Cécile Rallière

Ubiquitin-proteasome-dependent proteolysis in skeletal muscle

The ubiquitin-proteasome proteolytic pathway has recently been reported to be of major importance in the breakdown of skeletal muscle proteins. The first step in this pathway is the covalent attachment of polyubiquitin chains to the targeted protein. Polyubiquitylated proteins are then recognized and degraded by the 26S proteasome complex. In this review, we critically analyse recent findings in the regulation of this pathway, both in animal models of muscle wasting and in some human diseases. The identification of regulatory steps of ubiquitin conjugation to protein substrates and/or of the proteolytic activities of the proteasome should lead to new concepts that can be used to manipulate muscle protein mass. Such concepts are essential for the development of anti-cachectic therapies for many clinical situations.

Dynamic gene expression in fish muscle during recovery growth induced by a fasting-refeeding schedule.

BackgroundRecovery growth is a phase of rapid growth that is triggered by adequate refeeding of animals following a period of weight loss caused by starvation. In this study, to obtain more information on the system-wide integration of recovery growth in muscle, we undertook a time-course analysis of transcript expression in trout subjected to a food deprivation-refeeding sequence. For this purpose complex targets produced from muscle of trout fasted for one month and from muscle of trout fasted for one month and then refed for 4, 7, 11 and 36 days were hybridized to cDNA microarrays containing 9023 clones.ResultsSignificance analysis of microarrays (SAM) and temporal expression profiling led to the segregation of differentially expressed genes into four major clusters. One cluster comprising 1020 genes with high expression in muscle from fasted animals included a large set of genes involved in protein catabolism. A second cluster that included approximately 550 genes with transient induction 4 to 11 days post-refeeding was dominated by genes involved in transcription, ribosomal biogenesis, translation, chaperone activity, mitochondrial production of ATP and cell division. A third cluster that contained 480 genes that were up-regulated 7 to 36 days post-refeeding was enriched with genes involved in reticulum and Golgi dynamics and with genes indicative of myofiber and muscle remodelling such as genes encoding sarcomeric proteins and matrix compounds. Finally, a fourth cluster of 200 genes overexpressed only in 36-day refed trout muscle contained genes with function in carbohydrate metabolism and lipid biosynthesis. Remarkably, among the genes induced were several transcriptional regulators which might be important for the gene-specific transcriptional adaptations that underlie muscle recovery.ConclusionOur study is the first demonstration of a coordinated expression of functionally related genes during muscle recovery growth. Furthermore, the generation of a useful database of novel genes associated with muscle recovery growth will allow further investigations on particular genes, pathways or cellular process involved in muscle growth and regeneration.

Manipulation of the ubiquitin-proteasome pathway in cachexia: pentoxifylline suppresses the activation of 20S and 26S proteasomes in muscles from tumor-bearing rats

The development of pharmacological approaches for preventing the loss of muscle proteins would be extremely valuable for cachectic patients. For example, severe wasting in cancer patients correlates with a reduced efficacy of chemotherapy and radiotherapy. Pentoxifylline (PTX) is a very inexpensive xanthine derivative, which is widely used in humans as a haemorheological agent, and inhibits tumor necrosis factor transcription. We have shown here that a daily administration of PTX prevents muscle atrophy and suppresses increased protein breakdown in Yoshida sarcoma-bearing rats by inhibiting the activation of a nonlysosomal, Ca2+-independent proteolytic pathway. PTX blocked the ubiquitin pathway, apparently by suppressing the enhanced expression of ubiquitin, the 14-kDa ubiquitin conjugating enzyme E2, and the C2 20S proteasome subunit in muscle from cancer rats. The 19S complex and 11S regulator associate with the 20S proteasome and regulate its peptidase activities. The mRNA levels for the ATPase subunit MSS1 of the 19S complex increased in cancer cachexia, in contrast with mRNAs of other regulatory subunits. This adaptation was suppressed by PTX, suggesting that the drug inhibited the activation of the 26S proteasome. This is the first demonstration of a pharmacological manipulation of the ubiquitin-proteasome pathway in cachexia with a drug which is well tolerated in humans. Overall, the data suggest that PTX can prevent muscle wasting in situations where tumor necrosis factor production rises, including cancer, sepsis, AIDS and trauma.

Glucocorticoids regulate mRNA levels for subunits of the 19 S regulatory complex of the 26 S proteasome in fast-twitch skeletal muscles.

