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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 through disinhibition of PI3Kβ does not require AKT.Cultures of stably transfected 293 cells were treated with choline (Ch) or controls that (C) received no treatment. To parallel culture sets either the PI3K inhibitor LY294202 or the Akt inhibitor GSK690693 was either added or co-applied with the other agents as indicated. Cultures were harvested and the specific [3H]Eb binding measured. The error bars = +/- SEM and upregulation is indicated in the light grey and enhancement of upregulation is marked as dark grey. Western blot analysis of 293 cells receiving the treatment indicated were harvested at the time indicated post-addition of the treatment agents and assayed for the expression of total Akt (Akt) or antibodies specific to Akt phosphorylated at either Ser473 (Akt pSer473) or Thr308 (Akt pThr473).
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pone.0143319.g006: Upregulation through disinhibition of PI3Kβ does not require AKT.Cultures of stably transfected 293 cells were treated with choline (Ch) or controls that (C) received no treatment. To parallel culture sets either the PI3K inhibitor LY294202 or the Akt inhibitor GSK690693 was either added or co-applied with the other agents as indicated. Cultures were harvested and the specific [3H]Eb binding measured. The error bars = +/- SEM and upregulation is indicated in the light grey and enhancement of upregulation is marked as dark grey. Western blot analysis of 293 cells receiving the treatment indicated were harvested at the time indicated post-addition of the treatment agents and assayed for the expression of total Akt (Akt) or antibodies specific to Akt phosphorylated at either Ser473 (Akt pSer473) or Thr308 (Akt pThr473).

Mentions: An important target of the products of PI3K signaling is Akt (protein kinase B) and PDK1 [36]. Although Akt is not necessarily a major target of the PI3Kβ isoform [37–39], there remains the possibility this important kinase activity serves as a down-stream target of PI3Kβ. Further, choline promotes activation of Akt in human breast carcinoma cells through CK-mediated enhancement of Akt phosphorylation at Ser473 (AktpSer473;[40]). Thus, the suggestion that alterations in phosphorylation of Akt by inhibition of PI3Kβ correspond with or contribute directly to upregulation mechanisms was examined. Cultures of stably transfected 293 cells were established as described in previous experiments. One set of cultures received the Akt inhibitor, GSK690693 (1 μM; [41]) prior to the addition of either choline or LY294002. LY294002 was used to assure additional specificity of the PI3Kβ requirement since it is the PI3Kα isoform that dominates the Akt activation pathway whereas only a small if any contribution is made by PI3Kβ [38]. The results (Fig 6) reveal no evidence of an effect by Akt inhibition on upregulation. Also as part of the controls to measure Akt activity, western blot analysis of the phosphorylation of AktpSer473 or another phosphorylation site, AktpThr308, was used. Cell cultures were prepared in parallel, treated and then at various times thereafter (15, 30, 60, 90, 120, and 240 minutes, respectively) they were quickly rinsed in PBS and scraped into harvesting buffer (Methods). Cell lysate was then prepared for western blot analysis to measure the relative change in the total Akt, or phosphorylation of Akt at either AktpThr308 or AktpSer473, both markers of Akt activation. As shown in Fig 6B, the addition of choline produced an increase in Akt pSer473 phosphorylation that was evident within 15 minutes. The increase in phosphorylation continued steadily until reaching a maximum by 90 minutes as has been reported by others [41]. Choline addition did not result in modification of the phosphorylation status of AktpThr308. In the presence of the pan-inhibitor of PI3K, LY294002 (10 μM), the choline mediated increase in AktpSer473 was rapidly reduced and nearly absent 2 hours after initiating this treatment. A change in phosphorylation of AktpThr308 was not observed. Despite the substantial change in upregulation produced by choline, LY294002 alone or both agents together (Fig 6A), the phosphorylation status of Akt at the sites measured did not correlate with nor reflect the robust changes in upregulation being measured. Thus, no evidence of an effect by Akt activation or inhibition on upregulation was identified.


