Endometrial transporters and cytochrome P450 family ...

DOI: 10.5301/JE.2012.9232

Journal of Endometriosis 2012; 4 ( 1 ): 21-29

ORIGINAL ARTICLE

Endometrial transporters and cytochrome P450 family member in endometriosis Bianca De Leo1, Ramsey H. McIntire1, Lusine Aghajanova1, Felice Petraglia2, Linda C. Giudice1 1 2

Department of Obstetrics, Gynecology and Reproductive Sciences, UCSF, San Francisco, CA - USA Department of Pediatrics, Obstetrics and Reproductive Medicine, Siena - Italy

Abstract Purpose: To investigate expression of molecular transporters and CYP4B1 in endometrium from women with and without endometriosis. Methods: Expression of genes encoding proteins: ATP-binding cassette sub-family G member 1 (ABCG1), aquaporin 3 (AQP3), solute carrier family 16, member 6 (monocarboxylic acid transporter 7) (SLC16A6), and transmembrane emp24 protein transport domain containing 6 (TMED6), as well as a member of the cytochrome P450 family 4 subfamily B, polypeptide 1 (CYP4B1) were assessed by quantitative (Q )RT-PCR in proliferative (PE) and early secretory (ESE) endometrium from 27 normoovulatory women with and without endometriosis. Cellular localization of these proteins was determined by immunohistochemistry. Results: In eutopic endometrial tissue from women without endometriosis, mRNA expression of ABCG1, CYP4B1, and SLC16A6 was significantly up-regulated in the PE to ESE transition. CYP4B1 mRNA in endometrium from women with disease increased significantly compared to samples from women without disease, while there was no change in ABCG1 and SLC16A6 mRNA levels. However, immunodetection of ABCG1, AQP3, and TMED6 was similar across cycle phases, and there was no significant difference in immunostaining intensity or localization between women with and without endometriosis. Conclusions: Molecular transporters and CYP4B1 are expressed in human endometrium, and a role for transporters and CYP4B1 in normal endometrial function and in women with endometriosis remains to be determined. Key words: Autochrome P450, Endometriosis, Endometrium, Transporters Accepted: February 28, 2012

Introduction Endometriosis is a common pathologic condition characterized by the presence of endometrial-like tissue outside the uterine cavity. It affects up to 10% of women of reproductive age and up to 30% of women with infertility (1-4). The condition presents with numerous symptoms but generally causes pelvic pain and infertility. Numerous in vivo and in vitro studies, support the fact that eutopic endometrium from women with endometriosis differs from that of women without the disease (5-7). Global gene expression analysis of endometrial tissue biopsies

from women without versus with moderate/severe endometriosis throughout the normal menstrual cycle revealed molecular dysregulation of the proliferative-to-secretory transition in women with endometriosis with persistency of genes involved in cell survival and proliferation (5). Moreover, the significant dysregulation of progesterone regulated genes in the setting of endometriosis supports the hypothesis of a resistance to progesterone in endometrium of women with this disease (8-10). The human endometrium undergoes morphologic and functional changes during the menstrual cycle. Effects of estradiol (E2) dominating the proliferative phase (PE)

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are characterized by intense mitotic activity of epithelial cells, angiogenesis, and tissue growth. The progesterone (P4)-dominated secretory phase (SE) is characterized by considerable expansion of the endometrium because of stromal edema and glandular secretion as well as stromal fibroblast differentiation (decidualization) and growth of spiral arteries, which spread through the upper compartment of the spongy functional endometrial layer (11-14). Comparative analysis of the endometrial transcriptome in women with and without endometriosis revealed an impaired transition from PE to ESE in women with endometriosis showing a significant dysregulation in the expression of transcripts of genes encoding several transporters and molecules associated with metabolism during this transition period (5). Transporters are proteins involved in the movement of small molecules, or macromolecules and ions across a biological membrane. Transport proteins are integral membrane proteins, which transport substances by facilitated diffusion or active transport. Membrane transport proteins are physiologically associated with cellular secretion because they permit the synthesis of secretory components and substances (15). The cytochrome P450 family supports the oxidative, peroxidative, and reductive metabolisms of such endogenous and xenobiotic substrates as environmental pollutants, agrochemicals, steroids, fatty acids, and prostaglandins. In humans, cytochrome P450s are known for their central role in drug metabolism (16). In this study we analyzed four transporters previously shown to be dysregulated in the secretory phase of endometrium from women with severe endometriosis (5) and which may be potentially involved in the secretory function of the normal cycling endometrium. These are ATP-binding cassette, sub-family G, member 1 (ABCG1), aquaporin 3 (AQP3), solute carrier family 16-member 6 (monocarboxylic acid transporter 7, SLC16A6), and a transmembrane emp24 protein transport domain containing 6 (TMED6), as well as we analyzed a member of CYP450 family: cytochrome P450, family 4, subfamily B, polypeptide 1 (CYP4B1). Given the differences in gene expression observed during the key transition from PE to ESE (5), the objective of our study was to verify that the expression of several transporters and CYP4B1 in human endometrium changes in the proliferative and early secretory phases of the normal menstrual cycle and in the setting of endometriosis, where microarray analysis suggested dysregulation (5). 22

