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, 284 (26), 17868-76

Interleukin-32 Expression in the Pancreas

Affiliations

Interleukin-32 Expression in the Pancreas

Atsushi Nishida et al. J Biol Chem.

Abstract

Interleukin (IL)-32 is a recently described proinflammatory cytokine characterized by the induction of nuclear factor (NF)-kappaB activation. We studied IL-32 expression in human pancreatic tissue and pancreatic cancer cell lines. Tissue samples were obtained surgically. IL-32 expression was evaluated by standard immunohistochemical procedures. IL-32 mRNA expression was analyzed by Northern blotting and real time PCR analyses. IL-32 was weakly immunoexpressed by pancreatic duct cells. In the inflamed lesions of chronic pancreas, the ductal expression of IL-32 was markedly increased. A strong expression of IL-32alpha was detected in the pancreatic cancer cells. In pancreatic cancer cell lines (PANC-1, MIA PaCa-2, and BxPC-3 cells), the expression of IL-32 mRNA and protein was enhanced by IL-1beta, interferon (IFN)-gamma, and tumor necrosis factor (TNF)-alpha. An inhibitor of phosphatidylinositol 3-kinase (LY294002) significantly suppressed the IL-1beta-, IFN-gamma- and TNF-alpha-induced IL-32 mRNA expression. The blockade of NF-kappaB and activated protein-1 activation markedly suppressed the IL-1beta-, IFN-gamma-, and/or TNF-alpha-induced IL-32 mRNA expression. Furthermore, IL-32-specific small interfering RNA significantly decreased the uptake of [3H]thymidine and increased the annexin V-positive population (apoptotic cells) in PANC-1 cells. IL-32 knockdown also suppressed the mRNA expression of antiapoptotic proteins (Bcl-2, Bcl-xL, and Mcl-1). Pancreatic duct cells are the local source of IL-32, and IL-32 may play an important role in inflammatory responses and pancreatic cancer growth.

