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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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Upregulation related to inhibition of PI3Kb requires p38Mapk.A) Stably transfected 293 cells were either not treated (C) or treated with choline (Ch), or choline + hemicholinium-3 (HC3) in the presence of the p38Mapk inhibitor SB202190, the PI3Kβ inhibitor PI828 or both inhibitors as indicated. Specific [3H]Eb binding to crude cell membranes was measured at 24 hours post-treatment. Upregulation due to choline or nicotine is indicated in light grey and the ‘enhanced’ component in dark grey. 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. HC3 alone, as in other experiments, had not significant effect (data not shown). B) Western blot analysis of crude membranes prepared from cells in parallel with experiments in panel A. 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 β2/α4 ratio after normalization. The blot shown reflects a typical result for these experiments. C) The results of experiments performed in parallel with those in panel A and B only nicotine was substituted before measuring (C) [3H]Eb binding or (D) western blots of α4 or β2.
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pone.0143319.g003: Upregulation related to inhibition of PI3Kb requires p38Mapk.A) Stably transfected 293 cells were either not treated (C) or treated with choline (Ch), or choline + hemicholinium-3 (HC3) in the presence of the p38Mapk inhibitor SB202190, the PI3Kβ inhibitor PI828 or both inhibitors as indicated. Specific [3H]Eb binding to crude cell membranes was measured at 24 hours post-treatment. Upregulation due to choline or nicotine is indicated in light grey and the ‘enhanced’ component in dark grey. 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. HC3 alone, as in other experiments, had not significant effect (data not shown). B) Western blot analysis of crude membranes prepared from cells in parallel with experiments in panel A. 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 β2/α4 ratio after normalization. The blot shown reflects a typical result for these experiments. C) The results of experiments performed in parallel with those in panel A and B only nicotine was substituted before measuring (C) [3H]Eb binding or (D) western blots of α4 or β2.

Mentions: Choline-mediated upregulation is the summation of two processes distinguished by sensitivity to inhibition of choline kinase (CK) catalytic activity by hemicholinium-3 (HC3; [9]). Because CK impacts both p38Mapk and PI3K/Akt signaling [25], the possibility of interactions between PI3Kβ and the HC3-sensitive or HC3-insensitive [3H]Eb upregulation pathways, including acting through p38Mapk, were investigated. Cultures were prepared in parallel that received no treatment, choline, HC3 or choline plus HC3. As shown in Fig 3A and 3B and as reported previously [9,10], approximately 50–60% of the choline-mediated upregulation was sensitive to inhibition by HC3. Treatment with HC3 alone or in combination with other inhibitors such as PI828 produced no differences relative to the respective controls (not shown). In this and the experiments to be described, modification to the expression of β2 and alteration of the α4:β2 ratio was observed and it was again consistent with mechanism leading to altered [3H]Eb binding (Fig 3B).


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)

Upregulation related to inhibition of PI3Kb requires p38Mapk.A) Stably transfected 293 cells were either not treated (C) or treated with choline (Ch), or choline + hemicholinium-3 (HC3) in the presence of the p38Mapk inhibitor SB202190, the PI3Kβ inhibitor PI828 or both inhibitors as indicated. Specific [3H]Eb binding to crude cell membranes was measured at 24 hours post-treatment. Upregulation due to choline or nicotine is indicated in light grey and the ‘enhanced’ component in dark grey. 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. HC3 alone, as in other experiments, had not significant effect (data not shown). B) Western blot analysis of crude membranes prepared from cells in parallel with experiments in panel A. 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 β2/α4 ratio after normalization. The blot shown reflects a typical result for these experiments. C) The results of experiments performed in parallel with those in panel A and B only nicotine was substituted before measuring (C) [3H]Eb binding or (D) western blots of α4 or β2.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4664291&req=5

pone.0143319.g003: Upregulation related to inhibition of PI3Kb requires p38Mapk.A) Stably transfected 293 cells were either not treated (C) or treated with choline (Ch), or choline + hemicholinium-3 (HC3) in the presence of the p38Mapk inhibitor SB202190, the PI3Kβ inhibitor PI828 or both inhibitors as indicated. Specific [3H]Eb binding to crude cell membranes was measured at 24 hours post-treatment. Upregulation due to choline or nicotine is indicated in light grey and the ‘enhanced’ component in dark grey. 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. HC3 alone, as in other experiments, had not significant effect (data not shown). B) Western blot analysis of crude membranes prepared from cells in parallel with experiments in panel A. 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 β2/α4 ratio after normalization. The blot shown reflects a typical result for these experiments. C) The results of experiments performed in parallel with those in panel A and B only nicotine was substituted before measuring (C) [3H]Eb binding or (D) western blots of α4 or β2.
Mentions: Choline-mediated upregulation is the summation of two processes distinguished by sensitivity to inhibition of choline kinase (CK) catalytic activity by hemicholinium-3 (HC3; [9]). Because CK impacts both p38Mapk and PI3K/Akt signaling [25], the possibility of interactions between PI3Kβ and the HC3-sensitive or HC3-insensitive [3H]Eb upregulation pathways, including acting through p38Mapk, were investigated. Cultures were prepared in parallel that received no treatment, choline, HC3 or choline plus HC3. As shown in Fig 3A and 3B and as reported previously [9,10], approximately 50–60% of the choline-mediated upregulation was sensitive to inhibition by HC3. Treatment with HC3 alone or in combination with other inhibitors such as PI828 produced no differences relative to the respective controls (not shown). In this and the experiments to be described, modification to the expression of β2 and alteration of the α4:β2 ratio was observed and it was again consistent with mechanism leading to altered [3H]Eb binding (Fig 3B).

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