Induction of murine adenosine A2A receptor expression by LPS: analysis of the 5′ upstream promoter
Non-activated macrophages express low levels of A 2A Rs and lipopolysaccharides (LPS) upregulates A 2A R expression in an NF-κB-dependent manner. The murine A 2A R gene is encoded by three exons, m1, m2 and m3. Exons m2 and m3 are conserved, while m1 encodes the 5′ untranslated UTR. Three m1 variant...
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| creator | Elson, G Eisenberg, M Garg, C Outram, S Ferrante, C J Hasko, G Leibovich, S J |
| description | Non-activated macrophages express low levels of A
2A
Rs and lipopolysaccharides (LPS) upregulates A
2A
R expression in an NF-κB-dependent manner. The murine A
2A
R gene is encoded by three exons, m1, m2 and m3. Exons m2 and m3 are conserved, while m1 encodes the 5′ untranslated UTR. Three m1 variants have been defined, m1A, m1B and m1C, with m1C being farthest from the transcriptional start site. LPS upregulates A
2A
Rs in primary murine peritoneal and bone-marrow-derived macrophages and RAW264.7 cells by selectively splicing m1C to m2, through a promoter located upstream of m1C. We have cloned ∼1.6 kb upstream of m1C into pGL4.16(
luc2CP
/Hygro) promoterless vector. This construct in RAW 264.7 cells responds to LPS, and adenosine receptor agonists augmented LPS responsiveness. The NF-κB inhibitors BAY-11 and triptolide inhibited LPS-dependent induction. Deletion of a key proximal NF-κB site (402–417) abrogated LPS responsiveness, while deletion of distal NF-κB and C/EBPβ sites did not. Site-directed mutagenesis of CREB (309–320), STAT1 (526–531) and AP2 (566–569) sites had little effect on LPS and adenosine receptor agonist responsiveness; however, mutation of a second STAT1 site (582–588) abrogated this responsiveness. Further analysis of this promoter should provide valuable insights into regulation of A
2A
R expression in macrophages in response to inflammatory stimuli. |
| doi_str_mv | 10.1038/gene.2012.60 |
| format | Article |
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2A
Rs and lipopolysaccharides (LPS) upregulates A
2A
R expression in an NF-κB-dependent manner. The murine A
2A
R gene is encoded by three exons, m1, m2 and m3. Exons m2 and m3 are conserved, while m1 encodes the 5′ untranslated UTR. Three m1 variants have been defined, m1A, m1B and m1C, with m1C being farthest from the transcriptional start site. LPS upregulates A
2A
Rs in primary murine peritoneal and bone-marrow-derived macrophages and RAW264.7 cells by selectively splicing m1C to m2, through a promoter located upstream of m1C. We have cloned ∼1.6 kb upstream of m1C into pGL4.16(
luc2CP
/Hygro) promoterless vector. This construct in RAW 264.7 cells responds to LPS, and adenosine receptor agonists augmented LPS responsiveness. The NF-κB inhibitors BAY-11 and triptolide inhibited LPS-dependent induction. Deletion of a key proximal NF-κB site (402–417) abrogated LPS responsiveness, while deletion of distal NF-κB and C/EBPβ sites did not. Site-directed mutagenesis of CREB (309–320), STAT1 (526–531) and AP2 (566–569) sites had little effect on LPS and adenosine receptor agonist responsiveness; however, mutation of a second STAT1 site (582–588) abrogated this responsiveness. Further analysis of this promoter should provide valuable insights into regulation of A
2A
R expression in macrophages in response to inflammatory stimuli.</description><identifier>ISSN: 1466-4879</identifier><identifier>EISSN: 1476-5470</identifier><identifier>DOI: 10.1038/gene.2012.60</identifier><language>eng</language><publisher>London: Nature Publishing Group UK</publisher><subject>5' Untranslated Regions ; 631/208/200 ; 631/250/2504/342 ; 631/45/287/1190 ; Adenosine ; Agonists ; Biomedical and Life Sciences ; Biomedicine ; Bone marrow ; Cancer Research ; Cloning ; Cyclic AMP response element-binding protein ; Cytokines ; Exons ; Gene Expression ; Genes ; Genotype & phenotype ; Human Genetics ; Immunology ; Inflammation ; Lipopolysaccharides ; Macrophages ; NF-κB protein ; original-article ; Site-directed mutagenesis ; Stat1 protein ; Transcription ; Triptolide</subject><ispartof>Genes and immunity, 2013-04, Vol.14 (3), p.147-153</ispartof><rights>Macmillan Publishers Limited 2013</rights><rights>Copyright Nature Publishing Group Apr 2013</rights><rights>Macmillan Publishers Limited 2013.