Induction of murine adenosine A(2A) 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 va...
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| Veröffentlicht in: | Genes and immunity 2013-04, Vol.14 (3), p.147 |
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| 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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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><identifier>PMID: 23328845</identifier><language>eng</language><publisher>England: Nature Publishing Group</publisher><subject>Alternative Splicing ; Animals ; Base Sequence ; Binding Sites - genetics ; Cell Line ; Cells, Cultured ; Diterpenes - pharmacology ; Epoxy Compounds - pharmacology ; Exons - genetics ; Female ; Gene expression ; Genetic aspects ; Inflammation ; Lipopolysaccharides - pharmacology ; Luciferases - genetics ; Luciferases - metabolism ; Macrophages ; Macrophages - cytology ; Macrophages - drug effects ; Macrophages - metabolism ; Mice ; Mice, Inbred C57BL ; Molecular Sequence Data ; Mutagenesis, Site-Directed ; NF-kappa B - antagonists & inhibitors ; NF-kappa B - metabolism ; Nitriles - pharmacology ; Phenanthrenes - pharmacology ; Physiological aspects ; Promoter Regions, Genetic - genetics ; Protein Isoforms - genetics ; Purinergic P1 Receptor Agonists - pharmacology ; Receptor, Adenosine A2A - genetics ; Reverse Transcriptase Polymerase Chain Reaction ; STAT1 Transcription Factor - metabolism ; Sulfones - pharmacology ; Transcriptional Activation - drug effects</subject><ispartof>Genes and immunity, 2013-04, Vol.14 (3), p.147</ispartof><rights>COPYRIGHT 2013 Nature Publishing Group</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,780,784,27924,27925</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/23328845$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></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 A(2A) 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>Alternative Splicing</subject><subject>Animals</subject><subject>Base Sequence</subject><subject>Binding Sites - genetics</subject><subject>Cell Line</subject><subject>Cells, Cultured</subject><subject>Diterpenes - pharmacology</subject><subject>Epoxy Compounds - pharmacology</subject><subject>Exons - genetics</subject><subject>Female</subject><subject>Gene expression</subject><subject>Genetic aspects</subject><subject>Inflammation</subject><subject>Lipopolysaccharides - pharmacology</subject><subject>Luciferases - genetics</subject><subject>Luciferases - metabolism</subject><subject>Macrophages</subject><subject>Macrophages - cytology</subject><subject>Macrophages - drug effects</subject><subject>Macrophages - metabolism</subject><subject>Mice</subject><subject>Mice, Inbred C57BL</subject><subject>Molecular Sequence Data</subject><subject>Mutagenesis, Site-Directed</subject><subject>NF-kappa B - antagonists & inhibitors</subject><subject>NF-kappa B - metabolism</subject><subject>Nitriles - pharmacology</subject><subject>Phenanthrenes - pharmacology</subject><subject>Physiological aspects</subject><subject>Promoter Regions, Genetic - genetics</subject><subject>Protein Isoforms - genetics</subject><subject>Purinergic P1 Receptor Agonists - pharmacology</subject><subject>Receptor, Adenosine A2A - genetics</subject><subject>Reverse Transcriptase Polymerase Chain Reaction</subject><subject>STAT1 Transcription Factor - metabolism</subject><subject>Sulfones - pharmacology</subject><subject>Transcriptional Activation - drug effects</subject><issn>1466-4879</issn><issn>1476-5470</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2013</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNptkE1LAzEQhoMotlZvniXgQT1sTSa7ya63UvwoFBSr5yVNZutK94MkC-2_d0v1UJA5zMvwPHN4CbnkbMyZSO9XWOMYGIexZEdkyGMloyRW7HiXpYziVGUDcub9N2NccpmdkgEIAWkaJ0NiZ7XtTCibmjYFrTpX1ki1xbrxuzS5hckddWiwDY2juGkder-jl1s6f1s8UF3r9daXfqeHL6TJDe1aHxzqirauqZqA7pycFHrt8eJ3j8jn0-PH9CWavz7PppN5tIIYQmQEGpmiysAKnSlkYIwBUAg2Y6nFFBRPTFYw4GDtUhWi4MzyQmKiUYEWI3K9_7vSa8zLumiC06YqvcknArKsL0mKnhr_Q_VjsSpNU2NR9vcD4e5A6JmAm7DSnff5bPF-yF7t2bZbVmjz1pWVdtv8r3HxA0N2gG4</recordid><startdate>201304</startdate><enddate>201304</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</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>ISR</scope></search><sort><creationdate>201304</creationdate><title>Induction