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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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Choline-mediated upregulation does not correspond with a change in chaperone proteins 14-3-3eta or BiP, but may involve novel proteins identified by 2D-gel analysis.A) To examine if choline-mediated upregulation corresponds with changes in chaperone protein expression, western blot analysis was used to identify two candidate α4β2 chaperone proteins, 14-3-3, or the immunological heavy chain-binding protein, BiP, after cells were treated with either choline (Ch), hemichoinium-3 (HC30) or both. As seen in the results from these representative western blots, the changes in β2 expression, but stable α4 expression was as expected from previous experiments. In no case was any treatment-related change in BiP or 14-3-3eta measured relative to controls. B) To determine if other proteins are altered by choline and if identified changes are HC3-senstive, 2D-gel analysis of whole cell extract from stably transfected cells as identified were prepared and analyzed (see Text). 2D gels from control, choline or choline+HC3 treated cells are shown. Boxed regions (numbered 1–3) correspond to the same numbered enlargements shown in the next panel. C) Several spots that demonstrated changes in intensity or migration on 2D gels are shown at greater magnification (corresponding to the white boxes in B). The control (black arrow head) versus choline treatment (white arrow head) spots were removed from the blots, subjected to limited proteolysis and then subjected to sequencing using Mass-spectroscopy (see text). The identity of these three choline-responsive proteins shown is based upon protein-peptide sequence identity of 100% (sequences of proteolytic fragments are underlined and capital letters) and their sequence identity to the reference proteins noted taken from the NIH protein database (sequence match in bold). GTP-binding nuclear protein Ran (GI:5453555) maaqgepqvqfklvlvgdggtgkttfvkRHLTGEFEKkYVATLGVEVHPLVFHTNRgpikFNVWDTAGQEKfgglrDGYYIQAQCAIIMFDVTSRvtykNVPNWHRdlvrVCENIPIVLCGNKVDIKdrkvkaksivfhrkkNLQYYDISAKSNYNFEKPFLWLARkligdpnlefvampalappevvmdpalaaqyehdlevaqttalpdedddl//. Annexin A2, chain A (GI: 56966699) mstvheilcklslegdhstppsaygsvk AYTNFDAERDALNIETAIKtkGVDEVTIVNILTNRsneqrqdiafayqrrtkkelasalksalsghletvilgllktpaqydaselkasmkGLGTDEDSLIEIICSRtnqelqeinrvykemyktdlekdiisdtsgdfrklmvalakgrRAEDGSVIDYELIDQDAR.DLYDAGVKRkgtdvpkwisimtersvphlqkvfdryksyspydmlesirkevkgdlenaflnlvqciqnkplyfadrlydsmkgkgtrdkvlirimvsrsevdmlkirsefkrkygkSLYYYIQQDTKgdyqkallylcggdd//. Prefoldin 5 (GI:30583229) maqsinitelnlpqlemlkNQLDQEVEFLSTSIAQLK vvqtkyveakdclnvlnksnegkellvpltsssmyvpgkLHDVEHVLIDVGTGYYVEKtaedakdffkrkidfltkqmekiqpalqekhamkqavmemmsqtIQQLTALGAAQATAKA//. D) Subcellular distribution and expression of the cellular antigens identified by 2D-gel analysis and Mass spectroscopy using immunofluorescence of the 293 cells with protein-specific antibodies. All cells were treated, fixed, blocked and permeabilized and incubated with the indicated antibody overnight at 4°C. Cells were again washed and antibody binding revealed using an appropriate secondary antibody coupled to fluorescence. Random cell fields were then visualized and photographed as shown for each protein. Arrows points to representative cells of each field identify the typical appearance of antigen distribution under each condition.
