Zinc Sensing Receptor Signaling, Mediated by GPR39, Reduces Butyrate-Induced Cell Death in HT29 Colonocytes via Upregulation of Clusterin

Zinc enhances epithelial proliferation, protects the digestive epithelial layer and has profound antiulcerative and antidiarrheal roles in the colon. Despite the clinical significance of this ion, the mechanisms linking zinc to these cellular processes are poorly understood. We have previously identified an extracellular Zn2+ sensing G-protein coupled receptor (ZnR) that activates Ca2+ signaling in colonocytes, but its molecular identity as well as its effects on colonocytes' survival remained elusive. Here, we show that Zn2+, by activation of the ZnR, protects HT29 colonocytes from butyrate induced cell death. Silencing of the G-protein coupled receptor GPR39 expression abolished ZnR-dependent Ca2+ release and Zn2+-dependent survival of butyrate-treated colonocytes. Importantly, GPR39 also mediated ZnR-dependent upregulation of Na+/H+ exchange activity as this activity was found in native colon tissue but not in tissue obtained from GPR39 knock-out mice. Although ZnR-dependent upregulation of Na+/H+ exchange reduced the cellular acid load induced by butyrate, it did not rescue HT29 cells from butyrate induced cell death. ZnR/GPR39 activation however, increased the expression of the anti-apoptotic protein clusterin in butyrate-treated cells. Furthermore, silencing of clusterin abolished the Zn2+-dependent survival of HT29 cells. Altogether, our results demonstrate that extracellular Zn2+, acting through ZnR, regulates intracellular pH and clusterin expression thereby enhancing survival of HT29 colonocytes. Moreover, we identify GPR39 as the molecular moiety of ZnR in HT29 and native colonocytes

Uterine Foxl2 regulates the adherence of the Trophectoderm cells to the endometrial epithelium

Abstract Background Forkhead Transcription Factor L2 (FOXL2) is a member of the forkhead family with important roles in reproduction. Recent studies showed that FOXL2 is expressed in human and bovine endometrium and that its levels fluctuate during pregnancy. In this study, we aimed at evaluating the expression and function of FOXL2 in embryo implantation. Methods Mouse uteri at different days of pregnancy were isolated and analyzed for the expression and localization of FOXL2. A lentiviral strategy was further employed to either knockdown or overexpress FOXL2 in non-receptive human endometrial AN3-CA cells and in receptive Ishikawa cells, respectively. These genetically modified cells were compared to cells infected with a control lentivirus to determine the function of FOXL2 in trophectoderm cells adherence to Endometrial Epithelium was associated with the expression of genes known to be involved in acquisition of uterine receptivity. Results We report that FOXL2 is expressed in both, the luminal epithelium and the myometrium of the mouse uterus and that its expression declines prior to implantation. We found that endometrial cells expressing low FOXL2 levels, either endogenous or genetically manipulated, were associated with a higher attachment rate of mouse blastocysts or human Jeg3 spheroids and mouse blastocysts. In accordance, low-FOXL2 levels were associated with changes in the expression level of components of the Wnt/Fzd and apoptotic pathways, both of which are involved in uterine receptivity. Furthermore, FOXL2 expression was inversely correlated with G-protein signaling protein 2 (Rgs2) and cytokine expression. Conclusions These results suggest that FOXL2 interferes with embryo attachment. Better understanding of the function of FOXL2 in the uterus would possibly suggest novel strategies for treatment of infertility attributed to repeated implantation failure

Additional file 1: of Uterine Foxl2 regulates the adherence of the Trophectoderm cells to the endometrial epithelium

Figure S1. Embryos attachment to endometrial cells. Mouse blastocysts co-incubated with endometrial cell lines for 48 h (A). Attached embryos that stayed on the plate after the plates were washed and shaken (B). Thin arrows indicate attached embryos, thick arrow indicate unattached embryos. (C) The localization of osteopentin, an adhesion molecule, between the embryo and the endometrial cells. Osteopentin-green, DAPI-blue. (TIFF 2025 kb

Additional file 3: of Uterine Foxl2 regulates the adherence of the Trophectoderm cells to the endometrial epithelium

Figure S2. FOXL2 localization in the ovary. FOXL2 expressed by granulosa cells of early follicles declines at later stages of folliculogenesis. Blue = DNA staining (DAPI), RED = FOXL2. EA-Early antral, GF-Graffian Folicle. (TIFF 2025 kb

Activation of the Phenylpropanoid Pathway in <i>Nicotiana tabacum</i> Improves the Performance of the Whitefly <i>Bemisia tabaci</i> via Reduced Jasmonate Signaling

