The injectable-only contraceptive medroxyprogesterone acetate, unlike norethisterone acetate and progesterone, regulates inflammatory genes in endocervical cells via the glucocorticoid receptor
Yashini Govender, Chanel Avenant, Nicolette J D Verhoog, Roslyn M Ray, Nicholas J Grantham, Donita Africander, Janet P Hapgood, Yashini Govender, Chanel Avenant, Nicolette J D Verhoog, Roslyn M Ray, Nicholas J Grantham, Donita Africander, Janet P Hapgood
Abstract
Clinical studies suggest that the injectable contraceptive medroxyprogesterone acetate (MPA) increases susceptibility to infections such as HIV-1, unlike the injectable contraceptive norethisterone enanthate (NET-EN). We investigated the differential effects, molecular mechanism of action and steroid receptor involvement in gene expression by MPA as compared to NET and progesterone (P4) in the End1/E6E7 cell line model for the endocervical epithelium, a key point of entry for pathogens in the female genital mucosa. MPA, unlike NET-acetate (NET-A) and P4, increases mRNA expression of the anti-inflammatory GILZ and IκBα genes. Similarly, MPA unlike NET-A, decreases mRNA expression of the pro-inflammatory IL-6, IL-8 and RANTES genes, and IL-6 and IL-8 protein levels. The predominant steroid receptor expressed in the End1/E6E7 and primary endocervical epithelial cells is the glucocorticoid receptor (GR), and GR knockdown experiments show that the anti-inflammatory effects of MPA are mediated by the GR. Chromatin-immunoprecipitation results suggest that MPA, unlike NET-A and P4, represses pro-inflammatory cytokine gene expression in cervical epithelial cells via a mechanism involving recruitment of the GR to cytokine gene promoters, like the GR agonist dexamethasone. This is at least in part consistent with direct effects on transcription, without a requirement for new protein synthesis. Dose response analysis shows that MPA has a potency of ∼ 24 nM for transactivation of the anti-inflammatory GILZ gene and ∼ 4-20 nM for repression of the pro-inflammatory genes, suggesting that these effects are likely to be relevant at injectable contraceptive doses of MPA. These findings suggest that in the context of the genital mucosa, these GR-mediated glucocorticoid-like effects of MPA in cervical epithelial cells are likely to play a critical role in discriminating between the effects on inflammation caused by different progestins and P4 and hence susceptibility to genital infections, given the predominant expression of the GR in primary endocervical epithelial cells.
Conflict of interest statement
Competing Interests: The authors have declared that no competing interests exist.
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References
- Kaushic C, Ferreira VH, Kafka JK, Nazli A (2010) HIV infection in the female genital tract: discrete influence of the local mucosal microenvironment. Am J Reprod Immunol 63: 566–575.
- Kaushic C (2011) HIV-1 infection in the female reproductive tract: role of interactions between HIV-1 and genital epithelial cells. Am J Reprod Immunol 65: 253–260.
- Wira CR, Fahey JV, Sentman CL, Pioli PA, Shen L (2005) Innate and adaptive immunity in female genital tract: cellular responses and interactions. Immunol Rev 206: 306–335.
- Barclay C, Brennand J, Kelly R, Calder A (1993) Interleukin-8 production by the human cervix. Am J Obs Gynecol 169: 625–632.
- Fichorova RN, Anderson DJ (1999) Differential expression of immunobiological mediators by immortalized human cervical and vaginal epithelial cells. Biol Reprod 60: 508–514.
- Woodworth CD, Simpson S (1993) Comparative lymphokine secretion by cultured normal human cervical keratinocytes, papillomavirus-immortalized, and carcinoma cell lines. Am J Pathol 142: 1544–1555.
- Fichorova RN, Desai PJ, Gibson III C, Genco CA (2001) Distinct Proinflammatory Host Responses to Neisseria gonorrhoeae Infection in Immortalized Human Cervical and Vaginal Epithelial Cells Distinct Proinflammatory Host Responses to Neisseria gonorrhoeae Infection in Immortalized Human Cervical and Vaginal Ep. Infect Immun 69: 5840–5848.
- Rodriguez-Garcia M, Patel MV, Wira CR (2013) Innate and adaptive anti-HIV immune responses in the female reproductive tract. J Reprod Immunol 97: 74–84.
