C/EBPβ is a bZIP transcription factor known to be involved in various physiological processes, including adipogenesis, osteogenesis and liver development. Previous studies in this laboratory revealed an inhibition of myogenesis and reduced myogenic protein expression in 5-azacytidine treated mesenchymal stem cells retrovirally transduced to overexpress C/EBPβ. The goal of this thesis was to evaluate the role of C/EBPβ in myogenic differentiation by overexpression in C2C12 myoblasts and primary myoblasts. We demonstrate reduced MyoD protein expression and subsequent downregulation of myogenic proteins during differentiation following C/EBPβ overexpression. We localized C/EBPβ to the quiescent Pax7+ satellite cells associated with the muscle fiber. Upon satellite cell activation, we observed the downregulation of C/EBPβ protein expression prior to MyoD protein expression. Furthermore, the re-expression of C/EBPβ correlated with the loss of MyoD expression later in differentiation. Histological analysis of C/EBPβ-/- mice revealed smaller fibers and a reduced Pax7+ satellite cell population as compared to control animals. In this thesis, we propose that C/EBPβ is a negative regulator of skeletal muscle differentiation by inhibiting the expression of MyoD, thus impairing proper progression through the myogenic program. In addition, we propose a role for C/EBPβ in the maintenance of undifferentiatied satellite cells.

Skeletal muscle is one of the largest tissues in humans and provides many pivotal functions to support life. Abnormality in skeletal muscle functions can lead to disease. For example, insulin resistance in skeletal muscle leads to type II diabetes. The underlying mechanisms that control energy balance in skeletal muscle remain largely elusive, especially at the genetic level. Here in the second chapter, I showed that MyoD mediated the transcriptional regulation of ACSL5, a mitochondrial protein, in C2C12 myoblasts via two E-box elements. A SNP rs2419621 (T) created a de novo E-box that together with the two pre-existing proximal E-boxes strongly enhances ACSL5 expression in both CV1 and C2C12 cells. In the third chapter, I identified a novel VGLL4-interacting protein IRF2BP2 and verified the interaction with co-immunoprecipitation and mammalian two-hybrid assays. Functionally, overexpression of IRF2BP2 and transcription factor TEAD1 activates mouse VEGF-A promoter in CV1 cells and enhances the biosynthesis of VEGF-A in C2C12 myoblasts. In vivo studies showed that ischemia induced the expression of IRF2BP2 by more than three fold, suggesting that IRF2BP2 could play a pivotal role during tissue ischemia. IRF2BP2 is a nuclear protein in both mouse cardiac myocytes and C2C12 myoblasts as demonstrated by immunohistochemistry and immunocytochemistry, respectively. Therefore, I sought to delineate the mechanism for the nuclear shuttling of IRF2BP2 in the fourth chapter. With various DNA alternations, I mapped the NLS to an evolutionarily conserved sequence 354ARKRKPSP361 in IRF2BP2. Deletion of the positively charged amino acids resulted in the abolishment of the NLS signal. Next, I showed that phosphorylation of serine 360 (S360) mediates the nuclear import of the protein. Whereas an alanine substitution (S360A) at the site resulted in perinuclear accumulation of the protein, an aspartic acid substitution (S360D) forced the nuclear accumulation. Nevertheless, the forced accumulation of the S360D mutant did not enhance the activation of VEGF-A promoter in CV1 cells as did the wild-type protein. My studies revealed two novel mechanisms by which skeletal muscle could harvest energy, thus providing new insight into the energy metabolism in skeletal muscle

Avec leurs caractéristiques d'auto-renouvellement illimité et de pluripotence, les cellules souches embryonnaires humaines (hESC) représentent une source infinie de cellules pour la thérapie cellulaire de maladies, telle que la dystrophie musculaire de Duchenne. Des études ont démontré que les hESC pouvaient être différenciées en cellules musculaires squelettiques, mais les techniques employées sont longues et inefficaces. Ce mémoire décrit un nouveau protocole de différenciation des hESC en cellules musculaires squelettiques à l'aide d'un adénovirus exprimant le gène MyoD sous le contrôle du promoteur CAO (Ad.CAO-MyoD). L'efficacité de ce virus pour induire la myogenèse des hESC a été mise en évidence par la présence de divers marqueurs myogéniques. Ensuite, le potentiel de fusion de ces cellules a été illustré par le marquage de la MyHC et par l'observation de quelques myotubes. Ces résultats préliminaires semblent indiquer que l'Ad.CAO-MyoD est un outil prometteur pour différencier les hESC en cellules musculaires squelettiques.

