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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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Inhibition of PI3K is sufficient to upregulate [3H]Eb binding and enhance choline HC3-sensitive upregulation.Stably transfected 293 cells were either not treated (C) or treated with choline (Ch), hemicholinium-3 (HC3) or both (Ch+HC3) and separately nicotine (Nic) either in the presence or absence of LY294002 (LY) or Wortmannin (Wort), respectively. A) Cells were harvested 24 hours post treatment and specific binding by the α4β2 high affinity ligand [3H]Eb was measured (Methods). In all cases the results reflect the average of no less than 3 independent experiments. Upregulation is indicated in the light grey, enhancement of upregulation is in dark grey and stippled is the HC3-insensitive choline component. Error bars = +/- SEM. The significance values (P) are calculated from Student’s t-test of the indicated pairing. B) Western blot analysis of crude membranes prepared from choline-treated cells that received treatment as labeled and corresponded to those used for [3H]Eb binding to measure the relative expression of α4 or β2 subunits. The ratio of the band density for each subunit was measured, normalized to the control (1.0) and then plotted for each treatment. Above each lane is the change in the β2/α4 ratio calculated after normalization. The blot shown reflects a result that is typical for these experiments.
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pone.0143319.g001: Inhibition of PI3K is sufficient to upregulate [3H]Eb binding and enhance choline HC3-sensitive upregulation.Stably transfected 293 cells were either not treated (C) or treated with choline (Ch), hemicholinium-3 (HC3) or both (Ch+HC3) and separately nicotine (Nic) either in the presence or absence of LY294002 (LY) or Wortmannin (Wort), respectively. A) Cells were harvested 24 hours post treatment and specific binding by the α4β2 high affinity ligand [3H]Eb was measured (Methods). In all cases the results reflect the average of no less than 3 independent experiments. Upregulation is indicated in the light grey, enhancement of upregulation is in dark grey and stippled is the HC3-insensitive choline component. Error bars = +/- SEM. The significance values (P) are calculated from Student’s t-test of the indicated pairing. B) Western blot analysis of crude membranes prepared from choline-treated cells that received treatment as labeled and corresponded to those used for [3H]Eb binding to measure the relative expression of α4 or β2 subunits. The ratio of the band density for each subunit was measured, normalized to the control (1.0) and then plotted for each treatment. Above each lane is the change in the β2/α4 ratio calculated after normalization. The blot shown reflects a result that is typical for these experiments.

Mentions: In previous reports we noted that the potent class I phosphoinositide 3-kinases (PI3K) pan-inhibitor LY294002 was alone sufficient to produce significant upregulation of the 3H-epibadidine ([3H]Eb) sites associated with increased receptor expression in 293 cells stably transfected with the nicotinic acetylcholine receptor subunits α4 and β2 [9,10,14]. Also, in response to elevated choline or nicotine, the co-application of LY294002 greatly enhanced the upregulation of [3H]Eb produced by either agent alone. This result was confirmed and extended by measuring the effect of two widely used and well characterized as pan-inhibitors of class1 p110 PI3K catalytic activity; LY294002 and Wortmannin [16,19,20], on upregulation both alone and in the presence of choline (Fig 1A). Both inhibitors promoted upregulation of [3H]Eb binding in the absence of receptor ligand. Because the LY294002 effect was more consistent with less impact on cell morphology compared to wortmannin (Fig 1A and not shown), this agent was used for essentially all of the subsequent experiments. Choline alone produces upregulation through a hemicholinium-3 (HC3) sensitive, choline kinase (CK)-dependent [21], and a HC3-insensensitive pathway [10], we examined which of these pathways was most impacted by PI3K. The results reveal that LY294002-associated upregulation enhances choline-mediated upregulation of [3H]Eb. Further, both the HC3-sensitive and insensitive choline mediated upregulation pathways are enhanced by LY294002 and to a lesser extent by Wortmannin. In all cases, as before inhibition of PI3K activity using LY294002 or Wortmannin also enhances nicotine mediated upregulation (Fig 1A). These results are consistent with the conclusion that constitutive PI3K activity is a constitutive inhibitor of upregulation.


