Up-regulation of HIV coreceptor CXCR4 expression in human T lymphocytes is mediated in part by a cAMP-responsive element.
The chemokine and HIV receptor CXCR4 has been shown to play a role in chemotaxis and HIV-1 entry into T cells. Dibutyryl cAMP (DcAMP), an analog of cAMP, has been shown to increase CXCR4 cell surface expression and HIV-1 infectivity, but the molecular mechanism(s) responsible is unknown. Here we show that DcAMP treatment of purified human T lymphocytes increased transcription of CXCR4 mRNA as well as cell surface and intracellular CXCR4 protein expression. DcAMP-mediated stimulation of human PBL increased T-trophic HIV-1 (X4) fusion and viral replication as measured by syncytia formation and p24 levels, respectively. To determine the region(s) of the CXCR4 promoter required for cAMP responsiveness, truncations and point mutations of the CXCR4 promoter (nucleotides -1098 to +59) fused to luciferase were constructed and transiently transfected into human PBL. Deletional analysis demonstrated that the -1098 to -93 region of the CXCR4 promoter construct could be eliminated; the residual (-93 to +59) promoter retained cAMP responsiveness. Site-directed mutagenesis of a putative cAMP-responsive element (CRE) in the 5' UTR (+41 to +49) significantly and specifically attenuated the ability of DcAMP to drive the minimal CXCR4 promoter. Electrophoretic mobility shift assays demonstrated the formation of a complex between the CREB transcription factor and the putative CXCR4 CRE site. Our findings demonstrate a CRE element within the CXCR4 promoter that regulates CXCR4 transcription in response to changes in cAMP signaling. The cAMP-dependent up-regulation of CXCR4 mRNA results in increased CXCR4 intracellular and cell surface protein expression as well as increased HIV infectivity.
Cristillo
Anthony D
AD
Laboratory of Lymphocyte Biology, National Heart, Lung and Blood Institute, Bethesda, Maryland 20892, USA.
Highbarger
Helene C
HC
Dewar
Robin L
RL
Dimitrov
Dimiter S
DS
Golding
Hana
H
Bierer
Barbara E
BE
D003994:Bucladesine / Q000494:pharmacology; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000502:physiology*; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D015726:Giant Cells / Q000821:virology; D015497:HIV-1 / Q000254:growth & development; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000096:biosynthesis; D019718:Receptors, CXCR4 / Q000096:biosynthesis / Q000235:genetics*; D020218:Response Elements; D013601:T-Lymphocytes / Q000276:immunology / Q000378:metabolism / Q000821:virology*; D015533:Transcriptional Activation; D015854:Up-Regulation; D014779:Virus Replication
D017362:Cyclic AMP Response Element-Binding Protein; D046911:Macromolecular Substances; D012333:RNA, Messenger; D019718:Receptors, CXCR4; D003994:Bucladesine; D000242:Cyclic AMP