Epidermal growth factor signaling protects from cholestatic liver injury and fibrosis
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We have demonstrated that the signal transducer and activator of transcription 3 (STAT3) protects from cholestatic liver injury. Specific ablation of STAT3 in hepatocytes and cholangiocytes (STAT3∆hc) aggravated liver damage and fibrosis in the Mdr2−/− (multidrug resistance 2) mouse model for cholestatic disease. Upregulation of bile acid biosynthesis genes and downregulation of epidermal growth factor receptor (EGFR) expression were observed in STAT3∆hc Mdr2−/− mice but the functional consequences of these processes in cholestatic liver injury remained unclear. Here, we show normal canalicular architecture and bile flow but increased amounts of bile acids in the bile of STAT3∆hc Mdr2−/− mice. Moreover, STAT3-deficient hepatocytes displayed increased sensitivity to bile acid-induced apoptosis in vitro. Since EGFR signaling has been reported to protect hepatocytes from bile acid-induced apoptosis, we generated mice with hepatocyte/cholangiocyte-specific ablation of EGFR (EGFR∆hc) and crossed them to Mdr2−/− mice. Importantly, deletion of EGFR phenocopied deletion of STAT3 and led to aggravated liver damage, liver fibrosis, and hyperproliferation of K19+ cholangiocytes. Our data demonstrate hepatoprotective functions of the STAT3-EGFR signaling axis in cholestatic liver disease.
STAT3 is a negative regulator of bile acid biosynthesis.
STAT3 protects from bile acid-induced apoptosis and regulates EGFR expression.
EGFR signaling protects from cholestatic liver injury and fibrosis.
KeywordsEpidermal growth factor receptor EGFR Signal transducer and activator of transcription 3 STAT3 Cholestasis Bile acids Liver injury Hepatocyte apoptosis
Chronic cholestatic liver diseases are characterized by retention of bile acids in the liver which results in alterations of hepatobiliary bile acid transport and enzyme activities participating in bile acid biosynthesis. Hydrophobic bile acids are particularly toxic and promote cholangiocyte/hepatocyte damage, liver fibrosis, cirrhosis, and formation of hepatocellular carcinoma (HCC) under cholestatic conditions [1, 2, 3]. Deposition of collagen and other extracellular matrix components is an orchestrated event in cholestatic liver fibrosis and involves several cell types including Kupffer cells and stellate cells. The latter are activated by inflammatory cytokines and responsible for deposition of collagen together with portal myofibroblasts . Despite profound knowledge about cell types that promote cholestatic liver fibrosis and cirrhosis, little is known about hepatoprotective factors that modulate initial events of cholestatic liver injury. The transcription factor STAT3 is required for liver regeneration and hepatoprotection in various chronic liver diseases . STAT3 is mainly activated by IL-6 (interleukin 6) and IL-22 in hepatocytes. These cytokines bind to gp130 (glycoprotein 130) receptors and promote phosphorylation of STAT3 at tyrosine-705 (pY-STAT3) via Janus kinases (JAKs), STAT3 dimerization, and nuclear translocation .
Hepatoprotective functions of STAT3 signaling in cholestatic liver disease have been investigated in mice lacking STAT3, IL-6, gp130, or express pathway specific gp130 mutants. Bile duct ligation, cholic acid feeding, and genetic deletion of the Mdr2 gene (Mdr2−/−) resulted in aggravated cholestatic liver injury and fibrosis in hepatocyte-specific STAT3 knock-out mice and IL-6−/− mice [5, 6]. Moreover, mice with hepatocyte-specific deletion of gp130 (gp130hepa) displayed aggravated liver fibrosis and collagen deposition after DDC (3,5-diethoxycarbonyl-1,4-dihydrocollidine) feeding which is a chemical model for sclerosing cholangitis . The use of specific gp130 knock-in mutant alleles that lack either the region for STAT3 activation (gp130∆hepaSTAT) or carry a Y757F mutation that impedes activation of the Ras/MAPK pathway (gp130∆hepaRas)  demonstrated that the hepatoprotective activity of gp130 signaling was due to gp130-mediated STAT3 activation.
