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The Journal of Clinical Endocrinology & Metabolism 86(8):3924 –3928 Copyright © 2001 by The Endocrine Society
Expression of Scavenger Receptor Class B1 in Endometrium and Endometriosis SUMATHI RAMACHANDRAN, MINGQING SONG, ANA A. MURPHY, SAMPATH PARTHASARATHY
AND
Department of Gynecology and Obstetrics, Emory University School of Medicine, Atlanta, Georgia 30322 Endometriosis is characterized by the presence of endometrial glands and stroma within the peritoneum and other extrauterine sites. The presence of aromatase, a key enzyme in the biosynthesis of estradiol, has been demonstrated in eutopic endometrial samples of women with moderate to severe endometriosis, but not in those of disease-free women. Animal studies have shown that high density lipoprotein provides precursor cholesterol pool for steroidogenesis. Scavenger receptor class B1, a 82-kDa cell surface protein, is involved in the binding of high density lipoprotein to target cells and promotes cholesteryl ester uptake. In this study we detected the presence of scavenger receptor class B1 in eutopic and ectopic endometrium. There was more scavenger receptor class B1 protein associated with endometriosis compared with
E
NDOMETRIOSIS IS characterized by the presence of endometrial tissue in ectopic sites outside the uterus and is linked to pelvic pain and infertility. It is estimated to affect 2–10% of women of reproductive age (1). Endometriosis is an estrogen-responsive disease, and the pelvic pain associated with it improves when estrogen production is reduced with bilateral oophorectomy or chronic GnRH agonist treatment (2, 3). Ovarian steroids regulate, indirectly, the synthesis and secretion of a potent chemotactic and activating factor for monocytes/macrophages by endometrial cells of women with endometriosis and reveal a new mechanism for estradiol action (4). The transcripts for aromatase, an enzyme proximally involved in the biosynthesis of estrogen, was detected in the eutopic endometrial samples of women with endometriosis (5), and aromatase inhibitors are reported to suppress postmenopausal endometriosis (6). The presence of aromatase is necessary for tissues to generate estrogen. In mice, serum high density lipoprotein (HDL) serves an important function in providing cholesteryl esters (precursor for steroidogenesis) to cells (7), but its role in humans has not been well established. Scavenger receptor class B1 (SR-B1) is a newly described receptor that appears to regulate the entry of HDL into cells capable of steroidogenesis. SR-B1 expression correlates with both the selective transfer of cholesteryl ester into cells and cholesterol efflux from cells, the transfers probably mediated after docking of HDL at the cell surface. SR-B1 exhibits broad ligand specificity, and in animal models appears to be regulated by the action of pituitary hormones Abbreviations: G3PDH, Glyceraldehyde-3-phosphate dehydrogenase; HDL, high density lipoprotein; LDL, low density lipoprotein; SR-B1, scavenger receptor class B1.
matched endometrium. Two bands (82 and 45 kDa) were detected in the endometrium and endometriosis samples. Glycanase treatment indicated that the 45-kDa protein might be a nonglycosylated form of scavenger receptor class B1. Immunostaining of fixed tissues detected scavenger receptor class B1 in glandular epithelium of both tissues. Scavenger receptor class B1 and aromatase mRNA were increased in endometriosis tissues. Scavenger receptor class B1 expression in the endometrium and endometriosis supports a role for this receptor in the uptake of high density lipoprotein cholesterol, possibly supporting in situ estrogen production, which is detrimental in the progression of endometriosis. (J Clin Endocrinol Metab 86: 3924 –3928, 2001)
that stimulate steroidogenesis, suggesting an important role in supplying precursor cholesterol for steroid hormone production (8). Native low density lipoprotein (LDL), which does not bind to either class A receptors or CD36, binds with high affinity to SR-BI (9). The ligand binding specificity of SR-BI, determined by direct binding or competition assays, showed that this receptor also binds to modified LDL (acetylated LDL, oxidized LDL, and methylated BSA) (9, 10). Fielding and Fielding have shown that some LDL-associated free cholesterol is taken up by a selective mechanism that is independent of the LDL receptor, and this uptake may be mediated by SR-B1 (11). Tissue distribution of SR-B1 suggests that this receptor is predominantly expressed in the adrenal gland, ovaries, and liver (12, 13). Cholesterol-using cell types of the ovary, namely interstitial cells, thecal cells, and fully luteinized granulosa cells, are endowed with the HDL receptor mRNA, which provides credence to the functional significance of the role of HDL receptor SR-B1 in cholesterol transport and ovarian steroidogenesis (14). HDL plays a crucial role as a shuttle carrying cholesterol from peripheral cells to liver and steroidogenic tissues, the phenomena called reverse cholesterol transport (15). The presence of aromatase in the endometrium of endometriosis subjects (5) suggests that the endometrium may have the potential to synthesize estradiol. However, the source of precursor for estradiol synthesis, namely cholesterol, has not been studied. In endometriosis the peritoneum is bathed with lipoproteins, including mildly oxidized LDL (16), which may be a source of cholesterol for steroid synthesis. In the current study we present evidence to suggest the presence of the novel receptor SR-B1 in the endometrium and endometriosis tissues of patients with endometriosis,
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Ramachandran et al. • SR-B1 in Endometrium and Endometriosis The Journal of Clinical Endocrinology & Metabolism, August 2001, 86(8):3924 –3928 3925
possibly suggesting its role in promoting reverse cholesterol transport for steroid synthesis, which is detrimental to the disease.
