Differential expression of thyroid hormone receptor isoforms dictates the dominant negative activity of mutant Beta receptor

Mutations in the thyroid hormone receptor beta gene (TRbeta) cause resistance to thyroid hormone (RTH). Genetic analyses indicate that phenotypic manifestation of RTH is due to the dominant negative action of mutant TRbeta. However, the molecular mechanisms underlying the dominant negative action of mutants and how the same mutation results in marked variability of resistance in different tissues in vivo are not clear. Here we used a knock-in mouse (TRbetaPV mouse) that faithfully reproduces human RTH to address these questions. We demonstrated directly that TRbeta1 protein was approximately 3-fold higher than TRalpha1 in the liver of TRbeta(+/+) mice but was not detectable in the heart of wild-type and TRbetaPV mice. The abundance of PV in the liver of TRbeta(PV/PV) was more than TRbeta(PV/+) mice but not detectable in the heart. TRalpha1 in the liver was approximately 6-fold higher than that in the heart of wild-type and TRbetaPV mice. Using TR isoforms and PV-specific antibodies in gel shift assays, we found that in vivo, PV competed not only with TR isoforms for binding to thyroid hormone response elements (TRE) but also competed with TR for the retinoid X receptors in binding to TRE. These competitions led to the inhibition of the thyroid hormone (T(3))-positive regulated genes in the liver. In the heart, however, PV was significantly lower and thus could not effectively compete with TRalpha1 for binding to TRE, resulting in activation of the T(3)-target genes by higher levels of circulating thyroid hormones. These results indicate that in vivo, differential expression of TR isoforms in tissues dictates the dominant negative activity of mutant beta receptor, thereby resulting in variable phenotypic expression in RTH.     

Cardiac glucose utilization in mice with mutated alpha- and beta-thyroid hormone receptors

Abnormal thyroid function is usually associated with altered cardiac function. Mutations in the thyroid hormone (TH)-binding region of the TH beta-receptor (TRbeta) that eliminate its TH-binding ability lead to the thyroid hormone resistance syndrome (RTH) in humans, which is characterized by high blood TH levels, goiter, hyperactivity, and tachycardia. Mice with "knock-in" mutations in the TH alpha-receptor (TRalpha) or TRbeta that remove their TH-binding ability have been developed, and those with the mutated TRbeta (TRbeta(PV/PV)) appear to provide a model for RTH. These two types of mutants show different effects on cerebral energy metabolism, e.g., negligible change in glucose utilization (CMR(Glc)) in TRbeta(PV/PV) mice and markedly reduced CMR(Glc), like that found in cretinous rats, in the mice (TRalpha(PV/+)) with the knock-in mutation of the TRalpha gene. Studies in knockout mice have indicated that the TRalpha may also influence heart rate. Because mutations in both receptor genes appear to affect some parameters of cardiac function and because cardiac functional activity and energy metabolism are linked, we measured heart glucose utilization (HMR(Glc)) in both the TRbeta(PV/PV) and TRalpha(PV/+) mutants. Compared with values in normal wild-type mice, HMR(Glc) was reduced (-77 to -95%) in TRalpha(PV/+) mutants and increased (87 to 340%) in TRbeta(PV/PV) mutants, the degree depending on the region of the heart. Thus the TRalpha(PV/+) and TRbeta(PV/PV) mutations lead, respectively, to opposite effects on energy metabolism in the heart that are consistent with the bradycardia seen in hypothyroidism and the tachycardia associated with hyperthyroidism and RTH.     

An unliganded thyroid hormone beta receptor activates the cyclin D1/cyclin-dependent kinase/retinoblastoma/E2F pathway and induces pituitary tumorigenesis

Thyroid-stimulating hormone (TSH)-secreting tumors (TSH-omas) are pituitary tumors that constitutively secrete TSH. The molecular genetics underlying this abnormality are not known. We discovered that a knock-in mouse harboring a mutated thyroid hormone receptor (TR) beta (PV; TRbeta(PV/PV) mouse) spontaneously developed TSH-omas. TRbeta(PV/PV) mice lost the negative feedback regulation with highly elevated TSH levels associated with increased thyroid hormone levels (3,3',5-triiodo-l-thyronine [T3]). Remarkably, we found that mice deficient in all TRs (TRalpha1(-/-) TRbeta(-/-)) had similarly increased T3 and TSH levels, but no discernible TSH-omas, indicating that the dysregulation of the pituitary-thyroid axis alone is not sufficient to induce TSH-omas. Comparison of gene expression profiles by cDNA microarrays identified overexpression of cyclin D1 mRNA in TRbeta(PV/PV) but not in TRalpha1(-/-) TRbeta(-/-) mice. Overexpression of cyclin D1 protein led to activation of the cyclin D1/cyclin-dependent kinase/retinoblastoma protein/E2F pathway only in TRbeta(PV/PV) mice. The liganded TRbeta repressed cyclin D1 expression via tethering to the cyclin D1 promoter through binding to the cyclic AMP response element-binding protein. That repression effect was lost in mutant PV, thereby resulting in constitutive activation of cyclin D1 in TRbeta(PV/PV) mice. The present study revealed a novel molecular mechanism by which an unliganded TRbeta mutant acts to contribute to pituitary tumorigenesis in vivo and provided mechanistic insights into the understanding of pathogenesis of TSH-omas in patients.     

