Dominant inhibition of thyroid hormone action selectively in the pituitary of thyroid hormone receptor-beta null mice abolishes the regulation of thyrotropin by thyroid hormone

Thyroid hormones, T4 and T3, regulate their own production by feedback inhibition of TSH and TRH synthesis in the pituitary and hypothalamus when T3 binds to thyroid hormone receptors (TRs) that interact with the promoters of the genes for the TSH subunit and TRH. All TR isoforms are believed to be involved in the regulation of this endocrine axis, as evidenced by the massive dysregulation of TSH production in mice lacking all TR isoforms. However, the relative contributions of TR isoforms in the pituitary vs. the hypothalamus remain to be completely elucidated. Thus, to determine the relative contribution of pituitary expression of TR-alpha in the regulation of the hypothalamic-pituitary-thyroid axis, we selectively impaired TR-alpha function in TR-beta null mice (TR-beta-/-) by pituitary restricted expression of a dominant negative TR-beta transgene harboring a delta337T mutation. These animals exhibited 10-fold and 32-fold increase in T4 and TSH concentrations, respectively. Moreover, the negative regulation of TSH by exogenous T3 was completely absent and a paradoxical increase in TSH concentrations and TSH-beta mRNA was observed. In contrast, prepro-TRH expression levels in T3-treated TR-beta-/- were similar to levels observed in the delta337/TR-beta-/- mice, and ligand-independent activation of TSH in hypothyroid mice was equivalently impaired. Thus, isolated TR-beta deficiency in TRH paraventricular hypothalamic nucleus neurons and impaired function of all TRs in the pituitary recapitulate the baseline hormonal disturbances that characterize mice with complete absence of all TRs.     

Isometric force and endurance in soleus muscle of thyroid hormone receptor-alpha(1)- or -beta-deficient mice

The specific role of each subtype of thyroid hormone receptor (TR) on skeletal muscle function is unclear. We have therefore studied kinetics of isometric twitches and tetani as well as fatigue resistance in isolated soleus muscles of R-alpha(1)- or -beta-deficient mice. The results show 20-40% longer contraction and relaxation times of twitches and tetani in soleus muscles from TR-alpha(1)-deficient mice compared with their wild-type controls. TR-beta-deficient mice, which have high thyroid hormone levels, were less fatigue resistant than their wild-type controls, but contraction and relaxation times were not different. Western blot analyses showed a reduced concentration of the fast-type sarcoplasmic reticulum Ca(2+)-ATPase (SERCa1) in TR-alpha(1)-deficient mice, but no changes were observed in TR-beta-deficient mice compared with their respective controls. We conclude that in skeletal muscle, both TR-alpha(1) and TR-beta are required to get a normal thyroid hormone response.     

Beta 1 isoform-specific regulation of a triiodothyronine-induced gene during cerebellar development.

Although tissue-specific expression of the alpha 1 and beta 1 thyroid hormone receptors (TR-alpha 1 and TR-beta 1) suggests isoform-specific function, transfection studies to date have failed to show consistent differences in their ability to regulate gene expression. We here provide evidence that TR-beta 1 but not TR-alpha 1 regulates the expression of the gene coding for PCP-2 in cerebellar Purkinje cells during neonatal rat development and that such regulation appears to be both T3 dependent and T3 independent. Examination of neonatal rats revealed that the levels of three mRNAs expressed in cerebellar Purkinje cells (myoinositol-1,4,5-triphosphate receptor, calbindin, and PCP-2) rise from neonatal day 1 to day 15. This rise is preceded by the previously documented surge in brain T3 and TR-beta 1. Methimazole-induced hypothyroidism sharply reduces, but does not abolish, the rise in these mRNAs. Concomitant T3 administration normalizes the process. In order to establish more directly the role of TR-beta 1 and T3, cotransfection experiments were performed in CHO cells with PCP-2-lacZ construct and TR isoforms. These studies showed that TR-beta 1, even in the absence of T3, regulated the expression of the transfected PCP-2 construct. T3 augments the response to TR-beta 1 alone by 40% (P < .01). TR-alpha 1 had no effect on PCP-2-lacZ expression either in the presence or absence of T3.(ABSTRACT TRUNCATED AT 250 WORDS)     

