Gastroenterology

Gastroenterology

Volume 130, Issue 1, January 2006, Pages 137-149
Gastroenterology

Basic–alimentary tract
Reg IV Activates the Epidermal Growth Factor Receptor/Akt/AP-1 Signaling Pathway in Colon Adenocarcinomas

https://doi.org/10.1053/j.gastro.2005.10.001Get rights and content

Background & Aims: Reg IV, a secreted protein and member of the Reg multigene family, is up-regulated in malignancies of the human gastrointestinal tract, including colorectal carcinoma (CRC). However, in vitro signal transduction pathway(s) utilized by Reg IV are not yet known.

Methods: To determine the signaling pathway(s) responsive to Reg IV, we examined the effects of purified recombinant human Reg IV (rhR4) on HCT116 and HT29 colon adenocarcinoma cells.

Results: Addition of rhR4 to cultures led to a dose-dependent increase in cell number similar to that observed after treatment with epidermal growth factor (EGF). In addition, rhR4 treatment resulted in rapid phosphorylation of EGF receptor at Tyr992 and Tyr1068 and Akt at Thr308 and Ser473. Using luciferase reporter gene assays, we demonstrated that Reg IV signaling through EGF receptor and Akt results in increased activator protein-1 (AP-1) transcription factor activity. Real-time reverse-transcription polymerase chain reaction and Western blot analyses revealed quantitative increases in c-Jun, JunB, JunD, and FosB expression associated with increased AP-1 activity. Electrophoretic mobility shift assay further revealed significant increases in AP-1 binding activity in rhR4-treated cells, with increased supershift in the presence of antibodies to JunB, JunD, and FosB. Furthermore, rhR4 treatments led to the increased expression of Bcl-2, Bcl-XL, survivin, and matrilysin, genes associated with a poor prognosis in advanced CRC.

Conclusions: Reg IV is a potent activator of the EGF receptor/Akt/AP-1 signaling pathway in CRC. Disruption of Reg signaling may have utility as a therapeutic intervention for human gastrointestinal adenocarcinomas.

Section snippets

Cell Culture and Proliferation Assay

HCT116 and HT29 colon adenocarcinoma cells (American Type Culture Collection, Manassas, VA) were grown in Dulbecco’s modified Eagle medium (DMEM) containing 10% heat inactivated fetal bovine serum (Sigma Chemical Co, St Louis, MO). Endotoxin-free recombinant human Reg IV (rhR4) was produced in Pichia pastoris by fermentation and purified by tangential flow ultrafiltration and preparative high-performance liquid chromatography as previously described.45 Cells were placed overnight in serum-free

Reg IV Induces Proliferation of HCT116 and HT29 Cells Comparable to That of EGF

Reg IV is overexpressed in colorectal carcinomas16, 17, 18 and regenerating mucosa of patients with Crohn’s disease or ulcerative colitis,10, 47 suggesting a possible role in the regulation of cell proliferation and survival. To determine Reg IV effects on cell growth, we treated HCT116 and HT29 CRC cells with increasing concentrations of rhR4 (0–500 nmol/L) and EGF (0–10 nmol/L) for 72 hours. These cells were initially chosen for the study because they have a responsive EGFR system and are

Discussion

Advanced colorectal adenocarcinoma is generally poorly responsive to chemotherapy and radiation.70, 71 To improve treatment outcomes, a better understanding of the pathways that underlie the behavior of colorectal cancers and contribute to deregulated proliferation and resistance to chemotherapeutic agents and radiation is necessary. Members of the regenerating (Reg) multigene family are potential candidates for regulating these events. Recent studies confirmed the adverse prognostic

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    Supported by National Institutes of Health grants DK002457, DK060106, and DK52574 (to B.K.D.) and DK62265 (to S.A., a Research Scholar of the American Gastroenterological Association).

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