Ubp43 gene expression is required for normal Isg15 expression and fetal development

BACKGROUND: Isg15 covalently modifies murine endometrial proteins in response to early pregnancy. Isg15 can also be severed from targeted proteins by a specific protease called Ubp43 (Usp18). Mice lacking Ubp43 (null) form increased conjugated Isg15 in response to interferon. The Isg15 system has not been examined in chorioallantoic placenta (CP) or mesometrial (MM) components of implantation sites beyond 9.5 days post coitum (dpc). It was hypothesized that deletion of Ubp43 would cause disregulation of Isg15 in implantation sites, and that this would affect pregnancy rates. METHODS: Heterozygous (het) Ubp43 mice were mated and MM and CP implantation sites were collected on 12.5 and 17.5 days post-coitum (dpc). RESULTS: Free and conjugated Isg15 were greater on 12.5 versus 17.5 dpc in MM. Free and conjugated Isg15 were also present in CP, but did not differ due to genotype on 12.5 dpc. However, null CP had greater free and conjugated Isg15 when compared to het/wt on 17.5 dpc. Null progeny died in utero with fetal genotype ratios (wt:het:null) of 2:5:1 on 12.5 and 2:2:1 on 17.5 dpc. Implantation sites were disrupted within the junctional zone and spongiotrophoblast, contained less vasculature based on lectin B4 staining and contained greater Isg15 mRNA and VEGF protein in Ubp43 null when compared to wt placenta. CONCLUSION: It is concluded that Isg15 and its conjugates are present in implantation sites during mid to late gestation and that deletion of Ubp43 causes an increase in free and conjugated Isg15 at the feto-maternal interface. Also, under mixed genetic background, deletion of Ubp43 results in fetal death

Mutant mouse models and their contribution to our knowledge of corpus luteum development, function and regression

Abstract The corpus luteum is a unique organ, which is transitory in nature. The development, maintenance and regression of the corpus luteum are regulated by endocrine, paracrine and autocrine signaling events. Defining the specific mediators of luteal development, maintenance and regression has been difficult and often perplexing due to the complexity that stems from the variety of cell types that make up the luteal tissue. Moreover, some regulators may serve dual functions as a luteotropic and luteolytic agent depending on the temporal and spatial environment in which they are expressed. As a result, some confusion is present in the interpretation of in vitro and in vivo studies. More recently investigators have utilized mutant mouse models to define the functional significance of specific gene products. The goal of this mini-review is to identify and discuss mutant mouse models that have luteal anomalies, which may provide some clues as to the significance of specific regulators of corpus luteum function.</p

Deletion of the Isg15 Gene Results in Up-Regulation of Decidual Cell Survival Genes and Down-Regulation of Adhesion Genes: Implication for Regulation by IL-1β

The ubiquitin homolog interferon stimulated gene 15 (ISG15) is up-regulated in the endometrium in response to pregnancy in primates, ruminants, pigs, and mice. ISG15 covalently attaches to intracellular proteins (isgylation) and regulates numerous intracellular responses. We hypothesized that ISG15 depletion (Isg15−/−) alters decidual tissue gene expression and that IL-1β induces ISG15 expression and isgylation in cultured murine decidual explants and human uterine fibroblasts (HuFs). After studying the reproductive phenotype, contrary to earlier reports, up to 50% of the fetuses die between 7.5 and 12.5 d post coitum (dpc) in Isg15−/− mothers when mated to Isg15−/− fathers. Using microarray analysis, over 500 genes are differentially regulated in 7.5 dpc deciduas from Isg15−/− compared with Isg15+/+ mice. The gene for interferon-inducible protein 202b, which functions in cell-survival mechanisms, was up-regulated (mRNA and protein) in deciduas from Isg15−/− mice. Culture of Isg15+/+ mouse decidual explants (7.5 dpc) with IL-1β decreased Isg15 mRNA but increased free and conjugated ISG15. In predecidual HuF cells, IL-1β treatment increased ISG15 mRNA and isgylation. Additionally, IL-1β up-regulated expression of enzymes (HERC5, UBCH8) that coordinate the covalent conjugation of ISG15 to target proteins, as well as the gene that encodes the deisglyation enzyme UBP43 in HuF cells. In conclusion, deletion of Isg15 gene results in 50% fetal loss after 7.5 dpc, which can be explained through differential decidual gene expression that is functionally tied to cell survival and adhesion pathways. This fetal death also might relate to impaired IL-1β signaling, because ISG15 and isgylation are induced by IL-1β in human and murine endometrial stromal cells

Cytokeratin 18 expression inhibits Cytokine-induced death of cervical cancer cells

