Genes and Immunity (2008) 9, 419–430 & 2008 Macmillan Publishers Limited All rights reserved 1466-4879/08 $30.00 www.nature.com/gene ORIGINAL ARTICLE Prep1/Pbx2 complexes regulate CCL2 expression through the À2578 guanine polymorphism EK Wright Jr1,2, SH Page1,2, SA Barber3 and JE Clements2 1McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, USA; 2Department of Molecular and Comparative Pathobiology, Johns Hopkins University School of Medicine, Baltimore, MD, USA and 3Booz Allen Hamilton, Rockville, MD, USA CC-chemokine ligand 2 (CCL2) is the major chemoattractant protein that recruits monocytes to sites of inflammation and increased expression of CCL2 is associated with numerous inflammatory diseases including human immunodeficiency virus- associated dementia (HIV-D). The À2578 guanine polymorphism in the CCL2 promoter has been associated with increased expression of CCL2 as well as pathogenesis of HIV-D; however, the molecular mechanism of regulation is unknown. We propose a molecular model for À2578 G-regulated CCL2 expression in astrocytes, which are major producers of CCL2 in the brain. The À2578 G polymorphism creates a consensus-binding site for the transcriptional regulator Prep1, which along with binding partner Pbx2, preferentially binds the À2578 G allele. CCL2 promoters harboring the G allele under unstimulated conditions exhibit a lower basal activity compared to the ancestral A allele. Upon interleukin-1b stimulation, Prep1/Pbx2 complexes maintain the ability to bind À2578 G alleles, yet transcription levels from promoters that harbor the A or G allele are equally activated, suggesting that the À2578 region does not influence CCL2 transcription under proinflammatory conditions. Therefore, promoters that harbor the À2578 G allele undergo a higher fold induction and by extension, individuals homozygous for À2578 G would be expected to exhibit hyper-responsive CCL2 phenotypes during periods of inflammation. Genes and Immunity (2008) 9, 419–430; doi:10.1038/gene.2008.33; published online 15 May 2008 Keywords: TALE; CCL2; SNP; promoter; Prep1; Pbx2 Introduction promoters that harbor the À2578 G allele exhibit higher levels of activity than those harboring the A allele in CC-chemokine ligand 2 (CCL2, formally known as interleukin (IL)-1b-stimulated cells.11 Other studies de- monocyte chemoattractant protein 1), a member of the monstrated that the guanine polymorphism increased C-C chemokine family, is produced by macrophages, the levels of CCL2 produced in cultured peripheral blood microglia, activated astrocytes and endothelial cells to mononuclear cells, monocytes and hepatic stellate cells recruit circulating monocytes to sites of inflammation.1 providing evidence that the polymorphism regulates Owing to its importance in inflammatory responses, CCL2 expression in the context of the endogenous increased levels of CCL2 have been implicated in a promoter.11–16 variety of disease pathologies including arthersosclero- Almost 6 years ago, a provocative study associated the sis, cancer metastisis, multiple sclerosis, Alzheimer’s À2578 G allele with protection against initial HIV disease and human immunodeficiency virus-associated- infection; however, once individuals were infected, they dementia (HIV-D).2–7 In other settings, CCL2 appears to exhibited a more rapid progression to acquired immune be beneficial as it protects against initial HIV infection, is deficiency syndrome and a 4.5-fold increased chance of neuroprotective against HIV Tat and N-methyl-D-aspar- developing HIV-D compared to individuals carrying the tate-induced apoptosis during early HIV infection, and ancestral À2578 A allele.9 Preliminary molecular analysis promotes healing following myocardial infarct.8–10 Ge- in this study suggested that the region encompassing the netic analysis of the distal CCL2 promoter revealed a À2578 polymorphism shared homology with an inter- polymorphism at À2578 (alternatively designated feron-stimulated response element (ISRE) and that the À251811) with functional consequences in the context of transactivator interferon regulatory factor (IRF)-1 bound a luciferase reporter assay. Specifically, CCL2 distal the À2578 A allele with higher affinity than the À2578 G allele. Presently, numerous reports have associated the À2578 Correspondence: Dr JE Clements, Department of Molecular and G polymorphism with a wide variety of pathological Comparative Pathobiology, Johns Hopkins University School of sequelae such as progression of Mycobacterium turberculosis Medicine, 733 North Broadway, Broadway Research Building 831, infection, earlier age of onset of idiopathic immune- Baltimore, MD 21205, USA. E-mail: [email protected] mediated posterior segment uveitis, asthma severity, Received 15 