latex beads and zymosan are both readily phagocytosed by monocytes, only phagocytosis of zymosan is coupled with the production of inflammatory cytokines. Thus, phagocytosis per se is insufficient to initiate the induction of {beta}-catenin, whereas stimuli leading to cytokine production, such as LPS and zymosan, result in a drastic accumulation of the protein.



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  		FIGURE 2. Modulation of {beta}-catenin in human monocytes by LPS and zymosan, but not by latex beads. Human monocytes (106/ml) were incubated overnight in 24-well cell culture plates in the presence of 10% FCS before 100 ng/ml LPS (A), 200 µg/ml zymosan A (B), or latex beads (1 µm, 108/ml) (C) were added. After the times indicated, cells were lysed and {beta}-catenin was detected by Western blotting. {beta}-catenin in untreated cells was hardly detectable.

 
Adhesion-dependent accumulation of {beta}-catenin is inhibited by wortmannin

Since adherence factors, serum constituents, LPS, and zymosan are stimuli leading to various biological responses, we tested whether they share one common signal transduction pathway that might be responsible for the accumulation of {beta}-catenin.

It has been demonstrated convincingly that phosphorylation of {beta}-catenin by the serine-threonine kinase GSK-3{beta} is crucial for the destabilization of {beta}-catenin (10). Inhibition of GSK-3{beta} activity results in the accumulation of {beta}-catenin (49). As demonstrated by Delcommenne et al. (22), one enzyme capable of inhibiting GSK-3{beta} activity is ILK. Stimulation through integrins and growth factor receptors results in the activation of ILK activity (50) in a phosphatidylinositol 3-kinase (PI3-kinase)-dependent manner (22). All stimuli that induced a cellular accumulation of {beta}-catenin in our experiments have the potential to act via activation of ILK either through integrins or through growth factors. The latter may be involved in serum induction of {beta}-catenin. Adhesion to the extracellular matrix, which is partly mimicked by plastic adherence (51), involves integrin engagement leading to various signaling events (52). LPS has been shown to bind specifically to the {beta}2 integrin CD11a/CD18 (53), and the integrin receptor CD11b/CD18 participates in zymosan recognition and the subsequent TNF-{alpha} production by human monocytes (54). Assuming that the stimuli tested lead to an increase in ILK activity (via integrins or growth factor signaling) in a PI3-kinase-dependent manner, inhibition of PI3-kinase by wortmannin should prevent the accumulation of {beta}-catenin. As only adherence-dependent induction of {beta}-catenin was prevented by wortmannin (Fig. 3Go), accumulation of {beta}-catenin induced by the other stimuli was mediated through PI3-kinase-independent pathways and probably did not involve ILK signaling. Thus, diverse pathways seem to regulate the expression of {beta}-catenin in human monocytes.



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  		FIGURE 3. Adhesion-dependent accumulation of {beta}-catenin is inhibited by wortmannin. A and B, Human monocytes (106/ml) were incubated in the presence of the indicated concentrations of wortmannin for 5 h in 24-well cell culture plates in the absence of FCS (A) or in agarose-coated wells in the presence of 10% FCS (B). C and D, Cells (106/ml) were incubated overnight in 24-well cell culture plates in the presence of 10% FCS. Before adding 100 ng/ml LPS (C) or 200 µg/ml zymosan (D) for 2 h, cells were preincubated with the indicated concentrations of wortmannin for 15 min. Cell lysates were prepared and proteins were separated on SDS-PAGE. {beta}-catenin was detected by Western blotting.

 
No classical cadherin was detectable in human monocytes

Since {beta}-catenin has a crucial role in cell adhesion by interacting with classical cadherins, we investigated the expression of this protein family in human monocytes. Western blot analysis was performed using a pan-cadherin Ab that is directed against a common region of classical cadherins. As a positive control, the N-cadherin-expressing cell line KG-1a was used. Furthermore, by stimulating monocytes with LPS for 2 h, we measured the expression of cadherins under conditions that lead to an expression of {beta}-catenin. As can be seen in Fig. 4GoA, there was no positive signal for monocytes under the conditions tested.



