Dataset Information

Relative Role of Akt, ERK and CREB in Alcohol-Induced Microglia P2X4R Receptor Expression.

ABSTRACT:

Aims

Previously we have demonstrated altered microglia P2X4R expression in response to alcohol and pharmacological blockade with a selective P2X4R antagonist can reverse the action, suggesting that P2X4R play a role in mediating alcohol-induced effects on microglia. In the present study, we investigated the underlying signaling mediators, which may play a role in modulating P2X4R expression in microglia cells in response to alcohol.

Methods

Embryonic stem cell-derived microglia (ESdM) cells were used to investigate the potential mechanisms involved in the regulation of P2X4R in response to alcohol. Selective P2X4R antagonist and kinase inhibitors were used to further corroborate the signal transduction pathway through which alcohol modulates P2X4R expression in microglia.

Results

Alcohol (100 mM) suppressed phosphorylated AKT and ERK cascades in native ESdM cells. This alcohol-induced suppression was confirmed to be P2X4R-dependent through the use of a selective P2X4R antagonist and knockdown of P2XR4 by siRNA. Alcohol increased transcriptional activity of CREB. P2X4R antagonist blocked alcohol-induced effects on CREB, suggesting a P2X4R-mediated effect.

Conclusion

These findings provide important clues to the underlying mechanism of purinoceptors in alcohol-induced microglia immune suppression.

SUBMITTER: Gofman L

PROVIDER: S-EPMC5091293 | biostudies-literature | 2016 Nov

REPOSITORIES: biostudies-literature

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Publications

Relative Role of Akt, ERK and CREB in Alcohol-Induced Microglia P2X4R Receptor Expression.

Gofman Larisa L Fernandes Nicole C NC Potula Raghava R

Alcohol and alcoholism (Oxford, Oxfordshire) 20160305 6

<h4>Aims</h4>Previously we have demonstrated altered microglia P2X4R expression in response to alcohol and pharmacological blockade with a selective P2X4R antagonist can reverse the action, suggesting that P2X4R play a role in mediating alcohol-induced effects on microglia. In the present study, we investigated the underlying signaling mediators, which may play a role in modulating P2X4R expression in microglia cells in response to alcohol.<h4>Methods</h4>Embryonic stem cell-derived microglia (E ...[more]

PMID: 26946194

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Project description:Microglia are resident immune cells in the central nervous system that become activated and produce pro-inflammatory and neurotrophic factors upon activation of various cell-surface receptors. The P2X(4) receptor (P2X(4)R) is a sub-type of the purinergic ion-channel receptors expressed in microglia. P2X(4)R expression is up-regulated under inflammatory or neurodegenerative conditions, and this up-regulation is implicated in disease pathology. However, the molecular mechanism underlying up-regulation of P2X(4)R in microglia remains unknown. In the present study, we investigated the intracellular signal transduction pathway that promotes P2X(4)R expression in microglia in response to fibronectin, an extracellular matrix protein that has previously been shown to stimulate P2X(4)R expression. We found that in fibronectin-stimulated microglia, activation of phosphatidylinositol 3-kinase (PI3K)-Akt and mitogen-activated protein kinase kinase (MAPK kinase, MEK)-extracellular signal-regulated kinase (ERK) signalling cascades occurred divergently downstream of Src-family kinases (SFKs). Pharmacological interference of PI3K-Akt signalling inhibited fibronectin-induced P2X(4)R gene expression. Activation of PI3K-Akt signalling resulted in a decrease in the protein level of the transcription factor p53 via mouse double minute 2 (MDM2), an effect that was prevented by MG-132, an inhibitor of the proteasome. In microglia pre-treated with MG-132, fibronectin failed to up-regulate P2X(4)R expression. Conversely, an inhibitor of p53 caused increased expression of P2X(4)R, implying a negative regulatory role of p53. On the other hand, inhibiting MEK-ERK signalling activated by fibronectin suppressed an increase in P2X(4)R protein but interestingly did not affect the level of P2X(4)R mRNA. We also found that fibronectin stimulation resulted in the activation of the translational factor eIF4E via MAPK-interacting protein kinase-1 (MNK1) in an MEK-ERK signalling-dependent manner, and an MNK1 inhibitor attenuated the increase in P2X(4)R protein. Together, these results suggest that the PI3K-Akt and MEK-ERK signalling cascades have distinct roles in the up-regulation of P2X(4)R expression in microglia at transcriptional and post-transcriptional levels, respectively.

Project description:BackgroundAlcoholic liver disease (ALD) is commonly associated with intestinal permeability. An unanswered question is why only a subset of heavy alcohol drinkers develop endotoxemia. Recent studies suggest that circadian disruption is the susceptibility factor for alcohol-induced gut leakiness to endotoxins. The circadian protein PER2 is increased after exposure to alcohol and siRNA knockdown of PER2 in vitro blocks alcohol-induced intestinal barrier dysfunction. We have shown that blocking CYP2E1 (i.e., important for alcohol metabolism) with siRNA inhibits the alcohol-induced increase in PER2 and suggesting that oxidative stress may mediate alcohol-induced increase in PER2 in intestinal epithelial cells. The aim of this study was to elucidate whether a mechanism incited by alcohol-derived oxidative stress mediates the transcriptional induction of PER2 and subsequent intestinal hyperpermeability.MethodsCaco-2 cells were exposed to 0.2% alcohol with or without pretreatment with modulators of oxidative stress or PKA activity. Permeability of the Caco-2 monolayer was assessed by transepithelial electrical resistance. Protein expression was measured by Western blot and mRNA with real-time polymerase chain reaction. Wild-type C57BL/6J mice were fed with alcohol diet (29% of total calories, 4.5% v/v) for 8 weeks. Western blot was used to analyze PER2 expression in mouse proximal colon tissue.ResultsAlcohol increased oxidative stress, caused Caco-2 cell monolayer dysfunction, and increased levels of the circadian clock proteins PER2 and CLOCK. These effects were mitigated by pretreatment of Caco-2 cells with an antioxidant scavenger. Alcohol-derived oxidative stress activated cAMP response element-binding (CREB) via the PKA pathway and increased PER2 mRNA and protein. Inhibiting CREB prevented the increase in PER2 and Caco-2 cell monolayer hyperpermeability.ConclusionsTaken together, these data suggest that strategies to reduce alcohol-induced oxidative stress may alleviate alcohol-mediated circadian disruption and intestinal leakiness, critical drivers of ALD.

Dataset Information

Relative Role of Akt, ERK and CREB in Alcohol-Induced Microglia P2X4R Receptor Expression.

Aims

Methods

Results

Conclusion

Publications

Relative Role of Akt, ERK and CREB in Alcohol-Induced Microglia P2X4R Receptor Expression.

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