Material and methods Human subjects and tissue collection Endometrial biopsies were obtained from healthy fertile women without endometriosis (non-endo, n=15) and women with endometriosis (endo, n=12) (detailed patient characteristics presented in Tab. I) prior informed consent, under a protocol approved by the Committee on Human Research at the University of California San Francisco. Samples were also obtained through the UCSF NIH Human Endometrial Tissue and DNA Bank with appropriate institutional review, approvals, and written informed consents from all participating subjects. The diagnosis of endometriosis was based on visualization of lesions found during laparoscopy and was also confirmed by histology. A total of six (endo) and seven (non-endo) eutopic endometrial biopsy samples were obtained in the mid- to late-proliferative phase (peak circulating estradiol levels) and six (endo) and eight (non-endo) in the early-secretory phase (peak estradiol and progesterone levels) in women with and without endometriosis respectively. Controls had no history of endometriosis. All subjects had regular menstrual cycles (25-35 days), were documented not to be pregnant, and had not been on hormonal treatment for at least three months before tissue sampling (Tab. I). Samples were collected at room temperature in phosphate buffered saline (PBS). One portion of the tissue was snap frozen and stored in liquid nitrogen for future RNA isolation, and another portion was fixed in 4% formaldehyde followed by 70% ethanol after 24 hours and embedded in paraffin for immunohistochemistry.

Total RNA isolation Tissue samples were lysed in RLT-Plus lysis buffer (QIAGEN, Valencia, CA, USA)+ 0.1% β mercaptoethanol, purified using Qiagen RNeasy Plus Mini Kit (QIAGEN, Valencia, CA, USA) according to the manufacturer›s instructions and stored at -80ºC in RNase-free H2O until use. Each sample was quantified by spectroscopy and purity was analyzed by the 260/280 absorbance ratio.

Real-time quantitative RT-PCR For quantitative RT-PCR analysis, 1 μg of RNA previously isolated was reverse-transcribed to cDNA using the IScript cDNA Synthesis Kit (Bio-Rad Laboratories, Hercules,

© 2012 Wichtig Editore - ISSN 2035-9969

De Leo et al

Table I - Characteristics of patients donated endometrial biopsy samples for the study Patient

Cycle phase

Sample used in experiments

Diagnosis at laparoscopy

Age

Ethnicity

Endometriosis ST90

PE

Real time RT-PCR, IHC

Severe endometriosis

42

Caucasian

ST123

PE

Real time RT-PCR

Severe endometriosis, intramural fibroids

44

Caucasian

634

PE

Real time RT-PCR

Mild endometriosis, intramural fibroids

47

Asian

697

PE

Real time RT-PCR, IHC

Severe endometriosis

39

Caucasian

658

PE

Real time RT-PCR

Minimal endometriosis

36

Caucasian

606

PE

IHC

Mild-severe endometriosis

29

Caucasian

ST112

ESE

Real time RT-PCR, IHC

Severe endometriosis, pelvic pain

38

Caucasian

684

ESE

Real time RT-PCR, IHC

Moderate-severe endometriosis, intramural fibroids

36

Caucasian

ST113

ESE

Real time RT-PCR

Minimal endometriosis, pelvic pain, endometrial polyp

27

Caucasian

575

ESE

Real time RT-PCR

Severe endometriosis

26

Unknown

ST130

ESE

Real time RT-PCR

Severe endometriosis, adenomyosis

35

Caucasian

607

ESE

IHC

Severe endometriosis

23

Caucasian

PE

Real time RT-PCR, IHC

Pelvic organ prolapse

41

Asian

455

PE

Real time RT-PCR, IHC

Adenomyosis

39

Caucasian

359

PE

Real time RT-PCR

Pelvic pain, cervical stenosis

39

Asian

641

PE

Real time RT-PCR, IHC

Intramural fibroids

43

Black

691

PE

Real time RT-PCR

Uterine prolapse

40

Caucasian

No endometriosis UC12

693

PE

Real time RT-PCR

Laceration of bladder base

46

Caucasian

UC26

ESE

IHC

Intramural fibroids and pelvic pain

34

Caucasian

629

ESE

Real time RT-PCR

Intramural fibroids

46

Caucasian

664

ESE

Real time RT-PCR

Stress urinary incontinence, adenomyosis

44

Caucasian

650

ESE

Real time RT-PCR

Intramural fibroids

40

Caucasian

680

ESE

IHC

Uterovaginal prolapse, rectal sphincter incompetence, cervix, and vagina with hyperkeratosis and parakeratosis consistent with prolapse.

