Inducible Nitric Oxide Synthase Up-Regulates Notch-1 in Mouse Cholangiocytes: Implications for Carcinogenesis  Norihisa Ishimura, Steven F. Bronk, Gregory J. Gores  Gastroenterology  Volume 128, Issue 5, Pages 1354-1368 (May 2005) DOI: 10.1053/j.gastro.2005.01.055 Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 1 Notch-1 expression is up-regulated in PSC and CCA. (A) Immunohistochemical detection of Notch-1 in normal liver tissue, PSC, and CCA (200×). Weak expression was observed in normal ductal epithelium. In contrast, up-regulated expression of Notch-1 was observed frequently in PSC and CCA. (B) Staining intensity ratio of Notch-1. High-level staining intensity ratio of Notch-1 was calculated by the number of high-level stained cases (defined as a score of 2 on a scale of 0–2) relative to the number of total cases. High-level expression of Notch-1 was observed frequently in PSC and CCA. *P < .01, **P < .001 as compared with normal liver. (C) Immunoblot analysis of Notch-1 in human cholangiocarcinoma cell lines. Strong NICD expression was detected in all 3 human cholangiocarcinoma cell lines. Gastroenterology 2005 128, 1354-1368DOI: (10.1053/j.gastro.2005.01.055) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 2 Notch-1 expression in cholangiocytes is iNOS dependent. (A) Immunoreactive colocalization of Notch-1 and iNOS in large bile ducts obtained from the hilar region of an explanted PSC liver (100×; insets 40×). (B) Immunoblot analysis of Notch-1 in 603B and 603B-iNOS AS cells. Notch-1 protein was expressed constitutively by the 603B cells, but was expressed only minimally by the 603B-iNOS AS cells. fl, full-length Notch-1. *Nonspecific band. (C) Notch-1 mRNA expression in 603B and 603B-iNOS AS cells. Notch-1 mRNA was quantitated by real-time PCR. The expression was normalized as a ratio using 18S as a housekeeping gene. A value of 1 for this ratio was assigned arbitrarily to the data obtained from 603B. Notch-1 mRNA expression was 17-fold greater in 603B vs 603B-iNOS AS cells (*P < .01, as compared with 603B activity, n = 3 for each group). (D) Representative photomicrographs of Notch-1 immunoreactivity. basal, cells before treatment; jagged-1, cells after treatment with recombinant Jagged-1 (1 μg/mL); jagged-1 + dapt, cells after treatment with DAPT (5 μmol/L) and recombinant Jagged-1 (1 μg/mL). (E) Hes-1 mRNA expression in 603B and 603B-iNOS AS cells. Hes-1 mRNA was quantitated by real-time PCR as described earlier. Hes-1 mRNA expression was 4-fold greater in 603B vs 603B-iNOS AS cells. The expression in 603B cells was blocked with the same level as in 603B-iNOS AS cells by the γ-secretase inhibitor, DAPT (5 μmol/L). *P < .01, as compared with 603B activity, n = 3, for each group. Gastroenterology 2005 128, 1354-1368DOI: (10.1053/j.gastro.2005.01.055) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 2 Notch-1 expression in cholangiocytes is iNOS dependent. (A) Immunoreactive colocalization of Notch-1 and iNOS in large bile ducts obtained from the hilar region of an explanted PSC liver (100×; insets 40×). (B) Immunoblot analysis of Notch-1 in 603B and 603B-iNOS AS cells. Notch-1 protein was expressed constitutively by the 603B cells, but was expressed only minimally by the 603B-iNOS AS cells. fl, full-length Notch-1. *Nonspecific band. (C) Notch-1 mRNA expression in 603B and 603B-iNOS AS cells. Notch-1 mRNA was quantitated by real-time PCR. The expression was normalized as a ratio using 18S as a housekeeping gene. A value of 1 for this ratio was assigned arbitrarily to the data obtained from 603B. Notch-1 mRNA expression was 17-fold greater