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The Journal of Clinical Endocrinology and Metabolism logoLink to The Journal of Clinical Endocrinology and Metabolism
. 2010 Aug 11;95(11):E300–E309. doi: 10.1210/jc.2010-0459

Altered Retinoid Uptake and Action Contributes to Cell Survival in Endometriosis

Mary Ellen Pavone 1, Scott Reierstad 1, Hui Sun 1, Magdy Milad 1, Serdar E Bulun 1, You-Hong Cheng 1
PMCID: PMC2968735  PMID: 20702525

Abstract

Context: Retinoic acid (RA) controls multiple biological processes via exerting opposing effects on cell survival. Retinol uptake into cells is controlled by stimulated by RA 6 (STRA6). RA is then produced from retinol in the cytosol. Partitioning of RA between the nuclear receptors RA receptor α and peroxisome-proliferator-activated receptor β/δ is regulated by cytosol-to-nuclear shuttling proteins cellular RA binding protein 2 (CRABP2) and fatty acid binding protein 5 (FABP5), which induce apoptosis or enhance survival, respectively. The roles of these mechanisms in endometrium or endometriosis remain unknown.

Objective: The aim was to determine the regulation of retinoid uptake and RA action in primary stromal cells from endometrium (n = 10) or endometriosis (n = 10).

Results: Progesterone receptor was necessary for high STRA6 and CRABP2 expression in endometrial stromal cells. STRA6, which was responsible for labeled retinoid uptake, was strikingly lower in endometriotic cells compared to endometrial cells. CRABP2 knockdown in endometrial cells increased survival, and FABP5 knockdown in endometriotic cells decreased survival without altering the expression of downstream nuclear retinoic acid receptor α and peroxisome-proliferator-activated receptor β/δ.

Conclusions: In endometrial stromal cells, progesterone receptor up-regulates expression of STRA6 and CRABP2, which control retinol uptake and growth-suppressor actions of RA. In endometriotic stromal cells, decreased expression of these genes leads to decreased retinol uptake and dominant FABP5-mediated prosurvival activity.

In endometrium, progesterone receptor up-regulates expression of STRA6 and CRABP2; in endometriosis, decreased expression of these genes leads to decreased retinol uptake and pro-survival activity.

Endometriosis affects about 5–10% of reproductive-age women (1,2). It is a chronic condition characterized by the presence of endometrial implants outside of the uterus (1,2). Symptoms include dysmenorrhea, dyspareunia, and chronic pelvic pain, and endometriosis is a common cause of infertility (1,2). Estrogen plays a crucial role in both the establishment and maintenance of this condition (2,3,4).

Multiple studies have indicated that there is a differential expression of key molecules in eutopic endometrium when compared with ectopic endometriosis, particularly steroid hormone receptors (5,6). Compared with normal eutopic endometrium, endometriosis has significantly lower expression of both isoforms of progesterone receptor (PR), particularly PR-B (7,8). These findings support the concept of relative or functional progesterone resistance associated with endometriosis (2,9,10,11,12,13,14,15,16,17).

Estradiol (E2) is a well-defined mitogen for the growth and inflammation processes in endometriosis. We have demonstrated that in normal endometrium, progesterone acts on stromal cells to induce secretion of paracrine factors that in turn act on neighboring epithelial cells to induce the expression of the enzyme 17β-hydroxysteroid dehydrogenase type 2 (HSD17B2) (9,10,18,19). This enzyme catalyzes the conversion of E2 to estrone, a much less biologically potent estrogen (18). In endometriotic tissue, progesterone does not induce epithelial HSD17B2 expression because of a defect in the paracrine signaling from stromal cells, one of which was found to be retinoic acid (RA). This results in deficient metabolism of E2 giving rise to high local concentrations of E2 in endometriosis (9,10,18,19,20,21).

The gene stimulated by retinoic acid 6 (STRA6), a widely expressed multitransmembrane domain protein that binds the retinol binding protein (RBP) complex, has recently been identified as the main RBP receptor in cells and has revolutionized our understanding of RA biology (22,23,24,25,26,27,28,29,30,31,32,33,34). STRA6 protein is an integral cell-membrane protein that mediates the uptake of retinol into the cell (22,28,29). The biological actions of RA are mediated by RA receptors (RARα, -β, and -γ) that form heterodimers with retinoid X receptor at specific promoters and modulate transcription (35,36). The overall effect of RA regulates differentiation, cell cycle, and apoptosis (35,36). Because we hypothesize that endometriosis is RA-deficient tissue, we studied the regulation of STRA6 expression in endometriosis and normal endometrium (9,10,18,19).

It has recently been noted that RA may have dual effects on cell fate in a tissue-specific fashion (35,36). In normal cells, RA classically enhances differentiation and apoptosis and inhibits proliferation. On the other hand, in pathological cell types, RA paradoxically induces cell survival (35). In a pioneering study by Schug et al. (35), two lipid binding proteins, cellular RA binding protein 2 (CRABP2) and fatty acid binding protein 5 (FABP5), which shuttle RA from cytosol to the nucleus, were described. CRABP2 delivers RA to its classical nuclear receptor, RARα, which eventually enhances differentiation and apoptosis and inhibits proliferation. In contrast, FABP5 delivers RA to a different nuclear receptor, peroxisome-proliferator-activated receptor (PPAR) β/δ, which like RARα also heterodimerizes with retinoid X receptor but upon RA binding induces the expression of prosurvival genes. The cell fate was found to be determined by the CRABP2:FABP5 ratio in cells (35,36). Given that endometriosis is characterized by a resistance to apoptosis, we hypothesize that there are altered levels of CRABP2 and FABP5 in normal endometrium tissue compared to diseased endometriotic tissue. We also hypothesize that PR may be regulated by both retinoid uptake and action and that the PR resistance found in endometriosis leads to aberrant retinol uptake and action in endometriosis.