AS-TR phosphate. Positive staining of the cells appeared as red when visualized under the microscope.
Materials and Methods Tissue acquisition and processing
Having shown that SR-B1 is abundant in endometriotic implants compared with that in endometrium, studies were performed to determine whether there was de novo synthesis of this protein. For this, RNA was isolated from tissues using Tri-Reagent. The quality of the RNA was checked, 3 g RNA were reverse transcribed, and the product was PCR amplified using the primers for SR-B1 (5⬘-CTCATCAAGCAGCAGGT and 5⬘-CTTCGTTGGGTGGGTAGA) to obtain a PCR product 900 bp in length. The primers used for aromatase are 5⬘-TCATCATCACCATGGCGATGT and 5⬘-AGCATGCGGTACCAGCCTGT to yield a product size of length 250 bp. Sample sizes and number of amplification were optimized to produce measurements within a linear range. After denaturation at 95 C for 1 min, PCR was conducted as follows: 95 for 10 sec, annealing at 60 C for 15 sec and at 72 C for 15 sec, and extension at 72 C for 30 sec for 32 cycles. Glyceraldehyde-3-phosphate dehydrogenase (G3PDH) was amplified as an internal control.
Endometrium and endometriosis samples were collected from 18 subjects (20 –39 yr of age) undergoing surgery for endometriosis. They received no hormonal treatment, such as GnRH agonist or sex steroids, during the minimum 6-month period preceding surgery. All tissues were obtained with the patient’s fully informed consent. Collection of tissues and experimental protocol were approved by the human investigation committee of Emory University. The presence of glandular epithelium surrounded by stromal tissue was used to confirm the histopathalogical diagnosis of endometriosis and that there was no contamination with either ovarian tissue or peritoneum. After washing the tissues with ice-cold saline, tissues were snap-frozen and stored at ⫺70 C in Tri-Reagent for RNA and RT-PCR analyses and in lysis buffer containing protease inhibitors for protein analyses. Part of the tissue was fixed in 4% paraformaldehyde and embedded in paraffin for immunohistochemical studies. The same pair of endometrium and endometriosis tissues could not be used for all analyses (mRNA, immunohistochemistry, and Western blot) because the samples were relatively small in size.
Western blot analysis Using a polyclonal rabbit antibody raised against the C-terminal 14 amino acids of mouse SR-B1 (17), Western blotting was performed to estimate the protein levels. One hundred micrograms of the protein extract were size-fractionated on a 7.5% SDS-PAGE gel under reducing conditions, transferred to nitrocellulose, and immunoblotted with a polyclonal anti-SR-B1 antibody (provided by Helen H. Hobbs, University of Texas Southwestern Medical Center, Dallas, TX) at a 1:4000 dilution. The blot was incubated with horseradish peroxidase-conjugated secondary antibody and developed using the enhanced chemiluminescence detection system. Mouse ovary was used as a positive control. Ponceau-S staining was used to control for loading and transfer efficiency. Mouse ovary and endometriosis samples were subjected to glycanase enzyme treatment (0.5, 1, and 2 U) to evaluate glycosylation in these tissues.
Immunohistochemistry Tissues fixed in formal sucrose were sectioned and incubated with a 1:100 dilution of polyclonal rabbit antipeptide antibody. For the negative control, primary antibody was omitted. Mouse ovary was used as a positive control. After washing, tissue sections, including the negative control, were incubated with secondary antibody conjugated with alkaline phosphatase for 2 h. Sections were washed three times with PBS, and Fast Red was added as chromogen in conjunction with Naphthol
FIG. 1. Western blot analysis of the endometrium and endometriosis paired tissue samples. A: Lanes 1– 8, Endometrium samples; lanes 9 –16, paired endometriosis samples; lane 17, mouse ovary as a positive control. B, -Actin as an internal control.
RT-PCR analysis
Statistical analysis Densitometric scanning was performed, and the data were analyzed using two-tailed t test to evaluate statistical significance. P ⬍ 0.05 was considered significant.
