RESUMEN
We have identified two distinct Pax8 (a and b) mRNAs from the thyroid gland of the rainbow trout (Oncorhynchus mykiss), which seemed to be generated by alternative splicing. Both Pax8a and Pax8b proteins were predicted to possess the paired domain, octapeptide, and partial homeodomain, while Pax8b lacked the carboxy-terminal portion due to an insertion in the coding region of the mRNA. RT-PCR analysis showed each of Pax8a and Pax8b mRNAs to be abundantly expressed in the thyroid and kidney. In situ hybridization histochemistry further detected the expression of Pax8 mRNA in the epithelial cells of the thyroid follicles of the adult trout and in the thyroid primordial cells of the embryo. The functional properties of Pax8a and Pax8b were investigated by dual luciferase assay. The transcriptional regulation by the rat thyroid peroxidase (TPO) promoter was found to be increased by Pax8a, but not by Pax8b. Pax8a further showed synergistic transcriptional activity with rat Nkx2-1 for the human TPO upstream region including the enhancer and promoter. On the other hand, Pax8b decreased the synergistic activity of Pax8a and Nkx2-1. Electrophoretic mobility shift assay additionally indicated that not only Pax8a but also Pax8b can bind to the TPO promoter and enhancer, implying that the inhibitory effect of Pax8b might result from the lack of the functional carboxy-terminal portion. Collectively, the results suggest that for the trout thyroid gland, Pax8a may directly increase TPO gene expression in cooperation with Nkx2-1 while Pax8b may work as a non-activating competitor for the TPO transcription.
Asunto(s)
Empalme Alternativo , Autoantígenos/genética , Proteínas de Peces/metabolismo , Regulación de la Expresión Génica/fisiología , Yoduro Peroxidasa/genética , Proteínas de Unión a Hierro/genética , Oncorhynchus mykiss/genética , Factores de Transcripción Paired Box/genética , Glándula Tiroides/metabolismo , Secuencia de Aminoácidos , Animales , Autoantígenos/metabolismo , Secuencia de Bases , Clonación Molecular , Ensayo de Cambio de Movilidad Electroforética , Proteínas de Peces/genética , Hibridación in Situ , Yoduro Peroxidasa/metabolismo , Proteínas de Unión a Hierro/metabolismo , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Oncorhynchus mykiss/crecimiento & desarrollo , Filogenia , Regiones Promotoras Genéticas/genética , Isoformas de Proteínas , ARN Mensajero/genética , Ratas , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Glándula Tiroides/citología , Factor Nuclear Tiroideo 1 , Distribución Tisular , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Activación TranscripcionalRESUMEN
In an investigation aimed at clarifying the mechanism of crystal dissolution of the calcium carbonate lattice in otoconia (the mineral particles embedded in the otolithic membrane) of the endolymphatic sac (ELS) of the bullfrog, cDNAs encoding the A- and E-subunits of bullfrog vacuolar proton-pumping ATPase (V-ATPase) were cloned and sequenced. The cDNA of the A-subunit consisted of an 11-bp 5'-untranslated region (UTR), a 1,854-bp open reading frame (ORF) encoding a protein comprising 617 amino acids with a calculated molecular mass of 68,168 Da, and a 248-bp 3'-UTR followed by a poly(A) tail. The cDNA of the E-subunit consisted of a 72-bp 5'-UTR, a 681-bp ORF encoding a protein of 226 amino acids with a calculated molecular mass of 26,020 Da, and a 799-bp 3'-UTR followed by a poly(A) tail. Western blot and immunofluorescence analyses using specific anti-peptide antisera against the V-ATPase A- and E-subunits revealed that these subunits were present in the ELS, urinary bladder, skin, testes, and kidneys. In the ELS, positive cells were scattered in the follicular epithelium which, as revealed by electron microscopy, corresponds to the location of mitochondria-rich cells. These findings suggest that V-ATPase, including the A- and E-subunits, exists in mitochondria-rich cells of the ELS, which might be involved in dissolution of the calcium carbonate crystals in the lumen of the ELS.
Asunto(s)
ADN Complementario/química , Saco Endolinfático/enzimología , Rana catesbeiana , ATPasas de Translocación de Protón Vacuolares/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Western Blotting , Clonación Molecular , Saco Endolinfático/ultraestructura , Epitelio/enzimología , Técnica del Anticuerpo Fluorescente , Regulación Enzimológica de la Expresión Génica , Datos de Secuencia Molecular , Rana catesbeiana/genética , Rana catesbeiana/metabolismo , ATPasas de Translocación de Protón Vacuolares/químicaRESUMEN
Nkx2.1 (TTF-1), a homeodomain-containing transcription factor essential for specific gene expression in thyroid follicular cells, exists also in the thyroidal C cells that secrete calcitonin (CT). In this report, we examined the effect of Nkx2.1 on the CT gene transcription. Luciferase reporter assay using the 2kbp promoter sequence of rat CT/CGRP gene revealed that Nkx2.1 induced a significant increase in the promoter transcription. Furthermore, we detected Pax1 and/or Pax9 gene expression in mammalian medullary thyroid carcinoma cell lines, rat rMTC and human TT cells, and in mammalian thyroid glands by RT-PCR. The Pax9 mRNA, expressed in the TT cells and rat thyroid, was then isolated by cDNA cloning. Sequence analysis showed that both rat and human Pax9 proteins contained characteristic domains: i.e. the paired domain and octapeptide motif. Alternative transcripts encoding Pax9 isoforms were not identified in the rat thyroid or TT cells. Dual luciferase assay indicated that Pax9 did not increase transcription from the CT/CGRP promoter. Pax9 also showed no cooperative effects when it was co-transfected with Nkx2.1. The results suggest that CT gene expression could be directly activated by Nkx2.1, whereas Pax9 is not involved in transcription from the 2kbp CT promoter.