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J. Biol. Chem., Vol. 282, Issue 36, 26490-26502, September 7, 2007

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Inositol Pentakisphosphate Mediates Wnt/-Catenin Signaling^*

From the Department of Physiology and Biophysics, Diabetes and Metabolic Disease Research Center, School of Medicine, Health Sciences Center, State University of New York, Stony Brook, New York 11794-8661

Received for publication, March 12, 2007 , and in revised form, June 26, 2007.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Wnt3a stimulates lymphoid enhancer factor/T-cell factor protein-sensitive transcription, i.e. the canonical pathway, in mouse F9 embryonal tetratocarcinoma cells expressing rat Frizzled-1. We explored the potential roles for inositol polyphosphates as mediators of Wnt signaling in the canonical path-way. Wnt3a triggers G-protein-linked phosphatidylinositol signaling, transiently generating inositol polyphosphates, especially inositol pentakisphosphate (IP₅) accumulation. Knock-down of G_q abolishes, whereas expression of the Q209L constitutively active mutant of G_q mimics, the effects of Wnt3a on IP₅ generation and downstream signaling. Phospholipase C-1 and C-3 mediate the G protein signal to the level of phosphatidylinositol signaling. Knock-down and inhibitor studies of the enzymes responsible for generating IP₅ reveal inositol 1,4,5-trisphosphate 3-kinase and inositol polyphosphate multikinase as key mediators in the production of IP₅. Wnt3a stimulation of the canonical pathway requires accumulation of IP₅, which acts to inhibit the activity of glycogen synthase kinase-3, whereas stimulating casein kinase 2. Blockade of Wnt3a stimulation of IP₅ generation blocks -catenin accumulation, activation of lymphoid enhancer factor/T-cell factor protein-sensitive transcription, and promotion of primitive endoderm formation in response to Wnt3a. Phosphatidylinositol signaling mediates Wnt3a action in the canonical pathway, acting to generate inositol pentakisphosphate, a key second messenger of Wnt3a.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Wnt ligands are palmitoylated glycoproteins (1, 2) that regulate essential aspects of early development, including cell fate determination, embryonic patterning, and cell proliferation (2-4). Wnts are secreted and bind to cell-surface heptihelical receptors termed "Frizzleds" (5, 6), members of the superfamily of G protein-coupled receptors (7, 8). The Wnt canonical pathway is initiated by Wnt3a binding to Frizzled-1 and activation of heterotrimeric G proteins (e.g. G_q and G_o) and the phosphoprotein Dishevelled (Dvl) in mouse F9 teratocarcinoma cells (9) and Drosophila (10) as well. Activation of Dsh/Dvl inhibits glycogen synthase kinase 3 (GSK3),² which in the absence of Wnt catalyzes the phosphorylation and destabilization of -catenin. The Wnt3a stimulates nuclear accumulation of -catenin, enabling the activation of lymphoid enhancer factor/T-cell factor (Lef/Tcf)-dependent gene expression (11).

Casein kinase 2 (CK2), a family of protein kinases highly conserved in nature, has been reported to regulate many cellular processes, including gene expression, protein synthesis, cell proliferation, and apoptosis (12, 13). Several key components of the Wnt/-catenin/Lef-Tcf pathway are known substrates of CK2 in vitro, including Dsh/Dvl (14), the product of the adenomatous polyposis coli gene (APC) (15, 16), transcription elements Lef/Tcf (17-19), the product of the engrailed gene (20), and -catenin (21, 22). CK2 has been shown to act as a positive modulator of the Wnt/-catenin/Lef-Tcf pathway, suppressing -catenin degradation and -catenin binding to adenomatous polyposis coli (22, 23). Overexpression of CK2 in ventral blastomeres of Xenopus embryos stimulates dorsal axis formation, whereas expression of a kinase-dead CK2 mutant blocks ectopic axis formation in response to Xwnt8 (24).

Recently we reported that CK2 is activated by Wnt3a, operating downstream of Dvl and upstream of Lef/Tcf-sensitive gene transcription (23). Although earlier reported to act solely as "constitutively active" in cells, CK2 has been shown to be activated by Wnt3a (23). Overexpression or inhibition of CK2 affects early development via the Wnt/-catenin/Lef-Tcf-sensitive gene expression (24). CK2 enhanced -catenin stabilization by phosphorylating -catenin at Thr³⁹³ in the armadillo repeat region of -catenin in vitro (22). Although CK2 clearly mediates an essential aspect of Wnt3a signaling, the nature of this regulation of CK2 is unknown. CK2 activity has been shown in vitro to be sensitive to inositol polyphosphates (25). Consequently we hypothesized that Wnt may regulate CK2 (and thereby the Wnt/-catenin or canonical pathway) by influencing phosphatidylinositol signaling. In the current report, we investigate the validity of this hypothesis and probe the generation of water-soluble inositol polyphosphates in Wnt action. Our results show that Wnt3a stimulates phosphatidylinositol breakdown and the accumulation of inositol polyphosphates, particularly IP₅.IP₅ generation in response to Wnt3a is shown to be operating downstream of Frizzled-1, heterotrimeric G proteins (especially G_q), and PLC in the canonical Wnt/-catenin/Lef-Tcf pathway of mammalian cells. Inositol 1,4,5-trisphosphate 3-kinase (IP3K) and inositol polyphosphate multikinase (IPMK) are shown to be key mediators of the production of IP₅. Inhibiting the action of either enzyme blocks the ability of Wnt3a to accumulate -catenin, to activate Lef/Tcf-sensitive transcription, and to promote formation of primitive endoderm (PE).

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Materials—Adriamycin, ammonium aurintricarboxylate (ATA), chlorogenic acid (CGA), inositol 1,3,4,5,6-pentakisphosphate (Ins(1,3,4,5,6)P₅), and anti--catenin antibody were purchased from Sigma. Inositol 1,4,5-trisphosphate (Ins(1,4,5)P₃), inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P₄), and inositol hexakisphosphate (IP₆) were obtained from Calbiochem International (San Diego, CA). Antibodies against inositol 1,4,5-trisphosphate 3-kinase B (anti-IP3K-B, G-20) and G₁₁ (anti-G₁₁) as well as protein A/G Plus-agarose were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Other antibodies were purchase from following companies: anti-G_o was from Chemicon International (Temecula, CA); anti-G_q and anti-GSK3 were from BD Transduction Laboratories (San Diego, CA); anti-Glu-Glu monoclonal antibody was from Covance (Richmond, CA); and antibody TROMA-1 was from University of Iowa, Developmental Studies Hybridoma Bank (Iowa City, IA). Recombinant mouse Wnt3a and Wnt5a were provided by R&D System (Minneapolis, MN). Peptides (YRRAAVPPSPSLSRHSSPHQ(pS)EDEEE as GSK3 substrate and HRRRDDDSDDD as CK2 substrate) were obtained from Upstate%20Biotechnology">Upstate Biotechnology (Lake Placid, NY). myo-[³H]Inositol, [³H]Ins(1,4,5)P₃, and [³H]Ins(1,3,4,5)P₄ were purchased from American Radiolabeled Chemical (St. Louis, MO) and PerkinElmer Life Sciences. The synthesis of [³H]Ins(1,3,4,5,6)P₅ was made from [³H]Ins(1,4,5)P₃ by using human inositol polyphosphate multikinase (IPMK or IPK2) as described (26). [³²P]IP₆ was synthesized from [³²P]orthophosphate-labeled mung bean as described (26).