in 603B vs 603B-iNOS AS cells (*P < .01, as compared with 603B activity, n = 3 for each group). (D) Representative photomicrographs of Notch-1 immunoreactivity. basal, cells before treatment; jagged-1, cells after treatment with recombinant Jagged-1 (1 μg/mL); jagged-1 + dapt, cells after treatment with DAPT (5 μmol/L) and recombinant Jagged-1 (1 μg/mL). (E) Hes-1 mRNA expression in 603B and 603B-iNOS AS cells. Hes-1 mRNA was quantitated by real-time PCR as described earlier. Hes-1 mRNA expression was 4-fold greater in 603B vs 603B-iNOS AS cells. The expression in 603B cells was blocked with the same level as in 603B-iNOS AS cells by the γ-secretase inhibitor, DAPT (5 μmol/L). *P < .01, as compared with 603B activity, n = 3, for each group. Gastroenterology 2005 128, 1354-1368DOI: (10.1053/j.gastro.2005.01.055) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 3 NO donor induces Notch-1 expression in 603B-iNOS AS cells. (A) NO donor induces Notch-1 protein in 603B-iNOS AS cells. NICD expression was increased greatly by GSNO (500 μmol/L) in the 603B-iNOS AS cells in a time-dependent manner. Likewise, cleaved Notch-1 (c-notch-1) was increased at 24 hours after GSNO treatment. This effect was inhibited by DAPT (5 μmol/L). *Nonspecific band. (B) NO donor induces Notch-1 mRNA expression in 603B-iNOS AS cells. Notch-1 mRNA was quantitated by real-time PCR. The expression was normalized as a ratio using 18S as a housekeeping gene. A value of 1 for this ratio was assigned arbitrarily to the data obtained from nontreated 603B-iNOS AS cells. Data were expressed as mean ± SD. Notch-1 mRNA was increased significantly by GSNO (500 μmol/L) in a time-dependent manner. *P < .05, **P < .01, n = 3 for each group. (C) GSNO induces both expression and nuclear translocation of NICD in 603B-iNOS AS cells. 603B-iNOS AS cells were immunostained with Notch-1 antibody before and after GSNO treatment and examined by fluorescent microscopy. Not only the expression but also the nuclear translocation of Notch-1 was found in GSNO-treated 603B-iNOS AS cells. Nuclear translocation of Notch-1 was inhibited by DAPT (5 μmol/L). Gastroenterology 2005 128, 1354-1368DOI: (10.1053/j.gastro.2005.01.055) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 4 iNOS mediates Notch-1 expression by JNK1/2 signaling pathways. (A) Effect of signaling pathway inhibitors on Notch-1 protein expression in 603B cells. 603B cells were incubated for 8 hours with different inhibitors (SB203580 [20 μmol/L], SP600125 [20 μmol/L], PD98059 [20 μmol/L]). After incubation, cells were lysed, and immunoblot analysis for Notch-1 was performed. Inhibition of JNK1/2 blocked induction of Notch-1. *Nonspecific band. (B) Effect of inhibition of signaling pathways on Notch-1 mRNA expression in 603B cells. Notch-1 mRNA was quantitated by real-time PCR. Notch-1 mRNA was suppressed significantly by SP600125 (20 μmol/L). *P < .01, n = 3 for each group. Gastroenterology 2005 128, 1354-1368DOI: (10.1053/j.gastro.2005.01.055) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 5 iNOS via NO-mediated Notch-1 and COX-2 expression are independent. (A) Selective COX-2 inhibitor, NS398, did not attenuate Notch-1 protein expression. 603B cells were incubated in the presence or absence of the selective COX-2 inhibitor, NS398 (100 μmol/L), and Notch-1 protein expression was assessed by immunoblot analysis. (B) The γ-secretase inhibitor, DAPT, did not attenuate Notch-1 protein expression. 