Src kinases play a novel dual role in acute pancreatitis affecting severity but no role in stimulated enzyme secretion

Abstract

In pancreatic acinar cells, the Src family of kinases (SFK) is involved in the activation of several signaling cascades that are implicated in mediating cellular processes (growth, cytoskeletal changes, apoptosis). However, the role of SFKs in various physiological responses such as enzyme secretion or in pathophysiological processes such as acute pancreatitis is either controversial, unknown, or incompletely understood. To address this, in this study, we investigated the role/mechanisms of SFKs in acute pancreatitis and enzyme release. Enzyme secretion was studied in rat dispersed pancreatic acini, in vitro acute-pancreatitis-like changes induced by supramaximal COOH-terminal octapeptide of cholecystokinin (CCK). SFK involvement assessed using the chemical SFK inhibitor (PP2) with its inactive control, 4-amino-7-phenylpyrazol[3,4-d]pyrimidine (PP3), under experimental conditions, markedly inhibiting SFK activation. In CCK-stimulated pancreatic acinar cells, activation occurred of trypsinogen, various MAP kinases (p42/44, JNK), transcription factors (signal transducer and activator of transcription-3, nuclear factor-κB, activator protein-1), caspases (3, 8, and 9) inducing apoptosis, LDH release reflective of necrosis, and various chemokines secreted (monocyte chemotactic protein-1, macrophage inflammatory protein-1α, regulated on activation, normal T cell expressed and secreted). All were inhibited by PP2, not by PP3, except caspase activation leading to apoptosis, which was increased, and trypsin activation, which was unaffected, as was CCK-induced amylase release. These results demonstrate SFK activation is playing a dual role in acute pancreatitis, inhibiting apoptosis and promoting necrosis as well as chemokine/cytokine release inducing inflammation, leading to more severe disease, as well as not affecting secretion. Thus, our studies indicate that SFK is a key mediator of inflammation and pancreatic acinar cell death in acute pancreatitis, suggesting it could be a potential therapeutic target in acute pancreatitis.

Keywords: carboxy-terminal octapeptide of cholecystokinin; caspases; chemokines; pancreatic acini; signaling.

Fig. 1.

Effect of 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) and 4-amino-7-phenylpyrazol[3,4-d]pyrimidine (PP3) on COOH-terminal octapeptide of cholecystokinin (CCK)-mediated stimulation of the Src family of kinases (SFKs), p42/44, and JNK in pancreatic acinar cells. Rat pancreatic acinar cells were pretreated with no additions or with PP2 (10 μM) or PP3 (10 μM) for 1 h and then incubated with no additions (control) (all with final-0.1% DMSO) or with 100 nM CCK for 2 h and then lysed. Whole cell lysates were submitted to SDS-PAGE and transferred to nitrocellulose membranes. To determine SFK (A), p42/44 (B), and JNK (C) activity, we assessed the phosphorylation of Y416 Src, Y202/204 p42/44, and p-T183/Y185 JNK that correlate with their activity. Membranes were analyzed using anti-pY416 Src, anti-pY202/204 p42/44, and anti-p-T183/Y185 JNK antibodies. Total Src, JNK, and p42/44 antibodies were used to verify loading of equal amounts of protein. The bands were visualized using chemoluminescence, and quantification of phosphorylation was assessed using scanning densitometry. Top, a representative experiment of 3 others; bottom, each set is the mean of 5 experiments. Lanes in top are in the same order as shown for the means. *P < 0.05 vs. control, ∞P < 0.05 vs. PP3 alone, and $P < 0.05 comparing stimulants (CCK) vs. stimulants preincubated with PP2 or PP3, respectively.

Fig. 2.