Results Detection of SR-B1 protein in eutopic endometrium and endometriosis
In the Western analysis the presence of SR-B1 was detected in the endometrium, and the level was higher in endometriosis tissues compared with matched endometrium (Fig. 1A). Justification for equal loading of protein was achieved by detecting -actin levels (Fig. 1B). We detected two forms of the protein (82 and 45 kDa) in the endometrium and endometriosis samples unlike the mouse samples (only 82 kDa) used as positive control. The endometriosis tissue had high levels of SR-B1, especially the 45-kDa protein (P ⬍ 0.01), compared with the endometrium. To evaluate the significance of the two forms of the protein, the tissues were subjected to glycanase treatment at various concentrations. High levels of the 45-kDa protein detected after glycanase treatment of the human endometrium imply that the low molecular mass protein might be a nonglycosylated form of the receptor in humans (Fig. 2). Immunoreactive SR-B1 was detected in the endometrium and endometriosis fixed sections
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FIG. 2. Glycanase treatment of the mouse ovary and endometriosis tissues. Lanes 1–3, 0.5, 1.0, and 2.0 U, respectively, glycanase-treated mouse ovary; lane 4, untreated mouse ovary; lane 5, untreated endometriosis tissue; lanes 6 – 8, 0.5, 1.0, and 2.0 U, respectively, glycanase-treated endometriosis tissue.
(Fig. 3). SR-B1 localized focally in the glandular epithelium (red staining with Fast Red stain) and more diffusely in the surrounding stroma. There was no staining in the negative control without use of the primary antibody. Detection of SR-B1 and aromatase mRNA in eutopic endometrium and endometriosis
To determine whether the presence of SR-B1 was due to the endogenous synthesis of the protein, RT-PCR studies were performed using the primers described in Materials and Methods. SR-B1 message was expressed in the endometrium, and it was higher in the matched endometriosis tissues (P ⬍ 0.05; Fig. 4A). Aromatase was expressed more in the endometriotic tissues (P ⬍ 0.05) compared with eutopic endometrium (Fig. 4B). The housekeeping gene G3PDH indicated that an equal amount of RNA was amplified (Fig. 4C). Densitometric scanning of the Western blot indicated that SR-B1 (45-kDa protein) was elevated in the endometriosis tissues compared with endometrium (P ⬍ 0.01; Fig. 5). SR-B1 and aromatase expression were significantly higher (P ⬍ 0.05) in the eutopic endometrium compared with ectopic tissues (Fig. 6). On the average, SR-B1 was detected in 67% (mRNA in 65% and protein in 69% of tissues) of the normal endometrium and 94% of the endometriosis tissues (mRNA in 88% and protein in 100% of tissues; Table 1). Discussion
The present study demonstrates that SR-B1 is expressed in the endometrium and endometriosis tissues. Recently, data have been accumulated to show that SR-B1 might play an important role in both cholesterol efflux and influx. SR-B1 is coordinately regulated with steroidogenesis in the adrenal gland, ovaries, and testes (12, 18). Tropic hormones influence the expression of this receptor in the steroidogenic tissues. Cao et al. reported that the human SR-B1 gene contains the consensus site for steroidogenic factor-1, which controls the regulation of many of the genes in the steroidogenic pathway (19). Endometriosis is an estrogen-dependent disorder and is probably inherited in a polygenic manner with an etiology of complex multifactorial nature (20). Eutopic endometrium from extraovarian pelvic sites from patients contained P450 aromatase transcripts (5). Estrogen delivery to the endometriotic implants was classically viewed to be only via
FIG. 3. Immunohistological staining of the matched endometrium and endometriosis tissues. 1–3, Endometrium tissue sections; 4 – 6, endometriosis tissue sections.
circulating blood in an endocrine fashion. It was recently uncovered that an autocrine positive feedback mechanism favored the continuous production of estrogen in endometriotic stromal cells (21). Active steroidogenesis rapidly depleted endogenous cholesterol ester stores in these tissues and made them dependent on de novo cholesterol synthesis and/or the import of exogenous lipoproteins to maintain hormone biosynthesis. High doses of estrogen treatment to rats increased SR-B1 in adrenal gland and corpus luteal cells of the ovary and produced very low levels of circulating plasma lipoproteins (12). Studies from our laboratory have shown that the peritoneal cavity of women with endometriosis has a prooxidant milieu consisting of oxidized lipoproteins (22). Stangl et al. have shown that in the absence of LDL receptor, overexpression of SR-B1 can mediate sig-
Ramachandran et al. • SR-B1 in Endometrium and Endometriosis The Journal of Clinical Endocrinology & Metabolism, August 2001, 86(8):3924 –3928 3927
FIG. 4. RT-PCR detection of SR-B1 and aromatase in the endometrium and endometriosis tissues. Lane 1, 100-bp DNA molecular mass marker; lanes 2–9, endometrium samples; lanes 10 – 17, paired endometriosis samples; lane 18, mouse ovary as positive control. A, SR-B1; B, aromatase; C, coamplification of the housekeeping gene G3PDH.
