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Upregulation of Nicotinic Acetylcholine Receptor alph4+beta2 through a Ligand-Independent PI3Kbeta Mechanism That Is Enhanced by TNFalpha and the Jak2/p38Mapk Pathways.

Rogers SW, Gahring LC - PLoS ONE (2015)

Bottom Line: Further, these processes are impacted upon by an AG-490 sensitive Jak2-associated pathway.Upregulation through the PI3Kbeta pathway did not require Akt.The findings also suggest how illness and metabolic stress could alter the expression of this important nicotinic receptor and novel avenues to intercede in modifying its expression.

View Article: PubMed Central - PubMed

Affiliation: Salt Lake City Veteran's Administration Geriatric Research, Education and Clinical Center, Salt Lake City, Utah, 84148, United States of America.

ABSTRACT
High affinity nicotine-binding sites in the mammalian brain are neuronal nicotinic acetylcholine receptors (nAChR) assembled from at least alpha4 and beta2 subunits into pentameric ion channels. When exposed to ligands such as nicotine, these receptors respond by undergoing upregulation, a correlate of nicotine addiction. Upregulation can be measured using HEK293 (293) cells that stably express alpha4 and beta2 subunits using quantification of [3H]epibatidine ([3H]Eb) binding to measure mature receptors. Treatment of these cells with choline also produces upregulation through a hemicholinium3 (HC3)-sensitive (choline kinase) and an HC3-insensitive pathway which are both independent of the mechanism used by nicotine for upregulation. In both cases, upregulation is significantly enhanced by the pro-inflammatory cytokine tumor necrosis factor alpha (TNFα) which signals through its receptor Tnfr1 to activate p38Mapk. Here we report that the inhibition of class1 phosphoinositide 3-kinases isoform PI3Kbeta using the selective antagonist PI828 is alone sufficient to produce upregulation and enhance both nicotine and choline HC3-sensitive mediated upregulation. Further, these processes are impacted upon by an AG-490 sensitive Jak2-associated pathway. Both PI3Kbeta (negative) and Jak2 (positive) modulation of upregulation converge through p38Mapk and both overlap with TNFalpha enhancement of this process. Upregulation through the PI3Kbeta pathway did not require Akt. Collectively these findings support upregulation of endogenous alpha4beta2 as a balance among cellular signaling networks that are highly responsive to multiple environmental, inflammatory and metabolic agents. The findings also suggest how illness and metabolic stress could alter the expression of this important nicotinic receptor and novel avenues to intercede in modifying its expression.

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Diagram summarizing the findings of this study.Assembly of mature α4β2 nAChRs from a precursor pool of individual subunits can be upregulated by nicotine, possibly acting as a chaperone to increase α4+β2 subunit association and subcellular transport. The mechanisms through which choline and TNFα influence this process is to ultimately increase β2 subunit production (favoring pentameric α4+β2 assembly) through a p38MAPK mechanism that is inhibited by SB202190. The activity of p38MAPK is balanced by constitutive inhibition by PI3kβ (inhibited by PI828) and promoted by an increase in Jak2 kinase activity (inhibited by AG490). Jak2 activation is stimulated by choline acting through a choline kinase (CK) pathway that is distinguished by hemicholinium3-sensitivity (HC3) pathway or signaling by the pro-inflammatory cytokine tumor necrosis factor alpha (TNFα) acting through its receptor, TNFR1. The choline mediated upregulation mechanism through the HC3-insensitve pathway (CK-, in light grey) is not yet characterized but also produces upregulation through increased β2 production.
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pone.0143319.g008: Diagram summarizing the findings of this study.Assembly of mature α4β2 nAChRs from a precursor pool of individual subunits can be upregulated by nicotine, possibly acting as a chaperone to increase α4+β2 subunit association and subcellular transport. The mechanisms through which choline and TNFα influence this process is to ultimately increase β2 subunit production (favoring pentameric α4+β2 assembly) through a p38MAPK mechanism that is inhibited by SB202190. The activity of p38MAPK is balanced by constitutive inhibition by PI3kβ (inhibited by PI828) and promoted by an increase in Jak2 kinase activity (inhibited by AG490). Jak2 activation is stimulated by choline acting through a choline kinase (CK) pathway that is distinguished by hemicholinium3-sensitivity (HC3) pathway or signaling by the pro-inflammatory cytokine tumor necrosis factor alpha (TNFα) acting through its receptor, TNFR1. The choline mediated upregulation mechanism through the HC3-insensitve pathway (CK-, in light grey) is not yet characterized but also produces upregulation through increased β2 production.

