Human Cell 2006; 19: 38–42 doi: 10.1111/j.1749-0774.2005.00006.x

© 2006 The Authors38 Journal compilation © 2006 Japan Human Cell Society

Blackwell Publishing Ltd

2005 HUMAN CELL SOCIETY AWARD

Human endometrial cytodifferentiation by histone deacetylase inhibitors

Hiroshi UCHIDA, Tetsuo MARUYAMA, Takashi NAGASHIMA, Masanori ONO, Hirotaka MASUDA, Toru ARASE, Ikuko SUGIURA, Maki ONOUCHI, Takashi KAJITANI, Hironori ASADA and Yasunori YOSHIMURADepartment of Obstetrics and Gynecology, Keio University School of Medicine, Shinjuku, Tokyo, Japan

Abstract

Human uterine endometrium repeats proliferation, differentiation (decidualization) and tissuebreakdown during the menstrual period. Appropriate secretion of ovarian steroid hormones reg-ulates these sequential endometrial remodeling cycles. While progesterone replacement therapyis adopted for endometrial dysfunction of differentiation, including recurrent impairment ofimplantation, no obvious effective results are obtained. Histone reversible acetylation, regulatedby histone acetyltransferases and histone deacetylases plays a pivotal role in gene transcription.Although, in cells cultured with histone deacetylase inhibitors (HDACI), the expression of onlyabout 2% of expressed genes is changed twofold or more compared with untreated control cells.Numerous previous works have demonstrated that HDACI affect cell proliferation/apoptosis ina variety of types of cells. To date, several HDACI are in phase I or phase II clinical trials as anti-cancer drugs. However, no reports have been found that HDACI is useful for transdifferentiationin human endometrium. Recently, we reported that HDACI could induce the expression of differ-entiation marker proteins, morphological change and functional cytodifferentiation in both humanendometrial stromal and epithelial cells. In this review, we summarize the effect of HDACI againstthe human endometrial cytodifferentiation, indicating the possibility that HDACI can be used notonly as an anticancer drug, but also as a transdifferentiation reagent, based on a new strategy.

Key words: differentiation, endometrium, glycodelin, histone deacetylase inhibitor, migration.

HISTONE ACETYLATION AND GENE TRANSCRIPTION

Acetylation, which is one of histone modification, playsan important role in gene transcription.1–4 The acetylationstatus of core histones is reversibly and cooperativelyregulated by two groups of enzymes, histone acetyl-

transferases and histone deacetylases (HDAC). Whenlysine residues in the amino termini of histones areacetylated, neutralization of the positive charge results ina loosened nucleosome arrangement that makes promoterregions readily accessible to transcription factors. Theassociation of transcription factors with promoter regionsresults in defined gene transcription, which affectsmultiple cellular functions depending on the transcribedgene, such as cell proliferation or apoptosis, celldifferentiation and cell motility. Histone deacetylaseinhibitors (HDACI), such as trichostatin A (TSA) andsuberoylanilide hydroxamic acid (SAHA), maintain theacetylated status of gene by fitting into the pocket ofenzymatic reactive core in HDAC, resulting in a blockadepreventing the association between HDAC and acetylatedhistone protein.1,5

Correspondence: Dr Hiroshi Uchida, MD, PhD, Department of Obstetrics and Gynecology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku, Tokyo 160-8582, Japan. Email: uchida@sc.itc.keio.ac.jp

This report was presented at the 23rd Annual Meeting of the Japan Human Cell Society held on 25–27 August 2005 in Tsukuba, Japan.

Received 5 November 2005; accepted 15 November 2005.

Endometrial differentiation by HDACI

© 2006 The AuthorsJournal compilation © 2006 Japan Human Cell Society 39

HISTONE DEACETYLASE INHIBITORS AND HUMAN ENDOMETRIAL STROMAL CELLS

Human uterine endometrium repeats proliferation,differentiation, tissue breakdown and shedding during themenstrual cycle, regulated by the influence of estrogensand progestins. Coordinated and sequential productionof these ovarian steroid hormones is required for propercyclical changes of human endometrium. Inappropriateregulation of cyclical endometrial differentiation causesseveral problems, such as the elevation of the incidenceof endometrial carcinoma, osteoporosis and failure ofimplantation of fertilized eggs. While progesteronereplacement therapy is adopted for endometrial (luteal)dysfunction, no obviously effective results are obtained,especially in the cases of recurrent failure of in vitrofertilization and embryo transfer.

Although in cells cultured with TSA the expression ofonly about 2% of expressed gene is altered twofold ormore compared with untreated control cells, numerousprevious works have demonstrated that HDACI affect cellproliferation in a variety types of cells.2 Whereas severalworks have reported that HDACI can be used as a differ-entiation or transformation inducer in human erythro-leukemia cell line, K562 or v-ras transfected cells,6,7 noreports have mentioned the possibility of HDACI beingused to transdifferentiate endometrial cells. We have pre-viously reported that TSA can accelerate the ovariansteroid hormone-induced up-regulation of differentiationmarker proteins, including prolactin and insulin-likegrowth factor binding protein-1.8 In company with up-regulation of these proteins, TSA was able to induce mor-phological change (decidualization) in a similar way toovarian steroid hormones, and the effect on cell shape byTSA and steroid hormones were synergic.8 These findingsindicate that TSA may be able to be used as a transdiffer-entiation helper for human endometrial tissue.

