To understand whether extracellular calcium-sensing receptor (CaSR) expression on colonic myofibroblast cells (18Co) contributed to epithelial homeostasis, we activated the CaSR with 5 mM Ca2+, screened by RT-PCR Wnt family members, and measured their secretion. Transcripts for Wnt 1, 2, 2b, 3a, 4, and 7a were either absent or unchanged whereas Wnt3 decreased and Wnt5a increased. We assessed Wnt5a secretion by Western blot. High Ca2+ (5 mM) substantially increased Wnt5a secretion; small interfering RNA (siRNA) against the CaSR reduced this to constitutive amounts. Expression of Wnt5a plasmid but not Wnt1 or Wnt3a increased caudal homeodomain factor CDX2 transcripts and protein in HT-29 adenocarcinoma cells. Wnt5a increased activity of a sucrase-isomaltase (SI) promoter in Caco-2BBE cells. Wnt5a protein stimulation of CDX2 transcripts and protein and SI reporter were increased by overexpression of wild-type Ror2, a Wnt5a receptor, and reduced with siRNA against Ror2. CaSR activation of HT-29 cells increased Ror2 protein expression. Ror2 protein was expressed in mouse jejunum from crypt base to villus tip and in the colon on surface epithelia. Our results show that activation of a G protein-coupled receptor, the CaSR, stimulates secretion of Wnt5a from myofibroblasts. Stimulation of epithelia by the CaSR increased the expression of a receptor for Wnt5a, the tyrosine kinase Ror2, suggesting existence of a unique paracrine relationship for CDX2 homoeostasis in the intestine and revealing new contributions of CaSR-activated myofibroblasts to intestinal stem cell niche microenvironments.
- extracellular calcium-sensing receptor
wnt proteins are secreted cysteine-rich, palmitoylated glycoproteins that signal by interacting with coreceptors Frizzled and Lipoprotein-related peptide 5/6 (LRP5/6) to liberate β-catenin from its destruction complex and activate transcription of specific target genes (25, 39, 65). Intestinal homeostasis is critically dependent on Wnt signaling as demonstrated by experiments showing that transgenic expression of a Wnt inhibitor results in the loss of crypts or mice with constitutively stabilized β-catenin develop adenomas (19). Wnt signaling is a key regulatory pathway required for intestinal stem cell self-renewal and differentiation (68, 81), but there is little direct knowledge of how intestinal stem cell niches may be altered by G-protein receptor activation (58). Colon cancer is a disease of defective Wnt signaling (69, 76), yet there is little information about the molecular determinants of Wnt protein secretion or events that regulate expression of Wnt receptors in the normal intestine.
Because Ca2+ is a chemoprotective agent for colon cancer (4, 32, 47, 70), and the extracellular calcium-sensing receptor (CaSR) is expressed on the apical and basolateral membrane of colonic crypt epithelia (13, 15, 17, 24), we previously determined how CaSR activation of colon cancer cell lines influenced defective Wnt signaling (49). It was found that CaSR activation of cells with a truncated adenomatous polyposis coli (APC)-stimulated secretion of Wnt5a. The secreted Wnt5a worked in an autocrine manner to inhibit β-catenin signaling by increasing production of the E3-type ubiquitin ligase, Siah2. However, the overexpression of full-length APC prevented colon cancer cells from secreting Wnt5a after CaSR activation (49). Wnt5a protein expression in primary Dukes B colon cancer (22) is associated with a good prognosis, so understanding events that increase this protein's presence may be clinically relevant. In normal intestine Wnt5a expression is found in the stroma (26). Wnt5a is known to interact with several Frizzled receptors (2, 4, 5, and 7) as well as the orphan tyrosine kinase receptor Ror2 (18, 48, 55, 59, 74). Colonic myofibroblasts lie along the basement membrane and are physically poised to signal to the epithelia (63). It has been shown that the CaSR is expressed on colonic myofibroblasts and its activation will trigger secretion of bone morphogenetic protein (BMP)-2 (60). Therefore, we sought to determine whether CaSR stimulation of these myofibroblasts influenced Wnt5a homeostasis.
The transcription factor that plays a crucial role in the determination of the intestine is the homeobox gene CDX2, a homeodomain protein related to the Drosophila caudal gene (72). CDX2 stimulates differentiation and expression of sucrase-isomaltase (SI), lactase-phlorizin hydrolase, LI-cadherin, and Muc2 in intestinal epithelial cells (12, 31, 71, 79). BMP will increase CDX2 transcripts and protein in gastric cell lines (5). CDX2 expression is retained in the intestinal epithelium throughout adulthood but becomes reduced in colon cancer (21, 27, 52, 71). Reduced levels of CDX2 in Cdx2+/− mice result in increases in colon tumor progression (1, 10). Nevertheless, very little is known about extracellular determinants that can regulate expression of CDX2 transcript and protein in the intestine.