January 2008; revised 4 April 2008; accepted 7 April premature kidney graft failure and hepatitis C virus- 2008; published online 15 May 2008 related liver disease severity among others.12–14,17,18 Prep1/Pbx2 complexes bind CCL2 À2578 G region EK Wright et al 420 A number of studies have also correlated the À2578 G immunodeficiency virus (SIV) encephalitis and demen- polymorphism with increased expression of CCL2 tia.2 We utilized the same 929-bp distal human CCL2 in vivo. For example, plasma levels of CCL2 in promoter region as the previous study inserted into a unaffected-individuals and patients with tuberculosis, pGL4.11 firefly luciferase vector in conjunction with a Behcet’s and Alzheimer’s diseases were significantly pGL4.74 renilla luciferase vector in cotransfection experi- higher in individuals that harbor the À2578 G allele ments to control transfection efficiency. Extracts pre- compared to those that harbor the À2578 A allele.3,9,13,16,19 pared from transfected U87-MG cells that were either Further, levels of CCL2 in the cerebrospinal fluid of HIV stimulated with IL-1b or left unstimulated for 3 h were infected individuals were significantly higher in indivi- subjected to Dual-Luciferase Reporter Assay (Promega duals that harbored the G at À2578 compared to those Corporation, Madison, WI, USA) to quantitate both which harbored the A, indicating that this polymorph- firefly and renilla luciferase activity. A consistent reduc- ism influences in vivo central nervous system (CNS) tion in the basal level of transcription was observed in levels of CCL2.9 Finally, patients with systemic lupus cells transfected with vectors that contain the À2578 G erythematosus that carried the À2578 G allele demon- allele as compared to the À2578 A allele (Figure 1, strated an increase in interstitial macrophages in the P ¼ 0.0012). However, transcriptional levels were equiva- kidney, which is indicative of increased trafficking of lent after stimulation with IL-1b (Figure 1, P ¼ 0.88), monocytes into tissue, thereby providing biological reflecting a greater fold induction for the À2578 G allele significance of the À2578 G allele to increased levels of (Figure 1, P ¼ 0.016). These data suggested two possibi- CCL2.9 lities: (1) a transcription factor(s) binds preferentially to Because the majority of studies have demonstrated the À2578 A allele in unstimulated cells, providing a that the À2578 G allele confers increased expression of higher level of constitutive transcriptional activation CCL2 in cells that are stimulated with proinflammatory and/or (2) a transcription factor(s) with repressor cytokines as compared to the À2578 A allele, we were activity binds preferentially to the À2578 G promoter in puzzled by a potential regulatory mechanism involving unstimulated cells. decreased binding of a classical transactivator protein (IRF-1) and intrigued by the report of a specific complex The À2578 G region of the human CCL2 promoter exhibits able to bind the À2578 G allele in electrophoretic enhanced binding of a specific nuclear complex that mobility shift assay (EMSA) but not the A allele.12 Here does not contain IRF-1 or -2 we propose a molecular model by which the À2578 G Previous studies suggested that the À2578 region is an polymorphism regulates CCL2 expression in astrocytes ISRE and that the À2578 G polymorphism disrupts the and identify two proteins that bind as a complex binding of the transcription activator IRF-1.9 However, specifically to the À2578 G CCL2 allele. Neither protein aligning the consensus-binding elements for IRF-1 and -2 is a member of the IRF family (in fact, no definitive with the canonical ISRE, the CCL2 ISRE and the probes binding of IRF-1 or -2 to either À2578 allele could be used in this study (in reverse-complement orientation, demonstrated in our studies); rather, one protein was Figure 2a), revealed that an A or a G at position À2578 identified as Prep1 (also known as Pknox1), a member of disrupts the ISRE consensus site where ISRE, IRF-1 and the three amino-acid loop extension (TALE) family of IRF-2-binding sites are 100% conserved. Although this proteins in the subfamily MEINOX (Meis/Pknox1), and alignment suggests that the À2578 region is a poor ISRE, the second protein was identified as Pbx2, a second we nonetheless included IRF-1 and -2 (a known repressor TALE family member known to complex with Prep1. of ISREs) in our analyses in an effort to reconcile Both proteins are required to obtain binding of the previously reported studies. To examine the presence of complex to the À2578 G allele. With broader implications IRF-1 and -2 in U87-MG cells, we performed western blot perhaps relevant to the wide variety of diseases analysis on nuclear lysates, which demonstrated