Lack of whey acidic protein (WAP) four-disulfide core domain protease inhibitor 2 (WFDC2) causes neonatal death from respiratory failure in mice.

Respiratory failure is a life-threatening problem for pre-term and term infants, yet many causes remain unknown. Here, we present evidence that whey acidic protein (WAP) four-disulfide core domain protease inhibitor 2 (Wfdc2), a protease inhibitor previously unrecognized in respiratory disease, may be a causal factor in infant respiratory failure. Wfdc2 transcripts are detected in the embryonic lung and analysis of a Wfdc2-GFP knock-in mouse line shows that both basal and club cells, and type II alveolar epithelial cells (AECIIs), express Wfdc2 neonatally. Wfdc2-null-mutant mice display progressive atelectasis after birth with a lethal phenotype. Mutant lungs have multiple defects, including impaired cilia and the absence of mature club cells from the tracheo-bronchial airways, and malformed lamellar bodies in AECIIs. RNA sequencing shows significant activation of a pro-inflammatory pathway, but with low-quantity infiltration of mononuclear cells in the lung. These data demonstrate that Wfdc2 function is vitally important for lung aeration at birth and that gene deficiency likely causes failure of the lung mucosal barrier.

Kmt2b conveys monovalent and bivalent H3K4me3 in mouse spermatogonial stem cells at germline and embryonic promoters.

The mammalian male germline is sustained by a pool of spermatogonial stem cells (SSCs) that can transmit both genetic and epigenetic information to offspring. However, the mechanisms underlying epigenetic transmission remain unclear. The histone methyltransferase Kmt2b is highly expressed in SSCs and is required for the SSC-to-progenitor transition. At the stem-cell stage, Kmt2b catalyzes H3K4me3 at bivalent H3K27me3-marked promoters as well as at promoters of a new class of genes lacking H3K27me3, which we call monovalent. Monovalent genes are mainly activated in late spermatogenesis, whereas most bivalent genes are mainly not expressed until embryonic development. These data suggest that SSCs are epigenetically primed by Kmt2b in two distinguishable ways for the upregulation of gene expression both during the spermatogenic program and through the male germline into the embryo. Because Kmt2b is also the major H3K4 methyltransferase for bivalent promoters in embryonic stem cells, we also propose that Kmt2b has the capacity to prime stem cells epigenetically.

Expression of mucin 1 possessing a 3'-sulfated core1 in recurrent and metastatic breast cancer.

Breast cancer is the most frequent cancer threatening the lives of women between the ages of 30 and 64. The cancer antigen 15-3 assay (CA15-3) has been widely used for the detection of breast cancer recurrence; however, its sensitivity and specificity are inadequate. We previously found that the breast cancer cell line YMBS secretes mucin 1 possessing 3'-sulfated core1 (3Score1-MUC1) into the medium. Therefore, we here evaluated whether 3Score1-MUC1 is secreted into the blood streams of breast cancer patients, and whether it can serve as an improved breast cancer marker. We developed a lectin-sandwich immunoassay, called Gal4/MUC1, using a 3'-sulfated core1-specific galectin-4 and a MUC1 monoclonal antibody. Using the Gal4/MUC1 assay method, we found that 3Score1-MUC1 was profoundly expressed in the blood streams of patients with recurrent and/or metastatic breast cancer. The positive ratio of the Gal4/MUC1 assay was higher than that of the CA15-3 assay in both primary (n = 240) and relapsed (n = 43) patients, especially in the latter of which the positive ratio of Gal4/MUC1 was 86%. whereas that of CA15-3 was 47%. Furthermore, serum Gal4/MUC1 levels could more sensitively reflect the recurrence of primary breast cancer patients after surgery. Therefore, the Gal4/MUC1 assay should be an excellent alternative to the CA15-3 tumor marker for tracking the recurrence and metastasis of breast cancer.

Phosphorylation and externalization of galectin-4 is controlled by Src family kinases.

Galectin-4 is a cytosolic protein that lacks a signal sequence but is externalized and binds to 3-O-sulfated glycoconjugates extracellularly. The mechanism of subcellular localization and externalization of galectin-4 has not yet been determined. A preliminary experiment using pervanadate (PV) showed that galectin-4 is tyrosine-phosphorylated in cells and suggested that Src kinases are involved. Cell transfection with galectin-4 and active Src plasmids showed that galectin-4 can be tyrosine phosphorylated by members of the Src kinase family. The C-terminal peptide YVQI of galectin-4 was found to play an important role in its tyrosine phosphorylation, and the SH2 domains of Src and SHP2 were found to bind to this peptide. Immunofluorescence analysis showed that galectin-4 and phosphorylated proteins were intensely stained in the area of membrane protrusions of PV-treated or Src-activated cells. Furthermore, MUC1 derived from NUGC-4 cells was observed to bind to galectin-4, and externalization of the bound molecules from the cell to the medium increased in the hyperphosphorylated condition. Study of the transfection of the mutant galectin-4 which lacks the C-terminal peptide revealed that the phosphorylation status is important for externalization of galectin-4. These results suggest that externalization of galectin-4 can be regulated by signaling molecules and that it may function intracellularly as an adaptor protein serving to modulate the trafficking of glycoproteins.