Kentaro Nagaoka

A microRNA, miR-101a, controls mammary gland development by regulating cyclooxygenase-2 expression.

Mammary glands exhibit a series of developmental states that are typified by proliferation, differentiation, and involution. Here, we demonstrate that a microRNA (miRNA), miR-101a, plays an important role in the process of mammary gland development. We used miRNA microarray analysis to show that some miRNAs exhibit changes in their expression during mouse mammary gland epithelial cell (HC11) differentiation, which corresponds to the time when these cells acquire the milk-producing phenotype. In particular, we observed an increase of miR-101a expression throughout differentiation and involution in mammary gland tissue, as well as in HC11 cells. Overexpression experiments revealed that miR-101a suppressed the expression of beta-casein mRNA, a milk protein, and marker of cell differentiation, but its suppression was not mediated by transcriptional or direct post-transcriptional regulation of beta-casein mRNA. Overexpression of miR-101a also inhibited HC11 cell proliferation that could influence the differentiation state of the mammary gland. We speculate that a direct target of miR-101a is cyclooxygenase-2 (Cox-2) mRNA because there was an inverse relationship between these two genes during mammary gland development. Indeed, Cox-2 protein expression was suppressed by the overexpression of miR-101a, and the luciferase activity of reporter constructs containing the Cox-2 3UTR was also suppressed by miR-101a overexpression. As Cox-2 has been shown to mediate cell proliferation, it is possible that the inhibition of HC11 cell proliferation by miR-101a might be mediated by Cox-2. Taken together, these results suggest that miR-101a regulates cell proliferation via altering Cox-2 expression, which is critical for controlling mammary gland development.

A Chemokine, Interferon (IFN)-γ-Inducible Protein 10 kDa, Is Stimulated by IFN-τ and Recruits Immune Cells in the Ovine Endometrium

Abstract Proper distribution of immune cells in the uterus is a prerequisite for successful implantation and subsequent placentation, but biochemical signals that govern such events have not been well characterized. In the present study, the cDNA of a chemokine, interferon (IFN)-γ-inducible protein 10 kDa (IP-10), was identified from a cDNA subtraction study between uterine endometrial tissues from Day 17 pregnant and Day 15 cyclic ewes. The effect of IFN-τ on IP-10 expression and the involvement of IP-10 in the recruitment of immune cells were then investigated. Northern blot analysis revealed that large amounts of IP-10 mRNA were present during conceptus attachment to maternal endometrium and early placentation. IP-10 mRNA was localized to monocytes distributed in the subepithelial stroma of pregnant but not cyclic uteri. This finding was supported by the discovery of IP-10 mRNA expression in monocytes but not in lymphocytes, uterine epithelial cells, or stromal cells. Moreover, the expression of IP-10 mRNA by the monocytes was stimulated by IFN-α, IFN-γ, and IFN-τ in a dose-dependent manner, but the expression of IP-10 mRNA by the endometrial explants was most stimulated by IFN-τ. In a chemotaxis assay, migration of peripheral blood mononuclear cells was stimulated by the addition of IFN-τ stimulated-endometrial culture medium, and the effect was significantly reduced by neutralization with an anti-IP-10 antibody. These results suggest that endometrial IP-10 regulated by conceptus IFN-τ regulates recruitment and/or distribution of immune cells seen in the early pregnant uterus.

Induction of Endogenous Interferon Tau Gene Transcription by CDX2 and High Acetylation in Bovine Nontrophoblast Cells

Abstract Interferon tau gene (IFNT) is expressed only by mononuclear trophectoderm cells in ruminant ungulates. To our knowledge, its epigenetic regulation and interaction with trophectoderm lineage-specific caudal-related homeobox 2 transcription factor (CDX2) have not been characterized. Herein, we studied differences in chromatin structures and transcription of endogenous bovine IFNT in bovine trophoblast BT-1 and CT-1 cells and in nontrophoblast MDBK cells. Transcripts from endogenous IFNT and CDX2 genes were found in BT-1 and CT-1 cells but not in MDBK cells. Chromatin immunoprecipitation study revealed that CDX2 binding sites exist in proximal upstream regions of IFNT (IFN-tau-c1). Endogenous IFNT transcription in BT-1 cells was increased with CDX2 overexpression but was reduced with short interfering RNA specific for the CDX2 transcript. In chromatin immunoprecipitation studies, histone H3K18 acetylation of IFNT was higher in CT-1 cells than in MDBK cells, while histone H3K9 methylation was lower in CT-1 cells than in nontrophoblast cells. In MDBK cells (but not in CT-1 cells), histone deacetylases were bound to IFNT, which was reversed with trichostatin A treatment; treatment with trichostatin A and CDX2 then increased IFNT mRNA levels that resulted from abundant CDX2 mRNA expression. These data provide evidence that significant increase in endogenous IFNT transcription in MDBK cells (which do not normally express IFNT) can be induced through CDX2 overexpression and high H3K18 acetylation, but lowering of H3K9 methylation could also be required for the degree of IFNT transcription seen in trophoblast cells.