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)

Inhibition of PI3K is sufficient to upregulate [3H]Eb binding and enhance choline HC3-sensitive upregulation.Stably transfected 293 cells were either not treated (C) or treated with choline (Ch), hemicholinium-3 (HC3) or both (Ch+HC3) and separately nicotine (Nic) either in the presence or absence of LY294002 (LY) or Wortmannin (Wort), respectively. A) Cells were harvested 24 hours post treatment and specific binding by the α4β2 high affinity ligand [3H]Eb was measured (Methods). In all cases the results reflect the average of no less than 3 independent experiments. Upregulation is indicated in the light grey, enhancement of upregulation is in dark grey and stippled is the HC3-insensitive choline component. Error bars = +/- SEM. The significance values (P) are calculated from Student’s t-test of the indicated pairing. B) Western blot analysis of crude membranes prepared from choline-treated cells that received treatment as labeled and corresponded to those used for [3H]Eb binding to measure the relative expression of α4 or β2 subunits. The ratio of the band density for each subunit was measured, normalized to the control (1.0) and then plotted for each treatment. Above each lane is the change in the β2/α4 ratio calculated after normalization. The blot shown reflects a result that is typical for these experiments.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0143319.g001: Inhibition of PI3K is sufficient to upregulate [3H]Eb binding and enhance choline HC3-sensitive upregulation.Stably transfected 293 cells were either not treated (C) or treated with choline (Ch), hemicholinium-3 (HC3) or both (Ch+HC3) and separately nicotine (Nic) either in the presence or absence of LY294002 (LY) or Wortmannin (Wort), respectively. A) Cells were harvested 24 hours post treatment and specific binding by the α4β2 high affinity ligand [3H]Eb was measured (Methods). In all cases the results reflect the average of no less than 3 independent experiments. Upregulation is indicated in the light grey, enhancement of upregulation is in dark grey and stippled is the HC3-insensitive choline component. Error bars = +/- SEM. The significance values (P) are calculated from Student’s t-test of the indicated pairing. B) Western blot analysis of crude membranes prepared from choline-treated cells that received treatment as labeled and corresponded to those used for [3H]Eb binding to measure the relative expression of α4 or β2 subunits. The ratio of the band density for each subunit was measured, normalized to the control (1.0) and then plotted for each treatment. Above each lane is the change in the β2/α4 ratio calculated after normalization. The blot shown reflects a result that is typical for these experiments.
Mentions: In previous reports we noted that the potent class I phosphoinositide 3-kinases (PI3K) pan-inhibitor LY294002 was alone sufficient to produce significant upregulation of the 3H-epibadidine ([3H]Eb) sites associated with increased receptor expression in 293 cells stably transfected with the nicotinic acetylcholine receptor subunits α4 and β2 [9,10,14]. Also, in response to elevated choline or nicotine, the co-application of LY294002 greatly enhanced the upregulation of [3H]Eb produced by either agent alone. This result was confirmed and extended by measuring the effect of two widely used and well characterized as pan-inhibitors of class1 p110 PI3K catalytic activity; LY294002 and Wortmannin [16,19,20], on upregulation both alone and in the presence of choline (Fig 1A). Both inhibitors promoted upregulation of [3H]Eb binding in the absence of receptor ligand. Because the LY294002 effect was more consistent with less impact on cell morphology compared to wortmannin (Fig 1A and not shown), this agent was used for essentially all of the subsequent experiments. Choline alone produces upregulation through a hemicholinium-3 (HC3) sensitive, choline kinase (CK)-dependent [21], and a HC3-insensensitive pathway [10], we examined which of these pathways was most impacted by PI3K. The results reveal that LY294002-associated upregulation enhances choline-mediated upregulation of [3H]Eb. Further, both the HC3-sensitive and insensitive choline mediated upregulation pathways are enhanced by LY294002 and to a lesser extent by Wortmannin. In all cases, as before inhibition of PI3K activity using LY294002 or Wortmannin also enhances nicotine mediated upregulation (Fig 1A). These results are consistent with the conclusion that constitutive PI3K activity is a constitutive inhibitor of upregulation.

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