Figures

FIGURE 1.
FIGURE 1.
Representative immunohistochemical expression of IL-32 in the pancreas. A, IL-32 staining in normal pancreatic tissue (magnification ×100 and ×400), chronic pancreatitis (×100), pancreatic cancer (×100), and pancreatic cancer stained with control IgG. B, IL-32 staining in chronic pancreatitis. C, IL-32 staining in pancreatic cancer.
FIGURE 2.
FIGURE 2.
IL-32 mRNA and protein expression in human pancreatic cancer cell lines. A, IL-32 mRNA expression. The cells were stimulated with cytokines (100 ng/ml) for 12 h. IL-32 mRNA expression was analyzed by Northern blotting. Ribosomal RNA, stained by ethidium bromide, is shown in the lower panel. B, intracellular IL-32 protein expression. The cells were stimulated with each cytokine (IL-1β (10 ng/ml), TNF-α (100 ng/ml), and IFN-γ (100 ng/ml)) for 24 h and then lysed with lysis buffer. IL-32 protein was analyzed by Western blotting. C, combined effects of cytokines on IL-32 mRNA expression. PANC-1 cells were stimulated with IL-1β (10 ng/ml), TNF-α (100 ng/ml), IFN-γ (100 ng/ml), and combinations of these cytokines for 12 h, and then IL-32 mRNA expression was determined by Northern blotting.
FIGURE 3.
FIGURE 3.
Dose- and time-dependent induction of IL-32 mRNA in PANC-1 cells. Left, cells were incubated with different doses of each cytokine, and IL-32 mRNA expression was determined by real time PCR. The data were expressed as IL-32 mRNA expression relative to β-actin mRNA expression (mean ± S.D. from four different experiments). Right, cells were stimulated with cytokines (IL-1β (10 ng/ml), TNF-α (100 ng/ml), and IFN-γ (100 ng/ml)) for predetermined times, and IL-32 mRNA expression was sequentially analyzed by real time PCR.
FIGURE 4.
FIGURE 4.
Effects of MAPK inhibitors and a PI3K inhibitor on IL-32 mRNA expression in PANC-1 cells. A–C, the cells were stimulated with each cytokine (IL-1β (10 ng/ml), TNF-α (100 ng/ml), and IFN-γ (100 ng/ml)) in the presence or absence of MEK inhibitors (PD98059 (20 μm) and U0216 (12.5 μm)), p38 inhibitor (SB203580 (25 μm)), and PI3K inhibitor (LY294002 (25 μm)) for 12 h, and then IL-32 mRNA expression was determined by real time PCR. The data were expressed as IL-32α mRNA expression relative to β-actin mRNA expression (mean ± S.D. from four different experiments). **, p < 0.01. D–F, kinetics of Akt activation in PANC-1 cells. The cells were stimulated with cytokines (IL-1β (10 ng/ml), TNF-α (100 ng/ml), and IFN-γ (100 ng/ml)), and phosphorylated (p-) and total Akt were sequentially detected by Western blotting.
FIGURE 5.
FIGURE 5.
Effects of NF-κB and/or AP-1 inhibition on IL-32 mRNA expression. A–C, PANC-1 cells were infected with an adenovirus expressing the IκBΔN or DN-c-Jun, and at 48 h after infection, cells were stimulated with IL-1β (10 ng/ml), TNF-α (100 ng/ml), or IFN-γ (100 ng/ml) for 12 h. IL-32 mRNA expression was determined by real time PCR. Adenovirus expressing LacZ was used as a negative control. The data were expressed by IL-32 mRNA expression relative to β-actin mRNA expression (mean ± S.D. from four different experiments). **, p < 0.01. D and E, electrophoretic gel mobility shift assays for NF-κB and/or AP-1 DNA binding activities. PANC-1 cells were incubated with medium alone, IL-1β (10 ng/ml), TNF-α (100 ng/ml), or IFN-γ (100 ng/ml) with or without LY294002 (25 μm) for 1.5 h, and then nuclear extracts were prepared.
FIGURE 6.
FIGURE 6.
IL-32 mRNA interference experiments. A, PANC-1 cells were transfected with IL-32 siRNA or control siRNA, and IL-32 mRNA expression was determined by RT-PCR. Similarly, PANC-1 cells were transfected with IL-32α siRNA or control siRNA and then stimulated by TNF-α for 12 h. IL-32 mRNA expression was determined by RT-PCR. B, PANC-1 cells were transfected with IL-32 siRNA or control siRNA and then stimulated by TNF-α for 24 h. IL-32 protein expression was determined by Western blotting. C, [3H]thymidine incorporation assay. PANC-1 cells were transfected with IL-32 siRNA or control siRNA and then cultured for 12 h in the presence of [3H]thymidine. The data were expressed as disintegrations/min and as -fold stimulation over the control value (mean ± S.D. from four different experiments). **, p < 0.01. D, trypan blue dye exclusion assay. PANC-1 cells were transfected with IL-32 siRNA or control siRNA and cultured for 24 h. The number of trypan blue-positive cells (dead cells) was counted under microscope (mean ± S.D. from four different experiments). **, p < 0.01.
FIGURE 7.
FIGURE 7.
Knockdown of IL-32 gene stimulates apoptosis in PANC-1 cells. PANC-1 cells were transfected with IL-32α siRNA or control siRNA and cultured for 24 h. Apoptotic cells were analyzed by fluorescence-activated cell sorting using annexin V-fluorescein isothiocyanate. A, cytogram of fluorescence-activated cell sorting analyses. Annexin-V-positive cells are present in the upper and lower right. B, percentage of annexin V-positive cells (mean ± S.D. from four different experiments). **, p < 0.01.
FIGURE 8.
FIGURE 8.
Effects of IL-32 knockdown on the mRNA expression of proapoptotic and prosurvival (antiapoptotic) proteins. A, PANC-1 cells were transfected with IL-32α siRNA or control siRNA, and the mRNA expression of proapoptotic proteins (Bax, Bad, Bak, and Bid) was determined by RT-PCR. B, PANC-1 cells were transfected with IL-32 siRNA or control siRNA, and the mRNA expression of prosurvival proteins (Bcl-2, Bcl-xl, and Mcl-1) was determined by RT-PCR. C, PANC-1 cells were transfected with IL-32 siRNA or control siRNA, and Bcl-2 and Bax protein expression was determined by Western blotting.

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