The selenoenzyme plasma glutathione peroxidase (GPx3, pGPx) is highly expressed in thyroid, kidney, placenta and bronchio-epithelial tissue. In the thyroid gland, the enzyme is thought to regulate thyroid hormone production by controlling the availability of H2O2, the cosubstrate for thyroperoxidase (TPO) and to protect the thyrocyte against H2O2-induced oxidative damage. To explain the high thyroidal expression levels, we studied GPx3 promoter regulation. Sequence analysis of the GPx3 5′URS revealed putative binding sites for the thyroid-specific transcription factors Pax8 and Nkx2.1 (TITF-1). In electrophoretic mobility shift assays using nuclear extracts from the rat thyroid cell line FRTL5, the putative Pax8 binding site displayd the same binding pattern as the Pax8 site of the well characterized TPO promoter. 2530, 1824, 1271 and 578 bp fragments of the human GPx3 5′URS were inserted into the luciferase reporter vector pGL4 and analyzed by transient transfection of the thyroid carcinoma cell lines FTC-133, FTC-238 and ML1, the kidney cell line HKC-8 and the hepatocarcinoma cell line HepG2, all of human origin. Depending on the cell line, induction of luciferase activity by the constructs was 20 to 30 fold vs. the empty vector, but this basal activity decreased with increasing 5′URS fragment length in all cell lines. Cotransfection of an expression plasmid coding for wildtype Pax8 further increased GPx3 promoter activity significantly, which, in contrast, was not observed after cotransfection of two expression plasmids coding for Pax8 loss of function mutants. Our results provide a molecular basis for the high GPx3 expression levels in the thyroid gland, however, they are also relevant for regulated GPx3 expression in the kidney, since Pax8 is highly expressed in renal cells as well. Further experiments will demonstrate the effect of Nkx2.1 on GPx3 promoter activity, possibly even in concert with Pax8 as it is described for other thyroid-specifc genes like TPO and Tg.
Supported by DFG Priority Programme
01 - 05 Apr 2006
European Society of Endocrinology