Orientador: Maeli Dal Pai Silva / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-08T13:18:40Z (GMT). No. of bitstreams: 1 Almeida_FernandaLosiAlvesde_M.pdf: 1065788 bytes, checksum: 0b390cd2e5f289613db5ad2ca384e439 (MD5) Previous issue date: 2007 / Resumo: Nos peixes, o crescimento do tecido muscular ocorre por hipertrofia e/ou hiperplasia a partir da proliferação e diferenciação de mioblastos adultos ou células miossatélites, processos regulados pela expressão diferencial dos fatores de regulação miogênica (MRFs). O objetivo desse trabalho foi avaliar os mecanismos de crescimento muscular hiperplasico e hipertrofico e a expressão do MRF MyoD, na musculatura branca do pacu (Piaractus mesopotamicus), durante o crescimento. Exemplares juvenis (n=5) e adultos (n=5) de pacu foram anestesiados, sacrificados e determinados o peso corporal (g) e o comprimento total (cm). Fragmentos musculares brancos da região dorsal de cada exemplar, em cada fase estudada, foram congelados e imersos em nhexano congelado em nitrogenio liquido. Cortes histológicos (10 µm), obtidos em criostato, foram submetidos à  coloração hematoxilina-eosina para avaliação da morfologia e morfometria das fibras musculares brancas. Foi calculado o menor diametro de 100 fibras musculares brancas em cada animal de cada fase estudada. As fibras musculares foram distribuídas em classes, na dependência do seu diametro (<20, 20-50, >50 µm), para avaliar o grau de crescimento hipertrófico e hiperplá¡sico da musculatura. A expressão do MRF MyoD na musculatura branca foi analisada por Reação em Cadeia da Polimerase apos Transcrição Reversa (RT - PCR). Todos os produtos visualizados em gel de agarose a 1% foram clonados e sequenciados. A morfologia da musculatura dos exemplares juvenis e adultos foi semelhante, apresentando um padrão em mosaico caracterizado por fibras de diferentes diâmetros. Nos exemplares juvenis, foi observado um predomínio de fibras com diametro menor que 20 µm, caracterizando intensa hiperplasia. Nos exemplares adultos, houve o predomínio de fibras musculares com diâmetro maior que 50 µm, caracterizando intensa hipertrofia da musculatura. A expressão do RNAm para o gene MyoD foi significativamente maior na fase juvenil, se comparada com a fase adulta. Foi obtida a sequencia consenso parcial do gene MyoD (338 pares de bases) expresso na musculatura branca do pacu. Essa sequencia apresentou similaridade com as sequencias de MyoD de varias especies de vertebrados, incluindo peixes teleósteos. A expressão diferencial do MRF MyoD, observada nas fases de crescimento juvenil e adulta do pacu, possivelmente seja responsavel pelas diferenças observadas no padrão de crescimento, com a hiperplasia predominando nos juvenis e a hipertrofia, nos adultos / Abstract: Skeletal muscle growth in fish occurs by hypertrophy and hyperplasia and is dependent of the proliferation and differentiation of myogenic progenitor cells, events regulated by the diferential expression of the myogenic regulatory factors (MRFs). The aim of this study was to analyze the hyperplasia and hypertrophy processes and the MRF MyoD expression in the white muscle in pacu (Piaractus mesopotamicus) during growth. Juvenile (n=5) and adult (n=5) fishes were anaesthetized, sacrificed and the weight (g) and the total length (cm) were determined. White muscle samples from dorsal region of each sample, in each growth phase, were collected and and immersed in n- Hexane cooled in liquid nitrogen. Transverse sections (10 µm thick), obtained in a cryostat, were stained with Haematoxilin-Eosin to morphological and morphometric analysis. We calculated the smallest diameter from 100 white muscle fibres per animal in each group. White muscle fibers were grouped in three classes: <20, 20-50 and >50 µm to evaluate hypertrophy and hyperplasia in pacu white skeletal muscle. MyoD gene expression was determined by using RT-PCR. All PCR products visualized in 1% agarose gels were cloned and sequenced. Juvenile and adult pacu fish skeletal muscle showed similar morphology, with mosaic pattern characterized by fibers with different diameters. The great number of muscle fibers with diameter inferior 20 µm observed in juvenile fish confirms the active hyperplasic process. In adult fish, most fibers were over 50 µm diameter and denote the more intense muscle fiber hypertrophy. MyoD mRNA level in the juvenile fish was higher compared to adult fish. A consensus partial sequence for MyoD gene (338 bases pairs) was obtained. This sequence showed similarity with various vertebrate species, including teleost fishes. Differential expression of MyoD gene observed in white muscle of pacu possibly is related to differences in growth patterns during the phases analysed, with predominance of hyperplasia in juveniles and hypertrophy in adult fish / Mestrado / Histologia / Mestre em Biologia Celular e Estrutural