HISTONE DEACETYLASE INHIBITORS CAN INDUCE GLYCODELIN EXPRESSION VIA ACTIVATION OF GLYCODELIN PROMOTER IN ISHIKAWA CELLS

In the first step of the implantation mechanism, thefertilized egg initially makes contact with not endometrialstromal cells, but epithelial cells. In the case of clinical useof HDACI for cases of implantation failure, it is necessarythat HDACI be able to induce cytodifferentiation not onlyof stromal cells, but also epithelial cells. Glycodelin,

which is a 28-kDa globular secretory protein, shows aunique expression pattern in human endometrialepithelial cells. The expression is detected from LH+4(4 days after ovulation), reaching its peak at LH+10, andmarkedly decreasing during menstruation.9 It has beenreported that glycodelin inhibits human sperm–eggbinding and that has an immunosuppressive effect throughthe inactivation of natural killer cells or suppression ofT lymphocyte proliferation.10,11 These cellular functionswere thought to be the system to avoid multiple pregnancyand to contribute to protection of the embryonic semiallo-graft, respectively. The unique expression pattern, whichcoincides with the “implantation window” (appropriateperiod for implantation), suggests the possibility thatglycodelin functions to help implantation.

In the analysis of HDACI’s effect on endometrial epithelialcell cytodifferentiation, we adopted human adenocarci-noma cell line, Ishikawa (clone 3-H-12, estrogen receptorα and progesterone receptor [PR] are expressed)12 as amodel for human endometrial epithelial cells. In a RT-PCR and immunoprecipitation study, both mRNA andprotein of glycodelin were up-regulated by the treatmentof several HDACI belonging to the hydroxamic acid group,such as TSA and SAHA, in a similar way to the treatmentof ovarian steroid hormones.13 In Ishikawa cells, whichwere transfected with glycodelin promoter region (from−304 to 20 bp) containing luciferase expression vector,glycodelin promoter was activated by the addition ofE2P4, TSA or SAHA to the culture medium.13

In human adenocarcinoma cell line HEC-1B, ligand-activated PR has been demonstrated to stimulate glycodelingene expression through functional Sp1 sites present in theglycodelin promoter region between −304 to +20 bp.14

TSA and SAHA are known to activate the promoter ofp21/WAF1/Cip1 gene through Sp1 site in some types ofcells that do not express PRs.15 In agreement, we foundthat, in the absence of progesterone, TSA and SAHA couldactivate the glycodelin promoter harboring functionalSp1 sites. These results show that HDACI can induceglycodelin expression without ovarian steroid hormones.

HISTONE DEACETYLASE INHIBITORS CAN INDUCE MORPHOLOGICAL AND FUNCTIONAL CYTODIFFERENTIATION IN ISHIKAWA CELLS

To investigate whether cytodifferentiation is induced byHDACI, we analyzed the morphological change andglycogen synthesis in Ishikawa cells, stimulated by E2P4or HDACI. In microscopic observation study, widespread

H. Uchida et al.

© 2006 The Authors40 Journal compilation © 2006 Japan Human Cell Society

and flattened morphological changes were shown inIshikawa cells with stimulation of E2P4 or HDACI. Glycogensynthesis, which reflects the functional differentiation fromproliferative-phase to secretory-phase endometrium, werealso enhanced by these stimulations in Ishikawa cells.13

GLYCODELIN GENE SILENCING ABROGATES HISTONE DEACETYLASE INHIBITOR-INDUCED CYTODIFFERENTIATION

Our collective findings provoked in us the next question:whether glycodelin itself, induced by ovarian steroidhormones or HDACI, influence the cytodifferentiation ofIshikawa cells. To explore this question, we investigatedthe morphological change and glycogen synthesis ofIshikawa cells, suppressed glycodelin expression byglycodelin small interference RNA (siRNA). Glycodelingene silencing by siRNA represented almost completeabrogation of these cytodifferentiation effects by hormonesor HDACI. These results indicate that glycodelin plays animportant role in cytodifferentiation of human endometrialepithelial cells as a key molecule.