Several cellular and molecular mechanisms might account for the hepatoprotective activity of IL-6/gp130/STAT3 signaling in cholestatic liver injury . We demonstrated that enzymes for bile acid biosynthesis are upregulated in livers of Mdr2−/− mice, lacking STAT3 in hepatocytes and cholangiocytes (STAT3∆hc) , which might result in increased production of toxic bile and aggravated cholestatic liver damage. Moreover, expression of epidermal growth factor receptor (EGFR) was downregulated in livers of STAT3∆hc Mdr2−/− mice. EGFR signaling protects hepatocytes from bile acid-induced apoptosis which was demonstrated in vitro with hepatocytes harboring a dominant negative ERBB1 allele. A similar protective effect was observed after pretreatment of hepatocytes with the EGFR antagonist Iressa [8, 9, 10, 11].
Here, we employed Mdr2−/− mice to functionally test the hepatoprotective function of EGFR signaling in bile acid-induced liver injury and fibrosis. Importantly, conditional inactivation of EGFR in hepatocytes and cholangiocytes (EGFR∆hc) of Mdr2−/− mice led to severe jaundice and strongly aggravated liver damage and fibrosis. These data suggest a pivotal hepatoprotective function for EGFR signaling in cholestatic liver disease.
STAT3∆hc mice were generated by crossing mice carrying floxed alleles of STAT3  to AlfpCre transgenic mice . Furthermore, mice harboring floxed alleles of EGFR  were crossed to AlfpCre transgenic mice. Resulting AlfpCre EGFRflox/flox (EGFR∆hc) mice were bred with Mdr2−/− mice  to generate EGFR∆hc Mdr2−/− mice. Blood sera, bile, and liver tissue of a 7 week old male mouse were used for analyses. All mouse experiments were performed in accordance with Austrian and European laws and with the general regulations specified by the Good Science Practice guidelines of the Medical University of Vienna.
Histology and immunohistochemistry
Bile canaliculi, sinusoids, and central veins were visualized by immunofluorescence using a combination of antibodies directed against DPPIV/CD26, glutamine synthetase (GS), and secondary fluorochrome coupled antibodies as described previously . For other stainings, livers were formalin-fixed and paraffin-embedded. Hematoxylin/eosin and Sirius-red stainings were performed according to standard procedures. Immunohistochemical stainings were performed with antibodies for CD3 (Histocom, RM-9107), F4/80 (eBioscience, 14–4801-82), Ki67 (Novocastra, NCL-Ki67p), and K19 (monoclonal rat anti-Troma-III antibody provided by Rolf Kemler). For detection of apoptotic cells, ApopTag® Peroxidase in Situ Apoptosis Detection Kit (Millipore, S7100) was used.
Cell culture, MTT assay, and caspase 3/8 activity assay
Primary hepatocytes were isolated by collagenase liver perfusion using perfusion buffer (17,701–038, Applied Biosystems) and subsequent digestion buffer (17,703–034, Applied Biosystems). Cells were seeded in collagen coated wells and kept in Williams E medium (12,551–032, Applied Biosystems) overnight. On the following day, primary hepatocytes were treated with different concentrations of bile acids. Immortalized hepatocytes were cultured in RPMI supplemented with 80 ng/ml TGF-alpha (T7924, Sigma), 60 ng/ml IGF-II (I2526, Sigma), and 2.8 μM insulin (Novo Nordisk). Before stimulation with IL-6 (40 ng/ml, ImmunoTools, 12,340,065), immortalized hepatocytes were cultured over night without growth factors and treated with IL-6 for 2 h and 4 h. For viability testing, cells were incubated for 3 h at 37 °C with 1 mg/ml MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). The plates were dried and the crystals were dissolved. Absorbance was measured at 550 nm to assess cell viability. For detection of active caspases, cells were lysed and incubated with fluorescent substrates specific for caspase 3 (Ac-DEVD-AFC, ALX-260-032, Enzo Life Sciences) or caspase 8 (Ac-LETD-AFC, ALX-260-118, Enzo Life Sciences) for 1 h at 37 °C. Fluorescence was measured at an excitation wavelength of 400 nm and an emission wavelength of 505 nm.
