A novel mutation (E333D) in the thyroid hormone beta receptor causing resistance to thyroid hormone syndrome.

Resistance to thyroid hormone (RTH) is an inherited syndrome characterized by elevated serum thyroid hormones (TH), failure to suppress pituitary thyroid stimulating hormone (TSH) secretion, and variable peripheral tissue responsiveness to TH. The disorder is associated with diverse mutations in the thyroid hormone beta receptor (TRbeta). Here, we report a novel natural RTH mutation (E333D) located in the large carboxy-terminal ligand binding domain of TRbeta. The mutation was identified in a 22-year-old French woman coming to medical attention because of an increasing overweight. Biochemical tests showed elevated free thyroxine (T4: 20.8 pg/ml (normal, 8.5-18)) and triiodothyronine (T3: 5.7 pg/ml (normal, 1.4-4)) in the serum, together with an inappropriately nonsuppressed TSH level of 4.7 mU/ml (normal, 0.4-4). Her father and her brother's serum tests also showed biochemical abnormalities consistent with RTH. Direct sequencing of the TRbeta gene revealed a heterozygous transition 1284A>C in exon 9 resulting in substitution of glutamic acid 333 by aspartic acid residue (E333D). Further functional analyses of the novel TRbeta mutant were conducted. We found that the E333D mutation neither significantly affected the affinity of the receptor for T3 nor modified heterodimer formation with retinoid X receptor (RXR) when bound to DNA. However, in transient transfection assays, the E333D TRbeta mutant exhibited impaired transcriptional regulation on two distinct positively regulated thyroid response elements (F2- and DR4-TREs) as well as on the negatively regulated human TSHalpha promoter. Moreover, a dominant inhibition of the wild-type TRbeta counterpart transactivation function was observed on both a positive (F2-TRE) and a negative (TSHalpha) promoter. These results strongly suggest that the E333D TRbeta mutation is responsible for the RTH phenotype in the proposita's family.

Mice with a targeted mutation in the thyroid hormone beta receptor gene exhibit impaired growth and resistance to thyroid hormone.

Patients with mutations in the thyroid hormone receptor beta (TRbeta) gene manifest resistance to thyroid hormone (RTH), resulting in a constellation of variable phenotypic abnormalities. To understand the molecular basis underlying the action of mutant TRbeta in vivo, we generated mice with a targeted mutation in the TRbeta gene (TRbetaPV; PV, mutant thyroid hormone receptor kindred PV) by using homologous recombination and the Cre/loxP system. Mice expressing a single PV allele showed the typical abnormalities of thyroid function found in heterozygous humans with RTH. Homozygous PV mice exhibit severe dysfunction of the pituitary-thyroid axis, impaired weight gains, and abnormal bone development. This phenotype is distinct from that seen in mice with a null mutation in the TRbeta gene. Importantly, we identified abnormal expression patterns of several genes in tissues of TRbetaPV mice, demonstrating the interference of the mutant TR with the gene regulatory functions of the wild-type TR in vivo. These results show that the actions of mutant and wild-type TRbeta in vivo are distinct. This model allows further study of the molecular action of mutant TR in vivo, which could lead to better treatment for RTH patients.

Hormone binding induces rapid proteasome-mediated degradation of thyroid hormone receptors.

The thyroid hormone 3,3',5-triiodo-l-thyronine (T3) is essential for growth, differentiation, and development. Its biological activities are mediated by T3 nuclear receptors (TRs). At present, how T3 regulates TR proteins and the resulting functional consequences are still unknown. Immunofluorescence analyses of endogenous TR in the growth hormone-producing GC cells showed that the T3-induced rapid degradation of TR was specifically blocked by lactacystin, a selective inhibitor of the ubiquitin-proteasome degradation pathway. Immunoblots demonstrated that the transfected TRbeta1 was ubiquitinated and that the ubiquitination was T3 independent. Studies with a series of truncated TRbeta1 showed that the hormone-binding domain was sufficient for the T3-induced rapid degradation of TRbeta1 by the proteasome degradation pathway. T3 also induced rapid degradation of TRbeta2 and TRalpha1. In contrast, the stability of the non-T3-binding TRalpha2 and naturally occurring TRbeta1 mutants that do not bind T3 was not affected by T3 treatment, indicating that hormone binding to receptor was essential for the degradation of the wild-type receptors. In the presence of proteasome protease inhibitors, the levels of both total and ubiquitinated TRbeta1 protein increased, yet T3-dependent transcriptional activation and the expression of the growth hormone gene were diminished, suggesting that proteasome-mediated degradation played a novel role in modulating transcriptional activation by TR. The present study reveals a role of T3 in modulating the functions of TR by regulating its receptor level via the ubiquitin-proteasome degradation pathway.

