Rhythm and ROS: Hepatic Chronotherapeutic Features of Grape Seed Proanthocyanidin Extract Treatment in Cafeteria Diet-Fed Rats.

Polyphenols play a key role in the modulation of circadian rhythms, while the cafeteria diet (CAF) is able to perturb the hepatic biological rhythm and induce important ROS production. Consequently, we aimed to elucidate whether grape seed proanthocyanidin extract (GSPE) administration recovers the CAF-induced hepatic antioxidant (AOX) misalignment and characterize the chronotherapeutic properties of GSPE. For this purpose, Fischer 344 rats were fed a standard diet (STD) or a CAF and concomitantly treated with GSPE at two time-points (ZT0 vs. ZT12). Animals were euthanized every 6 h and the diurnal rhythms of hepatic ROS-related biomarkers, hepatic metabolites, and AOX gene expression were examined. Interestingly, GSPE treatment was able to recover the diurnal rhythm lost due to the CAF. Moreover, GSPE treatment also increased the acrophase of Sod1, as well as bringing the peak closer to that of the STD group. GSPE also corrected some hepatic metabolites altered by the CAF. Importantly, the differences observed at ZT0 vs. ZT12 due to the time of GSPE administration highlight a chronotherapeutic profile on the proanthocyanin effect. Finally, GSPE could also reduce diet-induced hepatic oxidative stress not only by its ROS-scavenging properties but also by retraining the circadian rhythm of AOX enzymes.

Time-of-Day Circadian Modulation of Grape-Seed Procyanidin Extract (GSPE) in Hepatic Mitochondrial Dynamics in Cafeteria-Diet-Induced Obese Rats.

Major susceptibility to alterations in liver function (e.g., hepatic steatosis) in a prone environment due to circadian misalignments represents a common consequence of recent sociobiological behavior (i.e., food excess and sleep deprivation). Natural compounds and, more concisely, polyphenols have been shown as an interesting tool for fighting against metabolic syndrome and related consequences. Furthermore, mitochondria have been identified as an important target for mediation of the health effects of these compounds. Additionally, mitochondrial function and dynamics are strongly regulated in a circadian way. Thus, we wondered whether some of the beneficial effects of grape-seed procyanidin extract (GSPE) on metabolic syndrome could be mediated by a circadian modulation of mitochondrial homeostasis. For this purpose, rats were subjected to "standard", "cafeteria" and "cafeteria diet + GSPE" treatments (n = 4/group) for 9 weeks (the last 4 weeks, GSPE/vehicle) of treatment, administering the extract/vehicle at diurnal or nocturnal times (ZT0 or ZT12). For circadian assessment, one hour after turning the light on (ZT1), animals were sacrificed every 6 h (ZT1, ZT7, ZT13 and ZT19). Interestingly, GSPE was able to restore the rhythm on clock hepatic genes (Bmal1, Per2, Cry1, Rorα), as this correction was more evident in nocturnal treatment. Additionally, during nocturnal treatment, an increase in hepatic fusion genes and a decrease in fission genes were observed. Regarding mitochondrial complex activity, there was a strong effect of cafeteria diet at nearly all ZTs, and GSPE was able to restore activity at discrete ZTs, mainly in the diurnal treatment (ZT0). Furthermore, a differential behavior was observed in tricarboxylic acid (TCA) metabolites between GSPE diurnal and nocturnal administration times. Therefore, GSPE may serve as a nutritional preventive strategy in the recovery of hepatic-related metabolic disease by modulating mitochondrial dynamics, which is concomitant to the restoration of the hepatic circadian machinery.

p43, a Truncated Form of Thyroid Hormone Receptor α, Regulates Maturation of Pancreatic ß Cells.

