EGF promotes human trophoblast cell invasion by downregulating CTGF expression via PI3K/AKT signaling

in Reproduction
Authors:
Jung-Chien Cheng Center for Reproductive Medicine, Henan Key Laboratory of Reproduction and Genetics, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China

Search for other papers by Jung-Chien Cheng in
Current site
Google Scholar
PubMed
Close
https://orcid.org/0000-0002-5399-9327
,
Yibo Gao Center for Reproductive Medicine, Henan Key Laboratory of Reproduction and Genetics, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China

Search for other papers by Yibo Gao in
Current site
Google Scholar
PubMed
Close
,
Jiaye Chen Center for Reproductive Medicine, Henan Key Laboratory of Reproduction and Genetics, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China

Search for other papers by Jiaye Chen in
Current site
Google Scholar
PubMed
Close
,
Qingxue Meng Center for Reproductive Medicine, Henan Key Laboratory of Reproduction and Genetics, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China

Search for other papers by Qingxue Meng in
Current site
Google Scholar
PubMed
Close
, and
Lanlan Fang Center for Reproductive Medicine, Henan Key Laboratory of Reproduction and Genetics, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China

Search for other papers by Lanlan Fang in
Current site
Google Scholar
PubMed
Close

Correspondence should be addressed to J-C Cheng; Email: jungchien.cheng@gmail.com

*(J-C Cheng and Y Gao contributed equally to this work)

Restricted access
Rent on DeepDyve

Sign up for journal news

In brief

Although the pro-invasive role of epidermal growth factor (EGF) has been reported in human trophoblast cells, the underlying mechanism remains largely unexplored. This work reveals that EGF-induced downregulation of connective tissue growth factor (CTGF) mediates the EGF-stimulated human trophoblast cell invasion.

Abstract

During the development of the placenta, trophoblast cell invasion must be carefully regulated. Although EGF has been shown to promote trophoblast cell invasion, the underlying mechanism remains largely undetermined. Our previous study using RNA-sequencing (RNA-seq) has identified that kisspeptin-1 is a downstream target of EGF in a human trophoblast cell line, HTR-8/SVneo, and mediates EGF-stimulated cell invasion. In the present study, after re-analysis of our previous RNA-seq data, we found that the CTGF was also downregulated in response to the EGF treatment. The inhibitory effects of EGF on CTGF mRNA and protein levels were confirmed in HTR-8/SVneo cells by reverse transcription quantitative real-time PCR and western blot, respectively. Treatment with EGF activated both PI3K/AKT and ERK1/2 signaling pathways. Using pharmacological inhibitors, our results showed that EGFR-mediated activation of PI3K/AKT signaling was required for the EGF-downregulated CTGF mRNA and protein levels. Matrigel-coated transwell invasion assays demonstrated that EGF treatment stimulated cell invasion. In addition, the invasiveness of HTR-8/SVneo cells was suppressed by treatment with recombinant human CTGF. By contrast, siRNA-mediated knockdown of CTGF increased cell invasion. Notably, the EGF-promoted HTR-8/SVneo cell invasion was attenuated by co-treatment with CTGF. This study provides important insights into the molecular mechanisms mediating EGF-stimulated human trophoblast cell invasion and increases the understanding of the biological functions of CTGF in the human placenta.

 

