The Molecular Basis For The Regulation Of Mhc Class Ii Expression In Thyrocytes

Only few studies have looked at the different levels of regulation of MHC class II gene expression in thyroid cells, although the phenomenon of MHC class II induction in autoimmune and malignant diseases is very well documented, as has been described before.

In IFN-y-induced macrophages the stability of MHC class II mRNA indicated long-lived transcripts. In contrast, in three different malignant thyroid cell lines the half-life value of HLA-DRa mRNA was measured between 1.3-7 h [63], This obvious reduction in the half-life values between immune cells and epithelial malignant cells may suggest that this level of regulation, not yet fully explored, may be an important distinct regulatory mechanism between the normal and aberrant expression of MHC class II molecules.

Polymorphism of MHC class II promoter may also influence their expression. However, when the sequence of the HLA-DRa promoter was determined in three malignant thyroid cell lines and in normal thyroid epithelial cells, which exhibited different levels of HLA-DR expression, no changes or point mutations could be found compared to the published sequence from B cell [63]. Hence, the structure of the promoter in thyroid cells was identical to that of immune cells, and no polymorphism of the promoter was found.

Recent evidence show that the same boxes and the same transcription factors that are used for MHC class II gene regulation in B cells and macrophages are also used in thyroid cells. The highly conserved S, X, X2 and Y boxes are necessary for IFN-y-induced expression of HLA-DR in the FRTL-5 rat thyroid cell line [162], as was demonstrated by transfection of reporter gene constructs. We have demonstrated the binding of protein complexes to these elements in human thyroid carcinoma cell lines by gel shift mobility experiments [84],

DNA-binding proteins that occupy the HLA-DRa promoter, which were identified in B cells and macrophages have been found in thyroid malignant cells. We have shown that specific proteins such as RFX5 or YB-1 were expressed constantly, and their levels were not significantly changed after the addition of IFN-y [unpublished data]. This can be explained by their ubiquitous expression and their involvement in the transcription of other genes. However, IFN-y had an effect on the binding of transcription factors to promoter elements. Addition of IFN-y to rat FRTL-5 thyroid cell line resulted in the formation of a novel complex on the HLA-DRa promoter [162], which could be suppresses by methimazole (MMI), previously mentioned as MHC class II inhibitor. Furthermore, the addition of IFN-y-induced an endogenous CIITA transcript, and overexpression of a cDNA construct coding for CIITA in these cells resulted in the increased formation of that novel complex. In contrast, the increase in MHC class II expression and DNAprotein complex formation induced by either IFN-y or CIITA could be reduced by the co-transfection of the specific thyroid Y box protein, TSEP-1, which is homologous to the human repressor protein YB-1 [163], Another protein termed SSBP-1 has been found to positively regulate HLA-DRa transcription in thyroid cells, as was observed when overexpression of its cDNA resulted in the increased expression of HLA-DRa [164], In addition, transfection of SSBP-1 together with CIITA additively increased endogenous HLA-DRa mRNA levels, which reached those induced by IFN-y alone. Addition of TSH to thyroid cells caused an increase in TSEP-1 levels and a decrease in SSBP-1 levels, whereas, the addition of IFN-y reversed this effect. Thus it seems that TSH prevents expression of MHC class II, in contrast to IFN-y, which simultaneously increases the levels of CIITA and SSBP-1 and decreases the levels of TSEP-1, resulting in the increased aberrant expression of HLA-DR. The opposite effects of TSH and IFN-y on the induction of the MHC class II promoter are in accord with inhibitory [47] rather than enhancing [44] effects o TSH on thyroid HLA-DR expression.

We have shown that the incubation of thyroid malignant cells with IFN-y increased the levels of CIITA mRNA, and addition of TNF-a had a synergistic effect (unpublished data). The malignant thyroid cell line mentioned earlier, which expresses HLA-DRa consti-tutively, showed very low levels of expression of the CIITA mRNA. This indicates that CIITA, although a key player in the regulation of MHC class II, does not solely determines MHC class II gene expression in thyroid cells, and other regulatory mechanisms may influence it as well (unpublished data, [165]).

In macrophages CIITA is expressed inducibly, and this expression is mediated by the activation of STAT1 by IFN-y, which leads to the binding of STAT1 to the CIITA promoter, together with the binding of USF-1 and IRF-1 [ 166]. In the malignant thyroid cell lines we have observed increased phosphorylation of STAT1 after addition of IFN-y, which lasts even after 18 h. In contrast, phosphorylation of STAT1 in the B-cell line Raji was faintly detected only ten minutes after addition of IFN-y. This indicates that activation of STAT1 in this immune cell line is a very rapid and transient event. The divergent regulation of HLA-DR molecules in noncancerous and cancerous thyrocytes mentioned before, also suggests different molecular pathways leading to HLA-DR expression.

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