603B cells were incubated in the presence or absence of the γ-secretase inhibitor, DAPT (5 μmol/L), and COX-2 protein expression was assessed by immunoblot analysis. Gastroenterology 2005 128, 1354-1368DOI: (10.1053/j.gastro.2005.01.055) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 6 Transient transfection with Notch-1 siRNA specifically down-regulates Notch-1 expression. (A) Immunoblot analysis of Notch-1 in mouse cholangiocytes. 603B cells were transfected transiently with siRNA expression vector targeting Notch-1 (notch-1 sirna). Transient transfection with Notch-1 siRNA shows its ability to reduce expression. (B) Densitometric analysis. Notch-1 protein expression was quantified relative to β-actin by densitometry. Notch-1 expression was 60% decreased in Notch-1 siRNA-transfected 603B cells. Gastroenterology 2005 128, 1354-1368DOI: (10.1053/j.gastro.2005.01.055) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 7 Transit transfection with Notch-1 siRNA did not significantly alter cell proliferation and DNA synthesis. (A) Effect of Notch-1 siRNA on cell proliferation. Cell proliferation was assessed by MTS assay. The optical density of 490 nm was measured by a microplate autoreader. Transient transfection with Notch-1 siRNA did not significantly alter cell proliferation rates in comparison with the 603B cells transfected with scramble (sc) RNA vector. ♦, Control; •, siRNA reagent; ▴, Notch-1 siRNA; ■, Sc-siRNA. (B) Effect of Jagged-1 on cell proliferation. Cell proliferation was assessed by MTS assay. Jagged-1 (1 μg/mL) did not significantly increase cell growth of 603B cells. ♦, Control; ▴, JAG. (C) Effect of Notch-1 siRNA on DNA synthesis. DNA synthesis was assessed by BrdU assay. The amount of BrdU was quantified spectrophotometrically with a microplate spectrophotometer at a wavelength of 450 nm with reference at a wavelength of 570 nm. Consistent with MTS assay, Notch-1 siRNA did not significantly alter DNA synthesis. Gastroenterology 2005 128, 1354-1368DOI: (10.1053/j.gastro.2005.01.055) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 8 Notch-1 inhibits TRAIL-induced apoptosis. (A) 603B cells that expressed Notch-1 constitutively were resistant to TRAIL-induced apoptosis. 603B cells and 603B cells transiently transfected with Notch-1 siRNA were incubated with 10 ng/mL TRAIL for 12 hours. Apoptosis was quantitated using DAPI staining and fluorescence microscopy. All data are expressed as mean ± SD. Pretreatment of DAPT sensitizes the 603B cell to TRAIL-mediated killing. *P < .0001, **P < .00001 as compared with 603B cells treated with TRAIL alone, n = 6 for each group. (B) DAPT also sensitized the human cholangiocarcinoma cell line, KMCH, to TRAIL-mediated killing. *P < .0001, as compared with KMCH cells treated with TRAIL alone, n = 6 for each group. Gastroenterology 2005 128, 1354-1368DOI: (10.1053/j.gastro.2005.01.055) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 9 Proposed model of signaling pathways for iNOS-mediated Notch-1 induction in mouse cholangiocytes. iNOS induces both COX-2 and Notch-1 expression; however, their expressions are independent. We have shown previously that iNOS induces COX-2 expression likely through a p38 MAPK and JNK1/2 pathway, and iNOS via COX-2 induction enhances cell proliferation. iNOS appears to induce Notch-1 expression by a JNK1/2-mediated pathway. The inhibition of Notch-1 signaling pathway increases the sensitivity of the cell to TRAIL-mediated apoptosis. In these processes, the continuous up-regulation of Notch-1 as well as COX-2 by iNOS may facilitate the tumorigenesis of nontumorigenic cells. Gastroenterology 2005 128, 1354-1368DOI: (10.1053/j.gastro.2005.01.055) Copyright © 2005 American Gastroenterological Association Terms and Conditions