Subjects and Methods

Tissue acquisition

Immediately after surgery, eutopic endometrium (n = 10) was obtained from women undergoing hysterectomy for benign indications other than endometriosis, and ectopic endometrium was obtained from the cyst walls of ovarian endometriomas (n = 10). Written informed consent was obtained before surgery, including a consent form for obtaining the tissue, which was approved by the Northwestern Institutional Review Board for Human Research. None of the patients had received any preoperative hormonal therapy. All samples were histologically confirmed. The average age of the subjects was 40.1 ± 6.1 yr for endometrium and 36.1 ± 3.1 for endometriosis, and this difference was not statistically significant. All samples were obtained during the proliferative stage of the menstrual cycle from premenopausal women. The phase of the menstrual cycle was determined by preoperative history as well as histological evaluation (endometrium).

Stromal cell isolation

Stromal cells were isolated from these two types of tissues using a protocol previously reported by Ryan et al. (37) with some minor modifications (37,38). Briefly, tissues were minced and digested with collagenase (Sigma, St. Louis, MO) and deoxyribonuclease (Sigma) at 37 C for 30 min, then with collagenase, deoxyribonuclease, pronase (Sigma), and hyaluronidase (Sigma) for an additional 20 min. Stromal cells were separated from epithelial cells by filtration through 70- and 20-μm sieves. They were then resuspended in DMEM/F12 1:1 (GIBCO/BRL, Grand Island, NY) containing 10% fetal bovine serum and grown in a humidified atmosphere with 5% CO2 at 37 C. All cells were passaged one time. After 16 h of serum starvation, endometrial stromal cells (ESC) and endometriotic stromal cells (OSIS) were incubated in serum-free DMEM/F12 medium containing 10−7 m R5020 for various periods of time.

RNA extraction and quantitative real-time RT-PCR

Total RNA from ESC or OSIS was isolated using TRIzol reagent (Sigma) and following the manufacturer’s protocol. One microgram of RNA was used to generate cDNA using q-script cDNA SuperMix (Quanta Biosciences, Gaithersburg, MD). Real-time quantitative PCR was performed using ABI 7900 Sequence Detection and the ABI Power Syber Green gene expression systems (Applied Biosystems, Foster City, CA). mRNA levels for total PR, STRA6, HSD17B2, CRABP2, FABP5, and 18S were quantified. 18S was used for normalization. Relative quantification of mRNA species was performed using the comparative threshold (CT) cycles method. For each sample, the gene CT value was normalized using the formula: ΔCT = CT gene of interest − CT 18S (housekeeping gene). To determine relative expression levels, the following formula was used: ΔΔCT = ΔCT sample − ΔCT calibrator. This value was used to plot the gene expression employing the formula: 2 − ΔΔCT.

For CRABP2 or FABP5 mRNA, commercially available primers were used (QIAGEN, Valencia, CA). For STRA6 mRNA, the following primers were used: forward, 5′-TGTTGTCCTGCTTACTCACCTTC-3′, and reverse, 5′-GAAGCTCATCCAACAGAATATGG-3′; for HSD17B2, forward, 5′-GCCAGTGCAGATAAAAGATGC-3′, and reverse, 5′-ATAAGAAGAAGCTCCCCATCAG-3′; for PR, forward, 5′-TCAGTGGGCAGAATGCTGTATTT-3′, and reverse, 5′-GCCACATGCTAAGGCATAATGA-3′; and for 18S, forward, 5′-AGGAATTCCCAGTAAGTGCG-3′, and reverse, 5′-GCCTCACTAAACCATCCAA-3′.

Small interfering RNA (siRNA) knockdown

ESC and OSIS were grown to approximately 50% confluency, then transfected with a nontargeting negative control siRNA (siCTL) (Dharmacon, Chicago, IL; or Ambion, Austin, TX) or siRNAs against PR (Dharmacon), CRABP2 (Dharmacon), or STRA6 (Ambion) at a final concentration of 100 or 250 μm using Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA). OSIS were transfected with control siRNA or siRNA against FABP5 (Dharmacon). Transfected cells were collected after 72 h and processed for real-time PCR or immunoblotting.

Immunoblotting

Cells were washed with ice-cold PBS and suspended in mammalian protein extraction reagent (Pierce, Rockford, IL). Lysates were cleared by centrifugation at 14,000 × g for 10 min. Equal amounts of protein (20 μg) were resolved on 4–20% Ready Gel Precast Gels (Bio-Rad Laboratories, Hercules, CA) for 90 min at 60 mAmp and transferred to a 0.45-mm pore nitrocellulose membrane at 150 mAmp for 2 h. The membranes were blotted for a minimum of 1 h in a solution of 1% dried milk in 4,5,6,7-tetrabromo-2H-benzotriazole (TBS) at room temperature and incubated with the primary antibodies at 4 C overnight. The membranes were then washed and incubated with the appropriate secondary antibodies for 1 h. Βeta-actin was used as a loading control. Detection was performed using a Supersignal West Femto Maximum Sensitivity Substrate System (Pierce). Quantification of the immunoblots was done using ImageJ software and normalized to actin.

Reproduction of 3H-retinol loaded RBP and retinol uptake assay

Human serum containing significant RBP was purchased from Invitrogen Life Technologies (New York, NY), and radioactive retinol ([11,12-3H(N)]retinol) was purchased from Perkin-Elmer Life and Analytical Sciences (Boston, MA). Human serum (100 μl) was incubated with 300 pmol (6 μCi) radioactive retinol at 24 C overnight. The reconstituted 3H-retinol-RBP was separated from excess radioactive retinol by gel filtration on a Quick Spin Column of Sephadex G-25 (Roche Diagnosis, Indianapolis, IN). The whole procedure was performed under dim red light using a protocol similar to that used by Kawaguchi et al. (22).

