529. PROPROTEIN CONVERTASE 6 PLAYS A CRITICAL ROLE IN MODULATING THE HUMAN ENDOMETRIAL EPITHELIUM FOR RECEPTIVITY AND IMPLANTATION

Successful embryo implantation is an important step in establishing pregnancy, requiring a healthy embryo and a receptive endometrium. Establishment of endometrial receptivity involves morphological and physiological changes initially in the endometrial epithelium, but the underlying molecular mechanisms are not fully understood. We have previously demonstrated that proprotein convertase 5/6 (PC6), a member of the proprotein convertase (PC) family, is up-regulated in the endometrium specifically at implantation in association with epithelial differentiation, in the human and monkey. PCs convert a range of precursor proteins of important functions into their bioactive forms; they are thus regarded as critical “master switch” molecules. The present study aimed to determine whether PC6 is a critical regulator in the endometrial epithelium for receptivity and implantation. We examined whether endometrial epithelial PC6 dys-regulation is associated with implantation failure in women and whether knockdown of PC6 by siRNA in human endometrial epithelial cells affects embryo adhesion in a cell culture model. Endometrial PC6 expression was assessed by immunohistochemistry in the mid-secretory phase of the menstrual cycle (receptive phase) in two unique clinical cohorts comprising women of known fertility and infertility (with no obvious gynecological disorders, and with fertile males). Endometrial epithelial PC6 levels were significantly lower in infertile vs fertile women in both cohorts. To further establish that PC6 is important for receptivity, a cell model relevant to human implantation was used involving co-culture of uterine epithelial cells with mouse embryos. The epithelial cells were stably transfected with PC6 siRNA and PC6 knock down was confirmed at the levels of mRNA, protein, and activity by real-time RT-PCR, Western blotting and activity assay respectively. Embryos readily adhered to normal epithelial cells, but the adhesion was significantly reduced in the PC6 knockdown epithelial cells. We are currently using proteomics technology to identify the pathways affected by PC6 knockdown. These results strongly suggest that PC6 plays a critical role in modulating the human endometrial epithelium for receptivity and implantation.

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156. IDENTIFICATION OF SCAFFOLDING PROTEINS AS PC6 SUBSTRATES IN THE HUMAN ENDOMETRIAL EPITHELIAL CELLS FOR EMBRYO IMPLANTATION

Reproduction Fertility and Development ◽

10.1071/srb10abs156 ◽

2010 ◽

Vol 22 (9) ◽

pp. 74

Author(s):

S. Heng ◽

Y. Li ◽

A. N. Stephens ◽

A. Rainczuk ◽

G. Nie

Keyword(s):

Epithelial Cells ◽

Molecular Mechanisms ◽

Embryo Implantation ◽

Scaffolding Proteins ◽

Proprotein Convertase ◽

Sirna Sequence ◽

C Terminus ◽

Precursor Proteins ◽

Endometrial Epithelium ◽

Endometrial Epithelial Cells

Successful embryo implantation is an important step in establishing pregnancy, which requires a healthy embryo and a receptive endometrium. Establishment of endometrial receptivity involves morphological and physiological changes initially in the endometrial epithelium, however the underlying molecular mechanisms are not fully understood. We have previously demonstrated that proprotein convertase 5/6 (PC6), a member of the proprotein convertase (PC) family, is up-regulated in the endometrium specifically at implantation in association with epithelial differentiation in the human and monkey. PCs convert a range of precursor proteins of important functions into their bioactive forms, they are thus regarded as critical ‘master switch’ molecules. The aim of this study was to identify target proteins of PC6 in the endometrial epithelial cells important for implantation. We used a HEC1A cell line in which PC6 was stably knocked down by siRNA approach (HEC1A-PC6). HEC1A cells that were similarly transfected with a scrambled siRNA sequence (HEC1A-control) were used as the control. Previous study confirmed that HEC1A-PC6 cells had much reduced capacity to adhere to blastocyst. A proteomic comparison between HEC1A-PC6 treated with or without human recombinant PC6 identified ezrin as a potential PC6 substrate. Ezrin is a cytoplasmic protein which is known to bind to ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50) thereby translocating to the plasma membrane.This complex has been associated with cytoskeletal re-organisation and changes in cell polarity. Co-immunoprecipitation of ezrin and EBP50 showed that knockdown of PC6 allowed the binding of ezrin to the C-terminus of EBP50 in HEC1A-PC6, whereas PC6 cleavage of EBP50 in HEC1A-control prevented the binding. This was also confirmed by immunofluorescence showing that ezrin and EBP50 were co-localized to the plasma membrane in HEC1A-PC6. This study thus identified that PC6 regulates scaffolding proteins such as EBP50 and ezrin in the endometrium for embryo implantation.