Mentions: When our results are placed into the context of cellular regulation there is the suggestion that quantitative modulation is a normal aspect of regulating α4β2 receptor numbers through the convergence of signaling networks that are responsive to diet (e.g., choline) and inflammation (Fig 8). This also suggest that upregulation to nicotine reflects an example of the impact by an exogenous agent that imbalances this finely-tuned regulatory interaction. Thus, continuous signaling through PI3Kβ is an important brake on producing high-affinity α4β2 sites whereas positive modulation through p38Mapk activation as during a pro-inflammatory event would be controlled by the TNFα/TNFR1 signaling intensity to Jak2 and enhanced activation of p38Mapk. Thus in terms α4β2 upregulation, p38Mapk serves as a metabolic ‘coincidence’ detector where convergence of Jak2 and PI3Kβ signaling is weighted and an outcome determined. The possibility that TNFR1 recruits and interacts directly with Jak, PI3K and p38Mapk has been proposed [58], and a direct interaction between PI3K and p38Mapk signaling is known [59]. The potential for TNFR1 forming the focus of such a cell-specific signaling complexes, especially recruitment of Jak2 into a complex that is sensitive to TNFα, is attractive [58,60–63]. Collectively, the enhancement of upregulation would include a summation of contributions by precursor pool status of α4 and β2, TNFR1/Jak2 upstream signaling to p38Mapk, signaling through the choline HC3-sensitive pathway to Jak2 and the direct disinhibition of p38Mapk by inhibiting constitutive PI3Kβ activity (Fig 8). While cross-talk between different cellular signaling pathways is common, they are also highly cell specific. Thus, confirmation of the relevance of this scheme to neuronal upregulation will be important towards understanding how this fits into the physiology contributing to the addiction phenotype. What does seem likely is that this important nicotinic receptor is subjected to significant modulation during periods of inflammatory illness and metabolic stress. Novel approaches to interfere with nicotine–mediated α4β2 upregulation are likely to be derived from a better understanding of these interactions.


Upregulation of Nicotinic Acetylcholine Receptor alph4+beta2 through a Ligand-Independent PI3Kbeta Mechanism That Is Enhanced by TNFalpha and the Jak2/p38Mapk Pathways.

Rogers SW, Gahring LC - PLoS ONE (2015)

Diagram summarizing the findings of this study.Assembly of mature α4β2 nAChRs from a precursor pool of individual subunits can be upregulated by nicotine, possibly acting as a chaperone to increase α4+β2 subunit association and subcellular transport. The mechanisms through which choline and TNFα influence this process is to ultimately increase β2 subunit production (favoring pentameric α4+β2 assembly) through a p38MAPK mechanism that is inhibited by SB202190. The activity of p38MAPK is balanced by constitutive inhibition by PI3kβ (inhibited by PI828) and promoted by an increase in Jak2 kinase activity (inhibited by AG490). Jak2 activation is stimulated by choline acting through a choline kinase (CK) pathway that is distinguished by hemicholinium3-sensitivity (HC3) pathway or signaling by the pro-inflammatory cytokine tumor necrosis factor alpha (TNFα) acting through its receptor, TNFR1. The choline mediated upregulation mechanism through the HC3-insensitve pathway (CK-, in light grey) is not yet characterized but also produces upregulation through increased β2 production.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4664291&req=5

