Fig. 1. Effect of caffeine and adenosine on Cyp6a8 and Cyp6a2 promoter activities in SL-2 cells and transgenic flies. (A) SL-2 cells, co-transfected with 0.2luc-A8, 0.8luc-A8 or 0.9luc-A2 and pRL null plasmids, were treated for 24 h with either 8 mM caffeine (caff) or with 8 mM adenosine (Ado). Plain media and 250 mM Tris, pH 8.0 used as solvent for caffeine and adenosine, respectively were used as controls. (B) Adult 0.2-luc4H-ry and 0.8-luc110H-ry females were treated (T) with 16 mM caffeine or with 8 mM adenosine for 24 h. Flies allowed to feed on media made with water or 250 mM Tris (pH 8.0) were used as untreated (UT) controls. Values represent mean ± S.E.M of triplicate samples. The p values for treated (T) and untreated (UT) samples compared are: a, 0.001; b, 1.0; c, 0.0002; d, 0.1; e, 0.0003; f, 0.08; g, 0.003; h, 0.18; i, 0.005; and j, 0.98.

Fig. 2. Effect of A1 adenosine receptor agonist (CPA) and antagonist (DPCPX) on Cyp6a2 and Cyp6a8 promoter activity. (A) SL-2 cells, co-transfected with 0.2luc-A8, 0.8luc-A8 or 0.9luc-A2 and pRL null plasmids, were treated (T) for 24 h with 5 μM DPCPX or 500 μM CPA made in DMSO. Cells treated with DMSO were used as controls (UT). (B) Adult 0.2-luc4H-ry and 0.8-luc110H-ry females were treated with 16 mM CPA or 1 mM DPCPX (T). DMSO treated flies were used as untreated control (UT). Values represent mean ± S.E.M of triplicate samples. The p values for treated and untreated samples compared are: a, 0.84; b, 0.29; c, 0.18; d, 0.93; e, 0.85; f, 0.07; g, 0.28; h, 0.13; I, 0.117; and j, 0.26.

Fig. 3. Effect of phosphodiesterase inhibitors on Cyp6a2 and Cyp6a8 promoter activities in SL-2 cells and in transgenic flies. (A) SL-2 cells, co-transfected with 0.2luc-A8, 0.8luc-A8 or 0.9luc-A2 and pRL null plasmids, were treated for 24 h with 500 μM rolipram (RP) or with 8 mM theophylline (TP). Cells incubated in DMSO containing or plain media were used as untreated (UT) controls for rolipram and theophylline, respectively. (B) Adult females from 0.2-luc4H-ry and 0.8-luc110H-ry reporter strains were treated with 16 mM theophylline or with 16 mM rolipram. Water or DMSO treated flies served as controls for theophylline and rolipram treatments, respectively. Values represent mean ± S.E.M of triplicate samples. The p values for treated and untreated samples compared are: a, 0.0002; b, 0.003; c, 0.009; d, 0.001; e, 0.0014; f, 0.003; g, 0.0001; h, 0.0001; I, 0.0004; and j, 0.006.

Fig. 4. Effect of X-linked dunce (dnc) mutation on Cyp6a8 promoter activity. Extracts prepared for the heads of dnc¹ mutant (dnc¹/Y; 0.8luc-A8/+) and dnc¹⁺ (dnc¹⁺ /Y; 0.8luc-A8/+) males carrying the 0.8luc-A8 reporter transgene were assayed for luc activity and protein concentration. Values represent mean ± S.E.M of triplicate samples (p = 0.0074).

Fig. 5. Effect of caffeine on cAMP-phosphodiesterase activity in vitro. Extracts prepared from SL-2 cells (A) and 0.8-luc110H-ry females (B) were incubated with different concentration of caffeine and assayed for cAMP-PDE activity using scintillation proximity assay. Values represent mean ± S.E.M of triplicate samples. The p values for caffeine-treated and untreated samples are: a, 0.0003; b, 0.0002; c, 0.05; and d, 0.0085.

Fig. 6. Effect of caffeine on intracellular cAMP level. SL-2 cells were incubated for 24 h in 100 μl of 1 mM caffeine containing medium. After incubation, extracts were prepared from treated and untreated cells, and cAMP levels were measured using ELISA. Values represent mean ± S.E.M of three independent experiments (p = 0.0049).

