Oxidative stress induces p38MAPK-dependent senescence in the feto-maternal interface cells

Jin Jin, Lauren Richardson, Samantha Sheller-Miller, Nanbert Zhong, Ramkumar Menon, Jin Jin, Lauren Richardson, Samantha Sheller-Miller, Nanbert Zhong, Ramkumar Menon

Abstract

Objective: This study tested the mechanism of the oxidative stress (OS)-induced senescence pathway at the feto-maternal interface cells.

Methods: Primary amnion mesenchymal cells (AMCs), chorion and decidual cells isolated from the placental membranes of women at normal term (not in labor) were exposed to OS-inducing cigarette smoke extract (CSE) for 48 h. Reactive oxygen species (ROS) was measured using 2'7'-dichlorodihydrofluorescein. Western blot analysis determined phosphorylated (P) p38MAPK and p53 expression. Senescence-associated β-Galactosidase (SA-β-Gal) and matrix metallopeptidase 9 (MMP9) histochemistry were used to measure senescence and inflammation respectively. Cotreatment of cells with the antioxidant, N-acetyl cysteine (NAC), or the p38MAPK inhibitor, SB203580 (SB), verified the activation specificity.

Results: CSE increased ROS production from AMCs, chorion cells, and decidual cells (P < 0.05) compared to controls. Western blot analysis determined that CSE induced p38MAPK activation (P < 0.05) and cotreatment with NAC inhibited ROS production and p38MAPK activation (P < 0.05) in all cell types. CSE did not increase p53 phosphorylation in any of the cells; however, AMCs showed constitutive P-p53 expression. CSE increased senescence in AMCs and chorion cells compared to controls (P = 0.01 and P = 0.003, respectively); however, senescence was not observed in decidual cells. Senescence was significantly reduced following cotreatment with SB and NAC (AMCs; P = 0.01 and chorion; P = 0.009). CSE increased MMP9 in all cells that was reduced by NAC.

Conclusion: OS induced p38MAPK activation and inflammation in all cell types that was associated with senescence in fetal cells but not in maternal cells.

Keywords: Amnion mesenchymal cells; Chorion; Decidua; Oxidative stress; Senescence; p38MAPK.

Copyright © 2018 Elsevier Ltd. All rights reserved.

