ト ピ ック ス

Role of epithelial-mesenchymal transitionin human adenomyosis
Department of Obstetrics and Gynecology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto, Japan

Khaleque N Khan, Jo Kitawaki!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!

in human endometriosis, lack the expression of E-cad-Introduction

herin［Adenomyosis is defined bythe］. Studies with breast cancer, bladder cancer

intramyometrial

and hepatoma cells indicated that over expression ofpresence of gland and stroma derived from basalis

hepatocyte growth factor（HGF）may down-regulate cad-endometrium surrounded by reactive hyperplastic or

herin-mediated cell-cell adhesion with consequent mi-

hypertrophic myometrium causing either focal or dif-gration and invasion into mesenchymal cells［ ］. Re-

fuse enlargement of the uterus［ ，］. It preferentiallycent reports demonstrated that estrogen-induced epi-affects parous women between the ages ofthelial-mesenchymal transition（EMT）of epithelial cells

andyears. The differences between endometriosis andmay contribute to the development of adenomyosis oradenomyosis are origin of endometrium（functionalis

may be involved in metastatic potential of cancer cells

or basalis）and anatomic site of the lesion（outside or［ ， ］. As an estromedin and pleiotropic growth fac-inside the uterus）［］. The exact physiopathology

tor, we speculated that similar to estrogen, HGF

and pathogenesis of adenomyosis are still elusive. Ac-

might play a similar role in EMT and in the invasion

cording to published literatures, there are differentof gland cells into myometriun in women with adeno-

proposals for pathogenesis of adenomyosis, such as,myosis.

de novo metaplasia of Müllerian remnants, invagina-

We demonstrated an inverse relationship between

tion through vascular or lymphatic channel, and stress

HGF and E-cadherin at the gene and protein level in.
，― ］

cells and tissues derived from women with adeno-

However, the most widely accepted opinion is thatmyosis［ ］. We also found that addition of HGF ex-reaction at the endo-myometrial interface［adenomyosis develops as a down-growth and invagina-

hibits scattering effect on endometrial epithelial cells

tion of the basalis endometrium into the myometrium（EECs）with down-regulation of E-cadherin expression］. However, a possible mechanism of gland invagi-in EECs［ ］. We investigated the possible ability ofnation from basalis endometrium deep into underlying

HGF to induce EMT in EECs and also in tissues de-myometrium is unknown. Recent reports demonstrated

rived from women with and without adenomyosis. In

the role of EMT in adenomyosis［ ，］. However mostorder to explore the possible mechanistic basis ofof them are confined to experiments in animal model

gland invagination deep into the myometrium, we ex-or using endometrial cell line. Information on EMT

amined three cellular aspects of EMT in response tousing human endometrial cells in primary culture andHGF such as cell-to-cell disruption, cell migration, andtissues is lacking.

cell morphological change. We further investigated［E-cadherin is a multigene family of transmembrane

gene and protein expression of two transcriptional re-glycoproteins and normally acts as an “adhesive zip-pressors of E-cadherin, Slug and Slain, in response toper” that means E-cadherin maintains tight cell-cell orestrogen and HGF either alone or in combination.

cell-matrix contact for epithelial cells［］. It has been

demonstrated that epithelial cells, potentially invasive

24Japanese Journal of Reproductive Endocrinology（2018）23 : 24-28

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Expression of E-cadherin and N-cadherin/
Vimentin in response to HGF
A dose-dependent cell separation/scattering effect ofcontralateral endometrium of focal adenomyosis.

Effect of HGF and estrogen in cell migration and morphological change of cells

HGF on EECs and an inverse relationship between

Boyden’s chamber assay indicated that HGF either

gene and protein expression of HGF and E-cadherinalone or in combination with estrogen was able towas observed and is described previously. In fact, asignificantly migrate EECs from upper chamber totime-dependent increase in the mRNA expression of E

lower chamber in a dose dependent（ ,-cadherin in EECs was found with maximum gene ex-fashion. An additive effect was observed in cell migra-pression attion between HGF and estrogen. When EECs wereandhrs（at pre-confluent to conflu-,ng/ml）

ent cells）and this increasing expression of E-cadherin

pre-treated with anti-HGF antibody and ICI

mRNA was decreased after treatment of pre-confluentER antagonist）, HGF and estrogen promoted cellular

EECs with HGF. In contrast, mRNA expression ofmigration of EECs were significantly decreased.