P43 is a truncated form of thyroid hormone receptor α localized in mitochondria, which stimulates mitochondrial respiratory chain activity. Previously, we showed that deletion of p43 led to reduction of pancreatic islet density and a loss of glucose-stimulated insulin secretion in adult mice. The present study was designed to determine whether p43 was involved in the processes of ß cell development and maturation. We used neonatal, juvenile, and adult p43-/- mice, and we analyzed the development of ß cells in the pancreas. Here, we show that p43 deletion affected only slightly ß cell proliferation during the postnatal period. However, we found a dramatic fall in p43-/- mice of MafA expression (V-Maf Avian Musculoaponeurotic Fibrosarcoma Oncogene Homolog A), a key transcription factor of beta-cell maturation. Analysis of the expression of antioxidant enzymes in pancreatic islet and 4-hydroxynonenal (4-HNE) (a specific marker of lipid peroxidation) staining revealed that oxidative stress occurred in mice lacking p43. Lastly, administration of antioxidants cocktail to p43-/- pregnant mice restored a normal islet density but failed to ensure an insulin secretion in response to glucose. Our findings demonstrated that p43 drives the maturation of ß cells via its induction of transcription factor MafA during the critical postnatal window.

Potential physio-pathological effects of branched fatty acid esters of hydroxy fatty acids.

Branched Fatty Acid Esters of Hydroxy Fatty Acids (FAHFAs) are a new endogenous lipid class with recently uncovered interesting biological effects and which have been detected in food of plant and animal origins. Some FAHFAs can improve glucose tolerance and insulin sensitivity, stimulate insulin secretion, and exert anti-inflammatory effects. Other beneficial health effects have also been suggested, in particular against some cancers. FAHFAs could therefore be a potential therapeutic target for the treatment of numerous metabolic disorders such as type II diabetes, hepatic steatosis, cardiovascular diseases and various cancers. Their recent discovery has generated a great interest in the field of human health. This short review aims at bringing together the information available to date in the literature concerning their chemical synthesis, biosynthesis and degradation pathways as well as their potential physio-pathological beneficial effects.

Regulation of mitochondrial activity controls the duration of skeletal muscle regeneration in response to injury.

Thyroid hormone is a major regulator of skeletal muscle development and repair, and also a key regulator of mitochondrial activity. We have previously identified a 43 kDa truncated form of the nuclear T3 receptor TRα1 (p43) which stimulates mitochondrial activity and regulates skeletal muscle features. However, its role in skeletal muscle regeneration remains to be addressed. To this end, we performed acute muscle injury induced by cardiotoxin in mouse tibialis in two mouse models where p43 is overexpressed in or depleted from skeletal muscle. The measurement of muscle fiber size distribution at different time point (up to 70 days) upon injury lead us to unravel requirement of the p43 signaling pathway for satellite cells dependent muscle regeneration; strongly delayed in the absence of p43; whereas the overexpression of the receptor enhances of the regeneration process. In addition, we found that satellite cells derived from p43-Tg mice display higher proliferation rates when cultured in vitro when compared to control myoblasts, whereas p43-/- satellites shows reduced proliferation capacity. These finding strongly support that p43 plays an important role in vivo by controling the duration of skeletal muscle regeneration after acute injury, possibly through the regulation of mitochondrial activity and myoblasts proliferation.

Glucocorticoid-dependent REDD1 expression reduces muscle metabolism to enable adaptation under energetic stress.

BACKGROUND: Skeletal muscle atrophy is a common feature of numerous chronic pathologies and is correlated with patient mortality. The REDD1 protein is currently recognized as a negative regulator of muscle mass through inhibition of the Akt/mTORC1 signaling pathway. REDD1 expression is notably induced following glucocorticoid secretion, which is a component of energy stress responses. RESULTS: Unexpectedly, we show here that REDD1 instead limits muscle loss during energetic stresses such as hypoxia and fasting by reducing glycogen depletion and AMPK activation. Indeed, we demonstrate that REDD1 is required to decrease O2 and ATP consumption in skeletal muscle via reduction of the extent of mitochondrial-associated endoplasmic reticulum membranes (MAMs), a central hub connecting energy production by mitochondria and anabolic processes. In fact, REDD1 inhibits ATP-demanding processes such as glycogen storage and protein synthesis through disruption of the Akt/Hexokinase II and PRAS40/mTORC1 signaling pathways in MAMs. Our results uncover a new REDD1-dependent mechanism coupling mitochondrial respiration and anabolic processes during hypoxia, fasting, and exercise. CONCLUSIONS: Therefore, REDD1 is a crucial negative regulator of energy expenditure that is necessary for muscle adaptation during energetic stresses. This present study could shed new light on the role of REDD1 in several pathologies associated with energetic metabolism alteration, such as cancer, diabetes, and Parkinson's disease.

