Volume 139, Issue 4, Pages 1385-1396.e8 (October 2010) FGF Receptors 1 and 2 Control Chemically Induced Injury and Compound Detoxification in Regenerating Livers of Mice  Friederike Böhm, Tobias Speicher, Claus Hellerbrand, Clive Dickson, Juha M. Partanen, David M. Ornitz, Sabine Werner  Gastroenterology  Volume 139, Issue 4, Pages 1385-1396.e8 (October 2010) DOI: 10.1053/j.gastro.2010.06.069 Copyright © 2010 AGA Institute Terms and Conditions

Figure 1 Verification of the loss of FGFR1 and FGFR2 in hepatocytes. (A) RNAs from total liver or primary hepatocytes of Alb-R1/R2 (ko) and control mice (ctrl) were analyzed for Fgfr or Gapdh expression with the use of qRT-PCR. Expression levels in control mice were arbitrarily set as 1. RNA from mouse epidermis was used as a positive control for fgfr3 expression. *P < .05; ns = not significant. (B) Lysates from total liver of Alb-R2-IIIb and control mice were analyzed by Western blotting for expression of FGFR2. Differently glycosylated forms of FGFR2 are indicated. (C) Sections from the liver of Alb-R2-IIIb and control mice were analyzed by immunohistochemistry for expression of FGFR2. (D) Primary hepatocytes from control and Alb-R1/R2 mice were serum-starved, treated with 10 ng/mL FGF7, harvested at the indicated time points, and analyzed by Western blotting for phosphorylated fibroblast growth factor receptor substrate 2 α (FRS2α) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Cells incubated for 60 minutes in the absence of FGF7 were used as control (60c). Gastroenterology 2010 139, 1385-1396.e8DOI: (10.1053/j.gastro.2010.06.069) Copyright © 2010 AGA Institute Terms and Conditions

Figure 2 Loss of FGFR1 and FGFR2 in hepatocytes protects from CCl4-induced inflammation. (A–E) Mice were injected once with CCl4 in mineral oil and killed at different time points. (A) Representative H&E-stained sections are shown. Necrotic area can be distinguished from normal liver tissue by the brighter color and the inflammatory cell infiltrates (encircled areas). It was determined by measuring 4–5 independent microscopic fields (n ≥ 5 per genotype) and is indicated as the percentage of total liver area. Bar, 100 μm. (B) AST and ALT levels were determined in the serum 48 hours after CCl4 injection (n ≥5 per genotype). (C) Liver sections from BrdU-injected animals were stained with an antibody against BrdU. Representative sections from Alb-R1/R2 mice and controls 36 hours after CCl4 injection are shown. The percentage of BrdU-positive cells was determined by counting 4–5 independent microscopic fields (n ≥5 mice per time point). Bar, 50 μm. (D) Liver sections were stained with antibodies against neutrophils (Ly6G) or macrophages (ER-MP23). Representative pictures are shown. Inflammatory cells were counted in 4–5 independent microscopic fields (200× magnification; n ≥5 per genotype). Bar, 50 μm. (E) RNAs from the liver of control and Alb-R1/R2 mice at different time points after CCl4 injection were analyzed by qRT-PCR for the levels of IL-1β, TNF-α, S100A8, S100A9, MIP-1α, or MIP-1β mRNAs. Rps29 was used for normalization. *P < .05; **P < .01; ***P < .001. Gastroenterology 2010 139, 1385-1396.e8DOI: (10.1053/j.gastro.2010.06.069) Copyright © 2010 AGA Institute Terms and Conditions

Figure 3 FGFR1 and FGFR2 cooperate to enhance survival of mice and to prevent hepatocyte necrosis after PH. Mice were subjected to PH. (A) The percentage of surviving animals at different time points after PH is indicated. Numbers above the bars indicate the number of animals observed at each time point per genotype. (B) AST and ALT levels in the serum were determined 6 hours after PH (n ≥5 per genotype). Macroscopic (C) and histologic analysis (D) showed the presence of necrotic lesions in the liver of Alb-R1/R2 mice 24 hours after PH (n ≥12 per genotype). Bar, 50 μm. **P < .01. Gastroenterology 2010 139, 1385-1396.e8DOI: (10.1053/j.gastro.2010.06.069) Copyright © 2010 AGA Institute Terms and Conditions

