Coordinate regulation of N-glycosylation gene DPAGT1, canonical Wnt signaling and E-cadherin adhesion

Abstract

SummaryThe metabolic pathway of protein N-glycosylation influences intercellular adhesion by affecting the composition and cytoskeletal association of E-cadherin protein complexes, or adherens junctions (AJs). In sparse cells, E-cadherin is modified extensively with complex N-glycans and forms nascent AJs, while in dense cultures, hypoglycosylated E-cadherin drives the assembly of mature AJs with increased levels of c-and a-catenins. N-glycosylation of E-cadherin is controlled by the DPAGT1 gene, a key regulator of the Nglycosylation pathway. DPAGT1 is a target of the canonical Wnt signaling pathway, with both b-and c-catenins binding to Tcf at its promoter. We now report that DPAGT1 senses cell density through canonical Wnt signaling. In dense cells, depletion of b-catenin from the DPAGT1 promoter correlated with downregulation of its cellular abundance, while loss of nuclear c-catenin reflected its greater recruitment to AJs. DPAGT1 itself affected canonical Wnt signaling, with forced changes in its expression resulting in corresponding changes in transcriptionally active b-catenin and canonical Wnt activity. Remarkably, a 2.4-fold increase in the DPAGT1 mRNA level resulted in increased N-glycosylation and reduced membrane localization of E-cadherin, coincident with dramatic changes in cell morphology. Lastly, we present evidence that N-glycosylation status of E-cadherin controls its antagonism of canonical Wnt signaling. Transfection of hypoglycosylated E-cadherin mutant, V13, but not fully N-glycosylated E-cadherin, into sparse cells inhibited canonical Wnt activity by depleting nuclear b-and c-catenins. Collectively, our studies show that cells coordinate DPAGT1 expression and protein N-glycosylation with canonical Wnt signaling and E-cadherin adhesion via positive and negative feedback mechanisms.

N-Glycosylation Induces the CTHRC1 Protein and Drives Oral Cancer Cell Migration

“…We conclude that a hyperglycosylated CTHRC1 glycoform exists in OSCC. Because CTHRC1 has only one N-glycan consensus sequence, and activation of the N-glycosylation pathway is typically accompanied by greater modification with complex and more highly branched N-glycans (15,17), our results indicate enhanced N-glycan heterogeneity on CTHRC1 in OSCC.…”

“…1E). Although GPT overexpression in these OSCC tumors averaged 4-fold, even a modest increase in DPAGT1 expression has been shown to result in a robust activation of canonical Wnt pathway activity and hyperglycosylation of proteins (17). Immunofluorescence analyses revealed low levels of CTHRC1 in AE, but prominent expression in OSCC (Fig.…”

“…Many components of canonical Wnt signaling are N-glycoproteins, and their N-glycosylation status controls Wnt signaling by regulating secretion and membrane localization of a canonical Wnt ligand, Wnt3a, and a co-receptor, LRP6 (17). In turn, canonical Wnt activates DPAGT1 expression via ␤-catenin at a transcriptional level.…”

“…In turn, canonical Wnt activates DPAGT1 expression via ␤-catenin at a transcriptional level. Furthermore, N-glycosylation inhibits E-cadherin adhesion and interferes with E-cadherin antagonism of canonical Wnt signaling (17,18). Aberrant activation of canonical Wnt and N-glycosylation pathways has been associated with tumorigenesis (9, 19 -21).…”

Endoplasmic reticulum stress and IRE-1 signaling cause apoptosis in colon cancer cells in response to andrographolide treatment

“…Cells were fixed in ice-cold methanol for 5 min, permeabilized with 0.2% Triton X-100 for 10 min, blocked with 6% BSA for 30 min and then incubated with primary antibodies Ki67 (Abcam, # 15580,) and GRP-78 overnight at 4°C. Cells were then incubated with Alexa Flour 488 labeled goat anti-mouse IgG (H+L) (1:200) (Invitrogen) and DAPI for 1 h. [31, 32] Sample images were taken with 200X magnification.…”