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pone.0143319.g007: Choline-mediated upregulation does not correspond with a change in chaperone proteins 14-3-3eta or BiP, but may involve novel proteins identified by 2D-gel analysis.A) To examine if choline-mediated upregulation corresponds with changes in chaperone protein expression, western blot analysis was used to identify two candidate α4β2 chaperone proteins, 14-3-3, or the immunological heavy chain-binding protein, BiP, after cells were treated with either choline (Ch), hemichoinium-3 (HC30) or both. As seen in the results from these representative western blots, the changes in β2 expression, but stable α4 expression was as expected from previous experiments. In no case was any treatment-related change in BiP or 14-3-3eta measured relative to controls. B) To determine if other proteins are altered by choline and if identified changes are HC3-senstive, 2D-gel analysis of whole cell extract from stably transfected cells as identified were prepared and analyzed (see Text). 2D gels from control, choline or choline+HC3 treated cells are shown. Boxed regions (numbered 1–3) correspond to the same numbered enlargements shown in the next panel. C) Several spots that demonstrated changes in intensity or migration on 2D gels are shown at greater magnification (corresponding to the white boxes in B). The control (black arrow head) versus choline treatment (white arrow head) spots were removed from the blots, subjected to limited proteolysis and then subjected to sequencing using Mass-spectroscopy (see text). The identity of these three choline-responsive proteins shown is based upon protein-peptide sequence identity of 100% (sequences of proteolytic fragments are underlined and capital letters) and their sequence identity to the reference proteins noted taken from the NIH protein database (sequence match in bold). GTP-binding nuclear protein Ran (GI:5453555) maaqgepqvqfklvlvgdggtgkttfvkRHLTGEFEKkYVATLGVEVHPLVFHTNRgpikFNVWDTAGQEKfgglrDGYYIQAQCAIIMFDVTSRvtykNVPNWHRdlvrVCENIPIVLCGNKVDIKdrkvkaksivfhrkkNLQYYDISAKSNYNFEKPFLWLARkligdpnlefvampalappevvmdpalaaqyehdlevaqttalpdedddl//. Annexin A2, chain A (GI: 56966699) mstvheilcklslegdhstppsaygsvk AYTNFDAERDALNIETAIKtkGVDEVTIVNILTNRsneqrqdiafayqrrtkkelasalksalsghletvilgllktpaqydaselkasmkGLGTDEDSLIEIICSRtnqelqeinrvykemyktdlekdiisdtsgdfrklmvalakgrRAEDGSVIDYELIDQDAR.DLYDAGVKRkgtdvpkwisimtersvphlqkvfdryksyspydmlesirkevkgdlenaflnlvqciqnkplyfadrlydsmkgkgtrdkvlirimvsrsevdmlkirsefkrkygkSLYYYIQQDTKgdyqkallylcggdd//. Prefoldin 5 (GI:30583229) maqsinitelnlpqlemlkNQLDQEVEFLSTSIAQLK vvqtkyveakdclnvlnksnegkellvpltsssmyvpgkLHDVEHVLIDVGTGYYVEKtaedakdffkrkidfltkqmekiqpalqekhamkqavmemmsqtIQQLTALGAAQATAKA//. D) Subcellular distribution and expression of the cellular antigens identified by 2D-gel analysis and Mass spectroscopy using immunofluorescence of the 293 cells with protein-specific antibodies. All cells were treated, fixed, blocked and permeabilized and incubated with the indicated antibody overnight at 4°C. Cells were again washed and antibody binding revealed using an appropriate secondary antibody coupled to fluorescence. Random cell fields were then visualized and photographed as shown for each protein. Arrows points to representative cells of each field identify the typical appearance of antigen distribution under each condition.