In the experiments described herein we show that activating the CaSR by Ca2+ or other recognized polyvalent agonists stimulated the secretion of Wnt5a from 18Co colonic myofibroblasts. This calcium-mediated secretion could be blocked by small-interfering RNA (siRNA) duplex against the CaSR. The secreted Wnt5a was biologically active in inhibiting β-catenin signaling in colon cancer cells with wild-type β-catenin and overexpressing full-length APC. Furthermore, Wnt5a transfection or recombinant Wnt5a protein addition to model epithelial cells increased synthesis and protein of the caudal homeodomain factor CDX2 as well as the activity of a SI promoter reporter. CaSR stimulation of colonic cancer cells increased expression of Ror2 protein, an orphan tyrosine kinase recently shown to work as a receptor for Wnt5a. Ror2 was expressed along the crypt-villus axis in mouse small intestine and only on surface epithelia of the colon. The effect of Wnt5a on CDX2 protein expression and was increased with Ror2 overexpression and attenuated with Ror2 knockdown. This CaSR-mediated Wnt5a/Ror2 axis may contribute to CDX2 homoeostasis in the adult intestine. CaSR-activation of colonic myofibroblasts generates a noncanonical Wnt signaling cascade that may enhance epithelial differentiation.
MATERIALS AND METHODS
Wnt-5a antibody was obtained from R&D systems (Minneapolis, MN). Monoclonal anti-β-actin was from Sigma (St. Louis, MO). Anti-Ror2 rabbit polyclonal antibody was purchased from Cell Signaling Technology (Boston, MA). A mouse monoclonal CDX2 antibody was obtained from Biogenex Laboratories (San Ramon, CA). Cell culture media McCoy's 5A medium modified and Dulbecco's modified Eagle's medium (DMEM), with or without calcium, were obtained from GIBCO-BRL (Grand Island, NY). The enhanced chemiluminescence supersignal kit was from Pierce (Rockford, IL). Protease inhibitors were from Boehringer Ingelheim.
The colonic myofibroblast cells CCD-18Co (18Co) was purchased from the American Type Culture Collection (ATCC; Rockville, MD). Primary cultures of human colonic myofibroblasts (hCMF) immortalized by telomerase were a generous gift of R. Mifflin (University of Texas Medical Branch, Galveston, TX). 18Co and hCMF cells were grown in minimum essential medium as previously described (60). The human colon adenocarcinoma Caco-2BBE and HT-29 cell lines were purchased from the ATCC. The HT-29-APC (APC inducible) was kindly provided by B. Vogelstein (The Johns Hopkins School of Medicine; Baltimore, MD). HT-29 and Caco-2 cells were cultured in DMEM supplemented with 10% or 20% FBS, respectively, and penicillin 100 μU/ml and were grown at 37°C in a humidified 5% CO2 atmosphere. The HT-29-APC line was cultured in McCoy's 5A medium modified supplemented with 10% FBS and penicillin 100 μU/ml. Cells were passaged weekly with 0.25% trypsin and used for experimentation within the first six passages as previously described (49).
siRNA duplex generation and transfection.
siRNA duplexes against the CaSR were synthesized following the protocol we previously reported (49, 60). Briefly, after BLAST analysis of target sites in the extracellular domain of the CaSR, we synthesized siRNA directed against nucleotides 371–390 utilizing an siRNA construction kit (Silencer siRNA construction kit, Ambion) according to the manufacturer's protocol. CCD-18Co cells were transfected with either 50 nM of siRNA against the CaSR or the scrambled sequence by using Superfect reagent following manufacturer's instructions (Qiagen, Valencia, CA). For altering Ror2 concentrations we used a commercially available siGENOME SMART pool set of four siRNA duplexes with a corresponding scrambled pool as our nonspecific control, synthesized by Dharmacon. We first tested the efficiency to suppress Ror2 concentration using RT-PCR, then Western blots in HT-29 cells after transfection with 10–200 nM of each pool, using Superfect as the transfection reagent. Maximal reduction of Ror2 protein, 48 h after transfection, was observed at 200 nM of the siRNA duplex compared with the scrambled control.
Transient transfections and luciferase reporter assays.
HT-29 and Caco-2 cells were seeded at equal amounts into 24-well tissue culture plates and grown in DMEM for 24 h. The cells were 50–60% confluent at the time of transfection. Cells were transfected with plasmid DNAs by using superfect transfection reagent (Qiagen, Mississauga, ON, Canada) according to manufacturer's recommendations. At least three different experiments were performed in triplicates. The following amounts of plasmids were used per well of a 24-well plate: 0.5 μg SI-Luc (N. Rivard, University of Sherbrooke, Sherbrooke, QC, Canada); 0.6, 1.0, 1.2, 1.8 μg Wnt5A, and 2 ng pRL-SV40 (Promega, Madison, WI). The control plasmid phRL-SV40 was used as internal control for transfection efficiency of SI promoter reporter. Eighteen hours posttransfection, cells were incubated in serum-free, Ca2+-free DMEM containing 4 mM l-glutamine, 0.2% BSA, penicillin 100 μU/ml, and 0.5 mM CaCl2 for 8 h. This medium was removed and substituted with the same medium supplemented with 0.5 mM extracellular Ca2+ (Ca2+o) for another 18 h. Cells were harvested by lysing in 100 μl of reporter lysis buffer (Promega). For TOPflash reporter assays, HT29-APC were transfected with 1 μg TOPflash and 2 ng phRL-null, and were cultured in the presence of 100 μM zinc for up to 18 h to induce full-length APC expression in the cells. Luciferase activity was measured via a Lumat LB9507 luminometer (Berthold Technologies, Bad Wilbad, Germany).
