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In earlier research, we identified a 43-kDa c-ErbAalpha1 protein (p43) in the mitochondrial matrix of rat liver. In the present work, binding experiments indicate that p43 displays an affinity for triiodothyronine (T3) similar to that of the T3 nuclear receptor. Using in organello import experiments, we found that p43 is targeted to the organelle by an unusual process similar to that previously reported for MTF1, a yeast mitochondrial transcription factor. DNA-binding experiments demonstrated that p43 specifically binds to four mitochondrial DNA sequences with a high similarity to nuclear T3 response elements (mt-T3REs). Using in organello transcription experiments, we observed that p43 increases the levels of both precursor and mature mitochondrial transcripts and the ratio of mRNA to rRNA in a T3-dependent manner. These events lead to stimulation of mitochondrial protein synthesis. In transient-transfection assays with reporter genes driven by the mitochondrial D loop or two mt-T3REs located in the D loop, p43 stimulated reporter gene activity only in the presence of T3. All these effects were abolished by deletion of the DNA-binding domain of p43. Finally, p43 overexpression in QM7 cells increased the levels of mitochondrial mRNAs, thus indicating that the in organello influence of p43 was physiologically relevant. These data reveal a novel hormonal pathway functioning within the mitochondrion, involving a truncated form of a nuclear receptor acting as a potent mitochondrial T3-dependent transcription factor.

The product of the B-cell translocation gene 1 (BTG1), a member of an antiproliferative protein family including Tis-21/PC3 and Tob, is thought to play an important role in the regulation of cell cycle progression. We have shown in a previous work that triiodothyronine (T3) stimulates quail myoblast differentiation, partly through a cAMP-dependent mechanism involved in the stimulation of cell cycle withdrawal. Furthermore, we found that T3 or 8-Br-cAMP increases BTG1 nuclear accumulation in confluent myoblast cultures. In this study, we report that BTG1 is essentially expressed at cell confluence and in differentiated myotubes. Whereas neither T3 nor cAMP exerted a direct transcriptional control upon BTG1 expression, we found that AP-1 activity, a crucial target involved in the triiodothyronine myogenic influence, repressed BTG1 expression, thus probably explaining the low BTG1 expression level in proliferating myoblasts. In transient transfection studies, we demonstrated that an AP-1-like sequence located in the BTG1 promoter was involved in this negative regulation. Our present data also bring evidence that the stimulation of BTG1 nuclear accumulation by T3 or 8-Br-cAMP probably results from an increased nuclear import or retention in the nucleus. Lastly, BTG1 overexpression in quail myoblasts mimicked the T3 or 8-Br-cAMP myogenic influence: (i) inhibition of myoblast proliferation due to an increased rate of myoblast withdrawal from the cell cycle; and (ii) stimulation of terminal differentiation. These data suggest that BTG1 is probably involved in T3 and cAMP myogenic influences. In conclusion, BTG1 is a T3 target involved in the regulation of myoblast differentiation., (Copyright 1999 Academic Press.)

We have previously shown that v-erb A stimulates quail myoblast differentiation in a T3 independent, cell-specific manner. In this work, we have studied the influence of v-erb B (the second oncogene carried in the AEV genome) upon quail myoblast proliferation and differentiation. v-erb B expression,moderately stimulates myoblast proliferation, and inhibits differentiation. Moreover, this oncoprotein fully inhibits the v-erb A myogenic influence. These data provide evidence that these two oncogenes do not cooperate in avian myoblasts. Consequently, in contrast to results obtained in other cell-types, coexpression of both oncogenes does not transform quail myoblasts.

We have previously shown that v-erb A expression strongly stimulates quail myoblast proliferation and differentiation without alteration of the triiodothyronine (T3) influence in this cell type. In order to understand the molecular basis of v-erb A action in myoblasts, we have studied the influence of this oncoprotein on c-erb A alpha1 encoded T3 nuclear receptor (TR alpha) activity. In transfection experiments, v-erb A did not inhibit the T3-dependent c-erb A alpha1 transcriptional activity in QM7 myoblasts in contrast to its action in HeLa cells. However, it repressed the retinoic acid receptor RAR alpha activity in both cell-types, indicating that v-erb A interactions with T3 or RA mediated transcription significantly differs. In EMSA experiments using a TREpa1 probe, T3R alpha binds as three complexes in HeLa cells. We have previously identified the slow migrating complex, undetectable in QM7 myoblasts, as a T3R/RXR heterodimer. Interestingly, v-erb A inhibited binding of this complex in HeLa cells, but did not affect binding of the two other complexes in QM7 myoblasts. Expression of RXR (gamma isoform), the TR alpha dimerization partner absent in proliferating QM7 cells, restored inhibition of c-erb A alpha1 transcriptional activity in these cells and abrogated the v-erb A myogenic influence. Lastly, v-erb A induced a T3-independent c-erb A alpha1 activity in QM7 cells when cotransfected in equimolar ratio with the receptor, by inhibiting AP-1 activity and stimulating transcription of a reporter gene driven by a TRE sequence.