Figure 4 Delayed activation of major signaling pathways and enhanced proliferation of hepatocytes after PH in surviving Alb-R1/R2 mice. (A) Cell proliferation in the liver at 48 hours after PH was assessed by BrdU incorporation. Representative sections from injured liver (48 hours after PH) are shown. Bar, 50 μm. (B) The percentage of proliferating cells was determined by counting 4–5 independent microscopic fields/per liver at 200× magnification, (n >6 per genotype and time point). *P < .05. (C) Liver lysates from control and Alb-R1/R2 mice before and at different time points after PH were analyzed by Western blotting for the levels of total and phosphorylated ERK1/2, p38, Akt, c-Jun-N-terminal kinases 1 and 2 (JNK1/2), signal transducer and activator of transcription 3 (STAT3), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Each lysate was obtained from pooled livers of 3 animals per time point and genotype. All results shown on this figure were obtained with the same lysates. Results were reproduced in an independent experiment with the use of lysates from different animals. Gastroenterology 2010 139, 1385-1396.e8DOI: (10.1053/j.gastro.2010.06.069) Copyright © 2010 AGA Institute Terms and Conditions

Figure 5 FGFR1 and FGFR2 signaling is important for compound detoxification in the regenerating liver. (A) Mice were subjected to PH. One group was anesthetized with ketamine/xylazine followed by buprenorphine treatment (K/X/B), in the second group ketamine/xylazine was replaced by isoflurane (Iso/B), and in the third group buprenorphine was omitted after ketamine/xylazine anesthesia (K/X). The percentage of surviving animals at different time points after PH is indicated. Numbers above the bars indicate the number of animals observed at each time point per genotype and treatment group. (B) Cell proliferation was assessed in mice of the Iso/B group 48 hours after PH with the use of BrdU incorporation (n >6 per genotype and treatment group). (C) Expression of Dbp, Tef, Cyp2a5, and Cyp2c38 was analyzed by qRT-PCR with RNAs from livers of untreated Alb-R1/R2 mice and control animals. RNAs from the liver tissue, which was removed on PH (0 hours PHx), or from the remaining liver 24 hours after PH were analyzed for comparison. **P < .01; ***P < .001. (D) Levels of Dbp and Tef mRNAs were analyzed by qRT-PCR in noninjured Alb-R1/R2 and control mice at different day times. Rps29 was used for normalization. Gastroenterology 2010 139, 1385-1396.e8DOI: (10.1053/j.gastro.2010.06.069) Copyright © 2010 AGA Institute Terms and Conditions