pone.0143319.g008: Diagram summarizing the findings of this study.Assembly of mature α4β2 nAChRs from a precursor pool of individual subunits can be upregulated by nicotine, possibly acting as a chaperone to increase α4+β2 subunit association and subcellular transport. The mechanisms through which choline and TNFα influence this process is to ultimately increase β2 subunit production (favoring pentameric α4+β2 assembly) through a p38MAPK mechanism that is inhibited by SB202190. The activity of p38MAPK is balanced by constitutive inhibition by PI3kβ (inhibited by PI828) and promoted by an increase in Jak2 kinase activity (inhibited by AG490). Jak2 activation is stimulated by choline acting through a choline kinase (CK) pathway that is distinguished by hemicholinium3-sensitivity (HC3) pathway or signaling by the pro-inflammatory cytokine tumor necrosis factor alpha (TNFα) acting through its receptor, TNFR1. The choline mediated upregulation mechanism through the HC3-insensitve pathway (CK-, in light grey) is not yet characterized but also produces upregulation through increased β2 production.
Mentions: When our results are placed into the context of cellular regulation there is the suggestion that quantitative modulation is a normal aspect of regulating α4β2 receptor numbers through the convergence of signaling networks that are responsive to diet (e.g., choline) and inflammation (Fig 8). This also suggest that upregulation to nicotine reflects an example of the impact by an exogenous agent that imbalances this finely-tuned regulatory interaction. Thus, continuous signaling through PI3Kβ is an important brake on producing high-affinity α4β2 sites whereas positive modulation through p38Mapk activation as during a pro-inflammatory event would be controlled by the TNFα/TNFR1 signaling intensity to Jak2 and enhanced activation of p38Mapk. Thus in terms α4β2 upregulation, p38Mapk serves as a metabolic ‘coincidence’ detector where convergence of Jak2 and PI3Kβ signaling is weighted and an outcome determined. The possibility that TNFR1 recruits and interacts directly with Jak, PI3K and p38Mapk has been proposed [58], and a direct interaction between PI3K and p38Mapk signaling is known [59]. The potential for TNFR1 forming the focus of such a cell-specific signaling complexes, especially recruitment of Jak2 into a complex that is sensitive to TNFα, is attractive [58,60–63]. Collectively, the enhancement of upregulation would include a summation of contributions by precursor pool status of α4 and β2, TNFR1/Jak2 upstream signaling to p38Mapk, signaling through the choline HC3-sensitive pathway to Jak2 and the direct disinhibition of p38Mapk by inhibiting constitutive PI3Kβ activity (Fig 8). While cross-talk between different cellular signaling pathways is common, they are also highly cell specific. Thus, confirmation of the relevance of this scheme to neuronal upregulation will be important towards understanding how this fits into the physiology contributing to the addiction phenotype. What does seem likely is that this important nicotinic receptor is subjected to significant modulation during periods of inflammatory illness and metabolic stress. Novel approaches to interfere with nicotine–mediated α4β2 upregulation are likely to be derived from a better understanding of these interactions.

Bottom Line: Further, these processes are impacted upon by an AG-490 sensitive Jak2-associated pathway.Upregulation through the PI3Kbeta pathway did not require Akt.The findings also suggest how illness and metabolic stress could alter the expression of this important nicotinic receptor and novel avenues to intercede in modifying its expression.

View Article: PubMed Central - PubMed

Affiliation: Salt Lake City Veteran's Administration Geriatric Research, Education and Clinical Center, Salt Lake City, Utah, 84148, United States of America.

ABSTRACT
High affinity nicotine-binding sites in the mammalian brain are neuronal nicotinic acetylcholine receptors (nAChR) assembled from at least alpha4 and beta2 subunits into pentameric ion channels. When exposed to ligands such as nicotine, these receptors respond by undergoing upregulation, a correlate of nicotine addiction. Upregulation can be measured using HEK293 (293) cells that stably express alpha4 and beta2 subunits using quantification of [3H]epibatidine ([3H]Eb) binding to measure mature receptors. Treatment of these cells with choline also produces upregulation through a hemicholinium3 (HC3)-sensitive (choline kinase) and an HC3-insensitive pathway which are both independent of the mechanism used by nicotine for upregulation. In both cases, upregulation is significantly enhanced by the pro-inflammatory cytokine tumor necrosis factor alpha (TNFα) which signals through its receptor Tnfr1 to activate p38Mapk. Here we report that the inhibition of class1 phosphoinositide 3-kinases isoform PI3Kbeta using the selective antagonist PI828 is alone sufficient to produce upregulation and enhance both nicotine and choline HC3-sensitive mediated upregulation. Further, these processes are impacted upon by an AG-490 sensitive Jak2-associated pathway. Both PI3Kbeta (negative) and Jak2 (positive) modulation of upregulation converge through p38Mapk and both overlap with TNFalpha enhancement of this process. Upregulation through the PI3Kbeta pathway did not require Akt. Collectively these findings support upregulation of endogenous alpha4beta2 as a balance among cellular signaling networks that are highly responsive to multiple environmental, inflammatory and metabolic agents. The findings also suggest how illness and metabolic stress could alter the expression of this important nicotinic receptor and novel avenues to intercede in modifying its expression.

Show MeSH
Related in: MedlinePlus