The thyroid hormone, 3,3, 5-triiod-L-thyronine (T3) is critically important for growth, differentiation and development. To test the hypothesis that T3, via its receptors (TRs), could mediate these diverse effects through a large network of co-regulators, we used human TRbeta1 as a bait to search for co-regulators in colon carcinoma RKO cells by the yeast two-hybrid system. Three positive clones were obtained. Sequence analysis shows that one of the clones is homologous to the human mitochondrial branched chain aminotransferase (HBCATm), which was designated as P3. The physical interaction between P3 and TRbeta1 was further confirmed by the dose-dependent specific binding of the in vitro translated 35S-labeled P3 to GST-TRbeta1. P3 was differentially expressed in different tissues and cultured cells. P3 binds to thyroid hormone response elements and heterodimerizes with TRbeta1. P3 represses the transactivation activity of TRbeta1. The extent of repression depends on the cell type in that the transfected TRbeta1 in CV1 cell is more sensitive to the repression effect of P3 than in colon carcinoma RKO cells. Thus, we have identified a novel TR co-repressor which could potentially modulate the functions of TRs in a tissue-dependent manner. These findings further strengthen the hypothesis that the diverse effects of TRs are mediated via a large network of co-regulatory proteins.Previously we have shown that the proliferation of growth hormone producing GC cells is stimulated by T3. The stimulatory effect is mediated by the increases both at the protein level of cyclin D1 and cyclin D1-associated kinase activity at the G1 phase of the cell cycle. We have now found that T3 stimulates the transcription rate of the cyclin D1 gene by direct interaction of TRbeta1 with the promoter of the cyclin D1 gene. A positive thyroid hormone response element was mapped to the ?935 to ?917 of the promoter. It is known that cyclin D1 is overexpressed in certain cancer cells. These results demonstrate that cyclin D1 is a TR target gene which would have important implications for our understanding of the biology of both normal and cancer cells.Resistance to thyroid hormone (RTH) is a syndrome characterized by refractoriness of the pituitary or peripheral tissues to the actions of T3. Mutations in the TRbeta gene result in TRbeta1 mutants that are responsible for the clinical phenotype by interfering with transcription of T3-regulated genes in a dominant negative manner. The molecular basis of RTH is difficult to study in humans due to limitations in tissue accessibility, and previously described mouse models of RTH have various limitations. Therefore, we have generated ?knockin? mice by targeting PV mutation into the TRbeta gene locus via homologous recombination. The PV mutant gene was derived from a heterozygous patient with stature (<5%tile), low body weight (<5%tile) and tachycardia. PV has a unique mutation in exon 10, a C-insertion at codon 448, which results in a frameshift mutation at the carboxyl-terminus of TRbeta. PV has no T3 binding activity and is one of the most potent dominant negative TRbeta mutants. A 17.5-kb targeting vector was constructed in which a C nucleotide was inserted at position 1642 of the human TRbeta1 cDNA, resulting in the same frameshift mutation as that characteristic of the PV mutant. The neomycin resistance gene (neoR) and the herpes simplex virus thymidine kinase (TK) gene were placed as selectable markers downstream of the PV site. In addition, neoR was flanked by two loxP sequences and placed in the opposite transcriptional orientation to that of the PV gene. This arrangement allows neoR to be subsequently excised with the Cre-LoxPPV. mRNA was detected in all tissues of PV mutant mice examined (brain, heart, lung, kidney, brown and white adipose tissues, muscle and liver). Using anti-PV antibody, PV protein was detected in the nuclei of the liver by immunocytochemical analysis. Compared to their wild-type litter mates (PV-/-), PV homozygous mice (PV+/+) had markedly elevated thyroid stimulating hormone (TSH) (20,000Y13.7 ng/dl vs 29.6Y4.5 ng/dl; n=8, p<0.01) and increased T4 levels (70.1 mg/dl vs 4.0Y1.3 mg/dl, n=18, p<0.01). In addition, PV homozygous mice had extraordinarily large thyroid glands histologically displaying extreme hyperplasia (PV+/+:46+25 mg; PV+/-:5+1.2 mg; PV-/-:4+0.9 mg, n=8). Furthermore, PV mutant mice exhibited delayed bone maturation as indicated by significantly shorter femoral and tibial bones. We examined the expression of T3 target genes in the pituitary gland and liver. Both the expression of the alpha and beta subunits of TSH were increased 15- and 1.5-fold in the homozygous PV mutant mice, respectively, indicating the dominant negative effect of the PV mutant. The expression of malic enzyme mRNA was repressed by 90% in the homozygous mice, which is consistent with the dominant negative effect of the PV mutant. However, the expression of S14 gene, which is a positively regulated T3 target gene in the liver, is increased by 2.5-fold in the homozygous mice. These results suggest a gain of function of the mutant PV gene. Our data indicate that the PV homozygous mice exhibit severe RTH phenotype which is entirely consistent with that seen in the patient. Moreover, the phenotype seen in PV mutant mice may be derived not only from the dominant negative action of the PV mutant gene, but could also be from a gain of function of the mutant gene. As far as we are aware, these mutant mice constitute the first true mouse model of RTH and they can now be used to study the molecular basis of RTH.
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