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  		FIGURE 4. No expression of classical cadherins in human monocytes. A, Classical cadherins were detected in cell lysates of the N-cadherin-positive cell line KG-1a and in human monocytes (mo) by Western blotting using a polyclonal pan-cadherin antiserum. Human monocytes (106/ml) were cultivated overnight in 24-well cell culture plates in medium containing 10% FCS, in the presence or absence of LPS (100 ng/ml) for the last 2 h of incubation. B, RT-PCR was performed as described in Materials and Methods. PCR products resulting from the specific amplification of cadherin cDNA have an expected size of approximately 320 bp. C, PCR products of the length expected were cloned and sequenced. The obtained sequences were identified by BLAST GenBank search. The number of clones, which correspond to the indicated sequence in relation to the total number of clones sequenced, are given in brackets.

 
As we could not exclude the possibility that there are cadherins whose epitopes were not efficiently recognized by the Ab, we applied a degenerate PCR approach using primers that were designed to detect all subclasses of classical cadherins. RT-PCR was conducted using cDNA from highly purified monocytes and the monocytic cell line THP-1. To increase the specificity of the detection method, we performed a nested PCR. All primers corresponded to conserved amino acid stretches within the cytoplasmic region of classical cadherins (55), as partly described previously (41). These primers were not intron spanning in all cadherin genes. Therefore, RNA was treated with DNase before reverse transcription. As positive controls, cDNA of DLD-1 and KG-1a cells, which are known to express cadherins, was used. Fig. 4GoB shows a PCR product of the length expected (~320 bp), resulting from the amplification of cDNA of both DLD-1 and KG-1a cells. Amplification of cDNA of highly purified monocytes and THP-1 cells resulted in a slightly longer PCR product. Cloning, sequencing, and subsequent BLAST search identified cadherin-7 and cadherin-3 as the encoded products of clones derived from the shorter PCR products of DLD-1 and KG-1a cDNA, respectively (Fig. 4GoC). In contrast, the longer PCR product amplified from cDNA of monocytes and THP-1 cells coded for annexin-11. There are no sequence similarities between annexin-11 and cadherins. The amplification of annexin-11 cDNA was due to mispriming of the degenerate primers designed to detect cadherins.

Identification of TCF-4

As the signaling role of {beta}-catenin is mediated through its interaction with LEF/TCF transcription factors, we investigated the expression of LEF/TCF in human monocytes. We applied RT-PCR to detect LEF/TCF mRNA in highly purified monocytes. Furthermore, we used the human monocytic cell line THP-1 to confirm the monocytic specificity of the PCR products obtained. A degenerate PCR approach was used to identify mRNA sequences of the LEF/TCF transcription factor family expressed in these cells. The degenerate primers encoding sequences in the highly conserved high mobility group domain of LEF/TCF were as described previously (40). In addition, we used a nested primer to increase the selectivity toward specific PCR products. Fig. 5GoA shows a PCR product of the length expected (~171 bp) resulting from the amplification of cDNA of both human monocytes and THP-1 cells. The longer PCR product obtained probably corresponded to the genomic DNA of LEF/TCF factors. The smaller PCR product was cloned and sequenced, and database searching was applied to identify the encoded products. As indicated in Fig. 4GoA, the predominant sequence that was identified proved to be TCF-4 mRNA. Whereas all clones sequenced from PCR products of monocytic cDNA encoded TCF-4 mRNA, four of five clones from THP-1 PCR endoded TCF-4 and one TCF-1.



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  		FIGURE 5. Detection of TCF-4 in human monocytes and THP-1 cells. A, RT-PCR was performed as described in Materials and Methods. PCR products of both monocyte (mo) and THP-1 cDNA were cloned. The identities of the clones sequenced for each cell type are shown. The number of clones, which correspond to the indicated sequence related to the number of total clones sequenced, are given in brackets. B, TCF-4 was detected by Western blotting in cell lysates of the human colon adenocarcinoma cell line DLD-1, human monocytes (mo), and THP-1 cells. Human monocytes were cultivated overnight in 24-well cell culture plates in medium containing 10% FCS, in the presence or absence of LPS (100 ng/ml) for the last 2 h of incubation. Due to different amounts of protein in lysates of DLD-1 cells, THP-1 cells, and monocytes subjected to SDS-PAGE, the signal intensities do not reflect the relative level of TCF-4 expressed in these cell types.