- Brabin L (2002) Interactions of the female hormonal environment, susceptibility to viral infections, and disease progression. AIDS Patient Care STDS 16: 211–221.
- MacDonald EM, Savoy A, Gillgrass A, Fernandez S, Smieja M, et al. (2007) Susceptibility of human female primary genital epithelial cells to herpes simplex virus, type-2 and the effect of TLR3 ligand and sex hormones on infection. Biol Reprod 77: 1049–1059.
- Hel Z, Stringer E, Mestecky J (2010) Sex Steroid Hormones, Hormonal Contraception, and the Immunobiology of Human Immunodeficiency Virus-1 Infection. Endocr Rev 31: 79–97.
- Gillgrass AE, Ashkar AA, Rosenthal KL, Kaushic C (2003) Prolonged Exposure to Progesterone Prevents Induction of Protective Mucosal Responses following Intravaginal Immunization with Attenuated Herpes Simplex Virus Type 2. J Virol 77: 9845–9851.
- Kaushic C, Ashkar AA, Reid LA, Rosenthal KL (2003) Progesterone Increases Susceptibility and Decreases Immune Responses to Genital Herpes Infection. J Virol 77: 4558–4565.
- MacLean R (2005) Injectable use may increase women’s odds of getting chlamydia or gonorrhea. Int Fam Plan Perspect 31: 45–46.
- Morrison CS, Bright P, Wong EL, Kwok C, Yacobson I, et al. (2004) Hormonal Contraceptive Use, Cervical Ectopy, and the Acquisition of Cervical Infections. Sex Transm Dis 31: 561–567.
- Trunova N, Tsai L, Tung S, Schneider E, Harouse J, et al. (2006) Progestin-based contraceptive suppresses cellular immune responses in SHIV-infected rhesus macaques. Virology 352: 169–177.
- Parr M, Kepple L, McDermott M, Drew M, Bozzola J, et al. (1994) A mouse model for studies of mucosal immunity to vaginal infection by herpes simplex virus type 2. Lab Invest 70: 369–380.
- WHO website. Hormonal Contraception and HIV: Technical Statment. Available: . Accessed 2014 April 29.
- fhi 360 website. Expanding access to injectable contraception. Available: . Accessed 2014 April 29.
- Smit JA, Beksinska ME (2013) Hormonal contraceptive continuation and switching in South Africa: Implications for evaluating the association of injectable hormonal contraceptive use and HIV. J Acquir Immune Defic Syndr 62: 363–365.
- Blish CA, Baeten JM (2011) Hormonal contraception and HIV-1 transmission. Am J Reprod Immunol 65: 302–307.
- Gray RH (2012) Correspondence Use of hormonal contraceptives and risk of HIV-1 transmission. Lancet Infect Dis 12: 507–508.
- Heffron R, Donnell D, Rees H, Celum C, Mugo N, et al. (2012) Use of hormonal contraceptives and risk of HIV-1 transmission: a prospective cohort study. Lancet Infect Dis 12: 19–26.
- Morrison CS, Nanda K (2012) Hormonal contraception and HIV: an unanswered question. Lancet Infect Dis 12: 2–3.
- Morrison CS, Chen P, Kwok C, Richardson BA, Chipato T, et al. (2010) Hormonal Contraception and HIV Acquisition: Reanalysis using Marginal Structural Modeling. AIDS 24: 1778–1781.
- Morrison CS, Skoler-Karpoff S, Kwok C, Chen P-L, van de Wijgert J, et al. (2012) Hormonal contraception and the risk of HIV acquisition among women in South Africa. AIDS 26: 497–504.
- Polis CB, Curtis KM (2013) Use of hormonal contraceptives and HIV acquisition in women: a systematic review of the epidemiological evidence. Lancet Infect Dis 13: 797–808.
- McCoy SI, Zheng W, Montgomery ET, Blanchard K, van der Straten A, et al. (2013) Oral and injectable contraception use and risk of HIV acquisition among women in sub-Saharan Africa. AIDS 27: 1001–1009.
- Wand H, Ramjee G (2012) The effects of injectable hormonal contraceptives on HIV seroconversion and on sexually transmitted infections. AIDS 26: 375–380.
- USAID website. Technical Brief: Hormonal Contraception and HIV. Available: . Accessed 2014 April 29.
