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. 2022 Feb;36(2):e22101.
doi: 10.1096/fj.202101262R.

Short-term tetrabromobisphenol A exposure promotes fibrosis of human uterine fibroid cells in a 3D culture system through TGF-beta signaling

Jingli Liu  1 , Linda Yu  1 , Lysandra Castro  1 , Yitang Yan  1 , Natasha P Clayton  2 , Pierre Bushel  3 , Norris D Flagler  2 , Erica Scappini  4 , Darlene Dixon  1
Affiliations

Short-term tetrabromobisphenol A exposure promotes fibrosis of human uterine fibroid cells in a 3D culture system through TGF-beta signaling

Jingli Liu et al. FASEB J. 2022 Feb.

Abstract

Tetrabromobisphenol A (TBBPA), a derivative of BPA, is a ubiquitous environmental contaminant with weak estrogenic properties. In women, uterine fibroids are highly prevalent estrogen-responsive tumors often with excessive accumulation of extracellular matrix (ECM) and may be the target of environmental estrogens. We have found that BPA has profibrotic effects in vitro, in addition to previous reports of the in vivo fibrotic effects of BPA in mouse uterus. However, the role of TBBPA in fibrosis is unclear. To investigate the effects of TBBPA on uterine fibrosis, we developed a 3D human uterine leiomyoma (ht-UtLM) spheroid culture model. Cell proliferation was evaluated in 3D ht-UtLM spheroids following TBBPA (10-6 -200 µM) administration at 48 h. Fibrosis was assessed using a Masson's Trichrome stain and light microscopy at 7 days of TBBPA (10-3 µM) treatment. Differential expression of ECM and fibrosis genes were determined using RT² Profiler™ PCR arrays. Network and pathway analyses were conducted using Ingenuity Pathway Analysis. The activation of pathway proteins was analyzed by a transforming growth factor-beta (TGFB) protein array. We found that TBBPA increased cell proliferation and promoted fibrosis in 3D ht-UtLM spheroids with increased deposition of collagens. TBBPA upregulated the expression of profibrotic genes and corresponding proteins associated with the TGFB pathway. TBBPA activated TGFB signaling through phosphorylation of TGFBR1 and downstream effectors-small mothers against decapentaplegic -2 and -3 proteins (SMAD2 and SMAD3). The 3D ht-UtLM spheroid model is an effective system for studying environmental agents on human uterine fibrosis. TBBPA can promote fibrosis in uterine fibroid through TGFB/SMAD signaling.

Keywords: TGFB; extracellular matrix; fibrosis; tetrabromobisphenol A; uterine fibroid.

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Conflict of interest statement

The authors declared no potential conflict of interest for the research, authorship, and publication of this article.

Figures

FIGURE 1
FIGURE 1
Histology and morphology of ht‐UtLM cells on day 7 in 2D and 3D cultures as well as human uterine fibroid tissues. (A) Morphology of ht‐UtLM cells in 2D and 3D cultures. Hematoxylin and eosin (H&E) staining (left); Masson's Trichrome staining (right). Blue color indicates collagen. (B) Morphology of 3D ht‐UtLM spheroids and primary human fibroid tissues. (C) Heat map of ECM and fibrosis‐associated gene expression in 3D compared to 2D by Fibrosis RT2 profiler PCR array. Fold change >2 and p < .05. Scale bar = 100 μm
FIGURE 2
FIGURE 2
Cell proliferation of ht‐UtLM spheroids by Cell Counting Kit (CCK8) assay. The ht‐UtLM spheroids were treated with TBBPA at different concentrations (10−6–200 μM) for 48 h. *p < .05 compared to control
FIGURE 3
FIGURE 3
Extracellular matrix (ECM) gene expression in 3D ht‐UtLM spheroids treated with TBBPA at 10−3 μM for 24 h. ECM gene expression was assessed by ECM RT2 profiler PCR array. (A) Heat map of significantly regulated ECM genes, upregulated (Red) or downregulated (Green). Fold regulation cutoff: 2, and p‐value cutoff: .05. (B) Differentially regulated expression of collagens mRNA. *p < .05 compared to control
FIGURE 4
FIGURE 4
Collagen accumulation in 3D ht‐UtLM spheroids treated with TBBPA for 7 days. (A) Hematoxylin and eosin (H&E) staining (top) and Masson's Trichrome staining (bottom). (B) Quantification of collagen content (blue). (C) Diameter of spheroids. *p < .05 compared to control. (D) PCNA expression and intensity quantification by western blotting. Scale bar = 100 μm
FIGURE 5
FIGURE 5
Upstream analysis of Fibrosis RT2 profiler PCR array in 3D ht‐UtLM spheroids induced by TBBPA at 24 h. (A) Upstream regulators analysis predicting the activation of TGFB signaling through the TGFB1 network. Upregulated genes are labeled red, and downregulated genes are labeled green. (B) Differentially expressed genes associated with TGFB signaling. *p < .05 compared to control
FIGURE 6
FIGURE 6
Upstream functional analysis of TBBPA‐activated TGFB1 network in Fibrosis RT2 profiler PCR array. The red relationship lines between TGFB1 network and organization of ECM predicted activation
FIGURE 7
FIGURE 7
Function analysis for networks of TGFB1, TGFBR1, and TGFBR2. The data of TGFB phospho‐protein array were analyzed by IPA in 3D ht‐UtLM spheroids with TBBPA treatment at 3 h. The red relationship lines between molecules in the networks and function predicted the activation of fibrogenesis
FIGURE 8
FIGURE 8
Activation of TGFB/SMAD signaling pathway induced by TBBPA in 3D ht‐UtLM spheroids at the protein level. TGF‐β phospho‐protein array was assessed at 3 h after treatment and analyzed by IPA. TGFB bound to the receptor complex and subsequently phosphorylated TGFBR1, which in turn phosphorylated SMAD2 and SMAD3. SMAD4 then bound to p‐SMAD2/3 and translocated to the nucleus to transcribe genes regulating fibrosis or early development
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