Temperature homeostasis in transgenic mice lacking thyroid hormone receptor-alpha gene products

We studied temperature homeostasis in male mice lacking all thyroid hormone receptor-alpha gene products (TRalpha-0/0). As other TRalpha-deficient mice, TRalpha-0/0 mice have lower core body temperature (T(C)) than cognate wild-type controls. We found that obligatory thermogenesis is normal in TRalpha-0/0 and that the lower T(C) at room temperature (RT, 20-22 C) is caused by a down setting of the hypothalamic thermostat. However, TRalpha-0/0 mice are cold intolerant due to impaired facultative thermogenesis. Norepinephrine-induced brown adipose tissue (BAT) thermogenesis is blunted, even though BAT-relevant genes and T(4) deiodinase respond normally to cold stimulation, as do serum T(3), serum glycerol (marker of lipolysis), and heart rate. BAT normally contributes to maintain T(C) at RT, 9 C below thermoneutrality, yet TRalpha-0/0 mice do not show signs of being cold stressed at 20-22 C. Instead, oxygen consumption is greater in TRalpha-0/0 than in wild-type mice at RT, suggesting the recruitment of an alternate, cold-activated form of thermogenesis to compensate for the lack of BAT thermogenesis. These results indicate that TRalpha is necessary for T(3) to modulate the central control of T(C) and for an essential step in norepinephrine activation of BAT thermogenesis but not to sustain obligatory thermogenesis. In addition, the results provide evidence for an alternate form of facultative thermogenesis, which probably originates in skeletal muscle and that is less effective and more energy demanding than BAT thermogenesis.     

Dickkopf 4 positively regulated by the thyroid hormone receptor suppresses cell invasion in human hepatoma cells

Thyroid hormone (T(3)) mediates cellular growth, development, and differentiation by binding to the nuclear thyroid hormone receptor (TR). Recent studies suggest that long-term hypothyroidism is associated with human hepatocellular carcinoma (HCC) independent from other major HCC risk factors. Dickkopf (DKK) 4, a secreted protein, antagonizes the Wnt signal pathway. In this study, we demonstrate that T(3) may play a suppressor role by inducing DKK4 expression in HCC cells at both the messenger RNA (mRNA) and protein levels. DKK4 was down-regulated in 67.5% of HCC cancerous tissues. The decrease in DKK4 levels was accompanied by a concomitant decrease in TR protein levels in the matched cancerous tissues in 31% of tissues compared by immunoblotting with the adjacent noncancerous tissues. Further, TR and DKK4 expression levels were positively correlated in both normal and cancerous specimens by tissue array analysis. In function assays, stable DKK4 transfected into J7 or HepG2 cells decreased cell invasion in vitro. Conversely, knocking down DKK4 restores cell invasiveness. DKK4-expressing J7 clones showed increased degradation of β-catenin, but down-regulation of CD44, cyclin D1, and c-Jun. To investigate the effect of DKK4 and TR on tumor growth in vivo, we established a xenograft of J7 cells in nude mice. J7-DKK4 and J7-TRα1 overexpressing mice, which displayed growth arrest, lower lung colony formation index, and smaller tumor size than in control mice, supporting an inhibitory role of DKK4 in tumor progression. CONCLUSION: Taken together, these data suggest that the TR/DKK4/Wnt/β-catenin cascade influences the proliferation and migration of hepatoma cells during the metastasis process and support a tumor suppressor role of the TR.     

Connexin40 messenger ribonucleic acid is positively regulated by thyroid hormone (TH) acting in cardiac atria via the TH receptor

