Molecular profiling demonstrates modulation of immune cell function and matrix remodeling during luteal rescue†.

The corpus luteum (CL) is essential for maintenance of pregnancy in all mammals and luteal rescue, which occurs around day 16-19 in the cow, is necessary to maintain luteal progesterone production. Transcriptomic and proteomic profiling were performed to compare the day 17 bovine CL of the estrous cycle and pregnancy. Among mRNA and proteins measured, 140 differentially abundant mRNA and 24 differentially abundant proteins were identified. Pathway analysis was performed using four programs. Modulated pathways included T cell receptor signaling, vascular stability, cytokine signaling, and extracellular matrix remodeling. Two mRNA that were less in pregnancy were regulated by prostaglandin F2A in culture, while two mRNA that were greater in pregnancy were regulated by interferon tau. To identify mRNA that could be critical regulators of luteal fate, the mRNA that were differentially abundant during early pregnancy were compared to mRNA that were differentially abundant during luteal regression. Eight mRNA were common to both datasets, including mRNA related to regulation of steroidogenesis and gene transcription. A subset of differentially abundant mRNA and proteins, including those associated with extracellular matrix functions, were predicted targets of differentially abundant microRNA (miRNA). Integration of miRNA and protein data, using miRPath, revealed pathways such as extracellular matrix-receptor interactions, abundance of glutathione, and cellular metabolism and energy balance. Overall, this study has provided a comprehensive profile of molecular changes in the corpus luteum during maternal recognition of pregnancy and has indicated that some of these functions may be miRNA-regulated.

Replication Study: Systematic identification of genomic markers of drug sensitivity in cancer cells.

In 2016, as part of the Reproducibility Project: Cancer Biology, we published a Registered Report (Vanden Heuvel et al., 2016), that described how we intended to replicate selected experiments from the paper 'Systematic identification of genomic markers of drug sensitivity in cancer cells' (Garnett et al., 2012). Here we report the results. We found Ewing's sarcoma cell lines, overall, were more sensitive to the PARP inhibitor olaparib than osteosarcoma cell lines; however, while the effect was in the same direction as the original study (Figure 4C; Garnett et al., 2012), it was not statistically significant. Further, mouse mesenchymal cells transformed with either the EWS-FLI1 or FUS-CHOP rearrangement displayed similar sensitivities to olaparib, whereas the Ewing's sarcoma cell line SK-N-MC had increased olaparib sensitivity. In the original study, mouse mesenchymal cells transformed with the EWS-FLI1 rearrangement and SK-N-MC cells were found to have similar sensitivities to olaparib, whereas mesenchymal cells transformed with the FUS-CHOP rearrangement displayed a reduced sensitivity to olaparib (Figure 4E; Garnett et al., 2012). We also studied another Ewing's sarcoma cell line, A673: A673 cells depleted of EWS-FLI1 or a negative control both displayed similar sensitivities to olaparib, whereas the original study reported a decreased sensitivity to olaparib when EWS-FLI1 was depleted (Figure 4F; Garnett et al., 2012). Differences between the original study and this replication attempt, such as the use of different sarcoma cell lines and level of knockdown efficiency, are factors that might have influenced the outcomes. Finally, where possible, we report meta-analyses for each result.

Changes in MicroRNA Expression During Maturation of the Bovine Corpus Luteum: Regulation of Luteal Cell Proliferation and Function by MicroRNA-34a.