of murine adenosine A(2A) receptor expression by LPS: analysis of the 5' upstream promoter</title><author>Elson, G ; Eisenberg, M ; Garg, C ; Outram, S ; Ferrante, C J ; Hasko, G ; Leibovich, S J</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-g242t-c3ec68e792d3a97e02ccc227e2d908de82715c9f0212ddb7f3f10d1f6e5ae72a3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2013</creationdate><topic>Alternative Splicing</topic><topic>Animals</topic><topic>Base Sequence</topic><topic>Binding Sites - genetics</topic><topic>Cell Line</topic><topic>Cells, Cultured</topic><topic>Diterpenes - pharmacology</topic><topic>Epoxy Compounds - pharmacology</topic><topic>Exons - genetics</topic><topic>Female</topic><topic>Gene expression</topic><topic>Genetic aspects</topic><topic>Inflammation</topic><topic>Lipopolysaccharides - pharmacology</topic><topic>Luciferases - genetics</topic><topic>Luciferases - metabolism</topic><topic>Macrophages</topic><topic>Macrophages - cytology</topic><topic>Macrophages - drug effects</topic><topic>Macrophages - metabolism</topic><topic>Mice</topic><topic>Mice, Inbred C57BL</topic><topic>Molecular Sequence Data</topic><topic>Mutagenesis, Site-Directed</topic><topic>NF-kappa B - antagonists & inhibitors</topic><topic>NF-kappa B - metabolism</topic><topic>Nitriles - pharmacology</topic><topic>Phenanthrenes - pharmacology</topic><topic>Physiological aspects</topic><topic>Promoter Regions, Genetic - genetics</topic><topic>Protein Isoforms - genetics</topic><topic>Purinergic P1 Receptor Agonists - pharmacology</topic><topic>Receptor, Adenosine A2A - genetics</topic><topic>Reverse Transcriptase Polymerase Chain Reaction</topic><topic>STAT1 Transcription Factor - metabolism</topic><topic>Sulfones - pharmacology</topic><topic>Transcriptional Activation - drug effects</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><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><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>Gale In Context: Science</collection><jtitle>Genes and immunity</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Elson, G</au><au>Eisenberg, M</au><au>Garg, C</au><au>Outram, S</au><au>Ferrante, C J</au><au>Hasko, G</au><au>Leibovich, S J</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Induction of murine adenosine A(2A) receptor expression by LPS: analysis of the 5' upstream promoter</atitle><jtitle>Genes and immunity</jtitle><addtitle>Genes Immun</addtitle><date>2013-04</date><risdate>2013</risdate><volume>14</volume><issue>3</issue><spage>147</spage><pages>147-</pages><issn>1466-4879</issn><eissn>1476-5470</eissn><abstract>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.</abstract><cop>England</cop><pub>Nature Publishing Group</pub><pmid>23328845</pmid><doi>10.1038/gene.2012.60</doi><tpages>7</tpages></addata></record> |
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| subjects | Alternative Splicing Animals Base Sequence Binding Sites - genetics Cell Line Cells, Cultured Diterpenes - pharmacology Epoxy Compounds - pharmacology Exons - genetics Female Gene expression Genetic aspects Inflammation Lipopolysaccharides - pharmacology Luciferases - genetics Luciferases - metabolism Macrophages Macrophages - cytology Macrophages - drug effects Macrophages - metabolism Mice Mice, Inbred C57BL Molecular Sequence Data Mutagenesis, Site-Directed NF-kappa B - antagonists & inhibitors NF-kappa B - metabolism Nitriles - pharmacology Phenanthrenes - pharmacology Physiological aspects Promoter Regions, Genetic - genetics Protein Isoforms - genetics Purinergic P1 Receptor Agonists - pharmacology Receptor, Adenosine A2A - genetics Reverse Transcriptase Polymerase Chain Reaction STAT1 Transcription Factor - metabolism Sulfones - pharmacology Transcriptional Activation - drug effects |
| title | Induction of murine adenosine A(2A) receptor expression by LPS: analysis of the 5' upstream promoter |
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