Figures

Figure 1. Characterization of placental membrane cells
Figure 1. Characterization of placental membrane cells
Bright-field and fluorescence microscopy showing the morphology of cultured primary amnion mesenchymal cells (AMCs), chorion cells, and decidual cells. Fluorescence microscopy showing vimentin (green) and cytokeratin-18 (CK-18; red) localization in AMCs, chorion cells, and decidual cells in vitro. Images were taken at 10X magnification.
Figure 2. ROS production in fetal membrane…
Figure 2. ROS production in fetal membrane cells
A) Cigarette smoke extract (CSE) treatment of amnion mesenchymal cells (AMCs) significantly increased ROS production at 10 minutes (P < 0.05) and 20 minutes (P < 0.05) compared to control (untreated) AMCs. Cotreatment with N-acetyl cysteine (NAC) and CSE significantly reduced ROS production in AMCs (P < 0.05 for all time points). B) CSE treatment of chorion cells significantly increased ROS production (P < 0.001) at all time points compared to control chorion cells. Cotreatment with NAC and CSE significantly prevented ROS production in chorion cells (P < 0.05 at all-time points). C) CSE treatment of decidual cells significantly increased ROS production (P < 0.001) at all time points compared to control decidual cells. Cotreatment with NAC and CSE significantly reduced ROS production in decidual cells (P < 0.001 at all-time points). An asterisk above the blue line represents a significant difference between control and CSE-treated cells, while an asterisk below the last line represents a significant difference between CSE and CSE and NAC cotreated cells.
Figure 3. Cigarette smoke extract (CSE) induces…
Figure 3. Cigarette smoke extract (CSE) induces p38MAPK activation in placental membrane cells
A–F) CSE treatment of amnion mesenchymal cells (AMCs), chorion cells, and decidual cells for up to 6 hours induced phosphorylation (P) of p38MAPK, as determined by western blots and immunofluorescence. A) CSE treatment induced higher levels of P-p38MAPK in AMCs than in control cells (P = 0.0005), while CSE + N-acetyl cysteine (NAC) reduced P-p38MAPK levels (P = 0.005). The treatment did not alter the total p38MAPK level. B) Nuclear translocation of P-p38MAPK increased following CSE treatment but was inhibited by the p38MAPK inhibitor, SB203580 (SB). C) CSE treatment induced higher levels of P-p38MAPK in chorion cells than in control cells (P = 0.009), while CSE + NAC reduced P-p38MAPK levels (P = 0.006). The treatment did not alter the total p38MAPK level. D) Nuclear localization of P-p38MAPK increased in chorion cells after CSE treatment, while CSE + SB treatment prevented this translocation. E) CSE treatment induced higher levels of P-p38MAPK in decidual cells compared to controls (P < 0.0001), while cotreatment with CSE + NAC reduced P-p38MAPK activation (P < 0.0001). The treatment did not alter the total p38MAPK level. F) Nuclear localization of P-p38MAPK in decidual cells was similar to that in AMCs. The relative fluorescence increased with CSE, while CSE + SB treatment reduced translocation. Bar graphs represent densitometry, and the y-axis represents relative densitometry units. Confocal microscopy images were captured at 63X magnification. Red staining indicates α-smooth muscle actin (α-SMA), green staining represents P-p38MAPK, and blue represents the DAPI nuclear stain.
Figure 4. Cigarette smoke extract (CSE) does…
Figure 4. Cigarette smoke extract (CSE) does not induce p53 activation in AMCs
Amnion mesenchymal cells (AMCs) constitutively expressed P-p53 and total p53 regardless of the treatment. Immunofluorescent staining documented nuclear localization of P-p53 after CSE treatment (white arrows). Confocal microscopy images were captured at 63X magnification. Red staining represents α-smooth muscle actin (α-SMA), green staining represents P-p53, and blue staining represents the DAPI nuclear stain.
Figure 5. Cigarette smoke extract (CSE) induces…
Figure 5. Cigarette smoke extract (CSE) induces senescence in placental membrane cells
A) Cigarette smoke extract (CSE) treatment (48 hours) induced senescence-associated-β-Galactosidase (SA-β-Gal) staining (blue stain; red arrows) in amnion mesenchymal cells (AMCs) and chorion cells but had little effect in decidual cells. B) CSE treatment significantly induced SA-β-Gal (P = 0.02) in AMCs compared to control cells, while CSE + N-acetyl cysteine (NAC) significantly reduced SA-β-Gal staining (P = 0.01). C) CSE treatment significantly induced SA-β-Gal (P = 0.003) in chorion cells compared to control cells, while CSE + NAC significantly inhibited SA-β-Gal staining (P = 0.009) compared to CSE alone. D) SA-β-Gal staining was not significantly different between CSE-treated and control decidual cells or after cotreatment with NAC. All bright-field images were captured after 48 hours of treatment and at 20X magnification. Data are documented in relative intensity units (RIU).
Figure 6. Cigarette smoke extract (CSE) induces…
Figure 6. Cigarette smoke extract (CSE) induces senescence-associated inflammation in placental membrane cells
A) Term not in labor (TNIL) explants that were cultured for 48 hours (controls) expressed no MMP9 in any placental membrane layers (i.e., amnion epithelial cells (AECs), amnion mesenchymal cells (AMCs), chorion cells, or decidual cells). B) CSE treatment of TNIL explants for 48 hours induced the MMP9 expression in all cell layers. C) Cotreatment with CSE + NAC reduced MMP9 expression.

