Volume 130, Issue 1, Pages 137-149 (January 2006) Reg IV Activates the Epidermal Growth Factor Receptor/Akt/AP-1 Signaling Pathway in Colon Adenocarcinomas  Kumar S. Bishnupuri, Qizhi Luo, Nabendu Murmu, Courtney W. Houchen, Shrikant Anant, Brian K. Dieckgraefe  Gastroenterology  Volume 130, Issue 1, Pages 137-149 (January 2006) DOI: 10.1053/j.gastro.2005.10.001 Copyright © 2006 American Gastroenterological Association Terms and Conditions

Figure 1 Reg IV induces proliferation of HCT116 and HT29 cells. HCT116 and HT29 cells incubated with increasing doses of rhR4 (0–500 nmol/L) and EGF (0–10 nmol/L) for 72 hours were analyzed for cell proliferation assay based on hexosaminidase enzyme activity. Treatment with Reg IV resulted in a dose-dependent increase in cell number of both cell lines and is comparable to that of EGF. Gastroenterology 2006 130, 137-149DOI: (10.1053/j.gastro.2005.10.001) Copyright © 2006 American Gastroenterological Association Terms and Conditions

Figure 2 Reg IV induces EGFR phosphorylation. Lysates from HCT116 cells incubated with (A and B) 200 nmol/L rhR4 or (C and D) 1 nmol/L EGF were analyzed by Western blotting for EGFR phosphorylation using rabbit anti–phospho-EGFR antibodies specific for phosphotyrosines at Tyr992 and Tyr1068. B and D are graphs of band intensities determined by densitometry scanning from 3 representative experiments and are depicted as relative to buffer-treated controls. Gastroenterology 2006 130, 137-149DOI: (10.1053/j.gastro.2005.10.001) Copyright © 2006 American Gastroenterological Association Terms and Conditions

Figure 3 Reg IV induces Akt phosphorylation, comparable and additive to EGF. HCT116 cells were incubated for 3 minutes either with rhR4 or EGF alone or in combination. Lysates were analyzed by Western blot using rabbit anti–phospho-Akt antibodies specific for phospho-Thr308 and Ser473. (A and B) Reg IV induces Akt phosphorylation. HCT116 cells exhibit a dose-dependent induction in Akt phosphorylation following treatment with rhR4. (C and D) EGF induces Akt phosphorylation. HCT116 cells exhibit a dose-dependent increase in Akt phosphorylation following treatment with EGF. (E and F) Reg IV induces Akt phosphorylation additive to EGF. HCT116 cells treated with 10 and 50 nmol/L rhR4 in combination with either 0.01 and 0.1 nmol/L EGF exhibit a significant and additive induction of Akt phosphorylation. (G and H) Inhibitor of EGFR inhibits Reg IV–induced Akt phosphorylation. Treatment of HCT116 cells with an increasing dose of a tyrosine kinase specific inhibitor of EGFR, AG1478, for 1 hour before addition of Reg IV (200 nmol/L) led to a dose-dependent inhibition of Akt phosphorylation. The left panels indicate the Western blots, and the right panels indicate the intensity of bands by densitometry scanning from 3 representative experiments. Gastroenterology 2006 130, 137-149DOI: (10.1053/j.gastro.2005.10.001) Copyright © 2006 American Gastroenterological Association Terms and Conditions

Figure 4 Reg IV induces AP-1 activity through EGFR signaling. (A) Plasmid pTAL and pAP-1. (B) Reg IV induces AP-1 activity. HCT116 cells transfected with pTAL or pAP-1 were treated overnight with rhR4 (200 nmol/L), heat-denatured rhR4, or vehicle. Lysates were subjected to dual-luciferase reporter assay analyses. Results were corrected for transfection efficiency using a Renilla luciferase coupled plasmid. We observed a significant increase in AP-1 activity following treatment with rhR4 but not in the presence of heat-denatured rhR4. (C) Reg IV induces AP-1–specific binding activity to its cognate binding site. Nuclear extracts from the rhR4-treated HCT116 cells were subjected to EMSAs using oligonucleotide probe containing a consensus AP-1 binding site. Cells treated with rhR4 exhibited 4-fold higher levels of AP-1 binding activity compared with untreated cells. Addition of a 50-fold molar excess of unlabeled AP-1 oligonucleotide led to the disappearance of the AP-1–specific band. However, addition of an unlabeled oligonucleotide probe containing a mutated AP-1 binding site did not demonstrate any change in band intensity. Data are based on 3 independent experiments. (D) Inhibitor of EGFR reduces Reg IV–induced AP-1 activity. HCT116 cells transfected with plasmid pAP-1 were treated with rhR4 and increasing doses of AG1478 or AG9. AG1478 significantly reduced the Reg IV–induced AP-1 activity. Gastroenterology 2006 130, 137-149DOI: (10.1053/j.gastro.2005.10.001) Copyright © 2006 American Gastroenterological Association Terms and Conditions

Figure 5 Treatment with Reg IV induces expression of AP-1 components. (A–D) mRNA expression. RNAs from HCT116 cells treated with rhR4 (200 nmol/L) were subjected to real-time RT-PCR analyses. c-Jun, JunB, JunD, and FosB transcripts increased following treatment with rhR4. (E and F) Protein expression. Cell lysates from rhR4-treated HCT116 cells were subjected to Western blot analysis for c-Jun, JunB, JunD, and FosB proteins (left panel). The right panel indicates the band intensity by densitometry scanning from 3 representative experiments. (G) Reg IV increases JunB-, JunD-, and FosB-induced supershift of AP-1–specific band in the EMSA assay. Nuclear extracts from untreated and rhR4-treated (1 hour) HCT116 cells were subjected to EMSA in the presence of specific antibodies to c-Jun, JunB, JunD, c-Fos, FosB, Fra-1, and Fra-2. Addition of antibodies to JunB, JunD, and FosB resulted in a supershift (SS), while antibodies to c-Jun, c-Fos, Fra-1, and Fra-2 had no effect. Furthermore, treatment with Reg IV increased the intensity of the supershift bands for JunB, JunD, and FosB. This is a representative figure of 3 duplicate experiments. cJ, c-Jun; JB, JunB; JD, JunD; cF, c-Fos; FB, FosB; F1, Fra-1; F2, Fra-2. Data are presented as mean ± SEM. *P < .05, **P < .01. Gastroenterology 2006 130, 137-149DOI: (10.1053/j.gastro.2005.10.001) Copyright © 2006 American Gastroenterological Association Terms and Conditions

Figure 6 Reg IV induces expression of Bcl-2, Bcl-XL, survivin, and matrilysin through EGFR signaling. (A) mRNA expression. Real time RT-PCR analyses exhibited that rhR4 treatment of HCT116 cells induced the expression of Bcl-2, Bcl-XL, survivin, and matrilysin. (B) Reg IV–mediated induction requires EGFR signaling. Reg IV induction of Bcl-2, Bcl-XL, survivin, and matrilysin was blocked in a dose-dependent fashion following addition of tyrosine kinase specific EGFR inhibitor AG1478, suggesting involvement of EGFR in Reg IV–mediated signal transduction pathways. Data are based on 3 independent experiments. Bars indicate the mean ± SEM. *P < .05, **P < .01. Gastroenterology 2006 130, 137-149DOI: (10.1053/j.gastro.2005.10.001) Copyright © 2006 American Gastroenterological Association Terms and Conditions