Thyroid hormone-induced cell proliferation in GC cells is mediated by changes in G1 cyclin/cyclin-dependent kinase levels and activity.

The thyroid hormone, 3,3', 5-triiodo-L-thyronine (T3), is essential for growth and regulation of metabolic functions. The biological activities of T3 are mediated by its interaction with the thyroid hormone nuclear receptors (TRs). The mechanism by which TRs mediate cell growth is unknown. We found that T3 stimulated cell growth in GC cells by shortening the doubling time approximately 3-fold. Flow cytometric analysis indicated that the growth stimulatory effect was mainly due to shortening of G1 phase accompanied by increases in S and G2/M phases of the cell cycle. These changes correlated with T3-induced increases in messenger RNA and protein levels of two key regulators of G1 progression, cyclins D1 and E, as well as cdk2. Furthermore, the kinase activities associated with cyclin D1 and E were activated up to 4-fold by T3, which led to increased phosphorylation of the retinoblastoma protein (Rb), the driving force in G1 to S cell cycle progression. These results show for the first time that the growth promoting effect of T3 in GC cells is mediated, at least in part, by increases in cyclin/cdk activities and the phosphorylation state of Rb. The functional link of T3 to Rb has important implications for the understanding of the biology of normal and cancer cells.

Transient expression of c-erbAbeta1 messenger ribonucleic acid and beta1 thyroid hormone receptor early in adipogenesis of Ob 17 cells.

In the murine Ob 17 preadipocyte cell line, the thyroid hormone T3 is an adipogenic factor necessary at an early stage for differentiation into adipocyte. We demonstrate here that this T3 dependence may involve a transient expression (at both the messenger RNA and the protein levels) of c-ErbA beta-type receptors (T3R), although a large body of T3R remained the product of the c-erbAalpha gene, as previously described. c-ErbAbeta1 (and not beta2) expression emerged significantly at growth arrest, peaked 2 days later, and almost disappeared in maturing adipocytes. This expression is related to the presence of T3, as total deprivation of culture medium from T3 prevented it, and the addition of 1.5 nM T3 to preconfluent cultures was able to restore it. When cells were cultured in the presence of T3 and thus were able to differentiate, the c-erbAbeta peak was accompanied by sequential rapid increases in CAAT/enhancer-binding protein-delta(C/EBPdelta), peroxisome proliferator-activated-gamma receptor (PPARgamma), and C/EBPalpha gene expressions. On the contrary, under thyroid hormone-deprived culture conditions that result in nondifferentiation of the preadipocytes, c-erbAbeta1, PPARgamma, and the large C/EBPalpha expressions were blunted, and a moderate early increase in c-erbAalpha1 transcripts was sustained for a longer period. Addition of T3 to T3-deprived preconfluent cells restored PPARgamma and C/EBPalpha expressions. Taken together, the results highlight the important role of T3 in the adipogenesis of Ob 17 cells through the involvement of both beta1 and alpha1 T3R subtypes.

Tissue-specific differential repression of gene expression by a dominant negative mutant of thyroid hormone beta1 receptor.