Cell Culture—The mouse F9 teratocarcinoma (F9) cells (from ATCC, Manassas, VA) were grown in Dulbecco's modified Eagle's medium supplemented with 15% fetal bovine serum (Hyclone, South Logan, UT) at 37 °C in a 5% CO₂ incubator. The human embryonic kidney HEK293 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and grown at 37 °C in a humidified incubator of 5% CO₂ and 95% air. F9 and HEK293 cells were co-transfected with an expression vector harboring rat Frizzled1 (Fz1) and a luciferase reporter construct, Super8X TOP-flash by using Lipofectamine Plus (Invitrogen) according to manufacturer's protocol. Stable clones were selected in the presence of 400 ng/ml G418, and maintained in the media containing 100 ng/ml G418 as previously described. For Wnt stimulation, cells were grown to monolayer and incubated in the absence of serum for 8 h prior to the treatment by either Wnt3a (15 ng/ml) or Wnt5a (60 ng/ml).

Analysis of Soluble Inositol Phosphates—Cells expressing Fz1 were plated on a 35-mm culture dish and incubated in inositol-free Dulbecco's modified Eagle's medium (Chemicon) containing myo-[³H]inositol (15 µCi/well) and 15% fetal bovine serum for 48 h. Cells were cultured in Dulbecco's modified Eagle's medium (without serum) for an additional 8 h before Wnt stimulation. Soluble inositol phosphates were extracted according to the method previously described (27). Briefly, cells were lysed in 500 µl of methanol and 0.5 N HCl mixture (methanol, 0.5 N HCl = 2:1) and inositol phosphates were extracted with 335 µl of chloroform. The aqueous phase containing inositol phosphates was neutralized by addition of 80 µl of saturated NaHCO₃. Equal counts of soluble inositol phosphates were applied to a strong anion-exchange, Partisil 10-SAX HPLC column (4.6 x 250 mm, Whatman, Florham Park, NJ) and eluted first with a linear gradient of 0.01-1.7 M ammonium phosphate (pH 3.5) over 30 min and followed by a 35-min step of 1.7 M ammonium phosphate (pH 3.5) at a flow rate of 0.5 ml/min. Fractions (0.5 ml/fraction) were collected and 5 ml of scintillation fluid were added into each tube. Radioactivity was measured by a scintillation counter. All data were normalized with respect to the total radioactivity measured in the lipid fraction. The elution times of internal standards, ADP and ATP, were monitored by a spectrophotometer at 260 nm. The identity of individual inositol phosphates was assigned on the basis of elution with known standards under the same condition.

Construction of Plasmids—pTrcHisA-hIPMK was a gift from Dr. Solomon Snyder (Johns Hopkins Medical School). To subclone the hIPMK into a mammalian expression vector, PCR was used to amplify inserted hIPMK in pTrcHisA-hIPMK by using the 5'-primer with EcoRI site (5'-GGAATTCGGATGGCCGCCGAGCCCCCAGC-3') and 3'-primer with BglII site (5'-ACAAGATCTTCAACTGTCCAAGATACTCCGAAG-3'). The PCR product encoding hIPMK was digested by EcoRI and BglII and subsequently subcloned into mammalian expression vectors, pCMV-HA and pCMV-MYC (BD Biosciences). The identity of the amplified sequences was confirmed by direct DNA sequencing.

Knock-down Target Proteins by Small Interfering RNA (siRNA)—F9 cells expressing Fz1 were grown to 60% confluency on 12-well plates. siRNA (100 nM, final concentration) was introduced into cells by using Lipofectamine 2000 (Invitrogen). Cells were cultured in the presence of siRNA for an additional 48-72 h according to manufacturer's recommendation. Sources of siRNA used in the study are listed as follows. siRNAs targeting G_q, G_o, G₁₁, and PLC₄ were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). siRNAs targeting PLC₁ and PLC₃ were synthesized by Ambion (Austin, TX) according to the sequence published by Kim et al. (28). All following siRNAs were obtained from Ambion: siRNA to IPMK (ID code 203326), siRNA to IP3K-A (ID code 170266), siRNA to IP3K-B (ID code 203096), siRNA to IP3K-C(ID code 171717), and siRNA to Dvl2 (ID code 61090). We used either immunoblotting or semi-quantitative RT-PCR analysis to determine the efficiency of knockdown on targeted molecules.

Semi-quantitative RT-PCR—Total RNA were collected from F9 control cells as well as siRNA-treated cells using STAT-60 (TEL-TEST, Inc., Friendswood, TX) according to the manufacturer's protocol. First strand cDNA was synthesized from 1 µg of total RNA by using a random hexamer primer and Superscript II reverse transcriptase (Invitrogen). The cDNA was subjected to PCR (94 °C for 45 s, 60 °C for 45 s, 72 °C for 30 s; for 20 cycles) using specific primer pairs to targeted genes and Taq DNA polymerase (Invitrogen). The primers used to target IPMK were 5'-GATTGGGCGGAAGAGCTACGA-3' and 5'-CTACCTTCTGAATACTGGCGGC-3'; the primers to target PLC₁ were 5'-CTTCTCTGGCCTGTTTGAGG-3' and 5'-TGCATACGTGTCTGGGACAT-3'; the primers to target PLC₂ were 5'-GACAAGCCTGAGAGGTCCT-3' and 5'-AAATGTCGAAGCGAGATGCT-3'; the primers to target PLC₃ were 5'-CAGGCCAGCACAGAGACATA-3' and 5'-GGTGTAGGGGTCACCTCAGA-3'; the primers to target PLC₄ were 5'-AGGTGCTACCACGAACATCC-3' and 5'-GCGTCTTCAAGTAGCCAAGG-3'. Cyclophylin A was amplified as a control.