CPEB-mediated ZO-1 mRNA localization is required for epithelial tight-junction assembly and cell polarity.

CPEB is a translational regulatory sequence-specific RNA binding protein that controls germ cell development. Here we show that CPEB heterozygous female mice are fertile but contain disorganized mammary epithelial cells in which ZO-1 and claudin-3, apical tight junction proteins, are mis-localized. CPEB depletion from mammary epithelial cells disrupts ZO-1 apical localization and tight junction distribution; conversely, ectopic expression of CPEB enhances ZO-1 localization. CPEB and ZO-1 mRNA are co-localized apically and ZO-1 3’UTR binding sites for CPEB are necessary for RNA localization. In a 3-dimensional culture system that models lumen-containing mammary ducts, depletion of CPEB or ZO-1 impairs central cavity formation, indicating a loss of cell polarity. Cavity formation in ZO-1 depleted cells is rescued when they are transduced with ZO-1 mRNA containing, but not lacking, CPEB binding sites. Our data demonstrates that CPEB-mediated ZO-1 mRNA localization is essential for tight junction assembly and mammary epithelial cell polarity.

Involvement of GATA transcription factors in the regulation of endogenous bovine interferon‐Tau gene transcription

Expression of interferon‐tau (IFNT), necessary for pregnancy establishment in ruminant ungulates, is regulated in a temporal and spatial manner. However, molecular mechanisms by which IFNT gene transcription is regulated in this manner have not been firmly established. In this study, DNA microarray/RT‐PCR analysis between bovine trophoblast CT‐1 and Mardin–Darby bovine kidney (MDBK) cells was initially performed, finding that transcription factors GATA2, GATA3, and GATA6 mRNAs were specific to CT‐1 cells. These mRNAs were also found in Days 17, 20, and 22 (Day 0u2009=u2009day of estrus) bovine conceptuses. In examining other bovine cell lines, ovary cumulus granulosa (oCG) and ear fibroblast (EF) cells, GATA2 and GATA3, but not GATA6, were found specific to the bovine trophoblast cells. In transient transfection analyses using the upstream region (−631 to +59u2009bp) of bovine IFNT gene (bIFNT, IFN‐tau‐c1), over‐expression of GATA2/GATA3 did not affect the transcription of bIFNT‐reporter construct in human choriocarcinoma JEG3 cells. Transfection of GATA2, GATA3, ETS2, and/or CDX2, however, was effective in the up‐regulation of the bIFNT construct transfected into bovine oCG and EF cells. One Point mutation studies revealed that among six potential GATA binding sites located on the upstream region of the bIFNT gene, the one next to ETS2 site exhibited reduced luciferase activity. In CT‐1 cells, endogenous bIFNT gene transcription was up‐regulated by over‐expression of GATA2 or GATA3, but down‐regulated by siRNA specific to GATA2 mRNA. These data suggest that GATA2/3 is involved in trophoblast‐specific regulation of bIFNT gene transcription. Mol. Reprod. Dev. 76: 1143–1152, 2009.

l-Amino acid oxidase plays a crucial role in host defense in the mammary glands

The innate immune system plays an important role in protecting organs that are continuous with the outer surface of the body from bacterial infection. The antibacterial factors involved in this system have been sought in exocrine glands, particularly in the mammary glands. Because milk produced in the mammary glands is enriched in various nutrients, supporting the proliferation of bacteria, mammary glands appear to be at the greatest risk of bacterial infection and proliferation. Here, we show that mouse milk contains L‐amino acid oxidase (LAO), a lactating mammary gland‐specific protein that displays antibacterial activity in vitro through the production of hydrogen peroxide from free amino acids. We produced LAO‐disrupted mouse lines to define the physiological properties and importance of the protein in vivo. The LAO‐knockout mice were healthy and had normal mammary gland development;however, the antibacterial activity normally observed in milk from wild‐type mice was absent from the milk of knockout mice. The content of free amino acids targeted by LAO was very low in wild‐type milk, whereas these amino acids were abundant in LAO‐knockout milk. Knockout mice exhibited weak resistance to an intramammary bacterial challenge compared to their wild‐type counterparts. Further, preadministration of wild‐type milk whey reduced the severity of bacterial infection in LAO‐knockout mice. These results demonstrate that milk LAO protects the mammary gland against bacterial infection, and this antibacterial effect may be due to the generation of hydrogen peroxide by using free amino acids abundantly present in milk. — Nagaoka, K.,Aoki, F., Hayashi, M., Muroi, Y., Sakurai, T., Itoh, K., Ikawa, M., Okabe, M., Imakawa, K., Sakai, S. L Amino acid oxidase plays a crucial role in host defense in the mammary glands. FASEBJ. 23, 2514–2520 (2009)

Changes in Immune Cell Distribution and IL‐10 Production are Regulated through Endometrial IP‐10 Expression in the Goat Uterus

Problem:u2002 Changes in distribution or redistribution of immune cells are required for the establishment and maintenance of pregnancy, but these changes during early pregnancy have been poorly understood in the ruminant ungulates. Expression of a chemokine, interferon‐γ (IFN‐γ)‐inducible protein 10u2003kDa (IP‐10, CXCL10), was identified in the endometrium of pregnant goats. Population and/or distribution of endometrial immune cells and their cytokine productions could be regulated by IP‐10 during the period of pregnancy establishment.