  • Collapse
  • Expand
  • Anteby EY, Greenfield C, Natanson-Yaron S, Goldman-Wohl D, Hamani Y, Khudyak V, Ariel I & Yagel S 2004 Vascular endothelial growth factor, epidermal growth factor and fibroblast growth factor-4 and -10 stimulate trophoblast plasminogen activator system and metalloproteinase-9. Molecular Human Reproduction 10 229235. (https://doi.org/10.1093/molehr/gah031)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Aplin JD 1991 Implantation, trophoblast differentiation and haemochorial placentation: mechanistic evidence in vivo and in vitro. Journal of Cell Science 99 681692. (https://doi.org/10.1242/jcs.99.4.681)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Awamleh Z, Gloor GB & Han VKM 2019 Placental microRNAs in pregnancies with early onset intrauterine growth restriction and preeclampsia: potential impact on gene expression and pathophysiology. BMC Medical Genomics 12 91. (https://doi.org/10.1186/s12920-019-0548-x)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Balaram P, John M, Rajalekshmy TN, Nair B, Schultz G & Nair K 1997 Expression of epidermal growth factor receptor in gestational trophoblastic diseases. Journal of Cancer Research and Clinical Oncology 123 161166. (https://doi.org/10.1007/BF01214668)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Bass KE, Morrish D, Roth I, Bhardwaj D, Taylor R, Zhou Y & Fisher SJ 1994 Human cytotrophoblast invasion is up-regulated by epidermal growth factor: evidence that paracrine factors modify this process. Developmental Biology 164 550561. (https://doi.org/10.1006/dbio.1994.1223)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Bradham DM, Igarashi A, Potter RL & Grotendorst GR 1991 Connective tissue growth factor: a cysteine-rich mitogen secreted by human vascular endothelial cells is related to the SRC-induced immediate early gene product CEF-10. Journal of Cell Biology 114 12851294. (https://doi.org/10.1083/jcb.114.6.1285)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Brigstock DR 1999 The connective tissue growth factor/cysteine-rich 61/nephroblastoma overexpressed (CCN) family. Endocrine Reviews 20 189206. (https://doi.org/10.1210/edrv.20.2.0360)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Burton GJ, Sebire NJ, Myatt L, Tannetta D, Wang YL, Sadovsky Y, Staff AC & Redman CW 2014 Optimising sample collection for placental research. Placenta 35 922. (https://doi.org/10.1016/j.placenta.2013.11.005)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Carpenter G 1987 Receptors for epidermal growth factor and other polypeptide mitogens. Annual Review of Biochemistry 56 881914. (https://doi.org/10.1146/annurev.bi.56.070187.004313)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Chang CC, Shih JY, Jeng YM, Su JL, Lin BZ, Chen ST, Chau YP, Yang PC & Kuo ML 2004 Connective tissue growth factor and its role in lung adenocarcinoma invasion and metastasis. Journal of the National Cancer Institute 96 364375. (https://doi.org/10.1093/jnci/djh059)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Chang CC, Yang MH, Lin BR, Chen ST, Pan SH, Hsiao M, Lai TC, Lin SK, Jeng YM & Chu CY et al.2013 CCN2 inhibits lung cancer metastasis through promoting DAPK-dependent anoikis and inducing EGFR degradation. Cell Death and Differentiation 20 443455. (https://doi.org/10.1038/cdd.2012.136)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Chaqour B, Yang R & Sha Q 2006 Mechanical stretch modulates the promoter activity of the profibrotic factor CCN2 through increased actin polymerization and NF-kappaB activation. Journal of Biological Chemistry 281 2060820622. (https://doi.org/10.1074/jbc.M600214200)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Chen CC & Lau LF 2009 Functions and mechanisms of action of CCN matricellular proteins. International Journal of Biochemistry and Cell Biology 41 771783. (https://doi.org/10.1016/j.biocel.2008.07.025)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Chen X, Liu Y, Xu X & Chen H 2012 Correlation of Cyr61 and CTGF in placentas from the late pre-eclamptic pregnancy. Journal of Perinatal Medicine 40 199200. (https://doi.org/10.1515/jpm-2011-0035)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Cheng JC, Chang HM, Fang L, Sun YP & Leung PC 2015 TGF-beta1 up-regulates connective tissue growth factor expression in human granulosa cells through Smad and ERK1/2 signaling pathways. PLoS One 10 e0126532. (https://doi.org/10.1371/journal.pone.0126532)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Cheng JC, Chang HM & Leung PCK 2017 TGF-beta1 inhibits human trophoblast cell invasion by upregulating connective tissue growth factor expression. Endocrinology 158 36203628. (https://doi.org/10.1210/en.2017-00536)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Cheng JC, Wang EY, Yi Y, Thakur A, Tsai SH & Hoodless PA 2018 S1P stimulates proliferation by upregulating CTGF expression through S1PR2-mediated YAP activation. Molecular Cancer Research 16 