Figure 1.
Exogenous GNAS increases cAMP levels and alters mucin gene expression in cells of pancreatic ductal lineage: the cell lines HPDE, PK-8, PCI-35, and MIA PaCa-2.
(A) Immunoblots of total lysates of cells transfected with the empty vector (Vec), wild-type GNAS-V5 (GW), or mutated GNAS-V5 (R201H; abbreviated as GM) are shown on the right. Double bands were observed in assays with the anti-G protein α antibody, where the upper bands indicate specific immunoreactivity of G protein α (arrowheads). (B) Cyclic AMP levels were determined using an enzyme immunoassay. (C and D) Quantitative real-time PCR analysis of MUC2 (C) and MUC5AC (D) expression. The data are shown on a logarithmic scale and values obtained are from independently duplicated experiments. Error bars indicate standard error. *p<0.05, **p<0.01.
Figure 2.
Exogenous GNAS does not stimulate cell proliferation or alterations of the cell cycle.
Colony formation of cells transfected with the empty vector (Vec), wild-type GNAS (GW), or mutated GNAS (R201H; abbreviated as GM; panels A and B). (A) Images of cells incubated with a selective medium for 4 weeks. Mock denotes untransfected cells. (B) The mean values of total area of surviving colonies are shown as relative values compared to the control (Vec). Each transfection consisted of 3 plates, and mean data from independently triplicated (PK-8 cells) or duplicated (PCI-35 and MIA PaCa-2 cells) experiments were plotted with a range of one standard error. (C) A colorimetric proliferation assay showing cell proliferation after transient exogenous GNAS expression. Relative absorbance values (Day 3/Day 1) were plotted and are presented as mean ± SEM. (D) The cell cycle assay of cells with exogenous GNAS expression. *p<0.05, **p<0.01.
Figure 3.
Alterations in gene expression related to signaling pathways as a result of exogenous mutated GNAS (R201H) expression in PK-8 (A), PCI-35 (B), and MIA PaCa-2 (C).
Genes in the PI3K-AKT and MAPK signaling pathways in KEGG Mapper (http://www.genome.jp/kegg/) [22] were mapped with label colors according to the ratio of expression in cells carrying mutated GNAS (GM) to that in cells transfected with the empty vector (Vec).
Figure 4.
MAPK activity contributes to expression of mucin genes under different state of G protein activity.
(A) Immunoblots of total lysates of cells transfected with the empty vector (Vec), wild-type GNAS-V5 (GW), and mutated GNAS-V5 (R201H; abbreviated as GM) with or without U0126, a potent mitogen-activated protein kinase kinase (MAP2K) inhibitor. (B) Cyclic AMP quantified using an enzyme immunoassay. U0126 treatment did not affect cAMP levels in PK-8 cells but downregulated cAMP in PCI-35 cells, except in the mutated GNAS transfectant. (C and D) A quantitative real-time PCR assay. (C) MUC2 was consistently downregulated by U0126 in PK-8 and PCI-35 cells, regardless of the presence of exogenous GNAS. (D) MUC5AC was consistently downregulated in PCI-35 cells, regardless of the presence of exogenous GNAS, and upregulated by U0126 in PK-8 cells expressing exogenous mutated GNAS. Values obtained from independently duplicated experiments were plotted. Error bars indicate standard error. *p<0.05; **p<0.01.
Figure 5.
PI3K-AKT activity influences mucin gene expression under different state of G protein activity.
(A) Immunoblots of total lysates of cells transfected with the empty vector (Vec), wild-type GNAS-V5 (GW), and mutated GNAS-V5 (R201H; abbreviated as GM) with or without LY294002, a specific inhibitor of PI3 kinase. (B) Cyclic AMP measured by means of an enzyme immunoassay. The cAMP production was not significantly affected by LY294002 in PK-8 cells but was upregulated in PCI-35 cells. (C and D) A quantitative real-time PCR assay. MUC2 is modestly downregulated by LY294002. The latter downregulated MUC5AC in PK-8 cells but upregulated it in PCI-35 cells; the effect was not significant in the PCI-35 clone expressing exogenous GNAS. Values obtained from independently duplicated experiments were plotted. Error bars indicate standard error. *p<0.05, **p<0.01.
Figure 6.
Diverse effects of signaling pathways on MUC2 and MUC5AC expression in PK-8 and PCI-35 cells.
(A) The regulation of MUC2 expression in PK-8 cells was predominantly mediated by the GPCR pathway, with synergistic effects of the MAPK pathway and additive effects of the PI3K pathway. Minimal interactions existed among the 3 signaling pathways themselves. (B) MUC2 expression in PCI-35 cells was regulated predominantly by the MAPK pathway and additively by the PI3K pathway, whereas the GPCR pathway was antagonistic. Active interactions existed among the signaling pathways: cAMP was upregulated by active MAPK and downregulated by active PI3K. (C) MUC5AC expression in PK-8 cells was regulated predominantly by the GPCR pathway, the MAPK pathway was antagonistic, and the PI3K pathway played a weak role. (D) MUC5AC expression in PCI-35 cells was regulated predominantly by the MAPK pathway, whereas both the PI3K and GPCR pathways were antagonistic.