Differential gene expression analysis was performed by semiquantitative PCR using a Mastercycler (Eppendorf, Hamburg, Germany). Total RNA of Co18 cells was isolated by the TRIzol reagent by following manufacturer's instructions. The recovered RNA was quantitated by spectrophotometry, and aliquots of 1 μg total RNA from low Ca2+o (0.5 mM) or high Ca2+o (5 mM), or cells treated with different CaSR agonists were used for cDNA synthesis at 37°C for 1 h in a volume of 20 μl containing the following reagents: 5 mM dNTP mix; 10 μM oligo(dT) primer; 10 U RNase inhibitor; 4 U Omniscript reverse transcriptase; 1 × buffer RT (all from Qiagen). The PCR reaction was prepared in a volume of 50 μl containing the following reagents: 0.2 mM dNTP mixture; 1.5 mM MgCl2; 1 μl template cDNA; 1.0 U platinum Taq DNA polymerase (Invitrogen, Carlsbad, CA). The PCR cycle conditions were initial denaturation at 94°C for 60 s followed by 30 amplification cycles denaturing at 94°C for 30 s, annealing at an amplicon dependent temperature for 30 s and elongation at 72°C for 1 min, followed by a final elongation at 72°C for 10 min. Primers for CaSR were 5′ CGG GGT ACC TTA AGC ACC TAC GGC ATC TAA 3′, antisense 5′ GCT CTA GAG TTA ACG CGA TCC CAA AGG GCT C 3′; the sequences used for CDX2 were sense 5′ AGA CCA ACA ACC CAA ACA GC 3′, antisense 5′ GTC ACC AGA GCT TCT TCT CTG GG 3′; the sequences used for Wnt1 were sense 5′ ACC CAA TCC CTC TCC ACT CT 3′, antisense 5′ GAT TCA AGG AAA AGC CAC CA 3′; the sequences used for Wnt2 were sense 5′ GTG GAT GCA AAG GAA AGG AA 3′, antisense 5′ CCC AGC CAG CAT GTC CTG AGA GT 3′; the sequences used for Wnt2b were sense 5′ GTG TCC TGG CTG GTT CCT TA 3′, antisense 5′ AGC TGG TGC AAA GGA AAG AA 3′; the sequences used for Wnt3 were sense 5′ TGT GAG GTG AAG ACC TGC TG 3′, antisense 5′ AAA GTT GGG GGA GTT CTC GT 3′; the sequences used for Wnt3a were sense 5′ GGA CAA AGC TAC CAG GGA GT 3′, antisense 5′ ACT CGA TGT CCT CGC TAC AG 3′; the sequences used for Wnt4 were sense 5′ CTC ATG AA C CTC CAC AAC AA 3′, antisense 5′ GCA CCA TCA AAC TTC TCC TT 3′; the sequences used for Wnt5a were sense 5′ TGT GTG CAA GTA GTG GGT GC 3′, antisense 5′ AAT ATT TTT CGT GGT TCC CAC C 3′; the sequences used for Wnt6 were sense 5′ GTC ACG CAG GCC TGT TCT AT 3′, antisense 5′ CGT CCA TAA AGA GCC TCG AC 3′; the sequences used for Wnt7a were sense 5′ TCT CAT GAA CTT GCA CAA CA 3′, antisense 5′ ACT TGT CCT TGA GCA CGT AG 3′. The resulting bands were visualized on a 1.0% agarose gel stained with ethidium bromide and compared with a 100-bp DNA ladder (Invitrogen) to confirm the predicted size. For positive amplification control we used the GAPDH gene, sense 5′ TTA GCA CCC CTG GCC AAG G 3′, antisense 5′ CTT ACT CCT TGG AGG CCA TG 3′.
Ror2 immunocytochemistry and immunohistochemistry.
HT-29 cells were cultured on coverslips for 24 h and then serum starved overnight in DMEM-0.2% BSA containing 0.5 mM Ca2+o, before Ca2+o challenge with 0.5, or 5 mM Ca2+o for 18 h. Cells were fixed with 4% formaldehyde for 20 min, washed three times with PBS, and blocked with 1% goat serum in PBS-0.2% Tween 20. Mouse small intestine and colon frozen sections were fixed at 4°C in Zamboni's fixative (4% paraformaldehyde, 0.2% picrinic acid in 0.1 M PBS) and processed for immunofluorescent analysis. Frozen sections of mouse jejunum or proximal colon were blocked with 5% goat serum in PBS-0.2% Tween 20 at room temperature for 1 h and then incubated overnight in a humidifying chamber at 4°C with 1:50 rabbit polyclonal antibody against Ror-2 (cat. no. 4105; Cell Signaling) diluted in antibody dilution fluid (ADR, Dako; Mississauga, ON, Canada). After being washed three times with PBS, tissue sections and coverslips were incubated for 2 h with 1:1,000 goat anti-rabbit in PBS-0.2% Tween 20 linked to Alexa 555 (Invitrogen, Burlington, ON, Canada). Negative controls were performed with mismatched secondary antibody. Hoechst counterstain was routinely used to screen for nuclear colocalization. Specimens were photographed by using an inverted fluorescence microscope (Olympus IMT-2, Markham, ON, Canada) and were analyzed with Image Pro-plus V6.0 software (Media Cybernetics, Silver Spring, MD).
Isolation of epithelia from mouse jejunum and colon by a perfusion-shear protocol.
We modified established protocols to generate intact epithelia with reduced mesenchymal contamination. Following cervical dislocation, the abdominal cavity of a mouse was opened and the jejunum, ileum, or proximal colon was flushed with saline (37°C) containing 0.5 mM dithiothreitol. The segment was then perfused with 5 ml of 30 mM EDTA in calcium-and magnesium-free Hanks' balanced salt solution (HBSS) (37°C) that was gassed with O2 with pH 7.4. Segments are then removed and perfused twice with 5 ml of the EDTA HBSS that were pooled and collected by centrifugation. Microscopic examination demonstrated sheets of detached epithelia. These collected sheets were resuspended in lysis buffer for Western blot analysis.
