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

The 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 γ- and α-catenins. N-glycosylation of E-cadherin is controlled by the DPAGT1 gene, a key regulator of the N-glycosylation pathway. DPAGT1 is a target of the canonical Wnt signaling pathway, with both ß- and γ-catenins binding to Tcf at its promoter. We now report that DPAGT1 senses cell density through canonical Wnt signaling. In dense cells, depletion of ß-catenin from the DPAGT1 promoter correlated with downregulation of its cellular abundance, while loss of nuclear γ-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 ß-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 ß- and γ-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.

Oral squamous cell carcinoma (OSCC) is one of the most pernicious malignancies, but the mechanisms underlying its development and progression are poorly understood. One of the key pathways implicated in OSCC is the canonical Wnt/ß-catenin signaling pathway. Previously, we reported that canonical Wnt signaling functions in a positive feedback loop with the DPAGT1 gene, a principal regulator of the metabolic pathway of protein N-glycosylation, to hyperglycosylate E-cadherin and reduce intercellular adhesion. Here, we show that in OSCC, DPAGT1 and canonical Wnt signaling converge to up-regulate CTHRC1 (collagen triple helix repeat containing 1), an N-glycoprotein implicated in tumor invasion and metastasis. We found that in human OSCC specimens, amplification of the levels of CTHRC1 was associated with its hyperglycosylation. Partial inhibition of DPAGT1 expression in OSCC CAL27 cells reduced CTHRC1 abundance by increasing protein turnover, indicating that N-glycosylation stabilizes CTHRC1. Additionally, canonical Wnt signaling promoted ß-catenin/T-cell factor transcriptional activity at the CTHRC1 promoter to further elevate CTHRC1 levels. We demonstrate that DPAGT1 promotes cell migration and drives the localization of CTHRC1 to cells at the leading edge of a wound front coincident with drastic changes in cell morphology. We propose that in OSCC, dysregulation of canonical Wnt signaling and DPAGT1-dependent N-glycosylation induces CTHRC1, thereby driving OSCC cell migration and tumor spread.

N-glycosylation gene DPAGT1 is a target of the Wnt/beta-catenin signaling pathway.

Protein N-glycosylation and the Wnt/ß-catenin signaling pathways play critical roles in development and cancer. Although N-glycosylation has been shown to influence Wnt signaling through its effects on Wnt ligands, it is unclear whether the Wnt/ß-catenin pathway impacts protein N-glycosylation. In this study, we show that promoters of the first N-glycosylation gene, DPAGT1, from Chinese hamster ovary (CHO), Madin-Darby canine kidney (MDCK), and human epidermoid carcinoma (A253) cells contain the T-cell factor/lymphoid enhancer-binding factor (TCF/LEF) consensus sequence. Treatment of cells with a Wnt activator, lithium chloride, up-regulated DPAGT1 transcript levels that correlated with an increase in the ß-catenin abundance. Furthermore, exposure of cells to a Wnt receptor ligand, Wnt3a, resulted in an increase in the DPAGT1 transcript levels that was abrogated by the Wnt inhibitor, Dickkopf-1. DNA mobility shift assays revealed specific protein complexes at the DPAGT1 TCF/LEF binding region that were competed off with antibodies to either Tcf3/4 or ß-catenin. Chromatin immunoprecipitation analysis confirmed the presence of ß-catenin at the DPAGT1 promoter in vivo. In addition, the DPAGT1 TCF/LEF sequence drove the expression of the luciferase reporter gene. Furthermore, up-regulation of DPAGT1 transcripts by Wnt3a led to altered N-glycosylation of E-cadherin. Interestingly, the DPAGT1 TCF/LEF sequence also interacted with γ-catenin, a close homologue of ß-catenin, although not in a lithium chloride-dependent manner. Our results provide the first evidence that the Wnt/ß-catenin signaling pathway regulates the metabolic pathway of protein N-glycosylation by targeting DPAGT1 expression. Moreover, they suggest the existence of another regulatory mechanism involving the interaction of Tcf with γ-catenin at the DPAGT1 promoter.

DNA hypermethylation near the transcription start site of collagen alpha2(I) gene occurs in both cancer cell lines and primary colorectal cancers.

Collagen production plays a significant role in tumor development, especially in breast cancer, hepatocarcinomas, and colorectal carcinoma. However, collagen production is decreased during oncogenic transformation of cells in culture. This study demonstrates that methylation of the collagen alpha2(I) gene transcription start site occurs frequently in human cancer cell lines (9 of 10), including breast cancer cell lines (MCF-7 and Hs578T), hepatocellular carcinoma cell lines (SNU387, SNU449, SNU398, and PLC/PRF/5), a fibrosarcoma cell line (HT1080), and colorectal carcinoma cell lines (HCT116, SW480, and SW620). In addition, the collagen gene is more methylated in colorectal cancer tissues compared with normal mucosa. The increased DNA methylation of the collagen gene in cell lines is inversely correlated with collagen mRNA steady-state levels. Most importantly, treatment of fibrosarcoma or breast carcinoma cells with a DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine, resulted in lower methylation and reactivation of the collagen gene in a dose-responsive manner. This is the first demonstration that the collagen alpha2(I) gene is methylated in multiple cancer cell lines correlating with loss of collagen expression and also methylated in primary cancer tissues. These data also suggest that methylation-induced repression of collagen transcription may be a frequent occurrence in cancer.

