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J. Biol. Chem., Vol. 275, Issue 23, 17894-17899, June 9, 2000

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Wnt Signaling to -Catenin Involves Two Interactive Components

GLYCOGEN SYNTHASE KINASE-3 INHIBITION AND ACTIVATION OF PROTEIN KINASE C^*

Rui-Hong Chen, W. Vivianne Ding, and Frank McCormick^§

From the University of California, San Francisco, School of Medicine, Cancer Research Institute, San Francisco, California 94115

Received for publication, July 8, 1999, and in revised form, February 28, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Wnt signaling involves inhibition of glycogen synthase kinase-3 (GSK-3) and elevation of cytoplasmic -catenin. This pathway is essential during embryonic development and oncogenesis. Previous studies on both Xenopus and mammalian cells indicate that lithium mimics Wnt signaling by inactivating GSK-3. Here we show that serum enhances accumulation of cytoplasmic -catenin induced by lithium in both 293 and C57MG cell lines and that growth factors are responsible for this enhancing activity. Growth factors mediate this effect through activation of protein kinase C (PKC), not through Ras or phosphatidylinositol 3-kinase. In addition, Wnt-induced accumulation of cytoplasmic -catenin is partially inhibited by PKC inhibitors and by chronic treatment of cells with phorbol ester. Both calphostin C, a PKC inhibitor, and a dominant negative PKC exhibit partial inhibition on Wnt-mediated transcriptional activation. We therefore propose that Wnt signaling to -catenin consists of two interactive components: one involves inhibition of GSK-3 and is mimicked by lithium, and the other involves PKC and serves to augment the effects of GSK-3 inhibition.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

-Catenin was originally identified as a cytoplasmic protein that binds to the cell adhesion molecule cadherin in cell-cell junctions where it connects cadherin to the actin cytoskeleton (1-3). It is also involved in the Wnt signaling pathway to regulate developmental processes in a variety of organisms, and in tumorigenesis (4-6). In normal or non-stimulated cells, the majority of the -catenin protein is present in cell-cell junctions with very little in cytoplasmic or nuclear fractions. This is due to rapid turnover of -catenin promoted by complexes containing the adenomatous polyposis coli protein, GSK-3,¹ and Axin/Conductin (7-14). The N-terminal clusters of serine and threonine residues of -catenin are putative GSK-3 phosphorylation sites (8), which serve as signals for ubiquitin-mediated degradation (15-17). Recently, an F-box protein -TrCP in a ubiquitin-ligase complex has been shown to be involved in the proteasome-mediated degradation of phosphorylated -catenin (18-20). However, in the presence of a Wnt signal, a Frizzled family receptor and the downstream component Dishevelled are activated. Dishevelled in turn leads to the inactivation of GSK-3, causing the accumulation of cytoplasmic -catenin. High levels of -catenin in the cytosol result in its translocation into the nucleus and subsequent interaction with the Tcf/Lef family of transcription factors to activate expression of Wnt-responsive genes (6). Recently, c-myc and cyclin D1 have been identified as target genes of -catenin (21, 22). In colon cancer cells, adenomatous polyposis coli protein is frequently mutated resulting in elevated levels of cytoplasmic -catenin (23). In addition, -catenin mutations have been detected in colon cancer cell lines with wild-type adenomatous polyposis coli protein and other types of cancer (24-28). The majority of these mutations are located in the putative GSK-3 phosphorylation sites (29, 30). Similarly, these mutations result in stabilization of the -catenin protein. Intracellular levels of -catenin are therefore regulated by different signaling pathways and play a central role in both development and tumorigenesis.

Lithium has marked effects on the embryonic development and patterning in different organisms including Xenopus and Dictyostelium (31). In Xenopus embryos, lithium causes axis duplication, resembling a phenotype caused by overexpression of Wnt, dominant negative GSK-3, or -catenin (32-35). In Dictyostelium, lithium alters cell fate determination, resulting in transformation of pre-spore cells into pre-stalk cells (36). In isolated rat adipocytes, treatment with lithium leads to increased glycogen synthesis (37). The action of lithium has been previously attributed to depletion of inositol, based on the observation that lithium inhibits inositol monophosphatase (38). However, recent observations suggest that lithium may mimic Wnt signaling by direct inhibition of GSK-3, leading to the accumulation of cytoplasmic -catenin (39-41). This is consistent with the fact that GSK-3 is a highly conserved serine/threonine protein kinase in evolution and plays a central role in diverse biological processes including those affected by lithium (34, 42-48).