References

    1. Dennery PA. Effects of oxidative stress on embryonic development. Birth Defects Res C Embryo Today. 2007;81(3):155–62.
    1. Agarwal A, Gupta S, Sharma RK. Role of oxidative stress in female reproduction. Reprod Biol Endocrinol. 2005;3:28.
    1. Myatt L, Cui X. Oxidative stress in the placenta. Histochem Cell Biol. 2004;122(4):369–82.
    1. Menon R. Oxidative stress damage as a detrimental factor in preterm birth pathology. Frontiers in Immunology. 2014;5:567.
    1. Chai M, Barker G, Menon R, Lappas M. Increased oxidative stress in human fetal membranes overlying the cervix from term non-labouring and post labour deliveries. Placenta. 2012;33(8):604–10.
    1. Sauer H, Wartenberg M, Hescheler J. Reactive oxygen species as intracellular messengers during cell growth and differentiation. Cell Physiol Biochem. 2001;11(4):173–86.
    1. Rinaldi SF, Hutchinson JL, Rossi AG, Norman JE. Anti-inflammatory mediators as physiological and pharmacological regulators of parturition. Expert Rev Clin Immunol. 2011;7(5):675–96.
    1. Agarwal A, Aponte-Mellado A, Premkumar BJ, Shaman A, Gupta S. The effects of oxidative stress on female reproduction: a review. Reprod Biol Endocrinol. 2012;10:49.
    1. Romero R, Grivel JC, Tarca AL, Chaemsaithong P, Xu Z, Fitzgerald W, Hassan SS, Chaiworapongsa T, Margolis L. Evidence of perturbations of the cytokine network in preterm labor. Am J Obstet Gynecol. 2015;213(6):836.e1–836.e18.
    1. Romero R, Miranda J, Chaiworapongsa T, Korzeniewski SJ, Chaemsaithong P, Gotsch F, Dong Z, Ahmed AI, Yoon BH, Hassan SS, Kim CJ, Yeo L. Prevalence and clinical significance of sterile intra-amniotic inflammation in patients with preterm labor and intact membranes. Am J Reprod Immunol. 2014;72(5):458–74.
    1. Biondi C, Pavan B, Lunghi L, Fiorini S, Vesce F. The role and modulation of the oxidative balance in pregnancy. Curr Pharm Des. 2005;11(16):2075–89.
    1. Saito S, Nakashima A, Myojo-Higuma S, Shiozaki A. The balance between cytotoxic NK cells and regulatory NK cells in human pregnancy. J Reprod Immunol. 2008;77(1):14–22.
    1. Menon R, Yu J, Basanta-Henry P, Brou L, Berga SL, Fortunato SJ, Taylor RN. Short fetal leukocyte telomere length and preterm prelabor rupture of the membranes. PLoS One. 2012;7(2):e31136.
    1. Feyaerts D, Benner M, van Cranenbroek B, van der Heijden OWH, Joosten I, van der Molen RG. Human uterine lymphocytes acquire a more experienced and tolerogenic phenotype during pregnancy. Sci Rep. 2017;7(1):2884.
    1. Menon R, Bonney EA, Condon J, Mesiano S, Taylor RN. Novel concepts on pregnancy clocks and alarms: redundancy and synergy in human parturition. Hum Reprod Update. 2016;22(5):535–60.
    1. Casey ML, Winkel CA, Porter JC, MacDonald PC. Endocrine regulation of the initiation and maintenance of parturition. Clin Perinatol. 1983;10(3):709–21.
    1. Gomez-Lopez N, Vega-Sanchez R, Castillo-Castrejon M, Romero R, Cubeiro-Arreola K, Vadillo-Ortega F. Evidence for a role for the adaptive immune response in human term parturition. Am J Reprod Immunol. 2013;69(3):212–30.
    1. Mendelson CR. Minireview: fetal-maternal hormonal signaling in pregnancy and labor. Mol Endocrinol. 2009;23(7):947–54.
    1. Romero R, Espinoza J, Goncalves LF, Kusanovic JP, Friel LA, Nien JK. Inflammation in preterm and term labour and delivery. Semin Fetal Neonatal Med. 2006;11(5):317–26.
