Objective
Uterine leiomyomas, the most common neoplasms of the female genital tract, are benign tumors of the uterus arising from the smooth muscle cells (SMCs) of the myometrium with an involvement of estrogen. Caveolin-1 (Cav-1), a major protein component in caveolae membrane lipid rafts, is down-regulated in several estrogen-related cancer cells, and overexpression of Cav-1 inhibits proliferation of cancer cells and vascular SMCs as well. Therefore, we hypothesize that Cav-1 is down-regulated in human uterine leiomyoma.
Results
Western blot using tissues from clinical patients showed that Cav-1 expression was significantly lower or undetectable in uterine leiomyoma compared with their matched myometrium ( P < .001). This finding was confirmed by immunohistochemistry and confocal microscopy. The cav-1 mRNA level in uterine leiomyomas was also significantly lower as detected by reverse transcription-quantitative polymerase chain reaction analysis ( P = .001). To further study the underlying mechanism, we performed primary cell culture, and found that the expression of Cav-1 remained low in cultured leiomyoma SMCs ( P = .009). Serum withdrawal did not change Cav-1 expression in leiomyoma SMCs, but increased expression in myometrial SMCs ( P = .006). 17-β estradiol inhibited the expression of Cav-1 protein ( P = .047) and mRNA ( P = .007) in leiomyoma SMCs, whereas it stimulated expression in myometrial SMCs ( P = .043). 17-β estradiol, although activating the mitogen-activated protein kinase pathway in both SMCs, did not stimulate their proliferation.
Conclusion
We conclude that human uterine leiomyomas in vitro express low levels of Cav-1, which may result from estrogen inhibition. This effect of estrogen may contribute to the pathogenesis of uterine leiomyoma. Further studies in vivo are needed to verify these results.
Uterine leiomyomas (fibroids), benign tumors that arise in the myometrium, are the most common gynecologic neoplasm, occurring in more than 70% of reproductive-age women. Evidence suggests that tumor growth is dependent on the presence of estrogen ; however, despite much investigation, including genetic and molecular approaches, the pathogenesis of uterine leiomyomas remains unclear.
Caveolin-1 (Cav-1), a major integral membrane protein component in caveolae, plays potential roles in cell cycle progression of several diseases, such as breast cancer, prostate cancer, and atherosclerosis. Both oncogenic and tumor suppressor function of Cav-1 have been demonstrated in breast cancer. However, Cav-1 may act as a tumor promoter in prostate cancer. Because overexpression of Cav-1 in Cav-1-deficient vascular smooth muscle cells (SMCs) inhibits cell proliferation, we speculate that Cav-1 may be down-regulated in leiomyoma SMCs. Because studies from several laboratories including ours have shown that SMCs cultured from uterine leiomyoma are less proliferative than those from the matched myometrium, it is likely that Cav-1 expression is up-regulated during in vitro culture.
Several lines of evidence support our hypothesis. First, caveolae identified by electron microscopy as omega-shaped invaginations of the plasma membrane play critical roles in regulation of smooth muscle (SM) tone and SMC proliferation, as seen in neointimal lesions and atherosclerosis. Vascular SMCs express all three Cav isoforms, Cav-1, -2 and -3. In cultured aortic SMCs, supplementation with serum stimulates cells to enter the cell cycle with a concomitant decrease of Cav-1 expression. Importantly, the expression of Cav-1 is significantly decreased in proliferating vascular SMCs of human atheroma. Genetic ablation of Cav-1 in mice stimulates SMC proliferation (neointimal hyperplasia) with concomitant activation of the p42/44 mitogen-activated protein kinase (MAPK) cascade and up-regulation of cyclin D1.
In addition, Cav-1, -2 and -3 are expressed in rat uterine SMCs. Large numbers of caveolae and elevated expression of Cav-1 and -2 isoforms were reported in the myometrium of ovariectomized rats. Treatment of ovariectomized rats with 17-β estradiol (E2) reduced the formation of caveolae by ∼90% with a reduction in Cav-1 and -2 protein levels.