Thyroid hormone (TH) regulates many cardiac genes via nuclear thyroid receptors, and hyperthyroidism is frequently associated with atrial fibrillation. Electrical activity propagation in myocardium depends on the transfer of current at gap junctions, and connexins (Cxs) 40 and 43 are the predominant junction proteins. In mice, Cx40, the main Cx involved in atrial conduction, is restricted to the atria and fibers of the conduction system, which also express Cx43. We studied cardiac expression of Cx40 and Cx43 in conjunction with electrocardiogram studies in mice overexpressing the dominant negative mutant thyroid hormone receptor-beta Delta337T exclusively in cardiomyocytes [myosin heavy chain (MHC-mutant)]. These mice develop the cardiac hypothyroid phenotype in the presence of normal serum TH. Expression was also examined in wild-type mice rendered hypothyroid or hyperthyroid by pharmacological treatment. Atrial Cx40 mRNA and protein levels were decreased (85 and 55%, respectively; P < 0.001) in MHC-mt mice. Atrial and ventricular Cx43 mRNA levels were not significantly changed. Hypothyroid and hyperthyroid animals showed a 25% decrease and 40% increase, respectively, in Cx40 mRNA abundance. However, MHC-mt mice presented very low Cx40 mRNA expression regardless of whether they were made hypothyroid or hyperthyroid. Atrial depolarization velocity, as represented by P wave duration in electrocardiograms of unanesthetized mice, was extremely reduced in MHC-mt mice, and to a lesser extent also in hypothyroid mice (90 and 30% increase in P wave duration). In contrast, this measure was increased in hyperthyroid mice (19% decrease in P wave duration). Therefore, this study reveals for the first time that Cx40 mRNA is up-regulated by TH acting in cardiac atria via the TH receptor and that this may be one of the mechanisms contributing to atrial conduction alterations in thyroid dysfunctions.     

Thyroxine-metabolizing rat uridine diphosphate-glucuronosyltransferase 1A7 is regulated by thyroid hormone receptor

Exposure of rats to microsomal enzyme inducers perturbs thyroid hormone (TH) homeostasis through a variety of mechanisms. Glucuronidation is an important metabolic pathway for TH and is catalyzed by uridine diphosphate-dibenzo-glucuronosyltransferase (UGT) family proteins. Administration of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to rats markedly increases the biliary clearance of glucuronidated T(4) and results in reduced plasma T(4) levels. Determination of the UGT1 isoforms responsible for glucuronidation of T(4) has yet to be conclusively established. We here provide evidence for the involvement of TCDD-inducible UGT1A7 in the glucuronidation of T(4) and TH-controlled UGT1A7 expression. Among a number of rat UGT1 isoenzymes examined in this study, UGT1A7 was the most active in catalyzing glucuronidation of T(4). Expression of UGT1A7 was positively regulated by T(4) through specific binding of TH receptor-retinoid X receptor heterodimers to a DR-5 sequence located between -109 and -93 in the UGT1A7 promoter. Overproduction of UGT1A7 protein decreased T(4) responsiveness of a reporter gene containing the T(4)-responsive UGT1A7 promoter sequence. These results raise the possibility that UGT1A7 plays a key role in the glucuronidation of T(4) leading to inactivation of T(4), functioning via feedback regulation to control T(4) levels in an autoregulatory manner, and that T(4) regulates its own metabolism and subsequent clearance from cells. Our findings also predict that accumulation of TCDD-inducible UGT1A7 proteins in TH-target cells might disrupt the TH signaling by lowering the intracellular pool of T(4).     

Modulation by oestrogen of thyroid hormone effects on thyrotrophin gene expression

The role of oestrogen in the regulation of TSH gene expression is unclear. We have examined the effect of administration of oestrogen in the rat on serum TSH, pituitary TSH content and pituitary cytoplasmic concentrations of mRNA encoding the TSH beta and alpha subunits, thus deriving measures of hormone release and synthesis. In addition, we have examined the effect of oestrogen on the binding of tri-iodothyronine (T3) to nuclear receptors in the anterior pituitary. Administration of oestrogen did not affect serum concentrations of TSH in euthyroid or untreated hypothyroid rats, but did augment the effects of T3 (1 and 2 micrograms) on serum TSH in hypothyroid animals 6 h after injection of T3. No influence of oestrogen or of thyroid status on pituitary content of TSH was seen. A marked increase in the concentrations of TSH beta and alpha mRNA in pituitary cytoplasm was found in hypothyroidism, compared with those in the euthyroid state. No effect of oestrogen on TSH mRNA was seen in euthyroid animals but concentrations of TSH beta and alpha mRNA were lower in hypothyroid animals than in vehicle-treated controls. A stimulatory influence of T3 on TSH mRNA was seen 6 h after injection of T3; this stimulation was absent in oestrogen-treated rats. No effect of oestrogen on the action of T3 was evident 72 h after beginning treatment with T3. In addition to effects on serum TSH and TSH mRNA, an increase in the number of pituitary nuclear receptors for T3 was seen after oestrogen treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

甲状腺激素调节脑发育的分子机制

本文复习了甲状腺功能减退时脑组织的病理变化和甲状腺激素影响脑发育的分子机制。     

Regulation of rat cardiac Kv1.5 gene expression by thyroid hormone is rapid and chamber specific.

