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Gastrin induces c-fos gene transcription via multiple signaling pathways
V M Stepan1, M Tatewaki, M Matsushima
1Department of Internal Medicine, University of Michigan Medical Center, Ann Arbor, Michigan 48109-0682, USA.
Abstract:
We previously observed that the trophic actions of gastrin (G17) on the AR42J rat acinar cell line are mediated by mitogen-activated protein kinase (MAPK)-induced c-fos gene transcription via protein kinase C (PKC)-dependent and -independent pathways. In this study, we further investigated the signaling pathways that target c-fos in response to G17. G17 led to a sixfold induction in luciferase activity in cells transfected with plasmids containing the -356+109 sequence of the murine c-fos promoter, which includes the Sis-inducible element (SIE), serum response element (SRE), and the Ca2+/cAMP response element (CRE) regulatory elements. Addition of either the selective PKC inhibitor GF-109203X or the MAPK/extracellular signal-regulated kinase inhibitor PD-98059 resulted in an 80% reduction in luciferase activity. G17 induced the transcriptional activity of both Elk-1 and Sap-1a, transcription factors that bind to the E26 transformation specific (Ets) DNA sequence of the SRE, and this effect was inhibited by both GF-109203X and PD-98059. Point mutations in the Ets sequence led to a 4-fold induction of c-fos transcription stimulated by G17 and to a 1.3-fold induction in response to epidermal growth factor (EGF). In contrast, mutations in the CA rich G (CArG) sequence of the SRE prevented transcriptional activation by both G17 and EGF. G17 induction of the Ets mutant construct was unaffected by either GF-109203X or PD-98059. Because activation of the SRE involves the small GTP-binding protein Rho A, we examined the role of Rho A in G17 induction of c-fos transcription. Inactivation of Rho A by either the specific inhibitor C3 or by expression of a dominant negative Rho A gene inhibited G17 induction of both the wild-type and the Ets mutant constructs by 60%. C3 also inhibited G17-stimulated AR42J cell proliferation. Thus G17 targets the c-fos promoter CArG sequence via Rho A-dependent pathways, and Rho A appears to play an important role in the regulation of the trophic action of G17.
Insights
Gastrin (G17) stimulates c-fos gene transcription in rat acinar cells through protein kinase C and MAPK pathways. Rho A signaling is crucial for G17
Area of Science:
- Molecular Biology
- Cell Signaling
- Gene Regulation
Background:
- Gastrin (G17) exerts trophic actions on AR42J rat acinar cells.
- These actions are mediated by mitogen-activated protein kinase (MAPK)-induced c-fos gene transcription.
- Protein kinase C (PKC)-dependent and -independent pathways are involved.
Purpose of the Study:
- To investigate the specific signaling pathways regulating c-fos transcription in response to G17.
- To elucidate the role of transcription factors Elk-1, Sap-1a, and Rho A in G17-mediated c-fos induction.
- To determine the contribution of specific promoter elements (SIE, SRE, CRE, Ets, CArG) to G17's effects.
Main Methods:
- Luciferase reporter assays using murine c-fos promoter constructs (-356+109) in AR42J cells.
- Pharmacological inhibition of PKC (GF-109203X) and MAPK/ERK (PD-98059).
- Site-directed mutagenesis of c-fos promoter elements (Ets, CArG) and Rho A manipulation (C3 inhibitor, dominant-negative mutant).
Main Results:
- G17 induced c-fos promoter activity sixfold, involving SIE, SRE, and CRE elements.
- PKC and MAPK inhibitors reduced G17-induced luciferase activity by 80%.
- G17 activated Elk-1 and Sap-1a, inhibited by GF-109203X and PD-98059.
- Mutations in the Ets sequence reduced G17 induction, while CArG mutations abolished it.
- Rho A inactivation inhibited G17 induction of both wild-type and Ets mutant constructs by 60%.
- Rho A inhibition also reduced G17-stimulated AR42J cell proliferation.
Conclusions:
- G17 targets the c-fos promoter's CArG sequence via Rho A-dependent pathways.
- Rho A plays a significant role in regulating the trophic actions of G17.
- This study clarifies the molecular mechanisms underlying gastrin's effects on cell growth and gene expression.