SUMMARY 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 between 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 an epigenetic role for HDAC6 in regulating gene expression during transitions between epithelial and mesenchymal phenotypes.

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.

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.

Innate immune proteins mannose‐binding lectin (MBL) and lysozyme were studied in channel catfish, Ictalurus punctatus, during two consecutive years at 2, 4, 6, 9, and 12 mos of age. Both groups maintained in indoor tanks in light/dark photoperiod at mean temperature 27 C. Dot‐blot enzyme linked immunosorbent assay (ELISA) for MBL and turbidometry lysozyme assay done to quantify the two innate proteins and means determined for two consecutive years. An increase in mean MBL was seen in 4‐mo catfish (26.9 ± 0.8 µg/mL) when compared with all other age groups. A decrease in mean MBL was seen in 6‐ and 9‐mo catfish when compared with all other age groups. Both 2‐mo (21 ± 1.4 µg/mL) and 12‐mo (19.9 ± 0.4 µg/mL) catfish were very similar in mean MBL concentrations. The greatest increase in mean lysozyme was seen at 4 mo (15 ± 5.0 µg/mL). Juvenile channel catfish could produce lysozyme at 2 mo equivalent to lysozyme concentrations found in 9‐ and 12‐mo catfish. The greatest increases for MBL and lysozyme were seen in 4‐mo catfish. Mean total protein of 26.7 mg/mL and mean albumin/globulin ratio of 0.7 were found for the two groups of channel catfish sera.

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