Wnt5a Western blotting.
For the determination of Wnt5a from 18Co cells, early passage cells were grown on 100-mm dishes. Cells were incubated for 18 h in serum-free, Ca2+-free DMEM (as above). This medium was removed and substituted with the same medium supplemented with 0.5 mM Ca2+ or 5 mM Ca2+, or 0.5 mM Ca2+ with different CaSR agonists or Gq/11 agonists for 18 h. At the end of the incubation period (18 h), conditioned medium was harvested and concentrated to 16× by Amicon Ultra filter devices (Millipore, Carrigtwohill, Ireland). Cells were washed twice with ice-cold PBS containing 1 mM sodium vanadate and 25 mM NaF, and then 100 μl of ice-cold lysis buffer was added (20 mM Tris·HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 25 mM NaF, 1% Triton X-100, 10% glycerol, 1 mM dithiothreitol, 1 mM sodium vanadate, 50 mM glycerophosphate), and a cocktail of protease inhibitors (10 μg/ml each of aprotinin, leupeptin, calpain, pepstatin, and soybean trypsin inhibitor as well as 100 μg/ml Pefabloc). NaF, sodium vanadate, and Pefabloc were freshly prepared on the same day of the experiments. After sonicating for 5 s, lysates were centrifuged at 10,000 g for 10 min at 4°C and processed as described (37). Equal amounts of protein from whole cellular lysates and conditioned medium were resolved on a gradient gel of 4–20% and were electrophoretically transferred to Immobilon membranes. Wnt5a was detected by using a 1:300 dilution of primary antibody in TBS containing 0.1% Tween 20 (vol/vol) and 1% BSA. Blots were washed for three 10-min periods at room temperature (TBS, 0.1% Tween 20) and then incubated for 1 h at room temperature with a secondary antibody conjugated to horseradish peroxidase (1:5,000) in blocking solution. Blots were then washed for a second time (3 × 10 min).
β-Actin was detected with 1:10,000 dilution of primary antibody in TBS-Tween 20 with 2.5% milk. Blots were washed for three 10-min periods at room temperature (1 × TBS, 0.1% Tween 20) and then incubated for 1 h with a secondary antibody conjugated to horseradish peroxidase (1:2,000) in blocking solution. Blots were then washed a second time (3 × 10 min). Bands were visualized by chemiluminescence as previously described. Quantitation of the Wnt5a was done by using NIH Image J v1.34 and a Personal Densitometer (Molecular Dynamics).
Data are presented as means ± SE of at least three separate experiments. Data were analyzed by Student's t-test or ANOVA when appropriate. P < 0.05 was set as the statistically significant difference level.
Effect of CaSR activation on Wnt5a secretion from colonic myofibroblasts.
We first determined by RT-PCR whether CaSR activation on 18Co colonic myofibroblasts by increased extracellular Ca2+ influenced expression of canonical and noncanonical Wnt family members (Fig. 1). Cells were treated with 5 mM Ca2+ for 18 h and compared with cells challenged with 0.5 mM Ca2+. Amplicons for canonical Wnts (1, 2, 2b, 3, 3a, 4) were either absent, not changed, or decreased (Wnt3) after the high-calcium treatment (data not shown). In contrast, as shown in Fig. 1A, increases in amplicons for the noncanonical Wnt, Wnt5a, were observed both in the 18Co cells and in primary cultures of hCMF, previously shown to express the CaSR (60). We focused our remaining experiments in determining whether Wnt5a protein was secreted from the colonic myofibroblasts after CaSR activation. We measured by Western blot, Wnt5a, in both conditioned media and cell lysates of cells transiently transfected with siRNA duplex against the CaSR compared with cells transfected with a scrambled siRNA duplex (Fig. 1B). After these transfections 18Co cells were challenged with either high (5 mM) or low (0.5 mM) Ca2+ for a further 18 h. Conditioned media and lysates were prepared and compared with the mobility of a recombinant Wnt5a protein (43 kDa). The increased Ca2+ increased the immunoreactive Wnt5a in the cell lysates. Treatment with siRNA against the CaSR reduced this increase compared with cells treated with the scrambled siRNA (Fig. 1B). High Ca2+ substantially increased the immunoreactive Wnt5a in the conditioned media from stimulated cells; siRNA against the CaSR reduced this increase to the unstimulated levels, compared with cells treated with the scrambled siRNA duplex (Fig. 1B). We observed that transient transfection with either active or scrambled siRNA duplex had no effect on the unstimulated Wnt5a secretion from the 18Co cells (data not shown). Treatment of the 18Co cells in low Ca2+ (0.5 mM) with other CaSR agonists such as neomycin sulfate (350 μM), spermine (1 mM) or polyarginine (1.5 μM) for 18 h also increased Wnt5a protein in the cell lysate (Fig. 1C) and stimulated Wnt5a secretion into the medium. We challenged these cells with other G protein-coupled receptor agonists to determine specificity of this stimulation. Angiotensin (1 μM), endothelin (10 nM), and ADP (1 μM) had no effect on the Wnt5a amplicon intensity; angiotensin had no effect on basal Wnt5a secretion, whereas endothelin and ADP both reduced it (data not shown). Because the siRNA duplex against the CaSR reduced the Ca2+ stimulation of Wnt5a secretion to unstimulated levels, our results strongly suggest that CaSR activation mediates increased Wnt5a secretion.