The corpus luteum (CL) develops from the remnants of the ovulatory follicle and produces progesterone, required for maintenance of pregnancy in mammals. The differentiation of granulosal and thecal cells into luteal cells is accompanied by hypertrophy and hyperplasia of cells. As the CL matures, growth ceases and in ruminants, the tissue acquires the ability to undergo regression in response to prostaglandin F2alpha. The regulators of this transition are poorly understood. MicroRNA, which are posttranscriptional regulators of tissue development and function, are expressed in the CL. However, the pattern of their expression and their function during the transition from developing to functional CL is not known. The objectives of this study were to profile the expression of miRNA in developing versus mature bovine CL and determine effects of miRNA on bovine luteal cell survival and function. Knockdown of Drosha in midcycle (MC) luteal cells decreased progesterone and increased luteal cell apoptosis in the presence or absence of proinflammatory cytokines. Microarray analysis demonstrated that a greater number of miRNA were expressed in MC compared to D4 CL. Ingenuity pathway analysis (IPA) predicted that D4-specific miRNA regulate pathways related to carbohydrate metabolism, while MC-specific miRNA regulate pathways related to cell cycle and apoptosis signaling. Both predictions are consistent with a switch in the CL from a growing phase to a maintenance phase. One of the MC specific miRNA, miR-34a, was selected for further analysis. Increased concentrations of miR-34a in MC luteal cells resulted in decreased luteal cell proliferation, increased progesterone production, and inhibition of Notch1 and YY1 translation, but had no effect on luteal cell apoptosis. In conclusion, these data support a role for miRNA in general, and miR-34a in particular, in luteal formation and function.

Regulating life or death: potential role of microRNA in rescue of the corpus luteum.

The role of miRNA in tissue biology has added a new level of understanding of gene regulation and function. The corpus luteum (CL) is a transitory endocrine gland; the dynamic nature of the CL makes it a candidate for regulation by miRNA. Rescue of the CL from luteolysis is essential for the maintenance of pregnancy in all eutherian mammals. Using next generation sequencing, we profiled miRNA expression in the bovine CL during maternal recognition of pregnancy. We identified 590 luteal miRNA, of which 544 were known and 46 were novel miRNAs. Fifteen (including 3 novel) miRNAs were differentially expressed between CL of pregnant vs. cyclic animals. Target analysis of the differentially expressed miRNA resulted in genes involved in regulating apoptosis and immune response, providing evidence that miRNAs regulate the intracellular pathways that lead to either luteal regression or survival.

Progesterone effects on lymphocytes may be mediated by membrane progesterone receptors.

Luteal cell-induced proliferation of T lymphocytes devoid of the nuclear progesterone receptor (PGR) is inhibited by progesterone. Functional effects of progesterone on bovine lymphocytes and the expression of membrane progesterone receptors (mPRs) alpha (PAQR7), beta (PAQR8), gamma (PAQR5), and progesterone receptor membrane component 1 (PGRMC1) mRNA were analyzed in corpus luteum (CL) and lymphocytes. Progesterone and a cell-impermeable progesterone conjugate caused a dose-dependent decrease in IL2 receptor α-subunit (IL2RA) mRNA and an increase in interleukin 2 (IL2) mRNA concentrations in cultured PBMCs. In luteal tissues, concentrations of PAQR7 and PAQR8 mRNA were lower in CL collected on day 11 compared with day 18, whereas PGRMC1 and PGR mRNA were greater on day 11 than on day 18. The mRNA of all three PAQRs and PGRMC1 were detected in bovine T lymphocytes, but not in B cells/monocytes. Progesterone increased intracellular Ca(++) and reduced the phosphorylation of zeta-chain-associated protein kinase 70 (Zap70). A specific, saturable, and single progesterone binding site with a steroid specificity characteristic of mPRs was demonstrated by saturation and competitive binding assays using T lymphocyte membranes, and PAQR7 receptors were localized on the plasma membranes by immunofluorescence. Thus, progesterone induces specific and rapid functional effects on T lymphocytes in the absence of PGR. The mPRs are potential intermediaries of the cell-surface actions of progesterone because they are expressed in lymphocytes, the actions of progesterone are mimicked by a cell-impermeable form of progesterone, and specific, saturable progesterone binding, which is characteristic of mPRs, is present on lymphocyte membranes.

Epidermal α6ß4 integrin stimulates the influx of immunosuppressive cells during skin tumor promotion.