 
To demonstrate that TCF-4 was expressed at the protein level, human TCF-4 was detected in cell lysates of human monocytes and THP-1 cells by Western blotting using an Ab directed against the N-terminal domain of human TCF-4. The human colon adenocarcinoma cell line DLD-1, which has been shown to express high levels of TCF-4 (56), served as a control. Fig. 5GoB shows that two isoforms of TCF-4 are expessed in human monocytes and THP-1 cells with different preferences. Stimulation of monocytes with LPS for 2 h has no influence on the expression of TCF-4 (Fig. 5GoB).

{beta}-catenin does not lead to LEF/TCF-dependent transcriptional activation in THP-1 cells

The presence of TCF-4 in human monocytes made us assume that accumulation of {beta}-catenin might result in the activation of the transcription factor. To approach this problem, we performed reporter gene assays in the monocytic cell line THP-1 using a luciferase reporter gene containing three optimal LEF/TCF sites (TOPFLASH) (14). The mutated reporter construct (FOPFLASH) served as a negative control. The transfection efficiencies were normalized to Renilla luciferase expression using a CMV promoter. A constitutively active cyclin B2-driven reporter plasmid was used as a positive control and was shown to be active under all conditions (Fig. 6GoB). In another set of control experiments in HeLa and DLD-1 cells, the TOPFLASH reporter gene was shown to be active (data not shown). Accumulation of {beta}-catenin in THP-1 cells was induced by lithium, which inhibits GSK-3{beta} (49) and thus prevents degradation of {beta}-catenin (Fig. 6GoA). Adding lithium in combination with the phorbol ester PMA resulted in a further increase in {beta}-catenin accumulation (Fig. 6GoA). The two substances have been shown to act synergistically in mammary epithelial cells (48). Although cellular {beta}-catenin was clearly increased after treatment with lithium (Fig. 6GoA), the LEF/TCF-dependent reporter gene TOPFLASH was neither activated by lithium alone nor by lithium together with PMA (Fig. 6GoB).



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  		FIGURE 6. Cellular {beta}-catenin does not activate a LEF/TCF-dependent promoter in THP-1 cells. THP-1 cells were transfected with the LEF/TCF-dependent Firefly luciferase reporter gene TOPFLASH, its mutated form FOPFLASH, or the constitutively active Firefly luciferase reporter gene driven by the cyclin B2 promoter. To normalize transfection efficiencies, cells were cotransfected with the CMV-driven Renilla luciferase reporter plasmid. After 46 h of incubation, cells were treated with lithium chloride (20 mM) or sodium chloride (20 mM) as a control and/or PMA (100 ng/ml) for 2 h. Cell lysates were prepared and analyzed for luciferase activity or assayed for {beta}-catenin by Western blotting. A, Proteins of cell lysates were separated on SDS-PAGE, and {beta}-catenin was detected by Western blotting. B, Relative luciferase activity of the cell lysates was determined. Values represent the mean ± SD of triplicate samples.

 
According to Staal et al. (35), inhibition of GSK-3{beta} proved to be insufficient for inducing {beta}-catenin-LEF/TCF-dependent transcription in Jurkat T cells. As cotransfection experiments with {beta}-catenin resulted in an activation of the TOPFLASH reporter gene in these cells, we tested whether cotransfection of THP-1 cells with {beta}-catenin led to similar results. To ensure high levels of TCF-4, we further cotransfected with the transcription factor. As shown in Fig. 7Go, none of the experimental conditions led to an activation of the TOPFLASH reporter gene. Consequently, to activate TCF-dependent transcription, another component in addition to {beta}-catenin and TCF-4 seems to be necessary.



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  		FIGURE 7. Exogenous {beta}-catenin and/or TCF-4 do not activate a LEF/TCF-dependent promoter in THP-1 cells. THP-1 cells were transfected with the LEF/TCF-dependent Firefly luciferase reporter gene TOPFLASH, its mutated form FOPFLASH, or the constitutively active Firefly luciferase reporter gene driven by the cyclin B2 promoter. Cotransfections were performed with {beta}-catenin and/or TCF-4. To normalize transfection efficiencies, cells were cotransfected with the CMV-driven Renilla luciferase reporter plasmid. After 48 h of incubation, cell lysates were prepared and relative luciferase activity was determined. Values represent means ± SD of triplicate samples.

