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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 the PI3Kβ isoform produces ligand-independent [3H]Eb receptor upregulation and enhances upregulation produced by choline and nicotine.A-C) The results from measurements of stably transfected 293 cells that were either not treated (C) or treated with the PI3K inhibitors LY294002 (LY, broad range), PIK75 (PI3Kα), PI828 (PI3Kβ), or AS60524 (PI3Kγ) as in (A), or in the presence of (B) choline or (C) nicotine as normalized to the control in (A). The specific [3H]Eb binding to membranes from these cells was measured for 3 independent experiments, the results normalized to the average no-treatment control value of 1.0 and all values from each experiment were then summed. D) Experiments similar to those in Panels A-C tested the impact to combining LY294002 and PI828 on upregulation of [3H]Eb binding or enhancement of upregulation produced by either choline or nicotine as indicated by + signs indicating addition of the agent. Upregulation relative to the control for each group is in light grey and enhancement of choline or nicotine upregulation is in dark grey. Error bars = +/- SEM. The significance values (P) are calculated from Student’s t-test of the indicated pairing. E) An example of a representative western blot showing the relative expression of α4 or β2 subunits in crude membrane fractions of cells after receiving the treatment as labeled. Nic, nicotine; Ch, choline. 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.
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pone.0143319.g002: Inhibition of the PI3Kβ isoform produces ligand-independent [3H]Eb receptor upregulation and enhances upregulation produced by choline and nicotine.A-C) The results from measurements of stably transfected 293 cells that were either not treated (C) or treated with the PI3K inhibitors LY294002 (LY, broad range), PIK75 (PI3Kα), PI828 (PI3Kβ), or AS60524 (PI3Kγ) as in (A), or in the presence of (B) choline or (C) nicotine as normalized to the control in (A). The specific [3H]Eb binding to membranes from these cells was measured for 3 independent experiments, the results normalized to the average no-treatment control value of 1.0 and all values from each experiment were then summed. D) Experiments similar to those in Panels A-C tested the impact to combining LY294002 and PI828 on upregulation of [3H]Eb binding or enhancement of upregulation produced by either choline or nicotine as indicated by + signs indicating addition of the agent. Upregulation relative to the control for each group is in light grey and enhancement of choline or nicotine upregulation is in dark grey. Error bars = +/- SEM. The significance values (P) are calculated from Student’s t-test of the indicated pairing. E) An example of a representative western blot showing the relative expression of α4 or β2 subunits in crude membrane fractions of cells after receiving the treatment as labeled. Nic, nicotine; Ch, choline. 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.

Mentions: Four major PI3K isotypes [16,17] regulate many cellular processes including cell growth and survival as well as vesicular and membrane trafficking including p110α and p110β (both widely expressed) or the more restricted expression of p110γ and p110δ (which is largely hematopoietic). To define which class1 PI3K isoform(s) promote upregulation, isoform-selective inhibitors were tested for their impact on PI3K-mediated upregulation of [3H]Eb binding (Fig 2). The 293 cells stably expressing α4 and β2 subunits were treated with different PI3K-specific inhibitors that included PIK-75 for PI3Kα [22], PI828 for PI3Kβ [23], and AS605240 for PI3Kγ [24]. Due to hematopoietic-preferred expression, PI3Kδ was not examined in these 293 cells (not shown). The results show that inhibition of PI3Kβ by PI828 was alone sufficient to impart upregulation whereas inhibitors of PI3Kα and PI3Kγ had no effect (Fig 2A). In some experiments the inhibition of PI3Kα by PIK75 could be associated with decreased α4β2 binding sites; although this effect was dose-dependent and increased as inhibitor concentrations reached 10x over the optimal KI (not shown). The use of additional inhibitors more specific to PI3Kα (e.g., PI-103; [22]) did not alter control level [3H]Eb binding (not shown). Thus, regulation of the α4β2 receptor expression and binding upregulation in the absence of ligand requires the PI3Kβ isoform.