In the present study, we characterized the expression profiles of Cav-1 in human uterine leiomyoma and matched myometrium, and further investigated its expression in response to 17-β estradiol in primary cultured SMCs. We have shown for the first time that Cav-1 expression level is low in uterine leiomyoma, and estrogen inhibits Cav-1 gene expression in cultured leiomyoma SMCs.
Materials and Methods
Tissue collection
Uterine leiomyoma (HL) and adjacent normal myometrial tissues (HM) were obtained from 24 patients (35 to 50 years old) undergoing hysterectomies at the Central Hospital for Obstetrics and Gynecology, Tianjin, China, with approval from the hospital ethics board and patients’ consent, as previously described. The endometrium of those patients was in the proliferative phase, which was diagnosed through the dilation and curettage procedure and by the pathologists in the Pathology Department of Tianjin Central Hospital for Obstetrics and Gynecology. Size of the tumors ranged from 3 to 12 cm in diameter. All patients did not take any medication or hormonal therapy for at least 3 months before surgery. Samples were excluded from the study if accurate menstrual cycle dates could not be assigned or if unexpected pathology was found (eg, endometriosis or other pelvic inflammatory diseases). All tissue samples used for this study were confirmed as histologically ordinary leiomyomas, with no cellular, epithelioid, bizarre, or plexiform variants present. After surgery, some tissues were collected and stored in liquid nitrogen for further extraction of protein and RNA, respectively. Another fraction of fresh tissues were used for primary cell culture as previously described.
Primary cell culture
Uterine leiomyoma and matched myometrial tissues from the same patient were cut into small pieces (∼1 mm 3 ) and placed into dissociation solution (dulbecco’s modified eagle medium [DMEM], 20% fetal bovine serum [FBS], 0.2% collagenase II [Invitrogen, Carlsbad, CA], and 50 mg/mL trypsin inhibitor [Sigma-Aldrich, St. Louis, MO]), followed by incubation for 2-6 hours at 37°C. Dissociated tissues were filtered through a sterile 100 μm mesh filter. Isolated cells were resuspended in phenol red-free DMEM (Hyclone, Logan, UT) with 10% heat-inactivated FBS (Hyclone, Logan, UT) and 1% antibiotic solution for further culture. Cells from passages 3-5 were used for all the experiments. Extraction of protein and RNA was performed after cells were treated with 17-β estradiol (Sigma, St. Louis, MO) dissolved in ethanol for 24 hours or 48 hours in the presence of 10% FBS. Cell proliferation was determined using hemocytometer counting or WST-1 cell proliferation assay as previously described.
Western blot
The homogenized tissues and cultured SMCs were lysed with ice-cold lysis buffer (150 mM NaCl, 50 mM Tris-Cl [pH 7.5], 1 mM EDTA, 1% NP-40, 0.1% sodium dodecyl sulfate, 0.25% sodium deoxycholate, 1 mM PMSF, 1 mM β-glycerophosphate, 1 mM NaF, 1 mM Na 3 VO 4 , and protease inhibitor cocktail tablets [Roche, Indianapolis, IN]), followed by centrifugation at 4°C for 15 minutes. Protein concentration was determined with a bicinchoninic acid Protein Assay Reagent (Pierce, Rockford, IL). Proteins were separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis, and transferred to polyvinylidene difluoride membranes. The membranes were blocked, and then probed for 1.5 hours or overnight with primary antibodies: Cav-1 (BD, San Diego, CA): 1:800; Cav-2 (BD): 1:1000; Cav-3 (BD): 1:1000; β-actin (Sigma, St. Louis, MO): 1:5000; α-tubulin (Sigma): 1:2000. The membranes were then incubated with horseradish peroxidase-conjugated secondary antibody (1:5000) for 1.5 hours. Protein signals were visualized using the West Pico Chemiluminescent Substrate Kit (Pierce, Rockford, IL). Images were acquired by a Molecular Image Chemidoc XRS System (Bio-Rad Laboratories) and analyzed using Quantity One soft-ware (Bio-Rad Laboratories).