Toxicity of Natural Deep Eutectic Solvent Betaine:Glycerol in Rats.

Natural deep eutectic solvents (NaDES) are new natural solvents in green chemistry that in some cases have been shown to allow better extraction of plant bioactive molecules compared to conventional solvents and higher phenolic compound absorption in rodents. However, there is a serious lack of information regarding their in vivo safety. The purpose of this study was to verify the safety of a NaDES (betaine:glycerol (1:2 mole ratio) of water) extract from green coffee beans, rich in polyphenols. Twelve 6-week-old male Wistar rats were randomized into two groups of 6 animals each and twice daily gavaged for 14 days either with 3 mL of water or 3 mL of phenolic NaDES extract. Oral administration of phenolic NaDES extract induced mortality in two rats. In addition, it induced excessive water consumption, reduced dietary intake and weight loss, hepatomegaly, and plasma oxidative stress associated with high blood lipid levels. In conclusion, this work demonstrated the toxicity of oral administration of the selected NaDES under a short-term condition. This occurs despite the fact that this NaDES extract contains polyphenols, whose beneficial effects have been shown. Therefore, complementary work is needed to find the best dose and formulation of NaDES that are safe for the environment and animals and ultimately for humans.

Mitochondrial MDM2 Regulates Respiratory Complex I Activity Independently of p53.

Accumulating evidence indicates that the MDM2 oncoprotein promotes tumorigenesis beyond its canonical negative effects on the p53 tumor suppressor, but these p53-independent functions remain poorly understood. Here, we show that a fraction of endogenous MDM2 is actively imported in mitochondria to control respiration and mitochondrial dynamics independently of p53. Mitochondrial MDM2 represses the transcription of NADH-dehydrogenase 6 (MT-ND6) in vitro and in vivo, impinging on respiratory complex I activity and enhancing mitochondrial ROS production. Recruitment of MDM2 to mitochondria increases during oxidative stress and hypoxia. Accordingly, mice lacking MDM2 in skeletal muscles exhibit higher MT-ND6 levels, enhanced complex I activity, and increased muscular endurance in mild hypoxic conditions. Furthermore, increased mitochondrial MDM2 levels enhance the migratory and invasive properties of cancer cells. Collectively, these data uncover a previously unsuspected function of the MDM2 oncoprotein in mitochondria that play critical roles in skeletal muscle physiology and may contribute to tumor progression.

Skeletal muscle expression of p43, a truncated thyroid hormone receptor α, affects lipid composition and metabolism.

Thyroid hormone is a major regulator of metabolism and mitochondrial function. Thyroid hormone also affects reactions in almost all pathways of lipids metabolism and as such is considered as the main hormonal regulator of lipid biogenesis. The aim of this study was to explore the possible involvement of p43, a 43 Kda truncated form of the nuclear thyroid hormone receptor TRα1 which stimulates mitochondrial activity. Therefore, using mouse models overexpressing p43 in skeletal muscle (p43-Tg) or lacking p43 (p43-/-), we have investigated the lipid composition in quadriceps muscle and in mitochondria. Here, we reported in the quadriceps muscle of p43-/- mice, a fall in triglycerides, an inhibition of monounsaturated fatty acids (MUFA) synthesis, an increase in elongase index and an decrease in desaturase index. However, in mitochondria from p43-/- mice, fatty acid profile was barely modified. In the quadriceps muscle of p43-Tg mice, MUFA content was decreased whereas the unsaturation index was increased. In addition, in quadriceps mitochondria of p43-Tg mice, we found an increase of linoleic acid level and unsaturation index. Last, we showed that cardiolipin content, a key phospholipid for mitochondrial function, remained unchanged both in quadriceps muscle and in its mitochondria whatever the mice genotype. In conclusion, this study shows that muscle lipid content and fatty acid profile are strongly affected in skeletal muscle by p43 levels. We also demonstrate that regulation of cardiolipin biosynthesis by the thyroid hormone does not imply p43.