HISTONE DEACETYLASE INHIBITORS CAN ALSO ENHANCE SINGLE/COLLECTIVE CELL MIGRATION VIA GLYCODELIN EXPRESSION

Histone deacetylase inhibitors have recently beendemonstrated to affect not only cell proliferation anddifferentiation, but also cell motility. In a study usinghepatic stellate cells, Rombouts et al. have shown thattreatment with TSA repressed wound healing activity incompany with down-regulation of expression of actin-related protein and Rho small GTPase, resulting in loss ofactin stress fiber formation.16 Furthermore, it has beendemonstrated that glycodelin is involved in regulation ofcell motility using HUVEC cells cultured in a mediumcontaining glycodelin partial peptides or antiglycodelinantibody.17 It is predicted that human endometrialepithelial cells acquire the ability to move into theappropriate positions for the formation of highly com-plicated gland structures and for remodeling the surfacelayer at the implantation site during the cytodifferentiationperiod (secretory-phase). We hypothesized that HDACIcan enhance the migration ability of Ishikawa cells in asimilar way to the stimulation of ovarian steroid hormones.To test our hypothesis, we analyzed Ishikawa cell migrationability using trans-well migration assay for single-cell

migration and wound-healing assay for collective cellmigration. The treatment with HDACI showed theenhancement of both single and collective cell migrationabilities in a dose-dependent manner. A relatively lowconcentration of HDACI was required to enhance cellmigration ability at a similar level to the enhancement byE2P4 (unpublished data, 2005). These results suggestthat HDACI can induce cell migration ability in companywith cytodifferentiation, avoiding induction of cellapoptosis, because the required concentration of HDACIfor alteration of cell migration ability or transdifferentiationis relative low, compared with the necessary dose for useas an anticancer drug. Moreover, this up-regulation of cellmigration ability was also blocked by glycodelin gene-silencing using siRNA (unpublished data, 2005). Usingan immunofluorescent technique, we found that actinstress fiber formation responded to treatment with E2P4or HDACI. The expression of the cadherin family wasuniquely altered, that is E-cadherin was down-regulatedand N-cadherin was up-regulated. This opposite response,namely “cadherin-switch”, was observed in the areas wherecells actively moved, such as organogenesis in embryo.18,19

These alterations of cadherin-family expression and actinstructure were observed only at the leading edge of themigrated wound, but not at the deeper zone far from thewound (unpublished data, 2005).

These HDACI-induced endometrial epithelial cytodif-ferentiations and cell migrations were mediated by gly-codelin expression, however, in the mechanism of signaltransduction, the relationship between HDACI andovarian steroid hormones remains unclear. MAPK phos-phorylates and activates nuclear steroid receptors, suchas estrogen receptor and PR.20 Since Zhong et al. havereported that HDACI can activate MAPK pathways,21

HDACI probably positively regulate the activity of thereceptors through MAPK activation. However, in experi-ments in glycodelin induction, the addition of HDACIcaused further up-regulation of expression of glycodelinin Ishikawa cells cultured in E2P4-containing medium.This synergistic effect indicates the possibility of the exist-ence of a glycodelin-mediated signal transduction pathwayfor cytodifferentiation including cell migration, inde-pendently from the steroid hormone-mediated pathway.

HISTONE DEACETYLASE INHIBITOR AS A DIFFERENTIATION INDUCER FOR WHOLE ENDOMETRIAL TISSUE (STROMA PLUS EPITHELIA)We have demonstrated the possibility of developing anew drug strategy for the treatment of endometrial

Endometrial differentiation by HDACI

© 2006 The AuthorsJournal compilation © 2006 Japan Human Cell Society 41

dysfunction, especially infertility, targeting glycodelin(Fig. 1). Since HDACI can induce decidualization-likedifferentiation of stromal cells, HDACI are predicted to behelpful for implantation steps after contact between eggand epithelia, that is the migration step of fertilized egginto stroma. Taken together, we conclude that HDACI,at lower concentration (to avoid induction of cellapoptosis), could be used as a total cytodifferentiation-inducing reagent for human uterine endometrial tissue.

ACKNOWLEDGMENTS

We are grateful for Ms Rei Sakurai for technical assistanceand Ms Shino Kuwabara for secretarial work. We alsothank Drs Masato Nishida (National Kasumigaura Hospi-tal, Ibaragi, Japan) for Ishikawa cells. This work wassupported in part by grants-in-aid for Scientific Research(C16591683 to H.U. and C13671743 to T.M.) and forExploratory Research (15659399 to T.M.) from JapanSociety for the Promotion of Science, grants from KeioUniversity for Encouragement of Young Medical Scien-

tists, Keio Medical Association (to H.U.), and MitsukoshiMedical Foundation (to T.M.).

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Figure 1 Hypothetical model – HDACI as an inducer of total endometrial tissue transdifferentiation. Reversible histone acetyla-tion is controlled by histone acetyl transferase (HAT) and histone deacetylase (HDAC). Acetylated status of histone proteins ismaintained by histone deacetylase inhibitor (HDACI)-derived blockade of HDAC. Electrostatic charged histone proteins byacetylation alter structural properties and create spaces for transcription factor (TF) to access target gene, followed by acceleratedspecific gene transcription. In human endometrial cells, HDACI up-regulate specific proteins, which are induced duringsecretory-phase in vivo, such as prolactin, insulin-like growth factor binding protein (IGFBP)-1, glycodelin and leukemiainhibitory factor (LIF). HDACI can also lead to morphological and functional changes in both human endometrial stromal andepithelial cells.

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