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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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TNFα enhancement and PI3Kβ inhibition of upregulation both act through p38Mapk.Experiments similar to those in Fig 3 were performed with the addition of treatment of cells with nicotine (Nic) or choline (Ch) and each with PI828, TNFα or both as indicated. At 24 hours post-treatment specific [3H]Eb binding by crude cell membranes was measured. Each bar is the average of no less than 3 independent experiments and error bars = +/- SEM. The significance values (P) are calculated from Student’s t-test of the indicated pairing. Upregulation is indicated in the light grey and enhancement of upregulation is in dark grey.
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pone.0143319.g004: TNFα enhancement and PI3Kβ inhibition of upregulation both act through p38Mapk.Experiments similar to those in Fig 3 were performed with the addition of treatment of cells with nicotine (Nic) or choline (Ch) and each with PI828, TNFα or both as indicated. At 24 hours post-treatment specific [3H]Eb binding by crude cell membranes was measured. Each bar is the average of no less than 3 independent experiments and error bars = +/- SEM. The significance values (P) are calculated from Student’s t-test of the indicated pairing. Upregulation is indicated in the light grey and enhancement of upregulation is in dark grey.

Mentions: In previous reports we demonstrated that TNFα through activation of TNFR1 leads to p38Mapk activity and the strong enhancement of α4β2 receptor ligand and choline-mediated upregulation [9,10,14]. This is similar to the results in Fig 3 where the enhancement of nicotine or the HC3-sensitive component of choline-mediated upregulation by inhibition of PI3Kβ is itself inhibited by the block of p38Mapk by SB202190. However, PI3Kβ inhibition differs in that it is alone sufficient to produce limited upregulation whereas TNFα does not do this [9,10,14]. Nevertheless the convergence of these activates at p38Mapk is notable and suggests that PI3Kβ activity as a negative upstream regulator of p38Mapk would also impact upon TNFα/TNFR1 singling in this system. To test this possibility, transfected 293 cells were treated as above with various combinations of PI828 with or without co-applications of TNFα, choline and nicotine, respectively. The results of these experiments, which in part also overlap with and reproduce those of Fig 3, are shown in Fig 4. As anticipated, PI828 alone produces an approximately 2-fold increase in upregulation but also in the presence of either nicotine or choline where upregulation is significantly enhanced over the effect of either of these agents alone. When TNFα is co-applied with nicotine, upregulation is increased from ~5.5-fold to ~8-fold or for choline from ~4-fold to ~6.5-fold. The same results were also obtained when nicotine or choline were combined with PI828 and/or TNFα, respectively (Fig 4). When the same experimental treatments included co-application of SB202190, all enhancements of nicotine or choline [3H]Eb binding produced by PI828, TNFα or a combination of these was lost (Fig 4). Thus, the conclusion is supported that both enhanced upregulation by either TNFα or PI3Kβ inhibition both require and act through p38Mapk.


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)

TNFα enhancement and PI3Kβ inhibition of upregulation both act through p38Mapk.Experiments similar to those in Fig 3 were performed with the addition of treatment of cells with nicotine (Nic) or choline (Ch) and each with PI828, TNFα or both as indicated. At 24 hours post-treatment specific [3H]Eb binding by crude cell membranes was measured. Each bar is the average of no less than 3 independent experiments and error bars = +/- SEM. The significance values (P) are calculated from Student’s t-test of the indicated pairing. Upregulation is indicated in the light grey and enhancement of upregulation is in dark grey.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0143319.g004: TNFα enhancement and PI3Kβ inhibition of upregulation both act through p38Mapk.Experiments similar to those in Fig 3 were performed with the addition of treatment of cells with nicotine (Nic) or choline (Ch) and each with PI828, TNFα or both as indicated. At 24 hours post-treatment specific [3H]Eb binding by crude cell membranes was measured. Each bar is the average of no less than 3 independent experiments and error bars = +/- SEM. The significance values (P) are calculated from Student’s t-test of the indicated pairing. Upregulation is indicated in the light grey and enhancement of upregulation is in dark grey.
Mentions: In previous reports we demonstrated that TNFα through activation of TNFR1 leads to p38Mapk activity and the strong enhancement of α4β2 receptor ligand and choline-mediated upregulation [9,10,14]. This is similar to the results in Fig 3 where the enhancement of nicotine or the HC3-sensitive component of choline-mediated upregulation by inhibition of PI3Kβ is itself inhibited by the block of p38Mapk by SB202190. However, PI3Kβ inhibition differs in that it is alone sufficient to produce limited upregulation whereas TNFα does not do this [9,10,14]. Nevertheless the convergence of these activates at p38Mapk is notable and suggests that PI3Kβ activity as a negative upstream regulator of p38Mapk would also impact upon TNFα/TNFR1 singling in this system. To test this possibility, transfected 293 cells were treated as above with various combinations of PI828 with or without co-applications of TNFα, choline and nicotine, respectively. The results of these experiments, which in part also overlap with and reproduce those of Fig 3, are shown in Fig 4. As anticipated, PI828 alone produces an approximately 2-fold increase in upregulation but also in the presence of either nicotine or choline where upregulation is significantly enhanced over the effect of either of these agents alone. When TNFα is co-applied with nicotine, upregulation is increased from ~5.5-fold to ~8-fold or for choline from ~4-fold to ~6.5-fold. The same results were also obtained when nicotine or choline were combined with PI828 and/or TNFα, respectively (Fig 4). When the same experimental treatments included co-application of SB202190, all enhancements of nicotine or choline [3H]Eb binding produced by PI828, TNFα or a combination of these was lost (Fig 4). Thus, the conclusion is supported that both enhanced upregulation by either TNFα or PI3Kβ inhibition both require and act through p38Mapk.

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