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Proprotein Convertase 5/6 Is Critical for Embryo Implantation in Women: Regulating Receptivity by Cleaving EBP50, Modulating Ezrin Binding, and Membrane-Cytoskeletal Interactions

Endocrinology ◽

10.1210/en.2011-1273 ◽

2011 ◽

Vol 152 (12) ◽

pp. 5041-5052 ◽

Cited By ~ 22

Author(s):

Sophea Heng ◽

Ana Cervero ◽

Carlos Simon ◽

Andrew N. Stephens ◽

Ying Li ◽

...

Keyword(s):

Plasma Membrane ◽

Cellular Localization ◽

Structural Changes ◽

Current Knowledge ◽

Embryo Implantation ◽

Critical Role ◽

Human Endometrium ◽

Scaffolding Protein ◽

Proprotein Convertase ◽

Proteomic Approach

Establishment of endometrial receptivity is vital for successful embryo implantation; its failure causes infertility. Epithelial receptivity acquisition involves dramatic structural changes in the plasma membrane and cytoskeleton. Proprotein convertase 5/6 (PC6), a serine protease of the proprotein convertase (PC) family, is up-regulated in the human endometrium specifically at the time of epithelial receptivity and stromal cell decidualization. PC6 is the only PC member tightly regulated in this manner. The current study addressed the importance and mechanisms of PC6 action in regulating receptivity in women. PC6 was dysregulated in the endometrial epithelium during the window of implantation in infertile women of three demographically different cohorts. Its critical role in receptivity was evidenced by a significant reduction in mouse blastocyst attachment of endometrial epithelial cells after PC6 knockdown by small interfering RNA. Using a proteomic approach, we discovered that PC6 cleaved the key scaffolding protein, ezrin-radixin-moesin binding phosphoprotein 50 (EBP50), thereby profoundly affecting its interaction with binding protein ezrin (a key protein bridging actin filaments and plasma membrane), EBP50/ezrin cellular localization, and cytoskeleton-membrane connections. We further validated this novel PC6 regulation of receptivity in human endometrium in vivo in fertile vs. infertile patients. These results strongly indicate that PC6 plays a key role in regulating fundamental cellular remodeling processes, such as plasma membrane transformation and membrane-cytoskeletal interface reorganization. PC6 cleavage of a crucial scaffolding protein EBP50, thereby profoundly regulating membrane-cytoskeletal reorganization, greatly extends the current knowledge of PC biology and provides substantial new mechanistic insight into the fields of reproduction, basic cellular biology, and PC biochemistry.

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A proteomic survey of microtubule-associated proteins in a R402H TUBA1A mutant mouse

PLoS Genetics ◽

10.1371/journal.pgen.1009104 ◽

2020 ◽

Vol 16 (11) ◽

pp. e1009104

Author(s):

Ines Leca ◽

Alexander William Phillips ◽

Iris Hofer ◽

Lukas Landler ◽

Lyubov Ushakova ◽

...

Keyword(s):