</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c2120-ea052aef6ea81ed9ead8356f3d675fc12bdd2c709a41807d76ea07513ee1bd1d3</citedby><cites>FETCH-LOGICAL-c2120-ea052aef6ea81ed9ead8356f3d675fc12bdd2c709a41807d76ea07513ee1bd1d3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://link.springer.com/content/pdf/10.1038/gene.2012.60$$EPDF$$P50$$Gspringer$$H</linktopdf><linktohtml>$$Uhttps://link.springer.com/10.1038/gene.2012.60$$EHTML$$P50$$Gspringer$$H</linktohtml><link.rule.ids>314,776,780,27901,27902,41464,42533,51294</link.rule.ids></links><search><creatorcontrib>Elson, G</creatorcontrib><creatorcontrib>Eisenberg, M</creatorcontrib><creatorcontrib>Garg, C</creatorcontrib><creatorcontrib>Outram, S</creatorcontrib><creatorcontrib>Ferrante, C J</creatorcontrib><creatorcontrib>Hasko, G</creatorcontrib><creatorcontrib>Leibovich, S J</creatorcontrib><title>Induction of murine adenosine A2A receptor expression by LPS: analysis of the 5′ upstream promoter</title><title>Genes and immunity</title><addtitle>Genes Immun</addtitle><description>Non-activated macrophages express low levels of A
2A
Rs and lipopolysaccharides (LPS) upregulates A
2A
R expression in an NF-κB-dependent manner. The murine A
2A
R gene is encoded by three exons, m1, m2 and m3. Exons m2 and m3 are conserved, while m1 encodes the 5′ untranslated UTR. Three m1 variants have been defined, m1A, m1B and m1C, with m1C being farthest from the transcriptional start site. LPS upregulates A
2A
Rs in primary murine peritoneal and bone-marrow-derived macrophages and RAW264.7 cells by selectively splicing m1C to m2, through a promoter located upstream of m1C. We have cloned ∼1.6 kb upstream of m1C into pGL4.16(
luc2CP
/Hygro) promoterless vector. This construct in RAW 264.7 cells responds to LPS, and adenosine receptor agonists augmented LPS responsiveness. The NF-κB inhibitors BAY-11 and triptolide inhibited LPS-dependent induction. Deletion of a key proximal NF-κB site (402–417) abrogated LPS responsiveness, while deletion of distal NF-κB and C/EBPβ sites did not. Site-directed mutagenesis of CREB (309–320), STAT1 (526–531) and AP2 (566–569) sites had little effect on LPS and adenosine receptor agonist responsiveness; however, mutation of a second STAT1 site (582–588) abrogated this responsiveness. Further analysis of this promoter should provide valuable insights into regulation of A
2A
R expression in macrophages in response to inflammatory stimuli.</description><subject>5' Untranslated Regions</subject><subject>631/208/200</subject><subject>631/250/2504/342</subject><subject>631/45/287/1190</subject><subject>Adenosine</subject><subject>Agonists</subject><subject>Biomedical and Life Sciences</subject><subject>Biomedicine</subject><subject>Bone marrow</subject><subject>Cancer Research</subject><subject>Cloning</subject><subject>Cyclic AMP response element-binding protein</subject><subject>Cytokines</subject><subject>Exons</subject><subject>Gene Expression</subject><subject>Genes</subject><subject>Genotype & phenotype</subject><subject>Human Genetics</subject><subject>Immunology</subject><subject>Inflammation</subject><subject>Lipopolysaccharides</subject><subject>Macrophages</subject><subject>NF-κB protein</subject><subject>original-article</subject><subject>Site-directed mutagenesis</subject><subject>Stat1 