Immunoblotting—Cells were harvested in lysis buffer containing protease and phosphatase inhibitors (20 mM Tris, pH 7.4, 150 mM NaCl, 1% Triton X-100, 200 µM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 mM NaF, 1 mM Na₃VO₄, and 100 nM okadaic acid) and the mixture were centrifuged at 15,000 x g for 20 min at 4 °C. The supernatant was collected and protein concentration was determined by the Lowry method (29). Equal amounts of proteins from samples were subjected to SDS-PAGE and separated proteins were transferred to nitrocellulose blots electrophoretically. The blots were incubated with 10% bovine serum albumin for 0.5 h, rinsed with water, and then probed with antibody against targeted proteins. To determine cytoplasmic levels of -catenin, cells were lysed in RIPA buffer supplemented with protease inhibitors (20 mM Tris, pH 7.4, 150 mM NaCl, 1% Triton X-100, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin) and cell lysate was incubated with ConA-Sepharose (Amersham Biosciences) for 1 h. The suspension was applied by centrifugation to remove membrane-associated -catenin. The supernatants were subjected to SDS-PAGE, followed by immunoblotting with anti--catenin antibody. Immune complexes were detected by the enhanced chemiluminescence method, as per the manufacturer's instructions.

Immunoprecipitation—F9 cells stably transfected with Fz1 were grown in 100-mm dishes and transiently transfected with 4 µg of pCMV-HA-IPMK. Twenty-four hours later, cells were treated with Wnt3a and then lysed at the indicated time with 3 ml of lysis buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 10 mM NaF, 1 mM Na₃VO₄, and 1% Triton X-100) containing protease inhibitors (200 µM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, and 10 µg/ml leupeptin). Whole cell lysates (2 mg) were incubated with anti-HA high affinity antibody (Roche Applied Science) immobilized on protein A/G-agarose at 4 °C for 4 h. Immune complexes were precipitated by centrifugation (10,000 x g) and dissolved in Laemmli's solution. Protein mixture was subjected to SDS-polyacrylamide gel electrophoresis followed by electrotransblotting. HA-tagged IPMK were analyzed by immunoblotting as described.

Lef/Tcf Reporter Assay—F9 clones stably transfected with Fz1 and Super8X TOPflash were cultured in 12-well plates and stimulated with Wnt3a for 5 h. Cell lysates were collected in reporter lysis buffer (Promega). Cell lysates (10 µl) were incubated for 10 s with 100 µl of reaction mixture containing 0.67 mM luciferin, 0.27 mM Coenzyme A, 0.1 mM EDTA, 1.1 mM MgCO₃, 4 mM MgSO₄, and 20 mM Tricine (pH 7.8). The intensity of luminescence was immediately measured using a luminometer (Lumat LB 9507, Berthold Technologies, Oak Ridge, TN). To study the effect of ATA, CGA, and adriamycin on Lef/Tcf-dependent transcription, inhibitors were added into culture media for 30 min prior to Wnt3a stimulation. Samples were assayed in triplicate and the luciferase activity was normalized based on protein concentration.

In Vitro CK2 Kinase Activity Assay—CK2 activity was analyzed, as previously described (30). Briefly, immunoprecipitated CK2 from whole cell lysates were washed with kinase assay buffer (20 mM MOPS, pH 7.2, 25 mM -glycerol phosphate, 5 mM EGTA, 1 mM sodium orthovanadate, 1 mM dithiothreitol, and 100 nM okadaic acid) and then incubated with 120 µM substrate peptide (HRRRDDDSDDD) in kinase assay buffer containing 10 µCi of [-³²P]GTP, 160 µM GTP, 25 mM MgCl₂ and with or without inositol phosphates at 30 °C for 10 min. The reaction was stopped by addition of 25 µl of 40% trichloroacetic acid. Samples were spotted onto a P81 Whatman filter membrane. Air-dried membranes were washed with 0.75% H₃PO₄ three times and incorporated -³²Pinsubstrates on the membrane was measured by liquid scintillation spectrometry.

In Vitro GSK3 Kinase Activity Assay—GSK3 activity was analyzed, as previously described (31). Briefly, GSK3 was immunoprecipitated from whole cell lysates followed by washing the pellet with kinase assay buffer (50 mM Tris, pH 7.5, 25 mM -glycerol phosphate, 5 mM EGTA, 1 mM sodium orthovanadate, and 1 mM dithiothreitol). The immunoprecipitated GSK3 was incubated with 10 µM substrate peptide (YRRAAVPPSPSLSRHSSPHQ(pS)EDEEE) in kinase assay buffer containing 10 µCi of [-³²P]ATP, 160 µM ATP, 25 mM MgCl₂, and with or without inositol phosphates at 30 °C for 10 min. The reaction was stopped by addition of 25 µl of 40% trichloroacetic acid. Samples were spotted onto a P81 Whatman filter membrane. Air-dried membranes were washed with 0.75% H₃PO₄ three times and incorporated -³²Pin substrates on the membrane was measured by liquid scintillation spectrometry.

Analysis of Primitive Endoderm Formation—F9 clones expressing Fz1 were propagated on 12-well plates. Cells were treated with control siRNA, or siRNA targeting IPMK in combination with siRNA targeting IP3K-B for 48 h and then challenged without or with Wnt3a. At the end of 4 days, clones were fixed with 3% paraformaldehyde for 5 min, stained with the mouse endoderm-specific marker, cytokeratin endo A, by the monoclonal antibody TROMA-1 (32). The fixed cells were examined using a Zeiss Axiovert 200M microscope for both phase-contrast and fluorescence images.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Inositol Metabolism in Wnt3a-treated Cells—Mouse F9 embryonic totipotent, teratocarcinoma cells stably transfected with the rat Fz1 display activation of the Wnt/-catenin/Lef/Tcf canonical pathway in response to treatment with Wnt3a, but not Wnt5a (9). More recently, we showed that Wnt3a activates CK2 and that this activation is obligate for Wnt3a stimulation of Lef/Tcf-sensitive gene transcription (23). Based upon the report that inositol phosphates (e.g. IP₄, IP₅, and IP₆) may regulate the activity of CK2 in vitro (25), we investigated the nature of water-soluble inositol polyphosphates accumulated in cells treated with Wnt3a for up to 60 min (Fig. 1A). F9 clones stably expressing either rat Fz1 or rat Frizzled-2 (Fz2) were labeled with [³H]inositol for 48 h and then treated with purified Wnt ligands for 0 to 60 min. F9 cells stably transfected with empty expressing vector (EV) were used as control cells. Anion exchange HPLC of either standard IP markers or water-soluble extracts from F9 cells expressing Fz1 and treated with Wnt3a (15 ng/ml) enabled analysis of the metabolism of inositol phosphates (Fig. 1A). The HPLC retention times, profiles, and the amplitude of the changes was highly reproducible under these conditions (not shown). Separation of inositol trisphosphate (IP₃), inositol 1,3,4,5-tetrakisphosphate (IP₄), inositol 1,3,4,5,6-pentakisphosphate (IP₅), and inositol hexakisphophate (IP₆) by anion exchange was excellent (Fig. 1, standards); IP₃ and IP₅ were readily identified in the cell extracts. In the absence of Wnt3a stimulation (+Wnt3a, at "0" min), the HPLC was able to detect IP₅. A slight elevation of IP₅ is present in the control (empty vector-transfected as well as wild-type) cells (data not shown). Thus, the baseline levels of IP₅ are not associated per se with expression of Fz1 in these cells. Within 10 min of Wnt3a treatment, the inositol phosphate profiles revealed increased IP₃ and IP₅ accumulation. Wnt3a stimulates an accumulation of IP₅ that reaches peak values within 15 min and declines thereafter. Quantification of intracellular IP₃, IP₄, and IP₅ in response to Wnt3a stimulation from multiple, separate preparations of [³H]inositol-labeled cells was performed to ascertain if the IP₃ response to Wnt3a was significant (Fig. 1B). The increase in IP₃ that peaks within 5 min was not significant, unlike the sharp increase in IP₅ accumulation that peaks within 15 min.