Neuronal Migration ◽

Molecular Mechanisms ◽

Mutant Mouse ◽

Critical Role ◽

Recurrent Mutation ◽

Intrinsic Defect ◽

Quantitative Mass Spectrometry ◽

Microtubule Associated Proteins ◽

Associated Proteins ◽

A Cell

Microtubules play a critical role in multiple aspects of neurodevelopment, including the generation, migration and differentiation of neurons. A recurrent mutation (R402H) in the α-tubulin gene TUBA1A is known to cause lissencephaly with cerebellar and striatal phenotypes. Previous work has shown that this mutation does not perturb the chaperone-mediated folding of tubulin heterodimers, which are able to assemble and incorporate into the microtubule lattice. To explore the molecular mechanisms that cause the disease state we generated a new conditional mouse line that recapitulates the R402H variant. We show that heterozygous mutants present with laminar phenotypes in the cortex and hippocampus, as well as a reduction in striatal size and cerebellar abnormalities. We demonstrate that homozygous expression of the R402H allele causes neuronal death and exacerbates a cell intrinsic defect in cortical neuronal migration. Microtubule sedimentation assays coupled with quantitative mass spectrometry demonstrated that the binding and/or levels of multiple microtubule associated proteins (MAPs) are perturbed by the R402H mutation including VAPB, REEP1, EZRIN, PRNP and DYNC1l1/2. Consistent with these data we show that the R402H mutation impairs dynein-mediated transport which is associated with a decoupling of the nucleus to the microtubule organising center. Our data support a model whereby the R402H variant is able to fold and incorporate into microtubules, but acts as a gain of function by perturbing the binding of MAPs.

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166. EMBRYO IMPLANTATION IS ASSOCIATED WITH SPECIFIC EXPRESSION OF PROPROTEIN CONVERTASE 6 IN THE RABBIT UTERUS

Reproduction Fertility and Development ◽

10.1071/srb10abs166 ◽

2010 ◽

Vol 22 (9) ◽

pp. 84

Author(s):

Y. Li ◽

P. K. Nicholls ◽

S. Heng ◽

Z. Sun ◽

J. Wang ◽

...

Keyword(s):

Mouse Models ◽

Embryo Implantation ◽

Attachment Site ◽

Biologically Active ◽

Pcr Analysis ◽

Proprotein Convertase ◽

Specific Expression ◽

Stromal Component ◽

Endometrial Epithelium ◽

Embryo Attachment

Proprotein convertase 5/6 (PC6) is a member of the proprotein convertase family that endoproteolytically cleave latent precursor proteins into their biologically active state. We have previously demonstrated that endometrial PC6 is critical for embryo implantation in mice and primates, including human. PC6 regulates the endometrial physiology specifically at implantation in association with epithelial differentiation during the establishment of endometrial receptivity (in human and monkey) and stromal cell decidualization (in the mouse, human and monkey). PC6 was further confirmed to be a unique PC member that is tightly regulated in the endometrium in relation to implantation. Our further studies (unpublished) suggest that PC6 regulates adhesion molecules in the endometrial epithelium for implantation in women. It is known that between the mouse and human, the endometrial stroma-mediated responses are similar whereas the epithelial cells behave differently. Because PC6 regulates primarily the stromal component (decidualization) in the mouse, in vivo mouse models are critical to investigate the roles of PC6 in decidualization. To address the function of PC6 in endometrial epithelium, non-mouse models relevant to human implantation are required. The rabbit is regarded as an excellent model to study the molecular events of embryo adhesion and attachment. The current study aimed to determine the expression pattern and localisation of PC6 in the rabbit uterus during early pregnancy. Quantitative RT-PCR analysis showed that PC6 mRNA expression was dynamically up-regulated in the rabbit uterus immediately prior to implantation. Western blotting and immunohistochemical analyses demonstrated that PC6 protein was predominantly localised to the basal glands throughout pregnancy, and up-regulated specifically in the epithelium at the embryo attachment site. These findings suggest that PC6 may play an essential role in rabbit implantation, and that the rabbit is a useful animal model to investigate the function of PC6 during embryo attachment.

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MO007ANALYSIS OF CALCIUM SIGNALING IN AUTOSOMAL DOMINANT POLYCYSTIC KIDNEY DISEASE (ADPKD)

Nephrology Dialysis Transplantation ◽

10.1093/ndt/gfab079.003 ◽

2021 ◽

Vol 36 (Supplement_1) ◽

Author(s):

Dorien Van Giel ◽

Jean-Paul Decuypere ◽

Djalila Mekahli ◽

Rudi Vennekens

Keyword(s):