BACKGROUND: Induction of α6ß4 integrin in the differentiated epidermal cell layers in skin is a hallmark of human cutaneous squamous cell carcinoma (SCC) pathogenesis and stimulates chemically induced SCC formation in Invα6ß4 transgenic mice, which exhibit persistent expression of α6ß4 in the suprabasal epidermal layers. However, the molecular basis for the support of SCC development by suprabasal α6ß4 is not fully understood. OBJECTIVE: We examined the relevance for suprabasal α6ß4 expression in the epidermis for the recruitment of immunosuppressive leukocytes during the early stages of tumor promotion. METHODS: In this study, we made use of the Invα6ß4 transgenic mouse model, which exhibits expression of α6ß4 integrin in the suprabasal layers of the epidermis driven by the involucrin promoter. First, we examined protein lysates from Invα6ß4 transgenic skin using a pro-inflammatory cytokine array panel. Next, we immunofluorescence labeling of murine skin sections was employed to immunophenotype tumor promoter-treated Invα6ß4 transgenic skin. Finally, a macrophage colony stimulating factor (M-CSF) neutralizing antibody strategy was administered to resolve Invα6ß4 transgenic skin inflammation. RESULTS: Employing the Invα6ß4 transgenic mouse model, we show that suprabasal α6ß4 integrin expression selectively alters the profile of secreted pro-inflammatory molecules by epidermal cells, in particular CXCL5 and M-CSF, in response to acute tumor promoter treatment. The induction of CXCL5 and M-CSF in Invα6ß4 transgenic epidermis was shortly followed by an exacerbated influx of CD200R(+) myeloid-derived suppressor cells (MDSCs), which co-expressed the M-CSF receptor, and FoxP3(+) Treg cells compared to wild-type mice. As a result, the levels of activated CD4(+) T lymphocytes were dramatically diminished in Invα6ß4 transgenic compared to wild-type skin, whereas similar levels of lymphocyte activation were observed in the peripheral blood. Finally, 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced CD200R(+) infiltrative cells and epidermal proliferation were suppressed in Invα6ß4 mice treated with M-CSF neutralizing antibodies. CONCLUSIONS: We conclude that aberrant expression of α6ß4 integrin in post-mitotic epidermal keratinocytes stimulates a pro-tumorigenic skin microenvironment by augmenting the influx of immunosuppressive granular cells during tumor promotion.

Inflammatory responses in epithelia: endotoxin-induced IL-6 secretion and iNOS/NO production are differentially regulated in mouse mammary epithelial cells.

BACKGROUND: IL-6 is a pro-inflammatory cytokine that signals via binding to a soluble or membrane bound receptor, while nitric oxide (NO), an oxidative stress molecule, diffuses through the cell membrane without a receptor. Both mediators signal through different mechanisms, yet they are dependent on NFκB. We proposed that both mediators are co-induced and co-regulated in inflamed mammary epithelial cells. METHODS: SCp2 mammary epithelial cells were treated with bacterial endotoxin (ET) for different time periods and analyzed for induction of IL-6 secretion and NO production by ELISA and Griess reaction, respectively. The expression of IL-6 and induced NO synthase (iNOS) was assayed by real time PCR and/or western immunoblots, and the activation of NFκB was assayed by immunobinding assay. To investigate the role of mammary cell microenvironment (cell-substratum or interaction of mammary epithelial cell types; critical to mammary development, function, and disease) in modulation of the inflammatory response, SCp2 cells were cultured with or without extracellular matrix (EHS) or in coculture with their myoepithelial counterpart (SCg6), and assayed for ET-induced IL-6 and NO. RESULTS: Endotoxin induced NFκB activation at 1 h after ET application. IL-6 secretion and NO production were induced, but with unexpected delay in expression of mRNA for iNOS compared to IL-6. NFκB/p65 activation was transient but NFκB/p50 activation persisted longer. Selective inhibition of NFκB activation by Wedelolactone reduced ET-induced expression of IL-6 mRNA and protein but not iNOS mRNA or NO production, suggesting differences in IL-6 and iNOS regulation via NFκB. SCp2 cells in coculture with SCg6 but not in presence of EHS dramatically induced IL-6 secretion even in the absence of ET. ET-induced NO production was blunted in SCp2/SCg6 cocultures compared to that in SCp2 alone. CONCLUSIONS: The differential regulation of IL-6 and iNOS together with the differential activation of different NFκB dimers suggest that IL-6 and iNOS are regulated by different NFκB dimers, and differentially regulated by the microenvironment of epithelial cells. The understanding of innate immune responses and inflammation in epithelia and linkage thereof is crucial for understanding the link between chronic inflammation and cancer in epithelial tissues such as the mammary gland.