View larger version (24K): [in this window] [in a new window]   FIGURE 1. Activation of Frizzled-1 by Wnt3a increases intracellular IP5. A, F9 clones stably expressing rat Fz1 were labeled with [3H]inositol for 48 h as described under "Experimental Procedures." Cells were treated with Wnt3a (15 ng/ml) for the time periods indicated. Radiolabeled inositol phosphates were isolated from cell lysates and separated by strong anion HPLC. Representative chromatograms are shown. The retention times for IP3, IP4, IP5, and IP6 standards were determined and are displayed. B, a quantitative analysis of intracellular IP3, IP4, and IP5 in response to Wnt3a stimulation is shown. *, p < 0.01; **, p < 0.001, versus corresponding intracellular IPs measured at time 0. C and D, F9 clones stably transfected with EV, or expressing vector harboring either rat Fz1 or rat Fz2 were grown in 35-mm dishes and labeled with [3H]inositol for 48 h. Empty vector-transfected cells and F9 clones expressing Fz1 were stimulated by Wnt3a (15 ng/ml) for up to 60 min. Fz2-expressing clones were stimulated by Wnt5a (60 ng/ml) for up to 60 min. Cells were lysed at the indicated time periods and radiolabeled inositol phosphates were extracted and separated. C, radioactivity of [3H]IP5 extracted from samples was counted and a quantitative analysis of intracellular IP5 in response to Wnt stimulation is shown. *, p < 0.01; **, p < 0.001, versus the values obtained from empty vector-transfected cells stimulated with Wnt3a at time 0. D, radioactivity of [3H]IP3 extracted from samples was counted and a plot of intracellular IP3 in response to Wnt stimulation is shown. *, p < 0.01; **, p < 0.001, versus the values obtained from empty vector-transfected cells stimulating with Wnt3a at time 0. The results are displayed as mean ± S.E. obtained from at least three separate experiments. E, Wnt3a induces transient accumulation of intracellular IP5 in HEK cells. HEK cells transiently transfected with Fz1 were labeled with [3H]inositol for 48 h. Radiolabeled inositol phosphates were extracted and separated by HPLC. Representative chromatograms obtained from cells without or with Wnt3a (15 ng/ml) treatment for either 10 or 15 min are compared. The data presented are representative of at least three separate determinations performed with separate cell lysates.

View larger version (34K): [in this window] [in a new window]   FIGURE 2. Gq mediates the increase in intracellular IP5 in response to Wnt3a. F9 clones stably expressing the Fz1 and Super8X TOPflash (as a luciferase-coupled gene reporter to measure Lef/Tcf-sensitive transcription) were either treated with siRNA for 60 h to knockdown Gq, Go, or G11 (A-C) or transiently transfected with constitutively activated G proteins (D). A, representative immunoblots (IB) of G-protein subunits in cells treated with siRNA to individually knockdown Gq, Go, or G11. Cells were treated for 60 h with siRNA. The cells were lysed and subjected to SDS-PAGE and immunoblotting. The blots of resolved proteins were stained for each subunit. -Actin was probed as a loading control. B, clones treated with siRNA were stimulated by Wnt3a for 6 h and Lef/Tcf-sensitive luciferase reporter gene activity was assayed. C, F9 clones stably expressing the Fz1 were metabolically labeled with [3H]inositol for a total 84 h. Twenty-four h after the start of the incubation in the media containing [3H]inositol, cells were treated with siRNA reagents targeting Gq, Go, or G11 in the same media for 60 h. Cells were serum-starved for 8 h prior to stimulation for 15 min with Wnt3a (15 ng/ml). Labeled inositol polyphosphates were isolated and quantified. The IP5 levels were determined. Results are displayed as mean ± S.E., derived from at least three separate experiments. *, p < 0.01; **, p < 0.001; versus control siRNA-treated and Wnt3a-stimulated groups; setting the value of control siRNA-treated cells without stimulation as "1." D, F9 clones were metabolically labeled with [3H]inositol for 48 h. Twenty-four hours after the start of the incubation in the media containing [3H]inositol, clones were transiently transfected with one of following plasmids; expression vectors harboring Q209LGq (QLGq), or Q205LGo (QLGo), or Q209LG11 (QLG11) (all with an EE-tag). Cellular [3H]IP3 and [3 H]IP5 were isolated and compared for cells transfected with EV or one of the CA mutant G protein subunits. Results are displayed as mean ± S.E., derived from at least three separate experiments. *, p < 0.01; **, p < 0.001 for the difference between QL mutant-expressing and EV control cells. Representative immunoblots of EE-tagged QLGq, QLGo, and QLG11 expressed in these cells are shown. -Actin was probed as a loading control.

G_q Mediates the Increase in Intracellular IP₅ in Response to Wnt3a Stimulation—Fz1 has been shown to be a member of the superfamily of seven transmembrane segment receptors that operate via heterotrimeric G proteins, from the mouse (9) to flies (10). We investigated if the Wnt3a-stimulated, Fz1-mediated accumulation of IP₅ observed in the mouse F9 cells was sensitive to the suppression (knockdown) or expression of constitutively active mutants (Gln to Leu substitutions) of those G proteins shown to mediate the canonical pathway in mouse cells and flies (Fig. 2). Knockdown of the individual subunits of G_q and G_o (as well as G₁₁ as a control) was accomplished using siRNA. The extent to which the siRNA treatments yield an effective knockdown was established by subjecting a sample of the whole cell lysates to SDS-PAGE and the resolved gel to immunoblotting (Fig. 2A). siRNA designed for each of the G subunits was shown to specifically knockdown the targeted G protein subunit, but not non-targeted G protein subunits, following a 60-h treatment with siRNA. Immunoblotting of -actin provides a measure of equivalent loading, being unaffected by siRNA treatment. Treatment with the control siRNA designed and provided by the commercial supplier, likewise showed no ability to knockdown either the G protein subunits or the -actin.

