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Comparative Study
. 2007 Mar 26:8:12.
doi: 10.1186/1471-2121-8-12.

Regulation of norrin receptor frizzled-4 by Wnt2 in colon-derived cells

Kestutis Planutis  1 , Marina Planutiene, Mary Pat Moyer, Anthony V Nguyen, Cherlyn A Pérez, Randall F Holcombe
Affiliations
Comparative Study

Regulation of norrin receptor frizzled-4 by Wnt2 in colon-derived cells

Kestutis Planutis et al. BMC Cell Biol. .

Abstract

Background: Norrin is a potent Wnt pathway ligand. Aberrant activation of this signaling pathway can result in colon tumors but the role of norrin-based signaling in the genesis of colon cancer, and its relationship to activation of the pathway by traditional Wnt ligands, is not defined.

Results: Fresh normal human colon tissue and all the cell lines studied expressed mRNA for Fz4, LRP5 and norrin, except Colo205 which lacked Fz4 expression. Canonical Wnt pathway throughput was increased slightly in NCM460 following treatment with Wnt3a CM but was inhibited by Wnt2 and Wnt1. The colon cancer cell line, RKO, responded to Wnt3a CM, Wnt2 and Wnt1 by increasing canonical Wnt pathway throughput. Wnt5a did not affect Wnt pathway throughput in either cell line. Wnt2, but not Wnt3a, abrogated Fz4 expression in NCM460, but not in RKO or another colon cancer cell line, HCT116. This effect on Fz4 was confirmed at both the RNA and protein levels via RT-PCR and a norrin binding assay. The expression of all others 9 Fz receptors did not change after treatment of NCM460 cells with Wnt2.

Conclusion: The data suggests that colonic mucosa and colon tumors may possess two autoregulatory positive Wnt feedback loops, one through canonical signals induced by Wnt:Fz interactions and one through signals resulting from norrin:Fz4 interactions. The latter interactions may be modulated via regulation of Fz4 expression by Wnt2. Retention of Fz4 by cancers, in contrast to the loss of Fz4 by normal mucosal cells, could provide a selective advantage to the tumor cells. Fz4 expression may play a critical role in responses to Wnt signaling in the tumor microenvironment.

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Figures

Figure 1
Figure 1
Expression of norrin signaling components. Panel A – RT-PCR for norrin signaling pathway components Fz4 (832 bp), norrin (400 bp), and LRP5 (300 bp) in normal colonic mucosa obtained fresh from colonoscopic biopsy. Molecular weight markers appear in lane 1. β-actin (544 bp) was utilized as a control to confirm RNA quality. Control PCR reactions without reverse transcriptase did not give any PCR products for any RNA sample with any pair of primers (data not shown). Identities of the PCR products were confirmed by sequence analysis. Panel B – Expression defined by RT-PCR of Fz4 (top row), norrin (3rd row) and LRP5 (bottom row) in various cell lines. Lane 1: no template control; lane 2: CSC1; lane 3: Colo205; lane 4: Colo320; lane 5: HCT116; lane 6: HCT116 with additional chromosome 3; lane 7: HT29; lane 8: NCM460 (normal mucosa-derived); lane 9: RKO; lane 10: EaHy926 (endothelial). Fz4, norrin and LRP5 mRNA was detected in all tested cell lines with the exception of Fz4 in Colo205. Actin control is depicted in 2nd row.
Figure 2
Figure 2
Responsiveness of NCM460 and RKO cells to Wnt stimulation. Wnt pathway throughput is measured following transient transfection of the luciferase-based SuperTopflash reporter construct. All experiments were performed in conjunction with Renilla luciferase determination to control for cell numbers, viability and transfection efficiency. All experiments were repeated at least three times and performed each time in triplicate. Panel A – Incubation with Wnt3a conditioned medium (CM) slightly, but significantly increased Wnt pathway throughput in NCM460 (p = 0.017) and very dramatically in RKO (p
Figure 3
Figure 3
RT-PCR results of Fz4 mRNA expression in normal mucosa-derived NCM460 cells and the colon cancer cell lines HCT116 and RKO following exposure to Wnt-producing cells in co-culture or cells transfected with a non-Wnt-producing pCMV control plasmid. The Fz4 RT-PCR product was 832 bp. All experiments were performed simultaneously with actin primers, producing a 544 bp product for quality control. Fz4 mRNA was downregulated by Wnt2 in normal colonic mucosa-derived NCM460 cells but not in the colon cancer cell lines. No effect on Fz4 expression following exposure to Wnt5a was seen. A. NCM460, HCT116 and RKO cells co-cultured using inserts with sham-transfected cells. B. NCM460, HCT116 and RKO co-cultured with Wnt2-producing cells. C. NCM460 co-cultured with Wnt5a-producing cells. D. Wnt 2 and Wnt5a production by transfected RKO; Western blot. A non-specific band is present in all 3 lanes, including control. The Wnt2 and Wnt5a specific bands are annotated with an arrow.
Figure 4
Figure 4
Norrin binding activity of NCM460 and RKO cells after partitioned co-culture. Norrin binding was downregulated by Wnt2, but not Wnt3a in normal colonic mucosa-derived NCM460 cells. Each experiment was repeated three times with six (panel A) or five (panel B) replicates for each data point. A. Comparison of norrin binding in NCM460 (left) and RKO (right) after exposure to sham-transfected cells and Wnt2-producing cells in partitioned co-culture (Pcc). The decrease in norrin binding in NCM460 is highly statistically significant (p
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