Prep1/Pbx2 complexes regulate CCL2 expression through the -2578 guanine polymorphism

Abstract

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 -2578 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 -2578 G-regulated CCL2 expression in astrocytes, which are major producers of CCL2 in the brain. The -2578 G polymorphism creates a consensus-binding site for the transcriptional regulator Prep1, which along with binding partner Pbx2, preferentially binds the -2578 G allele. CCL2 promoters harboring the G allele under unstimulated conditions exhibit a lower basal activity compared to the ancestral A allele. Upon interleukin-1 beta stimulation, Prep1/Pbx2 complexes maintain the ability to bind -2578 G alleles, yet transcription levels from promoters that harbor the A or G allele are equally activated, suggesting that the -2578 region does not influence CCL2 transcription under proinflammatory conditions. Therefore, promoters that harbor the -2578 G allele undergo a higher fold induction and by extension, individuals homozygous for -2578 G would be expected to exhibit hyper-responsive CCL2 phenotypes during periods of inflammation.

Figure 1

The −2578 G Polymorphism Influences Basal Activity of the CCL2 Distal Promoter. Distal promoters (929 bp) harboring either the −2578 G or A polymorphism were cloned directly upstream of the firefly luciferase gene in the pGL4.11 vector. Constructs were transfected into U87-MG cells (human astrocytic cell line) along with pGL4.74, a renilla luciferase construct that serves to control for transfection efficiency. [pGL4 vectors without inserts resulted in nominal background luciferase activity (not shown).] Transfected cells were either untreated or treated with human recombinant IL-1β (10 ng/mL) for 3 hours. Results are presented relative to the −2578 A construct. (A) Basal activity for the −2578 G construct is significantly lower than the −2578 A construct (p = 0.0012). (B) IL-1β stimulated activity is equal for constructs containing either the −2578 G or A allele (p = 0.88). (C) The fold induction is greater for the −2578 G construct (p = 0.016) as compared to the A construct. The results represent the mean of 4 individual experiments in which samples were processed in triplicate, controlled for transfection efficiency, and normalized to the −2578 A construct.

Figure 2

The −2578 G allele specifically binds a protein complex that does not contain IRF-1 or IRF-2. (A) Alignment of IRF-1, IRF-2 and ISRE consensus sites with the CCL2 ISRE (reverse-complement orientation) and both the −2578 A and G oligonucleotides, designed and used in EMSA experiments. Italicized nucleotides represent flexibility in the consensus sites (R = G or A; Y = C or T; S = C or G; W = A or T; and N = A, G, C or T). Bold nucleotides represent nucleotides in the CCL2 ISRE that do not match the consensus binding sequences. Bold/Italicized nucleotides highlight the location of the −2578 polymorphism, where neither polymorphism matches the consensus sequence. Western blot analysis to identify IRF-2 (B) and IRF-1 (C) in nuclear extracts from unstimulated U87-MG cells and U87-MG cells stimulated with 10 ng/ml IL-1β (a known activator of IRF-1) for 2.5 hours. EMSAs were performed using the unstimulated (D) and IL-1β stimulated (E) U87-MG nuclear lysates with labeled ISRE consensus oligonucleotide, −2578 A and G oligonucleotides. Antibodies were added to binding reactions as indicated above the gel lanes; ss, antibody complex resulting from supershift analysis using α-IRF-2 and α-IRF-1; IRF-2, indicates band that contains IRF-2 as part off the complex; IRF-1, indicates band that contains IRF-1 as part of the complex; GSC, protein complex that is specific to the −2578 G oligonucleotide. Competition EMSAs were performed using 200× unlabeled ISRE oligonucleotide (lane 2), −2578 A oligonucleotide (lane 3), −2578 G oligonucleotide (lane 4) and scrambled G oligonucleotide (lane 5, to account for nonspecific affects) with either unstimulated (F) or IL-1β stimulated (G) U87-MG nuclear extracts. To determine the specificity of the intense band present in the −2578 G lanes of the ISRE EMSA (D and E, lanes 7 and 8), competition EMSAs were performed using 100x unlabeled oligonucleotide of −2578 A oligonucleotide, −2578 G oligonucleotide, ISRE consensus oligonucleotide and scrambled G oligonucleotide against labeled-2578 A oligonucleotide (H) and labeled-2578 G oligonucleotide (I) using unstimulated U87-MG nuclear lysates. The G specific band is the only region shown for each EMSA because Figure 2D and 2E (lanes 7 and 8) clearly show that lanes containing the G specific band are void of nonspecific bands. Data are representative of at least three independent experiments.