STRA6 was knocked down in ESC as previously described. After overnight starvation, cells were cultured in fetal bovine serum-free media for 24 h. After washing twice with PBS, cells were incubated with 3H-retinol-RBP (5000 cpm) for 1 h. After washing twice with PBS, cells were solubilized with 1% Triton X-100 in PBS. Radioactivity was measured using a scintillation counter.

Statistical analysis

A one-tailed t test assuming unequal variance or ANOVA followed by the Tukey multiple comparison procedure was used. All values are given as the mean, with bars indicating sem, and a P value < 0.05 was considered significant.

Results

STRA6 and CRABP2 are differentially expressed in ESC vs. OSIS

We first investigated whether the genes that regulate retinoid uptake and action were differentially expressed in ESC and OSIS. We isolated ESC and OSIS and analyzed STRA6 and CRABP2 mRNA levels. The mRNA levels were normalized to 18S. We expressed the relative mRNA levels found in OSIS as a multiple of that found in the ESC.

As shown in Fig. 1, mean STRA6 and CRABP2 levels were significantly higher in ESC compared with OSIS. Protein levels determined by immunoblotting verified these mRNA results.

Figure 1.

Figure 1

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STRA6, CRABP2, and FABP5 mRNA and protein levels are differentially expressed in ESC and OSIS with higher levels seen in ESC. mRNA levels represent a minimum of 10 patients in each group. Protein data are a representative Western blot of experiments repeated at least 10 times. *, P < 0.05. NS, Nonsignificant. A, STRA6 mRNA and protein levels are significantly higher in ESC compared with OSIS. B, CRABP2 mRNA and protein levels are significantly higher in ESC compared with OSIS. C, FABP5 mRNA and protein levels are significantly higher in ESC compared with OSIS. D, Western blots verifying the mRNA results. There is a higher expression of STRA6, CRABP2, and FABP5 in ESC when compared with OSIS. E, Protein quantification using ImageJ software. The CRABP2:FABP5 ratio is integral in determining cell fate. The ratio of CRABP2:FABP5 differs in ESC (1:1) compared with OSIS (0.4:1).

PR, but not progesterone agonist, regulates STRA6 and CRABP2 expression

To understand the role of progesterone in transcriptional regulation of STRA6 and CRABP2 in cultured ESC, real-time quantitative PCR was performed after ESC were incubated with the progesterone agonist R5020 (10−7 m) for 24 h. STRA6 and CRABP2 mRNA levels were not significantly induced by progesterone (Fig. 2, A and B). Various time-course and dose-response experiments did not support any regulation by progesterone, E2, R5020, or the progesterone antagonist RU486 (data not shown).

Figure 2.

Figure 2

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Progesterone receptor, but not progesterone, regulates STRA6 and CRABP2 gene expression. NS, Nonsignificant. A and B, STRA6 and CRABP2 mRNA gene expression are not induced by progesterone treatment. Results represent the mean ± sem from six independent experiments using tissues from subjects.

To determine whether PR regulates the expression of CRABP2 and STRA6, PR was knocked down in ESC. PR knockdown (Fig. 3A) was found to significantly decrease STRA6 mRNA levels (Fig. 3B). PR knockdown was also found to significantly decrease CRABP2 mRNA levels (Fig. 3C). These results suggest that PR may regulate key genes involved in the uptake and action of RA in a ligand-independent fashion.

Figure 3.

Figure 3

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Knockdown of PR, with and without progesterone treatment, causes a significant decrease in STRA6 and CRABP2 expression. PR may regulate these genes in a ligand-independent manner. Results represent the mean ± sem from six independent experiments using tissues from different subjects. *, P < 0.05. NS, Nonsignificant. A, PR knockdown with and without treatment with R5020 in ESC. B, PR knockdown causes a decrease in the expression of STRA6 in ESC. C, PR knockdown causes a decrease in the expression of CRABP2 in ESC. siCTL, Nontargeting negative control siRNA; siPR, siRNA against PR.

STRA6 regulates retinol uptake into ESC

To determine whether STRA6 is a key modulator for cellular uptake of retinol/RBP in ESC, we used the siRNA technique to knock down endogenous STRA6 expression in the primary cultures of eutopic ESC (Fig. 4A) and assessed the effect on cellular uptake of vitamin A by 3H-retinol/RBP assay. As shown in Fig. 4C, knockdown of STRA6 significantly decreased cellular 3H-retinol uptake in eutopic ESC. Results represent the mean ± sem for six independent experiments using tissues from different subjects.

Figure 4.

Figure 4

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Knockdown of STRA6 significantly decreases HSD17B2, a gene known to be induced by RA. STRA6 is the main retinol receptor in cells and controls retinol uptake. Results represent the mean ± sem for six independent experiments using tissues from different subjects. *, P < 0.05. A, STRA6 knockdown in ESC. B, HSD17B2 gene expression decreases when STRA6 is knocked down. Because there is less retinol uptake in cells, less RA is available to induce HSD17B2. C, Knockdown of STRA6 markedly decreased 3H-retinol/RBP uptake in normal ESC. Radioactivity was measured with a scintillation counter. siCTL, Nontargeting negative control siRNA; siSTRA6, siRNA against STRA6.

HSD17B2, the enzyme that metabolizes and inactivates E2 in the endometrium, is a known RA-target gene. STRA6 knockdown ablated HSD17B2 expression, suggesting that retinol uptake is critical for RA action (Fig. 4B). Taken together, these data strongly support our hypothesis that STRA6 represents a major physiological mediator of cellular vitamin A uptake.

