@article{liu_paudel_flowers_piedrahita_wang_2023, title={Adrenomedullin Stimulates Proliferation, Migration and Adhesion of Porcine Trophectoderm Cells Via CALCRL-AKT-TSC2-MTORC1 Cell Signaling Pathway}, volume={101}, ISSN={["1525-3163"]}, DOI={10.1093/jas/skad068.040}, abstractNote={Abstract
               Adrenomedullin (ADM) as a highly conserved peptide hormone has been reported to increase significantly in the uterine lumen during the peri-implantation period of pregnancy in pigs, but its functional roles in growth and development of porcine conceptus (embryonic/fetus and its extra-embryonic membranes) as well as underlying mechanisms remain largely unknown. Therefore, we conducted in vitro experiments using our established porcine trophectoderm cell line (pTr1) isolated from day -12 porcine conceptuses to test the hypothesis that porcine ADM stimulates cell proliferation, migration and adhesion via AKT-TSC2-MTOR cell signaling pathway in pTr1 cells. The pTr1 cells were cultured in DMEM/F12 medium with 10% fetal bovine serum (FBS), 50 U/mL penicillin, 50 μg/mL streptomycin, 0.1 mM each for nutritionally nonessential amino acids, 1 mM sodium pyruvate, 2 mM glutamine, and 4 μg/mL insulin. Opti-MEM supplied with 2.5% (vol/vol) charcoal-stripped FBS was used for siRNA-mediated knockdown targeting non-treated control (siNTC) or specific genes including ADM (siADM) and its shared receptor component calcitonin-receptor-like receptor (CALCRL; siCALCRL). Cells were starved in FBS- and insulin-free medium for 24 hours before treatment. For proliferation assay, cell numbers were determined by staining with Janus-Green B after 48 h incubation. For migration assay, cells were treated with ADM after straight scratch in 6-well plates, and area of cell migration was calculated after 12 h treatment. For adhesion assay, cells were trypsinized in T-25 flasks and allowed for seeding in 96-well plates with density of 2×105 cells/0.2 mL/well, and the numbers of attached cells were determined after 12 h incubation. Western blot analyses were used to determine the expressions of target proteins at total and phosphorylated level. Porcine ADM at 10-7 M stimulated (P < 0.05) pTr1 cell proliferation, migration and adhesion by 1.4%-, 1.5%- and 1.2%-folds, respectively. These ADM-induced effects were abrogated (P < 0.05) by siADM and siCALCRL, as well as by rapamycin, the inhibitor of mechanistic target of rapamycin (MTOR). Using siRNA-mediated knockdown of CALCRL coupled with Western blot analyses, ADM signaling transduction was determined in which ADM binds to CALCRL to increase phosphorylation of MTOR, its downstream effectors (4EBP1, P70S6K, and S6), and upstream regulators (AKT and TSC2). Collectively, these results suggest that porcine ADM in histotroph act on its receptor component CALCRL to activate AKT-TSC2-MTOR, particularly MTORC1 signaling cascade, leading to elongation, migration and attachment of conceptuses. This research was supported by Agriculture and Food Research Initiative Competitive Grant no. 2022-67015-36491 from the USDA National Institute of Food and Agriculture.}, journal={JOURNAL OF ANIMAL SCIENCE}, author={Liu, Bangmin and Paudel, Sudikshya and Flowers, William L. and Piedrahita, Jorge A. and Wang, Xiaoqiu}, year={2023}, month={May} }
 @article{paudel_liu_cummings_wang_2023, title={Adrenomedullin: A Novel Peptide Hormone for Uterine Receptivity and Conceptus Elongation in Pigs?}, volume={101}, ISSN={["1525-3163"]}, DOI={10.1093/jas/skad068.049}, abstractNote={Abstract
               Adrenomedullin (ADM) is an evolutionarily conserved multi-functional peptide hormone in mammalian species. The implantation sites of conceptuses (embryo/fetus and its extra-embryonic membranes) in pregnant Adm+/− female mice are abnormally spaced leading to fetal crowding, intrauterine growth restriction (IUGR) and increases in pregnancy loss. As litter-bearing species, pigs exhibit a high incidence of early embryonic death (30-40%) and naturally occurring IUGR (15-25%), and significant numbers of stillborn piglets (3-9%). Therefore, it is imperative that the roles of ADM in regulation of uterine receptivity, as well as growth and development of conceptus in pigs be established. This study determined abundances of ADM in uterine luminal fluid, and the patterns of expression of ADM and its receptor components in uteri from cyclic and pregnant gilts, as well as conceptuses during the peri-implantation period of pregnancy. ADM receptor components include: calcitonin receptor-like receptor (CALCRL; G protein-coupled receptor bound by ADM), receptor activity modifying protein (RAMP2; dimerized with CALCRL to form ADM1 receptor) and RAMP3 (dimerized with CALCRL to form ADM2 receptor with lower specificity to ADM) and atypical chemokine receptor 3 (ACKR3; a decoy receptor that serves as a cell-autonomous molecular rheostat to dampen ADM signaling). Total recoverable ADM