Effect of Wnt5a on CDX2 transcript and protein in HT-29 adenocarcinoma cells.
The caudal homeodomain factor CDX2 is known to be central mediator of intestinal differentiation (72). To determine whether Wnt5a had a paracrine role in epithelial differentiation, we first determined whether there was a relationship between Wnt5a and CDX2 expression in a cell line previously shown to have very low endogenous expression of CDX2. HT-29 adenocarcinoma cells were transiently transfected with Wnt1, Wnt3a, or Wnt5a, and RT-PCR was performed for CDX2 amplicons. As illustrated in Fig. 2, only the cells transfected with Wnt5a manifested an increase in CDX2 amplicon intensity (Fig. 2A). We also screened other intestinal transcription factors (HNF-4α, GATA4, or ENT) and observed that Wnt5a had no effect on their expression (data not shown). We then examined whether transient transfection with Wnt5a influenced expression of CDX2 protein in these cells. The transfection of Wnt5a substantially increased CDX2 protein in the HT-29 cells (Fig. 2B). Comparable increases in CDX2 protein were also observed after Wnt5a transfection of subconfluent Caco-2/BBE cells (data not shown). Together these results demonstrate that Wnt5a will upregulate CDX2 transcripts and increase CDX2 protein in model epithelial cell lines.
Effect of Wnt5a on SI promoter activity in Caco-2BBE cells.
SI expression is regulated by CDX2. Because Wnt5a increased both CDX2 amplicons and protein, we assessed whether Wnt5a influenced the activity of a previously characterized SI promoter (12, 33) that had been used extensively in Caco-2BBE cells (Fig. 3). Cotransfection of different amounts of Wnt5a together with the SI promoter caused a significant (P < 0.05) increase in promoter activity that was dose responsive to the Wnt5a (Fig. 3A). A fourfold increase in activity was observed at the highest concentration of Wnt5a plasmid cotransfected. No stimulation of SI reporter activity was found with comparable cotransfection with either Wnt1 or Wnt3a (data not shown). We then measured Wnt5a levels in the media after cotransfection to demonstrate that Wnt5a was being secreted from the cells and the stimulation of the SI reporter was likely an autocrine stimulation rather then Wnt5a working via a cytosolic, receptor-independent mechanism. As illustrated in Fig. 3B, Western blots revealed that increasing the amount of Wnt5a plasmid increased the immunoreactive Wnt5a in both the lysate of the cells and the conditioned medium. The higher molecular weight of the Wnt5a suggested glycosylation of the secreted protein. Consistent with previous reports of SI reporter activation (33), we found that inclusion of an inhibitor of p38 MAP kinase (SB208503) reduced the Wnt5a stimulation [relative luciferase activity (RLA): 345 ± 23 vs. 190 ± 11, P < 0.05, n = 6, Fig. 3A]. Comparable stimulations of SI reporter activity by different amounts of cotransfected Wnt5a were also observed in HT-29 cells (data not shown). Thus, in addition to increasing CDX2 transcripts and protein, Wnt5a overexpression increased the activity of a CDX2 responsive gene, SI.
Activation of the CaSR stimulates increases in Ror2, a Wnt5a receptor, on intestinal epithelia.
We then determined whether the expression of the orphan tyrosine kinase, Ror2, that has a cysteine-rich domain shown to interact with Wnt5a (39, 59) was influenced by CaSR activation in the model epithelial cells. First, we measured Ror2 in the lysates of HT-29 cells treated with extracellular Ca2+ (0.5 vs. 5 mM) to activate the CaSR (Fig. 4). As illustrated, this challenge increased Ror2 protein in the HT-29 cells (Fig. 4A). Immunocytochemistry also demonstrated CaSR activation substantially increased Ror2 protein in these cells (Fig. 4B). The cells in low Ca2+ were labeled with Hoechst. The CaSR-stimulated increases in Ror2 were predominantly cytoplasmic with increases in punctate staining (Fig. 4B). In the remaining experiments we compared the effect of other CaSR agonists (neomycin sulfate, GdCl3, polyarginine). Each of the CaSR agonists added to cells in low calcium increased expression of Ror2 protein in the HT-29 adenocarcinoma cells (Fig. 4C). Together these results strongly suggested that CaSR activation of intestinal epithelia would increase the production of Ror2, a Wnt5a receptor.
Presence of Ror2 in mouse small intestine and colon.
To understand where Ror2 was expressed in the normal adult intestine we isolated epithelia by Ca2+-chelation technique and measured Ror2 protein by Western blot and performed immunohistochemistry of Ror2 on sections of jejunum and proximal colon (Fig. 5). In isolated epithelia from the jejunum, ileum, and colon, Ror2 protein was present (Fig. 5A). Alexa labeling of anti-Ror2 antibody in sections of the jejunum revealed Ror2 expression on all epithelia from the crypt base to the villus tip. Ror2 was also present throughout the lamina propria and on the circular smooth muscle (Fig. 5B). In contrast, in the proximal colon, Ror2 was found expressed only on the surface epithelia but not in the crypt base or sides. Little to no Ror2 expression was observed in the colonic lamina propria whereas some Ror2 expression was present on the circular smooth muscle (Fig. 5B). The alternate localization of colonic Ror2 compared with small intestinal Ror2 may suggest different functions in response to activation by Wnt5a.
