Effect of mitogen-activated protein kinases on chemokine synthesis induced by substance P in mouse pancreatic acinar cells

Abstract

Substance P, acting via its neurokinin 1 receptor (NK1 R), plays an important role in mediating a variety of inflammatory processes. Its interaction with chemokines is known to play a crucial role in the pathogenesis of acute pancreatitis. In pancreatic acinar cells, substance P stimulates the release of NFkappaB-driven chemokines. However, the signal transduction pathways by which substance P-NK1 R interaction induces chemokine production are still unclear. To that end, we went on to examine the participation of mitogen-activated protein kinases (MAPKs) in substance P-induced synthesis of pro-inflammatory chemokines, monocyte chemoanractant protein-1 (MCP-I), macrophage inflammatory protein-lalpha (MIP-lalpha) and macrophage inflammatory protein-2 (MIP-2), in pancreatic acini. In this study, we observed a time-dependent activation of ERK1/2, c-Jun N-terminal kinase (JNK), NFkappaB and activator protein-1 (AP-1) when pancreatic acini were stimulated with substance P. Moreover, substance P-induced ERK 1/2, JNK, NFkappaB and AP-1 activation as well as chemokine synthesis were blocked by pre-treatment with either extracellular signal-regulated protein kinase kinase 1 (MEK1) inhibitor or JNK inhibitor. In addition, substance P-induced activation of ERK 112, JNK, NFkappaB and AP-1-driven chemokine production were attenuated by CP96345, a selective NK1 R antagonist, in pancreatic acinar cells. Taken together, these results suggest that substance P-NK1 R induced chemokine production depends on the activation of MAPKs-mediated NFkappaB and AP-1 signalling pathways in mouse pancreatic acini.

1

Substance P stimulates ERK1/2 phosphorylation and NF_κB activation in a time-dependent manner. Substance P induces a time-dependent phosphorylation of ERK1/2 which coincides with a time-dependent activation of NF_κB. Freshly isolated pancreatic acini, obtained from three mice, were incubated with 1 μM substance P for 0, 3, 5, 10, 15, 45, 60, 120 min at 37°C. In some experiments, cells were lysed and cell proteins were subjected to Western blot analysis using antibodies against (A) phospho-ERK1/2, total ERK1/2 (B) Densitometric analysis of Western blot experiments from pancreatic acini. and (C) I_κB_α. In another experiment, the nuclear extract was used to isolate NF_κB and ELISA was carried out to detect activation of (D) NF_κB. The results are representative of three independent experiments. Results shown are the means + SE. # P≤0.05 when compared to 0 min control.

2

PD98059 dose-dependently decreases phosphorylation of ERK1/2 in pancreatic acini. PD98059 (an inhibitor of MEK1/2) effectively blocked phosphorylation of ERK1/2. Freshly isolated pancreatic acini, obtained from three mice, were pre-incubated with PD98059 at different doses of 10 μM, 30 μM, 50 μM, 100 μM for 1 hr at 37°C followed by stimulation with 1 μM substance P for 45 min at 37°C. Cells were subsequently lysed, and cell proteins were subjected to Western blot analysis using antibodies against (A) phospho-ERK1/2 and total ERK1/2. (B) Densitometric analysis of Western blot experiments from pancreatic acini. The results are representative of three independent experiments. Results shown are the means + SE. * P≤ 0.05 when compared to control, + P≤ 0.05 when compared to substance P (SP).

3

ERK1/2-mediated NF_κB activation is involved in substance P-induced chemokine synthesis. Substance Pinduced ERK1/2 phosphorylation and activation mediate NF_κB activation and chemokine production. Freshly isolated pancreatic acini, obtained from three mice, were pre-incubated with MEK1 inhibitor PD98059 for 1 hr followed by stimulation with 1 μM substance P for 45 min. Acini were separated from incubation medium by centrifugation. (A) The pellet (acini) was used for NF_κB extraction and detection whereas the supernatant was used to measure (B) MCP-1, (C) MIP-1 and (D) MIP-2 levels by ELISA. The results are representative of three independent experiments. Results shown are the means + SE. * P≤0.05 when compared to control, + P≤0.05 when compared to substance P (SP).

4

Substance P induces phosphorylation of JNK and AP-1 (c-Jun) activation in a time-dependent manner. Substance P induces a timedependent phosphorylation of JNK which is in line with a timedependent activation of AP-1 (c-Jun). Freshly isolated pancreatic acini, obtained from three mice, were incubated with 1 μM substance P for 0, 3, 5, 10, 15, 45, 60, 120 min at 37°C. In some experiments, cells were lysed, and cell proteins were subjected to Western blot analysis using antibodies against (A) phospho-JNK, total JNK. In another experiment, the nuclear extract was used to isolate AP-1 (c-Jun) and ELISA was carried out to detect activation of (C) AP-1 (c-Jun). (B) Densitometric analysis of Western blot experiments from pancreatic acini. The results are representative of three independent experiments. Results shown are the means + SE. #P ≤0.05 when compared to control (0 min).