PCR for genotyping
Genotyping was performed by PCR with primers 5′-gcgtctgactctacaacc-3′, 5′-agcctcatccttaggtact-3′ and 5′-gactgtgataaccttcagtg-3′ for floxed STAT3, 5′-aagtttaagaaaccccgctctact-3′, 5′-gcctgtgtccgggtctcgtcg-3′ and 5′-caaccagtgcacctagcctggc-3′ for floxed EGFR, 5′-cggtcgatgcaacgagtgatgagg-3′ and 5′-ccagagacggaaat-3′ for AlfpCre, 5′-cgagccgaggatgcataagt-3′, 5′-gataagcctgctcgatgcct-3′, 5′-tgtcaagaccgacctgtccg-3′ and 5′-tattcggcaagcaggcatcg-3′ for Mdr2.
Total RNA was isolated with TRIzol (Life Technologies, 15596-018) and reverse transcribed with QuantiTect Reverse Transcription Kit (Qiagen, 205313). qPCR was performed using Fast SYBR Green Mastermix (Thermo Fisher Scientific, 4385616) and an Applied Biosystems 7500 Fast Real Time PCR System with primers 5′-caccctcaagagcctgagtc-3′ and 5′-gttcgggctgatgtaccagt-3′ for COL1, 5′-caggtgaacccggcaagaacg-3′ and 5′-ggggaccagggcgaccact-3′ for COL3, 5′-tcctcttgttgctatcactgatagctt-3′ and 5′-cgctggtataaggtggtctcgtt-3′ for TIMP1, 5′-ccagaagaagagcctgaacca-3′ and 5′-gtccatccagaggcactcatc-3′ for TIMP2, 5′-agaggtcacccgcgtgctaa-3′ and 5′-tcccgaatgtctgacgtattga-3′ for TGFB1, 5′-tcgtccgctttgatgtctca-3′ and 5′-aaatctcgcctcgagctcttc-3′ for TGFB2, 5′-ccgaggactatgaccgggataa-3′ and 5′-cttgttgcccaggaaagtgaag-3′ for MMP2, 5′-atcccaccaaagtgagaacg-3′ and 5′-taatttccctccccggttac-3′ for CTGF, 5′-aatcccaggaccaactatggcagc-3′ and 5′-gaggcaaacttctgttccaatgg-3′ for EGFR, 5′-tgtttgtgatgggtgtg-3′ and 5′-tacttggcaggtttctc-3′ for GAPDH. The expression levels of transcripts were calculated with the comparative CT (threshold concentration) method. The individual RNA levels were normalized for GAPDH and are depicted as relative expression levels.
Serum levels of bilirubin, alanine aminotransferase (ALT), and alkaline phosphatase (ALP) were measured using the Reflotron® System (Roche Applied Science).
Bile flow measurement and bile composition
To measure bile flow, mice were anesthetised and kept on a heating plate during the experiment. The common bile duct was ligated using a string. The gall bladder was punctuated and a cannula was inserted and fixed. The bile was collected in a tube for 30 min, and afterwards the liver weight was measured. Bile acids in the bile were analyzed by isotope-dilution gas chromatography-mass spectrometry (GCMS) as described previously .
Livers were homogenized and hepatic hydroxyproline levels were measured as described previously .
Protein lysates were obtained according to standard procedures and analyzed by Western blot with antibodies for P-STAT3 (Cell Signaling, 9145) and β-actin (Sigma, A5316).
Significant differences were calculated with GraphPad Prism 5. Comparisons of the two groups were analyzed with unpaired t test or Mann-Whitney test. For more than two groups One-Way Analysis of Variance (ANOVA) and Bonferroni post test or Kruskal Wallis, and Dunns post test were used. Significant differences between experimental groups are stated as: *p < 0.05, **p < 0.01, or ***p < 0.001.
