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EGFR participates downstream of ERα in estradiol-17β-d-glucuronide-induced impairment of Abcc2 function in isolated rat hepatocyte couplets

  • Molecular Toxicology
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Abstract

Estradiol-17β-d-glucuronide (E17G) induces acute endocytic internalization of canalicular transporters, including multidrug resistance-associated protein 2 (Abcc2) in rat, generating cholestasis. Several proteins organized in at least two different signaling pathways are involved in E17G cholestasis: one pathway involves estrogen receptor alpha (ERα), Ca2+-dependent protein kinase C and p38-mitogen activated protein kinase, and the other pathway involves GPR30, PKA, phosphoinositide 3-kinase/AKT and extracellular signal-related kinase 1/2. EGF receptor (EGFR) can potentially participate in both pathways since it interacts with GPR30 and ERα. Hence, the aim of this study was to analyze the potential role of this receptor and its downstream effectors, members of the Src family kinases in E17G-induced cholestasis. In vitro, EGFR inhibition by Tyrphostin (Tyr), Cl-387785 or its knockdown with siRNA strongly prevented E17G-induced impairment of Abcc2 function and localization. Activation of EGFR was necessary but not sufficient to impair the canalicular transporter function, whereas the simultaneous activation of EGFR and GPR30 could impair Abcc2 transport. The protection of Tyr was not additive to that produced by the ERα inhibitor ICI neither with that produced by Src kinase inhibitors, suggesting that EGFR shared the signaling pathway of ERα and Src. Further analysis of ERα, EGFR and Src activations induced by E17G, demonstrated that ERα activation precedes that of EGFR and EGFR activation precedes that of Src. In conclusion, activation of EGFR is a key factor in the alteration of canalicular transporter function and localization induced by E17G and it occurs before that of Src and after that of ERα.

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Abbreviations

Abcc2:

Multidrug resistance-associated protein 2

E17G:

Estradiol 17β-d-glucuronide

EGFR:

Epidermal growth factor receptor

ERα:

Estrogen receptor alpha

GPR30:

G protein-coupled receptor 30

AKT:

Protein kinase B

CMFDA:

5-Chloromethylfluorescein diacetate

GS-MF:

Glutathione methylfluorescein

DMSO:

Dimethyl sulfoxide

IRHC:

Isolated rat hepatocyte couplets

SCRH:

Sandwich-cultured rat hepatocytes

cVA:

Canalicular vacuolar accumulation

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Acknowledgments

This work was supported by grants from Agencia Nacional de Promoción Científica y Tecnológica (PICTs 2010 No. 1197 and 2013 No. 1222) and Consejo Nacional de Investigaciones Científicas y Técnicas (PIP 0691 y PIP 0217).

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Authors and Affiliations

  1. Instituto de Fisiología Experimental (IFISE), Facultad de Ciencias Bioquímicas y Farmacéuticas (CONICET - U.N.R.), Suipacha 570, S2002LRL, Rosario, Argentina

    Ismael R. Barosso, Andrés E. Zucchetti, Gisel S. Miszczuk, Andrea C. Boaglio, Diego R. Taborda, Marcelo G. Roma, Fernando A. Crocenzi & Enrique J. Sánchez Pozzi

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  1. Ismael R. Barosso
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  2. Andrés E. Zucchetti
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  3. Gisel S. Miszczuk
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  4. Andrea C. Boaglio
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  5. Diego R. Taborda
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  6. Marcelo G. Roma
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  7. Fernando A. Crocenzi
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  8. Enrique J. Sánchez Pozzi
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Correspondence to Enrique J. Sánchez Pozzi.

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204_2015_1507_MOESM1_ESM.tif

Supplemental Figure 1. Panel A: Effect of submaximal concentration of ICI (0.1 µM) on the protection produced by increasing concentrations of Tyr (1.5–150 nM) on E17G-induced decrease in Abcc2 transport activity in IHRC. * Significantly different from Tyr+E17G. Data are expressed as mean ± standard error of the mean (SEM; n:3). Panel B: Effect of submaximal concentration of Tyr (15 nM) on the protection produced by increasing concentrations of ICI (0.01–1 nM) on E17G-induced decrease in Abcc2 transport activity in IHRC. * Significantly different from ICI+E17G. Data are expressed as mean ± standard error of the mean (SEM; n:3). Co-inhibition with submaximal concentrations of Tyr and ICI reached but could not surpass the maximal protection of Tyr or ICI alone confirming that the effect of the inhibitors was not additive (TIFF 1684 kb)

204_2015_1507_MOESM2_ESM.tif

Supplemental Figure 2. Panel A: Effect of submaximal concentration of IS (0.1 µM) on the protection produced by increasing concentrations of Tyr (1.5–150 nM) on E17G-induced decrease in Abcc2 transport activity in IHRC. * Significantly different from Tyr+E17G. Data are expressed as mean ± standard error of the mean (SEM; n:3). Panel B: Effect of submaximal concentration of Tyr (15 nM) on the protection produced by increasing concentrations of IS (0.01–1 nM) on E17G-induced decrease in Abcc2 transport activity in IHRC. * Significantly different from IS+E17G. Data are expressed as mean ± standard error of the mean (SEM; n:3). Co-inhibition with submaximal concentrations of Tyr and IS reached but could not surpass the maximal protection of Tyr or IS alone confirming that the effect of the inhibitors was not additive (TIFF 1596 kb)

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Barosso, I.R., Zucchetti, A.E., Miszczuk, G.S. et al. EGFR participates downstream of ERα in estradiol-17β-d-glucuronide-induced impairment of Abcc2 function in isolated rat hepatocyte couplets. Arch Toxicol 90, 891–903 (2016). https://doi.org/10.1007/s00204-015-1507-8

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