Normally, GSK-3 is highly active in resting cells. However, many extracellular stimuli result in the inhibition of its activity. For example, stimulation by insulin and growth factors activates the phosphatidylinositol 3-kinase (PI3K) pathway and the subsequent protein kinase B (PKB) activation leads to inhibition of GSK-3, probably via the N-terminal Ser-9 phosphorylation (49). Interestingly, Wnt stimulation also leads to similar degrees of inhibition of GSK-3 yet does not involve PI3K pathway, p70 S6 kinase, or MAP kinase pathway (50). Instead, TPA-sensitive isoforms of protein kinase C (PKC) have been shown to be involved in Wnt-induced inactivation of GSK-3 (50). PKC has been shown to phosphorylate and inactivate GSK-3 in vitro (51). However, the role of PKC activation in Wnt-induced -catenin accumulation remains unknown. Inactivation of GSK-3 by Wnt causes slower cytoplasmic turnover and increased nuclear translocation of -catenin, and leads to activation of Wnt target gene expression. However, there is no evidence that GSK-3 inhibition by insulin or growth factor stimulation leads to -catenin accumulation and Tcf/Lef-dependent transcriptional activation.

Here we provide evidence that growth factors can enhance lithium-induced -catenin accumulation and transactivation. This enhancement is through a distinct signaling pathway that involves TPA-sensitive isoforms of PKC. More importantly, our study suggests that two different components, one mimicked by lithium and the other mediated by PKC, contribute to Wnt-induced -catenin stablization and activation of gene expression.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- EGF, calphostin C, GF-109203X (bisindolylmaleimide I), and TPA (12-O-tetradecanoylphorbol-13-acetate) were obtained from Calbiochem. Insulin, platelet-derived growth factor, insulin-like growth factor-1, and L--lysophosphatidic acid (oleoyl) were purchased from Sigma. The monoclonal antibodies against -catenin and actin were purchased from Transduction Laboratories and Amersham Pharmacia Biotech, respectively. C57MG, C57MG-MV, C57MG-Wnt-1, Rat2-MV, and Rat2-Wnt-1 cell lines were generous gifts from Dr. Anthony Brown (Cornell University Medical College, New York) (52, 53). Conditioned Wnt media were made as described previously (67). 293-Wnt-1 cells were made from the pLNCX-Wnt-1 plasmid generously provided by Dr. Roel Nusse (Stanford University, CA). p110-CAAX, gag-PKB mammalian expression plasmids, and packaging cell lines for vector and Ras^Val-12 retroviruses were kindly provided by Dr. Pablo Rodriguez-Viciana. TOPFlash (a reporter plasmid containing multiple copies of wild-type Tcf-binding sites), FOPFlash (a reporter plasmid containing mutant Tcf-binding sites), full-length human Tcf4, and dominant negative Tcf4 were generous gifts from Dr. Hans Clevers (Utrecht University, The Netherlands) (54). PKC(T/A)₃, a dominant negative form of PKC (DN-PKC), was a gift from Dr. Peter Parker (Imperial Cancer Research Fund, United Kingdom) (55). AP-1-luciferase (AP-1-Luc) and p53-luciferase (p53-Luc) reporter plasmids were purchased from Stratagene. Cells were maintained in Dulbecco's modified Eagle's medium with 10% fetal calf serum and 1% penicillin/streptomycin. 1 mg/ml G418 (Life Technologies, Inc.) was added where selection was necessary.