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 through disinhibition of PI3Kβ does not require AKT.Cultures of stably transfected 293 cells were treated with choline (Ch) or controls that (C) received no treatment. To parallel culture sets either the PI3K inhibitor LY294202 or the Akt inhibitor GSK690693 was either added or co-applied with the other agents as indicated. Cultures were harvested and the specific [3H]Eb binding measured. The error bars = +/- SEM and upregulation is indicated in the light grey and enhancement of upregulation is marked as dark grey. Western blot analysis of 293 cells receiving the treatment indicated were harvested at the time indicated post-addition of the treatment agents and assayed for the expression of total Akt (Akt) or antibodies specific to Akt phosphorylated at either Ser473 (Akt pSer473) or Thr308 (Akt pThr473).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4664291&req=5

pone.0143319.g006: Upregulation through disinhibition of PI3Kβ does not require AKT.Cultures of stably transfected 293 cells were treated with choline (Ch) or controls that (C) received no treatment. To parallel culture sets either the PI3K inhibitor LY294202 or the Akt inhibitor GSK690693 was either added or co-applied with the other agents as indicated. Cultures were harvested and the specific [3H]Eb binding measured. The error bars = +/- SEM and upregulation is indicated in the light grey and enhancement of upregulation is marked as dark grey. Western blot analysis of 293 cells receiving the treatment indicated were harvested at the time indicated post-addition of the treatment agents and assayed for the expression of total Akt (Akt) or antibodies specific to Akt phosphorylated at either Ser473 (Akt pSer473) or Thr308 (Akt pThr473).
Mentions: An important target of the products of PI3K signaling is Akt (protein kinase B) and PDK1 [36]. Although Akt is not necessarily a major target of the PI3Kβ isoform [37–39], there remains the possibility this important kinase activity serves as a down-stream target of PI3Kβ. Further, choline promotes activation of Akt in human breast carcinoma cells through CK-mediated enhancement of Akt phosphorylation at Ser473 (AktpSer473;[40]). Thus, the suggestion that alterations in phosphorylation of Akt by inhibition of PI3Kβ correspond with or contribute directly to upregulation mechanisms was examined. Cultures of stably transfected 293 cells were established as described in previous experiments. One set of cultures received the Akt inhibitor, GSK690693 (1 μM; [41]) prior to the addition of either choline or LY294002. LY294002 was used to assure additional specificity of the PI3Kβ requirement since it is the PI3Kα isoform that dominates the Akt activation pathway whereas only a small if any contribution is made by PI3Kβ [38]. The results (Fig 6) reveal no evidence of an effect by Akt inhibition on upregulation. Also as part of the controls to measure Akt activity, western blot analysis of the phosphorylation of AktpSer473 or another phosphorylation site, AktpThr308, was used. Cell cultures were prepared in parallel, treated and then at various times thereafter (15, 30, 60, 90, 120, and 240 minutes, respectively) they were quickly rinsed in PBS and scraped into harvesting buffer (Methods). Cell lysate was then prepared for western blot analysis to measure the relative change in the total Akt, or phosphorylation of Akt at either AktpThr308 or AktpSer473, both markers of Akt activation. As shown in Fig 6B, the addition of choline produced an increase in Akt pSer473 phosphorylation that was evident within 15 minutes. The increase in phosphorylation continued steadily until reaching a maximum by 90 minutes as has been reported by others [41]. Choline addition did not result in modification of the phosphorylation status of AktpThr308. In the presence of the pan-inhibitor of PI3K, LY294002 (10 μM), the choline mediated increase in AktpSer473 was rapidly reduced and nearly absent 2 hours after initiating this treatment. A change in phosphorylation of AktpThr308 was not observed. Despite the substantial change in upregulation produced by choline, LY294002 alone or both agents together (Fig 6A), the phosphorylation status of Akt at the sites measured did not correlate with nor reflect the robust changes in upregulation being measured. Thus, no evidence of an effect by Akt activation or inhibition on upregulation was identified.

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