- Mostad SB, Kreiss JK, Ryncarz AJ, Mandaliya K, Chohan B, et al. (2000) Cervical shedding of herpes simplex virus in human immunodeficiency virus-infected women: effects of hormonal contraception, pregnancy, and vitamin A deficiency. J Infect Dis 181: 58–63.
- Wang CC, Mcclelland RS, Overbaugh J, Reilly M, Devange D, et al. (2004) The effect of hormonal contraception on genital tract shedding of HIV-1. AIDS 18: 205–209.
- Lavreys L, Baeten J, Martin Jr H, Overbaugh J, Mandaliya K, et al. (2004) Hormonal contraception and risk of HIV-1 acquisition: results of a 10-year prospective study. AIDS 18: 695–697.
- Abdool Karim Q, Abdool Karim SS, Frohlich JA, Grobler AC, Baxter C, et al. (2010) Effectiveness and safety of tenofovir gel, an antiretroviral microbicide, for the prevention of HIV infection in women. Science 329: 1168–1174.
- Gouws E, Stanecki KA, Lyerla R, Ghys PD (2008) The epidemiology of HIV infection among young people aged 15–24 years in southern Africa. AIDS 22: S5–S16.
- Hapgood JP, Koubovec D, Louw A, Africander D (2004) Not all progestins are the same: implications for usage. Trends Pharmacol Sci 25: 554–557.
- Africander D, Verhoog N, Hapgood JP (2011) Molecular mechanisms of steroid receptor-mediated actions by synthetic progestins used in HRT and contraception. Steroids 76: 636–652.
- Stanczyk FZ, Hapgood JP, Winer S, Mishell Jr DR (2013) Progestogens Used in Postmenopausal Hormone Therapy: Differences in Their Pharmacological Properties, Intracellular Actions, and Clinical Effects. Endocr Rev 34: 171–208.
- Bray J, Jelinsky S, Ghatge R, Bray J, Tunkey C, et al. (2005) Quantitative analysis of gene regulation by seven clinically relevant progestins suggests a highly similar mechanism of action through progesterone receptors in T47D breast cancer cells. J Steroid Biochem Mol Biol 97: 328–341.
- Sitruk-Ware R (2004) Pharmacological profile of progestins. Maturitas 47: 277–283.
- Koubovec D, Ronacher K, Stubsrud E, Louw A, Hapgood JP (2005) Synthetic progestins used in HRT have different glucocorticoid agonist properties. Mol Cell Endocrinol 242: 23–32.
- Stanczyk FZ, Roy S (1990) Metabolism of levonorgestrel, norethindrone, and structurally related contraceptive steroids. Contraception 42: 67–96.
- Africander D, Louw R, Hapgood JP (2013) Investigating the anti-mineralocorticoid properties of synthetic progestins used in hormone therapy. Biochem Biophys Res Commun 433: 305–310.
- Ronacher K, Hadley K, Avenant C, Stubsrud E, Simons SS, et al. (2009) Ligand-selective transactivation and transrepression via the glucocorticoid receptor: Role of cofactor interaction. Mol Cell Endocrinol 299: 219–231.
- Kadmiel M, Cidlowski JA (2013) Glucocorticoid receptor signaling in health and disease. Trends Pharmacol Sci 34: 518–530.
- Koubovec D, Vanden Berghe W, Vermeulen L, Haegeman G, Hapgood JP (2004) Medroxyprogesterone acetate downregulates cytokine gene expression in mouse fibroblast cells. Mol Cell Endocrinol 221: 75–85.
- Bamberger CM, Else T, Bamberger A, Beil FU, Schulte HM (1999) Dissociative Glucocorticoid Activity of Medroxyprogesterone Acetate in Normal Human Lymphocytes. J Clin Endocrinol Metab 84: 4055–4061.
- Hapgood JP (2013) Immunosuppressive biological mechanisms support reassessment of use of the injectable contraceptive medroxyprogesterone acetate. Endocrinology 154: 985–988.
- Huijbregts RPH, Helton ES, Michel KG, Sabbaj S, Richter HE, et al. (2013) Hormonal contraception and HIV-1 infection: medroxyprogesterone acetate suppresses innate and adaptive immune mechanisms. Endocrinology 154: 1282–1295.
- Fichorova RN, Rheinwald JG, Anderson D (1997) Generation of papillomavirus-immortalized cell lines from normal human ectocervical, endocervical, and vaginal epithelium that maintain expression of tissue-specific differentiation proteins. Biol Reprod 57: 847–855.
