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Fig. 6. CLIC4 is regulated by AP1 in keratinocytes undergoing Ca²⁺-induced differentiation. (A) Mouse keratinocytes in 0.05 mM Ca²⁺ medium were infected with -gal or A-Fos adenovirus for 12 hours prior to induction with 0.5 mM Ca²⁺ medium for 24 hours. Duplicate sets of cells were analyzed by real-time quantitative RT-PCR using involucrin, K1 and CLIC4 gene-specific primer sets. Bars represent standard error. (B) After 24 hours, the treated cells from A were analyzed by immunoblotting using K10, CLIC4 and actin antibodies. Asterisks (^*) in A-Fos lanes of both CLIC4 and K10 blots indicate `undetectable' CLIC4 and K10 bands that are within the linear exposure range of chemiluminescence. With longer exposures CLIC4 and K1 are detected in the A-Fos group (data not shown). (C) Triplicate sets of mouse keratinocytes were transfected with a reporter plasmid containing a 2.5 kb human CLIC4 promoter reporter construct prior to the treatment as described in A. The promoter responses were analyzed by measuring luciferase activity 16 hours after Ca²⁺ induction. (D) Schematic diagram of mouse and human CLIC4 promoters; solid black arrow indicates transcription start site, solid gray circles indicate AP1 binding sites and locations 5' upstream from the transcription start site. Two AP1 consensus sites are shown in the human promoter (A: –1507 and B: –840). (E) Luciferase reporter constructs containing the wild-type (WT), AP1-A site mutant (Mt A), AP1-B site mutant (Mt B), both AP1-A and AP1-B sites mutant (Mt A+B) of human CLIC4 promoter and an empty reporter construct (pGL3; Mock) were used to transfect mouse primary keratinocytes. Triplicate sets of transfected cells were induced with 0.5 mM calcium, and the promoter activities were measured by luciferase assay and presented as a fold increase in the promoter activities in high calcium versus low calcium-treated cells. (F) Biotinylated DNA oligos containing the wild-type (WT A and WT B) or the mutant (Mt A and Mt B) AP1 consensus binding sites derived from human CLIC4 promoter sequence were used to detect AP1 binding activities from the nuclear extracts generated from low or high calcium-treated cells in the absence or presence of A-Fos expression. AP1 proteins and the DNA oligo complex formed in the nuclear extracts were detected by ELISA-based assays as described in the Materials and Methods section, and the signals were normalized by the protein concentration of the nuclear extracts.

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