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

Ramnath RD, Sun J, Adhikari S, Bhatia M - J. Cell. Mol. Med. (2007 Nov-Dec)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, National University of Singapore, Singapore.

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.

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ERK1/2-mediated NFκB activation is involved in substance P-induced chemokine synthesis. Substance Pinduced ERK1/2 phosphorylation and activation mediate NFκB 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 NFκB 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).
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fig03: ERK1/2-mediated NFκB activation is involved in substance P-induced chemokine synthesis. Substance Pinduced ERK1/2 phosphorylation and activation mediate NFκB 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 NFκB 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).

Mentions: To verify whether the MEK1 inhibitor PD98059 blocks phosphorylation of ERK1/2 in substance P-treated cells, mouse pancreatic acinar cells were pre-incubated with PD98059 for 1 hr followed by stimulation with 1 μM substance P for 45 min. Cells were then lysed, and cell proteins were subjected to Western blot analysis. As shown in Figure 2A, PD98059 attenuated substance P-induced phosphorylation of ERK1/2 in a dose-dependent manner. In Figure 2B, densitometry showed that the four different doses of PD98059 namely 10 μM, 30 μM, 50 μM and 100 μM significantly blocked phosphorylation of ERK1/2 when compared to substance P-only treated group. To confirm the role of ERK1/2 in substance Pinduced NFκB activation and chemokine production, we pre-treated the pancreatic acini with PD98059 for 1 hr followed by stimulation with 1 μM substance P for 45 min [11]. Our results, in Figure 3A, showed that pre-treatment with PD98059 significantly inhibited substance P-induced NFκB activation in a dosedependent manner which was followed by a dosedependent decrease in CC chemokines (3B) MCP-1, (3C) MIP-1 μ and CXC chemokine (3D) MIP-2. The negative control in which pancreatic acini were pretreated with 10 μM (the dose sufficient to block substance P-mediated activation) of PD98059 for 1 hr followed by stimulation with placebo for 45 min had no significant effect on MAPK, NFκB and chemokine production when compared to unstimulated controls (data not shown). These results indicate that substance P induces the production of chemokines MCP-1, MIP-1α and MIP-2 viathe ERK1/2-mediated NFκB signalling pathway.


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

Ramnath RD, Sun J, Adhikari S, Bhatia M - J. Cell. Mol. Med. (2007 Nov-Dec)

ERK1/2-mediated NFκB activation is involved in substance P-induced chemokine synthesis. Substance Pinduced ERK1/2 phosphorylation and activation mediate NFκB 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 NFκB 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).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4401295&req=5

fig03: ERK1/2-mediated NFκB activation is involved in substance P-induced chemokine synthesis. Substance Pinduced ERK1/2 phosphorylation and activation mediate NFκB 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 NFκB 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).
Mentions: To verify whether the MEK1 inhibitor PD98059 blocks phosphorylation of ERK1/2 in substance P-treated cells, mouse pancreatic acinar cells were pre-incubated with PD98059 for 1 hr followed by stimulation with 1 μM substance P for 45 min. Cells were then lysed, and cell proteins were subjected to Western blot analysis. As shown in Figure 2A, PD98059 attenuated substance P-induced phosphorylation of ERK1/2 in a dose-dependent manner. In Figure 2B, densitometry showed that the four different doses of PD98059 namely 10 μM, 30 μM, 50 μM and 100 μM significantly blocked phosphorylation of ERK1/2 when compared to substance P-only treated group. To confirm the role of ERK1/2 in substance Pinduced NFκB activation and chemokine production, we pre-treated the pancreatic acini with PD98059 for 1 hr followed by stimulation with 1 μM substance P for 45 min [11]. Our results, in Figure 3A, showed that pre-treatment with PD98059 significantly inhibited substance P-induced NFκB activation in a dosedependent manner which was followed by a dosedependent decrease in CC chemokines (3B) MCP-1, (3C) MIP-1 μ and CXC chemokine (3D) MIP-2. The negative control in which pancreatic acini were pretreated with 10 μM (the dose sufficient to block substance P-mediated activation) of PD98059 for 1 hr followed by stimulation with placebo for 45 min had no significant effect on MAPK, NFκB and chemokine production when compared to unstimulated controls (data not shown). These results indicate that substance P induces the production of chemokines MCP-1, MIP-1α and MIP-2 viathe ERK1/2-mediated NFκB signalling pathway.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, National University of Singapore, Singapore.

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.

Show MeSH
Related in: MedlinePlus