ABSTRACT
Hepatic expression of cytochrome P450 2A6 (CYP2A6) varies widely in humans and is induced during hepatitis; however, the mechanism regulating CYP2A6 has not been established. The murine orthologue Cyp2a5 is regulated post-transcriptionally by mRNA stabilization. A 43-kDa protein that binds to the 3'-untranslated region (3'-UTR) of Cyp2a5 mRNA has been identified, but its role in mRNA stabilization is unclear. We hypothesized that similar interactions occur between cytosolic proteins in human liver and CYP2A6 3'-UTR mRNA. We identified, by RNA electrophoretic mobility shift assay, an hepatic cytosolic protein that binds specifically to sequences in the 3'-UTR of CYP2A6. Complexes did not form with denatured proteins and were eliminated with proteinase K digestion. Complex formation was inhibited with a molar excess of unlabeled CYP2A6 RNA but not by non-specific competitor RNA. Protein-mRNA interactions were not affected by probe denaturation, suggesting that RNA secondary structure is not essential for binding. UV cross-linking of complexes revealed RNA-binding proteins in both human and mouse liver cytosols with molecular masses of approximately 43 kDa. Using truncated RNA probes corresponding to various lengths of CYP2A6 mRNA, the protein-binding site was localized to a 50-nucleotide region between bases 1478 and 1527 of the 3'-UTR. Complex formation with hepatic cytosolic protein from four human subjects correlated with levels of hepatic CYP2A6 microsomal protein, suggesting a possible regulatory role. Further characterization of the RNA-binding protein, the primary binding site, and the influence of this interaction on CYP2A6 mRNA stability will help to elucidate the relevance of these findings to the post-transcriptional control of CYP2A6.
Subject(s)
3' Untranslated Regions/metabolism , Aryl Hydrocarbon Hydroxylases , Cytochrome P-450 Enzyme System/metabolism , Microsomes, Liver/metabolism , Mixed Function Oxygenases/metabolism , RNA-Binding Proteins/isolation & purification , Base Sequence , Cytochrome P-450 CYP2A6 , Cytochrome P-450 Enzyme System/genetics , Cytochrome P450 Family 2 , Cytosol/metabolism , Humans , In Vitro Techniques , Mixed Function Oxygenases/genetics , Molecular Sequence Data , Molecular Weight , RNA, Messenger/analysis , RNA, Messenger/metabolism , RNA-Binding Proteins/metabolismABSTRACT
Lymphocytes from acutely diabetic BB/W rats were transplanted into SWR/JM nu/nu athymic mice and nu/nu athymic rats. Despite the use of a wide variety of dose regimens and pretreatments, no recipient developed either insulitis, hyperglycemia, or glucose intolerance. These data contradict earlier reports of successful transfer of insulitis using similar methodology.
Subject(s)
Lymphoid Tissue/transplantation , Pancreatitis/immunology , Animals , Diabetes Mellitus, Experimental/immunology , Diabetes Mellitus, Type 1/immunology , Female , Glucose Tolerance Test , Islets of Langerhans/pathology , Male , Mice , Mice, Nude , Rats , Rats, Inbred StrainsABSTRACT
Weekly transfusions of whole blood from a nondiabetic subline of BB/W rats reduced the incidence of diabetes in susceptible BB/W rats from 39 to 0 percent and the incidence of pancreatic insulitis from 64 to 6 percent. Responsiveness of lymphocytes to concanavalin A was found to be low in rats with diabetes or insulitis. Transfusion restored concanavalin A responsiveness to levels observed in control rats free of diabetes or insulitis. These data suggest that whole blood alters the course of autoimmune BB/W rat diabetes.