Thyroid hormone affects the contractile and electrophysiological properties of the cardiac myocyte that result in part from changes in the expression of thyroid hormone-responsive cardiac genes, including those that regulate membrane ion currents. To determine the molecular mechanisms underlying this effect, expression of a voltage-gated K+ channel, Kv1.5, was measured in response to thyroid hormone. Using quantitative RT-PCR methodology, the content of Kv1.5 messenger RNA (mRNA) in left ventricles of euthyroid rats was 4.25+/-0.6x10(-20) mol/microg total RNA and was decreased by 70% in the hypothyroid rat ventricle to 1.27+/-0.80x10(-20) mol/microg RNA (P<0.01). Administration of T3 to hypothyroid animals restored ventricular Kv1.5 mRNA to control levels within 1 h of treatment, making this the most rapid T3-responsive cardiac gene reported to date. The half-life of Kv1.5 mRNA was 1.9 h and 2.0 h in euthyroid and hypothyroid ventricles, respectively, and T3 treatment of the rats did not alter its half-life. In atrial myocardium, expression of Kv1.5 mRNA (6.10+/-0.37x10(-20) mol/microg RNA) was unaltered by thyroid hormone status. The myocyte-specific and chamber-selective expression of Kv1.5 mRNA was confirmed in primary cultures of rat atrial and ventricular myocytes.     

p66Shc expression in proliferating thyroid cells is regulated by thyrotropin receptor signaling

It is almost unanimously accepted that thyrocyte proliferation is synergistically activated by TSH and insulin/IGF-I. Moreover, it was recently suggested that p66Shc, which is an adaptor molecule of the IGF-I receptor, might play a critical role in this synergistic effect. In this study, we undertook to confirm the role and the mechanism underlying the regulation of p66Shc expression via TSH receptor in thyrocytes. We have found that p66Shc expression is elevated in proliferating human thyroid tissues, including adenomatous goiter, adenoma, Graves' disease, and thyroid cancer, but not in normal thyroid. Among growth factors, TSH increased p66Shc expression both in vivo and in vitro; however, IGF-I, epidermal growth factor, or insulin did not. TSH and Graves' Ig increased the p66Shc expression via the TSH receptor-G(s)-cAMP pathway. However, interestingly, IGF-I or epidermal growth factor increased the tyrosine phosphorylations of p66Shc, and this was enhanced by TSH pretreatment. A similar synergism was observed during the DNA synthesis. When we measured the p66Shc levels induced by individual Igs from 130 patients with Graves' disease, TSH receptor stimulating activity and goiter size showed a weak correlation. We conclude that the expression of p66Shc is regulated by signaling through the TSH receptor in proliferating thyroid cells and that p66Shc appears to be an important mediator of the synergistic effect between TSH and IGF-I with respect to thyrocyte proliferation. Moreover, we suggest that TSH potentiates the regulatory effect of IGF-I on thyrocyte growth, at least in part, by increasing the expression of p66Shc.     

Analysis of thyroid hormone responsive gene expression in osteoblastic cells

Thyroid hormones regulate gene expression to influence the development and metabolism of many tissues including bone. The identification of genes that are regulated by thyroid hormones during skeletal development requires sensitive and quantitative techniques that are not limited by small amounts of available tissue and RNA. We have compared the efficiencies of differential display and poly A PCR subtraction hybridisation methods for the detection of thyroid hormone responsive genes expressed in osteoblastic cells. The utility of each technique was evaluated with respect to its sensitivity, specificity, cost and ability to identify novel genes. Subtraction hybridisation was rapid and more efficient in all categories. Poly A PCR facilitates quantitative and representative global amplification of cDNAs from low concentrations of RNA extracted from small tissue samples. The method, in combination with microarray analyses, may prove useful as an additional, complementary strategy to subtraction hybridisation for the analysis of differential gene expression in tissues where sample size is limiting.