Altered expression of skeletal muscle myosin isoforms in cancer cachexia

Cachexia is commonly seen in cancer and ischaracterized by severe muscle wasting, but little is known about theeffect of cancer cachexia on expression of contractile protein isoforms such as myosin. Other causes of muscle atrophy shift expression ofmyosin isoforms toward increased fast (type II) isoform expression. Weinjected mice with murine C-26 adenocarcinoma cells, a tumor cell linethat has been shown to cause muscle wasting. Mice were killed 21 daysafter tumor injection, and hindlimb muscles were removed. Myosin heavychain (MHC) and myosin light chain (MLC) content was determined inmuscle homogenates by SDS-PAGE. Body weight was significantly lower intumor-bearing (T) mice. There was a significant decrease in muscle massin all three muscles tested compared with control, with the largestdecrease occurring in the soleus. Although no type IIb MHC was detectedin the soleus samples from control mice, type IIb comprised 19% of thetotal MHC in T soleus. Type I MHC was significantly decreased in T vs. control soleus muscle. MHC isoform content was not significantly different from control in plantaris and gastrocnemius muscles. Thesedata are the first to show a change in myosin isoform expression accompanying muscle atrophy during cancer cachexia.

     

Effect of thyroid hormone on the myosin heavy chain isoforms in slow and fast muscles of the rat

The myosin heavy chain (MHC) was studied by biochemical methods in the slow-twitch (soleus) and two fast-twitch leg muscles of the triiodothyronine treated (hyperthyroid), thyroidectomized (hypothyroid) and euthyroid (control) rats. The changes in the contents of individual MHC isoforms(MHC-1, MHC-2A, MHC-2B and MHC-2X) were evaluated in relation to the muscle mass and the total MHC content. The MHC-1 content decreased in hyperthyreosis, while it increased in hypothyreosis in the soleus and in the fast muscles. The MHC-2A content increased in hyperthyreosis and it decreased in hypothyreosis in the soleus muscle. In the fast muscles hyperthyreosis did not affect the MHC-2A content, whereas hypothyreosis caused an increase in this MHC isoform content. The MHC-2X, present only in traces or undetected in the control soleus muscle, was synthesised in considerable amount in hyperthyreosis; in hypothyreosis the MHC-2X was not detected in the soleus. In the fast muscles the content of MHC-2X was not affected by any changes in the thyroid hormone level. The MHC-2B seemed to be not influenced by hyperthyreosis in the fast muscles, whereas the hypothyreosis caused a decrease of its content. In the soleus muscle the MHC-2B was not detected in any groups of rats. The results suggest that the amount of each of the four MHC isoforms expressed in the mature rat leg muscles is influenced by the thyroid hormone in a different way. The MHC-2A and the MHC-2X are differently regulated in the soleus and in the fast muscles; thyroid hormone seems to be necessary for expression of those isoforms in the soleus muscle.     

Thyroid hormone regulation of MHC isoform composition and myofibrillar ATPase activity in rat skeletal muscles.

Myosin heavy chain (MHC) isoform composition and Ca2+ Mg2+ dependent ATPase activity were determined in myofibrils prepared from skeletal muscles (diaphragm, soleus, plantaris and tibialis anterior) of euthyroid (C), hypothyroid (Tx) and hyperthyroid (T3) rats. Direct comparison between T3 and Tx gave an indication of the maximal effect of thyroid hormones. Significant differences in MHC-1 and MHC-2B proportions and in ATPase activity were found in all muscles. The difference in MHC-2A/X proportion was significant only in soleus, diaphragm and plantaris. When T3 and C were compared, significant variations in MHC isoform composition were found only in plantaris and diaphragm. The comparison between Tx and C showed significant differences in MHC isoform distribution and in ATPase activity in most muscles. The differences in ATPase activity among muscles and among thyroid states were consistent with those in MHC isoform distribution. From the correlations between ATPase activity and MHC isoform distribution the enzymatic activities of individual MHC isoforms were calculated. The results indicate that MHC isoform distribution is controlled by thyroid state in all skeletal muscles and that changes in MHC isoforms distribution are accompanied by proportional changes in ATPase activity.     