5

SP600125 decreases phosphorylation of JNK in pancreatic acini. SP600125 (JNK inhibitor) effectively blocked phosphorylation of JNK. Freshly isolated pancreatic acini, obtained from three mice, were pre-incubated with SP600125 at different doses of 10 μM, 25 μM, 50 μM, 100 μM for 1 hr at 37°C followed by stimulation with 1 μM substance P for 45 min at 37°C. Cells were subsequently lysed, and cell proteins were subjected to Western blot analysis using antibodies against (A) phospho-JNK and total JNK. (B) Densitometric analysis of Western blot experiments from pancreatic acini. The results are representative of three independent experiments. Results shown are the means + SE. * P≤0.05 when compared to control, + Pα0.05 when compared to substance P (SP).

6

JNK is involved in substance P-induced AP-1 (c-Jun) activation and chemokines synthesis. Substance Pinduced JNK phosphorylation and activation mediate AP-1 (c-Jun) activation and chemokine production. Freshly isolated pancreatic acini, obtained from three mice, were pre-incubated with JNK inhibitor SP600125 for 1 hr followed by stimulation with 1μM substance P for 45 min. Acini were separated from incubation medium by centrifugation. (A) The pellet (acini) was used for AP-1 (c-Jun) extraction and detection whereas the supernatant was used to measure (B) MCP-1, (C) MIP-1_κ and (D) MIP-2 levels by ELISA. The results are representative of three independent experiments. Results shown are the means + SE. * P≤0.05 when compared to control, + P≤0.05 when compared to substance P (SP).

7

Substance P-induced ERK1/2 and JNK cross activate NF_κ B and AP-1 (c-Jun). Freshly isolated pancreatic acini, obtained from three mice, were pre-incubated with either MEK1 inhibitor PD98059 or JNK inhibitor SP600125 for 1 hr followed by stimulation with 1 μM substance P for 45 min. Acini were separated from incubation medium by centrifugation. The pellet (acini) was used for extraction and detection of (A) NF_κB activation and (B) AP-1 (c-Jun) activation. The results are representative of three independent experiments. Results shown are the means + SE. *P≤0.05 when compared to control, +P≤0.05 when compared to substance P (SP).

8

Substance P-NK1R interaction is involved in ERK1/2 and JNK activation. Freshly isolated pancreatic acini, obtained from three mice, were pre-incubated with 1 μM CP96345 for half an hour at 37°C followed by stimulation with 1 μM substance P for 45 min for ERK1/2 and JNK at 37°C. Cells were subsequently lysed, and cell proteins were subjected to Western blot analysis using antibodies against (A) phospho-ERK, total ERK1/2 (B) phospho-JNK, total JNK. Corresponding densitometric analysis of Western blot experiments from pancreatic acini. The results are representative of three independent experiments. Results shown are the means + SE. * P≤ 0.05 when compared to control, + P ≤0.05 when compared to substance P (SP).

9

NK1R is involved in substance P-induced NF_κ B and AP-1 activation as well as MCP-1, MIP-1_α and MIP-2 production. Freshly isolated pancreatic acini, obtained from three mice, were pre-incubated with 1 μgM CP96345 for half an hour followed by stimulation with 1 μM substance P for 45 min. Acini were separated from incubation medium by centrifugation. The pellet was used for (A) NF_κB and (B) AP-1 extraction and NF_κB (p65) and AP-1 (c-Jun) DN-Abinding assays were carried out. The supernatant was used to measure (C) MCP-1, (D) MIP-1_α and (E) MIP-2 levels by ELISA. The results are representative of three independent experiments. Results shown are the means + SE. * P≤0.05 when compared to control, + P≤0.05 when compared to substance P (SP).

10

A schematic representation of the signalling cascade that mediates substance P-NK1-R induced chemokine production in pancreatic acinar cells. Substance P (SP) induces activation of MAP kinases ERK and JNK in pancreatic acinar cells. Moreover, substance P-induced phosphorylation of ERK and JNK mediates the activation of transcription factors NF_κB and AP-1, thus resulting in increased secretion of pro-inflammatory mediators MCP- 1, MIP-1_α and MIP-2 in acinar cells. Substance P-induced ERK1/2, JNK, NF_κB, AP-1 activation and chemokine synthesis are depended on NK1R.