Mitochondrial T3 receptor and targets.

The demonstration that TRα1 mRNA encodes a nuclear thyroid hormone receptor and two proteins imported into mitochondria with molecular masses of 43 and 28 kDa has brought new clues to better understand the pleiotropic influence of iodinated hormones. If p28 activity remains unknown, p43 binds to T3 responsive elements occurring in the organelle genome, and, in the T3 presence, stimulates mitochondrial transcription and the subsequent synthesis of mitochondrial encoded proteins. This influence increases mitochondrial activity and through changes in the mitochondrial/nuclear cross talk affects important nuclear target genes regulating cell proliferation and differentiation, oncogenesis, or apoptosis. In addition, this pathway influences muscle metabolic and contractile phenotype, as well as glycaemia regulation. Interestingly, according to the process considered, p43 exerts opposite or cooperative effects with the well-known T3 pathway, thus allowing a fine tuning of the physiological influence of this hormone.

Temperature homeostasis in mice lacking the p43 mitochondrial T3 receptor.

Thyroid hormones and Thra gene play a key role in energy expenditure regulation, temperature homeostasis, and mitochondrial function. To decipher the function of the mitochondrial TRα receptor in these phenomena, we used mice lacking specifically the p43 mitochondrial T3 receptor. We found that these animals were hypermetabolic, hyperphagic, and displayed a down setting of the core body temperature. However, p43-/- animals do not present cold intolerance or defect of facultative thermogenesis. In addition, the mitochondrial function of BAT is slightly affected in the absence of p43. Our study, therefore, suggests a complementarity of action between the mitochondrial receptor and other proteins encoded by the Thra gene in the control of basal metabolism, facultative thermogenesis, and determination of the set point of temperature regulation.

Chicoric acid is an antioxidant molecule that stimulates AMP kinase pathway in L6 myotubes and extends lifespan in Caenorhabditis elegans.

Chicoric acid (CA) is a caffeoyl derivative previously described as having potential anti-diabetic properties. As similarities in cellular mechanism similarities between diabetes and aging have been shown, we explored on L6 myotubes the effect of CA on the modulation of intracellular pathways involved in diabetes and aging. We also determined its influence on lifespan of Caenorhabditis elegans worm (C. elegans). In L6 myotubes, CA was a potent reactive oxygen species (ROS) scavenger, reducing ROS accumulation under basal as well as oxidative stress conditions. CA also stimulated the AMP-activated kinase (AMPK) pathway and displayed various features associated with AMPK activation: CA (a) enhanced oxidative enzymatic defences through increase in glutathion peroxidase (GPx) and superoxide dismutase (SOD) activities, (b) favoured mitochondria protection against oxidative damage through up-regulation of MnSOD protein expression, (c) increased mitochondrial biogenesis as suggested by increases in complex II and citrate synthase activities, along with up-regulation of PGC-1α mRNA expression and (d) inhibited the insulin/Akt/mTOR pathway. As AMPK stimulators (e.g. the anti-diabetic agent meformin or polyphenols such as epigallocatechingallate or quercetin) were shown to extend lifespan in C. elegans, we also determined the effect of CA on the same model. A concentration-dependant lifespan extension was observed with CA (5-100 µM). These data indicate that CA is a potent antioxidant compound activating the AMPK pathway in L6 myotubes. Similarly to other AMPK stimulators, CA is able to extend C. elegans lifespan, an effect measurable even at the micromolar range. Future studies will explore CA molecular targets and give new insights about its possible effects on metabolic and aging-related diseases.