 
Up-regulation of {beta}-catenin-TCF-4 target genes by LPS and zymosan

Since physiological inducers of {beta}-catenin, such as LPS and zymosan, could provide additional signals that are necessary for a functional interaction of {beta}-catenin and TCF-4 and that are absent when induction of {beta}-catenin is achieved by inhibiting its degradation, we tested whether these stimuli are capable of inducing target genes of {beta}-catenin-TCF-4 complexes. We chose to detect the mRNA of matrilysin (matrix metalloproteinase-7) and the mRNA of the receptor for the uPAR. Both genes have been described to be specifically induced by {beta}-catenin-TCF-4 in human colon cancer (21, 57). Fig. 8Go shows that induction of {beta}-catenin by both LPS and zymosan is accompanied by the induction of matrilysin and uPAR mRNA. In contrast, latex beads, which have no influence on the cellular {beta}-catenin concentration, had no effect on matrilysin and uPAR mRNA expression. The fact that lithium failed to induce {beta}-catenin-TCF-4 target gene expression in monocytes, although it induced {beta}-catenin accumulation (data not shown), agrees with our finding that treatment of THP-1 cells with lithium does not lead to {beta}-catenin-TCF-4-dependent transcription.



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  		FIGURE 8. Up-regulation of {beta}-catenin-TCF-4 target genes by LPS and zymosan. Human monocytes (106/ml) were incubated overnight in 24-well cell culture plates in the presence of 10% FCS before stimulating the cells with 100 ng/ml LPS, 200 µg/ml zymosan, 108/ml latex beads, and 20 mM lithium chloride for 3 h. mRNA of matrix metalloproteinase-7 (matrilysin), uPAR, and GAPDH were detected by using RT-PCR, as described in Materials and Methods. One representative experiment of three is shown.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The pivotal role of {beta}-catenin during embryogenesis and cancerogenesis has been described extensively. In this study, we have provided evidence that {beta}-catenin is involved in functions of primary immune cells. We found that {beta}-catenin accumulates in human monocytes upon adherence to plastic, after serum supplementation, and by stimulation with LPS and zymosan.

As high levels of {beta}-catenin may result in nuclear translocation of {beta}-catenin and subsequent target gene activation by its interaction with LEF/TCF transcription factors, this pathway may be used by adherence factors, serum constituents, LPS, and zymosan to induce gene expression. Among the four inducers of {beta}-catenin to date, only serum has been shown to be involved in LEF/TCF activation. In synergy with lithium, it promotes activation of these factors in mammary epithelial cells (48). The signaling cascade is independent of PI3-kinase signaling (48), a finding that correlates with our observations that show the failure of wortmannin to inhibit serum-induced {beta}-catenin accumulation.

Assuming that inactivation of GSK-3{beta} is a prerequisite for {beta}-catenin accumulation and that adherence-induced effects involve the participation of integrins, one could speculate that adhesion leads to activation of ILK, which could in turn mediate inactivation of GSK-3{beta}, as reported by Delcommenne et al. (22). The findings that ILK activation occurs in a PI3-kinase-dependent manner (22) and that adherence-induced {beta}-catenin accumulation is sensitive to wortmannin point to a role of PI3-kinase in {beta}-catenin accumulation probably as suggested above.

A role of {beta}-catenin in contributing to cellular adhesion processes by its interaction with classical cadherins was excluded, as we could not detect cadherins in monocytes.

Furthermore, a physical involvement of {beta}-catenin in phagocytosis by a direct contribution to cytoskeletal remodelling and adhesive functions seems unlikely since phagocytosis of latex beads had no effect. This view is further supported by the observation that inhibition of PI3-kinase, an enzyme contributing to the local assembly of the submembranous actin filament system leading to particle internalization (58), did not influence zymosan-induced induction of {beta}-catenin. Therefore, one could postulate the engagement of specific receptors in triggering the zymosan-induced accumulation of {beta}-catenin. Similarly, LPS may also act via specific receptors, resulting in gene induction via stabilization of {beta}-catenin.