CRABP2 knockdown influences apoptosis and proliferation in ESC

FABP5 is another intracytosolic lipid binding protein integral in determining cell fate (35,36). As shown in Fig. 1, mRNA and protein levels of both CRABP2 and FABP5 are higher in ESC compared with OSIS. However, the ratio of CRABP2:FABP5 differs. Western blot quantification relative to actin showed that whereas the ratio of CRABP2:FABP5 in ESC is 1:1, in OSIS, it is 0.4:1 (Fig. 1E). To investigate how the altered CRABP2:FABP5 ratio affects cell fate in our model of endometrium and endometriosis, we first determined mRNA and protein levels of CRABP2 and FABP5 in ESC and OSIS. As shown in Fig. 1, B and C, mRNA and protein levels of both CRABP2 and FABP5 are higher in ESC, compared with OSIS. However, the ratios of these genes are different, with a higher CRABP2:FABP5 ratio present in ESC. This is clearly evident by looking at the Western blot in Fig. 1D.

To investigate the effects of altering CRABP2 levels on apoptosis and proliferation, CRABP2 knockdown in normal ESC was performed (Fig. 5). We found that although the mRNA levels of the nuclear receptors RARα and PPARβ/δ remained unchanged (Fig. 5A), apoptosis and proliferation were significantly altered, as measured by BCL2, cleaved PARP, and PCNA protein activity (Fig. 5B). It appears that ablation of CRABP2 significantly decreases apoptotic activity and increases proliferation in ESC. Illustrated results are from a representative experiment reproduced in cells from at least three other subjects.

Figure 5.

Figure 5

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The CRABP2:FABP5 ratio is integral in determining cell fate. The ratio of CRABP2:FABP5 differs in ESC (1:1) compared with OSIS (0.4:1). Illustrated results are from a representative experiment reproduced in cells from at least three other subjects. *, P < 0.05. NS, Nonsignificant. A, Knockdown of CRABP2 in ESC does not change nuclear receptor mRNA levels. B, Knockdown of CRABP2 in ESC does affect genes involved in apoptosis and proliferation. Knockdown of CRABP2 causes an increase in genes involved in proliferation and antiapoptosis. C, Quantification of Western blots using ImageJ software. siCTL, Nontargeting negative control siRNA; siCRABP2, siRNA against CRABP2.

FABP5 knockdown regulates apoptosis and proliferation in OSIS

To further understand the role of CRABP2:FABP5 ratio on apoptosis and proliferation, FABP5 was knocked down in the pathological OSIS. We found that although the mRNA levels of the nuclear receptors RARα and PPARβ/δ were not significantly changed (Fig. 6A), apoptosis and proliferation markers were altered. Knockdown of FABP5 in OSIS significantly increased apoptotic activity, as seen by an increase in cleaved PARP activity and a decrease in BCL2, whereas decreasing proliferation, as seen by a decrease in PCNA (Fig. 6B). Thus, changing the levels of CRABP2 or FABP5 in either ESC or OSIS altered both apoptotic and proliferation markers (Fig. 6C). Again, illustrated results are from a representative experiment reproduced in cells from at least three other subjects.

Figure 6.

Figure 6

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Again, the CRABP2:FABP5 ratio is integral in determining cell fate. Illustrated results are from a representative experiment reproduced in cells from at least three other subjects. *, P < 0.05. NS, Nonsignificant. A, Knockdown of FABP5 in OSIS does not change nuclear receptor mRNA levels. B, Knockdown of FABP5 does affect genes involved in apoptosis and proliferation. Knockdown of FABP5 in ESC causes a decrease in genes involved in proliferation and antiapoptosis. C, Quantification of Western blots using ImageJ software. We proposed that in ESC, RA may signal through CRABP2, which shuttles RA to RARα, and promote proliferative actions. In OSIS, however, RA may signal through FABP5, shuttling RA to PPARβ/δ and mediating antiapoptotic activity. siCTL, Nontargeting negative control siRNA; siFABP5, siRNA against FABP5.

Discussion

Endometriosis is often described as a progesterone-resistant state, characterized by a decrease in the levels of total PR and PR-B, which in most human cells is the primary regulator of progesterone-responsive genes (7,39). This leads to a number of molecular and functional defects including HSD17B2 deficiency, leading to E2 excess in endometriotic tissue (10,12,19). We hypothesize that RA is a key downstream mediator of PR action in normal endometrium, whereas progesterone resistance in endometriosis is associated with a RA-deficient state. This study provides critical new mechanisms to support this notion. We suggest that deficient RA production because of defective retinol uptake and aberrant RA action via its shuttling to a prosurvival nuclear receptor are consequences of PR deficiency in endometriosis.

Traditionally, natural or synthetic ligands of PR were thought to be essential for its action in various tissues. Recent studies, however, indicate that steroid receptors, including PR, can regulate transcription in a ligand-independent manner by direct binding to a classic PR element (5,11,19), or nonclassically by tethering to other transcription factors including SP1 or AP1, or by stimulation of upstream cytoplasmic kinases that can then serve as direct inputs to transcription factor function, including c-Src and MAPKs (40,41,42,43,44,45,46,47,48,49). In breast cancer cells cultured in the absence of progesterone, up-regulation of endogenous PR decreased expression of genes including aromatase, COX-2, and HER-2/neu, whereas its knockdown induced aromatase and HER2/neu mRNA levels (44,45). Additionally, a ligand-independent action of PR to block nuclear factor-κB activation and expression of COX-2 has been demonstrated (50). Unliganded PR was shown to modulate the phenotype of both ER-negative and ER-positive breast cancer cells, suggesting that unliganded steroid receptors could be transcriptionally activated, which could regulate the expression of genes involved in proliferation control (41,43,46,47,48,49,50). Our findings suggest that PR may also regulate key genes involved in retinoid uptake and RA action, namely STRA6 and CRABP2, in endometrium and endometriosis, in a ligand-independent manner.