Kidney Disease ◽

Epithelial Cells ◽

Polycystic Kidney Disease ◽

Molecular Mechanisms ◽

Autosomal Dominant ◽

Trp Channels ◽

Cell Model ◽

Healthy Controls ◽

Polycystic Kidney ◽

Autosomal Dominant Polycystic Kidney

Abstract Background and Aims Autosomal Dominant Polycystic Kidney Disease (ADPKD) is an inheritable kidney disease characterized by the development of fluid-filled cysts in all nephron segments, leading to loss of renal function. Mutations in PKD1 or PKD2, which encode polycystin-1 and polycystin-2, are the most common cause of ADPKD. The molecular mechanisms underlying cystogenesis are poorly characterized but it is postulated that disturbed calcium homeostasis is a primary event in cystogenesis. The precise molecular players that cause this disturbance are still a poorly explored area, especially in relevant human cell types. We therefore aim to characterize the profile of calcium-coupled receptors and channels in a human renal epithelial cell model, to identify which receptors and channels are present and whether their function is affected in ADPKD. Method Human urine-derived conditionally immortalized proximal tubule epithelial cells (ciPTECs) of ADPKD patients and healthy controls were screened for calcium-coupled GPCRs, using a GPCR agonist library on Fura-2 loaded cell populations seeded in 96-well format using the Flexstation3 (Molecular Devices). Validation of specific hits was done using single-cell measurements with a fluorescence microscope and built-in perfusion system. The expression of TRP channels and STIM/Orai proteins was determined via qPCR. Results From a library of 418 GPCR agonists a selective amount of calcium-coupled GPCRs was found functionally active in ciPTECs. ciPTECs from both healthy controls and ADPKD patients were found to functionally express purinergic -, histamine -, serotonin and dopamine receptors. Through qPCR we found expression of various TRP channels, including TRPML1, TRPC1/3, TRPM3/4/7, TRPV4 and TRPA1, as well as high expression of STIM1/2 and Orai1/2/3. Conclusion We describe the first thorough characterization of molecular players involved in calcium signalling mechanisms in human renal epithelial cells, including the profile of calcium-coupled GPCRs and the expression of TRP channels and STIM/Orai proteins, of further interest to investigate disturbed calcium dynamics in ADPKD.

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Expression and steroid hormone regulation of TETs and DNMTs in human endometrium

Reproduction ◽

10.1530/rep-19-0562 ◽

2020 ◽

Vol 160 (2) ◽

pp. 247-257

Author(s):

Vishakha Mahajan ◽

Diana Osavlyuk ◽

Philip C Logan ◽

Satya Amirapu ◽

Anna P Ponnampalam

Keyword(s):

Epithelial Cells ◽

Stromal Cells ◽

Hormonal Regulation ◽

Dna Methyltransferases ◽

Hormone Regulation ◽

Secretory Phase ◽

Estrogen Exposure ◽

A Cell ◽

Endometrial Epithelium

DNA methyltransferases (DNMTs) and ten-eleven translocation proteins (TETs) facilitate methylation and hydroxymethylation of DNA, respectively. DNMTs are widely studied with conflicting results on their regulation in the endometrium. While the role of TETs in the endometrium remains relatively unexplored. Deregulated expression of TETs and DNMTs are associated with endometrial pathologies. The aim of this study is to characterize the temporal TET expression in endometrium and to determine the hormonal regulation of TETs in comparison to DNMTs. mRNA expressions were quantified by real-time PCR in endometrial tissues from cycling women and localization was determined by immunohistochemistry. Hormonal regulation was investigated in endometrial epithelial and stromal cell lines following a 24 and 48 h treatment cycle. TET1 and 3 mRNA expressions were significantly upregulated in the mid-secretory phase. TET protein expression was ubiquitous in endometrial epithelium throughout the menstrual cycle except during the late-secretory phase, while stromal staining was scattered. TET1 mRNA was significantly upregulated in response to estrogen in stromal cells. Transcriptions of all three TETs were induced in response to progesterone treatment in epithelial cells. Only DNMT3b in epithelial cells and DNMT1 in stromal cells were significantly upregulated upon 24-h estrogen exposure following a significant decrease of DNMT1 when treated with 24 h of estrogen and progesterone. This study suggests that TETs are expressed in a cell-specific, dynamic manner in the endometrium and are responsive to steroid hormones. Investigating the role of TETs individually and with respect to DNMTs, will help to elucidate gene regulatory mechanisms in endometrial biology and pathologies.