Resistance to thyroid hormone (RTH) is a genetic disease caused by the mutations of the thyroid hormone beta receptor (TRbeta) gene, producing receptors with a dominant negative action. The present study addressed the question as to whether tissue-specific factors modulate the dominant negative function in different tissues. We prepared stably transfected pituitary GH3 (GH3-PV) and liver SK-Hep-1 (SK-Hep-1-PV) cell lines with a potent dominant negative mutant, PV. The growth hormone (GH) and the malic enzyme genes (ME) in GH3 and SK-Hep-1, respectively, are directly regulated by the thyroid hormone, 3,3,'5-triiodo-L-thyronine (T3). The ratio of the expressed PV/endogenous TRbeta1 proteins was approximately 20 and 5 for GH3-PV and SK-Hep-1-PV cells, respectively. However, the T3-activated expression of the GH gene in GH3-PV and ME gene in SK-Hep-1-PV was repressed by approximately 30% and 90%, respectively, indicating the lack of correlation of PV/TRpbeta1 protein ratio with the dominant negative potency of mutant PV. Furthermore, the synergistic effect of the pituitary-specific factor 1 on the TR-mediated GH promoter activity was not repressed by mutant PV. Taken together, these results suggest that the dominant negative effect of mutant TR is variable in the tissues studied.

A novel resistance to thyroid hormone associated with a new mutation (T329N) in the thyroid hormone receptor beta gene.

Resistance to thyroid hormone (RTH) is a syndrome of elevated serum thyroxine, inappropriately "normal" serum thyrotropin (TSH) and reduced thyroid hormone responsiveness associated with point mutations in the thyroid hormone receptor-beta (TRbeta) gene. We describe a novel point mutation resulting in a cytosine for adenine substitution at nucleotide 1271 (exon 9) that results in the substitution of threonine for asparagine (T329N). This mutation was identified in a 30-year-old woman who was investigated for recurrent spontaneous abortions and was found to have RTH. Dextrothyroxine (D-T4) therapy was instituted. At 8 mg per day 2 pregnancies followed with the delivery of a healthy boy and an RTH-affected girl another miscarriage occurred on D-T4 treatment at 6 mg per day. The T329N mutation, which was also identified in the daughter, markedly reduces the affinity of TRbeta for triiodothyronine (T3). Formation of T329N mutant TR homodimers and heterodimers with RXRalpha on thyroid hormone response element F2 (TRE F2) was not affected, but the ability of T3 to interrupt T329N mutant TRbeta homodimerization was markedly reduced. The T329N mutant TRbeta was transcriptionally inactive in transient expression assays. In cotransfection assays with wild-type TRbeta1, the mutant TRbeta1 functioned in a dominant negative manner. The results suggest that the T329N mutation in the T3-binding domain of TRbeta is responsible for RTH in the proposita's family.

Calcitriol is a positive effector of adipose differentiation in the OB 17 cell line: relationship with the adipogenic action of triiodothyronine.

In a previous report, we showed that physiological concentrations of calcitriol (1 alpha,25-(OH)2 vitamin D3 or VD), markedly stimulated the terminal adipose differentiation of Ob 17 preadipocytes cultured under standard conditions with fetal calf serum (FCS), and increased the differentiating effect of triiodothyronine (T3) reported as a necessary adipogenic factor in these cells. Here, we demonstrate, for the first time, that VD is an intrinsic strong adipogenic factor for the Ob 17 preadipocytes cultured in thyroid hormone-deprived medium (adipogenic concentrations: 0.025-0.25 nM in the presence of stripped FCS, 1-10 pM under serum-free conditions). VD action was potentiated by the coaddition of either T3, or arachidonic acid, two agents which also bear proper adipogenic properties. The efficient concentration ranges of other vitamin D3 metabolites suggest a mediation through the VD nuclear receptor (VDR). An expression of the VDR gene is here demonstrated in the Ob 17 cells, and evidence is given that VDR mRNA level increased during the differentiation process and that this increase is moderately amplified under long term treatment with adipogenic concentrations of VD. Our results strongly suggest that adipose differentiation is under the control of different closely related nuclear receptors acting at an early preadipocyte step and probably in an interchangeable manner depending on the availability of their respective ligands. The existence of an interplay between these receptors in exerting their adipogenic action is suggested.

Cloning and initial characterization of human and mouse Spot 14 genes.

The intricate regulation of Spot 14 expression in rat lipogenic tissues has provided a useful tool in studying nutritional and hormonal factors involved in transcription. To gain insight into its function and its possible involvement in human lipid disorders, we cloned human and mouse Spot 14 genes that shared with the rat gene a strong homology concerning the deduced amino acid sequence (81 and 94%, respectively) as well as the promoter region. The mouse promoter was characterized by transfection studies, while quantitative RT-PCR and in situ hybridization experiments showed that Spot 14 is expressed in human liver and, at a high level, in multiple symmetric lipomatosis nodules.