It was proposed that PR deficiency may be responsible for increased proliferation and resistance to apoptosis seen in endometriosis (9,10,11). It was previously shown that when PR-B was knocked down in immortalized ESC, increased proliferation was noted, suggesting that PR-B may promote proliferation and confer antiapoptotic activities (51). This is consistent with our results because we show that PR regulates CRABP2 and CRABP2 is important for normal apoptosis.

It has been demonstrated that the dichotomous actions of RA can be explained by differences in the cytoplasmic lipid binding proteins CRABP2 and FABP5 (35,36). These transport proteins bind to RA and shuttle it to the appropriate nuclear receptors, RARα and PPARβ/δ, respectively. Recently, a low level of CRABP2 was found in a RA-resistant mouse model of breast cancer, whereby RA activates the nonclassic RA receptor PPARβ/δ (23). When CRABP2 levels were altered, RA was diverted from PPARβ/δ to RAR, and tumor growth was suppressed (23). This observation is consistent with our results and conclusions. Endometriosis is often described as a disease state characterized by inappropriately reduced apoptosis (9,10,51,52,53). We have shown that in endometriotic cells, an antiapoptotic state was mediated by decreased levels of CRABP2, compared with normal endometrial cells. Thus, RA may promote differentiation and apoptosis in normal endometrium via CRABP2, which shuttles RA to RARα. In endometriosis, on the other hand, this pathway is shifted toward FABP5, which shuttles RA to PPARβ/δ and mediates antiapoptotic activity (Fig. 6C). Additionally, knockdown of the intracytoplasmic nuclear binding proteins does not alter the expression of the nuclear proteins, demonstrating that this shuttling action is dependent on the presence of CRABP2 and FABP5 (Figs. 5 and 6).

It was recently reported that the expression of CRABP2 was correlated with the gain of ability to synthesize RA, which may be an important part of the process of embryo implantation (54,55). Taken together with our findings, this suggests that progesterone resistance found in endometriosis may impair implantation because of decreased levels of CRABP2. In fact, a decrease in implantation rate was reported clinically in women with endometriosis (14,17,56,57,58,59).

In summary, we demonstrated a novel role of PR in the expression of genes necessary for retinol uptake and action. There is decreased retinol uptake in OSIS. Additionally, the decreased ratio of cytosol-to-nucleus RA shuttling proteins CRABP2:FABP5 observed in OSIS contributes to a resistance to apoptosis in endometriosis. Currently, the use of RA analogs in a clinical setting has been limited secondary to potential teratogenicity as well as toxicity (60,61,62,63). However, agents that target the RA-shuttling system may prove to be useful therapeutic targets in the future, bypassing the toxicity and teratogenicity encountered with RA analogs (36).

Footnotes

This work was supported by National Institutes of Health Grant U54-HD40093, Friends of Prentice (to S.E.B.).

Disclosure Summary: M.E.P., H.S., M.M., Y.-H.C. have nothing to disclose. S.R. is currently employed by Life Technologies and is also a stock owner. S.E.B. received NIH Grant R37-HD36891 and has received consulting fees from Novartis, Endo, and Repros.

First Published Online August 11, 2010

Abbreviations: CRABP2, Cellular RA binding protein 2; CT, comparative threshold; E2, estradiol; ESC, endometrial stromal cell(s); FABP5, fatty acid binding protein 5; HSD17B2, 17β-hydroxysteroid dehydrogenase type 2; OSIS, endometriotic stromal cells; PPAR, peroxisome-proliferator-activated receptor; PR, progesterone receptor; RA, retinoic acid; RAR, RA receptor; RBP, retinol binding protein; siRNA, small interfering RNA; STRA6, stimulated by RA 6.

