Uterine Endoplasmic Reticulum Stress and Its Unfolded Protein Response May Regulate Caspase 3 Activation in the Pregnant Mouse Uterus

We have previously proposed that uterine caspase-3 may modulate uterine contractility in a gestationally regulated fashion. The objective of this study was to determine the mechanism by which uterine caspase-3 is activated and consequently controlled in the pregnant uterus across gestation. Utilizing the mouse uterus as our gestational model we examined the intrinsic and extrinsic apoptotic signaling pathways and the endoplasmic reticulum stress response as potential activators of uterine caspase-3 at the transcriptional and translational level. Our study revealed robust activation of the uterine myocyte endoplasmic reticulum stress response and its adaptive unfolded protein response during pregnancy coinciding respectively with increased uterine caspase-3 activity and its withdrawal to term. In contrast the intrinsic and extrinsic apoptotic signaling pathways remained inactive across gestation. We speculate that physiological stimuli experienced by the pregnant uterus likely potentiates the uterine myocyte endoplasmic reticulum stress response resulting in elevated caspase-3 activation, which is isolated to the pregnant mouse myometrium. However as term approaches, activation of an elevated adaptive unfolded protein response acts to limit the endoplasmic reticulum stress response inhibiting caspase-3 resulting in its decline towards term. We speculate that these events have the capacity to regulate gestational length in a caspase-3 dependent manne

Toxic effects of trace phenol/guanidine isothiocyanate (P/GI) on cells cultured nearby in covered 96-well plates

Background: A mixture of phenol and guanidine isothiocyanate (“P/GI”, the principal components of TRIzol™ and similar products) is routinely used to isolate RNA, DNA, and proteins from a single specimen. In time-course experiments of cells grown in tissue culture, replicate wells are often harvested sequentially and compared, with the assumption that in-well lysis and complete aspiration of P/GI has no effect on continuing cultures in nearby wells. Methods: To test this assumption, we investigated morphology and function of RAW 264.7 cells (an immortalized mouse macrophage cell line) cultured in covered 96-well plates for 4, 8, or 24 h at varying distances from a single control well or a well into which P/GI had been deposited and immediately aspirated completely. Results: Time- and distance-dependent disruptions resulting from proximity to a single well containing trace residual P/GI were seen in cell morphology (blebbing, cytoplasmic disruption, and accumulation of intracellular vesicles), cell function (pH of culture medium), and expression of genes related to inflammation (Tnfα) and autophagy (Lc3b). There was no transcriptional change in the anti-apoptotic gene Mcl1, nor the pro-apoptotic gene Hrk, nor in P/GI-unexposed control cultures. LPS-stimulated cells incubated near P/GI had lower expression of the cytokine Il6. These effects were seen as early as 4 h of exposure and at a distance of up to 3 well units from the P/GI-exposed well. Conclusions: Exposure to trace residual quantities of P/GI in covered tissue culture plates leads to substantial disruption of cell morphology and function in as little as 4 h, possibly through induction of autophagy but not apoptosis. This phenomenon should be considered when planning time-course experiments in multi-well covered tissue culture plates.</p

Phagocyte-like NADPH oxidase generates ROS in INS 832/13 cells and rat islets: role of protein prenylation

Recent evidence suggests that an acute increase in the generation of phagocyte-like NADPH-oxidase (Nox)-mediated reactive oxygen species (ROS) may be necessary for glucose-stimulated insulin secretion. Using rat islets and INS 832/13 cells, we tested the hypothesis that activation of specific G proteins is necessary for nutrient-mediated intracellular generation of ROS. Stimulation of β-cells with glucose or a mixture of mitochondrial fuels (mono-methylsuccinate plus α-ketoisocaproic acid) markedly elevated intracellular accumulation of ROS, which was attenuated by selective inhibitors of Nox (e.g., apocynin or diphenyleneiodonium chloride) or short interfering RNA-mediated knockdown of p47phox, one of the subunits of Nox. Selective inhibitors of protein prenylation (FTI-277 or GGTI-2147) markedly inhibited nutrient-induced ROS generation, suggesting that activation of one (or more) prenylated small G proteins and/or γ-subunits of trimeric G proteins is involved in this signaling axis. Depletion of endogenous GTP levels with mycophenolic acid significantly reduced glucose-induced activation of Rac1 and ROS generation in these cells. Other immunosuppressants, like cyclosporine A or rapamycin, which do not deplete endogenous GTP levels, failed to affect glucose-induced ROS generation, suggesting that endogenous GTP is necessary for glucose-induced Nox activation and ROS generation. Treatment of INS 832/13 cells or rat islets with pertussis toxin (Ptx), which ADP ribosylates and inhibits inhibitory class of trimeric G proteins (i.e., Gi or Go), significantly attenuated glucose-induced ROS generation in these cells, implicating activation of a Ptx-sensitive G protein in these signaling cascade. Together, our findings suggest a prenylated Ptx-sensitive signaling step couples Rac1 activation in the signaling steps necessary for glucose-mediated generation of ROS in the pancreatic β-cells

Estrogen receptor alpha isoform ERdelta7 in myometrium modulates uterine quiescence during pregnancyResearch in context

