Involvement of SRC family kinases in substance P-induced chemokine production in mouse pancreatic acinar cells and its significance in acute pancreatitis

Abstract

Substance P is known to play a key role in the pathogenesis of acute pancreatitis. Src family kinases (SFKs) are known to be involved in cytokine signaling. However, the involvement of SFKs in substance P-induced chemokine production and its role in acute pancreatitis have not been investigated yet. To that end, we have used primary preparations of mouse pancreatic acinar cells as our model to show that substance P/neurokinin 1 receptor (NK1R) induced activation of SFKs. SFKs mediated the activation of mitogen-activated protein kinases [extracellular signal-regulated kinase (ERK), c-Jun NH(2)-terminal kinase (JNK)], transcription factors [signal transducer and activator of transcription (STAT) 3, nuclear factor (NF) kappaB, activator protein-1 (AP-1)], and production of chemokines in pancreatic acinar cells. We further tested the significance of the SFK signaling pathway in acute pancreatitis. Our results show, for the first time, that treatment of mice with the potent and selective SFK inhibitor PP2 [4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo [3,4-D] pyrimidine], but not its negative inhibitor PP3 (4-amino-7-phenylpyrazol [3,4-D] pyrimidine), reduced the severity of pancreatitis. This was proven by significant attenuation of hyperamylasemia, pancreatic myeloperoxidase activity, chemokines, and water content. Histological evidence of diminished pancreatic injury also confirmed the protective effect of the inhibition of SFKs. Moreover, treatment with the substance P receptor antagonist CP96345 [(2S,3S)-cis-2-(diphenylmethyl)-N-((2-methoxyphenyl)-methyl)-1-azabicyclo(2.2.2.)-octan-3-amine] attenuated acute pancreatitis-induced activation of SFKs, ERK, JNK, STAT3, NFkappaB, and AP-1. The proposed signaling pathway through which substance P mediates acute pancreatitis is through substance P/NK1R-SFKs-(ERK, JNK)-(STAT3, NFkappaB, AP-1) chemokines. In light of our study, we propose that drugs targeting the substance P-mediated signaling pathways could prove beneficial in improving treatment efficacy in acute pancreatitis.

Fig. 1.

Substance P (SP)/NK1R induces a time-dependent increase and decrease in phosphorylation of Src family (Tyr416) in mouse pancreatic acinar cells. Freshly isolated pancreatic acini were incubated with either 1 μM SP/vehicle (saline) for 0, 3, 5, 10, 15, 30, and 45 min at 37°C or preincubated with 1 μM CP96345 for 30 min followed by stimulation with 1 μM SP for 10 min. The cells were lysed, and cell proteins were subjected to Western blot analysis using antibodies against phospho-Src family (Tyr416) and HPRT (a and c). Densitometric analysis of Western blot experiments were performed, and the group data from three independent preparations (n = 3) are presented in b and d. The results are representative of three independent (n = 3) experiments. Results shown are the means + S.E. ^*, P ≤ 0.05 compared with 0-min control; +, P ≤ 0.05 compared with SP.

Fig. 2.

Substance P (SP)-induced activation of ERK and JNK is mediated by SFKs in pancreatic acinar cells. Freshly isolated pancreatic acini were preincubated with PP2 at 1 or 10 μM or vehicle (dimethyl sulfoxide) for 30 min at 37°C followed by stimulation with 1 μM SP for 10 min at 37°C. Cells were subsequently lysed, and cell proteins were subjected to Western blot analysis using antibodies against phospho-Src family (Tyr416), phospho-ERK, phospho-JNK, and HPRT (a). The phosphorylated subunits, such as the p-Src family, p-44 ERK, p-42 ERK, p-54 JNK, and p-46 JNK, have been quantified. Densitometric analyses of Western blot experiments were performed, and the group data from three independent preparations (n = 3) are presented in b to d. Results shown are the means + S.E. ^*, P ≤ 0.05 compared with control; +, P ≤ 0.05 compared with SP.

Fig. 3.

SFKs are involved in SP-induced STAT3, NFκB, and AP-1 activation in pancreatic acinar cells. Freshly isolated pancreatic acini were preincubated with PP2 at 1 and 10 μM or vehicle (dimethyl sulfoxide) followed by stimulation with 1 μM SP for 45 min. The cells were separated from incubation medium by centrifugation. The pellet (cells) was used for STAT3 (a), NFκB (b), and AP-1 (c) nuclear extraction. STAT3, NFκB, and AP-1 DNA-binding assays were then carried out as described under Materials and Methods. The results are representative of three independent (n = 3) experiments. Results shown are the means + SE. ^*, P ≤ 0.05 compared with control; +, P ≤ 0.05 compared with SP.