Submitted by Nadir Basilio (nadirsb@uninove.br) on 2018-06-19T15:19:00Z No. of bitstreams: 1 Jacira Souza Ribeiro.pdf: 1136563 bytes, checksum: 6f35b1abf4cd3ca62f8560ee50069169 (MD5) / Made available in DSpace on 2018-06-19T15:19:00Z (GMT). No. of bitstreams: 1 Jacira Souza Ribeiro.pdf: 1136563 bytes, checksum: 6f35b1abf4cd3ca62f8560ee50069169 (MD5) Previous issue date: 2015-12-10 / The low-intensity pulsed ultrasound (LIPUS) has been used to promote muscle repair with better quality and in shorter time, however, there is no standardization for the parameters used in clinical practice. Thus, the aim of this study was to evaluate the effect of USPBI on the repair of skeletal muscle of rats after cryoinjury. Male Wistar rats (n=45) were divided into 3 groups: control; only injury; Injured and treated with LIPUS. The LIPUS application was performed daily, using the stationary mode, pulse 1: 4, 1 MHz frequency, intensity 0.4 W / cm2 for 3 minutes. The injured groups were euthanized at 1, 2, 3 and 7 days following injury induction. The tibialis anterior muscle (TA) was removed for morphological analysis and collagen remodeling, and the muscle sections stained with H&E and Picrosirus Red, respectively. Then, the slides were photographed and quantified using the program "Image J". The analysis of MyoD and myogenin gene expression was performed using real time PCR. The results showed that the USPBI promoted modulation of inflammatory responses with a decrease of inflammatory infiltrates after 1, 2, 3 and 7 days, and reduction of myonecrosis after 7 days, followed by an increase in the number of immature fibers after 3 and 7 days, and increase of blood vessels on days 2, 3 and 7 days. Regarding the deposition of collagen, the results showed better organization of the fibers in all experimental periods, and increased deposition of collagen fibers in the injured group and treated after 2 and 3 days. In addition, treatment with LIPUS promoted increased gene expression of MyoD reduction after 3 days and after 7 days. Regarding myogenin expression, the treated group showed increased expression after 7 days. In conclusion, the LIPUS induced positive effects on muscle repair process leading to reduced inflammation and myonecrosis, increased in the immature fibers and mature blood vessels, as well as modulation of Myod and miogenin in different periods. / O ultrassom pulsado de baixa intensidade (USPBI) tem sido utilizado por promover um reparo muscular de melhor qualidade e menor duração, porém, não há padronização quanto aos parâmetros utilizados na prática clínica. O objetivo deste estudo foi avaliar o efeito do USPBI sobre o reparo do músculo esquelético de rato após criolesão. Foram utilizados 45 ratos Wistar, machos, divididos em 3 grupos experimentais: controle; somente lesão; lesionado tratado com USPBI. A aplicação de USPBI foi realizada diariamente após indução da lesão, modo estacionário, pulsado 1:4, frequência 1 MHz, intensidade 0,4 W/cm2, durante 3 minutos. Os grupos lesionados foram eutanasiados após 1, 2, 3 e 7 dias da indução da lesão. O músculo tibial anterior (TA) foi removido para análise morfológica e de remodelamento do colágeno, sendo os cortes corados com H&E e Picrosirius Red, respectivamente. As lâminas foram fotografadas e quantificadas com auxílio do programa “Image J”. A expressão gênica de MyoD e miogenina foi obtida por PCR em tempo real. Os resultados evidenciaram que o USPBI promoveu modulação da resposta inflamatória, havendo redução do infiltrado inflamatório após 1, 2, 3 e 7 dias, e redução da mionecrose após 7 dias, seguido pelo aumento no número de fibras imaturas após 3 e 7 dias, e aumento dos vasos sanguíneos nos dias 2, 3 e 7 dias. Em relação à deposição de colágeno, os resultados evidenciaram melhor organização das fibras em todos os períodos experimentais, além de aumento da deposição de fibras colágenas no grupo lesionado e tratado após 2 e 3 dias. Além disso, o tratamento com USPBI promoveu aumento da expressão gênica de MyoD após 3 dias e redução após 7 dias. Em relação a expressão de miogenina, o grupo tratado demonstrou aumento da expressão após 7 dias. Em conclusão, o USPBI nos parâmetros utilizados induziu efeitos positivos ao processo de reparo muscular causando redução do processo inflamatório e mionecrose, aumento de fibras jovens vasos sanguíneos maduros, além de modulação de MyoD e miogenina nos diferentes períodos avaliados.

