Membrane proteome analysis of cerulein-stimulated pancreatic acinar cells: implication for early event of acute pancreatitis

Abstract

Background/aims: Cerulein pancreatitis is similar to human edematous pancreatitis with dysregulation of the production and secretion of digestive enzymes, edema formation, cytoplasmic vacuolization and the death of acinar cells. We hypothesized that membrane proteins may be altered as the early event during the induction of acute pancreatitis. Present study aims to determine the differentially expressed proteins in the membranes of cerulein-treated pancreatic acinar cells.

Methods: Pancreatic acinar AR42J cells were treated with 10(-8) M cerulein for 1 hour. Membrane proteins were isolated from the cells and separated by two-dimensional electrophoresis using pH gradients of 5-8. Membrane proteins were identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis of the peptide digests. The differentially expressed proteins, whose expression levels were more or less than three-fold in cerulein-treated cells, were analyzed.

Results: Two differentially expressed proteins (mannan-binding lectin-associated serine protease-2, heat shock protein 60) were up-regulated while four proteins (protein disulfide isomerase, gamma-actin, isocitrate dehydrogenase 3, seven in absentia homolog 1A) were down-regulated by cerulein treatment in pancreatic acinar cells. These proteins are related to cell signaling, oxidative stress, and cytoskeleton arrangement.

Conclusions: Oxidative stress may induce cerulein-induced cell injury and disturbances in defense mechanism in pancreatic acinar cells.

Keywords: Cerulein; Membrane proteome; Pancreatic acinar cells; Pancreatitis.

Fig. 1

Western blot analysis for aldolase A and Mox1 (A) and 2-DE gel map derived from non-treated and cerulein-treated cells (B). (A) The cells (2×10⁶/mL in a 100 mm culture) were treated with cerulein for the indicated time periods. Cytosolic and membrane fractions were isolated from the cells. Protein levels of aldolase 1 and Nox were determined by Western blotting using anti-aldolase A and anti-Nox antibodies. Aldolase A and Nox were used for the markers of cytosolic and membrane fractions, respectively. (B) The protein (300 µg in 300 µL) was applied to pH 5-8 linear IPG strips (17 cm), with 11% linear vertical SDS-PAGE as the second dimension. The gel was visualized by Coomassie staining. The numbered proteins are those that were differentially expressed between non-treated cells (none) and cerulein-treated cells (cerulein). The numbers I 1-4 indicate up-regulated proteins while the numbers D 1-2 denote down-regulated proteins by cerulean treatment. IEF, isoelectric focusing.

Fig. 2

Segments of 2-DE gel map derived from non-treated (none) and cerulein-treated cells (cerulein). The arrows indicate the differentially expressed proteins whose expression level was more than three times higher or lower in cerulein-treated cells than non-treated cells. The expression level was determined by the relative spot volume of the proteins compared with the total amount of the protein in the gel, and is expressed as the percentage volume (right panel). The proteins were identified by the MALDI-TOF MS analysis. A representative gel image and expression level (percentage volume) for each spot is shown. For each spot, the percentage volume was averaged and expressed as a mean±S.E. from three different experiments. ^*p<0.01 versus the none-treated cells. The proteins identified with MALDI-TOF MS were (I1) Hsp60, (I2) MASP-2, (D1) PDI, (D2) γ-actin, (D3) ICD3, and (D4) SIAH 1A. Hsp60, heat shock protein 60; PDI, protein disulfide isomerase; ICD3, isocitrate dehydrogenase 3; SIAH 1A, seven in absentia homolog 1A; MASP-2, mannan-binding lectin-associated serine protease-2.

Fig. 3

Western blot analysis for the differentially expressed proteins (A) and diagram according to the functional role of the proteins (B). (A) The differentially expressed proteins, determined by proteomics, were assessed by Western blot analysis. The cells (2×10⁶/mL in a 100 mm culture) were treated with cerulein for the indicated time periods. The membrane fractions were isolated from the cells. Protein levels of Hsp60, PDI, γ-actin, ICD3, SIAH 1A, and actin were determined by Western blotting. Since there is no commercially available antibody for MASP-2, the protein could not be verified by Western blotting. (B) Diagram of the proteins shows the overlap among the proteins whose expression levels were more than three-fold different between non-treated cells and cerulean-treated cells. The proteins having cell defense mechanism against cellular stress including oxidative stress include Hsp60, PDI and ICD3. The proteins related to cell signaling are MASP-2, SIAH 1A, Hsp60 and PDI. The protein showing a role in cytoskeleton arrangement is γ-actin. Some proteins share their functional roles. Hsp60 and PDI have functions in cellular defense mechanism and signal transduction. Hsp60, heat shock protein 60; PDI, protein disulfide isomerase; ICD3, isocitrate dehydrogenase 3; SIAH 1A, seven in absentia homolog 1A; MASP-2, mannan-binding lectin-associated serine protease-2.