Effect of Ror2 overexpression or silencing on Wnt5a production of CDX2 protein and SI reporter activity.
To further define a relationship between Wnt5a, Ror2, and CDX2 we used HT-29 cells that had endogenous levels of Ror2 expression, transient expression of overexpressed wild-type Ror2, or Ror2 knocked down with siRNA duplex, and added recombinant mouse (rm) Wnt5a protein and measured CDX2 protein (Fig. 6). Addition of rmWnt5a (200 ng/ml) to mock-transfected cells revealed an increase in CDX2 protein 1 h after addition that declined over the next hour (Fig. 6A). In cells that were overexpressing wild-type Ror2, addition of rmWnt5a protein also substantially increased CDX2 protein within 1 h, which remained elevated over the next hour. Notably, in the cells overexpressing Ror2 in the absence of Wnt5a addition, there was more CDX2 protein present compared with cells expressing endogenous levels of Ror2 (Fig. 6A). We then confirmed that siRNA against Ror2 would reduce Ror2 protein compared with cells transfected with a scrambled siRNA duplex (Fig. 6A, right) and added rmWnt5a protein to the Ror2 knockdown cells. As is illustrated in Fig. 6A, there was no CDX2 protein present before addition of rmWnt5a, and the increase after 1 h was reduced compared with the endogenously Ror2 expressing cells. These results suggested to us that Wnt5a interaction with Ror2 increased CDX2 protein levels and that, in the absence of Wnt5a activation, Ror2 alone could influence CDX2 protein expression in this model epithelia.
We then determined whether Wnt5a and Ror2 together influenced the activity of the SI promoter in the Caco-2BBE cells (Fig. 6B). As above, cotransfection with Wnt5a increased the activity of the SI promoter (RLA as % of 0.5 mM Ca2+: 180 ± 9). Cotransfection with wild-type Ror2 together with Wnt5a gave the highest stimulation in these experiments. This increase was attenuated by inclusion of a dominant-negative Ror2 (GPI-Ror2) construct (RLA: 246 ± 7 vs. 163 ± 5, P < 0.05, n = 6). Together these results suggest that Wnt5a can interact with Ror2 to increase CDX2 and SI promoter activity in intestinal epithelia.
CaSR-activated myofibroblast Wnt5a inhibits β-catenin signaling in full-length APC HT-29 cells.
In remaining experiments we assessed whether the CaSR-stimulated Wnt5a from the myofibroblasts was biologically active on intestinal epithelia by adding conditioned medium from the CaSR-activated 18Co cells to an HT-29 adenocarcinoma cell line with an inducible full-length APC and measuring activity of TOPflash, a well-characterized Tcf/β-catenin luciferase reporter (Fig. 7). After Zn2+ induction of APC expression, addition of an aliquot of conditioned medium from 18Co myofibroblasts that had been challenged with 5 mM Ca2+ (as above) inhibited the reporter activity (75 ± 8%, P < 0.05, n = 6) vs. Control (5 mM Ca2+, Fig. 7). Addition of the chimeric Frizzled-8 antibody (1:50), which is known to chelate Wnt protein, to the conditioned medium decreased the amount of inhibition of the TOPflash reporter (% inhibition of the Control: 41 ± 3 vs. 75 ± 8, P < 0.05, n = 6). Addition of comparable amounts of IgG had no effect on the Control activity. Addition of recombinant mouse Wnt5a protein (200 ng/ml) for 12 h reduced the Control activity by ∼62% (RLA: 220 ± 95 vs. 580 ± 62, P < 0.05, n = 6, Fig. 7). These results suggested to us that the Wnt5a produced from the colonic myofibroblasts after CaSR activation was biologically active in reducing the β-catenin reporter activity in a model epithelia expressing full-length APC.
The findings of the present study are summarized in the schematic illustrated in Fig. 8. Stimulation of the extracellular CaSR on subepithelial colonic myofibroblasts stimulated the secretion of the noncanonical Wnt family member Wnt5a. Activation of the CaSR on model epithelia increased expression of Ror2, an orphan tyrosine kinase that is a receptor for Wnt5a. The interaction of stromal Wnt5a with epithelial Ror2 increased the production of CDX2, a homeobox caudal domain transcription factor required for synthesis of several genes of absorptive epithelial differentiation. One example of these genes is SI, and the present experiments demonstrate that Wnt5a interacting with Ror2 will increase the activity of the SI promoter. The CaSR-stimulated Wnt5a from the myofibroblasts will also interact with epithelia expressing a full-length APC to inhibit β-catenin signaling. Our present findings are the first demonstration that a G protein-coupled receptor will stimulate Wnt5a secretion from a stromal cell. Our findings that the CaSR will mediate the secretion of a Wnt protein from the stroma and selectively increase expression of a receptor for that Wnt on the epithelia suggest existence of a new paracrine relationship. This paracrine relationship, which is mediated by the CaSR, may contribute to CDX2 homeostasis in the intestine. This selective production of Wnt5a and its receptor Ror2 on physically juxtaposed cell types is relevant to understanding determinants of the maintenance of the intestinal stem cell niche and subsequent epithelial differentiation (41, 43, 58, 68). Our present findings also suggest new mechanisms by which calcium-mediated chemoprevention from colon cancer may occur.
