ABSTRACT
Title
The activity of rMCP-5 is modulated by Palmitoylethanolamide via the regulation of Microphtalmia-Associated Transcription Factor.
Authors
M. Cipriano
Doctorate School in Pharmaceutical Science
Dept. of Experimental Pharmacology, University of Naples FEDERICO II – Italy
Endocannabinoid Research Group
Doctorate School in Pharmaceutical Science
Dept. of Experimental Pharmacology, University of Naples FEDERICO II – Italy
Endocannabinoid Research Group
Abstract
We have previously demonstrated that chymase protein, rat mast cell protease-5 (rMCP-5), exhibits pro-inflammatory and pro-angiogenic properties during λ-carrageenin-induced granuloma formation in rats. Several evidences show that rMCP-5 transcription is controlled by a basic helix–loop–helix leucine zipper (bHLH-Zip) DNA-binding protein, the Microphtalmia-associated Transcription factor (MITF). MITF is predominantly expressed in mast cells (MCs), where it controls specific cellular functions. Animals with a mutation inMITF gene exhibit a deficit in MCs number and develop several diseases, thus evidencing a close correlation between MITF, chymase and MCs.
Palmitoylethanolamide (PEA), an endogenous compound, belongs to a class of lipid mediators showing anti-nociceptive, immuno-modulating and anti-inflammatory properties. Different mechanisms of action are responsible of PEA effects; the most known is the one related to the Autacoid Local Injury Antagonism, according to which PEA modulates MCs activation. We have already demonstrated that PEA inhibited granuloma formation, a model of chronic inflammation sustained by MCs activation. Therefore, in the present study, we investigate the effect of PEA on MITF activity in λ -carrageenin-induced granuloma formation in rats.
MITF activity was evaluated in a in vivo model of granuloma, a feature of chronic inflammation, that was induced by subcutaneous λ-carrageenin-soaked sponge implants, on the back of male Wistar rats. PEA was injected into each sponge at the concentration of 200, 400, 800 μg/mL. After 96 hours, granulomas were detached and tissues were processed to evaluate various parameters: the effect of PEA on chymase and MITF expression were determined using Western Blot analysis, while semi-quantitative RT-PCR was used to evaluate mRNA levels of rMCP-5. EMSA essay was performed to evaluate PEA action on MITF binding to the DNA. Furthermore, immuno-precipitation assay was performed to evaluate the effect of phosphorylation of MITF’s serine sites.
Local administration of PEA caused a concentration-dependent reduction in rMCP-5 mRNA levels and in chymase expression, in granulomatous tissue, in confront to animals treated only with λ-carrageenin. Moreover, MITF protein expression was reduced in λ-carrageenin tissues, while the amounts of phosphorylated protein were increased. PEA treatment restored MITF protein expression and, in parallel, reduced the amounts of phosphorylated MITF. Finally, PEA reduced the MITF/DNA binding, that was increased in tissues treated with λ-carrageenin alone.
In conclusion, our results show that PEA is able to modulate the levels of rMCP-5, a pro-inflammatory and pro-angiogenic MC protease, and the transcriptional activity and stability of MITF in λ-carrageenin-induced granuloma in rats.
Therefore, PEA may be a considerable modulator of chronic inflammatory diseases sustained by MC activation.
Acknowledgments: this work was partially supported by Innovet and Epitech Group.
Palmitoylethanolamide (PEA), an endogenous compound, belongs to a class of lipid mediators showing anti-nociceptive, immuno-modulating and anti-inflammatory properties. Different mechanisms of action are responsible of PEA effects; the most known is the one related to the Autacoid Local Injury Antagonism, according to which PEA modulates MCs activation. We have already demonstrated that PEA inhibited granuloma formation, a model of chronic inflammation sustained by MCs activation. Therefore, in the present study, we investigate the effect of PEA on MITF activity in λ -carrageenin-induced granuloma formation in rats.
MITF activity was evaluated in a in vivo model of granuloma, a feature of chronic inflammation, that was induced by subcutaneous λ-carrageenin-soaked sponge implants, on the back of male Wistar rats. PEA was injected into each sponge at the concentration of 200, 400, 800 μg/mL. After 96 hours, granulomas were detached and tissues were processed to evaluate various parameters: the effect of PEA on chymase and MITF expression were determined using Western Blot analysis, while semi-quantitative RT-PCR was used to evaluate mRNA levels of rMCP-5. EMSA essay was performed to evaluate PEA action on MITF binding to the DNA. Furthermore, immuno-precipitation assay was performed to evaluate the effect of phosphorylation of MITF’s serine sites.
Local administration of PEA caused a concentration-dependent reduction in rMCP-5 mRNA levels and in chymase expression, in granulomatous tissue, in confront to animals treated only with λ-carrageenin. Moreover, MITF protein expression was reduced in λ-carrageenin tissues, while the amounts of phosphorylated protein were increased. PEA treatment restored MITF protein expression and, in parallel, reduced the amounts of phosphorylated MITF. Finally, PEA reduced the MITF/DNA binding, that was increased in tissues treated with λ-carrageenin alone.
In conclusion, our results show that PEA is able to modulate the levels of rMCP-5, a pro-inflammatory and pro-angiogenic MC protease, and the transcriptional activity and stability of MITF in λ-carrageenin-induced granuloma in rats.
Therefore, PEA may be a considerable modulator of chronic inflammatory diseases sustained by MC activation.
Acknowledgments: this work was partially supported by Innovet and Epitech Group.