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Comparison of dietary polyphenols for protection against molecular mechanisms underlying nonalcoholic fatty liver disease in a cell model of steatosis

Molecular Nutrition & Food Research ◽

10.1002/mnfr.201600781 ◽

2017 ◽

Vol 61 (9) ◽

pp. 1600781 ◽

Cited By ~ 9

Author(s):

Hossein Rafiei ◽

Kosar Omidian ◽

Brian Bandy

Keyword(s):

Liver Disease ◽

Fatty Liver ◽

Nonalcoholic Fatty Liver Disease ◽

Fatty Liver Disease ◽

Molecular Mechanisms ◽

Cell Model ◽

Nonalcoholic Fatty Liver ◽

Dietary Polyphenols ◽

A Cell

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Activation of SGK1 in Endometrial Epithelial Cells in Response to PI3K/AKT Inhibition Impairs Embryo Implantation

Cellular Physiology and Biochemistry ◽

10.1159/000447903 ◽

2016 ◽

Vol 39 (5) ◽

pp. 2077-2087 ◽

Cited By ~ 17

Author(s):

Madhuri S. Salker ◽

Jennifer H. Steel ◽

Zohreh Hosseinzadeh ◽

Jaya Nautiyal ◽

Zoe Webster ◽

...

Keyword(s):

Epithelial Cells ◽

Embryo Implantation ◽

Transport Processes ◽

Akt Inhibitor ◽

Down Regulation ◽

Uterine Lumen ◽

Akt Activation ◽

A Cell ◽

Phosphoinositide 3 Kinase ◽

Endometrial Epithelial Cells

Background: Serum & Glucocorticoid Regulated Kinase 1 (SGK1) plays a fundamental role in ion and solute transport processes in epithelia. In the endometrium, down-regulation of SGK1 during the window of receptivity facilitates embryo implantation whereas expression of a constitutively active mutant in the murine uterus blocks implantation. Methods/Results: Here, we report that treatment of endometrial epithelial cells with specific inhibitors of the phosphoinositide 3-kinase (PI3K)/AKT activity pathway results in reciprocal activation of SGK1. Flushing of the uterine lumen of mice with a cell permeable, substrate competitive phosphatidylinositol analogue that inhibits AKT activation (AKT inhibitor III) resulted in Sgk1 phosphorylation, down-regulation of the E3 ubiquitin-protein ligase Nedd4-2, and increased expression of epithelial Na+ channels (ENaC). Furthermore, exposure of the uterine lumen to AKT inhibitor III prior to embryo transfer induced a spectrum of early pregnancy defects, ranging from implantation failure to aberrant spacing of implantation sites. Conclusion: Taken together, our data indicate that the balanced activities of two related serine/threonine kinases, AKT and SGK1, critically govern the implantation process.

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Citrus Peels Phenolic Derivatives Alleviate Benzo[α]pyrene-Induced Inflammatory Injury of Human Intestinal Epithelial Cells Through NLRP3 and AhR Signaling Pathways

10.21203/rs.3.rs-1031787/v1 ◽

2021 ◽

Author(s):

Zhuwei Liang ◽

Huailing Wang ◽

Dan Luo ◽

Xiaoyu Liu ◽

Jie Liu

Keyword(s):

Epithelial Cells ◽

Signaling Pathways ◽

Intestinal Epithelial Cells ◽

Cell Model ◽

Intestinal Epithelial ◽

Human Intestinal Epithelial Cells ◽

Pathway Activation ◽

Citrus Peels ◽

A Cell ◽

Phenolic Derivatives

Abstract Benzo[α]pyrene (BaP) is ubiquitous in foods, and possesses a fatal cytotoxicity. In current study, ten Citrus peels (Chenpi) phenolic derivatives (CPDs) were isolated in a cell model of human intestinal epithelial (Caco-2) cells under BaP-exposure by a bio-assay guided method. Among them, methyl (3,4,5-trimethoxybenzoyl) valylphenylalaninate (Citrus peels phenolic derivative-2, CPD-2) performed the most protective activity by promoting the antiinflammatory potential on BaP-induced Caco-2 cells. CPD-2 inhibited BaP-induced intracellular ROS over-production and inflammatory epithelial cytokine, IL-4, IL-8, TNF-α, IL-1β and IL-18 over-expression, but not IL-6. CPD-2 also inhibited BaP-induced NLRP3 inflammasome and AhR signaling pathway activation. Overall, CPD-2 attenuates BaP-induced apoptotic death via promoting the antiinflammatory potentials by inhibiting the NLRP3 and AhR signaling pathways activation of Caco-2 cells. Finally, the Citrus peels phenolic derivatives was observed for the first time against BaP-induced inflammation and oxidative stress in human intestinal epithelial cells.

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