References

  1. Giudice LC, Kao LC 2004 Endometriosis. Lancet 364:1789–1799 [DOI] [PubMed] [Google Scholar]
  2. Bulun SE 2009 Endometriosis. N Engl J Med 360:268–279 [DOI] [PubMed] [Google Scholar]
  3. Osteen KG, Bruner-Tran KL, Eisenberg E 2005 Endometrial biology and the etiology of endometriosis. Fertil Steril 84:33–34; discussion 38–39 [DOI] [PubMed] [Google Scholar]
  4. Gurates B, Bulun SE 2003 Endometriosis: the ultimate hormonal disease. Semin Reprod Med 21:125–134 [DOI] [PubMed] [Google Scholar]
  5. Bulun SE, Utsunomiya H, Lin Z, Yin P, Cheng YH, Pavone ME, Tokunaga H, Trukhacheva E, Attar E, Gurates B, Milad MP, Confino E, Su E, Reierstad S, Xue Q 2009 Steroidogenic factor-1 and endometriosis. Mol Cell Endocrinol 300:104–108 [DOI] [PubMed] [Google Scholar]
  6. Burney RO, Talbi S, Hamilton AE, Vo KC, Nyegaard M, Nezhat CR, Lessey BA, Giudice LC 2007 Gene expression analysis of endometrium reveals progesterone resistance and candidate susceptibility genes in women with endometriosis. Endocrinology 148:3814–3826 [DOI] [PubMed] [Google Scholar]
  7. Attia GR, Zeitoun K, Edwards D, Johns A, Carr BR, Bulun SE 2000 Progesterone receptor isoform A but not B is expressed in endometriosis. J Clin Endocrinol Metab 85:2897–2902 [DOI] [PubMed] [Google Scholar]
  8. Wu Y, Strawn E, Basir Z, Halverson G, Guo SW 2006 Promoter hypermethylation of progesterone receptor isoform B (PR-B) in endometriosis. Epigenetics 1:106–111 [DOI] [PubMed] [Google Scholar]
  9. Bulun SE, Cheng YH, Pavone ME, Xue Q, Attar E, Trukhacheva E, Tokunaga H, Utsunomiya H, Yin P, Luo X, Lin Z, Imir G, Thung S, Su EJ, Kim JJ 2010 Estrogen receptor-β, estrogen receptor-α, and progesterone resistance in endometriosis. Semin Reprod Med 28:36–43 [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Bulun SE, Cheng YH, Pavone ME, Yin P, Imir G, Utsunomiya H, Thung S, Xue Q, Marsh EE, Tokunaga H, Ishikawa H, Kurita T, Su EJ 2010 17β-Hydroxysteroid dehydrogenase-2 deficiency and progesterone resistance in endometriosis. Semin Reprod Med 28:44–50 [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Bulun SE, Cheng YH, Yin P, Imir G, Utsunomiya H, Attar E, Innes J, Julie Kim J 2006 Progesterone resistance in endometriosis: link to failure to metabolize estradiol. Mol Cell Endocrinol 248:94–103 [DOI] [PubMed] [Google Scholar]
  12. Aghajanova L, Velarde MC, Giudice LC 2010 Altered gene expression profiling in endometrium: evidence for progesterone resistance. Semin Reprod Med 28:51–58 [DOI] [PubMed] [Google Scholar]
  13. Bruner-Tran KL, Ding T, Osteen KG 2010 Dioxin and endometrial progesterone resistance. Semin Reprod Med 28:59–68 [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Cakmak H, Taylor HS 2010 Molecular mechanisms of treatment resistance in endometriosis: the role of progesterone-hox gene interactions. Semin Reprod Med 28:69–74 [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Fazleabas AT 2010 Progesterone resistance in a baboon model of endometriosis. Semin Reprod Med 28:75–80 [DOI] [PubMed] [Google Scholar]
  16. Jones CJ, Nardo LG, Litta P, Fazleabas AT 2009 Peritoneal ectopic lesions from women with endometriosis show abnormalities in progesterone-dependent glycan expression. Fertil Steril 91:1608–1610 [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Wang C, Mavrogianis PA, Fazleabas AT 2009 Endometriosis is associated with progesterone resistance in the baboon (Papio anubis) oviduct: evidence based on the localization of oviductal glycoprotein 1 (OVGP1). Biol Reprod 80:272–278 [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Cheng YH, Imir A, Fenkci V, Yilmaz MB, Bulun SE 2007 Stromal cells of endometriosis fail to produce paracrine factors that induce epithelial 17β-hydroxysteroid dehydrogenase type 2 gene and its transcriptional regulator Sp1: a mechanism for defective estradiol metabolism. Am J Obstet Gynecol 196:391.e1–e7 [DOI] [PubMed] [Google Scholar]
  19. Cheng YH, Imir A, Suzuki T, Fenkci V, Yilmaz B, Sasano H, Bulun SE 2006 SP1 and SP3 mediate progesterone-dependent induction of the 17β hydroxysteroid dehydrogenase type 2 gene in human endometrium. Biol Reprod 75:605–614 [DOI] [PubMed] [Google Scholar]
  20. Zeitoun K, Takayama K, Sasano H, Suzuki T, Moghrabi N, Andersson S, Johns A, Meng L, Putman M, Carr B, Bulun SE 1998 Deficient 17β-hydroxysteroid dehydrogenase type 2 expression in endometriosis: failure to metabolize 17β-estradiol. J Clin Endocrinol Metab 83:4474–4480 [DOI] [PubMed] [Google Scholar]
  21. Bulun SE 2010 Progesterone resistance and endometrial disease. Preface. Semin Reprod Med 28:3 [DOI] [PubMed] [Google Scholar]
  22. Kawaguchi R, Yu J, Honda J, Hu J, Whitelegge J, Ping P, Wiita P, Bok D, Sun H 2007 A membrane receptor for retinol binding protein mediates cellular uptake of vitamin A. Science 315:820–825 [DOI] [PubMed] [Google Scholar]
  23. Blaner WS 2007 STRA6, a cell-surface receptor for retinol-binding protein: the plot thickens. Cell Metab 5:164–166 [DOI] [PubMed] [Google Scholar]
  24. Bouillet P, Sapin V, Chazaud C, Messaddeq N, Décimo D, Dollé P, Chambon P 1997 Developmental expression pattern of Stra6, a retinoic acid-responsive gene encoding a new type of membrane protein. Mech Dev 63:173–186 [DOI] [PubMed] [Google Scholar]