Mentions: A significant issue in this system to identify the cell signaling networks that lead to changes in β2 protein production and enhanced [3H]Eb density. One possibility is that a change in chaperoning of the receptor during assembly and transport through synthetic compartments leads to increased receptor expression [1,3]. Two candidate proteins that participate in this process are the immunological heavy chain-binding protein BiP [42] and 14-3-3eta [43]. To test the possibility that changes the expression of the 14-3-3eta or BiP correspond to upregulation related to PI3Kβ or Jak2 signaling, the expression levels of these chaperone proteins were measured. As above, subsequent to treatment of transfected 293 cells with choline, HC3 or choline and HC3 the whole cell lysates were analyzed using western blot. The typical results from 4 different experiments are shown in Fig 7A. Although the change in expression of β2 was as expected, in no case did treatment of the cells alter the expression of BiP or 14-3-3eta. The 14-3-3eta is shown because of its association to α4β2 [43], however no change in expression of other 14-3-3 proteins, as determined using additional antibodies or low isoform specificity, was observed (not shown). Thus, the expression of these candidate chaperone proteins did not vary with the treatments used it this study despite the change in α4β2 expression and 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)

Choline-mediated upregulation does not correspond with a change in chaperone proteins 14-3-3eta or BiP, but may involve novel proteins identified by 2D-gel analysis.A) To examine if choline-mediated upregulation corresponds with changes in chaperone protein expression, western blot analysis was used to identify two candidate α4β2 chaperone proteins, 14-3-3, or the immunological heavy chain-binding protein, BiP, after cells were treated with either choline (Ch), hemichoinium-3 (HC30) or both. As seen in the results from these representative western blots, the changes in β2 expression, but stable α4 expression was as expected from previous experiments. In no case was any treatment-related change in BiP or 14-3-3eta measured relative to controls. B) To determine if other proteins are altered by choline and if identified changes are HC3-senstive, 2D-gel analysis of whole cell extract from stably transfected cells as identified were prepared and analyzed (see Text). 2D gels from control, choline or choline+HC3 treated cells are shown. Boxed regions (numbered 1–3) correspond to the same numbered enlargements shown in the next panel. C) Several spots that demonstrated changes in intensity or migration on 2D gels are shown at greater magnification (corresponding to the white boxes in B). The control (black arrow head) versus choline treatment (white arrow head) spots were removed from the blots, subjected to limited proteolysis and then subjected to sequencing using Mass-spectroscopy (see text). The identity of these three choline-responsive proteins shown is based upon protein-peptide sequence identity of 100% (sequences of proteolytic fragments are underlined and capital letters) and their sequence identity to the reference proteins noted taken from the NIH protein database (sequence match in bold). GTP-binding nuclear protein Ran (GI:5453555) maaqgepqvqfklvlvgdggtgkttfvkRHLTGEFEKkYVATLGVEVHPLVFHTNRgpikFNVWDTAGQEKfgglrDGYYIQAQCAIIMFDVTSRvtykNVPNWHRdlvrVCENIPIVLCGNKVDIKdrkvkaksivfhrkkNLQYYDISAKSNYNFEKPFLWLARkligdpnlefvampalappevvmdpalaaqyehdlevaqttalpdedddl//. Annexin A2, chain A (GI: 56966699) mstvheilcklslegdhstppsaygsvk AYTNFDAERDALNIETAIKtkGVDEVTIVNILTNRsneqrqdiafayqrrtkkelasalksalsghletvilgllktpaqydaselkasmkGLGTDEDSLIEIICSRtnqelqeinrvykemyktdlekdiisdtsgdfrklmvalakgrRAEDGSVIDYELIDQDAR.DLYDAGVKRkgtdvpkwisimtersvphlqkvfdryksyspydmlesirkevkgdlenaflnlvqciqnkplyfadrlydsmkgkgtrdkvlirimvsrsevdmlkirsefkrkygkSLYYYIQQDTKgdyqkallylcggdd//. Prefoldin 5 (GI:30583229) maqsinitelnlpqlemlkNQLDQEVEFLSTSIAQLK vvqtkyveakdclnvlnksnegkellvpltsssmyvpgkLHDVEHVLIDVGTGYYVEKtaedakdffkrkidfltkqmekiqpalqekhamkqavmemmsqtIQQLTALGAAQATAKA//. D) Subcellular distribution and expression of the cellular antigens identified by 2D-gel analysis and Mass spectroscopy using immunofluorescence of the 293 cells with protein-specific antibodies. All cells were treated, fixed, blocked and permeabilized and incubated with the indicated antibody overnight at 4°C. Cells were again washed and antibody binding revealed using an appropriate secondary antibody coupled to fluorescence. Random cell fields were then visualized and photographed as shown for each protein. Arrows points to representative cells of each field identify the typical appearance of antigen distribution under each condition.