Aberrant amplification of the crosstalk between canonical Wnt signaling and N-glycosylation gene DPAGT1 promotes oral cancer.

Oral cancer is one of the most aggressive epithelial malignancies, whose incidence is on the rise. Previous studies have shown that in a subset of human oral squamous cell carcinoma (OSCC) tumor specimens, overexpression of the DPAGT1 gene, encoding the dolichol-P-dependent N-acetylglucoseamine-1-phosphate transferase, a key regulator of the metabolic pathway of protein N-glycosylation, drives tumor cell discohesion by inhibiting E-cadherin adhesive function. Recently, we reported that DPAGT1 was a target of the canonical Wnt signaling pathway. Here, we link overexpression of DPAGT1 in human OSCC tumor specimens to aberrant activation of canonical Wnt signaling. We report dramatic increases in ß- and γ-catenins at the DPAGT1 promoter and correlate them with reduced expression of a Wnt inhibitor, Dickkopf-1 (Dkk-1). Using human squamous carcinoma cell lines of the head and neck, we show that partial inhibition of DPAGT1 reduces canonical Wnt signaling, indicating that DPAGT1 and canonical Wnt signaling function in a positive feedback loop. We provide evidence that E-cadherin inhibits DPAGT1, canonical Wnt signaling and the OSCC cancer phenotype by depleting nuclear ß- and γ-catenins, with hypoglycosylated E-cadherin being the most effective. This suggests that in human OSCC, extensive N-glycosylation of E-cadherin compromises its ability to inhibit canonical Wnt signaling and DPAGT1 expression. Our studies reveal a novel interplay between DPAGT1/N-glycosylation and canonical Wnt signaling and suggest that dysregulation of this crosstalk is a key mechanism underlying OSCC. They also suggest that partial inhibition of DPAGT1 may represent an effective way to restore normal interactions among these essential pathways in oral cancer.

Regulatory factor for X-box family proteins differentially interact with histone deacetylases to repress collagen alpha2(I) gene (COL1A2) expression.

Our studies indicate that the regulatory factor for X-box (RFX) family proteins repress collagen alpha2(I) gene (COL1A2) expression (Xu, Y., Wang, L., Buttice, G., Sengupta, P. K., and Smith, B. D. (2003) J. Biol. Chem. 278, 49134-49144; Xu, Y., Wang, L., Buttice, G., Sengupta, P. K., and Smith, B. D. (2004) J. Biol. Chem. 279, 41319-41332). In this study, we examined the mechanism(s) underlying the repression of collagen gene by RFX proteins. Two members of the RFX family, RFX1 and RFX5, associate with distinct sets of co-repressors on the collagen transcription start site in vitro. RFX5 specifically interacts with histone deacetylase 2 (HDAC2) and the mammalian transcriptional repressor (mSin3B), whereas RFX1 preferably interacts with HDAC1 and mSin3A. HDAC2 cooperates with RFX5 to down-regulate collagen promoter activity, whereas HDAC1 enhances inhibition of collagen promoter activity by RFX1. Interferon-gamma promotes the recruitment of RFX5/HDAC2/mSin3B to the collagen transcription start site but decreases the occupancy by RFX1/mSin3A as manifested by chromatin immunoprecipitation assay. RFX1 binds to the methylated collagen sequence with much higher affinity than unmethylated sequence, recruiting more HDAC1 and mSin3A. The DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine, which inhibits DNA methylation, reduces RFX1/HDAC1 binding to the collagen transcription start site in chromatin immunoprecipitation assays. Finally, both RFX1 and RFX5 are acetylated in vivo. Trichostatin A stimulates the acetylation of RFX proteins and activates the collagen promoter activity. Collectively, our data strongly indicate two separate pathways for RFX proteins to repress collagen gene expression as follows: one for RFX5/HDAC2 in interferon-gamma-mediated repression, and the other for RFX1/HDAC1 in methylation-mediated collagen silencing.

The RFX family interacts at the collagen (COL1A2) start site and represses transcription.