  25. Chassaing N, Golzio C, Odent S, Lequeux L, Vigouroux A, Martinovic-Bouriel J, Tiziano FD, Masini L, Piro F, Maragliano G, Delezoide AL, Attié-Bitach T, Manouvrier-Hanu S, Etchevers HC, Calvas P 2009 Phenotypic spectrum of STRA6 mutations: from Matthew-Wood syndrome to non-lethal anophthalmia. Hum Mutat 30:E673–E681 [DOI] [PubMed] [Google Scholar]
  26. Golzio C, Martinovic-Bouriel J, Thomas S, Mougou-Zrelli S, Grattagliano-Bessieres B, Bonniere M, Delahaye S, Munnich A, Encha-Razavi F, Lyonnet S, Vekemans M, Attie-Bitach T, Etchevers HC 2007 Matthew-Wood syndrome is caused by truncating mutations in the retinol-binding protein receptor gene STRA6. Am J Hum Genet 80:1179–1187 [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Isken A, Golczak M, Oberhauser V, Hunzelmann S, Driever W, Imanishi Y, Palczewski K, von Lintig J 2008 RBP4 disrupts vitamin A uptake homeostasis in a STRA6-deficient animal model for Matthew-Wood syndrome. Cell Metab 7:258–268 [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Kawaguchi R, Yu J, Wiita P, Honda J, Sun H 2008 An essential ligand-binding domain in the membrane receptor for retinol-binding protein revealed by large-scale mutagenesis and a human polymorphism. J Biol Chem 283:15160–15168 [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Kawaguchi R, Yu J, Wiita P, Ter-Stepanian M, Sun H 2008 Mapping the membrane topology and extracellular ligand binding domains of the retinol binding protein receptor. Biochemistry 47:5387–5395 [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Pasutto F, Sticht H, Hammersen G, Gillessen-Kaesbach G, Fitzpatrick DR, Nürnberg G, Brasch F, Schirmer-Zimmermann H, Tolmie JL, Chitayat D, Houge G, Fernández-Martínez L, Keating S, Mortier G, Hennekam RC, von der Wense A, Slavotinek A, Meinecke P, Bitoun P, Becker C, Nürnberg P, Reis A, Rauch A 2007 Mutations in STRA6 cause a broad spectrum of malformations including anophthalmia, congenital heart defects, diaphragmatic hernia, alveolar capillary dysplasia, lung hypoplasia, and mental retardation. Am J Hum Genet 80:550–560 [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Segel R, Levy-Lahad E, Pasutto F, Picard E, Rauch A, Alterescu G, Schimmel MS 2009 Pulmonary hypoplasia-diaphragmatic hernia-anophthalmia-cardiac defect (PDAC) syndrome due to STRA6 mutations—what are the minimal criteria? Am J Med Genet A 149A:2457–2463 [DOI] [PubMed] [Google Scholar]
  32. West B, Bove KE, Slavotinek AM 2009 Two novel STRA6 mutations in a patient with anophthalmia and diaphragmatic eventration. Am J Med Genet A 149A:539–542 [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. White T, Lu T, Metlapally R, Katowitz J, Kherani F, Wang TY, Tran-Viet KN, Young TL 2008 Identification of STRA6 and SKI sequence variants in patients with anophthalmia/microphthalmia. Mol Vis 14:2458–2465 [PMC free article] [PubMed] [Google Scholar]
  34. Wu L, Ross AC 2010 Acidic retinoids synergize with vitamin A to enhance retinol uptake and STRA6, LRAT, and CYP26B1 expression in neonatal lung. J Lipid Res 51:378–387 [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Schug TT, Berry DC, Shaw NS, Travis SN, Noy N 2007 Opposing effects of retinoic acid on cell growth result from alternate activation of two different nuclear receptors. Cell 129:723–733 [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Schug TT, Berry DC, Toshkov IA, Cheng L, Nikitin AY, Noy N 2008 Overcoming retinoic acid-resistance of mammary carcinomas by diverting retinoic acid from PPARβ/δ to RAR. Proc Natl Acad Sci USA 105:7546–7551 [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Ryan IP, Schriock ED, Taylor RN 1994 Isolation, characterization, and comparison of human endometrial and endometriosis cells in vitro. J Clin Endocrinol Metab 78:642–649 [DOI] [PubMed] [Google Scholar]
  38. Noble LS, Takayama K, Zeitoun KM, Putman JM, Johns DA, Hinshelwood MM, Agarwal VR, Zhao Y, Carr BR, Bulun SE 1997 Prostaglandin E2 stimulates aromatase expression in endometriosis-derived stromal cells. J Clin Endocrinol Metab 82:600–606 [DOI] [PubMed] [Google Scholar]
  39. Igarashi TM, Bruner-Tran KL, Yeaman GR, Lessey BA, Edwards DP, Eisenberg E, Osteen KG 2005 Reduced expression of progesterone receptor-B in the endometrium of women with endometriosis and in cocultures of endometrial cells exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin. Fertil Steril 84:67–74 [DOI] [PubMed] [Google Scholar]
  40. Daniel AR, Faivre EJ, Lange CA 2007 Phosphorylation-dependent antagonism of sumoylation derepresses progesterone receptor action in breast cancer cells. Mol Endocrinol 21:2890–2906 [DOI] [PubMed] [Google Scholar]
  41. Carnevale RP, Proietti CJ, Salatino M, Urtreger A, Peluffo G, Edwards DP, Boonyaratanakornkit V, Charreau EH, Bal de Kier Joffé E, Schillaci R, Elizalde PV 2007 Progestin effects on breast cancer cell proliferation, proteases activation, and in vivo development of metastatic phenotype all depend on progesterone receptor capacity to activate cytoplasmic signaling pathways. Mol Endocrinol 21:1335–1358 [DOI] [PubMed] [Google Scholar]
  42. Daniel AR, Qiu M, Faivre EJ, Ostrander JH, Skildum A, Lange CA 2007 Linkage of progestin and epidermal growth factor signaling: phosphorylation of progesterone receptors mediates transcriptional hypersensitivity and increased ligand-independent breast cancer cell growth. Steroids 72:188–201 [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Faivre E, Skildum A, Pierson-Mullany L, Lange CA 2005 Integration of progesterone receptor mediated rapid signaling and nuclear actions in breast cancer cell models: role of mitogen-activated protein kinases and cell cycle regulators. Steroids 70:418–426 [DOI] [PubMed] [Google Scholar]