Figure 3

The −2578 G polymorphism creates a MEINOX consensus binding site (TGACAG). (A) Alignment of the MEINOX consensus site with the −2578 G CCL2 oligonucleotide (reverse complement) and the Chang oligonucleotide (an oligonucleotide developed to study the Meis1 consensus site here named Chang oligonucleotide 23). MEINOX family members share the same binding site consensus sequence (TGACAG). Bold nucleotides represent nucleotides in the Chang oligonucleotide that do not match the −2578 G CCL2 oligonucleotide sequence, while a bold/italicized nucleotide represents the location of the −2578 polymorphism. (B) EMSA using nuclear extracts from U87-MG cells comparing nuclear complex binding of the −2578 A oligonucleotide (lane 1), the −2578 G oligonucleotide (lane 2) and the Chang oligonucleotide (lane 3). Competition EMSAs using 100X concentration of unlabeled −2578 A oligonucleotide (lane 3),−2578 G oligonucleotide (lane 4). Chang oligonucleotide (lane 5) and scrambled G oligonucleotide (lane 6) against labeled-2578 G oligonucleotide (C) and labeled Chang oligonucleotide (D). High-affinity DNA binding sites for Meis1 (E). The results of binding site selection for in vitro-produced monomeric Meis1 displayed as the percent frequency of each nucleotide at each position. The consensus binding sites are listed below the percentages. (Adapted from Chang et al. 1997) (F) Alignment of the MEINOX consensus site with the −2578 G CCL2 oligonucleotide (reverse complement) and the mutant −2578 G CCL2 oligonucleotides (including −2578 A CCL2 oligonucleotide). EMSAs using the oligonucleotides from the first mutant panel (all transitions mutations) (G) and the second mutant panel (different mutations at positions 3 and 5) (H) bound to complexes from unstimulated U87-MG extracts. The G specific band is the only region shown for each EMSA because Figure 2D and 2E (lanes 7 and 8) clearly show that lanes containing the G specific band are void of nonspecific bands. Data are representative of at least three independent experiments.

Figure 4

Prep1 and Pbx2 complexes bind the −2578 G allele. Western blot analysis of nuclear extracts from unstimulated U87-MG cells and U87-MG cells stimulated with 10 ng/ml IL-1β for 2.5 hours for (A) Prep-1, (B) Pbx-2 (a known binding partner of Prep1) and (C) Meis-2; no protein expression could be visualized for Pbx1 (the other known binding partner for prep1, data not shown). (D) EMSA performed using the unstimulated U87-MG nuclear lysates with labeled −2578 A oligonucleotide (lane 1) or the −2578 G oligonucleotide (lanes 2−6). Antibodies were added to binding reactions as indicated above the gel lanes; ss, antibody complex resulting from supershift analysis using α-Pbx2. U87-MG cells were transfected with siRNA for Prep1, Pbx2 and Meis2 as well as with the Risk Free siGlo and Non-targeting controls. (E) Western blot analysis of whole cell lysates from transfected cells using antibody against Prep1, Pbx2, Meis2 and actin (loading control). (F) Nuclear lysates from U87-MG cells extracted 36 hours after siRNA tranfection were used in EMSA experiments with labeled-2578 G oligonucleotide. Labeled ISRE consensus oligonucleotide was added to each reaction to use the IRF-2 band for quantitation purposes. Figure 4F is representative of three independent experiments. (G) Prep1 (upper gel) and Pbx2 (bottom gel) were coimmunoprecipitated from unstimulated U87-MG nuclear lysates and detected by western blot. The G specific band is the only region shown in some EMSAs because Figure 2D and 2E (lanes 7 and 8) clearly show that lanes containing the G specific band are void of nonspecific bands. Data are representative of at least two independent experiments.