Quantitation of rat tissue thyroid hormone binding receptor isoforms by immunoprecipitation of nuclear triiodothyronine binding capacity.

A panel of anti-thyroid hormone receptor (TR) antisera were generated to allow direct assay of the concentrations of the alpha 1 and beta 1 receptor isoforms in nuclear extracts from adult rat liver, kidney, brain and heart, and fetal brain. An antiserum, immunoglobulin G (IgG)-beta 1, raised against amino acid sequence 62-92 of the rat TR-beta 1 specifically precipitated only TR-beta 1 in vitro translation products. A second antiserum, IgG-alpha 1/beta, generated against a sequence that is identical in the ligand binding region of rat TR-alpha 1 and TR-beta isoforms immunoprecipitated both TR-alpha 1 and -beta 1 translation products. These IgG preparations were used to specifically immunoprecipitate thyroid hormone receptor binding activity from nuclear extracts. IgG-beta 1 cleared almost 80%, and the IgG-alpha 1/beta immunoprecipitated nearly all binding from hepatic nuclear extracts. This distribution of TR protein, 80% beta 1 and 20% alpha 1, is the same as previously reported for their respective mRNAs in liver. In heart, kidney, and brain IgG-beta 1 cleared 45, 43, and 28% of total binding, respectively, and IgG-alpha 1/beta cleared all T3 binding activity from these tissues. In agreement with an earlier study, marked variations in specific protein/mRNA ratios were noted among these tissues. Consistent with our earlier report of the presence of only very low levels of TR-beta 1 mRNA in fetal brain, IgG-beta 1 cleared just 5% of binding in this tissue. Studies using an antiserum (IgG-ch) generated against homologous segments of the hinge region in both TR-alpha 1 and -beta 1 yielded results which contrasted sharply with those of IgG-alpha 1/beta. Whereas IgG-ch could also immunoprecipitate virtually all binding from hepatic extracts it cleared only 40-50% of binding from the other tissues, including fetal brain in which TR-alpha 1 accounts for greater than 90% of binding protein. The data suggest the presence of posttranslational modification of the TR-alpha 1 protein in the hinge region, consistent with the presence in this segment of potential phosphorylation sites.     

Sick euthyroid syndrome is associated with decreased TR expression and DNA binding in mouse liver

Infection is associated with low serum thyroid hormones and thyrotropin levels. Here we demonstrate that infection also reduces thyroid hormone receptor (TR) expression. In gel shift experiments, retinoid X receptor (RXR)/TR DNA binding was reduced in mouse liver by 60 and 77%, respectively, 4 and 16 h after lipopolysaccharide (LPS) administration. Surprisingly, LPS did not decrease either TR-alpha or TR-beta protein levels at 4 h, but by 16 h TR-alpha(1), TR-alpha(2), and TR-beta levels were reduced by 55, 87, and 41%, respectively. We previously reported that LPS rapidly decreases RXR protein levels in liver. Therefore, we added RXR-beta to hepatic nuclear extracts prepared 4 h after LPS treatment, which restored RXR/TR DNA binding to a level comparable to that of controls. A similar experiment conducted on extracts prepared 16 h after LPS administration did not restore RXR/TR DNA binding. We propose that decreased RXR expression is limiting for RXR/TR DNA binding at 4 h, whereas the reduction in both TR and RXR levels results in further decreased binding at 16 h.     

