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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 of HC3-sensitive choline [3H]Eb binding and TNFα enhancement of this process requires Janus kinase 2.Stably transfected 293 cells were exposed to (A) choline, Ch, or (B) nicotine, Nic, with or without addition of TNFα (25 ng/ml). In parallel cultures, inhibitors with differing specificities towards members of the Jak family were also added. The agents used were 1,2,3,4,5,6-Hexabromocyclohexane (1-6Hex; 50 μM); AG-490 (AG490, 25 μM), cucurbitacin I (Cucur, 100 nM); lestaurtnib (Lest, 300 nM), ZM 39923 (ZM399, 50 μM), ZM449829 (ZM449, 50 μM). For all plots the error bars are +/- SEM and upregulation is indicated in the light grey and enhancement of upregulation is in by dark grey. The significance values (P) are calculated from Student’s t-test of the indicated pairing. C) Experiments similar to those in panels A examined the impact of Jak inhibitors on choline-mediated upregulation. C, control; Ch, choline; HC3, hemicholinium-3. For this experiment the concentration of AG-490 was 100 μM (see text), but all other inhibitors were used as above. For choline upregulation, solid light grey is HC3-sensitive and light grey is HC3-insensitive. D) The impact of co-application of inhibitors of PI3K and those favoring Jak2 are shown. Experiments were conducted as described in prior figure legends and the text. The stippled area is equivalent to the HC3-independent choline mediated upregulation and the light grey is the HC3-dependent component. Dark grey is the component of upregulation associated with the indicated treatment.
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pone.0143319.g005: Upregulation of HC3-sensitive choline [3H]Eb binding and TNFα enhancement of this process requires Janus kinase 2.Stably transfected 293 cells were exposed to (A) choline, Ch, or (B) nicotine, Nic, with or without addition of TNFα (25 ng/ml). In parallel cultures, inhibitors with differing specificities towards members of the Jak family were also added. The agents used were 1,2,3,4,5,6-Hexabromocyclohexane (1-6Hex; 50 μM); AG-490 (AG490, 25 μM), cucurbitacin I (Cucur, 100 nM); lestaurtnib (Lest, 300 nM), ZM 39923 (ZM399, 50 μM), ZM449829 (ZM449, 50 μM). For all plots the error bars are +/- SEM and upregulation is indicated in the light grey and enhancement of upregulation is in by dark grey. The significance values (P) are calculated from Student’s t-test of the indicated pairing. C) Experiments similar to those in panels A examined the impact of Jak inhibitors on choline-mediated upregulation. C, control; Ch, choline; HC3, hemicholinium-3. For this experiment the concentration of AG-490 was 100 μM (see text), but all other inhibitors were used as above. For choline upregulation, solid light grey is HC3-sensitive and light grey is HC3-insensitive. D) The impact of co-application of inhibitors of PI3K and those favoring Jak2 are shown. Experiments were conducted as described in prior figure legends and the text. The stippled area is equivalent to the HC3-independent choline mediated upregulation and the light grey is the HC3-dependent component. Dark grey is the component of upregulation associated with the indicated treatment.

Mentions: Multiple inhibitors with varied specificity towards Jak1, Jak2 or Jak3 were examined. A particularly widely used and well-characterized inhibitor of Jak2 is the tyrphostin AG-490 [30]. Although this inhibitor is often viewed as specific to Jak2, at concentrations that achieved optimal Jak2 inhibition there could be additional off-site effects on other tyrosine kinase activities (e.g., EGFR;[31]). Given this caveat, we started by examining the impact of AG-490 towards choline or nicotine upregulation or its enhancement by TNFα. Transfected 293 cells were treated with either choline with or without TNFα as a control. As shown in Fig 5A, the anticipated increase in upregulation was achieved by both of these agents and they are enhanced further by TNFα. In the presence of AG-490 (1 to 25 μM, 10 μM shown), all TNFα-mediated enhancement of choline or nicotine upregulation was abolished with no effect on the upregulation by either agent alone.


