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 Table of Contents  
ORIGINAL ARTICLE
Year : 2018  |  Volume : 14  |  Issue : 9  |  Page : 276-281

The effect of TGF-β signaling on regulating proliferation of uterine leiomyoma cell via ERα signaling activated by bisphenol A, octylphenol and nonylphenol in vitro


Department of Obstetrics and Gynecology, School of Medicine, Zhongda Hospital, Southeast University, Nanjing 210009, China

Date of Web Publication29-Jun-2018

Correspondence Address:
Yang Shen
Department of Obstetrics and Gynecology, School of Medicine, Zhongda Hospital, Southeast University, Nanjing 210009
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0973-1482.235342

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 > Abstract 

Objectives: To study the transforming growth factor beta (TGF-β) signaling pathway in interactions with estrogen receptor alpha (ERα) signaling pathway mediating the growth of human uterine leiomyoma (UL) activated by phenolic environmental estrogens (EEs).
Methods: The subcultured UL cells were used to determine the validation of TGF-β3 for the viability of human UL cells using CCK-8 assay, mRNA expressions of ERα, and c-fos by quantitative reverse transcription polymerase chain reaction method, and expressions of p-Smad3, SnoN, and c-fos proteins by Western blot assay in each treatment group.
Results: Compared with each of EEs or TGF-β3 treatment, slightly decrease in the proliferation rate of UL was detected in the coexistence of each EE with TGF-β3. Interestingly, mRNA expressions of ERα and c-fos reduced in the setting of coexistence of TGF-β3 and EEs. Somehow, the expression of p-Smad3 and c-fos proteins significantly decreased in each of E2, bisphenol A (BPA), nonylphenol (NP), and octylphenol (OP) group, as well as the expression of SnoN protein significantly reduced only in BPA and NP groups, followed by TGF-β3 treatment. With the overlaid action of ICI 182,780, the expression of p-Smad3 protein significantly increased in OP group, but slightly increased in E2, BPA, NP, and OP groups. However, compared with the control group, the expression of SnoN and c-fos proteins significantly decreased in the same setting.
Conclusion: Both ERα signaling pathway and TGF-β signaling pathway have different roles in governing UL cell proliferation. The phenolic EEs can be a promoter to the proliferation of UL cells, which is mediated by ERα signaling pathway and cross-talked with TGF-β signaling pathway.

Keywords: bisphenol A, environmental estrogens, estrogen receptor, nonylphenol, octylphenol, transforming growth factor beta, uterine leiomyoma


How to cite this article:
Shen Y, Lu Q, Zhang P, Wu Y, Ren M. The effect of TGF-β signaling on regulating proliferation of uterine leiomyoma cell via ERα signaling activated by bisphenol A, octylphenol and nonylphenol in vitro. J Can Res Ther 2018;14:276-81

How to cite this URL:
Shen Y, Lu Q, Zhang P, Wu Y, Ren M. The effect of TGF-β signaling on regulating proliferation of uterine leiomyoma cell via ERα signaling activated by bisphenol A, octylphenol and nonylphenol in vitro. J Can Res Ther [serial online] 2018 [cited 2018 Sep 20];14:276-81. Available from: http://www.cancerjournal.net/text.asp?2018/14/9/276/235342


 > Introduction Top


Uterine leiomyoma (UL) is the most common benign tumors of the female reproductive tract in reproductive age, which has reported the prevalence of around 20–50%. However, women with UL usually present no clinical symptoms in the early stages, but then only 30% of women show clinical symptoms of varying severity.[1]

Currently, although the pathogenesis of UL is still not clear, there is growing evidence that the development of UL is influenced by many factors, including genetics, hormones, and environment.[2],[3] A number of studies suggested that environmental estrogens (EEs) have an impact on the development of UL, which also supported by our previous researches focused on the effects in vitro of EEs (bisphenol A [BPA] or nonylphenol [NP]) on the proliferation of UL can promote UL cells proliferation, and its possible mechanism associated with the increased expressions of insulin-like growth factor-1 (IGF-1), vascular endothelial growth factor (VEGF), and other tumor-promoting growth factors mediated by estrogen receptor alpha (ERα) signaling pathway.[4],[5],[6]

Also, the transforming growth factor beta (TGF-β) family plays a central role in the pathogenesis of UL,[7],[8] and TGF-β3 appears to be physiologically the most relevant TGF-β isoform in UL.[9] TGF-β3-induced profibrotic response in UL cells is mediated by Smad-dependent and Smad-independent phosphatidylinositol-3-kinase/Akt/mammalian target of rapamycin pathways.[10] Interestingly, some other studies suggested a co-regulation of the pathways and suggested that TGF-β may be able to restrict ERα- mediated proliferation.[11],[12] So, here we will further investigate the regulatory effect of TGF-β signaling pathway on UL proliferation promoted by phenolic EEs in this study.