34

Caucasian

UC24

ESE

Real time RT-PCR, IHC

History of low grade squamous intraepithelial lesion

45

Black

456

ESE

IHC

Intramural fibroids Leiomyoma

41

Caucasian

458

ESE

IHC

Intramural fibroids Leiomyoma

46

Caucasian

514

ESE

IHC

Incapacitating pelvic pain, intramural fibroidsleiomyoma

42

Caucasian

ESE, early secretory endometrium; IHC, immunohistochemistry; PE, proliferative endometrium; RT-PCR, reverse transcription-polymerase chain reaction

CA, USA). Real-time RT-PCR was first performed in triplicate in 25 μL reactions using SYBR GREEN master mix kit (BioRad) according to the manufacturer’s instructions, using selected oligo-primers pairs (Operon), designed using public databases. The resulting template cDNA was diluted four-fold for use in the PCR. The amplification protocol consisted of a “hot start” at

94°C for 15 min, followed by 40 cycles of denaturation, annealing at optimized temperature, and extension (94ºC, 60°C, and 72°C, each for 30 sec) using the Stratagene machine (Stratagene). All assays were optimized for primer concentration and PCR product specificity based on melting curve analysis. PCR products were analyzed by thermal dissociation

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(55°C–95°C) with a fluorescence measurement at every one-degree increment. For each assay, a no-template control was included to verify the quality and cDNA specificity of the primers (Tab. II). For the normalizer, ribosomal protein L19 (RPL19) and each gene of interest (GOI), a standard curve was generated using serial dilutions of template of DNA (1:4, 1:16, 1:64, 1:256, 1:1024). The template for each GOI standard curve was chosen based on the highest levels of overall expression during the menstrual cycle.

Immunohistochemistry Immunostaining was performed for AQP3, TMED, and ABCG1 using paraffin-embedded endometrial PE and ESE tissue samples from women with (n=6) and without (n=9) endometriosis. Paraffin embedded biopsies from the endometrium were sectioned to 4 μm and mounted on glass slides. Paraffin sections were de-paraffinized in Xylene (Sigma-Aldrich) and dehydrated in decreasing concentrations of ethanol. Antigen retrieval was performed by submerging slides in citrate buffer (1x Citra Plus, Vector Laboratories) at 90°C for 10 min. Slides were rinsed in 1x PBS and quenched for 10 min in peroxidase blocking solution (Dako Cytomation), and then blocked with 10% normal horse serum in PBS for 60 min. Sections were then incubated overnight at 4°C with primary antibodies to AQP3 (0.8 μg/mL, rabbit polyclonal, Millipore), or TMED6 (2 μg/mL, rabbit monoclonal, Sigma-Aldrich), or ABCG1 (0.66 μg/mL, rabbit monoclonal, Epitomics, Inc.). Non-immune rabbit IgG (2 μg/mL, Sigma-Aldrich) was used as a negative control. Binding of primary antibodies was detected using HRP-conjugated horse anti-rabbit antibody (Vector Laboratories Inc.) for 30 minutes at room tempera-

ture. Diaminobenzidine-hydrogen peroxide substrate (DAB, Vector Laboratories) was added to the slides followed by rinsing with distilled water. The slides were counterstained with hematoxylin (Vector Laboratories) and mounted with Clarion mounting medium (Sigma-Aldrich). A Leica microscope with camera (Leica Microsystems, Ltd., Wetzlar, Germany) was used to visualize the immunostaining and to photograph the results. The operators were blinded as to disease state and cycle phase during all stages of tissue preparation, staining, imaging, and analysis. Staining results were scored as positive (pos), weak, or negative (neg) within the luminal, glandular, stromal, and vascular compartments for each tissue sample. Each sample was analyzed twice. Commercially available antibodies against SLC16A6 and CYP4B1 were not validated for immunohistochemistry and did not provide reliable staining in the current study.

Statistical assessment Statistical analysis for the QPCR data was performed using the Kruskal-Wallis test (nonparametric ANOVA) with Dunn’s Multiple Comparison test. Statistical significance was determined at P