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 of HC3-sensitive choline [3H]Eb binding and TNFα enhancement of this process requires Janus kinase 2.Stably transfected 293 cells were exposed to (A) choline, Ch, or (B) nicotine, Nic, with or without addition of TNFα (25 ng/ml). In parallel cultures, inhibitors with differing specificities towards members of the Jak family were also added. The agents used were 1,2,3,4,5,6-Hexabromocyclohexane (1-6Hex; 50 μM); AG-490 (AG490, 25 μM), cucurbitacin I (Cucur, 100 nM); lestaurtnib (Lest, 300 nM), ZM 39923 (ZM399, 50 μM), ZM449829 (ZM449, 50 μM). For all plots the error bars are +/- SEM and upregulation is indicated in the light grey and enhancement of upregulation is in by dark grey. The significance values (P) are calculated from Student’s t-test of the indicated pairing. C) Experiments similar to those in panels A examined the impact of Jak inhibitors on choline-mediated upregulation. C, control; Ch, choline; HC3, hemicholinium-3. For this experiment the concentration of AG-490 was 100 μM (see text), but all other inhibitors were used as above. For choline upregulation, solid light grey is HC3-sensitive and light grey is HC3-insensitive. D) The impact of co-application of inhibitors of PI3K and those favoring Jak2 are shown. Experiments were conducted as described in prior figure legends and the text. The stippled area is equivalent to the HC3-independent choline mediated upregulation and the light grey is the HC3-dependent component. Dark grey is the component of upregulation associated with the indicated treatment.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0143319.g005: Upregulation of HC3-sensitive choline [3H]Eb binding and TNFα enhancement of this process requires Janus kinase 2.Stably transfected 293 cells were exposed to (A) choline, Ch, or (B) nicotine, Nic, with or without addition of TNFα (25 ng/ml). In parallel cultures, inhibitors with differing specificities towards members of the Jak family were also added. The agents used were 1,2,3,4,5,6-Hexabromocyclohexane (1-6Hex; 50 μM); AG-490 (AG490, 25 μM), cucurbitacin I (Cucur, 100 nM); lestaurtnib (Lest, 300 nM), ZM 39923 (ZM399, 50 μM), ZM449829 (ZM449, 50 μM). For all plots the error bars are +/- SEM and upregulation is indicated in the light grey and enhancement of upregulation is in by dark grey. The significance values (P) are calculated from Student’s t-test of the indicated pairing. C) Experiments similar to those in panels A examined the impact of Jak inhibitors on choline-mediated upregulation. C, control; Ch, choline; HC3, hemicholinium-3. For this experiment the concentration of AG-490 was 100 μM (see text), but all other inhibitors were used as above. For choline upregulation, solid light grey is HC3-sensitive and light grey is HC3-insensitive. D) The impact of co-application of inhibitors of PI3K and those favoring Jak2 are shown. Experiments were conducted as described in prior figure legends and the text. The stippled area is equivalent to the HC3-independent choline mediated upregulation and the light grey is the HC3-dependent component. Dark grey is the component of upregulation associated with the indicated treatment.
Mentions: Multiple inhibitors with varied specificity towards Jak1, Jak2 or Jak3 were examined. A particularly widely used and well-characterized inhibitor of Jak2 is the tyrphostin AG-490 [30]. Although this inhibitor is often viewed as specific to Jak2, at concentrations that achieved optimal Jak2 inhibition there could be additional off-site effects on other tyrosine kinase activities (e.g., EGFR;[31]). Given this caveat, we started by examining the impact of AG-490 towards choline or nicotine upregulation or its enhancement by TNFα. Transfected 293 cells were treated with either choline with or without TNFα as a control. As shown in Fig 5A, the anticipated increase in upregulation was achieved by both of these agents and they are enhanced further by TNFα. In the presence of AG-490 (1 to 25 μM, 10 μM shown), all TNFα-mediated enhancement of choline or nicotine upregulation was abolished with no effect on the upregulation by either agent alone.

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