Reverse transcription and real-time quantitative polymerase chain reaction
Total RNA in homogenized tissues or cultured cells was extracted using the RNeasy Mini Kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. RT reactions were carried out using the reverse transcriptase System (Promega, Madison, WI). All quantitative polymerase chain reaction (qPCR) experiments were performed on a real-time PCR machine (Bio-Rad Laboratories) with the QuantiTect SYBR Green PCR Kit and gene-specific primers purchased from Qiagen. Quantification of gene expression was assessed by the comparative cycle threshold (Ct) method. The relative amounts of mRNA for the target genes were determined by subtracting the Ct values for these genes from the Ct value for the housekeeping gene β-actin (ΔCt). Data are depicted as 2-ΔCt.
Immunohistochemical analysis
Tissue sections of leiomyoma and matched myometrium (5 μm) were prepared for immunohistochemistry. After being dewaxed and rehydrated in graded alcohol concentrations, tissue sections were treated with 0.5% Triton X-100 in phosphate buffer solution (PBS). Antigen retrieval was performed in heated 0.01 M citrate buffer (pH 6.4) at 0.15 MPa for 2 minutes. After blocking with 10% FBS, the sections were incubated with primary antibody diluted 1:200 containing 0.5% FBS overnight at 4°C. After washing in PBS, the sections were incubated with Alexa Fluor 488 goat-anti-mouse IgG (dilution: 1:200 in PBS; Invitrogen) for 1 hour. Nuclei were counterstained with propidium iodide (PI), followed by examination using an Olympus laser scanning confocal fluorescence microscope equipped with an argon laser that emits at 488 nm (Olympus IX81 with FluorView FV1000 software; Olympus, Mt. Waverley, Australia).
Data analysis
Analysis of variance and repeated measures analysis of variance with post-hoc testing as well as the 2-tailed Student t test were used. Data were expressed as mean ± SEM. Differences with P value < .05 were considered significant. Statistical analysis was performed using Instat 3.0 (Graphpad 12 Software; Graphpad Software Co., La Jolla, CA).
Results
Cav-1 expression is down-regulated in uterine leiomyomas
We first performed immunohistochemistry to visualize Cav-1 protein in leiomyomas. As shown in Figure 1 , A, Cav-1 signals appeared outside the nuclei and were much stronger in myometrium than in uterine leiomyoma, suggesting that Cav-1 expression was down-regulated in uterine leiomyoma.
To confirm this finding, we examined the expression of Cav-1 protein and mRNA in 24 patients in the proliferative phase of the menstrual cycle. Uterine leiomyomas and their matched myometrium were sampled for protein and RNA for Western blot and RT-qPCR analysis, respectively. In Figure 1 , B, Western blot analysis showed that the levels of Cav-1 protein in leiomyoma tissues were significantly lower than their matched myometrium ( P < .001). Consistently, the levels of cav-1 mRNA from these patients, as detected by RT-qPCR, were significantly lower than their matched myometrium ( P = .001), suggesting down-regulation of Cav-1 gene expression in uterine leiomyomas ( Figure 1 , D).
Cav-1 is known to form a stable hetero-oligomeric complex with Cav-2 in vivo. Knockout of Cav-1 results in deficiency of Cav-2 because of its spontaneous degradation in the absence of Cav-1. Therefore, we examined the expression of Cav-2. As expected, our results showed that the expression levels of Cav-2 protein ( P < .001; Figure 1 , C) and mRNA ( P = .002; Figure 1 , E) were also significantly lower in uterine leiomyoma tissues than in their matched myometrium. Cav-3 protein, although detected in the heart tissue of Sprague Dawley (SD) rats, was not detectable in either leiomyoma or matched myometrial tissues ( Appendix ; Supplemental Figure 1 ), which was consistent with a previous study showing no Cav-3 detection in mouse uterine tissues.
Cav-1 expression is also down-regulated in primary cultured leiomyoma SMCs
As reported previously, primary cultured leiomyoma SMCs have a lower proliferation capacity than those from matched myometrium. As mentioned in the Introduction, Cav-1 levels are inversely correlated with cell proliferation. Therefore, we wished to examine the expression levels of Cav-1 in cultured SMCs from uterine leiomyomas and matched myometrium. Primary culture of human myometrial and leiomyoma SMCs was performed as described in our previous study. Cells were maintained in phenol red-free medium containing 10% serum. The SMCs were characterized by immunostaining and Western blot analyses for SM α-actin and calponin. The SMCs from both myometrium and leiomyoma stained positive for these smooth muscle marker proteins ( Supplemental Figure 2, A ), as previously described. Western blot results showed less SM α-actin and calponin present in leiomyoma SMCs ( Supplemental Figure 2, B ). Cells from passages 3-5 in the presence of 10% serum were used for determination of Cav-1 protein and mRNA expression. As shown in Figure 2 , A, the expression level of Cav-1 protein in leiomyoma SMCs was ∼30% of that in SMCs derived from myometrium ( P = .009). Consistently, RT-qPCR results showed a low level of cav-1 mRNA in leiomyoma SMCs compared with matched myometrial SMCs ( P = .008; Figure 2 , B).