Cellular Fractionation, Electrophoresis, and Immunoblotting-- Cytoplasmic and membrane fractions were prepared according to a protocol described previously (56). Briefly, cells were washed twice and scraped in phosphate-buffered saline and the cell pellets were resuspended in ice-cold hypotonic buffer (20 mM Tris, pH 7.5, 25 mM sodium fluoride, and 1 mM EDTA) containing a protease inhibitor mixture tablet (Roche Molecular Biochemicals). Cells were lysed by incubating on ice for 20 min, followed by 30 strokes in a Dounce homogenizer. The lysates were subject to ultracentrifugation at 100,000 × g for 30 min at 4 °C. The supernatant and the pellet were collected as the cytoplasmic fraction (S100 fraction) and the membrane fraction (P100 fraction), respectively. Protein concentration of the cytoplasmic extracts was estimated by using Bradford reagent (Bio-Rad). Equal amounts of protein from each lysate was analyzed by 10 or 7.5% SDS-polyacrylamide gel electrophoresis. The protein bands were transferred to polyvinylidene difluoride membranes (Millipore), blocked in 2% bovine serum albumin, 1× TBS, 0.05% Tween 20. Membranes were incubated with appropriate primary antibodies for 1 h at room temperature or overnight at 4 °C and then probed with horseradish peroxidase-conjugated secondary antibodies. ECL reagents (Amersham Pharmacia Biotech) were used to visualize the protein bands on the membranes, Semi-Quantitation by Gel Doc 1000 (Bio-Rad).

Transfections and Luciferase Assay-- Transfections were performed by using LipofectAMINE (Life Technologies, Inc.) or FuGENE 6 (Roche Molecular Biochemicals) according to the manufactures instructions. Cells from one 10-cm dish were transfected with 5 µg of the TOPFlash reporter plasmid or 5 µg of activator plasmids plus 2.5 µg of the TOPFlash reporter plasmid. Ten hours after transfection, the cells were split into eight 6-well dishes. Cells were serum-starved overnight, followed by stimulation with different stimuli for another 16 h. Cells were washed once with phosphate-buffered saline and then lysed for 15 min at room temperature. The lysates were clarified by centrifugation at 14,000 rpm for 10 min and 20 µl of each lysate was used to measure luciferase reporter gene expression (luciferase assay kit, Promega). The luciferase activity was either normalized to Renilla luciferase activity from co-transfected internal control plasmid pRL-TK or normalized to protein concentration. All experiments were performed in duplicate at least 3 times.

Establishment of Stable C57MG-V12 Cell Line by Retroviral Infections-- Each 6-cm dish was seeded with 2 × 10⁵ C57MG cells the day before infection. The medium was removed from the dishes, and the virus stocks and Polybrene (final concentration of 8 µg/ml) (Sigma) were added to the cells and incubated for at least 5 h. Cells were allowed to recover in fresh media for 24 h before being split into G418 containing media for selection. After 2 weeks of selection, cells that are resistant to G418 were pooled together for further analysis.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Serum Stimulates Accumulation of Cytoplasmic -Catenin in the Presence of Lithium-- Regulation of cytoplasmic -catenin is critical for its signaling function. In colon carcinomas, cytoplasmic -catenin levels and Tcf/Lef-dependent transcription can be up-regulated by either the loss of adenomatous polyposis coli protein function or mutations of the putative GSK-3 phosphorylation sites in the N-terminal region of -catenin (27, 29, 30). Wnt stimulation also specifically causes repression of GSK-3 activity and subsequent -catenin accumulation in the cytosol and nucleus (6). We asked whether serum stimulation, which has been shown to decrease GSK-3 activity to a similar extent as Wnt stimulation (50, 57), would induce cytoplasmic -catenin accumulation. To test this possibility, cells were stimulated with serum, lysed in hypotonic buffer, and fractionated by ultracentrifugation. Normalized amounts of S100 lysates were separated by 10% SDS-polyacrylamide gel electrophoresis and cytoplasmic -catenin was visualized by immunoblotting. As shown in Fig. 1A, serum stimulation alone did not lead to cytoplasmic accumulation of -catenin. However, lithium did increase cytoplasmic -catenin levels, which is consistent with previous observations that lithium acts as a non-competitive inhibitor of GSK-3 and leads to the cytoplasmic accumulation of -catenin (39-41). Surprisingly, when cells were stimulated with both serum and lithium, a much more pronounced accumulation of cytoplasmic -catenin in C57MG and 293 cells was observed (Fig. 1A and data not shown). Furthermore, serum and lithium also synergistically enhanced transcriptional activation of a reporter gene with multiple copies of Tcf-binding sites (TOPFlash) (Fig. 1B). Whereas lithium activated the reporter gene only up to 3-fold, lithium plus serum gave rise to 10-fold activation in 293 cells. These results indicate that serum by itself does not have a significant effect on -catenin levels, yet can potentiate the effect of lithium on cytoplasmic -catenin accumulation and -catenin-dependent transcriptional activation.

