Intracellular mechanisms mediating the anti-apoptotic action of gastrin

https://doi.org/10.1016/j.bbrc.2004.08.059Get rights and content

Abstract

We previously reported that gastrin (G17) inhibits apoptosis of AR4-2J pancreatic adenocarcinoma cells, through the activation of Akt. We dissected the mechanisms responsible for this effect. D2, a CCKB receptor antagonist, inhibited G17 induction of Akt phosphorylation, measured by Western blots with anti-phospho-Akt antibodies. The intracellular calcium chelator BAPTA-AM, but not the PKC inhibitor GF109203X, blocked G17 induction of Akt. G17 stimulated BAD phosphorylation, measured by both Western blots with anti-phospho-BAD antibodies and by in vitro Akt kinase assays using recombinant BAD as substrate. G17 also induced FOXO3 phosphorylation assessed by Western blots with anti-phospho-FOXO3 antibodies, and BAPTA-AM inhibited this effect. Gastrin inhibited luciferase activity in cells transfected with FOXO1 together with a vector containing insulin-responsive sequences upstream of the luciferase reporter gene. In conclusion, G17 induces Akt through activation of CCKB receptors and of intracellular calcium-dependent, PKC-independent, pathways. This effect leads to BAD phosphorylation and to forkhead transcription factors inactivation.

Section snippets

Materials and methods

Plasmids. The plasmid expressing 3× IRS-Luc [13] was a gift of K. Guan (Ann Arbor, MI). The FOXO1 (FKHR) expressing vector was a gift from F.G. Barr (Philadelphia, PA) [13]. Gal4-ElkC [16] was a gift from R. Treisman (London, UK). pCMV-βGal was a gift from M. Uhler (Ann Arbor, MI). Recombinant BAD [12] was a gift from G. Nunez (Ann Arbor, MI).

Cell culture, transient transfection, and luciferase assays. For our experiments we used the rat exocrine pancreatic cell line AR4-2J (obtained from

Results

We previously observed that gastrin (G17) inhibits apoptosis of the AR4-2J rat acinar cell line through the phosphorylation and activation of protein kinase B/Akt [8]. In this study, we further dissected the mechanisms that mediate the anti-apoptotic action of gastrin in the AR4-2J cells. We confirmed the involvement of specific CCKB receptors in gastrin induction of Akt activation. D2 (10 nM), a specific CCKB receptor antagonist [5], inhibited gastrin (10 nM)-induced phosphorylation of Akt (Fig.

Discussion

The peptide hormone gastrin regulates numerous, complex cellular functions such as growth, proliferation, and secretion [1], [2], [3], [4]. In previous studies we reported that gastrin induces the Akt signal transduction pathway and that this effect leads to inhibition of AR4-2J cell apoptosis [8].

In this study, we show that gastrin induction of Akt leads to the phosphorylation, both in vitro and in vivo, of BAD, a well-characterized pro-apoptotic protein, which is phosphorylated and

Acknowledgments

The authors thank Thomas Witham, Daniel Miller, and Jace Nielsen for technical assistance. This work was supported by National Institute of Diabetes and Digestive and Kidney Disease (NIDDK) Grant RO1-DK-058312 (to A. Todisco) and by funds from the University of Michigan Gastrointestinal Peptide Research Center (NIH Grant P30DK34933).

References (19)

There are more references available in the full text version of this article.

Cited by (28)

  • Role of gastrin peptides in carcinogenesis

    2007, Cancer Letters
    Citation Excerpt :

    Proliferation of epithelial cells within Barrett’s oesophagus biopsies is also increased by treatment with G17, via the CCK-2R [111], suggesting a possible role for gastrin in promoting growth of cells within pre-malignant lesions. In addition to enhancing growth through positive signals, gastrin inhibits apoptosis [112]. This occurs through phosphorylation of phosphokinase B (PKB)/Akt via a pathway dependent on PI3K but independent of MAPK [113], and via a PKC-δ-dependent pathway that causes degradation of the inhibitor of NFκB, IκB-α [27,114].

  • Regulation of Gastric Acid Secretion

    2006, Physiology of the Gastrointestinal Tract
View all citing articles on Scopus
View full text