 > Materials and Methods Top


Tissue collection

In total, 15 ULs were sampled from premenopausal women (age 30–50 years) undergoing hysterectomy or myomectomy at the Zhongda Hospital between December 2013 and December 2014. Permission to use these samples was granted by the Ethics Committee of Zhongda Hospital (2014ZDSYLLO78.0). All samples were obtained after receiving written informed consent from the patients.

Observation of cell viability

The primary cell culture procedure was performed in accordance with reported methods.[13] UL cells were trypsinized, suspended, seeded in 96-well culture plates at a density of 2 × 105/well, and allowed to grow for 24 h at 37°C. Based on data in our previous research,[5],[6] the optimal dose of EEs was used in this study. The cells were incubated in 100 μl of medium with E2 (103 μmol/L, E2 group), BPA (10 μmol/L, BPA group), NP (32 μmol/L, NP group), octylphenol (OP) (8 μmol/L, OP group), or DMSO only (control group). Then TGF-β3 (5 ng/ml) was added to each well and mixed with the culture medium in each group set up above for the subsequent experiment. Five wells were used for each group.

Cell culture mixture was collected at 24, 48, and 72 h and centrifuged. The supernatant was discarded. Ten microliters of WST-8 was added to each well, followed by incubation for 0.5–4 h at 37°C in a humidified atmosphere of 95% air and 5% CO2. Subsequently, the proliferation rate was calculated according to the absorbance at 450 nm using an ELISA reader (VERSA man; Molecular Devices, Sunnyvale, CA, USA). The proliferation rate was calculated using the following formula: Cell proliferation inhibition rate = (average of value A from the experimental group − the average of value A from the control group)/(average of value A from the experimental group − average of value A from blank controller) ×100%.

mRNA expression of ERα and c-fos using real-time quantitative polymerase chain reaction

The mRNA expressions of ERα and c-fos in UL from each of EEs groups and EEs combined with TGF-β3 groups were identified as representative molecular of ER signaling pathway and TGF-β signaling pathway. Total RNA was extracted at 72 h using an RNA extraction kit (Qiagen, Valencia, CA, USA), according to the manufacturer's instructions. Total RNA concentration was measured using a spectrophotometer (Optizen, Mecasys, Dea-jeon, Korea) at 260 nm/280 nm. One microgram of total RNA was dissolved in diethyl pyrocarbonate and deionized water for cDNA synthesis.

One microgram of total RNA from each sample was reverse transcribed, and an equal amount of cDNA (40 ng) was used for amplification of ERα or c-fos (Applied Biosystems, Foster City, CA, USA) and detected using Bio-Rad IQ5. The real-time polymerase chain reaction system was designed as follows: SYBR green mix, 10 μl; upstream primer, 0.6 μl; downstream primer, 0.6 μl; ddH2O, 6.8 μl; and cDNA, 2 μl (total volume 20 μl). Meanwhile, the amplification reaction conditions were set for 35 cycles as follows: predenaturation at 94°C for 2 min, denaturation at 94°C for 45 s, annealing at 56°C for 45 s, and extension at 72°C for 45 s. Finally, fluorescence data were collected for analysis. The primer sequences used for ERα and c-fos quantification were as follows: ERα forward, 5'-GCC AGG CAC ATT CTA GAA GG-3'; ERα reverse, 5'-AGA CAT GAG AGC TGC CAA CC-3'; c-fos forward, 5'-GCC TCG TTC CTC CAG TCC GA-3'; c-fos reverse, 5'-TGC GAT GGA AAG GCC AGC CC-3'; β-actin forward, 5'-GAT GAC CCA GAT CAT GTT TGA G-3'; and β-actin reverse, 5'-AGG GCA TAC CCC TCG TAG AT-3'. Fold of difference relative to the reference gene (β-actin) was determined by conversion of 2−△△CT. △△CT = (CTobjective gene − CTreference gene) of experimental group − (CTobjective gene − CTreference gene) of control group.

