FIGURE 2.

Activity of the CCL2 promoter is positively regulated by TonEBP in NP cells. A, TonE predicted using Genomatix MatInspector in the first 2 kb of the proximal promoter of the rat CCL2 gene were compared with the human and mouse CCL2 promoters using Multiz alignments. Sites 1 and 2 show a high degree of conservation between all species compared. B, analysis using the Evolutionary Conserved Regions Browser demonstrates sequence conservation in the 2-kb CCL2 promoter between rat, human, canine, and rhesus. ECR threshold is set at 80%. CCL2 promoter: red, intergenic regions; green, transposons and simple repeats. Height of peaks represents percentage of identity between the compared genomes. C, diagrams showing predicted TonE #1–3 in the proximal promoter of the rat CCL2 luciferase reporter construct spanning −939/+59. Both wild-type (WT) and #2 TonE-mutated (Mutant) sequences are shown. D, CCL2 promoter activity is increased in NP cells transfected with wild-type CCL2 reporter and exposed to hypertonic medium (110 mm NaCl); TonE mutant construct shows no change in activity in response to hypertonicity. E, hypertonicity (Hyper)-dependent increase in wild-type CCL2 reporter activity is abolished by DN-TonEBP even at the lowest plasmid concentration of 50 ng, similar to inhibition of TauT reporter, a known TonEBP target. F, in cells cultured under isotonic (Iso) conditions, however, expression of DN-TonEBP does not affect CCL2 promoter activity. G, expression of TonEBP at all plasmid doses induces CCL2 promoter activity, similar to the effect on TauT. H, overexpression of TonEBP results in increased activity of only the wild-type, but not the TonE-mutated CCL2 promoter. Quantitative measurements represent mean ± S.E. of ≥3 biological replicates and 3 technical replicates per biological replicate. *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001. NS, not significant.