View larger version (14K): [in this window] [in a new window]   Fig. 1.   Serum enhancement of lithium-induced cytoplasmic -catenin accumulation and -catenin dependent transactivation. A, C57MG mammary epithelial cells were starved for 24 h, followed by stimulation with 10 mM lithium, 20% fetal bovine serum, and lithium plus serum for 3 h. Cells were then lysed and fractionated into S100 fractions. Equivalent amounts of protein from each S100 cytosolic fraction were analyzed by immunoblotting with a monoclonal antibody against -catenin. Quantified representation of the Western blot is shown as percentage of maximum at the bottom (Relative intensity). B, 293 cells were transiently transfected with the Tcf reporter plasmid (TOPFlash) and serum-starved for 16-24 h, and then stimulated with the indicated stimuli for another 16 h. Cell lysates, normalized to equal protein concentrations, were assayed for luciferase activity. The results are plotted as a percentage of maximum activity. Averages of three independent transfections are shown and the standard deviations are less than 10%.

Growth Factors Activate the Wnt/-Catenin Signal Transduction Pathway in the Presence of Lithium-- In order to find out what is responsible for the serum enhancement of lithium-induced -catenin stablization, we used L--lysophosphatidic acid and different growth factors, instead of serum, to stimulate cells. L--Lysophosphatidic acid neither caused the accumulation of -catenin by itself nor had any synergistic effect with lithium (data not shown). Combinations of growth factors (insulin, platelet-derived growth factor, insulin-like growth factor-1, and EGF) could substitute for serum stimulation (Fig. 2A). Among them, EGF partially substituted serum in -catenin accumulation (data not shown) and -catenin-dependent transcriptional activation (Fig. 2B). Therefore, growth factors play a major role in the cooperation of serum with lithium on the cytoplasmic -catenin stablization and -catenin-dependent transcriptional activation.

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Growth factors are responsible for cooperation with lithium in -catenin accumulation and transcriptional activation. A, combination of growth factors (GFs) (50 ng/ml EGF, 50 ng/ml platelet-derived growth factor, 50 ng/ml insulin-like growth factor-1, and 500 ng/ml insulin) was used to stimulate serum-starved 293 cells with or without 10 mM lithium. Cytosolic fractions were analyzed by immunoblotting with monoclonal antibodies against -catenin and actin. Normalized quantification of the Western blots is shown as percentage of maximum at the bottom (Relative ratio). B, EGF enhances lithium-induced -catenin-dependent transcriptional activation. Plasmid transfections and reporter gene assays were performed as described in the legend to Fig. 1. Cells were stimulated with EGF (50 ng/ml), lithium (10 mM), or with combination of EGF and lithium for 16 h. Relative luciferase activity is shown as a percentage of maximum activity.

Serum Effect on Cytoplasmic Levels of -Catenin Does Not Involve PI3K and Ras-- Since growth factors contributed to the serum effect on -catenin accumulation, we reasoned that growth factors might elicit their functions by feeding into GSK-3 via PI3K/PKB pathway (49, 58). Therefore, we examined if a constitutively active form of PI3K (p110-CAAX) (59) would substitute for serum in the reporter gene assay described earlier. p110-CAAX, co-transfected with the reporter TOPFlash, neither activated the reporter gene by itself nor synergized with lithium. Moreover, lithium-induced reporter gene activity could be further increased by serum stimulation in the p110-CAAX-transfected 293 cells (Fig. 3A). Likewise, co-transfection of gag-PKB, an activated form of PKB, with TOPFlash failed to enhance lithium-induced reporter gene activation (data not shown).

View larger version (21K): [in this window] [in a new window]   Fig. 3.   Neither PI3K nor Ras is involved in mediating serum enhancement of -catenin accumulation and transcriptional activation. A, 293 cells in 10-cm dishes were co-transfected with 5 µg of either pcDNA3 or p110-CAAX (a constitutively active form of PI3K) and 2.5 µg of the TOPFlash plasmid. The cells were then split into eight 6-well dishes, serum-starved, and stimulated as indicated for 16 h. Reporter gene activities were assayed and shown as percentages of maximum activity. B, accumulation of cytoplasmic -catenin in C57MG-Vector and C57MG-V12 cells. C57MG cells were infected with vector or RasVal-12 retroviruses and selected with 1 mg/ml G418 for 1-2 weeks. The G418-resistant cells were pooled and analyzed for cytoplasmic -catenin levels in response to serum, lithium, or both by immunoblotting.