15431555. (https://doi.org/10.1158/1541-7786.MCR-17-0681)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Cheng O, Thuillier R, Sampson E, Schultz G, Ruiz P, Zhang X, Yuen PS & Mannon RB 2006 Connective tissue growth factor is a biomarker and mediator of kidney allograft fibrosis. American Journal of Transplantation 6 22922306. (https://doi.org/10.1111/j.1600-6143.2006.01493.x)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Chien W, O'Kelly J, Lu D, Leiter A, Sohn J, Yin D, Karlan B, Vadgama J, Lyons KM & Koeffler HP 2011 Expression of connective tissue growth factor (CTGF/CCN2) in breast cancer cells is associated with increased migration and angiogenesis. International Journal of Oncology 38 17411747. (https://doi.org/10.3892/ijo.2011.985)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Chowdhury I & Chaqour B 2004 Regulation of connective tissue growth factor (CTGF/CCN2) gene transcription and mRNA stability in smooth muscle cells. Involvement of RhoA GTPase and p38 MAP kinase and sensitivity to actin dynamics. European Journal of Biochemistry 271 44364450. (https://doi.org/10.1111/j.1432-1033.2004.04382.x)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Fang L, Gao Y, Wang Z, Li Y, Yan Y, Wu Z, Cheng JC & Sun YP 2021 EGF stimulates human trophoblast cell invasion by downregulating ID3-mediated KISS1 expression. Cell Communication and Signaling 19 ID3-mediated. (https://doi.org/10.1186/s12964-021-00783-2)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Faxen M, Nasiell J, Blanck A, Nisell H & Lunell NO 1998 Altered mRNA expression pattern of placental epidermal growth factor receptor (EGFR) in pregnancies complicated by preeclampsia and/or intrauterine growth retardation. American Journal of Perinatology 15 913. (https://doi.org/10.1055/s-2007-993890)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Ferretti C, Bruni L, Dangles-Marie V, Pecking AP & Bellet D 2007 Molecular circuits shared by placental and cancer cells, and their implications in the proliferative, invasive and migratory capacities of trophoblasts. Human Reproduction Update 13 121141. (https://doi.org/10.1093/humupd/dml048)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Filla MS & Kaul KL 1997 Relative expression of epidermal growth factor receptor in placental cytotrophoblasts and choriocarcinoma cell lines. Placenta 18 1727. (https://doi.org/10.1016/s0143-4004(9790067-9)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Fitzgerald JS, Busch S, Wengenmayer T, Foerster K, de la Motte T, Poehlmann TG & Markert UR 2005 Signal transduction in trophoblast invasion. Chemical Immunology and Allergy 88 181199. (https://doi.org/10.1159/000087834)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Graham CH, Hawley TS, Hawley RG, MacDougall JR, Kerbel RS, Khoo N & Lala PK 1993 Establishment and characterization of first trimester human trophoblast cells with extended lifespan. Experimental Cell Research 206 204211. (https://doi.org/10.1006/excr.1993.1139)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Graness A, Cicha I & Goppelt-Struebe M 2006 Contribution of Src-FAK signaling to the induction of connective tissue growth factor in renal fibroblasts. Kidney International 69 13411349. (https://doi.org/10.1038/sj.ki.5000296)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Holbourn KP, Acharya KR & Perbal B 2008 The CCN family of proteins: structure-function relationships. Trends in Biochemical Sciences 33 461473. (https://doi.org/10.1016/j.tibs.2008.07.006)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Hynes NE & MacDonald G 2009 ErbB receptors and signaling pathways in cancer. Current Opinion in Cell Biology 21 177184. (https://doi.org/10.1016/j.ceb.2008.12.010)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Ivkovic S, Yoon BS, Popoff SN, Safadi FF, Libuda DE, Stephenson RC, Daluiski A & Lyons KM 2003 Connective tissue growth factor coordinates chondrogenesis and angiogenesis during skeletal development. Development 130 27792791. (https://doi.org/10.1242/dev.00505)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Jun JI & Lau LF 2011 Taking aim at the extracellular matrix: CCN proteins as emerging therapeutic targets. Nature Reviews. Drug Discovery 10 945963. (https://doi.org/10.1038/nrd3599)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Kubota S & Takigawa M 2015 Cellular and molecular actions of CCN2/CTGF and its role under physiological and pathological conditions. Clinical Science 128 181196. (https://doi.org/10.1042/CS20140264)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Ladines-Llave CA, Maruo T, Manalo AS & Mochizuki M 1991 Cytologic localization of epidermal growth factor and its receptor in developing human placenta varies over the course of pregnancy. American Journal of Obstetrics and Gynecology 165 13771382. (https://doi.org/10.1016/0002-9378(9190372-x)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Levitzki A & Gazit A 1995 Tyrosine kinase inhibition: an approach to drug development. Science 267 17821788. (https://doi.org/10.1126/science.7892601)