The transcription start site of the collagen alpha2(1) gene (COL1A2) has a sequence-specific binding site for a DNA methylation-responsive binding protein called regulatory factor for X-box 1 (RFX1) (Sengupta, P. K., Erhlich, M., and Smith, B. D. (1999) J. Biol. Chem. 274, 36649-36655). In this report, we demonstrate that RFX1 forms homodimers as well as heterodimers with RFX2 spanning the collagen transcription start site. Methylation at +7 on the coding strand increases RFX1 complex formation in gel shift assays. Methylation on the template strand, however, does not increase RFX1 complex formation. DNA from human fibroblasts contains minimal methylation on the coding strand (<4%) with variable methylation on the template strand. RFX1 acts as a repressor of collagen transcription as judged by in vitro transcription and co-transfection assays with an unmethylated collagen promoter-reporter construct. In addition, an RFX5 complex present in human fibroblasts interacts with the collagen RFX site, which is not sensitive to methylation. This is the first demonstration of RFX5 complex formation on a gene other than major histocompatibility complex (MHC) promoters. Also, RFX5 represses transcription of a collagen promoter-reporter construct in rat fibroblasts that have no detectable RFX5 complex formation or protein. RFX5 complex activates MHC II transcription by interacting with an interferon-gamma (IFN-gamma)-inducible protein, major histocompatibility class II trans-activator (CIITA). Collagen transcription is repressed by IFN-gamma in a dose-dependent manner in human but not in rat fibroblasts. IFN-gamma enhances RFX5 binding activity, and CIITA is present in the RFX5 complex of IFN-gamma-treated human fibroblasts. CIITA repressed collagen gene transcription more effectively in human fibroblasts than in rat fibroblasts, suggesting that the RFX5 complex may, in part, recruit CIITA protein to the collagen transcription start site. Thus the RFX family may be important repressors of collagen gene transcription through a RFX binding site spanning the transcription start site.

Major histocompatibility class II transactivator (CIITA) mediates repression of collagen (COL1A2) transcription by interferon gamma (IFN-gamma).

Interferon gamma (IFN-gamma) plays an important role during inflammation by repressing collagen and activating major histocompatibility class II (MHC-II) expression. Activation of MHC-II by IFN-gamma requires regulatory factor for X-box 5 (RFX5) complex as well as class II transactivator (CIITA). We have shown that the RFX family binds to the COL1A2 transcription start site, and the RFX5 complex represses COL1A2 gene expression during IFN-gamma response. In this report, we demonstrate that CIITA is a key mediator of COL1A2 repression by IFN-gamma. IFN-gamma up-regulates the expression of CIITA in a time-dependent manner in lung fibroblasts and promotes CIITA protein occupancy on COL1A2 transcription start site in vivo as judged by chromatin immunoprecipitation (ChIP) assays. There are coordinate decreases in the occupancy of RNA polymerase II on the collagen transcription start site with increasing CIITA occupancy during IFN-gamma treatment. In addition, we are able to specifically knockdown the IFN-gamma-stimulated expression of CIITA utilizing short hairpin interference RNA (shRNA) against CIITA. This leads to the alleviation of COL1A2 repression and MHC-II activation by IFN-gamma. RFX5 recruits CIITA to the collagen site as evidenced by DNA affinity chromatography. The presence of RFX5 complex proteins enhances the collagen repression by CIITA reaching levels occurring during IFN-gamma treatment. Co-expression of CIITA with deletion mutations and collagen promoter constructs demonstrates that CIITA represses collagen promoter mainly through its N-terminal region including the acidic domain and the proline/serine/threonine domain. Our data suggest that CIITA is a crucial member of a repressor complex responsible for mediating COL1A2 transcription repression by IFN-gamma.

Interferon gamma repression of collagen (COL1A2) transcription is mediated by the RFX5 complex.

Interferon gamma (IFN-gamma) plays an important physiological role during inflammation by down-regulating collagen gene expression and activating major histocompatibility II (MHC-II) complex. The activation of MHC-II by IFN-gamma requires activation of a trimeric DNA binding transcriptional complex, RFX5 complex, containing RFXB (also called RFXANK or Tvl-1), RFXAP, as well as RFX5 protein. Previously, we demonstrated that RFX5 binds to the collagen transcription start site and represses collagen gene expression (Sengupta, P. K., Fargo, J., Smith, B. D. (2002) J. Biol. Chem. 277, 24926-24937). In this report, we have examined the role of RFXB and RFXAP proteins within the RFX5 complex to regulate collagen gene expression. The data show that all three RFX5 complex proteins are required for maximum repression. Expression of proteins with mutations known to be important for RFX5 complex formation does not repress collagen promoter activity. Two mutated forms of RFX5 act as dominant negative proteins activating collagen expression and reversing IFN-gamma down-regulation of collagen expression in human lung fibroblasts. IFN-gamma increases expression and nuclear translocation of RFX5. RFXB has a naturally occurring splice variant isoform (RFX SV). Interferon increases expression of the long form of RFXB and decreases expression of RFX SV with the same kinetics as collagen gene expression. Overexpression of the splice variant form reverses the IFN-gamma induced collagen repression in human lung fibroblasts. Finally, all three RFX5 complex proteins increase at the collagen transcription start site with IFN-gamma treatment using chromatin immunoprecipitation analysis. Thus, these studies suggest an important role for RFX5 complex in collagen repression.