L’unité cellulaire du muscle squelettique est la myofibre, un syncytium hautement spécialisé générant la contraction musculaire. Au cours de la croissance et de la régénération musculaire, les cellules satellites quiescentes (cellules souches) du muscle squelettique adulte sont activées, prolifèrent puis fusionnent formant de nouvelles fibres. A l’aide d’un modèle murin de régénération et de cultures primaires, j’ai identifié TSHZ3 comme un nouveau marqueur des cellules satellites quiescentes et activées. Dans la lignée cellulaire C2C12, j’ai mis en évidence un effet répresseur spécifique de Tshz3 sur la différenciation myogénique. L’entrée des myoblastes dans la voie de différenciation terminale est déclenchée par le facteur Myogenin (MYOG). L’activation de la transcription du gène myogenin (Myog) est dépendante du facteur MYOD et fait intervenir le complexe de remodelage de la chromatine SWI/SNF. In vitro, TSHZ3 interagit avec BAF57 une sous unité du complexe SWI/SNF. TSHZ3 réprime l’activation dépendante de MYOD sur le promoteur proximal de Myog et cette répression dépend en partie de la présence de BAF57. L’activité répressive et la cinétique d’expression de Tshz3, indique que TSHZ3 pourrait empêcher l’activation prématurée du promoteur Myog lors de la prolifération des cellules satellites activées. TSHZ3 pourrait ainsi participer aux mécanismes de régulation permettant de contrôler l’équilibre entre prolifération, différenciation et renouvellement des progéniteurs myogéniques. / Skeletal muscles are made of several units called myofibers, a syncitium into which muscular contraction is generated. During the muscle growth and repair, the quiescent Satellite Cells (SCs; adult stem cells) become activated, proliferate and differentiate to form new multinucleated myofibers. In animal model and primary culture, I found that, Tshz3 was strongly expressed in the quiescent and activated satellite cells.In C2C12 myoblast cells, I showed a specific repressive effect of TSHZ3 on the myogenic differentiation. The terminal differentiation of the myoblastes is trigger by Myogenin (Myog). The transcriptional activation of Myog promoter involves MYOD and the SWI/SNF remodelling complex. In vitro, I showed that TSHZ3 interacts with BAF57, a subunit of the SWI/SNF complex. TSHZ3 represses the MYOD-dependant activation on the Myog promoter. This specific repression involves in part BAF57.The repressive activity of and the temporal dynamic of expression of Tshz3, indicated that TSHZ3 potentially is required to impede the premature activation of the Myog promotor during the SCs proliferation. These results suggest that TSHZ3 plays important roles in the molecular mechanisms operating in activated SCs when there are poised between proliferation, differentiation and self renewal of muscular progenitors.

The Hedgehog (Hh) signalling pathway is one of the key signalling pathways orchestrating intricate organogenesis, including the development of neural tube, heart and skeletal muscle. Yet, insufficient mechanistic understanding of its diverse roles is available. Here, we show the molecular mechanisms regulating the neurogenic, cardiogenic and myogenic properties of Hh signalling, via effector protein Gli2, in embryonic and adult stem cells. In Chapter 2, we show that Gli2 induces neurogenesis, whereas a dominant-negative form of Gli2 delays neurogenesis in P19 embryonal carcinoma (EC) cells, a mouse embryonic stem (ES) cell model. Furthermore, we demonstrate that Gli2 associates with Ascl1/Mash1 gene elements in differentiating P19 cells and activates the Ascl1/Mash1 promoter in vitro. Thus, Gli2 mediates neurogenesis in P19 cells at least in part by directly regulating Ascl1/Mash1 expression. In Chapter 3, we demonstrate that Gli2 and MEF2C bind each other’s regulatory elements and regulate each other’s expression while enhancing cardiomyogenesis in P19 cells. Furthermore, dominant-negative Gli2 and MEF2C proteins downregulate each other’s expression while imparing cardiomyogenesis. Lastly, we show that Gli2 and MEF2C form a protein complex, which synergistically activates cardiac muscle related promoters. In Chapter 4, we illustrate that Gli2 associates with MyoD gene elements while enhancing skeletal myogenesis in P19 cells and activates the MyoD promoter in vitro. Furthermore, inhibition of Hh signalling in muscle satellite cells and in proliferating myoblasts leads to reduction in MyoD and MEF2C expression. Finally, we demonstrate that endogenous Hh signalling is important for MyoD transcriptional activity and that Gli2, MEF2C and MyoD form a protein complex capable of inducing skeletal muscle-specific gene expression. Thus, Gli2, MEF2C and MyoD participate in a regulatory loop and form a protein complex capable of inducing skeletal muscle-specific gene expression. Our results provide a link between the regulation of tissue-restricted factors like Mash1, MEF2C and MyoD, and a general signal-regulated Gli2 transcription factor. We therefore provide novel mechanistic insights into the neurogenic, cardiogenic and myogenic properties of Gli2 in vitro, and offer novel plausible explanations for its in vivo functions. These results may also be important for the development of stem cell therapy strategies.

Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 53-58).

The Hedgehog (Hh) signalling pathway is one of the key signalling pathways orchestrating intricate organogenesis, including the development of neural tube, heart and skeletal muscle. Yet, insufficient mechanistic understanding of its diverse roles is available. Here, we show the molecular mechanisms regulating the neurogenic, cardiogenic and myogenic properties of Hh signalling, via effector protein Gli2, in embryonic and adult stem cells. In Chapter 2, we show that Gli2 induces neurogenesis, whereas a dominant-negative form of Gli2 delays neurogenesis in P19 embryonal carcinoma (EC) cells, a mouse embryonic stem (ES) cell model. Furthermore, we demonstrate that Gli2 associates with Ascl1/Mash1 gene elements in differentiating P19 cells and activates the Ascl1/Mash1 promoter in vitro. Thus, Gli2 mediates neurogenesis in P19 cells at least in part by directly regulating Ascl1/Mash1 expression. In Chapter 3, we demonstrate that Gli2 and MEF2C bind each other’s regulatory elements and regulate each other’s expression while enhancing cardiomyogenesis in P19 cells. Furthermore, dominant-negative Gli2 and MEF2C proteins downregulate each other’s expression while imparing cardiomyogenesis. Lastly, we show that Gli2 and MEF2C form a protein complex, which synergistically activates cardiac muscle related promoters. In Chapter 4, we illustrate that Gli2 associates with MyoD gene elements while enhancing skeletal myogenesis in P19 cells and activates the MyoD promoter in vitro. Furthermore, inhibition of Hh signalling in muscle satellite cells and in proliferating myoblasts leads to reduction in MyoD and MEF2C expression. Finally, we demonstrate that endogenous Hh signalling is important for MyoD transcriptional activity and that Gli2, MEF2C and MyoD form a protein complex capable of inducing skeletal muscle-specific gene expression. Thus, Gli2, MEF2C and MyoD participate in a regulatory loop and form a protein complex capable of inducing skeletal muscle-specific gene expression. Our results provide a link between the regulation of tissue-restricted factors like Mash1, MEF2C and MyoD, and a general signal-regulated Gli2 transcription factor. We therefore provide novel mechanistic insights into the neurogenic, cardiogenic and myogenic properties of Gli2 in vitro, and offer novel plausible explanations for its in vivo functions. These results may also be important for the development of stem cell therapy strategies.