Protein expression of p-Smad3, SnoN, and c-fos using Western blot analysis

UL cells were treated with each EEs or each EEs combined with estrogen antagonist ICI 182,780 for 72 h, and cell lysates were analyzed by Western blot as described elsewhere.[14],[15] Equal amounts of solubilized proteins from cultured cells (30–40 mg) were resolved in 8% and 10% SDS-polyacrylamide gel electrophoresis. The proteins were transferred onto polyvinylidene fluoride membranes (Bio-Rad, Hercules, CA, USA) and incubated with specific primary antibodies for 2 h at room temperature, followed by 1 h with appropriate horseradish peroxidase-conjugated secondary antibodies. Western blot analyses were performed using primary antibodies against p-Smad3 (1:500), SnoN (1:1000), and c-fos (1:500). The antigen-antibody complexes in Western blots were detected with an enhanced chemiluminescence detection system (Amersham Biosciences, Pittsburgh, PA, USA). Specific protein bands were visualized after exposure to autoradiography films by developing the films using an automatic X-ray developer. The intensity of each protein band was quantified using image analysis software and normalized against corresponding β-actin, detected by anti-b-actin antibody (1:5000). The normalized ratio was used to generate data graphs.

Statistical analysis

Statistical Package for the Social Sciences (SPSS 19.0, SPSS Inc., Chicago, IL, USA) was used for data analysis. All data are expressed as mean ± standard deviation. One-way analysis of variance and least significant difference methods were used for comparison between the groups. Bivariate correlation analysis was performed using Pearson's correlation coefficient method. P <0.05 was considered to indicate statistical significance.


 > Results Top


Effect of transforming growth factor beta 3 on uterine leiomyoma cells proliferation activated by phenolic environmental estrogens

The primary and passaged uterine fibroid cells were successfully established for the further experiment.[5],[6] According to our previous studies on the relationship between EEs and UL cell in vitro,[5],[6] we hereby try to evaluate whether TGF-β3 would regulate UL cells proliferation activated by three phenolic EEs. The effect curve demonstrated E2, BPA, and NP increased growth of UL cells when compared with untreated UL cells at different time (P < 0.05). Whereas, OP has significant effect on the UL cells proliferation at 48 h and 72 h (P < 0.05), but not at 24 h (P > 0.05) [Figure 1]. Also, TGF-β3 alone presented its effect on UL cells proliferation [Figure 2]. Thereafter, when compared with untreated UL cells, with the treatment of TGF-β3, the effect curve showed E2, BPA, and NP increased growth of UL cells at different time (P < 0.05). Whereas, OP has obvious effect on the UL cells proliferation at only 72 h (P < 0.05), but not at 24 h or 48 h (P > 0.05) [Figure 2]. Paradoxically, there was no obviously statistical difference in effects of EEs on UL proliferation between pre- and post-treatment of TGF-β3 (P > 0.05).
Figure 1: The effect curve of uterine leiomyoma cells treated by different environmental estrogens

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Figure 2: The effect curve of uterine leiomyoma cells treated by different environmental estrogens with transforming growth factor beta 3

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Alteration of estrogen receptor alpha and c-fos mRNA expression in uterine leiomyoma cells with treatment of environmental estrogens combined with or without transforming growth factor beta 3

To investigate the potential mechanism responsible for the interaction effects of TGF-β signaling pathway and ER signaling pathway, mRNA was isolated and ERα and c-fos mRNA expression was determined following treatment with EEs combined either with or without TGF-β3. As shown in [Figure 3] and [Figure 4], ERα, and c-fos mRNA was detected in each EEs group, but there is difference between E2 group and any of three EEs groups (P > 0.05) [Table 1]. Compared to the EEs group without treatment of TGF-β3, the expression of both ERα and c-fos mRNA decreased following treatment with TGF-β3 (P < 0.05), indicating their involvement in the TGF-β3 mediated cell effects.
Figure 3: Effect of environmental estrogens on estrogen receptor alpha and c-fos mRNA expression in uterine leiomyoma cells without transforming growth factor beta 3