The signaling role of {beta}-catenin in activating transcription factors of the LEF/TCF family has been studied in great detail in recent years (59, 60). LEF/TCF are believed to have a bipartite role in acting on transcriptional regulation. In the absence of {beta}-catenin, they recruit corepressors of the groucho/transducin-like enhancer of split family to the target gene enhancers and actively repress transcription of the genes (29). Transcriptional repression is believed to be relieved by the binding of {beta}-catenin to LEF/TCF. In addition to relieving repression, {beta}-catenin functions as a transactivator in the initiation of LEF/TCF-dependent transcription (61). Moreover, further coactivators, such as acetyltransferases (25, 26, 27) and pontin52 (28) as well as repressive proteins as the cAMP response element binding protein (60), a member of the C-terminal binding protein family of trancriptional corepressors (30), and presenilin 1 (62), seem to modulate trancriptional activation by {beta}-catenin-LEF/TCF complexes.

Although we could show that human monocytes and THP-1 cells express TCF-4, neither the inhibition of {beta}-catenin degradation nor cotransfection experiments with {beta}-catenin and TCF-4 resulted in the activation of a LEF/TCF-dependent promoter in THP-1 cells. The failure to detect LEF/TCF activation under our experimental conditions might have been due to missing coactivators and/or to the presence of repressive factors. In accordance with our data, Prieve et al. (36) also failed to detect {beta}-catenin-dependent activation of LEF/TCF in peripheral T lymphocytes, although both proteins were present in the nucleus.

Thus, the potential of {beta}-catenin-LEF/TCF to activate transcription in different cell types seems to be regulated by additional signals. Therefore, we thought it conceivable that physiological inducers of {beta}-catenin in monocytes could provide such additional signals, whereas a mere inhibition of {beta}-catenin degradation might have no effect. We could support this hypothesis by showing that the mRNA of matrilysin and uPAR, two {beta}-catenin-TCF-4 target genes, are induced by LPS and zymosan, but not by lithium in monocytes. In line with these findings, up-regulation of two other LEF/TCF-dependent genes has recently been reported in macrophages in response to LPS (37).

In conclusion, we provide evidence that {beta}-catenin is induced by diverse signaling pathways of human monocytes. Furthermore, we show for the first time that monocytes express transcription factors of the LEF/TCF family. The potential of TCF-4 to function both as a repressor or an activator of transcription points to hitherto undefined modes of gene regulation in monocytes. However, as suggested recently (63, 64), it cannot be excluded that {beta}-catenin may function as a transcriptional activator by interacting with other proteins than TCF-4.


   Acknowledgments
 
We thank Dr. B. Vogelstein for kindly providing us with the TOPFLASH and FOPFLASH reporter plasmids and the expression plasmids for {beta}-catenin and TCF-4. We thank C. Wohlenberg, Dr. T. Scholzen, and Dr. J. Gerdes for supporting us with their technical expertise. We are grateful to Dr. K. Wiebauer for very helpful discussions, and to Dr. R. N. Perham for critically reading the manuscript.


   Footnotes
 
1 This work was supported by a grant to K.E. from the Bundesministerium für Bildung und Forschung through the Innovation und Zukunftstechnologien für Krankheitsbehandlung und medizinischen Fortschritt. A.T. was a recipient of a fellowship of the Graduiertenförderung des Landes Sachsen. Back

2 Current address: Friedrich Miescher Institute, Maulbeerstrasse 66, CH-4058 Basel, Switzerland. Back

3 Address correspondence and reprint requests to Dr. Sunna Hauschildt, Institute of Zoology, Department of Immunobiology, University of Leipzig, Talstrasse 33, D-04103 Leipzig, Germany. E-mail address: shaus{at}rz.uni-leipzig.de Back

4 Abbreviations used in this paper: LEF, lymphoid enhancer factor; EGF, epidermal growth factor; GSK, glycogen synthase kinase; ILK, integrin-linked kinase; PI3-kinase, phosphatidylinositol 3-kinase; STBS, suspension TBS; TCF, T cell factor; uPAR, urokinase-type plasminogen activator. Back

Received for publication January 18, 2001. Accepted for publication October 5, 2001.


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 Introduction
 Materials and Methods
 Results
 Discussion
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