TTF-2 is involved in the nitric oxide-induced inhibition of thyroperoxidase (TPO) gene expression

LUCERO A, VÉLEZ M, NICOLA J, FOZZATTI L, MASINI-REPISO A

Santiago de Chile. Chile

Congreso; XII Latin-American Thyroid Congress; 2007

Latin-American Thyroid Society

Nitric oxide (NO) is a very effective signal transducer influencing a wide variety of biological processes. We have previously reported that the NO donor sodium nitroprusside (SNP), inhibited minimal rat TPO promoter activity in FRTL-5 thyroid cells. Minimal rat TPO promoter (420 bp) contains binding sites for TTF-1, TTF-2 and Pax-8 transcription factors. It is known that TTF-2 is crucial in the regulation of TPO gene expression by TSH. The present study was aimed to further analyze the mechanism involved in the inhibitory action of NO on TPO gene expression in TSH (0.5 mIU/mL) stimulated FRTL-5 cells. Data obtained by Northern Blot and RT-PCR assays indicated that TTF-2 mRNA level was reduced in FRTL-5 cells treated with SNP (100-500ìM, 24h). No evident changes in TTF-1 and Pax-8 mRNA levels were observed. In agreement with these results Western blot assays revealed that TTF-2 protein level was also diminished in SNP-treated cells (12-24 hs). Our previous observation indicated that NO inhibited the activity of 12ZTPOLUC, a construct containing 12 tandem repeats of Z site, the TTF-2 binding site at the TPO promoter. Values (arbitrary units) were: Basal= 1.00± 0.10; TSH= 3.09± 0.22; TSH+SNP(m M): 200= 1.61 ± 0.20**, 500= 1.03± 0.10*** (**p<0.01, ***p<0.001 vs TSH; n=6). In order to test changes in the binding of TTF-2 to Z site in TPO promoter we performed EMSA assays using oligoZ as a probe. In these experiments a reduction in TTF-2 binding activity to Z site was evidenced after treatment with SNP for 12-24 hs. From these results, we can conclude that TTF-2 plays an important role in mediating the inhibitory action of NO on TPO gene expression. The inhibitory effect of NO can be explained by a reduction of TTF-2 level that could be at least in part, the responsible of the decreased binding activity of the transcription factor to Z site. Since TPO is essential for thyroid hormone biosynthesis, these findings could be of pathophysiological interest, particularly in thyroid inflammatory states in which large quantities of NO are generated.