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. 2019 Nov 29;294(48):18337-18348.
doi: 10.1074/jbc.RA119.007758. Epub 2019 Oct 25.

Intestinal breast cancer resistance protein (BCRP) requires Janus kinase 3 activity for drug efflux and barrier functions in obesity

Jayshree Mishra  1 , Randall Simonsen  2 , Narendra Kumar  3
Affiliations

Intestinal breast cancer resistance protein (BCRP) requires Janus kinase 3 activity for drug efflux and barrier functions in obesity

Jayshree Mishra et al. J Biol Chem. .

Abstract

Breast cancer resistance protein (BCRP) is a member of ATP-binding cassette (ABC) transporter proteins whose primary function is to efflux substrates bound to the plasma membrane. Impaired intestinal barrier functions play a major role in chronic low-grade inflammation (CLGI)-associated obesity, but the regulation of BCRP during obesity and its role in maintaining the intestinal barrier function during CLGI-associated obesity are unknown. In the present study, using several approaches, including efflux assays, immunoprecipitation, immunoblotting, immunohistochemistry, paracellular permeability assay, FACS, cytokine assay, and immunofluorescence microscopy, we report that obese individuals have compromised intestinal BCRP functions and that diet-induced obese mice recapitulate these outcomes. We demonstrate that the compromised BCRP functions during obesity are because of loss of Janus kinase 3 (JAK3)-mediated tyrosine phosphorylation of BCRP. Our results indicate that JAK3-mediated phosphorylation of BCRP promotes its interactions with membrane-localized β-catenin essential not only for BCRP expression and surface localization, but also for the maintenance of BCRP-mediated intestinal drug efflux and barrier functions. We observed that reduced intestinal JAK3 expression during human obesity or JAK3 knockout in mouse or siRNA-mediated β-catenin knockdown in human intestinal epithelial cells all result in significant loss of intestinal BCRP expression and compromised colonic drug efflux and barrier functions. Our results uncover a mechanism of BCRP-mediated intestinal drug efflux and barrier functions and establish a role for BCRP in preventing CLGI-associated obesity both in humans and in mice.

Keywords: ATP-binding cassette subfamily 2 Junior blood group (ABCG2); JAK3 kinase; Janus kinase (JAK); breast cancer resistant protein (BCRP); chronic low grade inflammation (CLGI); efflux assay; gut homeostasis; metabolic disease; obesity; posttranslational regulation; tyrosine phosphorylation; tyrosine-protein kinase (tyrosine kinase).

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Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article