TABLE 1. Tissue SR-B1 mRNA and protein levels Case no.
FIG. 5. Densitometric scanning of the Western blot for SR-B1 to evaluate statistical significance. Comparisons are made between endometrium (E) and endometriosis (ES) tissues. **, P ⬍ 0.01.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Ratio %
FIG. 6. Densitometric scanning of SR-B1 and aromatase RT-PCR products to evaluate statistical significance. Comparisons are made between endometrium (E) and endometriosis (ES) tissues. *, P ⬍ 0.05.
nificant transport of sterols between lipoproteins and the endoplasmic reticulum of the cells (23). The current study demonstrates that SR-B1 is expressed in
mRNA
Protein
E
ES
E
ES
⫺ ⫾ ⫹ ⫺ ⫹⫹ ⫹⫹ ⫺ ⫾ ⫹ ⫺ ⫹⫹ ⫹⫹ ⫹⫹ ⫹ ⫺ ⫺ ⫾ ND
⫾ ⫹ ⫹⫹ ⫾ ⫹⫹ ⫺ ⫾ ⫹ ⫹⫹ ⫾ ⫹⫹ ⫺ ⫹⫹ ⫹⫹ ⫹ ⫹⫹ ⫹ ND
⫺ ⫹⫹ ⫺ ⫾ ⫹ ⫹⫹ ⫹ ⫹ ⫾ ND ND ND ND ND ⫾ ⫺ ⫾ ⫺
⫹ ⫹⫹ ⫹ ⫹⫹ ⫾ ⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹ ND ND ND ND ND ⫹ ⫹⫹ ⫹ ⫾
11/17 64.7
15/17 88.2
9/13 69
13/13 100
the endometrium, suggesting that it is very important to understand the biological utility of this molecule. It seems likely that the peritoneal lipoproteins supply cholesterol for steroid synthesis in the endometrium, and SR-B1 plays a role in the uptake of cholesterol. The results indicate that the endometrium expresses two forms of the receptor (glycosylated and nonglycosylated). However, it remains to be established which of the two forms of the protein would serve as a functional receptor. Increased intrinsic molecular alterations in pelvic endometriotic implants were proposed to contribute significantly to the development of endometriosis. Some of them include increased expression of cytokines (24), metalloproteinases (25, 26), and aromatase expression (21, 27). Our studies have indicated elevated levels of colony-stimulating growth factor-1 in the peritoneum of women with endometriosis compared with controls and dominant expression of colonystimulating growth factor-1, c-Fms (its receptor), and ER␣ in the endometriosis tissues, pointing to the fact that the cell
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cycle genes and other growth factor genes could also be up-regulated (28). Aberrant expression of SR-B1 and aromatase in the endometriosis subjects of our study may give rise to an increase in the concentration of bioactive estrogen in situ. Elevated local levels of estradiol might favor cell proliferation and growth of the endometriotic implants. Nevertheless, aromatase and SR-B1 alone are not enough to regulate estrogen production; other enzymes, such as 17hydroxysteroid dehydrogenase and cyclooxygenase-2, are also equally important. It would be worthwhile to study the presence of other enzymes on steroidogenesis in the endometriotic implants and their correlation with SR-B1 expression. Antiestrogens are used for effective management of endometriosis (29). Endometriosis is successfully suppressed using drugs such as danazol for estradiol deprivation or GnRH analogs (30). Control of pelvic pain using GnRH agonist is successful during and immediately after treatment, but pain associated with endometriosis returns later. Recently developed potent aromatase inhibitors are candidate drugs in the treatment of endometriosis that is resistant to standard treatments (6). In the light of such findings it would be worthwhile to study whether SR-B1 receptor blockers along with aromatase inhibitors may be an effective regimen to combat endometriosis. Acknowledgments We acknowledge the support provided by Dr. Kiran Gill, Dr. Rong Rong, and Raj Razdan. Received October 30, 2000. Accepted April 12, 2001. Address all correspondence and requests for reprints to: Sampath Parthasarathy, Ph.D., Department of Gynecology and Obstetrics, Emory University School of Medicine, Atlanta, Georgia 30322. E-mail:
[email protected]. This work was supported by Grant PO1-HD-35276-01A1 from the NICHHD, NIH (Bethesda, MD).
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