CD9 regulates transcription factor GCM1 and ERVWE1 expression through the cAMP/protein kinase A signaling pathway

ERVWE1 (SYNCYTIN-1), a membrane protein originating from the envelope gene of human endogenous retrovirus-W (HERV-W), mediates the fusion of mononucleated cytotrophoblasts into multinucleated syncytiotrophoblast. Though ERVWE1 has been characterized since its discovery, regulatory mechanisms associated with ERVWE1 expression have not been firmly established. We hypothesized that membrane protein CD9, involved in cell-cell fusion of fertilization and myogenesis, could be involved in the regulation of ERVWE1 gene expression. In this study, regulatory mechanisms of ERVWE1 expression were studied using human choriocarcinoma BeWo cells. Forskolin is an activator of adenylate cyclase, which increased CD9 and ERVWE1 expression. The increase in CD9 expression was inhibited by a protein kinase A (PKA) inhibitor, Rp-cAMPS. These results indicate that CD9 expression is regulated by the cAMP/PKA signaling pathway. Overexpression of CD9 increased expression levels of ERVWE1 as well as GCM1 (hGCMa), which is a transcription factor known to activate ERVWE1 gene transcription. However, high ERVWE1 expression induced by CD9 overexpression did not result in the increase in chorionic gonadotropin, beta polypeptide production. Moreover, CD9-induced increase in ERVWE1 and GCM1 expressions were inhibited by Rp-cAMPS. These results suggest that CD9 increases GCM1 expression via the cAMP/PKA signaling pathway, resulting in the increase in ERVWE1 expression.

Interleukin-1 receptor antagonist expression in the equine endometrium during the peri-implantation period.

To identify factors involved in the establishment of pregnancy in the mare, endometrium was collected from day 13 (day 0=day of ovulation) cyclic and day 13, 19, and 25 pregnant animals. From initial cDNA subtraction studies, interleukin-1 receptor antagonist (IL-1RN) mRNA was found as a candidate molecule expressed uniquely in the pregnant endometrium. Expression of IL-1RN mRNA was markedly increased in day 19 and 25 gravid endometrium. In situ hybridization analysis revealed that IL-1RN mRNA was localized to the glandular epithelium. Interleukin-1 receptor antagonist (IL-1RN) protein was found in the extracts of day 25 gravid endometrium and was immunochemically localized to the glandular epithelium/luminal cavity of the pregnant uterus. High concentrations of estradiol-17beta (E(2)) were detected in day 25 conceptuses. Concentrations of E(2) were higher in the gravid endometrial portion than in other endometrial regions. On the other hand, progesterone concentrations did not differ among endometrial samples analyzed. Furthermore, the expression of IL-1RN mRNA was up-regulated in endometrium culture samples treated with 10 ng/mL E(2) and 10 ng/mL progesterone. In the analysis of related gene expression, increased amounts of IL-1alpha and IL-6 mRNA were also found in the day 25 gravid endometrium; however, these expressions in endometrial culture samples were not up-regulated by the steroid treatment. These results indicate that expression of IL-1RN in the endometrium is likely regulated by E(2) and progesterone and suggest that IL-1RN regulates the degree of IL-1 signal transduction and thereby plays an important role in the establishment of equine pregnancy.

Mechanisms responsible for increase in circulating inhibin levels at the time of ovulation in mares

In female mammals, inhibin is secreted by the granulosa cells and selectively inhibits secretion of FSH. Although circulating immunoreactive (ir)-inhibin levels decrease after ovulation as a result of the disappearance of its main source, they abruptly increase at the time of ovulation in mares. To investigate the mechanisms responsible for this increase, 50 ml of equine follicular fluid (eFF) was administered into the abdominal cavity of mares during the luteal phase (eFF, n = 4). One hour after treatment, plasma levels of ir-inhibin and inhibin pro-alphaC (but not estradiol-17beta) were significantly higher in eFF-treated mares than in control mares (n = 4). The hormone profiles in eFF-treated mares were similar to those in mares with the spontaneous or hCG induced ovulations. The present study demonstrates that the release of follicular fluid into the abdominal cavity when the follicle ruptures is responsible for the ovulatory inhibin surge in the mare. These findings also suggest that circulating inhibin pro-alphaC may be useful for determining the time of ovulation in the mare.