Intestinal myofibroblasts are a resident cell type in the lamina propria (63). These cells sit under the basement membrane and are physically poised to signal to the intestinal epithelia. The 18Co human colonic myofibroblast cell line has been extensively used to show signaling cascades in response to inflammatory cytokines (63) and secretes TGF-β isoforms (54). Recently, the CaSR has been shown to be expressed on 18Co cells, as well as primary isolates of human colonic myofibroblasts. CaSR activation in that setting resulted in the stimulation of BMP-2 secretion and the extinction of the BMP antagonist noggin (60). Because Ca2+ supplementation can act as a chemoprotective agent against colon cancer (32, 47) and increasing dietary calcium reduces colonic adenoma recurrence (70), we sought to understand whether CaSR activation generated antagonists of Wnt signaling from the colonic myofibroblasts. Our evidence that the CaSR mediated the stimulation of Wnt5a secretion is from the interfering RNA experiments and the use of other well-defined agonists of the CaSR. Previously we have shown that siRNA against the CaSR blocked high Ca2+ stimulation of BMP-2 secretion, downregulated CaSR transcripts in 18Co cells (60), and caused a decrease of CaSR protein and transcript (49) in colon cancer cells. Additional evidence that the CaSR mediated the increased secretion of Wnt5a came from our experiments using other polyvalent CaSR agonists (13) that in low-Ca2+ medium increased Wnt5a in the lysates and conditioned medium of the treated cells. Because siRNA against the CaSR substantially reduced the secretion of this “noncanonical” Wnt family member to basal, presumably constitutive, levels, we conclude that CaSR activation mediated these increases.
Wnt5a has been shown to inhibit Wnt3a-stimulated β-catenin signaling by increasing the ubiquitin ligase Siah2 to result in increased degradation of β-catenin (73). In the present experiments the Wnt5a secreted in response to CaSR activation diminished β-catenin signaling in HT-29 cells engineered to express wild-type APC (49, 57). These cells have wild-type β-catenin (36). A previously characterized chimeric antibody that chelates Wnt protein (34) attenuated the reduction in β-catenin reporter activity caused by addition of conditioned medium from the myofibroblasts, consistent with the interpretation that this medium was biologically active and contained Wnt protein. This suggests to us that the Wnt5a (either after CaSR stimulation or constitutively secreted) has the capacity of reducing β-catenin activity in epithelial cells expressing wild-type APC and β-catenin. We therefore speculate that one mechanism of chemoprevention of colon cancer mediated by the CaSR is the transient stimulation of Wnt5a secretion from the underlying myofibroblasts.
It was previously shown that CaSR activation stimulates Wnt5a secretion only from intestinal cells expressing truncated APC; the secreted Wnt5a works in an autocrine manner to inhibit defective Wnt signaling by increasing Siah2 protein (49). Wnt5a transcripts may be increased in breast epithelia by PKC stimulation (38) and TNF-α will increase mRNA for Wnt5a in human macrophages (9). Secreted Wnt5a has been shown to regulate dopaminergic neurogenesis (14, 67), and indirect evidence suggests that N-methyl-aspartate receptor synaptic Wnt3a release may occur in the hippocampus during long-term potentiation (16). Basal Wnt secretion requires posttranslational modifications of Wnt in the endoplasmic reticulum, the activity of a retromer complex, and a highly conserved seven-pass transmembrane protein Wntless/Evi (3, 6, 7, 20, 23, 62). It is not completely understood how Wnt5a secretion is regulated or whether it may be activated by receptor stimulation (46, 56). The present studies clearly show that CaSR activation stimulated increases in Wnt5a protein secretion from the colonic myofibroblast cell line. The palmitoylation of Wnt5a is required for its binding to Fzd5, whereas glycosylation is required for secretion (45). We do not know which of these steps (palmitoylation, glycosylation, retromer activity, or Wntless activity) the CaSR mediates when stimulating Wnt5a secretion from the myofibroblasts. Additional studies are required to understand how CaSR activation is increasing Wnt5a secretion from these cells.
The demonstration that Wnt5a will signal canonically (to stimulate β-catenin signaling) or noncanonically (inhibit β-catenin signaling) depending on whether wild-type Ror2 was expressed (55) prompted us to investigate the status of Ror2 in the intestine. Immunocytochemistry and Western blot analysis demonstrated that CaSR activation could increase Ror2 expression in a model epithelial cell. Immunohistochemistry and Western blot analysis demonstrated Ror2 expression not only on the surface epithelia of the mouse colon but also in epithelia along the crypt-villus axis in the jejunum. Further experiments are required to understand the polarity of Ror2 expression in intestinal epithelia. Ror2 was expressed on the circular smooth muscle of both small and large intestine. The lamina propria of the small intestine was rich in cells expressing Ror2 whereas in the colon there was very little lamina propria Ror2 expression. Although epithelial Ror2 transcripts have been reported (53, 82), our study is the first demonstration that intestinal epithelia express Ror2 protein. Although epithelia of the small intestine and colon show transcripts for Frizzled 5 and Frizzled 7 (26), both of which may be activated by Wnt5a, we speculate that CaSR activation of the epithelia increases Ror2 expression to generate a “receptor context” for Wnt5a that favors noncanonical signaling. The ability to generate an appropriate receptor context may be homeostatic and may be altered with inflammation or colon cancer. If Wnt secretion from myofibroblasts has plasticity (i.e., different Wnts with inflammatory stimuli), then a comparable plasticity may exist with regard to the Wnt's cognate receptor on the epithelia. We note that CaSR activation on the subepithelia has produced the ligand (Wnt5a) for the receptor (Ror2) that CaSR activation has produced on the epithelia. Our in vitro studies suggest that noncanonical Wnt signaling exists in the mammalian intestine. We are not aware of studies of other paracrine relationships demonstrating such a specific choreography as the production of a ligand on one cell type and that ligand's receptor on the physically juxtaposed cell type.
