G. Zhou1, E. Rozengurt2, J. Sinnett-Smith2, S. Liu1, J. Yu1, J. Wu1, R. Sanchez1, F. C. Brunicardi1 1University Of California – Los Angeles,General Surgery/Surgery/David Geffen School Of Medicine,Los Angeles, CA, USA 2University Of California – Los Angeles,Digestive Diseases/Medicine/David Geffen School Of Medicine,Los Angeles, CA, USA
Introduction: Pancreatic and duodenal homeobox-1 (PDX-1) is a key pancreatic transcription factor known to be involved in tumorigenesis and markedly overexpressed in pancreatic ductal adenocarcinoma (PDAC). However, little is known about the related oncogenic signaling pathways causing the overexpression. The activation of the extracellular signal-regulated kinases (ERK) pathway driven by the K-RAS mutation plays a critical role in promoting survival, invasion and migration of PDAC cells. The purpose of this study is to determine the role of ERK in regulation of PDX-1 expression in PDAC.
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Methods: Antibody array screen was performed in GFP-PDX-1 stable HEK293 cells using a membrane filter arrayed with 400 antibodies, which was preblocked in a buffer containing 5% nonfat milk. After overnight incubation at 4°C, the presence of the antibody-antigen-PDX-1 complex was detected by horseradish peroxidase-conjugated anti-GFP antibody, followed by chemiluminescence. The ERK-PDX-1 interaction was confirmed in PDAC cells by performing immunoprecipitation/Western blotting using anti-ERK and anti-PDX-1 antibodies. Protein expression levels were determined by performing Western blotting using appropriate antibodies. Lipofectamine 2000 was used for transient transfection of PDX-1, ERK1, c-Jun N-terminal kinase 1 (JNK1) and Ubiquitin into HEK293 cells. PDAC cells were treated with various concentrations of epidermal growth factor (EGF) in the presence of 2% serum. Serine 268 residues of human PDX-1 were mutated into alanine by performing site-directed mutagenesis as instructed by the manufacture’s manual. The mutations were confirmed by standard PCR sequencing. Ubiquitination of PDX-1 was monitored by immunoprecipitating PDX-1, followed by followed by Western blotting using an antibody against ubiquitin.
Results:1) ERK1 and ERK2 were identified as PDX-1-interacting proteins in an antibody array screen; 2) The ERK-PDX-1 interaction was confirmed by immunoprecipitation/Western blotting in PDAC cells in response to EGF; 3) PDX-1 expression was specifically up-regulated by ERK1, but not by JNK1; 4) EGF enhanced PDX-1 expression in PDAC cells; 5) ERK inhibitor blocked EGF-stimulated PDX-1 expression in PDAC cells; and 6) Phosphorylation of proline-directed Ser 268 suppressed PDX-1 ubiquitination and stabilized PDX-1, thus, Ser 268 is a potential ERK phosphorylation site within PDX-1, whose phosphorylation contributes to the stabilization of PDX-1.
Conclusion:These data show that 1) ERK MAP kinase is a positive regulator of PDX-1; and 2) ERK up-regulates PDX-1 expression via a mechanism involving promoting phosphorylation of Ser 268, leading to decreased ubiquitination and enhanced stabilization of PDX-1. Thus, these data suggest that ERK MAP kinase plays an important role in PDX-1 overexpression in PDAC.