was greater in the uterine fluid of pregnant compared with cyclic gilts between days 10 and 16 post-estrus and was from uterine luminal epithelial (LE) and conceptus trophectoderm (Tr) cells. Uterine expression of CALCRL, RAMP2, and ACKR3 were affected by day (P < 0.05), pregnant status (P < 0.01) and/or day x status (P < 0.05). Within porcine conceptuses, expression of CALCRL, RAMP2 and ACKR3 increased between days 10 and 16 of pregnancy. Using an established porcine trophectoderm (pTr1) cell line isolated from elongating porcine conceptuses recovered on day 2 post-breeding, it was determined that 10-7 M ADM stimulated proliferation of pTr1 cells (P < 0.05) at 48 h, and increased phosphorylated mechanistic target of rapamycin (p-MTOR) and 4E binding protein (p-4EBP1) by 6.1- and 4.9-fold (P < 0.0001), respectively. These novel results indicate a significant role for ADM in uterine receptivity for implantation and conceptus growth and development in pigs. They also provide a framework for future studies of ADM signaling to affect proliferation and migration of Tr cells, spacing of blastocysts, implantation and placentation in pigs. This research was supported by Agriculture and Food Research Initiative Competitive Grant no. 2022-67015-36491 from the USDA National Institute of Food and Agriculture.}, journal={JOURNAL OF ANIMAL SCIENCE}, author={Paudel, Sudikshya and Liu, Bangmin and Cummings, Magdalina J. and Wang, Xiaoqiu}, year={2023}, month={May} }
 @article{liu_paudel_flowers_piedrahita_wang_2023, title={Uterine histotroph and conceptus development: III. Adrenomedullin stimulates proliferation, migration and adhesion of porcine trophectoderm cells via AKT-TSC2-MTOR cell signaling pathway}, volume={55}, ISSN={0939-4451 1438-2199}, url={http://dx.doi.org/10.1007/s00726-023-03265-6}, DOI={10.1007/s00726-023-03265-6}, abstractNote={Adrenomedullin (ADM) as a highly conserved peptide hormone has been reported to increase significantly in the uterine lumen during the peri-implantation period of pregnancy in pigs, but its functional roles in growth and development of porcine conceptus (embryonic/fetus and its extra-embryonic membranes) as well as underlying mechanisms remain largely unknown. Therefore, we conducted in vitro experiments using our established porcine trophectoderm cell line (pTr2) isolated from Day-12 porcine conceptuses to test the hypothesis that porcine ADM stimulates cell proliferation, migration and adhesion via activation of mechanistic target of rapamycin (MTOR) cell signaling pathway in pTr2 cells. Porcine ADM at 10–7 M stimulated (P < 0.05) pTr2 cell proliferation, migration and adhesion by 1.4-, 1.5- and 1.2-folds, respectively. These ADM-induced effects were abrogated (P < 0.05) by siRNA-mediated knockdown of ADM (siADM) and its shared receptor component calcitonin-receptor-like receptor (CALCRL; siCALCRL), as well as by rapamycin, the inhibitor of MTOR. Using siRNA-mediated knockdown of CALCRL coupled with Western blot analyses, ADM signaling transduction was determined in which ADM binds to CALCRL to increase phosphorylation of MTOR, its downstream effectors (4EBP1, P70S6K, and S6), and upstream regulators (AKT and TSC2). Collectively, these results suggest that porcine ADM in histotroph acts on its receptor component CALCRL to activate AKT-TSC2-MTOR, particularly MTORC1 signaling cascade, leading to elongation, migration and attachment of conceptuses.}, number={6}, journal={Amino Acids}, publisher={Springer Science and Business Media LLC}, author={Liu, Bangmin and Paudel, Sudikshya and Flowers, William L. and Piedrahita, Jorge A. and Wang, Xiaoqiu}, year={2023}, month={Apr}, pages={743–756} }
 @article{cummings_yu_paudel_hu_li_hemberger_wang_2022, title={Uterine-specific SIRT1 deficiency confers premature uterine aging and impairs invasion and spacing of blastocyst, and stromal cell decidualization, in mice}, volume={28}, ISSN={["1460-2407"]}, DOI={10.1093/molehr/gaac016}, abstractNote={Abstract
               A distinct age-related alteration in the uterine environment has recently been identified as a prevalent cause of the reproductive decline in older female mice. However, the molecular mechanisms that underlie age-associated uterine adaptability to pregnancy are not known. Sirtuin 1 (SIRT1), a multifunctional NAD+-dependent deacetylase that regulates cell viability, senescence and inflammation during aging, is reduced in aged decidua. Thus, we hypothesize that SIRT1 plays a critical role in uterine adaptability to pregnancy and that uterine-specific ablation of Sirt1 gene accelerates premature uterine aging. Female mice with uterine ablation of Sirt1 gene using progesterone receptor Cre (PgrCre) exhibit subfertility and signs of premature uterine aging. These Sirt1-deficient mothers showed decreases in litter size from their 1st pregnancy and became sterile (25.1 ± 2.5 weeks of age) after giving birth to the third