mesenchymal cell marker, N-cadherin, was upregu-（anIn an attempt to examine changes in cell morphol-lated in response to HGF treatment. The inverse rela-ogy of EECs, we found that HGF（tionship between HGF and E-cadherin protein expres-a sequential and time-dependent change in EECs mor-sion was significantly observed in the baslis en-

phology, from cobblestone-like appearance of EECs（atdometria

derivedfromwomen

withadenomyosis.

ng/ml）inducedhr）to a mesenchymal phenotype（elongated spindle

However, this inverse relationship between HGF and-shaped cells）at

E-cadherin protein expression was lost in the function-

served in response to estrogen either alone or in

alis/basalis endometria of control women and in thecombination with HGF.

functionalis endometria derived from women with adenomyosis. Although no visible tissue expression of Ncadherin was found in endometrium derived fromhr. A similar effect was also ob-Effect of HGF and estrogen on transcriptional
repressors of E-cadherin, Slug and Slain

women with and without adenomyosis, protein expres-Both basal and estrogen（E ）-stimulated and ER-

sion of another mesenchymal cell marker, Vimentin,

positive Ishikawa cells displayed c-Met（HGF receptor）

was significantly increased in the basalis endometria

expression. Estrogen and HGF was able to signifi-

derived from women with adenomyosis.

cantly up-regulate mRNA expression of SLUG andTissue concentration
myometrial interfaceofHGF

atendo-We collected tissues from the site of endo-myomaterial interface after hysterectomy of control women,

SLAIN in a dose-dependent manner（p＜ ．

vs. non-treated cells）and a synergistic effect was observed between Eand HGF. The individual effect of EandHGF was more prominent in the over expression of
SLUG gene comparing to SLAIN gene.

women with focal and diffuse adenomyosis. We ho-A similar pattern of E-cadherin and Slug immunoex-mogenized these tissues using Polytron homogenizerpression was observed in both functional and basal（Kinematics, Luzern, Switzerland）and tissue level ofendometria derived from contralateral side of focal

HGF（pg/μg protein）was measured by ELISA. A sig-adenomyosis. An overexpression of Slug was associ-nificantly higher tissue concentration of HGF wasated with a lower expression of E-cadherin at the ba-found at the endo-myometrial interface derived fromsal endometrium（ipsilateral side）and a higher expres-ipsilateral side of focal adenomyosis and anterior/pos-sion of both E-cadherin and Slug was observed at theterior walls of diffuse adenomyosis when comparedfunctional endometria（ipsilateral side）derived fromwith similar tissues derived from control women or

women with focal adenomyosis. Slain immunoreaction

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was not observed in either endometria. The detailtion in mind, we extended our experiments with twomaterials and methods and all these findings are al-additional cellular aspects of EMT, cell migration andready reported in our recent publication［ ］.

cell morphological change. We found that HGF wasable to induce a time-dependent sequence of morpho-Discussion

logical change of EECs from cobblestone appearance

We demonstrated here for the first time a possible
involvementof

HGF-inducedof EECs to spindle-shaped cells. This HGF-induced

epithelial-mesenchymal

EMT was further supported by HGF-induced cell mi-transition（EMT）in the development of human adeno-gration as we confirmed here by Boyden’s chamber

myosis. Unlike all previous studies, we used isolated

assay. These findings give us further information thatepithelial cells and endometrial tissues derived fromin addition to function as estromedin growth factor

women with both focal and diffuse adenomyosis for（regulated by E ）or multifunctional factor［ ， ］in

our current experimental evidence. We found an in-endometriosis, HGF also retains potential capacity toverse relationship in the gene and protein expression

induce EMT in EECs and in basal endometrium de-between HGF and E-cadherin in EECs and in gland

rived from women with adenomyosis.

cells of basalis endometrium derived from women

It has been reported that expression of E-cadherinwith both diffuse adenomyosis and ipsilateral side ofis negatively associated with the expression of a num-

focal adenomyosis. This means, a higher expression

ber of transcriptional repressors of E-cadherin such asof HGF in tissues was associated with a lower ex-

Slug, Slain, SIP― /Zeb― , E /E

pression of epithelial cell marker, E-cadherin. In con-. Among them, Slugtheir involvement in EMT［ ― ］

trast, an increased immunoexpression of mesenchymal

and Slain expressions are well described and they arecell marker, vimentin was observed.

found to interact with the proximal E-boxes of E-Cad-and Twist andWe could find an apparent increase in the gene ex-herin promoter［ ］. An inverse relationship between

pression of mesenchymal cell marker, N-cadherin in

the expression of E-cadherin and Slug/Slain has been

EECs, but N-cadherin protein expression was lost［ ］.

described in many different cell systems［ ， ）］.