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Figure 4: Effect of environmental estrogens on estrogen receptor alpha and c-fos mRNA expression in uterine leiomyoma cells with transforming growth factor beta 3

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Table 1: The effect of EEs on the expression of ERα and c-fos mRNA in UL cells with or without treatment of TGF-β3 (x̄±s, n=3)

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Alterations of p-Smad3, SnoN, and c-fos protein expression in uterine leiomyoma cells with treatment of environmental estrogens combined with or without treatment of estrogen agonist ICI 182,780

Western blot was performed to determine how ICI 182,780 may affect the protein levels of p-Smad3, SnoN, and c-fos. The intensity of protein bands was read by densitometry, and the standardized results with β-tubulin are summarized in [Table 2] and [Table 3]. The results indicated that following 72 h of treatment of E2, BPA, NP, and OP, expression of both p-Smad3 and c-fos proteins significantly decreased when compared with control group (P < 0.05). However, expression of SnoN protein significantly increased only in BPA group and NP group, but not in E2 group and OP group [Figure 5]. Interestingly, combined with the treatment of ICI 182,780, OP treated UL cells indicated obviously increased expression in p-Smad3 protein when compared with control group (P < 0.05). But slightly growth in the expression of p-Smad3 protein was showed in E2 group, BPA group, and NP group in the same setting (P > 0.05). Moreover, combined with the treatment of ICI 182,780, each EEs treated UL cells presented significantly reduced expression of SnoN and c-fos when compared with control group (P < 0.05) [Figure 6]. However, there were significant differences in expression of p-Smad3, SnoN, and c-fos proteins between pre- and post-treatment of ICI 182,780 (P < 0.05).
Table 2: Effect of EEs on expression of p-Smad3, SnoN, and c-fos proteins in UL cells (x̄±s, n=3)

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Table 3: Effect of EEs on expression of p-Smad3, SnoN, and c-fos proteins in UL cells treated with ICI 182,780 (x̄±s, n=3)

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Figure 5: Effect of environmental estrogens on expression of p-Smad3, SnoN and c-fos proteins in uterine leiomyoma cells Note: Lane 1: TGF-β; Lane 2: TGF-β+E2; Lane 3:TGF-β+BPA; Lane 4: TGF-β+NP; Lane 5: TGF-β+OP

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Figure 6: Effect of environmental estrogens on expression of p-Smad3, SnoN and c-fos proteins in uterine leiomyoma cells with combined treatment of ICI 182,780 Note: Lane 1: TGF-β+ICI182780; Lane 2: TGF-β++ICI182780+E2; Lane 3:TGF-β+ICI182780+BPA; Lane 4: TGF-β+ICI182780+NP; Lane 5: TGF-β+ICI182780+OP

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 > Discussion Top


UL is a common gynecological benign tumor that occurs in smooth muscle tissue. Despite the prevalence of these tumors, there is limited understanding of their pathogenesis and few successful therapeutic strategies.[1] Currently, the causes of UL remain unknown. Many clinical observations and experimental studies have reported that UL are estrogen-dependent.[16] UL may result from long-term stimulation of high levels of estrogens, in particular, EEs that enter the body via food intake and daily exposure.[17]

Phenolic EEs, widely used and thus easily exposed to humans, can mimic or disturb endogenous estrogen by binding to the estrogen receptor or affecting estrogen cell signaling pathway transduction by acting as an estrogen-like effect.[18] Our previous research, on quantitative detection of EEs in urine and blood samples collected from uterine fibroid patients and controls, suggests that the BPA exposure levels are significantly higher in the population of UL.[4],[19] Furthermore, we reported that EEs can promote proliferation of UL cells via updating ERα mRNA and protein, and the effect enhances followed with increased EEs concentration. In this study, we also found that the similar results either E2 or each EEs treatment can promote proliferation through overexpression of ERα could up-regulate expressions of IGF-1 and VEGF pathways.[5],[6] So far, we definitely propose EEs involve in the induction of ERα gene and its pathway genes, and consequently paly a tumorigenic effect in UL.