Calcitriol down-modulates the 3,5,3' triiodothyronine (T3) receptors and affects, in a biphasic manner, the T3-dependent adipose differentiation of Ob 17 preadipocytes.

In previous reports, we showed that T3 is required for terminal differentiation of the murine Ob 17 preadipocytes, and that it partially down-modulates the abundance of its own nuclear receptor sites (T3R). We also reported that a profound depletion of the T3R was produced by all-trans-retinoic acid at concentrations that inhibit adipose differentiation. Here, we report that calcitriol (VD), which activates a nuclear receptor (VDR) closely related to the T3R and retinoid receptors, also markedly affects nuclear T3 binding and T3-induced differentiation of Ob 17 cells. Within a nearly physiological concentration range (0.1-2.5 nM), calcitriol profoundly down-modulated T3R abundance without altering the affinity for T3. The T3R depletion was a fast event, sustained under VD and reversed within 48 h of VD withdrawal. The order of efficient concentration ranges of VD and analogs suggests an involvement of the VDR. The T3R-depleting effect of VD was observed at every stage of adipose differentiation and was additive to the depleting effect of T3. Within the 0.1-2.5 nM VD concentration range, the c-erbA alpha and -alpha 1 messenger RNA levels (only c-erbA alpha gene products were detected in these cells) were poorly decreased; VD also did not alter a protein band specifically detected with specific anti-c-erbA alpha 1 antibodies in Western blots of nuclear extracts. VD accelerated the T3R disappearance rate; the results suggest that this would probably involve sequestration, rather than degradation, events. Interestingly, calcitriol added to the culture medium of Ob 17 preadipocytes markedly influenced the adipose differentiation, exerting a clear-cut stimulation at levels of 0.25 nM or less and profound inhibition at concentrations above 0.25 nM. Both effects were observed provided that VD was added within an early critical period of the differentiation process, as we previously reported for T3. The stimulations caused by low concentrations of VD and 1.5 nM T3 were additive. Increasing the VD concentration produced a progressive attenuation, then a suppression, of the stimulating effect of T3. Comparative analyses of VD-related changes in adipose differentiation and T3R abundance suggest that a correlation may exist between optimal differentiation and a partial depletion of the T3R, whereas a profound depletion of the T3R occurred at inhibitory concentrations of VD. The present results sustain the concept that T3R play a role in the differentiation of Ob 17 preadipocytes. Moreover, the results suggest that there may be a T3 receptor site concentration optimal for efficient differentiation. A regulation of this concentration involves ligands of other closely related receptors and, thus, probably the interplays that exist between these receptors.

Nuclear factors specifically favor thyroid hormone binding to c-ErbA alpha 1 protein (thyroid hormone receptor alpha) over-expressed in E. coli.

A recombinant rat thyroid hormone receptor alpha (TR alpha or c-ErbA alpha 1) was produced in E. coli as a non-mutated, nonfusioned protein and obtained as an efficient DNA and T3 binding protein that could be easily handled in a buffer-soluble state (rec-TR alpha). It was found that nuclear extracts (NE) added to rec-TR alpha markedly amplified not only DNA binding, which has been well documented, but also T3 binding (increased binding site concentration), which has not yet been reported. This T3 binding amplifying effect on rec-TR alpha occurs at low NE protein concentrations that produce no or minimal endogenous TR with respect to rec-TR, while similar concentrations of other proteins (e.g. ovalbumin or cytosol) only moderately enhanced T3 binding. The T3 binding amplifying nuclear factors, which are partly heat-labile, appeared as necessary auxiliaries in the analyses of partially purified rec-TR alpha. A protective effect of NE against a loss of affinity for T3 under the action of antibodies directed to certain sequences in the TR alpha D domain suggests that nuclear factors help rec-TR alpha to acquire and/or stabilize a conformation that allows the high affinity T3 binding. The nature of this nuclear amplifying factor is still unknown: RXR alpha which, produced in vitro, could amplify binding of the rec-TR alpha to a DNA thyroid response element, was unable to display such a rescue of high affinity binding sites.