Melanophilin, a novel aldosterone-induced gene in mouse cortical collecting duct cells.

The molecular mechanisms of aldosterone-regulated Na+ transport are not entirely clear. The goal of this study was to identify aldosterone-induced genes potentially involved in the trafficking of the epithelial Na+ channel (ENaC). We report that the transcript levels of melanophilin (MLPH), a protein involved in vesicular trafficking in melanocytes, are rapidly increased by aldosterone in cortical collecting duct (CCD) cells. This effect was near maximal at physiological aldosterone concentrations, indicating that it is mediated by the mineralocorticoid receptor. De novo protein synthesis is not required for the induction of MLPH mRNA by aldosterone. To determine whether this induction has functional consequences on transepithelial Na+ current, we generated clonal CCD cell lines that express a tetracycline-inducible MLPH. Induction of MLPH in these cells led to a relatively modest, but statistically significant, increase in amiloride-sensitive Na+ current, suggesting the MLPH may be involved in ENaC trafficking. MyosinVc, the epithelial-specific class V myosin that is highly homologous to MyosinVa, another component of the melanosome trafficking complex, has putative consensus sites for serum and glucocorticoid-induced kinase 1 (SGK1), an early aldosterone-induced kinase that mediates some of aldosterone's effects on Na+ transport. Our results indicate that MyosinVc is phosphorylated by endogenous SGK1, suggesting that this complex may be involved in the aldosterone-regulated trafficking of ENaC in the CCD. These results suggest potential mechanisms by which aldosterone may regulate Na+ transport both directly, by increasing the abundance of MLPH, and indirectly by increasing the transcription of SGK1, which in turn regulates the activity of MyosinVc.

Granulocyte-macrophage colony-stimulating-factor increases interferon-tau protein secretion in bovine trophectoderm cells.

PROBLEM: Uterine-derived factors are required for optimal conceptus development and secretion of the maternal recognition of pregnancy factor, interferon-tau (IFN-tau). Identifying these factors may lead to the development of schemes for increasing pregnancy success in cattle. METHOD OF STUDY: The objectives were to examine the effects of granulocyte-macrophage colony-stimulating-factor (GM-CSF) on trophectoderm proliferation rates and IFN-tau production, and verify the appropriateness of using an in vitro model of bovine trophectoderm (CT-1 cell). RESULTS: Rate of [(3)H]-thymidine incorporation into DNA was increased by supplementation of CT-1 medium with 10 or 100 ng/mL porcine (po) GM-CSF. GM-CSF supplementation to CT-1 medium also increased IFN-tau secretion. When results were normalized to account for number of CT-1 cells, 10 and 100 ng/mL poGM-CSF increased antiviral activity and IFN-tau concentrations (using an IFN-tau-specific enzyme-linked immunosorbent assay) in CT-1 conditioned medium compared with controls. CONCLUSIONS: These findings indicate that GM-CSF increases proliferation and IFN-tau production in bovine trophectoderm.

Fibroblast growth factor-2 is expressed by the bovine uterus and stimulates interferon-tau production in bovine trophectoderm.

Uterine-derived factors are essential for conceptus development and secretion of the maternal recognition-of-pregnancy factor, interferon-tau (IFNT), in ruminant species. The objectives of this study were to determine whether fibroblast growth factor-2 (FGF-2) is expressed in the bovine uterus during early pregnancy in cattle and to determine whether FGF-2 supplementation affects IFNT mRNA and protein abundance in bovine trophectoderm. FGF-2 mRNA was present in endometrium throughout the estrous cycle and was localized to the luminal and glandular endometrial epithelium at d 17-18 after estrus in pregnant and nonpregnant cows. Immunoreactive FGF-2 protein was detected within the endometrium and in the uterine lumen at d 17-18 after estrus, and concentrations did not differ based on pregnancy status. In a bovine trophectoderm cell line, CT-1, supplementation of medium with at least 1 ng/ml FGF-2 increased the incorporation of [(3)H]thymidine into DNA. Similarly, IFNT secretion from CT-1 cells increased after FGF-2 supplementation (1-100 ng/ml) for 72 h. Abundance of IFNT mRNA in CT-1 cells increased after 24 h exposure to 1, 10, or 100 ng/ml FGF-2. In bovine blastocysts, FGF-2 supplementation did not affect cell number after 72 h of culture but did stimulate IFNT protein concentrations in conditioned medium. In summary, FGF-2 is present in the uterine lumen during early pregnancy and increases IFNT mRNA and protein abundance in trophectoderm. The magnitude by which FGF-2 stimulates IFNT expression suggests that this uterine-derived factor plays an active role in regulating the establishment and maintenance of pregnancy in ruminants.