Marked potentiation of the dominant negative action of a mutant thyroid hormone receptor beta in mice by the ablation of one wild-type beta allele

Mutations in the thyroid hormone receptor (TR) beta gene result in resistance to thyroid hormone (RTH), characterized by reduced sensitivity of tissues to thyroid hormone. To understand which physiological TR pathways are affected by mutant receptors, we crossed mice with a dominantly negative TRbeta mutation (TRbetaPV) with mice carrying a TRbeta null mutation (TRbeta(-/-)) to determine the consequences of the TRbetaPV mutation in the absence of wild-type TRbeta. TRbeta(PV/-) mice are distinct from TRbeta(+/-) mice that did not show abnormalities in thyroid function tests. TRbeta(PV/-) mice are also distinct from TRbeta(PV/+) and TRbeta(-/-) mice in that the latter shows mild dysfunction in the pituitary-thyroid axis, whereas the former exhibit very severe abnormalities, including extensive papillary hyperplasia of the thyroid epithelium, indistinguishable from that observed in TRbeta(PV/PV) mice. Similar to TRbeta(PV/PV) mice, TRbeta(PV/-) mice exhibited impairment in weight gain. Moreover, the abnormal regulation patterns of T3-target genes in the tissues of TRbeta(PV/-) and TRbeta(PV/PV) mice were strikingly similar. Using TR isoforms and PV-specific antibodies in gel shift assays, we found that in vivo, PV competed with TRalpha1 for binding to thyroid hormone response elements in TRbeta(PV/-) mice as effectively as in TRbeta(PV/PV) mice. Thus, the actions of mutant TRbeta are markedly potentiated by the ablation of the second TRbeta allele, suggesting that interference with wild-type TRalpha1-mediated gene regulation by mutant TRbeta leads to severe RTH.     

Contrasting skeletal phenotypes in mice with an identical mutation targeted to thyroid hormone receptor alpha1 or beta

Thyroid hormone (T(3)) regulates bone turnover and mineralization in adults and is essential for skeletal development. Surprisingly, we identified a phenotype of skeletal thyrotoxicosis in T(3) receptor beta(PV) (TRbeta(PV)) mice in which a targeted frameshift mutation in TRbeta results in resistance to thyroid hormone. To characterize mechanisms underlying thyroid hormone action in bone, we analyzed skeletal development in TRalpha1(PV) mice in which the same PV mutation was targeted to TRalpha1. In contrast to TRbeta(PV) mice, TRalpha1(PV) mutants exhibited skeletal hypothyroidism with delayed endochondral and intramembranous ossification, severe postnatal growth retardation, diminished trabecular bone mineralization, reduced cortical bone deposition, and delayed closure of the skull sutures. Skeletal hypothyroidism in TRalpha1(PV) mutants was accompanied by impaired GH receptor and IGF-I receptor expression and signaling in the growth plate, whereas GH receptor and IGF-I receptor expression and signaling were increased in TRbeta(PV) mice. These data indicate that GH receptor and IGF-I receptor are physiological targets for T(3) action in bone in vivo. The divergent phenotypes observed in TRalpha1(PV) and TRbeta(PV) mice arise because the pituitary gland is a TRbeta-responsive tissue, whereas bone is TRalpha responsive. These studies provide a new understanding of the complex relationship between central and peripheral thyroid status.     

Thyroid status during skeletal development determines adult bone structure and mineralization

Childhood hypothyroidism delays ossification and bone mineralization, whereas adult thyrotoxicosis causes osteoporosis. To determine how effects of thyroid hormone (T3) during development manifest in adult bone, we characterized TRalpha1(+/m)beta(+/-) mice, which express a mutant T3 receptor (TR) alpha1 with dominant-negative properties due to reduced ligand-binding affinity. Remarkably, adult TRalpha1(+/m)beta(+/-) mice had osteosclerosis with increased bone mineralization even though juveniles had delayed ossification. This phenotype was partially normalized by transient T3 treatment of juveniles and fully reversed in compound TRalpha1(+/m)beta(-/-) mutant mice due to 10-fold elevated hormone levels that allow the mutant TRalpha1 to bind T3. By contrast, deletion of TRbeta in TRalpha1(+/+)beta(-/ -) mice, which causes a 3-fold increase of hormone levels, led to osteoporosis in adults but advanced ossification in juveniles. T3-target gene analysis revealed skeletal hypothyroidism in TRalpha1(m/+)beta(+/-) mice, thyrotoxicosis in TRalpha1(+/+)beta(-/-) mice, and euthyroidism in TRalpha1(+/)beta(-/-) double mutants. Thus, TRalpha1 regulates both skeletal development and adult bone maintenance, with euthyroid status during development being essential to establish normal adult bone structure and mineralization.     