Background: Circulating estrogen (E2) levels are high throughout pregnancy and increase towards term, however its local tissue specific actions vary across gestation. For example, myometrial E2 regulated uterotonic action is disabled until term, whereas it's proliferative function is maintained in the breast. We have identified gestationally regulated splicing events, mediated by hnRNPG and modulated by E2 that generate alternatively spliced estrogen receptor alpha (ERα) variants (ERΔ7 and ERα46) in the myometrium. These variants allow for differential, gestationally regulated, modulation of the uterotonic action of E2. Methods: Human myometrium isolated from preterm and term non-laboring and laboring pregnant women were analyzed for ERα isoforms and splice factor levels. Lentiviral mediated shRNA knockdown of hnRNPG and overexpression of ERΔ7 were performed in human myometrial (hTERT-HM) cells. Functional 3D collagen contraction assays were executed. Findings: ERΔ7 acts as a dominant negative repressor of the uterotonic action of ERα66 and ERα46 isoforms through the regulation of the myometrial gap junction protein GJA1. Elimination of hnRNPG inhibits the generation of ERΔ7 while overexpression of ERΔ7 inhibited GJA1 expression. Moreover in vivo human myometrial hnRNPG levels decline at term in an E2 dependent manner resulting in a withdrawal of ERΔ7 levels and its tocolytic action at term. Interpretation: Our findings implicate the unique role of ERΔ7 as a modulator of myometrial quiescence and define the mechanism of ERΔ7 generation, through hormonally regulated splicing events. Fund: This study was supported by NIH OPRU U01 supplement (HD047905), University of Pittsburgh and Wayne State University Perinatal Research Initiative (USA). Keywords: Estrogen receptor alpha, Alternative splicing, hnRNPG, Myometrium, Parturition, GJA

Maternal plasma and salivary anelloviruses in pregnancy and preterm birth

Introduction: Human anelloviruses, including torque teno virus (TTV) and torque teno mini virus (TTMV), are ubiquitous in the general population and have no known pathogenicity. We investigated the prevalence and viral load of TTV and TTMV in plasma and saliva over pregnancy, and assessed their association with spontaneous or medically indicated preterm birth. Methods: This is a secondary analysis of the Measurement of Maternal Stress (MOMS) study, which recruited 744 individuals with singleton pregnancies from 4 US sites (Chicago, Pittsburgh, San Antonio, and rural Pennsylvania). Baseline outpatient visits took place in the second trimester (between 12′0 and 20′6/7  weeks’ gestation), and follow-up visits in the third trimester (between 32′0 and 35′6/7  weeks’ gestation). In a case-control study design, participants who delivered preterm ( Results: TTV was detected in plasma from 81% (second trimester) and 77% (third trimester) of participants, and in saliva from 64 and 60%. Corresponding detection rates for TTMV were 59 and 41% in plasma, and 35 and 24% in saliva. TTV and TTMV concentrations were similar between matched plasma and saliva samples. TTV prevalence and concentrations were not significantly different between groups (sPTB, iPTB, and controls). However, plasma TTMV in the third trimester was associated with sPTB and earlier gestational age at delivery. The iPTB group was not different from either the sPTB or the control group. In saliva, concentrations of TTV and TTMV were similar among the three groups. Both TTV and TTMV were more prevalent with increasing parity and were more common in Black and Hispanic participants compared to non-Hispanic White participants. Conclusion: Anellovirus presence (specifically, TTMV) in the third trimester may be associated with preterm birth. Whether this association is causative remains to be determined.</p

Gestational regulation of ER stress signaling in the pregnant mouse uterus from E6–E19.

<p>A) Analysis of protein expression of DDIT3 and XBP1(S) in the nuclear fraction of a gestational series of mouse uteri from E6–19. NCOA3 was used as a loading control. Graphs of ROD of B) DDIT3 and C) XBP1(S) normalized to NCOA3. D) Representative western blots of cleaved (CL) and full length (FL) CASP12 in the cytoplasmic fraction across gestation. PDI was used as a loading control. ROD of E) CL CASP12 and F) FL CASP12 normalized to PDI. Data are represented as Mean ROD ± SE. N = 3 for each time point. Data labeled with different letters are significantly different from each other (P<0.05).</p

List of Primers Used for QPCR.

<p>A list of the primers used for mRNA expression analysis of genes in the ER Stress and Unfolded Protein Response in the pregnant mouse uterus across gestation.</p

Examination of uterine CASP8 cleavage across gestation from E6–E19.

<p>A) Western blot analysis of the cytoplasmic fraction in a gestational series of mouse uteri immunoblotted for full length (FL) and cleaved (CL) CASP8. Recombinant active CASP8 was utilized as a positive control (+) and PDI was used as a loading control B) Graphs of ROD of full length CASP8 normalized to PDI. Data are represented as Mean ROD ± SE. N = 3 for each time point.</p

Localization of ERS and UPR markers in the pregnant mouse uterus.

<p>Immunoflouresence studies were used to determine the localization of A) active CASP3 (Red) B) DDIT3 (Red) C) and HSPA5 (Green) at E 6, 8, 11, 13, 15, 17 and 19.</p

Transcriptional regulation of the ERSR and UPR in the pregnant mouse uterus from E6–E19.

<p>Gestational regulation of A) <i>Atf4,</i> B) <i>Ddit3</i>, C) <i>Casp12,</i> D) <i>Atf6</i> and E) <i>Hspa5</i> in the pregnant mouse uterus from E6–19 as analyzed by Q-PCR. Expression was normalized to <i>Rplp0,</i> which has been found to remain constant across gestation. Data are represented as mean fold change in expression ± SE. N = 3 for each time point. Data labeled with different letters are significantly different from each other (P<0.05).</p