Ethanol augments PDGF-induced NADPH oxidase activity and proliferation in rat pancreatic stellate cells

Abstract

Background/aims: Activated stellate cells are considered the principal mediators of chronic alcoholic pancreatitis/fibrosis. However the mechanisms of alcohol action on pancreatic stellate cells (PaSCs) are poorly understood. The aims of this study were to determine the presence and role of the NADPH oxidase system in mediating alcohol effects on PaSCs with specific emphasis on proliferation.

Methods: PaSC NADPH oxidase components mRNA and protein were determined by RT-PCR and Western blot. The NADPH oxidase activity was measured by detecting the production of reactive oxygen species using lucigenin-derived chemiluminescence assay. PaSC DNA synthesis, a measure of proliferation, was performed by determining the [3H] thymidine incorporation into DNA.

Results: mRNA for NADPH oxidase components Nox1, gp91(phox), Nox4, p22(phox), p47(phox) and p67(phox) and protein for NADPH oxidase subunits gp91(phox), p22(phox), p47(phox) and p67(phox) are present in PaSCs. Treatment with platelet-derived growth factor (PDGF) significantly increased the NADPH oxidase activity and DNA synthesis in cultured PaSCs. Alcohol treatment markedly augmented both the NADPH oxidase activity and the DNA synthesis caused by PDGF, which was prevented by antioxidant N-acetyl-L-cysteine, ROS scavenger tiron, and the NADPH oxidase inhibitor diphenylene iodium. The effects of PDGF on NADPH oxidase activity and DNA synthesis were prevented in PaSCs isolated from the pancreas of mice with a genetic deficiency of p47(phox).

Conclusions: Ethanol causes proliferation of stellate cells by augmenting the activation of the cell's NADPH oxidase system stimulated by PDGF. These results provide new insights into the mechanisms of alcohol-induced fibrosing disorders.

Fig. 1.

Pancreatic stellate cells express mRNA and protein for NADPH oxidase components. Pancreatic stellate cells, passage 1 or 2, were used for mRNA extraction for RT-PCR analysis, and protein preparation for Western blot as described in ‘Materials and Methods’. A RT-PCR of mRNA for NADPH oxidase subunits. B Western blot analysis for protein expression of NADPH oxidase subunits. These results are representative of two independent experiments.

Fig. 2.

NADPH oxidase activity located in the membrane fraction of pancreatic stellate cells. Pancreatic stellate cells were lysed in lysis buffer and the membrane and cytosolic fractions were separated by centrifugation. The activity of NADPH oxidase in each fraction was analyzed as described in ‘Materials and Methods’. DPI was added to the membrane fraction 10 min prior to activity assay. The results represent the means and SD of the values of three independent experiments.

Fig. 3.

NADPH oxidase measurement is specific for NADPH. The pancreatic stellate cell membrane fraction was used to examine the chemiluminescence generation with the addition of either NADPH or NADH. The results are the means and SD of the values of three independent experiments.

Fig. 4.

PDGF treatment increases NADPH oxidase activity in pancreatic stellate cells in a dose- and time-dependent fashion. Pancreatic stellate cells were treated with PDGF at the indicated concentrations for the indicated times. The membrane fraction was used for NADPH oxidase activity assay. A Cells were treated with rat PDGF BB at indicated concentrations for 24 h. B Cells were treated with PDGF 20 ng/ml for indicated times. The results represent the mean and SD of the values of four independent experiments.

Fig. 5.

PDGF treatment increases NADPH oxidase activity in the membrane fraction of pancreatic stellate cells. Pancreatic stellate cells were treated without or with rat PDGF BB 10 ng/ml for 24 h. The membrane and cytosolic fractions were separated as described in ‘Materials and Methods’. The results shown are means and SD of the values of three to four independent experiments. ∗∗ p < 0.001 as compared to control.

Fig. 6.

Ethanol treatment augments PDGF-induced NADPH oxidase activity in pancreatic stellate cells. Activity is inhibited by DPI. Pancreatic stellate cells were treated with or without ethanol (100 mM) for 3 days and rat PDGF BB (10 ng/ml) and DPI (5 μM) for the last 24 h of incubation. The results represent means and SD of the values of three to four independent experiments.^a p < 0.0001 as compared to control;^b p < 0.05 as compared to PDGF alone;^c p < 0.01 as compared to ethanol + PDGF.

Fig. 7.

Ethanol treatment augments the effect of PDGF on DNA synthesis. Activity is inhibited by DPI, NAC and tiron. Pancreatic stellate cells were treated with or without 100 m M ethanol for 72 h and rat PDGF BB (10 ng/ml) or in combination with DPI (5 μM), NAC (10 mM), or tiron (1.0 mM) for 24 h. The results represent means and SD of the values of three to four independent experiments.^a p < 0.001 as compared to basal;^b p < 0.001 as compared to PDGF alone;^c p < 0.001 as compared to ethanol + PDGF.

Fig. 8.

Ethanol augments the effect of NADPH oxidase activity in pancreatic stellate cells grown on Matrigel. Activity is inhibited by DPI, NAC and tiron. Pancreatic stellate cells grown on Matrigel were treated with ethanol (100 mM) for 72 h and rat PDGF BB (10 ng/ml), DPI (5 μM), NAC (10 mM), or tiron (1.0 mM) for the last 24 h of incubation. The results represent means and SD of the values of three independent experiments.^a p < 0.01 as compared to basal;^b p < 0.001 as compared to PDGF alone;^c p < 0.001 as compared to ethanol + PDGF.

Fig. 9.

Ethanol augments the effect of PDGF on DNA synthesis in pancreatic stellate cells grown on Matrigel and inhibition by DPI, NAC and tiron. Pancreatic stellate cells grown on Matrigel were treated with ethanol for 72 h, and rat PDGF BB (10 ng/ml), DPI (5 μM), NAC (10 mM) alone or tiron (1.0 mM) in the presence of 1.0 μCi [³ H] thymidine for 24 h. The results represent means and SD of the values of three independent experiments.^{a, b} p < 0.001 as compared to control and PDGF alone, respectively;^c p < 0.01 as compared to ethanol + PDGF.

Fig. 10.

PDGF activation of NADPH oxidase requires the p47^phox subunit of NADPH oxidase in pancreatic stellate cells. Pancreatic stellate cells were isolated from wild-type (WT) and p47^phox genetically deficient mice as indicated in ‘Materials and Methods‘. The cells were treated with hPDGF BB at the indicated concentration for 24 h. The results represent means and SEM of the values of three independent experiments. ∗∗ p < 0.001 as compared to basal.

Fig. 11.

NADPH oxidase p47^phox subunit mediates PDGF-induced DNA synthesis in pancreatic stellate cells. Pancreatic stellate cells isolated from wild-type and p47^phox genetically deficient mice were grown as indicated in ‘Materials and Methods’. The cells were treated with hPDGF BB at indicated concentrations for 24 h. The results represent means and SEM of the values of three independent experiments. ∗ p < 0.05 and ∗∗ p < 0.01 as compared to basal.