Depletion of the p43 mitochondrial T3 receptor increases Sertoli cell proliferation in mice.

Among T3 receptors, TRα1 is ubiquitous and its deletion or a specific expression of a dominant-negative TRα1 isoform in Sertoli cell leads to an increase in testis weight and sperm production. The identification of a 43-kDa truncated form of the nuclear receptor TRα1 (p43) in the mitochondrial matrix led us to test the hypothesis that this mitochondrial transcription factor could regulate Sertoli cell proliferation. Here we report that p43 depletion in mice increases testis weight and sperm reserve. In addition, we found that p43 deletion increases Sertoli cell proliferation in postnatal testis at 3 days of development. Electron microscopy studies evidence an alteration of mitochondrial morphology observed specifically in Sertoli cells of p43-/- mice. Moreover, gene expression studies indicate that the lack of p43 in testis induced an alteration of the mitochondrial-nuclear cross-talk. In particular, the up-regulation of Cdk4 and c-myc pathway in p43-/- probably explain the extended proliferation recorded in Sertoli cells of these mice. Our finding suggests that T3 limits post-natal Sertoli cell proliferation mainly through its mitochondrial T3 receptor p43.

Protein sequences involved in the mitochondrial import of the 3,5,3'-L-triiodothyronine receptor p43.

The major effect of T3 on mitochondrial activity has been partly explained by the discovery of p43, a T3-dependent transcription factor of the mitochondrial genome. P43 is imported into mitochondria in an atypical manner which is not yet fully understood. Our aim was to characterize the p43 sequences inducing its mitochondrial import, using in organello import experiments with wild-type or mutated proteins and validation in CV1 cells. We find that several sequences define the mitochondrial addressing. Two alpha helices in the C-terminal part of p43 are actual mitochondrial import sequences as fusion to a cytosolic protein induces its mitochondrial translocation. Helix 5 drives the atypical mitochondrial import process, whereas helices 10/11 induce a classical import process. However, despite its inability to drive a mitochondrial import, the N-terminal region of p43 also plays a permissive role as in the presence of the C-terminal import sequences different N-terminal regions determine whether the protein is imported or not. These results can be extrapolated to other mitochondrial proteins related to the nuclear receptor superfamily, devoid of classical mitochondrial import sequences.

Oxidative stress, inflammation, and autophagic stress as the key mechanisms of premature age-related hearing loss in SAMP8 mouse Cochlea.

AIMS: In our aging society, age-related hearing loss (ARHL) or presbycusis is increasingly important. Here, we study the mechanism of ARHL using the senescence-accelerated mouse prone 8 (SAMP8) which is a useful model to probe the effects of aging on biological processes. RESULTS: We found that the SAMP8 strain displays premature hearing loss and cochlear degeneration recapitulating the processes observed in human presbycusis (i.e., strial, sensory, and neural degeneration). The molecular mechanisms associated with premature ARHL in SAMP8 mice involve oxidative stress, altered levels of antioxidant enzymes, and decreased activity of Complexes I, II, and IV, which in turn lead to chronic inflammation and triggering of apoptotic cell death pathways. In addition, spiral ganglion neurons (SGNs) also undergo autophagic stress and accumulated lipofuscin. INNOVATION AND CONCLUSION: Our results provide evidence that targeting oxidative stress, chronic inflammation, or apoptotic pathways may have therapeutic potential. Modulation of autophagy may be another strategy. The fact that autophagic stress and protein aggregation occurred specifically in SGNs also offers promising perspectives for the prevention of neural presbycusis.

E2F transcription factor-1 regulates oxidative metabolism.

