Role for protein kinase C delta in the functional activity of human UGT1A6: implications for drug-drug interactions between PKC inhibitors and UGT1A6

Abstract

Many UDP-glucuronosyltransferases (UGTs) require phosphorylation by protein kinase C (PKC) for glucuronidation activity. Inhibition of UGT phosphorylation by PKC inhibitor drugs may represent a novel mechanism for drug-drug interactions. The potential for PKC-mediated inhibition of human UGT1A6, an isoform involved in the glucuronidation of drugs such as acetaminophen (paracetamol) and endogenous substrates including serotonin, was evaluated using various cell model systems. Of ten different PKC inhibitors screened for their effects on acetaminophen glucuronidation by human LS180 colon cells, only rottlerin (PKC delta selective inhibitor; IC(50) = 9.0 +/- 1.2 microM) and the non-selective PKC inhibitors (calphostin-C, curcumin and hypericin) decreased glucuronidation by more than 50%. Using UGT1A6-infected Sf9 insect cells, calphostin-C and hypericin showed three times more potent inhibition of serotonin glucuronidation in treated whole cells versus cell lysates. However, both curcumin and rottlerin showed significant direct inhibition and so (indirect) PKC effects could not be differentiated in this model system. Of nine PKC isoforms co-expressed with UGT1A6 in human embryonic kidney 293T cells only PKC delta increased protein-normalized UGT1A6-mediated serotonin glucuronidation significantly (by 63% +/- 4%). These results identify an important role for PKC delta in UGT1A6-mediated glucuronidation and suggest that PKC delta inhibitors could interfere with glucuronidation of UGT1A6 substrates.

Figure 1.. PKC inhibitor screen evaluating the effect of ten different PKC isoform selective and non-selective inhibitors on acetaminophen glucuronidation in LS180 cells.

Calphostin-C, curcumin, hypericin, and valproic acid are considered pan PKC inhibitors, while the other inhibitors selected have some selectivity for specific PKC isoforms. NPC-15437 at 100 μM resulted in the detachment of the LS180 cells from the culture plates after 2 hours with complete cell lysis by 26 hours (overt toxicity). Data points correspond to the mean of duplicate determinations performed on the same day.

Figure 2.. Concentration-dependent inhibition of acetaminophen glucuronidation by the PKC inhibitors calphostin-C (A), hypericin (B), curcumin (C), and rottlerin (D) in human liver microsomes, LS180 cell lysates, and intact LS180 cells.

In panel A, B, and D, the effect of selective PKC inhibitors, calphostin-C (A) and hypericin (B), and PKC δ selective inhibitor, rottlerin (D) on acetaminophen glucuronidation was evaluated using both intact LS180 cells and human liver microsomes. In panel C, inhibition of acetaminophen glucuronidation by curcumin was compared between intact LS180 cells, homogenates of (washed and untreated) LS180 cells, and human liver microsomes. Data points correspond to the mean ± standard error of three determinations performed on the same day.

Figure 3.. Concentration-dependent inhibition of serotonin and acetaminophen (APAP) glucuronidation by the PKC inhibitors calphostin-C (A), hypericin (B), curcumin (C), and rottlerin (D) in human liver microsomes, UGT1A6-expressing Sf9 insect cell lysates, and intact UGT1A6-expressing Sf9 cells.

Sf9 cells were infected with UGT1A6-expressing baculovirus for three days after which the UGT1A6-expressing cells were treated for 2 hours with PKC inhibitor. Cell lysates were then prepared and either serotonin or acetaminophen glucuronidation was monitored ex cellulo. Untreated UGT1A6-expressing cell lysates and pooled HLM were also prepared and subsequently treated with PKC inhibitor to monitor direct inhibition of UGT1A6. Acetaminophen glucuronidation activity was only monitored in intact UGT1A6-expressing Sf9 cells. Data points correspond to the mean ± SE of three independent determinations.

Figure 4.. Co-expression of nine different PKC isoforms with UGT1A6 in HEK293T cells.

A) Immunoblot of the catalytic domain of different PKC isoforms (α, β1, β2, δ, ε, γ, ι, η, and ζ) labeled with a hemagglutinin (HA) epitope tag and co-expressed with UGT1A6 (and β-galactosidase as a transfection control) in HEK293T cells. Cell lysates were prepared 48 hours post-transfection and subjected to immunoblot analysis with an anti-HA antibody. Similar results were observed in two other independent experiments. Shown below the PKC blot are the results of blotting different amounts of a positive control protein (glutathione-S-transferase fused with an HA tag). B) β-galactosidase activities measured in the lysates of cells (same as in A) that were cotransfected with plasmids expressing UGT1A6, β-galactosidase, and 9 different PKC isoforms. Instead of PKC, some cells were cotransfected with the PKC vector control (labeled UGT1A6 control) or with a non-specific protein control (labeled TMED7).

Figure 5.. Effect of overexpression of nine different PKC isoforms on UGT1A6 protein levels in HEK293T cells.

A) Immunoblot of UGT1A6 coexpressed with the catalytic domains of different PKC isoforms (α, β1, β2, δ, ε, γ, ι, η, and ζ) and β-galactosidase (as a transfection control) in HEK293T cells. Instead of PKC, some cells were cotransfected with the PKC vector control (labeled UGT1A6 control) or with a non-specific protein control (labeled TMED7). Cell lysates were prepared 48 hours post-transfection and subjected to immunoblot analysis with an anti-UGT1A peptide antibody. A UGT1A6 standard (6 to 100 ng per lane) provided with the WB-UGT1A6 kit (BD Gentest) was used as a standard for quantitation purposes. Shown is a representative blot from 3 independent experiments. B) Quantification of UGT1A6 protein expression by immunoblot analysis (representative blot shown in A). Data points correspond to the mean ± standard error of three independent experiments performed on separate days. C) UGT1A6 protein data (from B) normalized to β-galactosidase activities (shown in Figure 4B) Data points on B and C correspond to the mean ± SE of three independent experiments performed on separate days. Statistical comparisons were made using one-way ANOVA followed by a Dunnett’s post hoc test. * indicates that the UGT1A6 protein level is significantly different (P<0.05) from the UGT1A6 control value.

Figure 6.. Effect of overexpression of nine different PKC isoforms on UGT1A6 specific activity in HEK293T cells.

The catalytic domains of nine different PKC isoforms (α, β1, β2, δ, ε, γ, ι, η, and ζ) were transiently co-expressed with UGT1A6 in HEK293T cells. Instead of PKC, some cells were cotransfected with the PKC vector control (labeled UGT1A6 control) or with a non-specific protein control (labeled TMED7). Cell lysates were prepared 48 hours post-transfection and assayed for serotonin glucuronidation. Serotonin glucuronidation activities were standardized to UGT1A6 protein (shown in Figure 5) and reported as a percentage of UGT1A6 control (without PKC) activity. Data points correspond to the mean ± standard error of three independent experiments performed on separate days. Statistical comparisons were made using one-way ANOVA followed by a Dunnett’s post hoc test. * indicates that UGT1A6 specific activity is significantly different from the UGT1A6 control value.