Figure 6 FGF7 activates a cytoprotective response in the liver. (A) Liver lysates were prepared from control and Alb-R1/R2 mice 10 and 30 minutes after injection of saline or 5 μg FGF7 and analyzed by Western blotting for the levels of phosphorylated FRS2α and total and phosphorylated ERK1/2, p38, Akt, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Each lysate was obtained from a single mouse. For statistical analysis see Supplementary Figure S6. (B) Control mice were intraperitoneally injected with 0.5 or 5 μg of FGF7 or vehicle and killed 10 minutes after injection, and RNAs from the liver were analyzed by qRT-PCR for expression of Dbp and Tef. (C and D) Alb-R1/R2 mice and control littermates were injected intraperitoneally with saline or 5 μg of FGF7. They were killed 10 or 30 minutes after injection, and RNAs from the liver were analyzed by qRT-PCR for expression of Dbp and Tef (C) or Cyp2a5 and Cyp2c38 (D). Rps29 was used for normalization. *P < .05. Gastroenterology 2010 139, 1385-1396.e8DOI: (10.1053/j.gastro.2010.06.069) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 1 FGFR1 and FGFR2 on hepatocytes are dispensable for liver homeostasis. (A) Primary hepatocytes from control and Alb-R1/R2 mice were serum-starved, treated with 10 ng/mL epidermal growth factor (EGF), harvested at the indicated time points and analyzed by Western blotting for total and phosphorylated Erk1/2. Cells incubated for 60 min in the absence of EGF were used as control (60c). (B) Paraffin sections from non-injured liver of control and Alb-R1/R2 mice (11 weeks of age) were stained with H&E. Representative sections are shown. Bar represents 50 μm. (C) Serum analysis of control and Alb-R1/R2 was performed according to standard methods. Concentrations of albumin, alkaline phosphatase, AST, and ALT were analyzed in at least 3 mice per genotype. Error bars indicate mean ± SEM. ns = not significant. Gastroenterology 2010 139, 1385-1396.e8DOI: (10.1053/j.gastro.2010.06.069) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 2 Loss of FGFR1 and FGFR2 in hepatocytes does not affect fibrosis, inflammation, liver weight, cell proliferation, and serum transaminases after prolonged CCl4 treatment. Mice were subjected to long-term CCl4 treatment as described in Materials and Methods. (A) Liver sections were stained with sirius red; fibrotic areas appear red. The fibrotic area was determined in 4–5 independent sections (200x magnification; n = 12 per genotype). Bar, 100 μm. (B and C) Liver sections from these animals were stained with antibodies against Ly6G and ER-MP23. Representative sections stained with ER-MP23 are shown in panel B. Bar, 50 μm. (C) The number of Ly6G+ and ER-MP23+ cells was counted in 4–5 independent microscopic fields (n ≥ 6). (D) The liver weight-to-body weight ratio was determined in 12 control mice and 12 Alb-R1/R2 mice after long-term CCl4 treatment. (E) Cell proliferation in the liver of Alb-R1/R2 mice and control mice after long-term CCl4 treatment was assessed by BrdU incorporation. The percentage of proliferating cells was determined by counting 4–5 independent microscopic fields/per liver at 200x magnification (n > 6 per genotype and time point). Error bars indicate mean ± SEM. (F) Serum levels of ALT and AST are shown (n > 6 per genotype for CCl4; n = 3 for oil controls). Error bars represent mean ± SEM. Gastroenterology 2010 139, 1385-1396.e8DOI: (10.1053/j.gastro.2010.06.069) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 3 Expression of FGFs and other growth factors after PH in Alb-R1/R2 mice and control animals. (A) RNA was isolated from the liver and spleen of wild-type animals before and at different time points after PH as indicated. Samples of 20 μg of RNA were analyzed by RNase protection assay for the expression of different growth factors and of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). tRNA (20 μg) was used as a negative control. (B) Expression of different growth factors in the liver of Alb-R1/R2 and control mice before and after PH is indicated in a table. The +++ indicates strong expression; ++, intermediate expression; +, low expression. Gastroenterology 2010 139, 1385-1396.e8DOI: (10.1053/j.gastro.2010.06.069) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 4 Liver cell proliferation in single FGFR knockout mice after PH. Cell proliferation in the liver of Alb-R1 (A), Alb-R2-IIIb (B) mice, and control littermates at different time points after PH was assessed by BrdU incorporation. The percentage of proliferating cells was determined by counting 4–5 independent microscopic fields/per liver at 200 × magnification (n >6 per genotype and time point). Error bars represent mean ± SEM. *P < .05; ns = not significant. Gastroenterology 2010 139, 1385-1396.e8DOI: (10.1053/j.gastro.2010.06.069) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 5 Regulation of Dbp, Tef, and their Cyp targets in response to ketamine/xylazine, isoflurane, or liver surgery. (A) RNAs from liver of untreated Alb-R1/R2 mice and control animals as well as from the liver tissue, which was removed on PH (0 hour PH), were analyzed for expression of Dbp and Tef by qRT-PCR. Anesthesia was performed by isoflurane (Iso) inhalation as indicated. (B) Mice were anesthetized with ketamine/xylazine (K/X) or isoflurane (Iso) as indicated. They were killed before or 10 minutes after anesthesia. RNAs from the liver were analyzed for expression of Dbp, Tef, Cyp2a5, and Cyp2c38 by qRT-PCR. Rps29 was used for normalization. *P < .05; **P < .01; ns = not significant. Gastroenterology 2010 139, 1385-1396.e8DOI: (10.1053/j.gastro.2010.06.069) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 6 FGF7-induced activation of major signaling pathways in the liver of control and Alb-R1/R2 mice. Liver lysates were prepared from control and Alb-R1/R2 mice 10 and 30 minutes after injection of saline or 5 μg of FGF7 and analyzed by Western blotting for the levels of phosphorylated FRS2α and total and phosphorylated ERK1/2, p38, and Akt. Each lysate was obtained from a single mouse. Band intensities were determined and normalized to the intensity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or total levels of the corresponding signaling protein as indicated on the y-axis. Expression levels in the liver of saline-injected control animals were arbitrarily set as 1. Gastroenterology 2010 139, 1385-1396.e8DOI: (10.1053/j.gastro.2010.06.069) Copyright © 2010 AGA Institute Terms and Conditions