Ras is a major mediator in the growth factor signaling pathways (60, 61). We wondered if Ras might be involved in synergizing with lithium in -catenin accumulation. We made a stable C57MG-V12 cell line expressing activated Ras^Val-12. The C57MG-V12 cells showed typical morphology of Ras-transformed cells (data not shown). We then examined if Ras^Val-12 could substitute for serum in potentiation of -catenin accumulation by lithium. The C57MG-V12 cells did not accumulate more cytoplasmic -catenin than the vector cells when treated with lithium. In addition, the cytoplasmic -catenin levels in both the vector and the C57MG-V12 cells could be further elevated to a similar extent by treatment with serum and lithium (Fig. 3B). Therefore, expression of Ras^Val-12 cannot substitute for serum in potentiation of -catenin accumulation.

PKC Is Necessary for Serum Potentiation of Lithium-induced -Catenin Accumulation-- One of the other candidate pathways activated by serum and growth factors is the PKC pathway. To investigate if PKC is involved in the accumulation of -catenin, we tested TPA, a potent activator of certain isoforms of PKC, in conjunction with lithium to induce -catenin accumulation. TPA alone had little effect on the cytoplasmic -catenin levels. Interestingly, TPA plus lithium stimulated further the accumulation of -catenin (Fig. 4A). The level of stimulation by TPA is comparable to that of serum. These results suggest that although activation of PKC alone is not sufficient for activation of Wnt signaling pathway, it can enhance Wnt signaling by cooperating with lithium. We postulated that the serum cooperation might be due to the activation of PKC. To test this, we pretreated cells with calphostin C, a PKC inhibitor, before being stimulated with serum and lithium. As shown in Fig. 4B, treatment of calphostin C inhibited serum potentiation of -catenin accumulation. Furthermore, long-term treatment of cells with TPA ablated the serum potentiation of lithium-stimulated -catenin accumulation (data not shown). Chronic treatment of cells with TPA has been shown to down-regulate the TPA-sensitive isoforms of PKC. Therefore, the TPA-sensitive isoforms of PKC might be responsible for the enhancement of lithium-induced -catenin accumulation by serum.

View larger version (18K): [in this window] [in a new window]   Fig. 4.   PKC contributes to the serum enhancement of lithium-induced -catenin accumulation. A, TPA enhances lithium-induced -catenin accumulation. 293 cells were serum-starved for 24 h and then stimulated with 100 ng/ml TPA, 10 mM lithium or both. Cytoplasmic -catenin levels were analyzed as described. B, calphostin C inhibits the serum enhancement of lithium-induced -catenin accumulation. Serum-starved 293 cells were pretreated with calphostin C (1 µM) for 30 min before stimulation. Cytoplasmic fractions were analyzed for -catenin and actin levels. Normalized quantification of the Western blots is shown at the bottom of each panel.

TPA-sensitive PKCs Are Necessary But Not Sufficient for the Full Accumulation of -Catenin Induced by Wnt-1-- Wnt signaling also leads to cytoplasmic -catenin accumulation by inhibiting GSK-3 activity (62, 63). Since lithium mimics the Wnt signaling in terms of inhibiting the activity of GSK-3, we examined if serum could also enhance the Wnt signaling by increasing Wnt-stimulated -catenin accumulation. C57MG-Wnt-1 cells showed elevated levels of cytoplasmic -catenin (52, 53). However, serum starvation or serum stimulation of C57MG-Wnt-1 cells did not affect the levels of cytoplasmic -catenin accumulation (Fig. 5A). Therefore, serum does not seem to further enhance -catenin accumulation induced by Wnt-1 expression. This observation suggests that lithium is not the surrogate for the Wnt signaling, and serum or growth factors can provide additional signal(s) that leads to further accumulation of -catenin.