A dominant negative peroxisome proliferator-activated receptor-gamma knock-in mouse exhibits features of the metabolic syndrome

Peroxisome proliferator-activated receptor-gamma (PPARgamma), a member of the nuclear hormone receptor family, is a master regulator of adipogenesis. Humans with dominant negative PPARgamma mutations have features of the metabolic syndrome (severe insulin resistance, dyslipidemia, and hypertension). We created a knock-in mouse model containing a potent dominant negative PPARgamma L466A mutation, shown previously to inhibit wild-type PPARgamma action in vitro. Homozygous PPARgamma L466A knock-in mice die in utero. Heterozygous PPARgamma L466A knock-in (PPARKI) mice exhibit hypoplastic adipocytes, hypoadiponectinemia, increased serum-free fatty acids, and hepatic steatosis. When subjected to high fat diet feeding, PPARKI mice gain significantly less weight than controls. Hyperinsulinemic-euglycemic clamp studies in PPARKI mice revealed insulin resistance and reduced glucose uptake into skeletal muscle. Female PPARKI mice exhibit hypertension independent of diet. The PPARKI mouse provides a novel model for studying the relationship between impaired PPARgamma function and the metabolic syndrome.     

Altered growth in male peroxisome proliferator-activated receptor gamma (PPARgamma) heterozygous mice: involvement of PPARgamma in a negative feedback regulation of growth hormone action

The peroxisome proliferator-activated receptor gamma (PPARgamma) plays a major role in fat tissue development and physiology. Mutations in the gene encoding this receptor have been associated to disorders in lipid metabolism. A thorough investigation of mice in which one PPARgamma allele has been mutated reveals that male PPARgamma heterozygous (PPARgamma +/-) mice exhibit a reduced body size associated with decreased body weight, reflecting lean mass reduction. This phenotype is reproduced when treating the mice with a PPARgamma- specific antagonist. Monosodium glutamate treatment, which induces weight gain and alters body growth in wild-type mice, further aggravates the growth defect of PPARgamma +/- mice. The levels of circulating GH and that of its downstream effector, IGF-I, are not altered in mutant mice. However, the IGF-I mRNA level is decreased in white adipose tissue (WAT) of PPARgamma +/- mice and is not changed by acute administration of recombinant human GH, suggesting an altered GH action in the mutant animals. Importantly, expression of the gene encoding the suppressor of cytokine signaling-2, which is an essential negative regulator of GH signaling, is strongly increased in the WAT of PPARgamma +/- mice. Although the relationship between the altered GH signaling in WAT and reduced body size remains unclear, our results suggest a novel role of PPARgamma in GH signaling, which might contribute to the metabolic disorder affecting insulin signaling in PPARgamma mutant mice.     

Expression of human and mouse adenine nucleotide translocase (ANT) isoform genes in adipogenesis

Adenine nucleotide translocases (ANTs) are mitochondrial proteins encoded by nuclear DNA that catalyze the exchange of ATP generated in the mitochondria for ADP produced in cytosol. There are four ANT isoforms in humans (hANT1-4) and three in mice (mANT1, mANT2 and mANT4), all encoded by distinct genes. The aim of this study was to quantify expression of ANT isoform genes during the adipogenesis of mouse 3T3-L1 and human Simpson–Golabi–Behmel syndrome (SGBS)-derived preadipocytes. We also studied the effects of the adipogenesis regulators, insulin and rosiglitazone, on ANT isoform expression in differentiated adipocytes and examined the expression of ANT isoforms in subcutaneous and visceral white adipose tissue (WAT) from mice and humans. We found that adipogenesis was associated with an increase in the expression of ANT isoforms, specifically mANT2 in mouse 3T3-L1 cells and hANT3 in human SGBS cells. These changes could be involved in the increases in oxidative metabolism and decreases in lactate production observed during differentiation. Insulin and rosiglitazone induced mANT2 gene expression in mature 3T3-L1 cells and hANT2 and hANT3 gene expression in SGBS adipocytes. Furthermore, human WAT expressed greater amounts of hANT3 than hANT2, and the expression of both of these isoforms was greater in subcutaneous WAT than in visceral WAT. Finally, inhibition of ANT activity by atractyloside or bongkrekic acid impaired proper adipocyte differentiation. These results suggest that changes in the expression of ANT isoforms may be involved in adipogenesis in both human and mouse WAT.