STAT3 inhibits bile acid production
STAT3 protects hepatocytes from bile acid-induced apoptosis
STAT3 protects from cholestatic liver injury via regulation of EGFR
STAT3 protects from cholestatic liver fibrosis via regulation of EGFR
Hepatic fibrosis is due to chronic liver injury and partially reversible which puts hepatoprotective factors for anti-fibrotic therapies into the limelight. Genetically modified mouse models for liver fibrosis [24, 25] have unraveled effector molecules such as TGF-ß (transforming growth factor beta) , PDGF-B  (platelet derived growth factor b), PDGF-C (platelet derived growth factor c) , or TIMP-1 (tissue inhibitor of metalloproteinase 1) , but hepatoprotective factors are not well characterized. We have recently shown that the cytokine IL-6 and the cytokine-inducible transcription factor STAT3 protect from cholestatic liver injury and fibrosis in the Mdr2−/− mouse model for cholestatic liver disease . Both, IL-6−/− Mdr2−/− and STAT3∆hc Mdr2−/− mice showed aggravated liver damage and deposition of collagen in the periportal areas. Gene expression profiling demonstrated that genes for bile acid biosynthesis enzymes were upregulated whereas, EGFR was downregulated in STAT3∆hc and STAT3∆hc Mdr2−/− mice. The mode how STAT3 represses bile acid biosynthesis genes and the implication of known regulators such as FXR-α (farnesoid X receptor alpha) or HNF-4 (hepatocyte nuclear factor 4) [30, 31] has to be determined. We show that blunted repression of bile acid biosynthesis genes at the mRNA levels is reflected by a more than two-fold increase of total bile acid concentrations in the bile of STAT3∆hc mice. Mdr2−/− mice lack a phospholipid pump in the canalicular membrane which prevents formation of mixed micelles between bile acids and phospholipids. The free bile acids are cytotoxic and damage cholangiocytes leading to mild cholestasis and periportal fibrosis . Therefore, elevated bile acid concentrations, as observed in STAT3∆hc mice, are particularly harmful in an Mdr2-deficient genetic background which contributes to aggravated liver damage and fibrosis in STAT3∆hc Mdr2−/− mice.
Aggravated liver damage and formation of bile infarcts have been observed in cholic acid-treated STAT3∆hc mice but hepatocyte-intrinsic effects of STAT3 on cell survival have not been addressed. Here, we show that primary and immortalized hepatocytes, derived from STAT3∆hc mice, were more sensitive to bile acid-induced apoptosis than control hepatocytes. Reduced expression of EGFR, as observed in hepatocytes of STAT3∆hc and STAT3∆hc Mdr2−/− mice, was maintained in immortalized STAT3-deficient hepatocytes. It has been shown that expression of a dominant negative ERBB1 sensitized hepatocytes to bile acid-induced apoptosis. A similar effect was observed after pre-treatment of hepatocytes with the EGFR antagonist Iressa [8, 9, 10, 11]. Therefore, our results suggest that STAT3 prevents bile acid-induced apoptosis via positive regulation of EGFR expression in a hepatocyte-intrinsic manner.
The functional consequence of EGFR signaling in cholestatic liver disease was investigated in Mdr2−/− mice lacking EGFR expression in hepatocytes and cholangiocytes. Importantly, EGFR∆hc Mdr2−/− mice phenocopied aggravated liver damage and fibrosis of STAT3∆hc Mdr2−/− mice albeit liver damage and deposition of collagen was less severe. This was reflected by the extended survival of EGFR∆hc Mdr2−/− mice when compared to STAT3∆hc Mdr2−/− mice which died prematurely due to severe jaundice . STAT3 regulates additional hepatoprotective factors such as IGF-1 (insulin like-growth factor 1)  which might explain more severe cholestatic liver damage in STAT3∆hc Mdr2−/− mice. Compensatory hepatocyte proliferation and apoptosis were not increased in EGFR∆hc Mdr2−/− mice. Hepatocyte proliferation might be limited by ablation of EGFR which is implicated in liver regeneration after partial hepatectomy . Moreover, bile acid-induced liver damage in Mdr2−/− mice might be rather due to a necrotic mechanism than apoptosis . Our study suggests that activation of EGFR signaling might represent a therapeutic strategy to interfere with cholestatic liver injury and fibrosis. It also emphasizes the need to monitor adverse hepatic effects of EGFR inhibitors in cancer patients that simultaneously suffer from cholestatic liver disease.
This work was supported by the Ludwig Boltzmann Gesellschaft (LBG); the Austrian Science Fund (FWF) Doktoratskolleg-plus grant “Inflammation and Immunity;” the FWF grants P25925-B20, P26908-B20, P25599-B19, and P29222-B28; and the Comprehensive Cancer Center (CCC) Vienna Research Grant. Open access funding provided by Medical University of Vienna.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
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