Effect of PP2 and PP3 on CCK-mediated stimulation of nuclear transcription factors [nuclear factor-κB (NF-κB, A), signal transducer and activator of transcription (STAT)-3 (B), and activator protein (AP-1, C)] in pancreatic acinar cells. Freshly isolated pancreatic acini were preincubated with no additions or with PP2 (10 μM) or PP3 (10 μM) followed by stimulation with 100 nM CCK for 2 h. The nuclear extracts were used for STAT-3, NF-κB, and AP-1 DNA-binding assays that were carried out as described in materials and methods. ns, Not significant. The results are means ± SE from 5 independent experiments.

Fig. 3.

Effect of PP2 and PP3 on CCK-mediated secretion of macrophage inflammatory protein (MIP)-2 or monocyte chemotactic protein-1 (MCP-1) in pancreatic acinar cells. Freshly isolated rat pancreatic acini were preincubated with either 10 μM PP2 or PP3 for 1 h followed by stimulation with 100 nM CCK for 2 h as described above. The supernatant was used to measure MIP-2 (A) and MCP-1 (B) levels by ELISA as described in materials and methods. Results shown are means ± SE of 4 independent experiments. The basal absolute values for chemokine release in the pancreatic acinar were 0.75 ± 0.21 ng/ml for MIP-2 and 0.62 ± 0.11 ng/ml for MCP-1. *P < 0.05 for CCK, PP2-CCK, or PP3-CCK compared with control, PP2, or PP3 alone, respectively. #P < 0.05, PP2 preincubated cells vs. control.

Fig. 4.

Effect of PP2 and PP3 on CCK-mediated secretion of MIP-1α or regulated on activation, normal T cell expressed and secreted (RANTES) in pancreatic acinar cells. Freshly isolated rat pancreatic acini were preincubated with either 10 μM PP2 or PP3 for 1 h followed by stimulation with 100 nM CCK for 2 h (MIP-1α) or 30 min (RANTES). The supernatant was used to measure MIP-1α (A) and RANTES (B) levels by ELISA as described in materials and methods. Results shown are means ± SE of 4 independent experiments. The basal absolute values for chemokine release in the pancreatic acinar were 73.4 ± 12.9 pg/ml for MIP-1-α and 1.91 ± 0.36 ng/ml for RANTES. *P < 0.05 for CCK, PP2-CCK, or PP3-CCK compared with control, PP2, or PP3 alone, respectively.

Fig. 5.

Effect of inhibition of SFK on CCK-mediated lactate dehydrogenase (LDH) release in pancreatic acini. Freshly isolated rat pancreatic acini were preincubated with either 10 μM PP2 or PP3 for 1 h followed by stimulation with 100 nM CCK for 2 h. Acinar cell necrosis was measured by the percentage of total LDH released in the medium, as described in materials and methods. Results are representative of 4 independent (n = 4) experiments. Results shown are means ± SE. *P < 0.05 comparing control, PP2, or PP3 alone vs. CCK, PP2-CCK, or PP3-CCK, respectively.

Fig. 6.

Effect of inhibition of SFK on CCK-mediated caspase activation in pancreatic acini. Freshly isolated rat pancreatic acini were preincubated with either 10 μM PP2 or PP3 for 1 h followed by stimulation with 100 nM CCK for 4 h. Caspase-3, -8, and -9 activities were measured as described in materials and methods in the lysates of isolated pancreatic acini. Results shown are means ± SE of 4 independent experiments. *P < 0.05 vs. control, #P < 0.05 vs. PP2 alone, ∞P < 0.05 vs. PP3 alone, and **P < 0.05 comparing CCK alone vs. PP2-CCK.

Fig. 7.

Effect of inhibition of SFK on CCK-mediated trypsin activation in pancreatic acini. Freshly isolated rat pancreatic acini were preincubated with either 10 μM PP2 or PP3 for 1 h followed by stimulation with 100 nM CCK for 20 min. Trypsin activity was measured as described in materials and methods in the lysates of isolated pancreatic acini. Results shown are means of 4 independent experiments. *P < 0.05 vs. control, #P < 0.05 vs. PP2 alone, and ∞P < 0.05 vs. PP3 alone.