This could be due to post-translational degradation ofBolos et al.［ ］demonstrated that ectopic expression

protein at tissue level. The increased tissue expres-of Slug/Slain in epithelial cells caused EMT by thesion of HGF was coincided with significantly higher

aid of migratory and invasive behaviors. A recent

tissue concentrations of HGF in endometria derived

study［ ］reported that estrogen regulates Snail andfrom ipsilateral side of focal adenomyosis and anterior

Slug in the down-regulation of E-cadherin and induces/posterior walls of endometria derived from womenmetastatic potential of ovarian cancer cells. As an es-with diffuse adenomyosis. Interestingly, tissue level oftrogen growth factor, we found in our current study

HGF was remarkably low in the endometrium of con-that similar to estrogen, HGF has the optimal capac-

trol women and in the endometrium derived from le-ity to up-regulate the gene expression of Slug/Slain

sion free contralateral side of focal adenomyosis.

and there was an additive effect between HGF and E

According to published literatures, lower tissue ex-

in further modulation of Slug/Slain expression inpression of E-cadherin and increased tissue expression

Ishikawa cells. We also found an inverse relationshipof Vimentin or N-cadherin dictates a transitional cas-in the protein expression between Slug and E-cad-

cade from an epithelial cell phenotype to mesenchy-

herin but not between Slain and E-cadherin. This ef-

mal cell phenotype. In other words, a phenomenon offect was predominant in the basal endometrium de-

EMT that was reported to be involved in the metas-

rived from the ipsilateral side of focal adenomyosis.

tatic invasion of cancer cells［ ］might also work inHowever, this inverse relationship between Slug andthe pathogenesis of adenomyosis. With this specula-

E-Cadherin was not observed in functional layer or in26

Japanese Journal of Reproductive Endocrinology Vol.23 2018

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E2/HGF

EMT

Slug/Slain

epithelial cell marker

mesenchymal cell markers

E-cadherin

N-cadherin/Vimentin

induction
cell changes/cell migration

EMT

possible basalis gland invagination into myometrium

adenomyosis
FigureFigure legend
A diagrammatic representation of hepatocyte growth factor（HGF）-and estrogen（E ）-induced pathways in the occurrence of epithelial-mesenchymal transition（EMT）in adenomyosis. Up-regulation of SLUG and SNAIL, two transcriptional repressors of E-cadherin, inresponse to HGF and E is associated with decreased expression of E-cadherin（epithelialcell marker）and increased expression of N-cadherin/Vimentin（mesenchymal cell markers）
causing disruption of tight cell-cell contact. These cellular events in endometrial cells andin intact tissues trigger morphological changes of endometrial epithelial cells（EECs）to a
mesenchymal phenotype and induced increased cell migration, essential components ofEMT that was induced by HGF and E either alone or in combination. All these eventsof EMT may be involved in the invagination of glandular epithelial cells from basalis endometrium into the myometrium and finally result in the development of adenomyosis.

similar tissues derived from contralateral side of focal

gland invagination deep into myometrium by inducingadenomyosis.

EMT at the endo-myometrial interface in adenomyosis

Collectively, we found higher tissue levels of HGF

and this effect was augmented by the local estrogen.

in endometrium, HGF-mediated up-regulation of Slug/

Estrogen-induced EMT has found to be abrogated af-

Slain with consequent down-regulation of E-cadherin,

ter treatment with raloxifene, a selective estrogen re-

HGF-induced morphological change to a fibroblast-like

ceptor modulator［ ］. Possible changes in HGF-in-phenotype, a shift from epithelial marker expression

duced cellular cascade of EMT in response to estro-

to mesenchymal marker expression and HGF-induced

gen suppressing agents can be the future target of in-increased cell migration, All these biological events investigation. Further studies are needed to strengthen

response to HGF support in favor of EMT in adeno-our current findings.

myosis［ ］. A diagrammatic representation of different
cascades of EMT involved in the development of human adenomyosis is shown in Figure. We conclude

that similar to estrogen, HGF may be involved inReferences．Bergeron C, Amant F, Ferenczy A（）Pathology andpathophysiology of adenomyosis. Best Practice & Research

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