Serum starvation increases Cav-1 protein expression in primary cultured HM-SMCs, but not HL-SMCs
Previous reports showed that serum withdrawal causes an increase in Cav-1 protein levels. Expression of the cav-1 gene in vascular SMCs is down-regulated by treatment with 10% serum. It is also reported that cav-1 gene expression can be up-regulated in NIH 3T3 cells by withdrawal of serum from culture medium, suggesting an inhibitory effect of serum factors on cav-1 gene expression. Therefore, we examined whether the expression of Cav-1 in both HM- and HL-SMCs was also altered in response to serum withdrawal. As expected, serum withdrawal for 24 hours up-regulated the expression of Cav-1 protein ( P = .006) and mRNA ( P = .010) levels in HM-SMCs, which suggests that the cav-1 gene is inducible and its expression can be down-regulated by growth factor(s) or hormone(s) in the serum ( Figure 2 , C and E). In HL-SMCs, serum withdrawal increased mRNA expression ( P = .042), but did not significantly increase Cav-1 protein ( Figure 2 , D and F). This dissociation between mRNA and protein levels of Cav-1 was reported in a previous study, in which platelet-derived growth factor treatment for 24 hours induces a 3-fold increase in the cav-1 mRNA level in vascular SMCs but a decrease in the Cav-1 protein level. Serum withdrawal for 24 hours also significantly increased cav-2 mRNA expression in both HM- ( P = .028) and HL-SMCs ( P = .047) without a significant increase in Cav-2 protein ( Supplemental Figure 3 ).
17-β estradiol decreases Cav-1 expression in HL-SMCs, but increases expression in HM-SMCs
It is well known that estrogen plays a critical role in the development of uterine leiomyoma. A previous study showed that estrogen down-regulated Cav-1 in the uteri of female rats receiving ovariectomies, suggesting potential regulation of cav-1 gene expression in SMCs by estrogen. Given that high estrogen levels are present in uterine leiomyoma tissues because of increased biosynthesis, we determined the effects of estrogen on cav-1 gene expression in cultured HM- and HL-SMCs.
To do so, primary cultured HM- and HL-SMCs were first treated with different concentrations of 17-β estradiol for 24 hours in the presence of 10% serum, followed by Western blot and RT-qPCR analyses. As shown in Figure 3 , A, treatment with 17-β estradiol concentration-dependently down-regulated the expression of Cav-1 protein in primary cultured HL-SMCs, but significantly stimulated the expression of Cav-1 protein in cultured HM-SMCs, as detected by Western blot. Consistently, RT-qPCR results showed a decreased level of cav-1 mRNA ( P = .007) treated with 1 nm 17-β estradiol for 24 hours in leiomyoma SMCs, but increased level of cav-1 mRNA ( P = .044) in matched myometrial SMCs ( Figure 3 , B). As shown in Figure 3 , C and D, treatment with 1 nM 17-β estradiol for 48 hours still significantly increased the expression of cav-1 mRNA ( P = .041) and protein ( P = .018) in myometrial SMCs, but deceased the expression of cav-1 mRNA ( P = .031) and protein ( P = .016) in leiomyoma SMCs. Up-regulation of Cav-1expression in HM-SMCs by 17-β estradiol was inhibited by treatment with ICI 182780 (0.1 μM), an estrogen receptor (ER) antagonist ( P = .031; Figure 3 , E), suggesting involvement of classical estrogen receptors. However, administration of ICI 182,780 not only compensated for the inhibitory effect of E2, but further increased the level of Cav-1 in HL-SMCs ( P = .011).
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