GALNT3 MAINTAINS THE EPITHELIAL STATE BY PROMOTING O-GalNAC MODIFICATION OF E-CADHERIN IN TROPHOBLAST STEM CELLS AND HUMAN MAMMARY EPITHELIAL CELLS

GALNT3 MAINTAINS THE EPITHELIAL STATE BY PROMOTING O-GalNAC MODIFICATION OF E-CADHERIN IN TROPHOBLAST STEM CELLS AND HUMAN MAMMARY EPITHELIAL CELL

MAP3K4 Controls the Chromatin Modifier HDAC6 during Trophoblast Stem Cell Epithelial-to-Mesenchymal Transition

The first epithelial-to-mesenchymal transition (EMT) occurs in trophoblast stem (TS) cells during implantation. Inactivation of the serine/threonine kinase MAP3K4 in TS cells (TSKI4 cells) induces an intermediate state of EMT, where cells retain stemness, lose epithelial markers, and gain mesenchymal characteristics. Investigation of relationships among MAP3K4 activity, stemness, and EMT in TS cells may reveal key regulators of EMT. Here, we show that MAP3K4 activity controls EMT through the ubiquitination and degradation of HDAC6. Loss of MAP3K4 activity in TSKI4 cells results in elevated HDAC6 expression and the deacetylation of cytoplasmic and nuclear targets. In the nucleus, HDAC6 deacetylates the promoters of tight junction genes, promoting the dissolution of tight junctions. Importantly, HDAC6 knockdown in TSKI4 cells restores epithelial features, including cell-cell adhesion and barrier formation. These data define a role for HDAC6 in regulating gene expression during transitions between epithelial and mesenchymal phenotypes

Innate Immune Proteins and Early Innate Immune Response of Channel Catfish (Ictalurus Punctatus)

The channel catfish (Ictalurus punctatus) is susceptible to bacterial and viral infections acqired from its pond environment. The innate immune proteins mannose-binding lectin (MBL) and lysozyme were studied in two different groups of channel catfish aged 2, 4, 6, 9, and 12 months-old. The two groups were maintained at a mean temperature of 270C and were one-year apart in their bleedings. Dot-blot enzyme linked immunosorbent assay for MBL and turbidometry lysozyme quantitive assays were done to determine the two innate immune proteins. The greatest increases in mean MBL and mean lysozyme concentrations were seen at 4 months. Two month-old catfish were comparable with 12 month-old catfish in their concentrations of MBL and lysozyme (p \u3c 0.05). A decrease was seen at 6 and 9 months of MBL and at 9 and 12 months for lysozyme. Mean protein of 26.7 mg/ml and mean albumin/globulin ratio of 0.7 were determined for 4, 6, 9, and 12 month-old catfish. This study provided a foundation for understanding these innate immune proteins and the early immune response of channel catfish

Coordinated regulation of Rel expression by MAP3K4, CBP, and HDAC6 controls phenotypic switching

Coordinated gene expression is required for phenotypic switching between epithelial and mesenchymal phenotypes during normal development and in disease states. Trophoblast stem (TS) cells undergo epithelial-mesenchymal transition (EMT) during implantation and placentation. Mechanisms coordinating gene expression during these processes are poorly understood. We have previously demonstrated that MAP3K4-regulated chromatin modifiers CBP and HDAC6 each regulate thousands of genes during EMT in TS cells. Here we show that CBP and HDAC6 coordinate expression of only 183 genes predicted to be critical regulators of phenotypic switching. The highest-ranking co-regulated gene is the NF-κB family member Rel. Although NF-κB is primarily regulated post-transcriptionally, CBP and HDAC6 control Rel transcript levels by binding Rel regulatory regions and controlling histone acetylation. REL re-expression in mesenchymal-like TS cells induces a mesenchymal-epithelial transition. Importantly, REL forms a feedback loop, blocking HDAC6 expression and nuclear localization. Together, our work defines a developmental program coordinating phenotypic switching

MAP3K4 Controls the Chromatin Modifier HDAC6 during Trophoblast Stem Cell Epithelial-to-Mesenchymal Transition

The first epithelial-to-mesenchymal transition (EMT) occurs in trophoblast stem (TS) cells during implantation. Inactivation of the serine/threonine kinase MAP3K4 in TS cells (TSKI4 cells) induces an intermediate state of EMT, where cells retain stemness, lose epithelial markers, and gain mesenchymal characteristics. Investigation of relationships among MAP3K4 activity, stemness, and EMT in TS cells may reveal key regulators of EMT. Here, we show that MAP3K4 activity controls EMT through the ubiquitination and degradation of HDAC6. Loss of MAP3K4 activity in TSKI4 cells results in elevated HDAC6 expression and the deacetylation of cytoplasmic and nuclear targets. In the nucleus, HDAC6 deacetylates the promoters of tight junction genes, promoting the dissolution of tight junctions. Importantly, HDAC6 knockdown in TSKI4 cells restores epithelial features, including cell-cell adhesion and barrier formation. These data define a role for HDAC6 in regulating gene expression during transitions between epithelial and mesenchymal phenotypes

GALNT3 Maintains the Epithelial State in Trophoblast Stem Cells

Summary: O-GalNAc glycosylation is initiated in the Golgi by glycosyltransferases called GALNTs. Proteomic screens identified >600 O-GalNAc-modified proteins, but the biological relevance of these modifications has been difficult to determine. We have discovered a conserved function for GALNT3 in trophoblast stem (TS) cells, blastocyst trophectoderm, and human mammary epithelial cells (HMECs). The loss of GALNT3 expression in these systems reduces O-GalNAc glycosylation and induces epithelial-mesenchymal transition. Furthermore, Galnt3 expression is reduced in aggressive, mesenchymal claudin-low breast cancer cells. We show that GALNT3 expression controls the O-GalNAc glycosylation of multiple proteins, including E-cadherin in both TS cells and HMECs. The loss of GALNT3 results in the intracellular retention of E-cadherin in the Golgi. Significantly, re-expression of GALNT3 in TS cells increases O-GalNAc glycosylation and restores the epithelial state. Together, these data demonstrate the critical biological role of GALNT3 O-GalNAc glycosylation to promote the epithelial phenotype in TS cells, blastocyst trophectoderm, and HMECs. : Raghu et al. demonstrate that O-GalNAc glycosylation is critical for epithelial state maintenance in trophoblast stem cells and HMECs. MAP3K4 promotes GALNT3 O-GalNAc modification of E-cadherin. Loss of GALNT3 results in the retention of E-cadherin in the Golgi. GALNT3 re-expression restores cell surface localization of E-cadherin, protecting the epithelial state. Keywords: epithelial-to-mesenchymal transition, EMT, MAP3K4, trophoblast stem cells, blastocyst, GALNT3, O-GalNAc glycosylation, E-cadherin, histone deacetylase, HDAC