- Cairns C, Cairns W, Okret S (1993) Inhibition of Gene Expression by Steroid Hormone Receptors Via a Negative Glucocorticoid Response Element: Evidence for the Involvement of DNA-Binding and Agonistic Effects of the Antiglucocorticoid/Antiprogestin RU486. DNA Cell Biol 12: 695–702.
- Arriza JL, Weinberger C, Cerelli G, Glaser TM, Handelin BL, et al. (1987) Cloning of human mineralocorticoid receptor complementary DNA: structural and functional kinship with the glucocorticoid receptor. Science 237: 268.
- Brinkmann AO, Faber P, van Rooij HC, Kuiper GGJ, Ris C, et al. (1989) The human androgen receptor: Domain structure, genomic organization and regulation of expression. J Steroid Biochem 34: 307–310.
- Flouriot G, Brand H, Denger S, Metivier È, Kos M, et al. (2000) Identification of a new isoform of the human estrogen receptor-alpha (hER- a) that is encoded by distinct transcripts and that is able to repress hER- a activation function 1. EMBO J 19: 468–470.
- Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29: 2002–2007.
- Verhoog N, Du Toit A, Avenant C, Hapgood JP (2011) Glucocorticoid-independent Repression of TNFa-stimulated IL-6 Expression by the Glucocorticoid Receptor: A potential Mechanism For the Protection Againsts an Excessive Inflammatory Response. J Biol Chem 286: 19297–19310.
- Ma H, Shang Y, Lee D, Stallcup M (2003) Study of nuclear receptor-induced transcription complex assembly and histone modification by chromatin immunoprecipitation assays. Methods Enzym 364: 284–296.
- Mosmann T (1983) Rapid Colorimetric Assay for Cellular Growth and Survival: Application to Proliferation and cytotoxicity assays. J Immunol Methods 12: 16.
- Ayroldi E, Riccardi C (2009) Glucocorticoid-induced leucine zipper (GILZ): a new important mediator of glucocorticoid action. FASEB 23: 3649–3658.
- Commins SP, Borish L, Steinke JW (2010) Immunologic messenger molecules: cytokines, interferons, and chemokines. J Allergy Clin Immunol 125: S53–72.
- Hermoso MA, Cidlowski JA (2003) Putting the brake on inflammatory responses: the role of glucocorticoids. IUBMB Life 55: 497–504.
- Muzikar KA, Nickols NG, Dervan PB (2009) Repression of DNA-binding dependent glucocorticoid receptor-mediated gene expression. Proc Natl Acad Sci U S A 106: 16598–16603.
- McKay LI, Cidlowski JA (1999) Molecular control of immune/inflammatory responses: interactions between nuclear factor-kappa B and steroid receptor-signaling pathways. Endocr Rev 20: 435–459.
- De Bosscher K, Van Craenenbroeck K, Meijer OC, Haegeman G (2008) Selective transrepression versus transactivation mechanisms by glucocorticoid receptor modulators in stress and immune systems. Eur J Pharmacol 583: 290–302.
- Avenant C, Kotitschke A, Hapgood JP (2010) Glucocorticoid Receptor Phosphorylation Modulates Transcription Efficacy through GRIP-1 Recruitment. Biochemistry 49: 972–985.
- King EM, Chivers JE, Rider CF, Minnich A, Giembycz MA, et al. (2013) Glucocorticoid repression of inflammatory gene expression shows differential responsiveness by transactivation- and transrepression-dependent mechanisms. PLoS One 8: e53936.
- Hadley KE, Louw A, Hapgood JP (2011) Differential nuclear localisation and promoter occupancy play a role in glucocorticoid receptor ligand-specific transcriptional responses. Steroids 76: 1176–1184.
- Auphan N, DiDonato JA, Rosette C, Helmberg A, Karin M (1995) Immunosuppression by Glucocorticoids: Inhibition of NF-kB Activity Through Induction of IkB. Science 270: 286–290.
- Scheinman RI, Cogswell PC, Lofquist AK, Baldwin Jr AS (1995) Role of Transcriptional Activation of IkBa in Mediation of Immunosuppression by Glucocorticoids. Science 270: 283–286.
- Heck S, Bender K, Kullmann M, Gottlicher M, Herrlich P, et al. (1997) I κ B α -independent downregulation of NF- κ B activity by glucocorticoid receptor. EMBO J 16: 4698–4707.