We investigated first if the knockdown of G protein subunits influenced the ability of the Fz1-expressing F9 cells to respond to Wnt3a stimulation, measuring the activation of Lef/Tcf-sensitive transcription as the read-out (Fig. 2B). siRNA treatments of the cells with the control siRNA or siRNA targeting G₁₁ have no influence on the ability of the cells to activate Lef/Tcf-sensitive transcription in response to Wnt3a. Knockdown of G_q, in contrast, virtually abolishes the ability of these cells to activate the canonical Wnt pathway in response to purified Wnt3a. Similarly, knockdown of G_o led to suppression of the ability of Wnt3a to activate the canonical Wnt3a/-catenin/Lef-Tcf pathway, in good agreement with earlier studies in these cells (9) and in Drosophila (10). Having established the biology of the response and the ability of the knockdown of specific G protein subunits to suppress the response, we investigated the effects of knockdown of the -subunits of G_q, G_o, and G₁₁ on the accumulation of tritiated IP₅ from [³H]inositol-labeled cells (Fig. 2C). Knockdown of G_q abolishes the ability of Wnt3a to accumulate IP₅ in the Fz1-expressing cells. Knockdown of G_o, to a lesser extent than the knockdown of G_q, also suppresses the ability of Wnt3a to stimulate IP₅ accumulation. Knockdown of G₁₁, in contrast, has no effect on Wnt3a to stimulate the IP₅ response.

View larger version (19K): [in this window] [in a new window]   FIGURE 3. PLC1/PLC3 mediates Wnt3a-stimulated IP5 accumulation. A, RNA was isolated from F9 clones stably transfected with rat Fz1 and Super8X TOPflash without (upper panel) or with (lower panel) siRNA for 48 h. RT was employed followed by PCR amplification using primers specific for mRNA of each individual PLC isoform. Representative results of RT-PCR amplifications are displayed. Cyclophylin A mRNA was amplified as a control. B, Fz1-expressing cells were treated with siRNA for 48 h prior to the stimulation of Wnt3a (15 ng/ml) and luciferase reporter activity was assayed 6 h after the administration of Wnt3a. C, F9 clones stably expressing the Fz1 were metabolically labeled with [3H]inositol for 72 h. Twenty-four hours after the start of the incubation in the media containing [3H]inositol, cells were treated with either control siRNA or siRNA targeting both PLC1 and PLC3 in combination in the same media for 48 h. Cells were serum-starved for 8 h prior to stimulation with Wnt3a (15 ng/ml). Fifteen minutes after Wnt3a administration, inositol polyphosphates were isolated from whole cell extracts, separated, and quantified as described. The results are displayed as mean ± S.E., derived from at least three separate experiments. **, p < 0.001, versus Wnt3a-treated, control groups; #, p < 0.01, versus Wnt3a-treated cells pretreated with siRNA to PLC1 or PLC3, individually.

PLC Mediates Wnt3a-stimulated IP₅ Accumulation—The best known downstream effectors of G_q are members of the PLC -family of isoforms (36). To circumvent the absence of antibodies that could reliably stain the various isoforms of PLC expressed in these cells, we resorted to the use of RT-PCR with primers specifically designed to amplify the mRNAs for each of the mouse PLC isoforms to evaluate expression in F9 cells (Fig. 3A). PLC₁, PLC₃, and PLC₄ mRNAs are detected in the F9 cells, whereas PLC₂ is not. siRNA were designed to suppress each of the isoforms and cells were treated with an siRNA for 48 h prior to analysis of F9 cells transiently expressing Fz1 (Fig. 3, A and B). RT-PCR amplication of the mRNAs from the F9 cells was performed using primers for all three isoforms and RNA from cells pretreated with either control siRNA or an individual siRNA targeting a specific isoform. The results from the amplification indicate that the siRNAs designed for each isoform indeed were specific, reducing the amount of mRNA of a targeted isoform to almost below detection, whereas not significantly reducing the mRNA encoding the other isoforms or the cyclophilin A control (Fig. 3A). Assay of the ability of Wnt3a to activate Lef/Tcf-sensitive gene transcription was assayed in cells in which knockdown of one of three isoforms of PLC had been targeted (Fig. 3B). knockdown of either PLC₁ or PLC₃, but not PLC₄ provokes an attenuation of the ability of Wnt3a to activate gene transcription. When used in tandem, the siRNA targeting PLC₁ and PLC₃ in combination yield a profound suppression of Wnt3a action on the Lef/Tcf-sensitive transcription (Fig. 3B). Having established that both PLC₁ and PLC₃ were participating at downstream effects for Wnt3a activation of Frizzled-1, we probed if suppression of these two PLC isoforms would impact the ability of Wnt3a to stimulate the accumulation of IP₅ (Fig. 3C). Knockdown of PLC₁ and PLC₃, in tandem, resulted in substantial reduction in the amount of IP₅ accumulated in response to Wnt3a in the F9 cells expressing Fz1. The PLC inhibitor 1-octadecyl-rac-glycero-3-phosphocholine (ET-18-OH) also effectively blocks accumulation of inositol polyphosphates (not shown).

View larger version (36K): [in this window] [in a new window]   FIGURE 4. Wnt3a-induced accumulation of intracellular IP5 is derived from the action of both IP3K and IPMK. A, schematic diagram of proposed pathways for IP5 synthesis in vivo. Activation of PLC hydrolyzes PI(4,5)P2 and generates diacylglycerol (DAG) and inositol 1,4,5-P3. IP3K and IPMK convert inositol (1,4,5)P3 to inositol 1,3,4,5-P4 and inositol 1,4,5,6-P4, respectively. IPMK further converts both inositol 1,3,4,5-P4 and inositol 1,4,5,6-P4 to inositol 1,3,4,5,6-P5. Adriamycin inhibits IP3K, CGA inhibits IPMK, whereas ATA inhibits both IP3K and IPMK. B, inhibition of either IPMK or IP3K blocks Wnt3a-induced IP5 accumulation. Mouse F9 clones expressing Fz1 were metabolically labeled with [3H]inositol for 48 h. Clones were treated without or with ATA (2 µM), CGA (40 µM), or adriamycin (20 µM) for 30-60 min prior to the addition of the Wnt3a (15 ng/ml). Fifteen min after Wnt3a stimulation, intracellular IP5 accumulation was measured. The results are displayed as mean ± S.E., derived from at least three separate experiments. *, p < 0.001, versus correspondent control groups. C, suppression of IP3K and IPMK using siRNA reagents: analysis by immunoblotting (IB)(IP3K) and RT-PCR amplification (IMPK). -Actin was employed as a loading control for immunoblotting, whereas cyclophilin A was employed in the same set of samples amplified by RT-PCR. D, F9 clones expressing Fz1 were metabolically labeled with [3H]inositol for 72 h. Twenty-four hours after the start of the incubation in the media containing [3H]inositol, cells were treated with either control siRNA or siRNA reagents targeting either IPMK or IP3K-B, or siRNA targeting both IPMK and IP3K-B in combination. Cells were serum-starved for 8 h prior to stimulation for 15 min without or with Wnt3a (15 ng/ml). Intracellular accumulation of IP3 and IP5 were monitored by HPLC. The results are displayed as mean ± S.E., derived from at least three separate experiments. *, p < 0.001, versus correspondent, control siRNA-treated cells. #, p < 0.001, versus cells treated with the same siRNA in the absence of Wnt3a (-Wnt3a). &, p < 0.01, versus siRNA-treated cells in which either IPMK or IP3K-B alone were suppressed.

