Cigarette smoke toxin hydroquinone and misfolding pancreatic lipase variant cooperatively promote endoplasmic reticulum stress and cell death

Abstract

Mutation-induced protein misfolding of pancreatic secretory enzymes and consequent endoplasmic reticulum stress can cause chronic pancreatitis. A recent study revealed that cigarette smoke also increases the risk of the disease through endoplasmic reticulum stress. Here, we investigated the cumulative cellular effect of the G233E misfolding human pancreatic lipase variant and hydroquinone; a main toxic constituent of cigarette smoke, using mammalian cell lines. We found that hydroquinone reduces cell viability on a dose-dependent manner through programmed cell death, and diminishes lipase secretion without affecting its expression. Interestingly, hydroquinone decreased the viability more markedly in cells expressing the G233E lipase variant, than in cells producing wild-type lipase. The more substantial viability loss was due to increased endoplasmic reticulum stress, as demonstrated by elevated levels of X-box binding protein 1 mRNA splicing and immunoglobulin binding protein, NAD(P)H:quinone oxidoreductase 1 and C/EBP homologous protein expression. Unresolved endoplasmic reticulum stress, and especially up-regulation of the pro-apoptotic transcription factor C/EBP homologous protein were likely responsible for the increased cell death. Our observations demonstrated that the combination of hydroquinone and misfolding pancreatic lipase variant promote increased levels of endoplasmic reticulum stress and cell death, which may predispose to chronic pancreatitis.

Fig 1. Viability of HEK 293AD cells in the presence of main smoke constituents.

A) Cells were treated with indicated concentrations of acrolein, crotonaldehyde or hydroquinone (HQ). After 24 h incubation at 37 ˚C the levels of cellular metabolic activity were determined by MTT assay as described in Materials and Methods. Values are expressed as percent of untreated cells. Data points represent the average of two or three independent experiments with SD. B) Cells were exposed to indicated concentrations of HQ. After 24 h incubation at 37 ˚C, cells were stained with FITC annexin V and propidium iodide, and analyzed by flow cytometry as described in Materials and Methods. Fluorescence of FITC annexin V and propidium iodide are shown on the x and y axes, respectively. Representative pictures of three experiments are shown.

Fig 2. Effect of hydroquinone (HQ) on PNLIP secretion in HEK 293AD cells.

One day post-transfection, cells were incubated in the absence or presence of 25 μM HQ for 24 h at 37 ˚C. A) PNLIP protein in the conditioned media was analyzed by SDS-PAGE and Coomassie staining as described in Materials and Methods. B) PNLIP and GAPDH proteins in cell lysates were monitored by SDS-PAGE and immunoblotting as described previously [16].

Fig 3. Effect of hydroquinone (HQ) on the viability of wild-type and G233E mutant PNLIP expressing HEK 293AD cells.

One day post-transfection, cells were incubated in the absence or presence of 25 μM HQ for 24 h at 37 ˚C, and the levels of cellular metabolic activity were analyzed by MTT assay as described in Materials and Methods. Cells transfected with empty pcDNA3.1(-) vector were used as control. Data points represent the average of 6 independent experiments with SD. **P ≤ 0.01; ***P ≤ 0.001.

Fig 4. Effect of hydroquinone (HQ) on the levels of ER stress markers in wild-type and G233E mutant PNLIP expressing HEK 293AD cells.

One day post-transfection, cells were incubated in the absence or presence of 25 μM HQ for 24 h at 37 ˚C. The mRNA levels of A) XBP1 splicing, B) BiP, C) CHOP and D) NQO1 expressions were assessed as described in Materials and Methods. Cells transfected with empty pcDNA3.1(-) vector were used as control. Data points represent the average of 3 independent experiments with SD. * P ≤ 0.05; **P ≤ 0.01.

Fig 5. Viability of AR42J cells in the presence of hydroquinone (HQ).

Cells were treated with indicated concentrations of HQ for 24 h at 37 ˚C. A) The levels of cellular metabolic activity were analyzed by MTT assay as described in Materials and Methods. Values are expressed as percent of untreated cells. Data points represent the average of three independent experiments with SD. B) Annexin V and propidium iodide stainings of cells were monitored by flow cytometry as described in Materials and Methods. Annexin V and propidium iodide fluorescence are shown on the x and y axes, respectively. Representative images of three experiments are shown.

Fig 6. Effect of hydroquinone (HQ) on PNLIP secretion in AR42J cells.

One day post-transduction, cells were incubated in the absence and presence of 40 μM HQ for 24 h at 37 ˚C. A) PNLIP protein in the conditioned media was analyzed by SDS-PAGE and Coomassie staining as described in Materials and Methods. B) PNLIP and GAPDH proteins in cell lysates were monitored by SDS-PAGE and immunoblotting as described previously [16].

Fig 7. Effect of hydroquinone (HQ) on the viability of wild-type and G233E mutant PNLIP expressing AR42J cells.

One day post-transduction, cells were incubated in the absence or presence of 40 μM HQ for 24 h at 37 ˚C, and the levels of cellular metabolic activity were analyzed by MTT assay as described in Materials and Methods. Cells transfected with empty adenovirus vector were used as control. Data points represent the average of four independent experiments with SD. * P ≤ 0.05; ***P ≤ 0.001.

Fig 8. Effect of hydroquinone (HQ) on the levels of ER stress markers in wild-type and G233E mutant PNLIP expressing AR42J cells.

One day post-transduction, cells were incubated in the absence or presence of 40 μM HQ for 24 h at 37 ˚C. The mRNA levels of A) XBP1 splicing, B) BiP, C) CHOP and D) NQO1 expressions were assessed as described in Materials and Methods. Cells infected with empty adenovirus vector were used as control. Data points represent the average of three independent experiments with SD. * P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.