    1. Shynlova O, Lee YH, Srikhajon K, Lye SJ. Physiologic uterine inflammation and labor onset: integration of endocrine and mechanical signals. Reprod Sci. 2013;20(2):154–67.
    1. Smith R, Mesiano S, McGrath S. Hormone trajectories leading to human birth. Regul Pept. 2002;108(2–3):159–64.
    1. Nunes V, Cross J, Speich JE, Morgan DR, Strauss JF, 3rd, Ramus RM. Fetal membrane imaging and the prediction of preterm birth: a systematic review, current issues, and future directions. BMC Pregnancy Childbirth. 2016;16(1):387.
    1. Bracchitta G, Catalfo A, Martineau S, Sage E, De Guidi G, Girard PM. Investigation of the phototoxicity and cytotoxicity of naproxen, a non-steroidal anti-inflammatory drug, in human fibroblasts. Photochem Photobiol Sci. 2013;12(5):911–22.
    1. Akasaka E, Takekoshi S, Horikoshi Y, Toriumi K, Ikoma N, Mabuchi T, Tamiya S, Matsuyama T, Ozawa A. Protein oxidative damage and heme oxygenase in sunlight-exposed human skin: roles of MAPK responses to oxidative stress. Tokai J Exp Clin Med. 2010;35(4):152–64.
    1. Menon R, Boldogh I, Urrabaz-Garza R, Polettini J, Syed TA, Saade GR, Papaconstantinou J, Taylor RN. Senescence of primary amniotic cells via oxidative DNA damage. PLoS ONE [Electronic Resource] 2013;8(12):e83416.
    1. Polettini J, Behnia F, Taylor BD, Saade GR, Taylor RN, Menon R. Telomere Fragment Induced Amnion Cell Senescence: A Contributor to Parturition? PLoS ONE [Electronic Resource] 2015;10(9):e0137188.
    1. Hirota Y, Daikoku T, Tranguch S, Xie H, Bradshaw HB, Dey SK. Uterine-specific p53 deficiency confers premature uterine senescence and promotes preterm birth in mice. J Clin Invest. 2010;120(3):803–15.
    1. Menon R, Mesiano S, Taylor RN. Programmed Fetal Membrane Senescence and Exosome-Mediated Signaling: A Mechanism Associated With Timing of Human Parturition. Front Endocrinol (Lausanne) 2017;8:196.
    1. Canzoneri BJ, Feng L, Grotegut CA, Bentley RC, Heine RP, Murtha AP. The chorion layer of fetal membranes is prematurely destroyed in women with preterm premature rupture of the membranes. Reprod Sci. 2013;20(10):1246–54.
    1. Menon R, Boldogh I, Hawkins HK, Woodson M, Polettini J, Syed TA, Fortunato SJ, Saade GR, Papaconstantinou J, Taylor RN. Histological evidence of oxidative stress and premature senescence in preterm premature rupture of the human fetal membranes recapitulated in vitro. American Journal of Pathology. 2014;184(6):1740–51.
    1. Itahana K, Campisi J, Dimri GP. Methods to detect biomarkers of cellular senescence: the senescence-associated beta-galactosidase assay. Methods Mol Biol. 2007;371:21–31.
    1. Dixon CL, Richardson L, Sheller-Miller S, Saade G, Menon R. A distinct mechanism of senescence activation in amnion epithelial cells by infection, inflammation, and oxidative stress. Am J Reprod Immunol. 2017
    1. Behnia F, Taylor BD, Woodson M, Kacerovsky M, Hawkins H, Fortunato SJ, Saade GR, Menon R. Chorioamniotic membrane senescence: a signal for parturition? Am J Obstet Gynecol. 2015;213(3):359.e1–16.
    1. Mor G, Cardenas I, Abrahams V, Guller S. Inflammation and pregnancy: the role of the immune system at the implantation site. Ann N Y Acad Sci. 2011;1221:80–7.
    1. Bonney EM, Ramkamar Differential senescence in feto-maternal tissues during mouse pregnancy, placenta. 2017

Source: PubMed

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