protein</subject><subject>Transcription</subject><subject>Triptolide</subject><issn>1466-4879</issn><issn>1476-5470</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2013</creationdate><recordtype>article</recordtype><sourceid>BENPR</sourceid><recordid>eNp90L1OwzAQB3ALgUQpbDyAJVYSfE5sJ2xVxUelSiABs-XGl5KqiYOdSHTjmXgknoREZWBATHfD7z70J-QcWAwsya7W2GDMGfBYsgMygVTJSKSKHY69lFGaqfyYnISwYQwkyHxC7KKxfdFVrqGupHXvqwapsdi4MHYzPqMeC2w75ym-tx5DGO1qR5ePT9fUNGa7C1UYh7tXpOLr45P2beg8mpq23tWuQ39KjkqzDXj2U6fk5fbmeX4fLR_uFvPZMio4cBahYYIbLCWaDNDmaGyWCFkmVipRFsBX1vJCsdykkDFl1QCZEpAgwsqCTabkYr93OPzWY-j0xvV-eDFoLlNQiRqy-U9Bkoos46DEoC73qvAuBI-lbn1VG7_TwPSYth7T1mPaWrKBR3seBtas0f9a-pf_BrVjgqs</recordid><startdate>20130401</startdate><enddate>20130401</enddate><creator>Elson, G</creator><creator>Eisenberg, M</creator><creator>Garg, C</creator><creator>Outram, S</creator><creator>Ferrante, C J</creator><creator>Hasko, G</creator><creator>Leibovich, S J</creator><general>Nature Publishing Group UK</general><general>Nature Publishing Group</general><scope>AAYXX</scope><scope>CITATION</scope><scope>3V.</scope><scope>7T5</scope><scope>7X7</scope><scope>7XB</scope><scope>88A</scope><scope>88E</scope><scope>8AO</scope><scope>8C1</scope><scope>8FD</scope><scope>8FE</scope><scope>8FH</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>ABUWG</scope><scope>AFKRA</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BHPHI</scope><scope>CCPQU</scope><scope>DWQXO</scope><scope>FR3</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>H94</scope><scope>HCIFZ</scope><scope>K9.</scope><scope>LK8</scope><scope>M0S</scope><scope>M1P</scope><scope>M7P</scope><scope>P64</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>PRINS</scope><scope>RC3</scope></search><sort><creationdate>20130401</creationdate><title>Induction of murine adenosine A2A receptor expression by LPS: analysis of the 5′ upstream promoter</title><author>Elson, G ; 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2A
Rs and lipopolysaccharides (LPS) upregulates A
2A
R expression in an NF-κB-dependent manner. The murine A
2A
R gene is encoded by three exons, m1, m2 and m3. Exons m2 and m3 are conserved, while m1 encodes the 5′ untranslated UTR. Three m1 variants have been defined, m1A, m1B and m1C, with m1C being farthest from the transcriptional start site. LPS upregulates A
2A
Rs in primary murine peritoneal and bone-marrow-derived macrophages and RAW264.7 cells by selectively splicing m1C to m2, through a promoter located upstream of m1C. We have cloned ∼1.6 kb upstream of m1C into pGL4.16(
luc2CP
/Hygro) promoterless vector. This construct in RAW 264.7 cells responds to LPS, and adenosine receptor agonists augmented LPS responsiveness. The NF-κB inhibitors BAY-11 and triptolide inhibited LPS-dependent induction. Deletion of a key proximal NF-κB site (402–417) abrogated LPS responsiveness, while deletion of distal NF-κB and C/EBPβ sites did not. Site-directed mutagenesis of CREB (309–320), STAT1 (526–531) and AP2 (566–569) sites had little effect on LPS and adenosine receptor agonist responsiveness; however, mutation of a second STAT1 site (582–588) abrogated this responsiveness. Further analysis of this promoter should provide valuable insights into regulation of A
2A
R expression in macrophages in response to inflammatory stimuli.</abstract><cop>London</cop><pub>Nature Publishing Group UK</pub><doi>10.1038/gene.2012.60</doi><tpages>7</tpages><oa>free_for_read</oa></addata></record> |
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| source | Springer Nature - Complete Springer Journals; Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals |
| subjects | 5' Untranslated Regions 631/208/200 631/250/2504/342 631/45/287/1190 Adenosine Agonists Biomedical and Life Sciences Biomedicine Bone marrow Cancer Research Cloning Cyclic AMP response element-binding protein Cytokines Exons Gene Expression Genes Genotype & phenotype Human Genetics Immunology Inflammation Lipopolysaccharides Macrophages NF-κB protein original-article Site-directed mutagenesis Stat1 protein Transcription Triptolide |
| title | Induction of murine adenosine A2A receptor expression by LPS: analysis of the 5′ upstream promoter |
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