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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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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-1κ 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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fig06: 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-1κ 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 make sure that SP600125, an inhibitor of JNK which prevents the phosphorylation of JNK substrates by blocking the ATP-binding domain of JNKs, inhibits phosphorylation of JNK in substance P-treated cells, mouse pancreatic acinar cells were pre-incubated with SP600125 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 5A, SP600125 attenuated substance P-induced phosphorylation of JNK. In Figure 5B, densitometry showed that 10 μM, 25 μM, 50 μM and 100 μM of SP600125 significantly blocked phosphorylation of JNK when compared to substance P- only treated group. To determine if substance P-induced synthesis of chemokines MCP-1, MIP-1α and MIP-2 and AP-1 (c-Jun) activation are mediated through JNK, pancreatic acini were pretreated with SP600125 for 1 hr followed by stimulation with 1 μM substance P for 45 min. Our data in Figure 6A showed that SP600125 inhibited substance P-induced AP-1 (c-Jun) activation in a dosedependent manner. As shown in Figure 6B, C and D, pre-treatment with SP600125 caused a concentration- dependent attenuation of substance P-induced production of MCP-1, MIP-1α and MIP-2. The negative control in which pancreatic acini were pre-treated with 10 μM (the dose sufficient to block substance P-mediated activation) of SP600125 for 1 hr followed by stimulation with placebo for 45 min had no significant effect on JNK, AP-1 (c-Jun) and chemokine production when compared to unstimulated controls (data not shown). These results demonstrate that substance P-induced synthesis of MCP-1, MIP-1α and MIP-2 is mediated by JNK/AP-1 (c-Jun) 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)

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-1κ 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

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getmorefigures.php?uid=PMC4401295&req=5

fig06: 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-1κ 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 make sure that SP600125, an inhibitor of JNK which prevents the phosphorylation of JNK substrates by blocking the ATP-binding domain of JNKs, inhibits phosphorylation of JNK in substance P-treated cells, mouse pancreatic acinar cells were pre-incubated with SP600125 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 5A, SP600125 attenuated substance P-induced phosphorylation of JNK. In Figure 5B, densitometry showed that 10 μM, 25 μM, 50 μM and 100 μM of SP600125 significantly blocked phosphorylation of JNK when compared to substance P- only treated group. To determine if substance P-induced synthesis of chemokines MCP-1, MIP-1α and MIP-2 and AP-1 (c-Jun) activation are mediated through JNK, pancreatic acini were pretreated with SP600125 for 1 hr followed by stimulation with 1 μM substance P for 45 min. Our data in Figure 6A showed that SP600125 inhibited substance P-induced AP-1 (c-Jun) activation in a dosedependent manner. As shown in Figure 6B, C and D, pre-treatment with SP600125 caused a concentration- dependent attenuation of substance P-induced production of MCP-1, MIP-1α and MIP-2. The negative control in which pancreatic acini were pre-treated with 10 μM (the dose sufficient to block substance P-mediated activation) of SP600125 for 1 hr followed by stimulation with placebo for 45 min had no significant effect on JNK, AP-1 (c-Jun) and chemokine production when compared to unstimulated controls (data not shown). These results demonstrate that substance P-induced synthesis of MCP-1, MIP-1α and MIP-2 is mediated by JNK/AP-1 (c-Jun) 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