El gen UCP3 s'expressa majoritàriament al múscul esquelètic i al TAM en rosegadors, i pràcticament de manera exclusiva al múscul esquelètic en humans. El gen s'activa en resposta a diferents estímuls, entre els quals trobem l'àcid retinoic, els àcids grassos no esterificats i les hormones tiroïdals. L'estudi de la regulació de la transcripció del gen UCP3 en cèl·lules musculars ens ha permès obtenir informació sobre els mecanismes moleculars responsables de l'expressió d'UCP3 al múscul esquelètic i de la modulació d'aquesta expressió deguda als estímuls esmentats. L'estudi de la regulació de l'expressió d'UCP3in vivo ens ha permès establir la importància d'alguns d'aquests mecanismes en un context fisiològic. D'acord amb l'expressió específica d'UCP3 al múscul, el factor de transcripció miogènic MyoD és necessari per l'activitat basal del promotor del gen UCP3. MyoD regula l'expressió del gen humà UCP3 a través d'unes seqüències semblants a Ebox properes al lloc d'inici de la transcripció (-29/-9). A més a més, l'activació del promotor del gen humà UCP3 per MyoD és necessària per tal que l'àcid retinoic, els àcids grassos o les hormones tiroïdals en modulin l'activitat. L'àcid retinoic, un conegut activador transcripcional de l'expressió dels gens UCP1 i UCP2, activa l'expressió del gen UCP3 en cèl·lules musculars diferenciades. La resposta del gen UCP3 humà a l'àcid retinoic està mitjançada pels receptors d'àcid retinoic (RAR-RXR) i l'element de resposta a hormones DR1 (AGGTTTCAGGTCA) situat a la regió proximal (-71/-59) del promotor d'UCP3. Per altra banda, l'activació del gen UCP3 pels àcids grassos es dóna a través de PPARalfa o PPARdelta (receptors activats per proliferadors peroxisomals) i de l'element DR1, in vitro i in vivo. En ratolins PPAR-alfa-KO s'ha observat una necessitat diferencial de PPAR-alfa per regular l'expressió del gen UCP3, en funció del teixit (cor o múscul esquelètic) i de l'estadi del desenvolupament (nounats i adults). A nivell molecular, els processos d'acetilació són importants per l'activació del promotor del gen UCP3. El coactivador p300 és capaç de coactivar la resposta dependent de lligand de PPAR-alfa en el promotor, i l'activitat acetiltransferasa de p300 és necessària per aquesta coactivació. Tant l'estat d'acetilació de les histones com de MyoD són importants per l'activació del promotor del gen UCP3. Finalment, s'ha observat que les hormones tiroïdals activen l'expressió del gen UCP3 humà i de ratolí al múscul esquelètic in vivo i en cèl·lules musculars en cultiu. Les hormones tiroïdals activen el promotor d'UCP3 a través dels receptors d'hormones tiroïdals (TR) i la regió del DNA que conté l'element DR1. Per tant, l'element DR1 present en la regió proximal del promotor del gen UCP3 és un element multihormonal que mitjança l'activació del gen UCP3 per l'àcid retinoic, les hormones tiroïdals i els àcids grassos. En el futur, seria interessant estudiar la relació que s'estableix entre aquestes vies de senyalització in vivo. / UCP3 gene is mainly expressed in skeletal muscle and brown adipose tissue in rodents, and almost exclusively in skeletal muscle in humans. The gene is activated in response to different stimulus, such as retinoic acid, fatty acids and thyroid hormones. In the present study we investigate the molecular mechanisms responsible for UCP3 gene expression in skeletal muscle and for the retinoic acid, fatty acids and thyroid hormones-dependent activation. Studying UCP3 gene regulation in vivo has allowed to establish the importance of some of these mechanisms in a physiological context. In agreement with the specific expression of human UCP3 in muscle, the myogenic transcription factor MyoD is needed for UCP3 promoter basal activity. MyoD regulates the expression of the human UCP3 gene through Ebox-like sequences near the initiation transcription site (-29/-9). Moreover, MyoD is necessary for retinoic acid, fatty acid or thyroid hormone-dependent activation of the UCP3 promoter. Retinoic acid, a transcriptional activator of UCP1 and UCP2 gene expression, activates UCP3 gene expression in differentiated skeletal muscle cells. Human UCP3 gene response to retinoic acid is mediated by retinoic acid receptors (RAR-RXR) through a hormone response element DR1 (AGGTTTcAGGTCA) located in the proximal region of the promoter (-71/-59). In addition, UCP3 gene activation by fatty acids is achieved by PPAR-alpha or PPAR-delta (peroxisome proliferator activated receptor) through the previously described DR1, in vitro and in vivo. Studies in PPAR-alpha-KO mice has revealed that PPAR-alpha is differentially required for UCP3 gene expression, depending on tissues (heart or skeletal muscle) and development stages (newborns and adults).Finally, thyroid hormones activate human and mouse UCP3 gene expression in vivo and in vitro. This activation is mediated by thyroid hormone receptor (TR) through the DNA region that contains the DR1 element, in both human and mouse UCP3 promoter. In conclusion, the DR1 element located in the proximal region of UCP3 gene promoter is a multihormonal response element able to mediate retinoic acid, thyroid hormone and fatty acid-dependent activation of UCP3 gene. In the future, it should be interesting to study the relationship between these signalling pathways in vivo.