<div><p>Background</p><p>Phloem-feeding insects can manipulate plant-induced resistance and are able to suppress effective jasmonic acid/ethylene (JA/ET) defenses by the induction of inefficient salicylic acid (SA) based responses. As a result, activation of the phenylpropanoid biosynthesis pathway in transgenic plants is anticipated to cause complex interactions between phloem-feeding insects and their host plants due to predicted contradiction between two defense forces: the toxicity of various phenylpropanoids and the accumulation of SA via a branch of the activated pathway.</p><p>Methodology/Principal Findings</p><p>Here, we investigated the effect of activating the phenylpropanoids pathway in <i>Nicotiana tabacum</i>, by over-expression of the PAP1 transcription factor, on the whitefly <i>Bemisia tabaci</i>, a phloem-feeding insect model. Our performance assays indicated that the over-expression made the transgenic plants a more suitable host for <i>B. tabaci</i> than wild-type (WT) plants, although these plants accumulated significantly higher levels of flavonoids. Transcription analyses of indicator genes in the SA (<i>PR1a</i>) and JA/ET (<i>ERF1</i>, <i>COI1</i> and <i>AOC</i>) pathways followed by quantification of the SA and JA hormone levels, indicated that <i>B. tabaci</i> infestation periods longer than 8 hours, caused higher levels of activity of SA signaling in transgenic plants and higher levels of JA/ET signaling in WT plants.</p><p>Conclusions/Significance</p><p>Taken together, these results emphasize the important role JA/ET-induced defenses play in protecting plants from successful infestation by <i>B. tabaci</i> and likely other phloem-feeding insects. It also indicates the necessity of phloem feeders to suppress these defenses for efficient utilization of plant hosts. Our data also indicate that the defensive chemistry produced by the phenylpropanoids pathway has only a minor effect on the insect fitness.</p></div

Hyaluronan control of the primary vascular barrier during early mouse pregnancy is mediated by uterine NK cells

Successful implantation is associated with a unique spatial pattern of vascular remodeling, characterized by profound peripheral neovascularization surrounding a periembryo avascular niche. We hypothesized that hyaluronan controls the formation of this distinctive vascular pattern encompassing the embryo. This hypothesis was evaluated by genetic modification of hyaluronan metabolism, specifically targeted to embryonic trophoblast cells. The outcome of altered hyaluronan deposition on uterine vascular remodeling and postimplantation development were analyzed by MRI, detailed histological examinations, and RNA sequencing of uterine NK cells. Our experiments revealed that disruption of hyaluronan synthesis, as well as its increased cleavage at the embryonic niche, impaired implantation by induction of decidual vascular permeability, defective vascular sinus folds formation, breach of the maternal-embryo barrier, elevated MMP-9 expression, and interrupted uterine NK cell recruitment and function. Conversely, enhanced deposition of hyaluronan resulted in the expansion of the maternal-embryo barrier and increased diffusion distance, leading to compromised implantation. The deposition of hyaluronan at the embryonic niche is regulated by progesterone-progesterone receptor signaling. These results demonstrate a pivotal role for hyaluronan in successful pregnancy by fine-tuning the periembryo avascular niche and maternal vascular morphogenesis

Chemical analyses of SA and JA hormone levels.

<p>A) SA levels in WT (gray bars) and PAP green (black bars) plants before and after 8 h, 48 h, 5 days and 18 days of <i>B. tabaci</i> infestation. B) JA levels in WT (gray bars) and PAP green (black bars) plants during the same infestation periods. Values are means of ng SA or JA per g fresh weight (FW) ± standard error. Asterisks indicate significant differences (<i>P</i>≤0.05) between WT and PAP green plants in each time point. The hat symbol indicates nearly significant differences (<i>P</i> = 0.07).</p

The proportion of progeny that had reached advanced stages of development (late 4^th nymphs and adults) by day 25 on WT (gray bars) and PAP green (black bars) plants under the three spraying regimes: SA, MeJA and control (DDW/0.015% Tween-20).

<p>Sequential Bonferroni a priori (planned) comparisons (log-likelihood ratio test) were performed on four selected pairs, using the conservative Dunn-Sidak method. A) MeJA-WT vs. MeJA-PAP green. B) SA-WT vs. SA-PAP green. C) Control-WT vs. MeJA-WT. D) Control-PAP green vs. MeJA-PAP green. Asterisks indicate significant differences (<i>P</i>≤0.05) between pairs.</p

Time-course analyses of SA and JA pathway marker genes expression in WT (gray bars) and PAP green (black bars) plants before and after 8 h, 48 h, 5 days and 18 days of <i>B. tabaci</i> infestation.

<p>A) <i>PR1a</i>. B) <i>ERF1</i>. C) <i>COI1</i>. D) <i>AOC</i>. Values represent mean 2^(−ΔCt) ± standard error. Asterisks indicate significant differences (<i>P</i>≤0.05) between WT and PAP green plants at each time point as detected by <i>a priori</i> and orthogonal comparisons. The hat symbol indicates nearly significant differences (<i>P</i> = 0.07).</p

No-Choice performance experiments on WT and PAP green plants.

<p>A) Proportion of surviving adults on WT (gray bars) and PAP green (black bars) plants over a period of 72 h. B) The mean daily number of oviposited eggs per female on WT (gray bars) and PAP green (black bars) plants. C) The mean survival of progeny on WT and on PAP green plants. D) The proportion of progeny that had reached different stages of development (2^nd to early 4^th nymphs, late 4^th nymphs and adults) at day 18. Asterisks indicate significant differences (<i>P</i>≤0.05) between WT and PAP green plants. Error bars represent standard error of the means.</p