Low doses of bisphenol A promote human seminoma cell proliferation by activating PKA and PKG via a membrane G-protein-coupled estrogen receptor

Abstract

Background: Fetal exposure to environmental estrogens may contribute to hypofertility and/or to testicular germ cell cancer. However, many of these xenoestrogens have only a weak affinity for the classical estrogen receptors (ERs,) which is 1,000-fold less potent than the affinity of 17beta-estradiol (E(2)). Thus, several mechanisms have been suggested to explain how they could affect male germ cell proliferation at low environmental relevant concentrations.

Objectives: In this study we aimed to explore the possible promoting effect of bisphenol A (BPA) on human testicular seminoma cells. BPA is a well-recognized estrogenic endocrine disruptor used as a monomer to manufacture poly carbonate plastic and released from resin-lined food or beverage cans or from dental sealants.

Methods and results: BPA at very low concentrations (10(-9) to 10(-12) M) similar to those found in human fluids stimulated JKT-1 cell proliferation in vitro. BPA activated both cAMP-dependent protein kinase and cGMP-dependent protein kinase pathways and triggered a rapid (15 min) phosphorylation of the transcription factor cAMP response-element-binding protein (CREB) and the cell cycle regulator retinoblastoma protein (Rb). This nongenomic activation did not involve classical ERs because it could not be reversed by ICI 182780 (an ER antagonist) or reproduced either by E(2) or by diethylstilbestrol (a potent synthetic estrogen), which instead triggered a suppressive effect. This activation was reproduced only by E(2) coupled to bovine serum albumin (BSA), which is unable to enter the cell. As with E(2)-BSA, BPA promoted JKT-1 cell proliferation through a G-protein-coupled nonclassical membrane ER (GPCR) involving a Galpha(s) and a Galpha(i)/Galpha(q) subunit, as shown by the reversible effect observed by the corresponding inhibitors NF449 and pertussis toxin.

Conclusion: This GPCR-mediated nongenomic action represents--in addition to the classical ER-mediated effect--a new basis for evaluating xenoestrogens such as BPA that, at low doses and with a high affinity for this GPCR, could interfere with the developmental programming of fetal germ cell proliferation and/or differentiation when they cross the placenta.

Keywords: ER; GPCR; PKA; PKG; bisphenol A; estrogen receptor; testicular germ cell cancer.

Figure 1

Stimulation of JKT-1 cell proliferation by 24-hr exposure to (A) various doses of BPA or (B ) E₂ (10⁻⁹ M), E₂-BSA (10⁻⁹ M), or DDT (10⁻⁹ M). Values shown are the percent change in cell number compared with control (steroid-free medium containing DMSO for BPA or medium containing ethanol for estrogens) given as the mean ± SE of three independent experiments. *p < 0.05.

Figure 2

Effects of 24-hr exposure to E₂, E₂-BSA, or DES alone (each at 10⁻⁹ M) or combined with BPA (10⁻⁹ M) on cell proliferation in JKT-1 cells. Values shown are percent change in cell number compared with control (steroid-free medium containing DMSO for bisphenol A or medium containing ethanol for estrogens and DDT) given as the mean ± SE of three independent experiments. *p < 0.05.

Figure 3

Promotion of cell proliferation in JKT-1 cells through activation of the PKA pathway. (A) Exposure to BPA or E₂-BSA (both at 10⁻⁹ M) with or without inhibition of PKA by H89 (5 × 10⁻⁶ M) for 90 min; values shown are the percent change in cell number (mean ± SE) compared with control (steroid-free medium with DMSO for BPA or medium containing ethanol for E₂-BSA). (B) Western blot analysis of CREB phosphorylation (pCREB; top) and actin expression in each sample (bottom) during exposure to 10⁻⁹ M BPA. Forskolin (25 μM) was used as a positive control for CREB activation. (C) Bands from three experiments were quantified by densitometry, and results were normalized to actin expression in each sample, plotted with SE, and compared with control (time 0). *p < 0.05.

Figure 4

Activation of Rb phosphorylation during BPA activation. (A) Western blot analysis of Rb phosphorylation (pRb) during activation with BPA (10⁻⁹ M) for the indicated time points. (B) Bands from three experiments quantified by densitometry and compared with control (steroid-free medium containing ethanol). *p < 0.05.

Figure 5

Effect of 90-min pretreatment with ICI on cell proliferation in JKT-1 cells treated for 24 hr with E₂, DES, E₂-BSA, BPA (all at 10⁻⁹ M). Values shown are percent change in cell number compared with control (steroid-free medium containing DMSO for BPA or medium containing ethanol for estrogens) given as the mean ± SE of three independent experiments. *p < 0.05.

Figure 6

Effect of 90-min pretreatment with KT5823 (10⁻⁶ M) or NF449 (10⁻⁵ M) on BPA promotion of JKT-1 cell proliferation via a GPCR that involves both Gα_s and Gα_i/Gα_q subunits. Cells were exposed to 10⁻⁹ M BPA for 24 hr. Values shown are percent change in cell number compared with control (steroid-free medium with DMSO) given as the mean ± SE of three independent experiments. *p < 0.05.

Figure 7

Effect of 90-min pretreatment with PTX (100 ng/mL) on GPCR-mediated activation of JKT-1 cell proliferation induced by 24-hr exposure to BPA (10⁻⁹ M). Values shown are percent change in cell number compared with control (steroid-free medium with DMSO) given as the mean ± SE of three independent experiments. *p < 0.05.

Figure 8

BPA promotion of JKT-1 cell proliferation is not ERK1/2 dependent. After 90-min pretreatment with PD (10⁻⁶ M), cells were exposed to E₂-BSA or BPA (10⁻⁹ M) for 24 hr. Values shown are percent change in cell number compared with control (steroid-free medium containing DMSO for BPA and medium containing ethanol for E₂-BSA) given as the mean ± SE of three independent experiments. *p < 0.05.

Figure 9

Proposed model showing the process by which BPA stimulates JKT-1 cell proliferation by activating PKA and PKG pathways via a nonclassical GPCR. Solid lines represent demonstrated steps, and dotted lines represent possible pathways yet to be demonstrated. AC, adenylate cyclase.