We study the regulation of UDP-glucuronosyltransferase (UGT, transferase) genes at the molecular level using the human system. UGT isozymes form the primary system for detoxifying potentially noxious chemicals taken into the body. After having completed the description of the novel UGT1A complex locus that includes 13 different genes that share 4 common exons, we discovered 5 are strategically expressed in the mucosal surface throughout the gastrointestinal (GI) tract in a differential pattern. Isozymes utilize different optimal conditions for catalysis on a per chemical basis to metabolize a broad array of substrates found to be endogenous metabolites, chemicals taken in from the environment and diet, and agents consumed for therapeutic reasons. UGT1A10, distributed primarily in the lower GI, was shown to be the primary metabolizer of estrogens and phytoestrogens, nonsteroidal anti-inflammatory agents, and acids derived from dietary plants. We also expanded on our earlier discovery that UGTs require phosphorylation by using UGT1A7 and UGT1A10 as models. UGT1A7 is distributed primarily in esophageal squamous cells. Protein kinase C (PKC) isozymes were shown to carry out phosphorylation of UGT1A family members, whereas tyrosine kinase supported activity for at least one UGT2B family member studied. We demonstrated immunoprecipitable [33P] orthophosphate labeling of UGT and inhibition of labeling and activity with curcumin, a general kinase inhibitor, and calphostin-C, a specific PKC inhibitor. Through mutation analysis of PKC sites in UGT combined with immunoreactivity of anti-phospho-serine, inhibition of UGT activity with PKCepsilon-translocation inhibitor peptide, and colocalization of UGT and activated PKCepsilon, we established that the latter phosphorylates a select phospho-group in UGT1A7. While individual mutants at 2 PKC sites in UGT1A7 caused null activity, mutants at a third site elicited a dramatic pH shift from 8.5 to 6.4, at least 4 new substrates, and loss of 4 substrates; UGT1A7 also metabolized the new substrates if PKCepsilon was inhibited in cell culture with its specific translocation inhibitor or by either of 3 general kinase inhibitors. To the contrary, UGT2B7 was unaffected by PKC inhibitors; it was inhibited, however, between 50 and 60% with the tyrosine kinase inhibitors, herbimycin-C and genistein. Alteration of one or all three PKC sites in UGT2B7 had no effect on activity, while a single or double mutation at the two tyrosine kinase sites caused null activity. To determine the implication of UGT inhibition by curcumin, a highly used condiment in certain populations, we sought to determine whether we could enhance the action of the promising immunosuppressant, mycophenolic acid (MPA), which is widely prescribed for renal transplant patients and autoimmune diseases. Because there are serious side-effects associated with high MPA dosage requirements due to extensive glucuronidation, we sought to inhibit its in-vivo glucuronidation with preadministration of curcumin. Whereas our demonstration that the primary metabolizers, UGT1A7, 1A8, 1A9, and 1A10, of MPA are located in the GI mucosal cells and that each requires between 0.8 to 2.9 millimolar concentrations to reach saturation kinetics, which contribute to high oral-dose requirements, we uncovered that oral curcumin (100 mg/Kg b.w.)-pretreatment of antigen-treated mice caused a 6-fold enhancement of MPA immunosuppression of cytotoxic T-lymphocyte proliferation.
