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MONTANI, VALERIA,TANIGUCHI, SHIN-ICHI,SHONG, MINHO,SUZUKI, KOICHI,OHMORI, MASAYUKI,GIULIANI, CESIDIO,NAPOLITANO, GIORGIO,SAJI, MOTOYASU,FIORENTINO, BRUNO,REIMOLD, ANDREAS M.,TING, JENNY P.-Y,KOHN, LEO 충남대학교 생물공학연구소 1999 생물공학연구지 Vol.7 No.-
Aberrant expression of major histocompatibility complex(MHC) classⅡ proteins on thyrocytes, which is associated with autoimmune thyroid disease, is mimicked by γ-interferon (γ-IFN). To define elements and factors that regulate classⅡ gene expression in thyrocytes and that might be involved in aberrant expression, we have studied γ-IFN-induced HLA-DRα gene expression in rat FRTL-5 thyroid cells. The present report shows that classⅡ expression in FRTL-5 thyrocytes is positively regulated by the classⅡ transactivator (CIITA), and that CIITA mimics the action of γ-IFN. Thus as is the case for γ-IFN, several distinct and highly conserved elements on the 5'-flanking region of the HLA-DRα gene, the S, X_1, X_2, and Y boxes between -137 to -65 bp, are required for classⅡ gene expression induced by pCIITA transfection in FRTL-5 thyroid cells. CIITA and γ-IFN do not cause additive increases in HLA-DRα gene expression in FRTL-5 cells, consistent with the possibility that CIITA is an intermediate factor in the γ-IFN pathway to increased classⅡ gene expression. Additionally, γ-IFN treatment of FRTL-5 cells induces an endogenous CIITA transcript; pCIITA transfection mimics the ability of γ-IFN treatment of FRTL-5 thyroid cells to increase the formation of a specific and novel protein/DNA complex containing CBP, a coactivator of CRE binding proteins important for cAMP-induced gene expression; and the action of both γ-IFN and CIITA to increase classⅡ gene expression and increase complex formation is reduced by cotransfection of a thyroid Y box protein, which suppresses MHC classⅠ gene expression in FRTL-5 thyroid cells and is a homolog of human YB-1, which suppresses MHC classⅡ expression in human glioma cells. we conclude that CIITA and TSH receptor suppressor element binding protein-1 are components of the γ-IFN-regulated transduction system which, respectively, increase or decrease classⅡ gene expression in thyrocytes and may, therefore, be involved in aberrant classⅡ expression associated with autoimmune thyroid disease. (Endocrinology 139: 280-289, 1998)
BALDUCCI-SILANO, PINA L.,SUZUKI, KOICHI,OHTA, MASANORI,SAITO, JUN,OHMORI, MASAYUKI,MONTANI, VALERIA,NAPOLITANO, GIORGIO,SHONG, MINHO,TANIGUCHI, SHIN-ICHI,PIETRARELLI, MICHELE,LAVARONI, STEFANO,MORI, A 충남대학교 생물공학연구소 1999 생물공학연구지 Vol.7 No.-
The single strand binding protein (SSBP-1) is a positive regulator of TSH receptor gene expression and binds to an element with a GXXXXG motif. The S box of the mouse major histocompatibility classⅡ gene has multiple GXXXXG motifs and can also bind SSBP-1. The S box is one of four highly conserved elements on the 5'-flanking region of classⅡ genes that are necessary for interferon-γ (IFNγ) to overcome the normally suppressed state of the gene and induce aberrant classⅡ expression. In this report we show that SSBP-1, when overexpressed in FRTL-5 thyroid cells, is a positive regulator of human leukocyte antigen (HLA)-DRα classⅡ gene expression, as is IFNγ or the classⅡ trans-activator (CIITA). This is evidenced by increased exogenous promoter activity, increased endogenous RNA levels, and increased endogenous antigen expression after transfecting full-length SSBP-1 complementary DNA together with a HLA-DRα promoter-reporter gene chimera into TSH-treated FRTL-5 thyroid cells whose endogenous SSBP-1 levels are low. IFNγ reverses the ability of TSH to decrease endogenous SSBP-1 RNA levels. Also, whereas SSBP-1 transfection does not cause any increase in IFNγ-induced exogenous promoter activity, transfection of SSBP-1 and CIITA additively increases endogenous classⅡ RNA levels to levels measured in cells treated with IFNγ. Further, competition studies show that SSBP-1 binding is necessary for formation of the double strand protein/DNA complexes that are seen in electrophoretic mobility shift assays when the classⅡ 5'-flanking region is incubated with extracts from IFNγ-treated FRTL-5 cells and that have been previously associated with IFNγ-induced aberrant classⅡ expression. These data suggest that SSBP-1 is involved in the action of IFNγ to overcome the normally suppressed state of the classⅡ gene; it functions together with CIITA, whose expression is independently increased by IFNγ. The effect of SSBP-1 as a positive regulator of classⅡ promoter activity is lost in cells maintained without TSH, in which endogenous SSBP-1 RNA levels are already high in the absence of aberrant classⅡ gene expression. These data suggest that high levels of endogenous SSBP-1 are insufficient to cause aberrant classⅡ expression, but, rather, TSH or IFNγ treatment additionally modulates the cell, albeit differently, such that transfected or endogenous SSBP-1, respectively, can express its positive regulatory activity. The effect of TSH is consistent with reports indicating that TSH enhances the ability of IFNγ to increase classⅡ gene expression despite the fact IFNγ increases endogenous SSBP-1 to only the same levels as in cells untreated with TSH. Finally, the effect of SSBP-1 as a positive regulator is lost when GXXXXG motifs, which exist on both the coding and noncoding strands of the S box, are mutated. Consistent with this, mutation and oligonucleotide competition studies show that GXXXXG motifs are necessary for either strand of the S box to bind protein/DNA complexes containing SSBP-1 in FRTL-5 cell extracts or to bind to recombinant SSBP-1. They also suggest that the SSBP-1-binding sites on either strand of the HLA-DRα S box are functionally distinct. We conclude from these data that the positive regulatory action of SSBP-1 on classⅡ gene expression involves GXXXXG motifs on each strand of the highly conserved S box of the classⅡ 5'-flanking region. As SSBP-1 is modulated by IFNγ and is involved in classⅠ and TSH receptor as well as classⅡ gene expression in FRTL-5 cells, the sum of the data supports the hypotheses that common transcription factors regulate all three genes, and their altered activities may contribute to the development of autoimmunity. (Endocrinology 139: 2300-2313, 1998)