Myosin heavy chain isoform composition in striated muscle after denervation and self-reinnervation

The total content of myosin heavy chains (MHC) and their isoform pattern were studied by biochemical methods in the slow-twitch (soleus) and fast-twitch (extensor digitorum longus) muscles of adult rat during atrophy after denervation and recovery after self-reinnervation. The pattern of fibre types, in terms of ultrastructure, was studied in parallel. After denervation, total MHC content decreased sooner in the slow-twitch muscle than in the fast-twitch. The ratio of MHC-1 and the MHC-2B isoforms to the MHC-2A isoform decreased in the slow and the fast denervated muscles, respectively. After reinnervation of the slow muscle, the normal pattern of MHC recovered within 10 days and the type 1 isoform increased above the normal. In the reinnervated fast muscle, the 2B/2A isoform ratio continued to decrease. Traces of the embryonic MHC isoform, identified by immunochemistry, were found in both denervated and reinnervated slow and fast muscles. A shift in fibre types was similar to that found in the MHC isoforms. Within 2 months of recovery a tendency to normalization was observed. The results show that (a) MHC-2B isoform and the morphological characteristics of the 2B-type muscle fibres are susceptible to lack of innervation, similar to those of type 1, (b) during muscle recovery induced by reinnervation the MHC isoforms and muscle fibres shift transiently to type 1 in the soleus and to type 2A in the extensor digitorum longus muscles, and (c) the embryonic isoform of MHC may appear in the adult skeletal muscles if innervation is disturbed.     

Cross-talk between thyroid hormone receptor and liver X receptor regulatory pathways is revealed in a thyroid hormone resistance mouse model

Hypercholesterolemia is found in patients with hypothyroidism and resistance to thyroid hormone. In this study, we examined cholesterol metabolism in a thyroid hormone receptor beta (TR-beta) mutant mouse model of resistance to thyroid hormone. Whereas studies of cholesterol metabolism have been reported in TR-beta knock-out mice, generalized expression of a non-ligand binding TR-beta protein in this knock-in model more fully recapitulates the hypothyroid state, because the hypothyroid effect of TRs is mediated by the unliganded receptor. In the hypothyroid state, a high cholesterol diet increased serum cholesterol levels in wild-type animals (WT) but either did not change or reduced levels in mutant (MUT) mice relative to hypothyroidism alone. 7alpha-Hydroxylase (CYP7A1) is the rate-limiting enzyme in cholesterol metabolism and mRNA levels were undetectable in the hypothyroid state in all animals. triiodothyronine replacement restored CYP7A1 mRNA levels in WT mice but had minimal effect in MUT mice. In contrast, a high cholesterol diet markedly induced CYP7A1 levels in MUT but not WT mice in the hypothyroid state. Elevation of CYP7A1 mRNA levels and reduced hepatic cholesterol content in MUT animals are likely because of cross-talk between TR-beta and liver X receptor alpha (LXR-alpha), which both bind to a direct repeat + 4 (DR+4) element in the CYP7A1 promoter. In transfection studies, WT but not MUT TR-beta antagonized induction of this promoter by LXR-alpha. Electromobility shift analysis revealed that LXR/RXR heterodimers bound to the DR+4 element in the presence of MUT but not WT TR-beta. A mechanism for cross-talk, and potential antagonism, between TR-beta and LXR-alpha is proposed.     

Contractile properties and myosin expression in rats born and reared in hypergravity

The effects of hypergravity (HG) on soleus and plantaris muscles were studied in Long Evans rats aged 100 days, born and reared in 2-g conditions (HG group). The morphological and contractile properties and the myosin heavy chain (MHC) content were examined in whole muscles and compared with terrestrial control (Cont) age-paired rats. The growth of HG rats was slowed compared with Cont rats. A decrease in absolute muscle weight was observed. An increase in fiber cross-sectional area/muscle wet weight was demonstrated, associated with an increase in relative maximal tension. The soleus muscle changed into a slower type both in contractile parameters and in MHC content, since HG soleus contained only the MHC I isoform. The HG plantaris muscle presented a faster contractile behavior. Moreover, the diversity of hybrid fiber types expressing multiple MHC isoforms (including MHC IIB and MHC IIX isoforms) was increased in plantaris muscle after HG. Thus the HG environment appears as an important inductor of muscular plasticity both in slow and fast muscle types.     

