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Activation of insulin-like growth factor 1 receptor participates downstream of GPR30 in estradiol-17β-d-glucuronide-induced cholestasis in rats

  • Molecular Toxicology
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Abstract

Estradiol-17β-d-glucuronide (E17G), through the activation of different signaling proteins, induces acute endocytic internalization of canalicular transporters in rat, including multidrug resistance-associated protein 2 (Abcc2) and bile salt export pump (Abcb11), generating cholestasis. Insulin-like growth factor 1 receptor (IGF-1R) is a membrane-bound tyrosine kinase receptor that can potentially interact with proteins activated by E17G. The aim of this study was to analyze the potential role of IGF-1R in the effects of E17G in isolated perfused rat liver (IPRL) and isolated rat hepatocyte couplets. In vitro, IGF-1R inhibition by tyrphostin AG1024 (TYR, 100 nM), or its knock-down with siRNA, strongly prevented E17G-induced impairment of Abcc2 and Abcb11 function and localization. The protection by TYR was not additive to that produced by wortmannin (PI3K inhibitor, 100 nM), and both protections share the same dependency on microtubule integrity, suggesting that IGF-1R shared the signaling pathway of PI3K/Akt. Further analysis of the activation of Akt and IGF-1R induced by E17G indicated a sequence of activation GPR30-IGF-1R-PI3K/Akt. In IPRL, an intraportal injection of E17G triggered endocytosis of Abcc2 and Abcb11, and this was accompanied by a sustained decrease in the bile flow and the biliary excretion of Abcc2 and Abcb11 substrates. TYR did not prevent the initial decay, but it greatly accelerated the recovery to normality of these parameters and the reinsertion of transporters into the canalicular membrane. In conclusion, the activation of IGF-1R is a key factor in the alteration of canalicular transporter function and localization induced by E17G, and its activation follows that of GPR30 and precedes that of PI3K/Akt.

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Abbreviations

Abcc2:

Multidrug resistance-associated protein 2

Abcb11:

Bile salt export pump

E17G:

Estradiol 17β-d-glucuronide

EGFR:

Epidermal growth factor receptor

ERα:

Estrogen receptor alpha

GPR30:

G protein-coupled receptor 30

IGF:

Insulin-like growth factor 1

PI3K:

Phosphoinositide 3-kinase

Akt:

Protein kinase B

CMFDA:

5-Chloromethylfluorescein diacetate

GS-MF:

Glutathione methylfluorescein

CGamf:

Cholyl-glycylamido-fluorescein

DMSO:

Dimethyl sulfoxide

IRHC:

Isolated rat hepatocyte couplets

SCRH:

Sandwich-cultured rat hepatocytes

cVA:

Canalicular vacuolar accumulation

IPRL:

Isolated perfused rat liver

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Acknowledgements

We thank J. Pellegrino for assistance with confocal microscopy.

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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, Gisel S. Miszczuk, Nadia Ciriaci, Romina B. Andermatten, Paula M. Maidagan, Valeria Razori, 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. Gisel S. Miszczuk
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  10. Enrique J. Sánchez Pozzi
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Correspondence to Enrique J. Sánchez Pozzi.

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Financial support

This work was supported by grants from Agencia Nacional de Promoción Científica y Tecnológica (PICTs 2013 N° 1222 and 2013 N° 0974) and Consejo Nacional de Investigaciones Científicas y Técnicas (PIP 0217 and PUE IFISE 0089).

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204_2017_2098_MOESM1_ESM.eps

Additional Fig. 1. Activation studies of insulin-like growth factor receptor-I (IGF-IR). IRHCs were incubated with IGF-I alone (0.1-20 nM) (a) and together with the EGFR agonist EGF (b); cVA of GS-MF was calculated as the percentage of couplets displaying visible fluorescence in their canalicular vacuoles from a total of at least 200 couplets per preparation, referred to as control cVA values. Data are expressed as mean ± SEM (n = 3). * Significantly different of control (EPS 169 kb)

204_2017_2098_MOESM2_ESM.eps

Additional Fig. 2. Image analysis of Abcc2 and Abcb11 in IRHC. a. Distributions of Abcc2 (a, left) and actin (a, right) fluorescence intensity. b. Distributions of Abcb11 (b, left) and actin (b, right) fluorescence intensity. The densitometric analysis of the distribution of Abcc2 and Abcb11 fluorescence intensity was performed along an 8-μm line perpendicular to the canalicular vacuole (4 µM to each side of the vacuole center) using the ImageJ 1.48 software (National Institutes of Health, Bethesda, MD) with the RGB profile plot plugin. F-actin-associated fluorescence was used to delimit the canalicular space. Each line profile measurement was normalized to the sum of all intensities of the respective measurement. The distribution of transporter-associated fluorescence (green channel), expressed as a percentage of the total, was then calculated for each canaliculi and compared statistically using the Mann–Whitney test; any difference among groups thus reflects changes in localization along the 8-μm line. Analysis of confocal microscopy data was performed in a blinded manner. Results are expressed as mean ± SEM. n = 6–8 canalicular vacuoles per preparation, from three independent preparations. Statistical analysis of the profiles revealed a significant internalization of Abcc2 and Abcb11 under E17G treatment (p < 0.05 vs control), which was completely abolished by TYR (p < 0.05 vs E17G). Note that none of the treatments affected the normal distribution of actin, which showed similar profile (EPS 2079 kb)

204_2017_2098_MOESM3_ESM.eps

Additional Fig. 3. Image analysis of Abcc2 and Abcb11 in PRLs. Distributions of Abcc2 (a, left)- and actin (a, right)-associated fluorescence intensity. b. Distributions of Abcb11 (b, left)- and actin (b, right)-associated fluorescence intensity. Graphs represent the intensity of fluorescence associated with the transporters along an 8-µm line (from -4 µm to +4 µm of the canalicular center) perpendicular to the canaliculus. Occludin-associated fluorescence was used to delimit the canalicular space. The variances of the densitometric profiles of Abcb11 and Abcc2 localization were compared with the Mann–Whitney U test. In control livers, transporter-associated fluorescence was concentrated in the canalicular space. E17G-induced internalization of transporters from the canalicular membrane (P < 0.01 versus control) was detected as a decrease in the fluorescence intensity in the canalicular area together with an increased fluorescence at a greater distance from the canaliculus. Distribution profiles of livers treated with E17G+TYR were similar to control and indicated a significantly decreased of Abcb11 and Abcc2 internalization (P < 0.01 versus E17G). (n = 20-50 canaliculi per preparation, three independent preparations). Statistical analysis of the distribution profiles of occludin showed no changes in the normal distribution by any of the treatments (EPS 2384 kb)

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Barosso, I.R., Miszczuk, G.S., Ciriaci, N. et al. Activation of insulin-like growth factor 1 receptor participates downstream of GPR30 in estradiol-17β-d-glucuronide-induced cholestasis in rats. Arch Toxicol 92, 729–744 (2018). https://doi.org/10.1007/s00204-017-2098-3

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