Beyond the use of enzyme inhibitors, we made use of siRNA to knockdown both IP3K and IPMK (Fig. 4C). IP3K has three isoforms in mammals, designated IP3K-A, -B, and -C (46). On the basis of RT-PCR amplification studies, all three are found to be expressed in mouse F9 cells (not shown). Treating cells with siRNA targeting the IP3K-B isoform effectively suppresses the expression of this enzyme, as shown by immunoblotting. Suitable antibodies to IP3K-A and -C isoforms were not available, but the siRNAs for each isoform were found to effectively suppress mRNA levels (not shown). Similar knockdown studies were performed targeting IPMK, again using RT-PCR amplification of mRNA in the absence of a suitable antibody for detection of IPMK by immunoblotting. The levels of IPMK mRNA in cells treated with the control siRNA versus the IPMK-targeting siRNA were established. The RT-PCR amplification data demonstrate effective suppression of IPMK mRNA through the use of these siRNA reagents (Fig. 4C). Having established conditions directed toward knockdown of both enzymes, we evaluated the ability of siRNA-mediated suppression to modulate the IP₃ and IP₅ response to Wnt3a stimulation of the canonical pathway (Fig. 4D). In the absence of stimulation by Wnt3a, cells display little change in intracellular IP₃ or IP₅ levels when IPMK is suppressed by siRNA. Earlier studies report dramatic reductions in IP₅ accumulation in HEK293 and HeLa cells (45), as well as in Rat-1 cells (38), when IPMK expression was suppressed with siRNA. Unlike these earlier studies performed with serum-fed cells, our studies required that we eliminate serum to study the Wnt response, likely the basis for the differences in the effects observed in response to IPMK suppression.

Suppression of IP3K-B (Fig. 4D), but not that of either IP3K-A or IP3K-C (not shown), leads to an increase in IP₃ (likely a reflection of blocked utilization of IP₃), but not IP₅. Suppression of both IP3K-B and IPMK in tandem yields a reduction in intracellular IP₅, as predicted (Fig. 4D). The ability of the suppression of IP3K-B to increase the intracellular IP₃ level is amplified in cells treated with as compared without Wnt3a (Fig. 4D). To provide independent verification of the ability of the siRNA reagents for IP3K-B and IPMK to block IP₅ accumulation, we assayed the effects of these same siRNA reagents on IP₃, IP₄, and IP₅ levels in serum-starved F9 cells that were metabolically labeled and then stimulated with serum for 10 min (see supplemental data Fig. S3). The results demonstrate that knock-down of either enzyme blocks IP₅ accumulation in response to an independent agonist (i.e. serum). Wnt3a stimulates a marked increase in the accumulation of IP₅ in the cells treated with control siRNAs. Suppression of either IP3K or IPMK, or both enzymes, substantially reduces the ability of Wnt3a to stimulate IP₅ accumulation. These data, taken together, suggest that the source of the IP₅ generated in response to Wnt3a includes roles of both IP3K and IPMK.

IPMK and IP3K Are Essential for Wnt3a/-Catenin Canonical Signaling to PE Formation—It was essential to probe further downstream in the Wnt/-catenin pathway, to establish the linkage between IPMK/IP3K and changes in IP₅ extended to the regulation of intracellular accumulation of -catenin, activation of Lef/Tcf-sensitive gene transcription, and PE formation. At the level of -catenin accumulation, treatment of cells with inhibitors, either adriamycin (IP3K-selective) or ATA (inhibits both IP3K and IPMK) attenuates Wnt3a action (Fig. 5A). The Lef/Tcf-sensitive transcriptional response to Wnt3a stimulation is similarly sensitive to each of these inhibitors. A brief treatment with adriamycin, CGA, or ATA essentially abolishes the ability of Wnt3a to signal to the level of gene transcription (i.e. Lef/Tcf-sensitive gene activation) providing data in good agreement with the results from study of -catenin accumulation (Fig. 5B). Treatment of cells with siRNA targeting either IP3K-B or IPMK provokes a substantial, but not complete, reduction in the ability of Wnt3a to activate Lef/Tcf-sensitive gene transcription (Fig. 5C). Pretreating the Fz1-expressing cells with siRNAs that target both IP3K and IPMK in combination achieved greater suppression of the Wnt3a-stimulated gene activation than does either siRNA treatment alone (Fig. 5C).

Knockdown of IPMK suppresses -catenin accumulation and Lef/Tcf-sensitive gene transcription in response to Wnt3a stimulation, prompting the query, what effect might overexpression of IPMK have on the transcriptional response? Clones expressing Fz1 were transiently transfected with expression vector harboring a Myc-tagged version of IPMK, enabling assay of the level of relative expression by immunoblotting (Fig. 5D). Analysis of Wnt3a-stimulated activation of Lef/Tcf-sensitive reporter gene activity reveals increasing reporter gene activity in response to Wnt3a as the level of exogenous IMPK increases (Fig. 5D). Likewise, the knockdown of IPMK provoked by siRNA treatment targeting this enzyme blunts Wnt3a action at this level; expression of the exogenous IPMK "rescues" the Wnt response (Fig. 5E). Finally, if the sources of IP₅ involve IP3K and IPMK and the accumulation of IP₅ is essential to the operation of the Wnt/-catenin canonical signaling pathway, one would predict that suppression of the expression of IPMK and IP3K-B would suppress not only IP₅ accumulation and gene reporter activity, but also the ability of Wnt3a to promote the formation of PE (Fig. 5F). We tested this hypothesis, making use of the monoclonal antibody to stain for the expression of the PE marker protein TROMA-1 antigen (Cytokerotin Endo A). In the absence of Wnt3a stimulation, TROMA-1 antigen is not present, because of the embryonic character of these cells (Fig. 5F). After treatment of the Fz1-expressing cells with Wnt3a (15 ng/ml) and a 4-day period post-treatment, positive staining of the cells demonstrates Wnt3a-induced PE formation. In those cells treated with siRNA targeting both IPMK and IP3K in combination, Wnt3a stimulates little IP₅ accumulation (Fig. 4D), little Lef/Tcf-sensitive gene transcription (Fig. 5C), and virtually no PE formation (Fig. 5F).