View larger version (31K): [in this window] [in a new window]   Fig. 5.   PKC is necessary for full activation of Wnt/-catenin pathway. A, Wnt-1 activates -catenin accumulation in the absence of serum. C57MG-MV (vector) and C57MG-Wnt-1 cells were starved or restimulated with 20% fetal calf serum. Cytoplasmic fractions were analyzed for -catenin accumulation. B, calphostin C partially inhibits -catenin accumulation in the C57MG-Wnt-1 cells. The C57MG-MV or C57MG-Wnt-1 cells were treated with increasing concentrations of calphostin C (0.2 µM to 1 µM) for 3 h, harvested, fractionated, and analyzed for cytoplasmic -catenin levels. C, chronic treatment of C57MG cells with TPA partially blocks their responses to conditioned Wnt-1 media. C57MG cells were treated with dimethyl sulfoxide, TPA at 0.2, 1, or 5 µM for 24 h. The cells were then stimulated with either control or Wnt-1 media for 3 h. Cytoplasmic fractions were analyzed for -catenin and actin levels. Normalized quantification is shown at the bottom of panels B and C.

PKC has been shown to participate in the inactivation of GSK-3 by Wnt (50). To investigate whether PKC is involved in the -catenin accumulation induced by Wnt-1, we treated the Wnt-1 cells with different PKC inhibitors and examined the cytoplasmic -catenin accumulation by immunoblotting. Treatment of the C57MG-Wnt-1 or 293-Wnt-1 cells with increasing concentrations of calphostin C for 3 h resulted in gradual reduction of the cytoplasmic -catenin (Fig. 5B and data not shown). Similarly, chronic treatment of C57MG-Wnt-1 or 293-Wnt-1 cells with TPA or treatment of the C57MG-Wnt-1 cells with GF-109203X (1 µM) also led to partial inhibition of -catenin accumulation (data not shown). Furthermore, -catenin accumulation stimulated by conditioned Wnt media was also partially inhibited by treatment of calphostin C, as well as by chronic treatment of TPA in C57MG and 293 cells (Fig. 5C and data not shown). To test whether the inhibition of -catenin accumulation by these inhibitors has any functional significance, we assayed the Tcf reporter activity in 293-Wnt-1 cells. The reporter activity was 10-fold higher in 293-Wnt-1 cells compared with 293-Vector cells and was partially inhibited by calphostin C in a dose-dependent manner in 293-Wnt-1 cells (Fig. 6A). To further strengthen the conclusion from the PKC inhibitor experiments, we employed PKC(T/A)₃, a dominant negative PKC (DN-PKC), to test whether it has any effect on Wnt-mediated transcriptional activation. In control experiments, DN-PKC blocked 70% of TPA-stimulated AP-1 luciferase reporoter activity (Fig. 6B, left panel) and it had no significant effect on p53-mediated transcriptional activation (Fig. 6B, right panel). The expression of DN-PKC caused partial loss of Wnt-induced transcriptional activation in a dose-dependent manner, although it was not as strong as the dominant negative Tcf4 (DN-Tcf4) (Fig. 6B, middle panel). Since activation of PKC by TPA alone did not result in any accumulation of cytoplasmic -catenin, we conclude that activation of PKC is not sufficient for transduction of Wnt signal yet it is an important component of Wnt signaling.

View larger version (31K): [in this window] [in a new window]   Fig. 6.   PKC participates in Wnt-mediated activation of transcription. A, calphostin C partially inhibits Wnt-associated transcriptional activation. 293-Wnt-1 or 293-Vector cells were transfected with the reporter plasmid TOPFlash and the internal control plasmid pRL-TK for 24 h. Cells were treated with increasing concentrations of calphostin C (0.1 to 1 µM) for another 16 h and then harvested for luciferase assay. The activity is expressed as a percentage of maximum luciferase activity. B, in 12 wells, 293-Wnt-1 cells were co-transfected with 0.2 µg of each reporter plasmid (AP-1-Luc, TOPFlash, or p53-Luc) and 1 µg of the dominant negative PKC (DN-PKC). For AP-1 luciferase assay, the cells were stimulated with 200 ng/ml TPA for 16 h; for TOPFlash luciferase assay, 0.5 or 1 µg of DN-PKC or the dominant negative Tcf4 (DN-Tcf4) was co-transfected; for p53 luciferase assay, 0.3 µg of p53 was used as an activator. The luciferase activity is expressed as a percentage of the maximum activity in each assay and represents the average of triplicates of three independent experiments.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