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Show All Figures
getmorefigures.php?uid=PMC4664291&req=5

pone.0143319.g007: Choline-mediated upregulation does not correspond with a change in chaperone proteins 14-3-3eta or BiP, but may involve novel proteins identified by 2D-gel analysis.A) To examine if choline-mediated upregulation corresponds with changes in chaperone protein expression, western blot analysis was used to identify two candidate α4β2 chaperone proteins, 14-3-3, or the immunological heavy chain-binding protein, BiP, after cells were treated with either choline (Ch), hemichoinium-3 (HC30) or both. As seen in the results from these representative western blots, the changes in β2 expression, but stable α4 expression was as expected from previous experiments. In no case was any treatment-related change in BiP or 14-3-3eta measured relative to controls. B) To determine if other proteins are altered by choline and if identified changes are HC3-senstive, 2D-gel analysis of whole cell extract from stably transfected cells as identified were prepared and analyzed (see Text). 2D gels from control, choline or choline+HC3 treated cells are shown. Boxed regions (numbered 1–3) correspond to the same numbered enlargements shown in the next panel. C) Several spots that demonstrated changes in intensity or migration on 2D gels are shown at greater magnification (corresponding to the white boxes in B). The control (black arrow head) versus choline treatment (white arrow head) spots were removed from the blots, subjected to limited proteolysis and then subjected to sequencing using Mass-spectroscopy (see text). The identity of these three choline-responsive proteins shown is based upon protein-peptide sequence identity of 100% (sequences of proteolytic fragments are underlined and capital letters) and their sequence identity to the reference proteins noted taken from the NIH protein database (sequence match in bold). GTP-binding nuclear protein Ran (GI:5453555) maaqgepqvqfklvlvgdggtgkttfvkRHLTGEFEKkYVATLGVEVHPLVFHTNRgpikFNVWDTAGQEKfgglrDGYYIQAQCAIIMFDVTSRvtykNVPNWHRdlvrVCENIPIVLCGNKVDIKdrkvkaksivfhrkkNLQYYDISAKSNYNFEKPFLWLARkligdpnlefvampalappevvmdpalaaqyehdlevaqttalpdedddl//. Annexin A2, chain A (GI: 56966699) mstvheilcklslegdhstppsaygsvk AYTNFDAERDALNIETAIKtkGVDEVTIVNILTNRsneqrqdiafayqrrtkkelasalksalsghletvilgllktpaqydaselkasmkGLGTDEDSLIEIICSRtnqelqeinrvykemyktdlekdiisdtsgdfrklmvalakgrRAEDGSVIDYELIDQDAR.DLYDAGVKRkgtdvpkwisimtersvphlqkvfdryksyspydmlesirkevkgdlenaflnlvqciqnkplyfadrlydsmkgkgtrdkvlirimvsrsevdmlkirsefkrkygkSLYYYIQQDTKgdyqkallylcggdd//. Prefoldin 5 (GI:30583229) maqsinitelnlpqlemlkNQLDQEVEFLSTSIAQLK vvqtkyveakdclnvlnksnegkellvpltsssmyvpgkLHDVEHVLIDVGTGYYVEKtaedakdffkrkidfltkqmekiqpalqekhamkqavmemmsqtIQQLTALGAAQATAKA//. D) Subcellular distribution and expression of the cellular antigens identified by 2D-gel analysis and Mass spectroscopy using immunofluorescence of the 293 cells with protein-specific antibodies. All cells were treated, fixed, blocked and permeabilized and incubated with the indicated antibody overnight at 4°C. Cells were again washed and antibody binding revealed using an appropriate secondary antibody coupled to fluorescence. Random cell fields were then visualized and photographed as shown for each protein. Arrows points to representative cells of each field identify the typical appearance of antigen distribution under each condition.
Mentions: A significant issue in this system to identify the cell signaling networks that lead to changes in β2 protein production and enhanced [3H]Eb density. One possibility is that a change in chaperoning of the receptor during assembly and transport through synthetic compartments leads to increased receptor expression [1,3]. Two candidate proteins that participate in this process are the immunological heavy chain-binding protein BiP [42] and 14-3-3eta [43]. To test the possibility that changes the expression of the 14-3-3eta or BiP correspond to upregulation related to PI3Kβ or Jak2 signaling, the expression levels of these chaperone proteins were measured. As above, subsequent to treatment of transfected 293 cells with choline, HC3 or choline and HC3 the whole cell lysates were analyzed using western blot. The typical results from 4 different experiments are shown in Fig 7A. Although the change in expression of β2 was as expected, in no case did treatment of the cells alter the expression of BiP or 14-3-3eta. The 14-3-3eta is shown because of its association to α4β2 [43], however no change in expression of other 14-3-3 proteins, as determined using additional antibodies or low isoform specificity, was observed (not shown). Thus, the expression of these candidate chaperone proteins did not vary with the treatments used it this study despite the change in α4β2 expression and 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