IP₅ Enhances CK2 Activity—We sought to explore the link between Wnt3a-stimulated accumulation of IP₅ and the activation of CK2, following up earlier reports that inositol polyphosphates can regulate the activity of CK2 in vitro.We tested the effects of specific enzyme inhibitors that regulate the Wnt3a-stimulated accumulation of IP₅ on the activity of CK2 in Fz1-expressing F9 cells (Fig. 6). The following enzymes were targeted with inhibitors: PLC (1-octadecyl-rac-glycero-3-phosphocholine, ET-18-OH), IPMK (CGA), and IP3K/IPMK (ATA). Cells were treated with the inhibitors for 30 min prior to stimulation with purified Wnt3a (Fig. 6A). As reported earlier (23), Wnt3a stimulates CK2 activity in F9 cells expressing Fz1 (Fig. 6A). Inhibition of the generation of IP₅ at the levels of PLC, IP3K, or IPMK abolishes the ability of Wnt3a to increase CK2 activity (Fig. 6A). In vitro experiments were performed in parallel, making use of CK2 pulled-down from whole cell lysates and assayed for the effects of various inositol polyphosphates on CK2 activity (Fig. 6B). IP₄, IP₅, and IP₆, but not IP_3, stimulated increased activity of CK2 (Fig. 6B), confirming earlier studies (25). Finally, we examined the concentration dependence of the activation of CK2 by IP₅ (0-80 µM, Fig. 6C). Increasing concentrations of IP₅ stimulates a concentration-dependent increase in CK2 activity. Half-maximal stimulation of CK2 activity is observed at 10 µM IP₅.

IP₅ Suppresses the Activity of GSK3—To complete our analysis of likely targets for regulation by inositol polyphosphates, we performed experiments parallel to those performed on CK2, only targeting instead GSK3. GSK3 plays a pivotal role in the Wnt canonical pathway (48-50). Although there was no indication in the literature that GSK3 was regulated by inositol polyphosphates, we tested this possibility experimentally. Wnt3a acts to inhibit GSK3, the enzyme responsible for the phosphorylation of -catenin, catalyzing a pathway culminating in the destruction of the phospho--catenin (51). Treating the Fz1-expressing F9 cells with Wnt3a suppresses the activity of GSK3 (Fig. 7A). Treating the cells with inhibitors of PLC (ET-18-OH), IPMK (CGA), and IP3K/IPMK (ATA) prior to treatment with Wnt3a abolished the ability of Wnt3a to inhibit GSK3 activity (Fig. 7A), much as these inhibitors abolished the ability of Wnt3a to stimulate CK2 activity (Fig. 6A).

View larger version (37K): [in this window] [in a new window]   FIGURE 5. Signaling in Wnt canonical pathway to -catenin, gene transcription, and PE formation. F9 clones expressing the Fz1 and Super8X TOPflash were stimulated by Wnt3a. Lef/Tcf-dependent transcription (B-E) and accumulation of cytoplasmic -catenin (A) were measured. The results are displayed as mean ± S.E., obtained from at least three separate experiments. Endoderm formation (F) was analyzed at day 4 after Wnt3a treatment. A, effects of pretreating cells with either ATA or adriamycin (as described in the legend to Fig. 4) on the accumulation of cytoplasmic -catenin in the absence versus the presence of Wnt3a. B, clones pre-treated with ATA, CGA, and adriaymycin were stimulated without or with Wnt3a and the Lef/Tcf-sensitive luciferase reporter activity determined. **, p < 0.001, for the difference from cells treated only with Wnt3a (no inhibitor). C, clones were treated with siRNA for 48 h to knockdown either IPMK or IP3K-B, or both prior to Wnt3a stimulation. The Lef/Tcf-sensitive luciferase reporter activity was measured. **, p < 0.001, versus cells treated with Wnt3a only, not treated with siRNAs targeting IP3K, IPMK, or both in tandem. #, p < 0.01, versus cells treated with either "siRNA to IP3K-B" alone or "siRNA to IPMK" alone. D, Fz1-expressing cells were transiently transfected for 24 h with increasing amounts of plasmid harboring Myc-tagged IPMK as indicated. Clones were stimulated by Wnt3a and Lef/Tcf-sensitive luciferase transcriptional activity was measured. The expression of Myc-tagged IPMK in transfected clones was analyzed by immunoblotting (IB) with anti-Myc antibody (lower panel). *, p < 0.01; **, p < 0.001, for the difference from Wnt3a-treated control cells. E, Fz1-expressing cells were treated with siRNA for 24 h to knockdown IPMK. These knockdown cells were transiently transfected to express exogenous, Myc-tagged IPMK. Cells were stimulated without or with Wnt3a and the Lef/Tcf-sensitive luciferase reporter gene activity measured. *, p < 0.01; **, p < 0.001 versus Wnt3a-treated control cells. F, cells were treated with either control siRNA or siRNAs targeting IPMK as well as IP3K-B in combination for 48 h. Cells then were treated without or with Wnt3a (15 ng/ml) for 4 days and the formation of PE examined by immunostaining of the fixed cells with TROMA-1 antibody, a secondary fluorescently labeled antibody, in tandem with indirect immunofluoresence. PC, phase-contrast. IIF, indirect immunofluorescence. Bar, 20 µm.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Metabolism of membrane-anchored phosphatidylinositol 4,5-bisphosphate by PLC yields the second messengers diacylglycerol and a water-soluble 1-myo-inositol polyphosphate IP₃. PLC is an effector for many members of the superfamily of G protein-coupled receptors (36). Frizzleds display seven transmembrane segments, N-terminal sequences on the exofacial side of the cell membrane, and C-terminal sequences on the cytoplasmic face of the cell membrane where they can act as substrates for various protein kinases involved in signaling. Despite the level of homology that exists between Frizzleds and other members of the G protein-coupled receptors, it was only recently demonstrated that Frizzleds are bona fide G protein-coupled receptors themselves (7). Genetic evidence from Drosophila (10) and biochemical data from mammalian cells (9) demonstrate the essential role of heterotrimeric G proteins in coupling Frizzleds to their effectors in Wnt signaling pathways, including the Wnt/-catenin canonical pathway (9), the non-canonical Wnt/cGMP, Ca²⁺ pathway (35, 52), and planar cell polarity (10, 53, 54). In the current work, we show that Wnt/-catenin signaling mediated by Frizzled-1 provokes the breakdown of phosphatidylinositol 4,5-bisphosphate and elevation of intracellular levels of IP₅.

