Proteome variations in pancreatic stellate cells upon stimulation with proinflammatory factors

Abstract

Pancreatic stellate cells are key mediators in chronic pancreatitis and play a central role in the development of pancreatic fibrosis, stromal formation, and progression of pancreatic cancer. This study was aimed at investigating molecular changes at the level of the proteome that are associated with the activation of pancreatic stellate cells by proinflammatory factors, namely TNF-α, FGF2, IL6, and chemokine (C-C motif) ligand 4 (CCL4). They were added individually to cells growing in serum-free medium next to controls in medium supplemented with serum, thus containing a mixture of them all, or in serum-free medium alone. Variations were detected by means of a microarray of 810 antibodies targeting relevant proteins. All tested factors triggered increased proliferation and migration. Further analysis showed that TNF-α is the prime factor responsible for the activation of pancreatic stellate cells. CCL4 is associated with cellular neovascularization, whereas FGF2 and IL6 induction led to better cellular survival and decreased apoptotic activity of the stellate cells. The identified direct effects of individual cytokines on human pancreatic stellate cells provide new insights about their contribution to pancreatic cancer promotion.

Keywords: Antibody Microarray; Microarray; Pancreatic Cancer; Pancreatic Stellate Cell; Proteomics; Stromal Cell; Tumor Necrosis Factor (TNF).

FIGURE 1.

Venn diagram showing the number of proteins that were found regulated upon growth of PSCs in the presence of TNF-α, CCL-4, IL-6, and FGF-2. A yellow background indicates the number of proteins uniquely regulated at one condition only. Light gray and medium gray stand for molecules that were regulated at two or three growth conditions, respectively. The number of proteins that were differentially expressed under all four conditions is shown in the area with the darkest background.

FIGURE 2.

Expression level variations of the 45 commonly regulated proteins, presented as the logarithm of fold change (Log-FC).

FIGURE 3.

Immunohistochemical analysis of normal and PDAC tissues. Tissue sections were analyzed with antibodies binding FLNA, MMP3, and cortactin (CTTN) in normal versus PDAC tissue sections. Cytokeratin 19 (CK19) and SMA were used to stain ductal and stellate cells, respectively. Original magnification was 100- and 663-fold for the insets.

FIGURE 4.

Immunoblot analysis showing the expression of α-SMA in PSCs under various treatment conditions. Ctrl, control.

FIGURE 5.

FACS sorting analysis revealed the percentage of PSCs (% of events) in S phase. For each condition, three independent measurements were performed.

FIGURE 6.

Proliferation of PSCs under various incubation conditions. The assay measured the incorporation of a fluorescent dye to the DNA that was proportional to the number of cells in the system. The fluorescence intensities are shown in arbitrary units (AU). The p values were calculated in comparison with the control cells grown in serum-free medium.

FIGURE 7.

Migration assay. PSCs were grown to confluence. A gap was generated by physically scraping off cells. The number of cells was determined before and after 48 h of growth under the indicated incubation conditions.

FIGURE 8.

Apoptosis assay. The fluorescence signal is representative for the mitochondrial membrane potential, which, in turn, is a function of reduced apoptotic activity. The higher the signal, the smaller the apoptosis activity. The measurements were done relative to analyses in serum-free medium. AU, arbitrary units.

FIGURE 9.

Analysis of the level of reactive oxygen species. The cellular level of reactive oxygen species was determined as a function of the conversion of 2′,7′-dichlorofluorescein to its oxidized and then fluorescent version. AU, arbitrary units; Ctrl, growth in serum-free medium.

FIGURE 10.

Schematic summary of the different effects of TNF-α, FGF2, IL6, and CCL4 on PSCs.