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 the PI3Kβ isoform produces ligand-independent [3H]Eb receptor upregulation and enhances upregulation produced by choline and nicotine.A-C) The results from measurements of stably transfected 293 cells that were either not treated (C) or treated with the PI3K inhibitors LY294002 (LY, broad range), PIK75 (PI3Kα), PI828 (PI3Kβ), or AS60524 (PI3Kγ) as in (A), or in the presence of (B) choline or (C) nicotine as normalized to the control in (A). The specific [3H]Eb binding to membranes from these cells was measured for 3 independent experiments, the results normalized to the average no-treatment control value of 1.0 and all values from each experiment were then summed. D) Experiments similar to those in Panels A-C tested the impact to combining LY294002 and PI828 on upregulation of [3H]Eb binding or enhancement of upregulation produced by either choline or nicotine as indicated by + signs indicating addition of the agent. Upregulation relative to the control for each group is in light grey and enhancement of choline or nicotine upregulation is in dark grey. Error bars = +/- SEM. The significance values (P) are calculated from Student’s t-test of the indicated pairing. E) An example of a representative western blot showing the relative expression of α4 or β2 subunits in crude membrane fractions of cells after receiving the treatment as labeled. Nic, nicotine; Ch, choline. 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.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0143319.g002: Inhibition of the PI3Kβ isoform produces ligand-independent [3H]Eb receptor upregulation and enhances upregulation produced by choline and nicotine.A-C) The results from measurements of stably transfected 293 cells that were either not treated (C) or treated with the PI3K inhibitors LY294002 (LY, broad range), PIK75 (PI3Kα), PI828 (PI3Kβ), or AS60524 (PI3Kγ) as in (A), or in the presence of (B) choline or (C) nicotine as normalized to the control in (A). The specific [3H]Eb binding to membranes from these cells was measured for 3 independent experiments, the results normalized to the average no-treatment control value of 1.0 and all values from each experiment were then summed. D) Experiments similar to those in Panels A-C tested the impact to combining LY294002 and PI828 on upregulation of [3H]Eb binding or enhancement of upregulation produced by either choline or nicotine as indicated by + signs indicating addition of the agent. Upregulation relative to the control for each group is in light grey and enhancement of choline or nicotine upregulation is in dark grey. Error bars = +/- SEM. The significance values (P) are calculated from Student’s t-test of the indicated pairing. E) An example of a representative western blot showing the relative expression of α4 or β2 subunits in crude membrane fractions of cells after receiving the treatment as labeled. Nic, nicotine; Ch, choline. 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.
Mentions: Four major PI3K isotypes [16,17] regulate many cellular processes including cell growth and survival as well as vesicular and membrane trafficking including p110α and p110β (both widely expressed) or the more restricted expression of p110γ and p110δ (which is largely hematopoietic). To define which class1 PI3K isoform(s) promote upregulation, isoform-selective inhibitors were tested for their impact on PI3K-mediated upregulation of [3H]Eb binding (Fig 2). The 293 cells stably expressing α4 and β2 subunits were treated with different PI3K-specific inhibitors that included PIK-75 for PI3Kα [22], PI828 for PI3Kβ [23], and AS605240 for PI3Kγ [24]. Due to hematopoietic-preferred expression, PI3Kδ was not examined in these 293 cells (not shown). The results show that inhibition of PI3Kβ by PI828 was alone sufficient to impart upregulation whereas inhibitors of PI3Kα and PI3Kγ had no effect (Fig 2A). In some experiments the inhibition of PI3Kα by PIK75 could be associated with decreased α4β2 binding sites; although this effect was dose-dependent and increased as inhibitor concentrations reached 10x over the optimal KI (not shown). The use of additional inhibitors more specific to PI3Kα (e.g., PI-103; [22]) did not alter control level [3H]Eb binding (not shown). Thus, regulation of the α4β2 receptor expression and binding upregulation in the absence of ligand requires the PI3Kβ isoform.

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