Upregulation of Salmonella-induced IL-6 production in Caco-2 cells by PJ-34, PARP-1 inhibitor: involvement of PI3K, p38 MAPK, ERK, JNK, and NF-kappaB

Abstract

Following Salmonella invasion, intestinal epithelial cells release a distinct array of proinflammatory cytokines. Interleukin (IL)-6 produced by enterocytes may have anti-inflammatory and cell-protective effects, and may counteract some of the injurious effects of sepsis and endotoxemia. Recent studies in a variety of rodent models of experimental colitis by using PJ-34, a potent poly (ADP-ribose) polymerase-1 (PARP-1) inhibitor, support the concept that the marked beneficial effect of PJ-34 can be exploited to treat human inflammatory diseases. The present study was to investigate the effect of PJ-34 on Salmonella-induced enterocyte IL-6 production and its mechanisms. We found that PJ-34 enhanced Salmonella-induced IL-6 production in Caco-2 cells, either secreted protein or mRNA expression. PJ-34 treatment enhanced the activity of NF-kappaB in Salmonella-infected Caco-2 cells. Besides, the involvement of PJ-34 in up-regulating IL-6 production in S. typhimurium-infected Caco-2 cells might be also through the ERK but not p38 MAPK, JNK or PI3K/Akt pathways, as demonstrated by Western blot of phosphorylated ERK, p38, JNK and Akt proteins. It suggests that PJ-34 may exert its protective effect on intestinal epithelial cells against invasive Salmonella infection by up-regulating IL-6 production through ERK and NF-kappaB but not P38 MAPK, JNK or PI3K/Akt signal pathways.

Figure 1

(a) Effect of PJ-34 on Salmonella-induced IL-6 protein secretion. Caco-2 cells were left untreated (control), or treated with 5, 10, 20, 40, or 80 μM PJ-34 (5, 10, 20, 40, 80PJ), or with a volume of DMSO (D) equivalent to the highest concentration of PJ-34. They were then uninfected (control and PJ only) or infected with the wild-type S. typhimurium strain SL1344 for 1 hour. Supernatant was analyzed by ELISA 6 hours later for IL-6. The amount of IL-6 produced is shown as the fold increase over uninfected, control cells. The results are representative of the results of two similar experiments. The data are the means ± standard deviations for three determinations. *P < .05. (b) Effect of PJ-34 on Salmonella-induced IL-6 mRNA. Caco-2 cells were left untreated, or treated with 5, 10, 20, 40, or 80 μM PJ-34. They were then uninfected or infected with the wild-type S. Typhimurium strain SL1344 for 1 hour. Total RNA was prepared 3 hours later and analyzed by real-time quantitative PCR to estimate amounts of IL-6 transcript. The amount of IL-6 mRNA produced, normalized to the corresponding amount of GAPDH transcript, is shown as the fold increase over uninfected, control cells. The results are representative of the results of two similar experiments. The data are the means ± standard deviations for three determinations. *P < .05.

Figure 2

Effect of PJ-34 on Salmonella-induced activation of NF- κ B. NF-κB is a central regulator of the intestinal epithelial cell innate immune response induced by infection with enteroinvasive bacteria. The Western blots illustrate (a) the expression of Iκ-Bα proteins in cytosolic extracts of Caco-2 cells exposed to wild-type S. Typhimurium strain SL1344 in the absence or presence of PJ-34. Actin works as a normalization of nuclear protein and (b) the expression of p65 NF-kB proteins in nuclear extracts of Caco-2 cells exposed to wild-type S. Typhimurium strain SL1344 in the absence or presence of PJ-34. Actin and LaminA/C work as a normalization of cytosolic and nuclear protein, respectively. The results shown are representative of 3 separate experiments.

Figure 3

Effect of PJ-34 on Salmonella-activated intracellular signals. Caco-2 cells were left untreated, or treated with 40 μM PJ-34, and then infected with wild-type S. Typhimurium strain SL1344 for the times indicated. Activations of the ERK, JNK, and p38 were analyzed in whole cell protein by immunoblotting with antibodies to phosphorylated (p) ERK, JNK, and p38 and total ERK and p38. The activation of ERK was enhanced by PJ-34 but not the JNK and p38. The results shown are representative of 3 separate experiments.

Figure 4

Effect of PJ-34 on Salmonella-activated intracellular signals. Caco-2 cells were left untreated, or treated with 40μM PJ-34, and then infected with wild-type S. Typhimurium strain SL1344 for the times indicated. Activation of the AKT pathway was analyzed in cell fraction (membrane part) by immunoblotting with antibodies to phosphorylated (p) AKT and total AKT. E-cadherin works as a normalization of membranous protein. The results shown are representative of 3 separate experiments. The activation and recruitment of Akt were not significantly suppressed by PJ-34.