Numerous data suggest that mitochondrial activity is involved in the regulation of cell growth and differentiation. Therefore, we have studied the changes in mitochondrial activity in avian myoblast cultures (QM7 line) undergoing differentiation or in BrdU-treated, differentiation-deficient cells. As we have previously shown that triiodothyronine and v-erb A expression stimulate myogenic differentiation, we have also observed their influence upon mitochondrial activity. Comparison of control and BrdU-treated myoblasts indicated that precocious differentiation events were associated with a stimulation of citrate synthase and cytochrome oxidase activities. They also induced a transient decrease in mitochondrial membrane potential assessed by rhodamine 123 uptake. In control myoblasts, a general stimulation of mitochondrial activity was recorded at cell confluence, prior to terminal differentiation. These events did not occur in BrdU-treated myoblasts, thus indicating that they were tightly linked to myoblast commitment. Whereas no significant triiodothyronine influence could be detected upon mitochondrial activity, we observed that v-erb A expression significantly depresses the mitochondrial membrane potential in control myoblasts. This action was not observed in BrdU-treated myoblasts, thus suggesting that it involves an indirect pathway linked to differentiation. Moreover, the oncoprotein abrogated the decrease in E2-PDH subunit level observed at cell confluence. These data underline that changes in mitochondrial activity occurred prior to myoblast terminal differentiation and could be involved in the processes regulating myogenesis. In addition, they provide the first evidence that the v-erb A oncoprotein influences mitochondrial activity.

In a previous work, we have shown that T3 induces a potent stimulation of avian myoblast differentiation. In this study, we demonstrated that this hormone did not affect MyoD and myogenin expression. As numerous data suggest that T3 could affect the cAMP pathway, we have studied its involvement in the myogenic activity of triiodothyronine on quail myoblast. In agreement with Zalin and Montagues (Cell 2, 103-108 (1974)), we observed a transient rise in myoblast intracellular cAMP level some hours before the onset of terminal differentiation. Interestingly, this rise occurred earlier in T3-treated than in control myoblasts, and cAMP production was significantly increased by the hormone. Moreover, T3 increased CREB transcriptional activity, thus suggesting that the entire cAMP signaling pathway was stimulated by this hormone. In addition, we observed that addition of an inhibitor of adenylate cyclase activity prior to the cAMP rise dramatically inhibited myoblast differentiation. Last, we showed that cAMP mimicked all T3 actions upon myoblast differentiation: (1) T3 and cAMP reduced myoblast proliferation by increasing the number of postmitotic myoblasts at cell confluence; (2) T3 and cAMP increased BTG1 nuclear accumulation; (3) T3 and cAMP stimulated terminal differentiation only when added during the proliferative phasis. These data strongly suggest that the transient rise in cAMP production could be essential for myoblast terminal differentiation. In addition, it appears that, at least in avian myoblasts, T3 stimulation of terminal differentiation involves the cAMP pathway.

The v-erbA oncoprotein represents a mutated version of a thyroid hormone receptor, responsible for the induction of a differentiation arrest in chicken erythroid cells. We have studied the influence of v-erbA on proliferation and differentiation of avian myoblasts. Secondary quail myoblast cultures were infected either with an avian retrovirus carrying the v-erbA oncogene in association with the neomycin resistance gene, or with a control deleted v-erbA/neoR alpha retrovirus. We report here that v-erbA expression led to an increase in myoblast proliferation and to a surprising stimulation of quail myoblast terminal differentiation. In addition, these effects occurred in the presence or absence of T3, and v-erbA did not suppress T3 influence on myoblasts. Transient transfection assays demonstrated that, in contrast to its action in HeLa cells, v-erbA was unable to repress the transcriptional activation of a TRE-CAT reporter gene by liganded c-erbA alpha receptors in quail myoblasts. We also observed that the AP-1/c-erbA/v-erbA interactions are not functional in quail myoblasts. These data suggest that, in these cells, v-erbA action does not interfere with T3 induced mechanisms. They also demonstrate a cell specificity for the v-erbA pathway. Lastly, expression of c-erbA/v-erbA chimeric proteins and of the S61G v-erbA mutant indicates that the DNA binding domain of v-erbA, and more specifically serine 61, is directly involved in the enhancement of myoblast differentiation by the oncoprotein.