  • Lin BR, Chang CC, Che TF, Chen ST, Chen RJ, Yang CY, Jeng YM, Liang JT, Lee PH & Chang KJ et al.2005 Connective tissue growth factor inhibits metastasis and acts as an independent prognostic marker in colorectal cancer. Gastroenterology 128 923. (https://doi.org/10.1053/j.gastro.2004.10.007)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Liu Y, Fan X, Wang R, Lu X, Dang YL, Wang H, Lin HY, Zhu C, Ge H & Cross JC et al.2018 Single-cell RNA-seq reveals the diversity of trophoblast subtypes and patterns of differentiation in the human placenta. Cell Research 28 819832. (https://doi.org/10.1038/s41422-018-0066-y)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Livak KJ & Schmittgen TD 2001 Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25 402408. (https://doi.org/10.1006/meth.2001.1262)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Maruo T, Matsuo H, Otani T & Mochizuki M 1995 Role of epidermal growth factor (EGF) and its receptor in the development of the human placenta. Reproduction, Fertility, and Development 7 14651470. (https://doi.org/10.1071/rd9951465)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Morrish DW, Dakour J & Li H 1998 Functional regulation of human trophoblast differentiation. Journal of Reproductive Immunology 39 179195. (https://doi.org/10.1016/s0165-0378(9800021-7)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Oda K, Matsuoka Y, Funahashi A & Kitano H 2005 A comprehensive pathway map of epidermal growth factor receptor signaling. Molecular Systems Biology 1 2005.0010. (https://doi.org/10.1038/msb4100014)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Oh SY, Song SE, Seo ES, Kim KH, Choi SJ, Suh YL, Sadovsky Y & Roh CR 2009 The expression of connective tissue growth factor in pregnancies complicated by severe preeclampsia or fetal growth restriction. Placenta 30 981987. (https://doi.org/10.1016/j.placenta.2009.08.006)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Pollheimer J & Knofler M 2005 Signalling pathways regulating the invasive differentiation of human trophoblasts: a review. Placenta 26(Supplement A) S21S30. (https://doi.org/10.1016/j.placenta.2004.11.013)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Qiu Q, Yang M, Tsang BK & Gruslin A 2004a Both mitogen-activated protein kinase and phosphatidylinositol 3-kinase signalling are required in epidermal growth factor-induced human trophoblast migration. Molecular Human Reproduction 10 677684. (https://doi.org/10.1093/molehr/gah088)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Qiu Q, Yang M, Tsang BK & Gruslin A 2004b EGF-induced trophoblast secretion of MMP-9 and TIMP-1 involves activation of both PI3K and MAPK signalling pathways. Reproduction 128 355363. (https://doi.org/10.1530/rep.1.00234)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Rayego-Mateos S, Rodrigues-Diez R, Morgado-Pascual JL, Rodrigues Diez RR, Mas S, LaVoz C, Alique M, Pato J, Keri G, Ortiz A, Egido J & Ruiz-Ortega M 2013 Connective tissue growth factor is a new ligand of epidermal growth factor receptor. Journal of Molecular Cell Biology 5 3 233 35. (https://doi.org/10.1093/jmcb/mjt030)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Ryseck RP, Macdonald-Bravo H, Mattei MG & Bravo R 1991 Structure, mapping, and expression of fisp-12, a growth factor-inducible gene encoding a secreted cysteine-rich protein. Cell Growth and Differentiation 2 225233.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Samarin J, Cicha I & Goppelt-Struebe M 2009a Cell type-specific regulation of CCN2 protein expression by PI3K-AKT-FoxO signaling. Journal of Cell Communication and Signaling 3 7984. (https://doi.org/10.1007/s12079-009-0055-5)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Samarin J, Rehm M, Krueger B, Waschke J & Goppelt-Struebe M 2009b Up-regulation of connective tissue growth factor in endothelial cells by the microtubule-destabilizing agent combretastatin A-4. Molecular Cancer Research 7 180188. (https://doi.org/10.1158/1541-7786.MCR-08-0292)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Schmittgen TD & Livak KJ 2008 Analyzing real-time PCR data by the comparative C(T) method. Nature Protocols 3 11011108. (https://doi.org/10.1038/nprot.2008.73)