Cells respond to stress by coordinating proliferative and metabolic pathways. Starvation restricts cell proliferative (glycolytic) and activates energy productive (oxidative) pathways. Conversely, cell growth and proliferation require increased glycolytic and decreased oxidative metabolism levels. E2F transcription factors regulate both proliferative and metabolic genes. E2Fs have been implicated in the G1/S cell-cycle transition, DNA repair, apoptosis, development and differentiation. In pancreatic ß-cells, E2F1 gene regulation facilitated glucose-stimulated insulin secretion. Moreover, mice lacking E2F1 (E2f1(-/-)) were resistant to diet-induced obesity. Here, we show that E2F1 coordinates cellular responses by acting as a regulatory switch between cell proliferation and metabolism. In basal conditions, E2F1 repressed key genes that regulate energy homeostasis and mitochondrial functions in muscle and brown adipose tissue. Consequently, E2f1(-/-) mice had a marked oxidative phenotype. An association between E2F1 and pRB was required for repression of genes implicated in oxidative metabolism. This repression was alleviated in a constitutively active CDK4 (CDK4(R24C)) mouse model or when adaptation to energy demand was required. Thus, E2F1 represents a metabolic switch from oxidative to glycolytic metabolism that responds to stressful conditions.

P43-dependent mitochondrial activity regulates myoblast differentiation and slow myosin isoform expression by control of Calcineurin expression.

We have previously shown that mitochondrial protein synthesis regulates myoblast differentiation, partly through the control of c-Myc expression, a cellular oncogene regulating myogenin expression and myoblast withdrawal from the cell cycle. In this study we provide evidence of the involvement of Calcineurin in this regulation. In C2C12 myoblasts, inhibition of mitochondrial protein synthesis by chloramphenicol decreases Calcineurin expression. Conversely, stimulation of this process by overexpressing the T3 mitochondrial receptor (p43) increases Calcineurin expression. Moreover, expression of a constitutively active Calcineurin (ΔCN) stimulates myoblast differentiation, whereas a Calcineurin antisense has the opposite effect. Lastly, ΔCN expression or stimulation of mitochondrial protein synthesis specifically increases slow myosin heavy chain expression. In conclusion, these data clearly suggest that, partly via Calcineurin expression, mitochondrial protein synthesis is involved in muscle development through the control of myoblast differentiation and probably the acquisition of the contractile and metabolic phenotype of muscle fibres.

A polyphenol extract modifies quantity but not quality of liver fatty acid content in high-fat-high-sucrose diet-fed rats: possible implication of the sirtuin pathway.

High-fat or high-fat-high-sucrose diets are known to induce non-alcoholic fatty liver disease and this is emerging as one of the most common liver diseases worldwide. Some polyphenols have been reported to decrease rat hepatic lipid accumulation, in particular those extracted from red grapes such as resveratrol. The present study was designed to determine whether a polyphenol extract (PPE), from red grapes, modulates liver fatty acid composition and desaturase activity indexes in rats fed a high-fat-high-sucrose (HFHS) diet, and to explore whether sirtuin-1 deacetylase activation was implicated in the effect of the PPE against liver steatosis. The effect of this PPE on mitochondriogenesis and mitochondrial activity was also explored. The PPE decreased liver TAG content in HFHS+PPE diet-fed rats in comparison with HFHS diet-fed rats. The PPE had no effect on liver fatty acid composition, desaturase activity indexes and stearoyl-CoA desaturase 1 (SCD1) gene expression. Sirtuin-1 deacetylase protein expression was significantly increased with the PPE; AMP kinase protein expression was higher with the PPE in comparison with the HFHS rats, but no modification of phosphorylated AMP kinase was observed. Protein expression of phospho-acetyl-CoA carboxylase was decreased in HFHS rats and returned to basal values with the PPE. Finally, the PPE modulated PPARγ coactivator-1α (PGC-1α) but did not modify mitochondriogenesis and mitochondrial activity. In conclusion, the PPE partially prevented the accumulation of TAG in the liver by regulating acetyl-CoA carboxylase phosphorylation, a key enzyme in lipid metabolism, probably via sirtuin-1 deacetylase activation. However, the PPE had no effect on the qualitative composition of liver fatty acids.