View larger version (13K): [in this window] [in a new window]   FIGURE 6. IP5 enhances CK2 activity in vitro. A, F9 clones expressing Fz1 were pre-treated with inhibitors to PLC (ET-18-OH), IPMK (CGA), or IPMK and IP3K (ATA) for 20 min prior to the stimulation by Wnt3a. Following immunoprecipitation of CK2, CK2 activity was measured by in vitro kinase assay as described under "Experimental Procedures." The results are displayed as mean ± S.E., derived from at least three separate experiments. *, p < 0.001 versus untreated control cells; #, p < 0.001 versus Wnt3a-treated control cells. B and C, the CK2 was immunoprecipitated from whole cell lysates prepared from wild-type F9 cells. The immune complex was resuspended in kinase reaction buffer and aliquots with equal volumes were made. Kinase reactions were conducted in the absence (-) or presence of 20 µM of inositol phosphates (IP3, IP4, IP5, or IP6 as indicated) (B), or in the presence of increasing concentrations of IP5 as indicated (C). CK2 activities were compared and the CK2 activity obtained from samples in the absence of inositol phosphates was set as 1. *, p < 0.01; **, p < 0.001 versus samples untreated with inositol polyphosphates.

It was an earlier observation from in vitro studies of CK2 activity (23, 25) that prompted us to explore the effects of Wnt3a on phosphatidylinositol signaling in F9 cells, cells amenable to biochemical manipulation and analysis. We observed that Wnt3a stimulates a dose-dependent and time-dependent accumulation of IP₅ in cells, a process that could be blocked by enzyme inhibitors at the level of PLC, IP3K, and IPMK. How then, we asked, is the accumulation of IP₅ related to the output of the Wnt canonical pathway? IP₃ has a remarkable and diverse set of functions in cell signaling and physiology (55, 56); far less is known about the functions of IP₅. Accumulation of inositol polyphosphates has been shown to function in cell proliferation, apoptosis, differentiation (57-59), and control of left-right symmetry in zebrafish development (60). The current work expands upon these earlier observations, providing a demonstration that Wnt canonical signaling includes phosphatidylinositol signaling to the level of IP₅.

View larger version (13K): [in this window] [in a new window]   FIGURE 7. IP5 suppresses GSK3 activity in vitro. A, F9 clones expressing Fz1 were pre-treated with inhibitors to PLC (ET-18-OH), IPMK (CGA), or IPMK and IP3K (ATA) for 20 min prior to the stimulation by Wnt3a. GSK3 was immunoprecipitated from whole lysates and its activity was measured by in vitro kinase assay as described under "Experimental Procedures." *, p < 0.001 versus untreated, control cells; #, p < 0.001 versus Wnt3a-treated control cells. B and C, the GSK3 was immunoprecipitated from whole cell lysates prepared from wild-type F9 cells. The immune complex was resuspended in kinase reaction buffer and aliquots with equal volumes were made. B, kinase reactions were conducted in the absence (-) or presence of 20 µM inositol phosphates (IP3, IP4, IP5, or IP6 as indicated). C, kinase activity was measured in the presence of increasing concentrations of IP5 as indicated. GSK3 activity obtained from untreated control cells was set as 1. **, p < 0.001, versus samples untreated with inositol polyphosphates.

View larger version (29K): [in this window] [in a new window]   FIGURE 8. IP5: pivotal regulation of GSK3 and CK2 in Frizzled1/-catenin/Lef-Tcf signaling pathway. Wnt3a binds to Frizzled1 (FZ1), a member of the superfamily of G protein-coupled receptors, and thereby activates Gq, which in turn activates PLC. Activation of PLC leads to hydrolysis of phosphatidylinositol 4,5-bisphosphate and generates IP3 and diacylglyceride (DAG). IP3 is quickly converted to IP5, the combined action of IPMK and IP3K, and does not appreciably bind to the IP3 receptor altering intracellular Ca2+. Increase of intracellular IP5 accumulation stimulated (indirectly) the activation of CK2 while inhibiting (indirectly) GSK3 activity. Wnt-stimulated -catenin stabilization and Lef/Tcf-sensitive transcriptional activation by Wnt3a are dependent upon the accumulation of IP5. KD, knock down by siRNA; IP3R, IP3 receptor.

This work reveals several novel aspects of Wnt canonical signaling. Our studies are the first to show the existence of a Wnt/-catenin "canonical" pathway regulating phosphatidylinositol signaling. Wnt5a regulation of phosphatidylinositol signaling via a Frizzled-2 mediated "non-canonical" pathway was reported earlier (34, 47). IP₅ accumulates in response to Wnt3a, whereas IP₃ accumulates in response to Wnt5a signaling. Not only does IP₅ accumulation occur in response to Wnt3a, but it also is an essential component of signaling of the canonical pathway to the level of -catenin accumulation, Lef/Tcf-sensitive transcription, and PE formation in F9 cells. Finally, we show that IP₅ plays a critical role at the level of CK2 and GSK3, stimulating the former while inhibiting the latter. Thus, phosphatidylinositol signaling is essential to Wnt signaling via the canonical pathway.

	FOOTNOTES

 
^* This work was supported by Grant GM 069375 from the NIGMS, National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1-S4.

¹ To whom correspondence should be addressed. Tel.: 631-444-3489; Fax: 631-444-3432; E-mail: wangh{at}pharm.stonybrook.edu.

² The abbreviations used are: GSK3, glycogen synthase kinase 3;IP₃, inositol trisphosphate; IP₄, inositol 1,3,4,5-tetrakisphosphate; IP₅, inositol 1,3,4,5,6-pentakisphosphate; IP₆, inositol hexakisphosphate; CA, constitutively active; IP3K, inositol 1,4,5-trisphosphate 3-kinase; ATA, aurintricarboxylic acid; CGA, chlorogenic acid; PE, primitive endoderm; RT, reverse transcription; PI(4,5)P₂, phosphatidylinositol 4,5-bisphosphate; Lef, lymphoid enhancer factor; Tcf, T-cell factor; CK2, casein kinase 2; PLC, phospholipase C; IPMK, inositol polyphosphate multikinase; siRNA, small interfering RNA; HPLC, high performance liquid chromatography; EV, expressing vector; MOPS, 4-morpholinepropanesulfonic acid; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; HA, hemagglutinin; Fz1, Frizzled1; HEK, human embryonic kidney.

	ACKNOWLEDGMENTS

 
We thank Dr. Randall Moon (HHMI, Department of Pharmacology, University of Washington, Seattle, WA) for generous gifts of reporter plasmid Super8X TOPflash and mutated reporter plasmid Super8X FOPflash. We thank Dr. Mario Rebecchi (Department of Anesthesiology, SUNY at Stony Brook) for the Partisil 10-SAX HPLC column used in analysis of inositol polyphosphates.

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