  44. Hardy DB, Janowski BA, Chen CC, Mendelson CR 2008 Progesterone receptor inhibits aromatase and inflammatory response pathways in breast cancer cells via ligand-dependent and ligand-independent mechanisms. Mol Endocrinol 22:1812–1824 [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Hardy DB, Janowski BA, Corey DR, Mendelson CR 2006 Progesterone receptor plays a major antiinflammatory role in human myometrial cells by antagonism of nuclear factor-κB activation of cyclooxygenase 2 expression. Mol Endocrinol 20:2724–2733 [DOI] [PubMed] [Google Scholar]
  46. Jacobsen BM, Richer JK, Sartorius CA, Horwitz KB 2003 Expression profiling of human breast cancers and gene regulation by progesterone receptors. J Mammary Gland Biol Neoplasia 8:257–268 [DOI] [PubMed] [Google Scholar]
  47. Jacobsen BM, Schittone SA, Richer JK, Horwitz KB 2005 Progesterone-independent effects of human progesterone receptors (PRs) in estrogen receptor-positive breast cancer: PR isoform-specific gene regulation and tumor biology. Mol Endocrinol 19:574–587 [DOI] [PubMed] [Google Scholar]
  48. Pierson-Mullany LK, Lange CA 2004 Phosphorylation of progesterone receptor serine 400 mediates ligand-independent transcriptional activity in response to activation of cyclin-dependent protein kinase 2. Mol Cell Biol 24:10542–10557 [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Pierson-Mullany LK, Skildum A, Faivre E, Lange CA 2003 Cross-talk between growth factor and progesterone receptor signaling pathways: implications for breast cancer cell growth. Breast Dis 18:21–31 [DOI] [PubMed] [Google Scholar]
  50. Auricchio F, Migliaccio A, Castoria G 2008 Sex-steroid hormones and EGF signalling in breast and prostate cancer cells: targeting the association of Src with steroid receptors. Steroids 73:880–884 [DOI] [PubMed] [Google Scholar]
  51. Wu Y, Shi X, Guo SW 2008 The knockdown of progesterone receptor isoform B (PR-B) promotes proliferation in immortalized endometrial stromal cells. Fertil Steril 90:1320–1323 [DOI] [PubMed] [Google Scholar]
  52. Béliard A, Noël A, Foidart JM 2004 Reduction of apoptosis and proliferation in endometriosis. Fertil Steril 82:80–85 [DOI] [PubMed] [Google Scholar]
  53. Nasu K, Yuge A, Tsuno A, Nishida M, Narahara H 2009 Involvement of resistance to apoptosis in the pathogenesis of endometriosis. Histol Histopathol 24:1181–1192 [DOI] [PubMed] [Google Scholar]
  54. Bucco RA, Zheng WL, Wardlaw SA, Davis JT, Sierra-Rivera E, Osteen KG, Melner MH, Kakkad BP, Ong DE 1996 Regulation and localization of cellular retinol-binding protein, retinol-binding protein, cellular retinoic acid-binding protein (CRABP), and CRABP II in the uterus of the pseudopregnant rat. Endocrinology 137:3111–3122 [DOI] [PubMed] [Google Scholar]
  55. Bucco RA, Zheng WL, Davis JT, Sierra-Rivera E, Osteen KG, Chaudhary AK, Ong DE 1997 Cellular retinoic acid-binding protein (II) presence in rat uterine epithelial cells correlates with their synthesis of retinoic acid. Biochemistry 36:4009–4014 [DOI] [PubMed] [Google Scholar]
  56. Barnhart K, Dunsmoor-Su R, Coutifaris C 2002 Effect of endometriosis on in vitro fertilization. Fertil Steril 77:1148–1155 [DOI] [PubMed] [Google Scholar]
  57. Aghajanova L, Hamilton A, Kwintkiewicz J, Vo KC, Giudice LC 2009 Steroidogenic enzyme and key decidualization marker dysregulation in endometrial stromal cells from women with versus without endometriosis. Biol Reprod 80:105–114 [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Jackson KS, Brudney A, Hastings JM, Mavrogianis PA, Kim JJ, Fazleabas AT 2007 The altered distribution of the steroid hormone receptors and the chaperone immunophilin FKBP52 in a baboon model of endometriosis is associated with progesterone resistance during the window of uterine receptivity. Reprod Sci 14:137–150 [DOI] [PubMed] [Google Scholar]
  59. Wei Q, St Clair JB, Fu T, Stratton P, Nieman LK 2009 Reduced expression of biomarkers associated with the implantation window in women with endometriosis. Fertil Steril 91:1686–1691 [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Bollag W, Majewski S, Jablonska S 1994 Cancer combination chemotherapy with retinoids: experimental rationale. Leukemia 8:1453–1457 [PubMed] [Google Scholar]
  61. Molin L, Thomsen K, Volden G, Jensen P, Knudsen E, Nyfors A, Schmidt H 1987 Retinoids and systemic chemotherapy in cases of advanced mycosis fungoides. A report from the Scandinavian Mycosis Fungoides Group. Acta Derm Venereol 67:179–182 [PubMed] [Google Scholar]
  62. Nandan R 2006 Promising results achieved with a combination of chemotherapy and two retinoids in patients with advanced non-small-cell lung cancer. Lung Cancer 51:387–388 [DOI] [PubMed] [Google Scholar]
  63. Norsa A, Martino V 2007 Somatostatin, retinoids, melatonin, vitamin D, bromocriptine, and cyclophosphamide in chemotherapy-pretreated patients with advanced lung adenocarcinoma and low performance status. Cancer Biother Radiopharm 22:50–55 [DOI] [PubMed] [Google Scholar]

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