[Triiodothyronine mitochondrial receptors: import and molecular mechanisms]. / Les récepteurs mitochondriaux de la triiodothyronine: import et mécanismes d'action.

Thyroid hormone exerts a diversity of physiological influences over developmental and metabolic processes. Searching for receptors able to mediate this extended regulation led to the identification of triiodothyronine (T3) nuclear receptors encoded by two different genes, c-erbA alpha (TR alpha) and c-erbA beta (TR beta). More recently, two N-terminally truncated forms of the triiodothyronine nuclear receptor TR alpha 1, with molecular weights of 43 and 28 kDa, have been discovered in mitochondria. Synthesized through the use of internal initiation sites of translation occurring in the TR alpha 1 transcript, they are addressed into mitochondria according to an atypical process. Two mitochondrial import sequences have been characterized in the C-terminal part of these proteins; in addition, their N-terminal part, devoid of negative charges, plays a permissive role in this import. Whereas the function of p28 remains unknown, p43 is a T3-dependent transcription factor of the mitochondrial genome, acting through dimeric complexes involving at least two other truncated forms of nuclear receptors, mtRXR and mtPPAR. P43 activation by T3 stimulates mitochondrial protein synthesis, respiratory chain activity and mitochondriogenesis. Through the mitochondrial/nuclear crosstalk, this direct T3 mitochondrial pathway influences the expression of nuclear genes involved in the regulation of cell proliferation and differentiation. In particular, in myoblasts, p43 overexpression stimulates terminal differentiation and induces a preferential expression of slow myosin, by down-regulating c-Myc expression and up-regulating calcineurin and myogenin expression. Comparison of the respective influences of the nuclear and mitochondrial T3 pathways demonstrates either both additivity (myoblast differentiation), complementarity (mitochondriogenesis, myoblast differentiation) or opposite influences (myosin expression), thus indicating that these two pathways introduce a fine-tuning of the hormone influence.

Avian MyoD and c-Jun coordinately induce transcriptional activity of the 3,5,3'-triiodothyronine nuclear receptor c-ErbAalpha1 in proliferating myoblasts.

Although physical interactions with other receptors have been reported, heterodimeric complexes of T(3) nuclear receptors (TR) with retinoid X receptors (RXRs) are considered as major regulators of T(3) target gene expression. However, despite the potent T(3) influence in proliferating myoblasts, RXR isoforms are not expressed during proliferation, raising the question of the nature of the complex involved in TRalpha transcriptional activity. We have previously established that c-Jun induces TRalpha1 transcriptional activity in proliferating myoblasts not expressing RXR. This regulation is specific to the muscle lineage, suggesting the involvement of a muscle-specific factor. In this study, we found that MyoD expression in HeLa cells stimulates TRalpha1 activity, an influence potentiated by c-Jun coexpression. Similarly, in the absence of RXR, MyoD or c-Jun overexpression in myoblasts induces TRalpha1 transcriptional activity through a direct repeat 4 or an inverted palindrome 6 thyroid hormone response element. The highest rate of activity was recorded when c-Jun and MyoD were coexpressed. Using c-Jun-negative dominants, we established that MyoD influence on TRalpha1 activity needs c-Jun functionality. Furthermore, we demonstrated that TRalpha1 and MyoD physically interact in the hinge region of the receptor and the transactivation and basic helix loop helix domains of MyoD. RXR expression (spontaneously occurring at the onset of myoblast differentiation) in proliferating myoblasts abrogates these interactions. These data suggest that in the absence of RXR, TRalpha1 transcriptional activity in myoblasts is mediated through a complex including MyoD and c-Jun.