Also, ERα signaling pathway discussed above, most importantly, the possible role of TGF-β pathway and its downstream gene Smad3 in the pathogenesis of human UL was ascertained.[20] TGF-β is a major regulator of many essential cellular processes including proliferation, differentiation, migration, immune response, and apoptosis. Previously, both Lee and Nowak, Arici and Sozen found that TGF-β3 mRNA was overexpression in leiomyoma compared to normal myometrium.[21],[22] Furthermore, Lee and Nowak found that a TGF-β neutralizing antibody decreases levels of types I and III collagen mRNA in leiomyoma and myometrial cells.[21] Therefore, it is indicated that TGF-β3 stimulates proliferation in both leiomyoma and myometrial cells.[23] TGF-β family members pass signals through the cell membrane receptors to induce a cascade of positive and negative regulatory steps that end in activation of transcriptional activator complexes. The signaling pathway cascade is mediated by the phosphorylation of receptor-regulated Smads (R-Smads), which are represented by Smad2 and Smad3 for the TGF-β family.[24] Smad3 is one of two homologous proteins that involved in signaling pathway from TGF-β to modulate gene transcription. Increased expression of Smad3 may account for the development of UL and its matrix components.[20] Moreover, Salama et al. demonstrated that TGF-β3 induces profibrotic effects (expression of types I and III collagen and others) on leiomyoma cells through Smad and non-Smad pathways.[25] The UL tissue was found to overexpress TGFBR1, TGFBR2, Smad3, Smad4, and phosphorylated Smad3 compared to myometrium.[26] Taken together, all of these studies mentioned above point to a potential role for aberrant TGF-β-Smad signaling in UL. Now our study found that TGF-β3 definitely stimulated the UL proliferation through up-regulating c-fos on behalf of TGF-β signaling pathway activation, which is consistent with other previous researches.

It is reported that ERα and TGF-β regulatory pathways intersect, and ERα blocks TGF-β signaling pathway by multiple means, including direct interactions of its signaling components, Smads in breast cancer.[27] In this study, interestingly, we examined whether the TGF-β signaling pathway is associated with ERα signaling in UL cells, as well as we determined whether the exposure to combined TGF-β3 and EEs may induce UL progression by affecting molecular cross-talk between ERα and TGF-β signaling pathways. Herein, we surprised to find that no additive effect but slightly downward trend on UL proliferation was observed following the interaction of TGF-β3 and EEs, which indicated that there may be some relationship between EEs and TGF-β3 functioning in UL occurrence and development. Furthermore, we investigated the variation of downstream factors in ERα signaling and TGF-β signaling in order to elaborate the weird observations post co-effect of EEs and TGF-β3. The results showed the expression of both ERα and c-fos mRNA significantly decreased following treatment with TGF-β3 in each EEs group, which again determine TGF-β signaling pathway cross-talking with ERα signaling pathway on regulating the growth of UL activated by phenolic EEs. The results indicated that expression of both p-Smad3 and c-fos proteins significantly decreased following treatment of E2, BPA, NP, and OP, as well as SnoN protein significantly increased following treatment of BPA and NP, when compared with control group. Subsequently, combination with the treatment of ICI 182,780, an estrogen agonist to block the ERα signaling pathway, all of the EEs treated UL cells presented the trend of growth in expression of p-Smad3 and reduced expression of SnoN and c-fos. Consequently, we could infer from the result that the classical ERα pathway together with TGF-β signaling pathway co-regulate the growth of UL cells, but above all, ERα signaling pathway used to inhibit TGF-β signaling pathway under the effect of estrogen or estrogen-like. Also, intracellular signaling activated by TGF-β also includes non-Smad pathways which have not yet been settled in our research.


 > Conclusion Top


Our findings raise again that both ERα signaling pathway and TGF-β signaling pathway have not only played very important impacts, but also exerted different roles on the incidence of UL induced by phenolic EEs, which will pave the road for future research on elucidating the pathogenesis and making clinical prophylactic decision of UL. Both less exposure to EEs and blockade of TGF signaling pathway are necessary strategy to prevent UL.

Financial support and sponsorship

This work was supported by Maternal and Child Healthcare Project of Jiangsu Province Health Department (F201407), Youth Fund Project of Jiangsu Province Health Department (Q201305), Science and Technology Project of Nanjing City (201201054), National Natural Science Pre-research Project Funds of Southeast University (3290001102), and SRTP Project of Southeast University (T11431001).

Conflicts of interest

There are no conflicts of interest.

 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]



 

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