Objectives: In cervical cancer, increased cytokeratin 18 (CK18) filament expression is associated with disease progression. However, it may also provide resistance to cytokine-induced apoptosis. The present study tested whether CK18 expression influences susceptibility to cytokine-induced apoptosis. Methods: The cervical cancer cell lines C-4II (high CK18 expression), ME-180 (low CK18 expression), and 2 subtypes of HeLa cells containing or lacking CK18 expression (CK18(+) and CK18(-) cells, respectively) were exposed to vehicle (control), Fas ligand (FasL) (50 ng/mL), or tumor necrosis factor alpha (TNF-alpha; 10 ng/mL) without/with cycloheximide (CHX; 2.5 mu g/mL) to test the hypothesis that diminished CK18 expression increases susceptibility to cytokine-induced apoptosis. Results: Flow cytometric analysis of cell death via TUNEL staining revealed that cytokine-induced apoptosis was 2-fold greater in ME-180 cells than C-4II cells in response to FasL+CHX or TNF-alpha+CHX (P \u3c 0.05). Similarly, there was a higher incidence of FasL-induced apoptosis in CK18(-) HeLa cells (23% and 91% apoptotic for FasL and FasL+CHX, respectively) than CK18(+) HeLa cells (1% and 11%, respectively; P \u3c 0.05). Surprisingly, TNF-alpha had no effect on either CK18(+) or CK18(-) HeLa cells (P \u3c 0.05). Caspase 3 activity was greater in CK18(-) HeLa cells than in CK18(+) HeLa cells at 8 and 18 hours after FasL treatment (P \u3c 0.05), an effect abrogated by the caspase 8 inhibitor IETD-fmk (P \u3c 0.05). Conclusions: Cervical cancer cells with diminished CK18 expression are more susceptible to cytokine-induced apoptosis, particularly in response to FasL treatment. These observations suggest that relative CK18 expression is an important factor when considering therapeutic strategies to enhance immune cell-mediated death of cervical cancer cells

Maternal and embryonic control of uterine sphingolipid-metabolizing enzymes during murine embryo implantation

During early gestation in invasively implanting species, the uterine stromal compartment undergoes dramatic remodeling, defined by the differentiation of stromal fibroblast cells into decidual cells. Lipid signaling molecules from a number of pathways are well-established functional components of this decidualization reaction. Because of a correlation in the events that transpire in the uterus during early implantation with known functions of bioactive sphingolipid metabolites established from studies in other organ systems, we hypothesized that uterine sphingolipid metabolism would change during implantation. By a combination of Northern blot, Western blot, and immunohistochemical analyses, we establish that enzymes at each of the major catalytic steps in the sphingolipid cascade become transcriptionally up-regulated in the uterus during decidualization. Each of the enzymes analyzed was up-regulated from Days of Pregnancy (DOP) 4.5-7.5. When comparing embryo-induced decidualization (decidual) with mechanically induced decidualization (deciduomal), sphingomyelin phosphodiesterase 1 (Smpd1) mRNA and sphingosine kinase 1 (SPHK1) protein were shown to be dually regulated in the endometrium by both maternal and embryonic factors. As measured by the diacyl glycerol kinase assay, ceramide levels rose in parallel with Smpd1 gene expression, suggesting that elevated transcription of sphingolipid enzymes results in heightened catalytic activity of the pathway. Altogether, these findings place sphingolipids on a growing list of lipid signaling molecules that become increasingly present at the maternal-embryonic interface

The Involvement of Proline-Rich 15 in Early Conceptus Development in Sheep1

The ruminant conceptus undergoes a period of elongation that is required for maternal recognition of pregnancy, prior to attaching to the endometrium. The purpose of these studies was to investigate the role of proline-rich 15 (PRR15) in the sheep conceptus by examining mRNA expression, protein localization, and the effect of PRR15 mRNA degradation. Conceptuses were collected on Days 11, 13, 15, 16, 17, 21, and 30 after mating. Quantitative RT-PCR showed expression of PRR15 mRNA corresponded with the process of trophoblast elongation, with peak expression occurring on Days 15 and 16. A recombinant ovine PRR15 was generated and used to create polyclonal antibodies against PRR15. Immunohistochemistry of a Day 15 conceptus indicated that PRR15 was localized predominantly in the nucleus of the trophectoderm and extraembryonic primitive endoderm. To test whether PRR15 was required during early conceptus development, RNA interference was employed. Blastocysts collected on Day 8 after mating were infected with a lentivirus expressing a short-hairpin RNA (shRNA) that targeted PRR15 mRNA for degradation, an shRNA containing a three-nucleotide mismatch to PRR15 mRNA, or a lentivirus expressing no shRNA. After infection, blastocysts were transferred into recipient ewes and collected back on Day 15 of gestation. Although the majority of the control and mismatched shRNA-treated conceptuses elongated and survived to Day 15, none of the embryos treated with the lentivirus expressing shRNA against PRR15 mRNA elongated, and most died. In conclusion, expression of PRR15 mRNA occurred during a narrow window of conceptus development, and degradation of PRR15 mRNA led to conceptus demise or abnormal development