  • Stahl PL, Salmen F, Vickovic S, Lundmark A, Navarro JF, Magnusson J, Giacomello S, Asp M, Westholm JO & Huss M et al.2016 Visualization and analysis of gene expression in tissue sections by spatial transcriptomics. Science 353 7882. (https://doi.org/10.1126/science.aaf2403)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Staun-Ram E, Goldman S, Gabarin D & Shalev E 2004 Expression and importance of matrix metalloproteinase 2 and 9 (MMP-2 and -9) in human trophoblast invasion. Reproductive Biology and Endocrinology : RB&E 2 59. (https://doi.org/10.1186/1477-7827-2-59)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Staun-Ram E, Goldman S & Shalev E 2009 p53 Mediates epidermal growth factor (EGF) induction of MMP-2 transcription and trophoblast invasion. Placenta 30 10291036. (https://doi.org/10.1016/j.placenta.2009.09.010)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Suzuma K, Naruse K, Suzuma I, Takahara N, Ueki K, Aiello LP & King GL 2000 Vascular endothelial growth factor induces expression of connective tissue growth factor via KDR, Flt1, and phosphatidylinositol 3-kinase-Akt-dependent pathways in retinal vascular cells. Journal of Biological Chemistry 275 4072540731. (https://doi.org/10.1074/jbc.M006509200)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Waddell JM, Evans J, Jabbour HN & Denison FC 2011 CTGF expression is up-regulated by PROK1 in early pregnancy and influences HTR-8/Svneo cell adhesion and network formation. Human Reproduction 26 6775. (https://doi.org/10.1093/humrep/deq294)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Wang FM, Yu F, Tan Y, Liu G & Zhao MH 2014 The serum levels of connective tissue growth factor in patients with systemic lupus erythematosus and lupus nephritis. Lupus 23 655664. (https://doi.org/10.1177/0961203314524291)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Winterhager E & Gellhaus A 2014 The role of the CCN family of proteins in female reproduction. Cellular and Molecular Life Sciences 71 22992311. (https://doi.org/10.1007/s00018-014-1556-9)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Yang DH, Kim HS, Wilson EM, Rosenfeld RG & Oh Y 1998 Identification of glycosylated 38-kDa connective tissue growth factor (IGFBP-related protein 2) and proteolytic fragments in human biological fluids, and up-regulation of IGFBP-rP2 expression by TGF-beta in Hs578T human breast cancer cells. Journal of Clinical Endocrinology and Metabolism 83 25932596. (https://doi.org/10.1210/jcem.83.7.5097)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Yu Y, Fang L, Wang S, Li Y, Guo Y & Sun YP 2019 Amphiregulin promotes trophoblast invasion and increases MMP9/TIMP1 ratio through ERK1/2 and Akt signal pathways. Life Sciences 236 116899. (https://doi.org/10.1016/j.lfs.2019.116899)

    • PubMed
    • Search Google Scholar
    • Export Citation