THYROTROPIN (TSH) INCREASE NITRIC OXIDE (NO) SYNTHASE III (NOS III) GENE EXPRESSION AT TRANSCRPTIONAL LEVEL BY INVOLVING cAMP-RESPONSIVE ELEMENTS IN FRTL-5 THYROID CELLS

FOZZATTI, L; VÉLEZ, ML; NICOLA, JP; PELLIZAS, CG; LUCERO, AM; MASINI-REPISO, AM

Santiago de Chile. Chile

Congreso; XII Congreso de la Sociedad Latinoamericana de Tiroides; 2007

NO is a free radical that mediates cell communication and signal transduction in many tissues. It is generated from L-arginine with additional L-citrulline production by at least three different NOS isoforms (I-III). NOS expression has been demonstrated in thyroid cells being the highest level of expression displayed by NOS III. Although NOS III is a Ca2+-dependent constitutive enzyme, several factors modify its expression in several cell types. We have previously reported that endogenous thyrocyte-produced NO acts as a negative signal on TSH-induced thyroid specific genes expression and proliferation in the thyroid cell. The aim of this work was to analyze the action of TSH on the regulation of NOS III in FRTL-5 cells. Treatment of cells with different concentrations of TSH for 12, 24 y 48h increased NO generation (14C-citrulline; maximum TSH 200µIU/ml, 2-fold, 24h, p<0.05) indicating a TSH-dependent NO production. We evaluated the action of TSH on NOS III gene expression. An increment of NOS III mRNA (in situ hibridization) was induced by 200µIU/ml TSH (2-fold, 48h, p<0.01). In order to examinate the mechanism involved, we measured the effect of TSH on the functional activity of the 5´-upstream human NOS III promoter region (-1193/+109) by transfection assays. This construct contains two cAMP-responsive elements (CRE) in its 5´-upstream region. We observed that TSH increased the activity of the -1193/+109 human NOS III promoter in a concentration-dependent manner. To test the importance of the CRE sequences in the stimulatory effect of TSH on NOS III promoter activity, we analyzed a shorter fragment of the human NOS III promoter (-265/+109), where CRE sites were absent. This construct (-265/+109) was unresponsive to TSH, suggesting that the CRE sites could be involved in the effect of TSH on NOS gene expression. In conclusion, our data provide evidence that the increase induced by TSH on NO production in the thyrocyte could be explained, at least in part, by a TSH-induced stimulation of NOS III gene expression at transcriptional level possibly mediated by cAMP. A novel role of TSH in the regulation of NOS III gene expression could be proposed.