How CDX2 gene expression is regulated during cell differentiation and progression of colonic adenocarcinomas is not understood. In fact, little is known about the regulation of CDX2 activity. Definitive studies have shown that phosphorylation of CDX2, by p38 MAP kinase, will regulate its transcriptional activity and modulate its interactions with other cofactors (8, 11, 12, 33, 35, 64). The targeted degradation of CDX2 following its phosphorylation by Cdk2 has demonstrated that CDX2 homeostasis may be regulated by cell cycle machinery (12). Whereas earlier studies suggested that PTEN and PI3K might regulate CDX2 expression (40), others (31) were unable to demonstrate that PI3K had a role in the repression of CDX2 expression in colon cancer cell lines. BMP will activate CDX2 expression in gastric cancer cells (5); we do not know whether Wnt5a interaction with Ror2 can stimulate SMAD signaling. Our present experiments clearly show that noncanonical Wnt5a can increase CDX2 transcripts and protein in a model epithelial cell line.
We selected for our experiments the HT-29 adenocarcinoma cell line since it has been reported to have considerably reduced CDX2 transcript levels in contrast with CDX2-expressing cell lines such as Caco-2 and DLD-1 (31). Insight into how Wnt5a increases CDX2 in the HT-29 cells was found in our experiments overexpressing wild-type Ror2 or reducing Ror2 expression with interfering RNA. We observed that overexpression of Ror2 in the absence of Wnt5a increased CDX2 protein expression. Stimulation of filopodia generation by overexpression of Ror2 in the absence of Wnt5a has been noted by others (59). Because Ror2 is found expressed in epithelia along the crypt-villus axis in the small intestine, we speculate that in the absence of Wnt5a, Ror2 alone might lead to increases in CDX2 expression, but further experiments are required to define this relationship. However, knocking down Ror2 expression with siRNA reduced the Wnt5a stimulation of CDX2, consistent with the interpretation that Wnt5a interaction with Ror2 will increase CDX2 protein expression. A target of CDX2, the SI promoter, was also stimulated by Wnt5a and overexpressed Ror2; this stimulation was inhibited by a previously characterized dominant-negative construct of Ror2, GPI-Ror2 (55). Wnt5a has been shown to activate PKC (77), calcium-calmodulin kinase 11 (37), and JNK (78); activate glycogen synthetase kinase (80); and induce hyperphosphorylation of Disheveled (39, 67). During Xenopus development Wnt5a stimulates protocadherin expression requiring Ror2, utilizing a pathway that signals through PI3K and cdc42 to activate JNK (66). The sensitivity of the Wnt5a stimulation of the SI reporter in the present study to a pharmacological inhibitor of p38 MAP kinase suggests to us that TAK-1/p38 kinases may be involved. Nevertheless, the signaling cascade activated by Wnt5a interacting with Ror2 to increase CDX2 transcripts in intestinal epithelia is currently not known.
Our present findings provide additional insight into how CaSR-expressing pericryptal myofibroblasts could contribute to the maintenance of an intestinal stem cell niche and facilitate epithelial differentiation. The stem cell niche can be modeled as a microenvironment where stem cells compete for extracellular factors, the relative concentrations of which will dictate a balance between self-renewal and differentiation of these cells (58). Because of their physical proximity, intestinal myofibroblasts, together with other stromal cells, may define this microenvironment (41, 43). Key regulatory pathways known to be involved in intestinal stem cell renewal and differentiation are the Wnt, BMP, and Hedgehog pathways (2, 28–30, 42, 58, 68, 81). Activation of the CaSR on colonic myofibroblasts may influence Wnt, BMP, and Hedgehog signaling. Previously we have reported that CaSR activation of primary and 18Co colonic myofibroblasts stimulated secretion of BMP-2 and extinction of noggin, which together lead to increased intestinal barrier differentiation (60). We also have observed that CaSR activation of the 18Co cells stimulated synthesis and secretion of Ihh but extinction of Sonic hedgehog (50). Our present results demonstrate that CaSR activation increases secretion of Wnt5a from colonic myofibroblasts, which after interaction with its receptor, Ror2, on a model epithelium, increased CDX2 and a CDX2-target gene, SI, as well as inhibited defective Tcf4 signaling on this epithelium. There is little information about the intestinal stem cell niche microenvironment provided by the underlying myofibroblasts (43). We conclude that our present findings strongly support the interpretation that CaSR activation of the myofibroblasts provides a microenvironment that supports, through noncanonical Wnt signaling, the differentiation of the intestinal epithelia. How this novel paracrine relationship of CaSR-mediated Wnt5a secretion from the stroma and CaSR-mediated Ror2 on the intestinal epithelia becomes altered with inflammation or the development of colon cancer remains to be determined.
This work was funded by an operating grant from the Crohn's and Colitis Foundation of Canada and the Kingston General Hospital Foundation to R. J. MacLeod. We acknowledge a CIHR/CAG/Ferring fellowship to I. Pacheco.
We thank R. Nusse for GPI-Ror2, Y. Minami for wild-type Ror2 plasmids, and N. Rivard for sucrase-isomaltase promoter. We also acknowledge the technical assistance of C. Spencer.
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