The influence of triiodothyronine (T3) on avian myoblast proliferation and differentiation was studied in secondary cultures using plating densities of 2,500 and 7,000 cells/cm2. Culture media were depleted of T3 (control myoblasts) and increasing amounts were then added to concentrations of 0.6, 3 and 15 nM T3 (treated myoblasts). Independent of the cell density, T3 induced a dose-related decrease in myoblast proliferation measured by cell number, doubling time and 3H-thymidine incorporation. However, with the lower plating density, this influence was delayed, occurring only after the third day of culture for 0.6 nM T3-treated myoblasts and simultaneous with the onset of myosin heavy chain accumulation. Moreover, when myoblasts were exposed to BrdU for 48 h, the T3 growth inhibitory effect disappeared, thus showing that this effect was clearly linked to differentiation. In addition, we have shown that T3 induced an early fusion of myoblasts: 65% of the maximal value of the fusion index was reached on day 3 in the T3-treated cells in comparison to 25% in the control myoblasts. This hormone also enhanced accumulation of muscle-specific proteins (connectin, acetylcholine receptors, myosin heavy chain), tested by cytoimmunofluorescence, ELISA, binding experiments and Western blot. All these results show that T3 increased myoblast differentiation through a pathway including myoblast withdrawal from the cell cycle. The influence of T3 could partly explain its previously reported positive effect on the number of muscle fibers.

Thyroid function was studied in small for gestational age (SGA) or control newborn lambs. Neonatal changes in plasma concentrations of TSH, T3, rT3, total and free T4 were monitored, and thyroid scintigraphs were performed. Responsiveness of the hypothalamic-pituitary-thyroid axis to cold exposure and TRH or TSH administration was assessed. In addition, T4 and T3 kinetic studies were performed. In agreement with results obtained in babies, plasma T3, total T4 and free T4 concentrations were depressed in low birth weight animals, whereas TSH and rT3 levels were not affected. Thyroid size expressed relatively to the body weight was higher in SGA animals, thus suggesting that a partial compensation for low thyroid hormone levels had occurred during the fetal life. Plasma TSH and T4 concentrations increased by a same extent after exposure to cold and TRH or TSH administration in SGA and control lambs; however, the rise in T3 levels was depressed in the former in all stimulation tests. T3 and T4 production rates were similar in the two experimental groups. In SGA lambs, the metabolic clearance rate and the total distribution space of these two hormones were significantly increased; the fast T3 pool was higher, and the slow T3 pool lower than in control animals. All these results demonstrate that, despite low circulating thyroid hormone concentrations, SGA lambs are not hypothyroid. An increased T4 and T3 storage in the extravascular compartment is probably the major factor involved in the occurrence of this plasma deficiency.(ABSTRACT TRUNCATED AT 250 WORDS)

Several experiments were performed to study the mechanisms inducing the neonatal rises in plasma iodothyronine concentrations in lambs. TSH levels rose during the first 4 to 8h of life, whereas plasma T4 an T3 concentrations increased only from birth to respectively 2 and 1h; the rise in free T4 levels was longer and more important than the rise in total T4. Only T4 changes were strongly related to the extent of TSH increase. The neonatal TSH surge was inhibited by delaying the first milk intake, indicating a great importance of the early nutritional status; in these conditions, the neonatal T4 rise did not occur, whereas the T3 increase was not affected; therefore, in contrast to T4, the T3 increase occurring at birth is not TSH-dependent. As in thyroidectomized lambs continuously infused with T4, plasma T3 concentrations did not increase at birth, it appears that the neonatal T3 surge probably has a thyroidal origin. These results raise the possibility of the existence of a specific stimulator of thyroidal T3 secretion, at least in the newborn lamb. In addition, comparison of the respective T4 increases, at birth or after TSH stimulation in 24 h-old animals, suggests that the ability of the thyroid to respond to a sustained stimulation is strongly reduced at birth. Lastly, neonatal changes in the affinity and/or capacity of carrier proteins for T4, perhaps partly induced by the observed simultaneous rise in free fatty acid levels, could explain that plasma T3 concentrations remained elevated despite a decrease in total T4 levels from 2 h after birth.