From genetic and biochemical studies of Drosophila and Xenopus, a linear pathway has been elucidated from the extracellular ligand Wnt to the transcription factor Tcf/Lef. Wnt binds to its receptor Frizzled, triggering the activation of Dishevelled, which in turn leads to the inactivation of GSK-3 (6). Inactivation of GSK-3 abolishes the phosphorylation and ubiquitin-mediated degradation of -catenin (18-20). Nevertheless, the molecular mechanisms underlying the inactivation of GSK-3 and -catenin accumulation have not been fully understood. It has been proposed that PKC might be involved in the inactivation of GSK-3 because certain inhibitors of PKC are capable of antagonizing Wnt-mediated GSK-3 inactivation (50). Our data further demonstrated that Wnt/-catenin pathway could comprise two components, one is mimicked by lithium and the other involves PKC.

It is interesting that Wnt-1 and growth factors inactivate GSK-3 to a similar extent (50, 57), yet only Wnt-1 can induce cytoplasmic -catenin accumulation (Figs. 1 and 5). Earlier studies have indicated that signaling from growth factors (e.g. insulin) to GSK-3 is wortmannin sensitive and involves PKB (49, 64, 65) whereas that from Wnt to GSK-3 is insensitive to wortmannin treatment (50). Instead, Wnt-1-induced GSK-3 inactivation is completely blocked by certain PKC inhibitors (50). Therefore, the pathway from GSK-3 inhibition to cytoplasmic -catenin accumulation appears to be fundamentally different between Wnt signaling and growth factor signaling. In the case of insulin signaling, GSK-3 inhibition is mediated by phosphorylation of Ser-9 on GSK-3 (66-68); in Wnt signaling, GSK-3 activity is inhibited to 50% of its normal activity but little is known about the mechanism of the inhibition. It is of interest to study whether Wnt-mediated GSK-3 inhibition is accomplished via covalent modification and whether different pools of GSK-3 are involved in Wnt signaling.

Lithium has profound effects on morphogenesis in diverse organisms (31). One action of lithium has been recently shown to mimic Wnt signaling by direct inhibition of GSK-3 causing the accumulation of cytoplasmic -catenin (39-41). In this study, we confirmed the lithium effect on GSK-3 inhibition (data not shown) and -catenin accumulation in both 293 and C57MG cells. We further showed that serum or growth factors could cooperate with lithium to induce -catenin stablization (Figs. 1 and 2). This raised a possibility that Wnt might also synergize with serum or growth factors to induce maximum accumulation of -catenin. However, our results indicated that Wnt-induced -catenin accumulation is independent of serum (Fig. 5). These observations imply that lithium might only partially mimic the Wnt signaling pathway.

Which serum-activated pathways are involved in the cooperation with lithium to induce cytoplasmic -catenin accumulation? We ruled out the involvement of PI3K/PKB and Ras in the synergistic stablization of -catenin (Fig. 3). Since PKC is implicated as a candidate component in the Wnt signal transduction, we examined PKC inhibitors on serum enhancement of lithium-induced -catenin accumulation. Indeed, PKC inhibitors completely block the serum enhancement function. The role of PKC is further supported by the observation that TPA, a potent activator of certain PKC isoforms, can also cooperate with lithium (Fig. 4). In conclusion, we identified a pathway involving PKC that enhances lithium-induced -catenin accumulation. It is noteworthy that a synergy between lithium and insulin-like growth factor-1 is reported in the stimulation of granule neuron progenitor proliferation, a process involving inhibition of GSK-3 (69). Questions emerge as what is the target(s) of growth factors/PKC pathway. One possibility is that growth factors/PKC may cooperate with lithium in the inactivation of GSK-3. To test this hypothesis, we immunoprecipitated GSK-3 kinase from resting cells or serum-stimulated cells and performed in vitro kinase assay in the presence of increasing amounts of lithium. We did not observe any cooperative inhibition of GSK-3 by serum and lithium (data not shown). However, considering the nature of the in vitro kinase assay, we still cannot rule out the possibility of cooperative inhibition of GSK-3 by serum and lithium in vivo. Further study is needed to dissect the converging point from activation of PKC to -catenin accumulation.

