Regulation of major histocompatibility class II gene expression in FRTL-5 thyrocytes: Opposite effects of interferon and methimazole

Abstract

Aberrant expression of major histocompatibility complex (MHC) class II antigens is associated with autoimmune thyroid disease; aberrant expression duplicating the autoimmune state can be induced by interferon-gamma (IFN gamma). We have studied IFN gamma-induced human leukocyte antigen (HLA)-DR alpha gene expression in rat FRTL-5 thyroid cells to identify the elements and factors important for aberrant expression. Using an HLA-DR alpha 5'-flanking region construct from -176 to +45 bp coupled to the chloramphenicol acetyltransferase reporter gene, we show that there is no basal class II gene expression in FRTL-5 thyroid cells, that IFN gamma can induce expression, and, as is the case for antigen-presenting cells from the immune system, that IFN gamma-induced expression requires several highly conserved elements on the 5'-flanking region, which, from 5' to 3', are the S, X-1, X-2, and Y boxes. Methimazole (MMI), a drug used to treat patients with Graves' disease and experimental thyroiditis in rats or mice, can suppress the IFN gamma-induced increase in HLA-DR alpha gene expression as a function of time and concentration; MMI simultaneously decreases IFN gamma-induced endogenous antigen presentation by the cell. Using gel shift assays and the HLA-DR alpha 5'-flanking region from -176 or -137 to +45 bp as radiolabeled probes, we observed the formation of a major protein-DNA complex with extracts from FRTL-5 cells untreated with IFN gamma, termed the basal or constitutive complex, and formation of an additional complex with a slightly faster mobility in extracts from cells treated with IFN gamma. MMI treatment of cells prevents IFN gamma from increasing the formation of this faster migrating complex. Formation of both complexes is specific, as evidenced in competition studies with unlabeled fragments between -137 and -38 bp from the start of transcription; nevertheless, they can be distinguished in such studies. Thus, high concentrations of double stranded oligonucleotides containing the sequence of the Y box, but not S, X-1, or X-2 box sequences, can prevent formation of the IFN gamma-increased faster migrating complex, but not the basal complex. Both complexes involve multiple proteins and can be distinguished by differences in their protein composition. Thus, using specific antisera, we show that two cAMP response element-binding proteins, activating transcription factor-1 and/or -2, are dominant proteins in the upper or basal complex. The upper or basal complex also includes c-Fos, Fra-2, Ets-2, and Oct-1. A dominant protein that distinguishes the IFN gamma-increased lower complex is CREB-binding protein (CBP), a coactivator of cAMP response element-binding proteins. We, therefore, show that aberrant expression of MHC class II in thyrocytes, induced by IFN gamma, is associated with the induction or increased formation of a novel protein-DNA complex and that its formation as well as aberrant class II expression are suppressed by MMI, a drug used to treat human and experimental autoimmune thyroid disease. Its component proteins differ from those in a major, basal, or constitutive protein-DNA complex formed with the class II 5'-flanking region in cells that are not treated with IFN gamma and that do not express the class II gene.