Plasma thyroxine levels were detectable in rat fetuses from day 17 of pregnancy and increased exponentially from 3.48 +/- 0.13 (S.E.M.) nmol/l to 11.33 +/- 0.90 nmol/l at birth. Ligation of the maternal uterine vein and artery, performed on day 17 of pregnancy, significantly reduced both the fetal growth rate, observed initially on day 19 of pregnancy (controls: 2.24 +/- 0.02 g; fetuses from ligated horns: 1.71 +/- 0.03 g, P less than 0.001) and plasma thyroxine levels, observed initially on day 20 of pregnancy (controls: 6.56 +/- 0.13 nmol/l; fetuses from ligated horns: 5.41 +/- 0.13 nmol/l, P less than 0.001). In addition we observed positive relationships between plasma thyroxine levels and body weights of the two groups on days 20 and 21 of pregnancy and at birth. These results suggest that relationships observed between plasma thyroxine levels and body weight in fetuses or newborn animals may be explained by the common effect of the placental blood flow mediated by fetal nutrition.

The changes in plasma levels of thyroxine (T4), free thyroxine (free T4), triiodothyronine (T3), free triiodothyronine (free T3), and reverse triiodothyronine (rT3) were observed in intact or hypophysectomized lamb foetuses during the last third of gestation. Hypophysectomy was performed at two stages: 100 and 120 days of gestation. Cord blood was collected after Caesarean section. In intact foetuses, T4 and free T4 levels increased significantly between day 100 and 120 of gestation, but decreased from day 132 to birth. T3 levels, low until day 132, rose abruptly during the last 12 days of gestation, as did the T3/free T4 ratio. Free T3 levels, relatively high on day 100, decreased until day 130 to rise during the last days of gestation. Reverse T3 levels, high since day 100, increased sharply until day 132 as did the reverse T3/free T4 ratio, and then declined. In hypophysectomized foetuses, undetectable or very low levels of T4, free T4 and rT3 were observed on day 120 and 144. T3 levels, although decreased by pituitary destruction, remained at 39.7% (120 days) and 21.4% (144 days) of the values recorded in intact foetuses. Free T3 levels, normally depressed at the end of gestation, were not affected by hypophysectomy on day 120. When hypophysectomy was performed later, the levels of T4, free T4 and rT3 were slightly increased on day 144 as compared with those of the other hypophysectomized foetuses. This study confirms the changes in the plasma iodothyronine levels observed previously in the lamb foetus.(ABSTRACT TRUNCATED AT 250 WORDS)

Neonatal endocrine status was studied in 14 lambs born 7 days before term, after estrogen injection into the ewes, and in 15 full-term animals. Plasma cortisol and triiodothyronine (T3) levels were depressed during the first hours of life in preterm lambs and plasma reverse T3 levels were significantly higher than in controls. The rise in plasma cortisol levels after Synacthen injection was significantly lowered by prematurity, suggesting reduced sensitivity of the adrenal cortex to ACTH. After ovine TSH injection, plasma thyroxine (T4) levels increased during a shorter period of time in preterm lambs, resulting in a lowered T4 rise; the T3 response was not affected by prematurity. After TRH injection, the rises in plasma T3 and T4 levels were significantly higher in preterm than in full-term lambs, suggesting a pituitary hypersensitivity to TRH linked to prematurity. Moreover it appeared that the response of reverse T3 to TSH or TRH was very weak.

The influence of food restriction, applied from birth to 36 h post partum, on neonatal thyroid function was studied in newborn Limousin x Romanov lambs. The control animals (n = 18) had free access to the mother and suckled ad libitum. Restricted lambs (n = 16) were removed from the mother and received limited amounts of colostrum in proportion to birth weight; 8 lambs were supplemented with lactose (30 g/l of colostrum). Plasma glucose and free fatty acid levels were significantly lower in restricted lambs, whereas urea levels were elevated. Plasma cortisol concentrations in control animals decreased during the period studied, but rose during the first 8 h of life in restricted lambs. Lactose supplementation only partially restored glucose and urea levels. Food restriction induced considerable modifications in neonatal thyroid function. The postpartum rise in plasma thyroid-stimulating hormone (TSH), thyroxine (T4) and free T4 levels occurring in control lambs was inhibited in restricted animals. However, the surge in plasma triiodothyronine (T3) levels was not affected, suggesting that this change was not related to physiological neonatal TSH hypersecretion. Thereafter, thyroid hormone concentrations decreased sharply during food restriction, whereas reverse T3 levels remained higher than in the controls. In response to these T4 and T3 deficiencies, plasma TSH levels rose only in lactose-supplemented animals. In agreement with the significant modifications in the values of the T3/free T4 and reverse T3/free T4 ratios, the abrupt changes in T3 and reverse T3 levels suggest that food restriction affected the peripheral conversion of T4 into T3 and reverse T3.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of various amounts of thyrotropin-releasing hormone (TRH) injected subcutaneously or intravenously (alone or in combination with growth hormone-releasing factor: GRF 1-44) on growth hormone (GH), thyroid-stimulating hormone (TSH), thyroxine (T4) and triiodothyronine (T3) were studied in the plasma of 2-week, 2-month and 3-month old lambs. After subcutaneous TRH administration, increases in plasma TSH, T4 and T3 levels were equivalent, whatever the amount of TRH used (1,2,5 or 10 micrograms/kg). These responses lasted longer after 5 and 10 micrograms/kg. After intravenous TRH administration in 2-week old lambs, the maximal increase in plasma TSH levels occurred after the injection of 0.25 microgram/kg. However, plasma T4 and T3 responses were not different, whatever the amount used. As previously, the amount of TRH affected the duration of these responses more than the magnitude of the pituitary-thyroid axis response. Whatever the injection route, amount used or animal age, TRH alone did not increase GH secretion in lambs. However, it slightly delayed the GH response to GRF. GRF did not affect the response of TSH and T4 to TRH; however it could inhibit T3 increase. In conclusion, in contrast to results obtained in calves by Hodate et al. (1985), TRH did not enhance GH secretion in lambs but, as expected, induced sharp increases in plasma thyroid hormone levels. Its classification as a "growth factor" is therefore questionable, at least in lambs.