The role of PKC in the Wnt signaling pathway remains controversial. In vitro, PKC has been shown to phosphorylate and inactivate GSK-3 (51). An earlier report showed that a PKC inhibitor Ro31-8220 or chronic treatment with TPA resulted in complete block of Wnt-mediated inhibition of GSK-3 in mouse 10T1/2 fibroblasts (50). In that study, the authors did not determine the effect of these PKC inhibitors on Wnt-induced -catenin accumulation. On the other hand, others reported that treatment with the PKC inhibitors GF-109203X or Ro31-8220 caused -catenin accumulation in certain human breast epithelial cell lines, whereas treatment with the PKC inhibitor calphostin C or TPA-induced down-regulation of PKCs has no significant effect on -catenin accumulation (15). Our data showed that Wnt-dependent -catenin stablization was partially inhibited by certain PKC inhibitors (Fig. 5 and data not shown). The discrepancy may be explained by the differences of these inhibitors on various isoforms of PKCs or their nonspecific effects on other kinases. It is possible that TPA-sensitive PKCs are involved in the Wnt-induced GSK-3 inactivation whereas atypical PKCs contribute to -catenin degradation (15, 50). However, the most plausible explanation is from the recent publication by Hers and colleagues (70), who showed that the PKC inhibitors GF-109203X and Ro31-8220 also inhibit GSK-3, potentially slowing down -catenin degradation.

The importance of PKCs in colon cancer has been demonstrated in transgenic PKC _II mice that exhibit hyperproliferation and increased Wnt signaling in the colonic epithelium (71). Recently, certain Wnt and Frizzled homologs have been reported to modulate PKC but have no effect on expression of -catenin target genes, whereas other Frizzled homologs capable of activating -catenin target genes do not activate PKC (72). Consistent with the report, we found that molecules such as growth factors or TPA that activate PKCs do not stimulate any detectable -catenin accumulation and transcriptional activation. In contrast, using inhibitors and the dominant negative PKC mutant, we showed that PKCs do participate in Wnt/-catenin signaling, although they are not the essential components of this pathway. For example, calphostin C-mediated PKC inhibition or TPA-induced PKC down-regulation results in partial inhibition of Wnt-induced -catenin accumulation. Furthermore, both calphostin C and the dominant negative PKC inhibit partially the Wnt-mediated Tcf/Lef reporter activation. These results strengthened the notion that PKC activation alone is not sufficient to cause stablization of -catenin, but it is a component of Wnt pathway that leads to maximum accumulation of -catenin and transcriptional activation. Elevation of -catenin levels in the cytosol and nucleus as well as its transcriptional activity is strongly correlated with tumorigenesis. Therefore, understanding the regulation of free -catenin pools by different signaling pathways could facilitate the development of new strategies to regulate -catenin signaling.

	ACKNOWLEDGEMENTS

We thank Dr. P. Polakis, Dr. H. Clevers, Dr. A. Brown, Dr. P. Parker, Dr. P. Rodriguez-Viciana, and Dr. H. Jiang for plasmids, retroviruses, and cell lines. We thank Dr. D. Stokoe and Dr. P. Sabbatini for critical reading of the manuscript. We also thank members of Dr. Frank McCormick's laboratory for discussions and help.

	FOOTNOTES

^* This work was supported in part by the Daiichi Cancer Research Program.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Recipient of the Carol Franc Buck Fellowship of the Cancer Research Institute at University of California, San Francisco.

^§ To whom correspondence should be addressed: University of California, San Francisco, School of Medicine, Cancer Research Institute, 2340 Sutter Street, San Francisco, CA 94115. Tel.: 415-502-1710; Fax: 415-502-3179; E-mail: mccormick@cc.ucsf.edu.

Published, JBC Papers in Press, April 3, 2000, DOI 10.1074/jbc.M905336199

	ABBREVIATIONS

The abbreviations used are: GSK-3, glycogen synthase kinase-3; PKB, protein kinase B; PI3K, phosphatidylinositol 3-kinase; TPA, 12-O-tetradecanoylphorbol-13-acetate; PKC, protein kinase C; EGF, epidermal growth factor; DN, dominant negative.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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