Chondroitin 4-O-Sulfotransferase-1 Modulates Wnt-3a Signaling through Control of E Disaccharide Expression of Chondroitin Sulfate

Satomi Nadanaka,Miho Ishida,Masami Ikegami,Hiroshi Kitagawa

doi:10.1074/jbc.m802997200

Satomi Nadanaka, Miho Ishida + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m802997200

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Abstract

Wnt-3a is a ligand that activates the beta-catenin-dependent pathway in Wnt signaling, which is implicated in numerous physiological events such as morphogenesis. So far, heparan sulfate (HS) proteoglycans have been highlighted as a low affinity receptor for morphogens containing Wnts. Here we show the importance of chondroitin sulfate (CS) proteoglycans in the efficient signaling of Wnt-3a and the structural features of CS required for the regulation of Wnt-3a signaling. Wnt-3a signaling was depressed in a mouse L cell mutant, called sog9, which is defective in the EXT1 gene encoding the HS-synthesizing enzyme and the chondroitin 4-O-sulfotransferase (C4ST-1) gene compared with parental L cells. The transfection of sog9 cells with C4ST-1 resulted in the recovery of Wnt-3a signaling, whereas the expression of EXT1 in sog9 cells could not restore Wnt-3a signaling. In addition, the expression level of introduced C4ST-1 correlated with the recovery of Wnt-3a signaling accompanied by the increased expression of the E disaccharide unit of CS. Interestingly, molecular interaction analyses using Biacore revealed that squid CS-E (rich in the E disaccharide unit) bound strongly to Wnt-3a (K(d)=13.2 nm) to the same extent as heparin from bovine lung (K(d)=8.43 nm). In contrast, other CS isoforms as well as HS isolated from bovine kidney showed little binding activity to Wnt-3a. Moreover, exogenously added CS-E potently inhibited the accumulation of beta-catenin induced by Wnt-3a. These results suggest that CS-E-like structures synthesized by C4ST-1 participate in Wnt-3a signaling and modulate the physiological events caused by Wnt-3a signals.

Highlights

Introduction ofC4ST-1 into sog9Cells Rescues the Reduced Response sog9/CSase to Wnt-3a—As described, synthesis of both heparan sulfate (HS) and chondroitin sulfate (CS) was prominently suppressed in sog9 cells because of the deficiency of EXT-1 and C4ST-1(10, 19)
Sog9 Cells Deficient in EXT1 and C4ST-1 Show a Diminished Response to Wnt-3a—To examine whether Wnt-3a signaling is regulated by CS-PGs in mouse fibroblast L cells as well as chondrocytes [9], the effects of removing CS chains using chondroitinase ABC on Wnt-3a signaling were examined by measuring the stabilization of ␤-catenin in L cells as a direct response to canonical Wnt stimulation
We have shown that C4ST-1 can modulate Wnt-3a signaling by controlling the fine structures of CS chains of PGs

Summary

EXPERIMENTAL PROCEDURES

Materials—Purified recombinant mouse Wnt-3a was obtained from R & D Systems (Minneapolis, MN). Conditioned Media—Mouse L-Wnt-3a cells (American Type Culture Collection, Manassas, VA) were grown in DMEM containing 10% fetal bovine serum, 0.4 mg/ml G418, 100 units/ml penicillin, and 100 ␮g/ml streptomycin sulfate. Chondroitinase ABC was mixed with protease inhibitor mixture (Nacalai Tesque, Kyoto, Japan) and added to cultured cells. Cells were washed and incubated with 4 ␮g/ml Alexa Fluor 488 goat antirabbit IgG (HϩL) (Invitrogen) for 2 h at room temperature. Recombinant mouse Wnt-3a (4.8 ng) was incubated with rabbit anti-Wnt-3a antibody (20 ng) for 30 min at room temperature. Wnt-3a-anti-Wnt-3a antibody complexes were added to cells, incubated for 30 min at 4 °C, and washed with PBS. After cells were fixed with acetone, they were incubated with 2 ␮g/ml of Alexa Fluor 488 goat anti-rabbit IgG (HϩL) for 60 min at room temperature. Immunofluorescence was visualized on an all-in-one type fluorescence microscope BZ-8000 (Keyence, Osaka, Japan)

RESULTS

DISCUSSION

Findings

C L rWnt-3a anti-Wnt-3a Ab