Figures

Figure 1.
Figure 1.
Intestinal drug transport functions are compromised during obesity. A, colons from normal and obese mice were excised out, ligated in Petri dishes, injected with Hoechest 33342, and the intracellular accumulation of Hoechst 33342 measured. Contribution of BCRP and P-glycoprotein in intestinal drug efflux functions in WT mice were determined by treating with or without novobiocin or verapamil or BCRP siRNAs prior to Hoechest 33342 loading. Knockdown of BCRP expression was determined by Western blot analysis of the tissue lysates from WT mouse treated with scramble or BCRP siRNA and probed with BCRP antibody. B, paracellular permeability was determined using luminal retention of FITC-inulin probe in the ligated mice colons from WT, obese, WT treated with novobiocin or verapamil or BCRP siRNA or siBCRP-scram. C–E, colonic tissue sections (upper panels) or lysates (lower panels) from WT and obese mice were either immunostained or immunoblotted using indicated primary antibodies. Representative images are shown (n = 6) for immunostaining. Green indicates BCRP/MRP2/BCRP. Scale bar 550 μm. Colonic tissue lysates (lower panels) from the tissues of upper panels were analyzed using IB for indicated proteins using β-actin as controls. Representative blots (n = 3) are shown. Quantification of the resulting bands (lower right graphs) was done by densitometry analysis using Bio-Rad Gel Doc System equipped with ImageLab software and the results were normalized against β-actin. β-actin panels were reused in D and E. A–E, the data are representative of at least three independent experiments. Bar graphs, values are mean ± S.D. * denotes comparison with WT mice. A, obese, p = 0.0003; WT+Novo, p = 0.005; WT+siBCRP, p = 0.002. B, obese, p = 0.002; WT+Novo, p = 0.0003; WT+siBCRP, p = 0.0002. E, obese, p = 0.0004.
Figure 2.
Figure 2.
Intestinal BCRP expression is decreased in obese humans. A, immunofluorescence staining of colonic mucosa of healthy and obese human subjects were done using BCRP antibody. Representative images are shown (n = 6). Arrows indicate the differences in localization of BCRP in the luminal surfaces (white) and crypts (yellow) of normal and obese subjects. Scale bar 550 μm. B, fluorescent intensity per unit area (using normal as 100%) for BCRP images (n = 10) were calculated using NIS Element software. C, colonic tissue lysates from normal and obese male and female human subjects were analyzed using IB for BCRP proteins and β-actin controls. Representative blots (n = 3) are shown. D, quantification of the resulting bands from (C) was done using densitometry. The data are representative of at least three independent experiments. B and D, Values are mean ± S.D. * denotes p < 0.05 compared with normal subjects. B, obese, p = 0.0002; D, obese male, p = 0.0003; obese female, p = 0.006.
Figure 3.
Figure 3.
Tyrosine phosphorylation of BCRP is affected during obesity. A, total RNA was extracted using TRIzol (Invitrogen) from colons of healthy and obese subjects and WT and obese group of mice. Total RNA was reverse transcribed into cDNA using Transcriptor First Strand cDNA Synthesis Kit (Roche Biochemicals). Equal amounts of cDNA as estimated using spectrophotometer were subjected to reverse transcriptase PCR using PCR machine (Bio-Rad) and analyzed using agarose gel electrophoresis. Band intensity was measured using Bio-Rad imaging system using 18SRNA as control, and relative expression of BCRP mRNA is shown. For real-time PCR, 2 μg of total RNA was reverse transcribed into cDNA using Transcriptor First Strand cDNA Synthesis Kit (Roche Biochemicals). Equal amounts of cDNA as measured using NanoDrop were subjected to real-time PCR analysis using ABI PRISM 7600. A plot of CT value with respect to housekeeping gene 18S RNA are shown for absolute quantification of the BCRP gene expression as determined using ABI PRISM software (n = 3 experiments). B–C, co-IP followed by IB studies were done using colonic tissue lysates either from normal and obese mice (B) or humans (C) probed with the indicated antibodies using β-actin as controls. Representative blots (n = 3) are shown. Densitometry analysis of the Western images were done using ImageLab software (Bio-Rad), and ratios of average densities (n = 3 experiments) between pBCRP and BCRP are shown. * indicate statistically significant differences (B, p = 0.004; C, p = 0.002).
Figure 4.
Figure 4.
Compromised JAK3 expression is responsible for decreased BCRP phosphorylation during obesity. A and D, colonic tissue sections from normal or obese human subjects (A) were immunostained using JAK3 antibody whereas colonic tissue sections from WT or JAK3 KO mice (D) were immunostained using BCRP antibody. Representative images (n = 10) are shown from each group (n = 6). B and E, Western blot analysis was done using tissue lysates from normal and obese human (upper panel, male; lower panel, female) subjects (B) or WT and JAK3 KO mice (E) using indicated antibodies with β-actin as control. Representative blots (n = 3) are shown. C and F, quantification of the resulting bands in B and E were done by densitometry and the results were normalized against controls. Values are mean ± S.D. * denotes p < 0.05 compared with healthy human subjects (C) or WT mice (F). C, obese male, p = 0.003; obese female, p = 0.005. F, JAK3 KO, p = 0.002. G, intracellular accumulation of Hoechst 33342 was measured to determine the BCRP-mediated drug efflux function in colons from WT mice, JAK3 KO mice, WT mice treated with tofacitinib, WT mice treated with scramble and WT mice treated with BCRP siRNA (n = 6 each group). H, colonic IL-2 was determined using colonic tissue lysates from WT and KO and mouse using Multi-Analyte cytokine assay kit (Qiagen), and mean values from each group (n = 6 mice per group) are shown. All the data are representative of at least three independent experiments. Values are mean ± S.D. * denotes p < 0.05 compared with WT mice. G, JAK3 KO, p = 0.0002; WT+Tof, p = 0.0005; WT+siBCRP, p = 0.0002. H, JAK3 KO, p = 0.003.
Figure 5.
Figure 5.
JAK3 association with BCRP regulates intestinal drug transport and barrier functions. A and B, upper panels, colonic tissue sections from WT mice (A) or normal human subjects (B) were immunostained using BCRP and JAK3 primary antibodies followed by FITC- and Cy3-conjugated secondary antibodies, respectively. Representative images are shown (n = 10). A and B, lower panels, co-IP followed by IB were done using tissue lysates from WT mice (A) or normal human subjects (B) and indicated antibodies with β-actin as control. C, top panels, HT-29 Cl-19a cells grown on cover slips were immunostained using indicated antibodies. C, bottom panels, and D, HT-29 Cl-19a cells grown on Petri dishes were lysed using lysis buffer and equal (input control) amounts of JAK3 (C) or BCRP (D) proteins from the lysates of HT-29 Cl-19a cells either serum starved (−IL-2/negative control) or IL-2 treated, or IL-2 treated in the presence of JAK3 inhibitor tofacitinib were subjected to IP followed by IB using indicated antibodies. E, fluorescence-activated cell sorting (FACS) analysis is presented as dot plots to measure intracellular accumulation of Hoechst using HT-29 Cl-19a cells under similar experimental conditions as in D. Relative fluorescence intensities are shown on the x axis and the cell counts for 10,000 events on the y axis. F, experiments in (E) were repeated (n = 5) and mean ± S.D. values are shown. * denotes p = 0.004 compared with serum starved (−IL-2) cells. G, paracellular permeability of confluent monolayer of HT-29 Cl-19a cells under similar experimental conditions as in D was determined and apical inulin retentions are shown. Values are mean ± S.D. * denotes p = 0.0002 (IL-2), p = 0.006 (IL-2+Tof.) compared with serum-starved (−IL-2) cells. All the Western blots shown are representative (n = 3). The data are representative of at least three independent experiments.
Figure 6.
Figure 6.
JAK3-mediated tyrosine phosphorylation of BCRP promotes membrane localization of BCRP. A–C and E, the effects of JAK3 activation on co-localization of BCRP and β-catenin was determined using confluent HT-29 Cl-19a cells and indicated primary antibodies under serum starved (control) (A), or IL-2 treated (B), or IL-2 treated in the presence of JAK3-inhibitor tofacitinib (C), or IL-2 treated in presence or absence of β-catenin-shRNA (E) conditions. Representative images (n = 10) are shown. Scale bar 14 μm. D(i), co-IP followed by IB studies were done to determine BCRP interactions with β-catenin using equal amounts of BCRP (input control) from IL-2–treated HT-29 Cl-19a cell lysates and indicated antibodies. Representative blots (n = 3) are shown. D(ii), equal amounts (panels below the black bar) of recombinant and purified proteins of BCRP and JAK3 were subjected to in vitro kinase assay in presence or absence of ATP (U. S. patent 9739779B2) followed by incubation with equal amounts recombinant and purified His-tagged β-catenin proteins. The reaction mixture were subjected to co-IP followed by IB with indicated antibodies (panels above the black bar). Representative blots (n = 3 experiments) are shown. F, intracellular accumulation of Hoechst was determined using HT-29 Cl-19a cells either serum starved (−IL-2) control or IL-2 treated or IL-2 treated in the presence of JAK3 inhibitor tofacitinib or IL-2 in presence of β-catenin-shRNA (n = 6 each groups). Values are mean ± S.D. * denotes p = 0.0005 compared with serum-starved (−IL-2) cells. The data are representative of at least three independent experiments.
Figure 7.
Figure 7.
BCRP interactions with B-catenin are compromised during human obesity. A and B), co-IP followed by IB studies were done to determine BCRP interactions with β-catenin in human colons using tissue lysates from healthy (A) and obese (B) subjects and indicated antibodies with β-catenin as controls. Representative blots (n = 3 blots/subject) are shown. C, co-immunofluorescence staining of colonic mucosa of healthy and obese human subjects are shown using indicated antibodies. Representative images (n = 10) are shown from each group. Scale bar 100 μm. The data are representative of at least three independent experiments.
Figure 8.
Figure 8.
Proposed models for BCRP phosphorylation mediated mucosal barrier function and predisposition to obesity.
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