Aberrant expression of myosin isoforms in skeletal muscles from mice lacking the rev-erbAalpha orphan receptor gene

The rev-erbAalpha orphan protein belongs to the steroid nuclear receptor superfamily. No ligand has been identified for this protein, and little is known of its function in development or physiology. In this study, we focus on 1) the distribution of the rev-erbAalpha protein in adult fast- and slow-twitch skeletal muscles and muscle fibers and 2) how the rev-erbAalpha protein influences myosin heavy chain (MyHC) isoform expression in mice heterozygous (+/-) and homozygous (-/-) for a rev-erbAalpha protein null allele. In the fast-twitch extensor digitorum longus muscle, rev-erbAalpha protein expression was linked to muscle fiber type; however, MyHC isoform expression did not differ between wild-type, +/-, or -/- mice. In the slow-twitch soleus muscle, the link between rev-erbAalpha protein and MyHC isoform expression was more complex than in the extensor digitorum longus. Here, a significantly higher relative amount of the beta/slow (type I) MyHC isoform was observed in both rev-erbAalpha -/- and +/- mice vs. that shown in wild-type controls. A role for the ratio of thyroid hormone receptor proteins alpha1 to alpha2 in modulating MyHC isoform expression can be ruled out because no differences were seen in MyHC isoform expression between thyroid hormone receptor alpha2-deficient mice (heterozygous and homozygous) and wild-type mice. Therefore, our data are compatible with the rev-erbAalpha protein playing an important role in the regulation of skeletal muscle MyHC isoform expression.     

Single-fiber myosin heavy chain polymorphism during postnatal development: modulation by hypothyroidism

The primary objective of this study was to follow the developmental time course of myosin heavy chain (MHC) isoform transitions in single fibers of the rodent plantaris muscle. Hypothyroidism was used in conjunction with single-fiber analyses to better describe a possible linkage between the neonatal and fast type IIB MHC isoforms during development. In contrast to the general concept that developmental MHC isoform transitions give rise to muscle fibers that express only a single MHC isoform, the single-fiber analyses revealed a very high degree of MHC polymorphism throughout postnatal development. In the adult state, MHC polymorphism was so pervasive that the rodent plantaris muscles contained approximately 12-15 different pools of fibers (i.e., fiber types). The degree of polymorphism observed at the single-fiber level made it difficult to determine specific developmental schemes analogous to those observed previously for the rodent soleus muscle. However, hypothyroidism was useful in that it confirmed a possible link between the developmental regulation of the neonatal and fast type IIB MHC isoforms.     

Distribution of myosin heavy chain isoforms in non-weight-bearing rat soleus muscle fibers

Talmadge, Robert J., Roland R. Roy, and V. Reggie Edgerton.Distribution of myosin heavy chain isoforms in non-weight-bearing rat soleus muscle fibers. J. Appl.Physiol. 81(6): 2540-2546, 1996.The effects of14 days of spaceflight (SF) or hindlimb suspension (HS) (Cosmos 2044)on myosin heavy chain (MHC) isoform content of the rat soleus muscleand single muscle fibers were determined. On the basis ofelectrophoretic analyses, there was a de novo synthesis of type IIx MHCbut no change in either type I or IIa MHC isoform proportions aftereither SF or HS compared with controls. The percentage of fiberscontaining only type I MHC decreased by 26 and 23%, and the percentageof fibers with multiple MHCs increased from 6% in controls to 32% inHS and 34% in SF rats. Type IIx MHC was always found in combinationwith another MHC or combination of MHCs; i.e., no fibers contained typeIIx MHC exclusively. These data suggest that the expression of thenormal complement of MHC isoforms in the adult rat soleus muscle isdependent, in part, on normal weight bearing and that the absence ofweight bearing induces a shift toward type IIx MHC protein expression in the preexisting type I and IIa fibers of the soleus.