Fig. 7. Effect of dibutyryl cAMP (db-cAMP) on Cyp6a2 and Cyp6a8 promoter activities. (A) SL-2 cells were transfected with different plasmids as described in Fig. 1. The transfected cells were then incubated for 24 h in plain (untreated) or 4 mM dibutyryl cAMP containing (treated) media. Extracts were prepared and assayed for F-luc and R-luc activities as described in Materials and methods. (B) 0.2-luc4H-ry and 0.8-luc110H-ry female flies were allowed to feed on medium containing 4 mM dibutyryl cAMP or water for 24 h. Extracts were prepared from these flies were assayed for firefly luciferase activity and protein content. Values represent mean ± S.E.M of triplicate samples. The p values for treated and untreated samples compared are: a, 0.03; b, 0.004; c, 0.0001; and d, 0.0002.

Fig. 8. Western blot analysis of the endogenous D-JUN protein level in caffeine-treated and untreated SL-2 cells and 0.8-luc110H-ry transgenic female flies. SL-2 cells and transgenic flies were treated for 24 h with 8 mM and 16 mM caffeine, respectively. Extracts prepared from treated (T) and untreated (UT) cells and flies were fractionated by SDS-PAGE, and blotted onto PVDF membrane. The resulting blots were probed with D-jun and actin antibodies, and the immunoreactive bands of SL-2 cell (A) and transgenic fly (B) extracts were quantified by using Bio-Rad “Quantity one” software program. The data were normalized using actin as internal control and the results are shown in panels C and D. The star represents a cross reacting protein band. Values represent mean ± S.E. M of triplicate samples. The p values for treated and untreated samples compared are: a, 0.005 and b, 0.0095 for D.

Fig. 9. Effect of D-jun sense expression on constitutive and caffeine-induced activities of Cyp6a8 promoter. SL-2 cells transfected with 0.8luc-A8 plasmid alone or co-transfected with 0.8luc-A8 and D-jun (S)/Tub plasmids were incubated for 24 h in plain media or media containing 8 mM caffeine. Extracts prepared from these cells were assayed and compared for luciferase activity. Three transfection experiments were done with three different batches of cells and three different isolates of 0.8luc-A8 and D-jun (S)/Tub plasmids on different days. Each bar represents mean ± S.E. M of three experiments. The p values for the means compared are: a, 0.0013 and b, 0.007.

Fig. 10. Effect of D-jun antisense expression of Cyp6a8 promoter activity and D-jun level. (A). SL-2 cells co-transfected with D-jun (AS)/HS and 0.8luc-A8 reporter plasmids were transferred to plain or 8 mM caffeine containing media, and heat-shocked at 37 °C with three 30 min pulses as described in Materials and methods. Extracts prepared from the transfected cells were assayed for luciferase activities and protein concentration. Values represent mean ± S.E. M of three transfections with three batches of cells. The p value for the means compared (a) is 0.0075. (B). Western blot analysis of D-jun levels in S2 cells. Control — cells transfected with 0.8luc-A8 reporter plasmid alone; +D-jun (AS) — cells co-transfected with 0.8luc-A8 and D-jun (AS)/HS plasmids. Two experiments were done with two different batches of S2 cells. The cell extracts were analyzed on the same Western blot as described in Materials and methods. Numbers of cells used in two experiments were not identical.

Fig. 11. Effect of caffeine on the steady-state D-JUN mRNA levels in adult females of different strains of D. melanogaster. Total RNA isolated from female flies were fractionated on 2.2 M formaldehyde–1.2% agarose gel, blotted and hybridized with D-jun and RP-49 gene probes. The levels of hybridization were determined by using a radioanalytical imager as described in Materials and methods. Levels of RP-49 mRNA were used to normalize the data. Values represent mean ± S.E.M of triplicate samples. The p values for the means compared are: a, 0.374; b, 0.209; and c, 0.267.

Fig. 12. Effect of caffeine on D-jun promoter activity. SL-2 cells were transfected with D-jun/luc reporter plasmid carrying − 540/+1 DNA of D-jun gene in front of the luciferase reporter gene in pGL2(N) basic vector. The transfected cells were incubated in the presence or absence of 8 mM caffeine, and luciferase activity and protein concentration were measured as described in Materials and methods. Each bar shows the mean ± S.E. M of triplicate samples. p = 0.104.

Fig. 13. Model summarizing the mechanism of Cyp6a8 promoter induction by caffeine. (A). Caffeine inhibits the cAMP-phosphodiesterase causing elevated levels of intracellular cAMP, which in turn stimulates the Cyp6a8 promoter activity. (B). Caffeine decreases the overall levels of D-jun (oval) and thereby, relieves the D-jun-mediated repression of Cyp6a8 promoter activity. D-jun acts as a repressor of Cyp6a8 promoter activity either by binding to AP-1 site (black diamond) or to a hitherto unknown binding site.