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

Understanding m6A’s role in the myometrium during gestation

Epigenetic modifications, such as DNA methylation, mainly affects the function of genes through regulating the transcription or translation processes, without altering the DNA sequences. The regulation of m6A modification is dynamic and reversible and is established by m6A methyltransferases (“writers”), such as methyltransferase-like protein 3 (METTL3). It is removed by m6A demethylases (“erasers”), such as α-ketoglutarate-dependent dioxygenase alkB homolog 5 (ALKBH5). The effects of m6A modification on RNA metabolism depend on the recognition by different m6A -binding proteins (“readers”), including the YT521-B homology (YTH) domain family and the heterogeneous nuclear ribonucleoproteins (HNRNPs). We believe that splicing directed by m6A methylation plays a significant role in regulating the physiology of the pregnant myometrium through alternative splicing of pre-mRNAs, facilitating rapid expansion and plasticity of the genome, producing an altered active proteome allowing for the transition from a quiescent to laboring myometrial compartment. Western blot analysis of pregnant mouse uteri from mid gestation to term in labor revealed distinct and corollary gestational modifications in the reader and writers METTL3, hnRNPC and YTHDC1, which spiked at mid gestation and again at term correlating with m6A labeling and previously observed increases in myometrial splicing events. In contrast as expected the eraser ALKBH5 levels declined towards term. Future analysis will examine if these gestational changes are hormonally regulated. We believe that alternative splicing and changes in the epi-transcriptome through m6A regulation are an underlying mechanism involved in normal term labor and understanding these phenomena may lead to development of an effective therapeutic for preterm labor

Understanding m6A’s role in the myometrium during gestation

Epigenetic modifications, such as DNA methylation, mainly affects the function of genes through regulating the transcription or translation processes, without altering the DNA sequences. The regulation of m6A modification is dynamic and reversible and is established by m6A methyltransferases (“writers”), such as methyltransferase-like protein 3 (METTL3). It is removed by m6A demethylases (“erasers”), such as α-ketoglutarate-dependent dioxygenase alkB homolog 5 (ALKBH5). The effects of m6A modification on RNA metabolism depend on the recognition by different m6A -binding proteins (“readers”), including the YT521-B homology (YTH) domain family and the heterogeneous nuclear ribonucleoproteins (HNRNPs). We believe that splicing directed by m6A methylation plays a significant role in regulating the physiology of the pregnant myometrium through alternative splicing of pre-mRNAs, facilitating rapid expansion and plasticity of the genome, producing an altered active proteome allowing for the transition from a quiescent to laboring myometrial compartment. Western blot analysis of pregnant mouse uteri from mid gestation to term in labor revealed distinct and corollary gestational modifications in the reader and writers METTL3, hnRNPC and YTHDC1, which spiked at mid gestation and again at term correlating with m6A labeling and previously observed increases in myometrial splicing events. In contrast as expected the eraser ALKBH5 levels declined towards term. Future analysis will examine if these gestational changes are hormonally regulated. We believe that alternative splicing and changes in the epi-transcriptome through m6A regulation are an underlying mechanism involved in normal term labor and understanding these phenomena may lead to development of an effective therapeutic for preterm labor

The transcription factors steroidogenic Factor-1 and SOX9 regulate expression of Vanin-1 during mouse testis development

We previously showed, using differential expression screening and in situ hybridization that Vanin-1, which encodes a glycosylphosphatidylinositol-linked membrane-associated pantetheinase, is expressed in a sex-specific manner during fetal gonad development in mice (Bowles, J., Bullejos, M., and Koopman, P. (2000) Genesis 27, 124-135). In the present study we investigate in detail the expression and regulation of Vanin-1 in the fetal testis. Vanin-1 is co-expressed with the transcription factors steroidogenic factor-1 (SF-1) and SOX9 in Sertoli cells and, at a lower level, with SF-1 in Leydig cells in developing testes. SF-1 is able to activate the transcription of the Vanin-1 promoter in in vitro reporter assays, and this activation is further augmented by SOX9. We found that SF-1 is able to bind to two sites in the Vanin-1 promoter, whereas SOX9 can bind to a single interposed site defined by DNA footprinting. Mutation of the SF-1 or SOX9 sites disrupts the binding of these factors and activation of transcription. The expression of Vanin-1 was abolished in Leydig cells of a mouse mutant lacking SF-1 in that cell type. Our findings account for the sex-and cell-type-specific expression of Vanin-1 in the developing mouse gonad in vivo, which we suggest is required to provide an appropriate environment for male germ cell development

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

Examination of BID cleavage and translocation in the pregnant mouse uterus from E6–E19.

<p>Western blot analysis of BID and tBID in the cytosolic fraction (A) and tBID in the mitochondrial fraction (B). GAPDH and COX4I1 were utilized as a loading control for the cytosolic and mitochondrial fraction respectively. C) Graphs of ROD of BID normalized GAPDH. Data are represented as Mean ROD ± SE. N = 3 for each time point.</p

Mitochondrial CYCS retention in pregnant mouse uterus from E6–E19.

<p>Representative western blots of a gestational series of mouse uteri immunoblotted for A) CYCS in the mitochondrial fraction and B) in the cytosolic fraction. C) ROD of CYCS in the mitochondrial fraction normalized to COX4I1. Levels of GAPDH and COX4I1 were used as controls to determine the loading and purity of the cytosolic and mitochondrial fractions respectively. Data are represented as Mean ROD ± SE. N = 3 for each time point.</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