- Newton R, Hart LA, Stevens DA, Bergmann M, Donnelly LE, et al. (1998) Effect of dexamethasone on interleukin-1 β - (IL-1 β) -induced nuclear factor- κ B (NF- κ B) and κ B-dependent transcription in epithelial cells. Eur J Biochem 254: 81–89.
- Adcock IM, Nasuhara Y, Stevens DA, Barnes PJ (1999) Ligand-induced differentiation of glucocorticoid receptor (GR) trans-repression and transactivation: preferential targetting of NF- k B and lack of I- k B involvement. Br J Pharmacol 127: 1003–1011.
- Cvoro A, Yuan C, Paruthiyil S, Miller OH, Yamamoto KR, et al. (2011) Cross Talk between Glucocorticoid and Estrogen Receptors Occurs at a Subset of Proinflammatory Genes. J Immunol. 186 (7): 4354–4360.
- Tomasicchio M, Avenant C, Du Toit A, Ray RM, Hapgood JP (2013) The progestin-only contraceptive medroxyprogesterone acetate, but not norethisterone acetate, enhances HIV-1 Vpr-mediated apoptosis in human CD4+ T cells through the glucocorticoid receptor. PLoS One 8: e62895.
- Elovitz M, Wang Z (2004) Medroxyprogesterone acetate, but not progesterone, protects against inflammation-induced parturition and intrauterine fetal demise. Am J Obstet Gynecol 190: 693–701.
- Africander D, Louw R, Verhoog N, Noeth D, Hapgood JP (2011) Differential regulation of endogenous pro-inflammatory cytokine genes by medroxyprogesterone acetate and norethisterone acetate in cell lines of the female genital tract. Contraception. 84 (4): 423–435.
- Pudney J, Quayle AJ, Anderson DJ (2005) Immunological Microenvironments in the Human Vagina and Cervix: Mediators of Cellular Immunity Are Concentrated in the Cervical Transformation Zone. Biol Reprod 73: 1253–1263.
- Hapgood JP, Ray RM, Govender Y, Avenant C, Tomasicchio M (2014) Differential glucocorticoid receptor-mediated effects on immunomodulatory gene expression by progestin contraceptives: implications for HIV-1 pathogenesis. Am J Reprod Immunol. doi:.
- Al-Hendy A, Salama SA (2006) Ethnic distribution of estrogen receptor-alpha polymorphism is associated with a higher prevalence of uterine leiomyomas in black Americans. Fertil Steril 86: 686–693.
- Vladic-Stjernholm Y, Vladic T, Blesson CS, Ekman-Ordeberg G, Sahlin L (2009) Prostaglandin treatment is associated with a withdrawal of progesterone and androgen at the receptor level in the uterine cervix. Reprod Biol Endocrinol 7: 116.
- Hapgood JP, Africander D, Louw R, Ray RM, Rohwer JM (2013) Potency of progestogens used in hormonal therapy: Toward understanding differential actions. J Steroid Biochem Mol Biol: 1–9.
- Kirton K, Cornette JC (1974) Return of ovulatory cyclicity following an intramuscular injection of medroxyprogesterone acetate (Provera). Contraception 10: 39–45.
- Fotherby K (1983) Variability of pharmacokinetic parameters for contraceptive steroids. Steroid Biochem 19: 817–820.
- Janeway Jr CA, Travers P, Walport M (2012) Immunobiology. 8th Editio. Murphy K, editor New York: Garland Science.
- Lee B, Montaner LJ (1999) Chemokine immunobiology in HIV-1 pathogenesis. J Leukoc Biol 65: 552–565.
- Coleman JS, Hitti J, Bukusi EA, Mwachari C, Muliro A, et al. (2007) Infectious correlates of HIV-1 shedding in the female upper and lower genital tracts. AIDS 21: 1569–1578.
- Sha BE, Zariffard MR, Wang QJ, Chen HY, Bremer J, et al. (2005) Female genital-tract HIV load correlates inversely with Lactobacillus species but positively with bacterial vaginosis and Myco- plasma hominis. J Infect Dis 191: 25–32.
- Morrison C, Fichorova R, Mauck C, Chen P, Kwok C, et al. (2014) Cervical Inflammation and Immunity Associated with Hormonal Contraception, Pregnancy and HIV-1 Seroconversion. JAIDS. doi:.
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