Neonatal changes in plasma total and free iodothyronine levels were monitored in 18 Limousin X Romanov suckling lambs (experiment 1) and 24 Limousin X Romanov animals bottle-fed in standardized conditions (experiment 2). In the two experiments, plasma free T3 levels were closely related to total T3 levels, whereas some differences could be observed between total and free plasma T4 levels. The neonatal rise in free T4 in particular was higher than that observed for total T4 levels. Consequently, the enhancement of T4 availability for T3-generating cells could partly explain the neonatal T3 surge. In addition, plasma reverse T3 levels decreased progressively during the first 48 h of life in suckling lambs. These results suggest also that hypotrophy and/or nutritional status could affect neonatal thyroid function: plasma active iodothyronine levels decreased sharply in bottle-fed lambs (birthweight : 2.29 Kg) compared to suckling ones (birthweight : 3.57 Kg), whereas reverse T3 levels increased.

Neonatal changes in plasma free and total iodothyronines, cortisol, glucose and urea levels have been studied in 8 control (birthweight greater than or equal to 2.5 kg) and 16 hypotrophic lambs (birthweight less than 2.5 kg) receiving limited amounts of colostrum during the first 36 h of life and then fed ad libitum. During the period of colostrum feeding, plasma glucose levels were low in both groups and increased after the onset of ad libitum feeding; they were significantly lower in hypotrophic animals from birth to 36 h. Plasma urea levels increased during the period of colostrum feeding and decreased thereafter in all animals. At birth, they were significantly higher in hypotrophic lambs. Over the entire period studied (20 d), plasma levels of total T4, free T4, total T3 and free T3 were markedly lowered in hypotrophic lambs without alterations in the values of the T3/free T4 ratio. No differences could be observed in plasma reverse T3 and cortisol levels. For all blood parameters recorded, the neonatal changes were parallel in the two groups of lambs. In agreement with hypoglycemia and hyperuremia observed at birth in hypotrophic lambs, with the litter size recorded for each experimental group and with previous results, placental insufficiency linked to a large litter size gestation could be at the origin of low thyroid hormone levels.

The influence of a 7 days prematurity, induced by oestrogen or dexamethasone injection to the mothers, on neonatal changes in plasma T4, T3, reverse T3 (rT3), TSH and cortisol levels was studied in 6 full term, 6 oestrogen preterm and 6 dexamethasone preterm lambs. In addition, the pituitary-thyroid axis sensitivity was assessed by the magnitude of the response to TRH administration. At birth, plasma cortisol and T3 levels, as the value of the T3/T4 ratio, were significantly lower in the two groups of preterm lambs than in full term animals; however, whereas plasma T3 concentrations and values of the T3/T4 ratio remained low in oestrogen lambs, they were quickly restored and elevated T3 levels associated to high T4 levels could be even observed in dexamethasone lambs; in this last group, these abrupt changes could be a consequence of raised TSH plasma concentrations recorded at birth. Moreover, if plasma rT3 levels and values of the rT3/T4 ratio were similar during the first hours of life in dexamethasone and full-term lambs, they were significantly higher in oestrogen animals. The responsiveness of the pituitary-thyroid axis to TRH was normal in dexamethasone animals, but was significantly enhanced in oestrogen ones, probably as a consequence of low T3 levels.