litter. We report that uterine-specific Sirt1 deficiency impairs invasion and spacing of blastocysts, and stromal cell decidualization, leading to abnormal placentation. We found that these problems traced back to the very early stages of hormonal priming of the uterus. During the window of receptivity, Sirt1 deficiency compromises uterine epithelial–stromal crosstalk, whereby estrogen, progesterone and Indian hedgehog signaling pathways are dysregulated, hampering stromal cell priming for decidualization. Uterine transcriptomic analyses also link these causes to perturbations of histone proteins and epigenetic modifiers, as well as adrenomedullin signaling, hyaluronic acid metabolism, and cell senescence. Strikingly, our results also identified genes with significant overlaps with the transcriptome of uteri from aged mice and transcriptomes related to master regulators of decidualization (e.g. Foxo1, Wnt4, Sox17, Bmp2, Egfr and Nr2f2). Our results also implicate accelerated deposition of aging-related fibrillar Type I and III collagens in Sirt1-deficient uteri. Collectively, SIRT1 is an important age-related regulator of invasion and spacing of blastocysts, as well as decidualization of stromal cells.}, number={7}, journal={MOLECULAR HUMAN REPRODUCTION}, author={Cummings, Magdalina J. and Yu, Hongyao and Paudel, Sudikshya and Hu, Guang and Li, Xiaoling and Hemberger, Myriam and Wang, Xiaoqiu}, year={2022}, month={Jun} }
 @article{paudel_wu_wang_2021, title={Amino Acids in Cell Signaling: Regulation and Function}, volume={1332}, ISBN={["978-3-030-74179-2"]}, ISSN={["2214-8019"]}, DOI={10.1007/978-3-030-74180-8_2}, abstractNote={Amino acids are the main building blocks for life. Aside from their roles in composing proteins, functional amino acids and their metabolites play regulatory roles in key metabolic cascades, gene expressions, and cell-to-cell communication via a variety of cell signaling pathways. These metabolic networks are necessary for maintenance, growth, reproduction, and immunity in humans and animals. These amino acids include, but are not limited to, arginine, glutamine, glutamate, glycine, leucine, proline, and tryptophan. We will discuss these functional amino acids in cell signaling pathways in mammals with a particular emphasis on mTORC1, AMPK, and MAPK pathways for protein synthesis, nutrient sensing, and anti-inflammatory responses, as well as cell survival, growth, and development.}, journal={AMINO ACIDS IN NUTRITION AND HEALTH: AMINO ACIDS IN GENE EXPRESSION, METABOLIC REGULATION, AND EXERCISING PERFORMANCE}, author={Paudel, Sudikshya and Wu, Guoyao and Wang, Xiaoqiu}, year={2021}, pages={17–33} }
 @article{paudel_liu_cummings_quinn_bazer_caron_wang_2021, title={Temporal and spatial expression of adrenomedullin and its receptors in the porcine uterus and peri-implantation conceptuses}, volume={105}, ISSN={["1529-7268"]}, DOI={10.1093/biolre/ioab110}, abstractNote={Abstract
               Adrenomedullin (ADM) is an evolutionarily conserved multifunctional peptide hormone that regulates implantation, embryo spacing, and placentation in humans and rodents. However, the potential roles of ADM in implantation and placentation in pigs, as a litter-bearing species, are not known. This study determined abundances of ADM in uterine luminal fluid, and the patterns of expression of ADM and its receptor components (CALCRL, RAMP2, RAMP3, and ACKR3) in uteri from cyclic and pregnant gilts, as well as conceptuses (embryonic/fetus and its extra-embryonic membranes) during the peri-implantation period of pregnancy. Total recoverable ADM was greater in the uterine fluid of pregnant compared with cyclic gilts between Days 10 and 16 post-estrus and was from uterine luminal epithelial (LE) and conceptus trophectoderm (Tr) cells. Uterine expression of CALCRL, RAMP2, and ACKR3 were affected by day (P < 0.05), pregnant status (P < 0.01) and/or day x status (P < 0.05). Within porcine conceptuses, the expression of CALCRL, RAMP2, and ACKR3 increased between Days 10 and 16 of pregnancy. Using an established porcine trophectoderm (pTr1) cell line, it was determined that 10−7 M ADM stimulated proliferation of pTr1 cells (P < 0.05) at 48 h, and increased phosphorylated mechanistic target of rapamycin (p-MTOR) and 4E binding protein 1 (p-4EBP1) by 6.1- and 4.9-fold (P < 0.0001), respectively. These novel results indicate a significant role for ADM in uterine receptivity for implantation and conceptus growth and development in pigs. They also provide a framework for future studies of ADM signaling to affect proliferation and migration of Tr cells, spacing of blastocysts, implantation, and placentation in pigs.}, number={4}, journal={BIOLOGY OF REPRODUCTION}, author={Paudel, Sudikshya and Liu, Bangmin and Cummings, Magdalina J. and Quinn, Kelsey E. and Bazer, Fuller W. and Caron, Kathleen M. and Wang, Xiaoqiu}, year={2021}, month={Oct}, pages={876–891} }