Fig. 4.

SFKs are involved in the secretion of CC and CXC chemokines in pancreatic acinar cells. Freshly isolated pancreatic acini were preincubated with either PP2 at 1 and 10 μM or PP3 at 1 μM for 30 min followed by stimulation with 1 μM SP for 45 min. The supernatant was used to measure MCP-1 (a), MIP-1α (b), and MIP-2 (c) levels by ELISA as described under Materials and Methods. The results are representative of three independent (n = 3) experiments. Results shown are the means + S.E. ^*, P ≤ 0.05 compared with control; +, P ≤ 0.05 compared with SP.

Fig. 5.

Effects of prophylactic and therapeutic PP2 administration on the severity of acute pancreatitis. Mice (n = 10 in each group) were given 10 hourly injections of caerulein (50 μg/kg i.p.). PP2 or PP3 was administered to mice intraperitoneally either prophylactically (1 h before) or therapeutically 1 h after the first caerulein injection. One hour after the last caerulein injection, mice were sacrificed by an intraperitoneal injection of a lethal dose of pentobarbitone. Plasma amylase activity (a and b) and pancreatic edema (water content; c and d) were determined as described under Materials and Methods. Results shown are the means + S.E. ^*, P ≤ 0.05 when caerulein- or PP3-treated animals were compared with placebo-treated animals. +, P ≤ 0.05 when PP2-treated animals were compared with caerulein-treated animals.

Fig. 6.

Morphological changes in mouse pancreas on induction of acute pancreatitis with/without prophylactic and therapeutic treatment with PP2 or PP3. Representative micrographs from each group were shown (×400 magnifications). A, control, no pancreatitis. B, caerulein-induced acute pancreatitis. C, caerulein-induced acute pancreatitis in mice administered 1 mg/kg PP3 (prophylactic). D, caerulein-induced acute pancreatitis in mice administered 1 mg/kg PP3 (therapeutic). E, caerulein-induced acute pancreatitis in mice administered 1 mg/kg PP2 (prophylactic). F, caerulein-induced acute pancreatitis in mice administered 1 mg/kg PP2 (therapeutic).

Fig. 7.

Involvement of SFKs in the mobilization of pancreatic neutrophils and chemokine in acute pancreatitis. Mice (n = 10 in each group) were given 10 hourly injections of caerulein (50 μg/kg i.p.). PP2 was administered in mice at doses of 0.5, 1, and 1.5 mg/kg i.p. 1 h before or at a dose of 1 mg/kg 1 h after the first caerulein injection. One hour after the last caerulein injection, mice were sacrificed by an intraperitoneal injection of a lethal dose of pentobarbitone, and pancreatic MPO (a), MCP-1 (b), MIP-1α (c), and MIP-2 (d) levels were measured as described under Materials and Methods. Results shown are the means + S.E. ^*, P ≤ 0.05 when caerulein-treated animals were compared with placebo-treated animals. +, P ≤ 0.05 when PP2-treated animals were compared with caerulein-treated animals.

Fig. 8.

Inhibition of SFKs attenuated the activation of pancreatic STAT3, NFκB, AP-1, and MAP kinases in acute pancreatitis. Mice (n = 10 in each group) were given 10 hourly injections of caerulein (50 μg/kg i.p.). PP2 was administered to mice at a dose of 1 mg/kg i.p. 1 h before or 1 h after the first caerulein injection. One hour after the last caerulein injection, mice were sacrificed by an intraperitoneal injection of a lethal dose of pentobarbitone. The activation of pancreatic STAT3 (a), NFκB (b), AP-1 (c), and MAP kinases (d-f) was quantified as described under Materials and Methods. Results shown are the means + S.E. ^*, P ≤ 0.05 when caerulein-treated animals were compared with placebo-treated animals. +, P ≤ 0.05 when PP2-treated animals were compared with caerulein-treated animals.

Fig. 9.

Blockade of NK1R reduced acute pancreatitis-induced activation of MAP kinases and transcription factors STAT3, NFκB, and AP-1 in the pancreas. Mice (n = 6 in each group) were given 10 hourly injections of caerulein (50 μg/kg i.p.). CP96345 was administered to mice at a dose of 2.5 mg/kg i.p., 1 h before or 1 h after the first caerulein injection. One hour after the last caerulein injection, mice were sacrificed by an intraperitoneal injection of a lethal dose of pentobarbitone. The activation of pancreatic STAT3 and MAP kinases (a) and the densitometry analysis (c and d) were described under Materials and Methods. Results shown are the means + S.E. ^*, P ≤ 0.05 when caerulein-treated animals were compared with placebo-treated animals. +, P ≤ 0.05 when CP96345-treated animals were compared with caerulein-treated animals.