Circulating levels of glucocorticoids are increased in many traumatic and muscle-wasting conditions that include insulin-dependent diabetes, acidosis, infection, and starvation. On the basis of indirect findings, it appeared that these catabolic hormones are required to stimulate Ub (ubiquitin)-proteasome-dependent proteolysis in skeletal muscles in such conditions. The present studies were performed to provide conclusive evidence for an activation of Ub-proteasome-dependent proteolysis after glucocorticoid treatment. In atrophying fast-twitch muscles from rats treated with dexamethasone for 6 days, compared with pair-fed controls, we found (i) increased MG132-inhibitable proteasome-dependent proteolysis, (ii) an enhanced rate of substrate ubiquitination, (iii) increased chymotrypsin-like proteasomal activity of the proteasome, and (iv) a co-ordinate increase in the mRNA expression of several ATPase (S4, S6, S7 and S8) and non-ATPase (S1, S5a and S14) subunits of the 19 S regulatory complex, which regulates the peptidase and the proteolytic activities of the 26 S proteasome. These studies provide conclusive evidence that glucocorticoids activate Ub-proteasome-dependent proteolysis and the first in vivo evidence for a hormonal regulation of the expression of subunits of the 19 S complex. The results suggest that adaptations in gene expression of regulatory subunits of the 19 S complex by glucocorticoids are crucial in the regulation of the 26 S muscle proteasome.

Muscle fiber differentiation in fish embryos as shown by in situ hybridization of a large repertoire of muscle-specific transcripts.

Skeletal muscles are composed of different fiber types, largely defined by differential expression of protein isoforms involved in myofibrillogenesis or metabolism. To learn more about the gene activations that underlie the differentiation and the diversification of embryonic fish myotomal fibers, we investigated the developmental expression of 25 muscle genes in trout embryos by in situ hybridization of muscle‐specific transcripts. The earliest event of muscle differentiation, at approximately the 25‐somite stage, was the expression of a variety of muscle‐specific genes, including slow‐twitch and fast‐twitch muscle isoforms. The activation of these muscle genes started in the deep somitic domain, where the slow muscle precursors (the adaxial cells) were initially located, and progressively spread laterally throughout the width of the myotome. This mediolateral progression of gene expression was coordinated with the lateral migration of slow adaxial cells, which specifically expressed the slow myosin light chain 1 and the SLIM1/FHL1 genes. Subsequently, the fast and slow skeletal muscle isoforms precociously expressed in the course of the mediolateral wave of muscle gene activation became down‐regulated in the superficial slow fibers and the deep fast fibers, respectively. Finally, several muscle‐specific genes, including troponins, a slow myosin‐binding protein C, tropomodulins, and parvalbumin started their transcription only in late embryos. Taken together, these findings show in fish embryos that a common myogenic program is triggered in a mediolateral progression in all muscle cells. The acquisition of the slow phenotype involves the additional activation of several slow‐specific genes in migrating adaxial muscle cells. These events are followed by sequential gene activations and repressions in fast and slow muscle cells. Developmental Dynamics 233:659–666, 2005.

Expression of subunits of the 19S complex and of the PA28 activator in rat skeletal muscle.

A precise knowledge of the role of subunits of the 19S complex and the PA28 regulator, which associate with the 20S proteasome and regulate its peptidase activities, may contribute to design new therapeutic approaches for preventing muscle wasting in human diseases. The proteasome is mainly responsible for the muscle wasting of tumor-bearing and unweighted rats. The expression of some ATPase (MSS1, P45) and non ATPase (P112-L, P31) subunits of the 19S complex, and of the two subunits of the PA28 regulator, was studied in such atrophying muscles. The mRNA levels for all studied subunits increased in unweighted rats, and analysis of MSS1 mRNA distribution profile in polyribosomes showed that this subunit entered active translation. By contrast, only the mRNA levels for MSS1 increased in the muscles from cancer rats. Thus, gene expression of the proteasome regulatory subunits depends on a given catabolic state. Torbafylline, a xanthine derivative which inhibits tumor necrosis factor production, prevented the activation of protein breakdown and the increased expression of 20S proteasome subunits in cancer rats, without reducing the elevated MSS1 mRNA levels. Thus, the increased expression of MSS1 is regulated independently of 20S proteasome subunits, and did not result in accelerated proteolysis.

Developmental program expression of myosin alkali light chain and skeletal actin genes in the rainbow trout Oncorhynchus mykiss

We have isolated MLC1(F) (tMLC1(F)), MLC3(F) (tMLC3(F)) and skeletal actin cDNAs from the teleost Oncorhynchus mykiss. Sequence analysis indicates that tMLC1(F) and tMLC3(F) are not produced from differentially spliced mRNAs as reported in avians and rodents but are encoded by different genes. Results from RNase protection analysis showed that the corresponding transcripts are expressed in fast skeletal muscles. Whole-mount in situ hybridisation revealed distinct expression patterns of the myosin alkali light chains and skeletal actin genes during skeletal muscle development in the embryo.

Identification of novel genes including Dermo-1, a marker of dermal differentiation, expressed in trout somitic external cells

SUMMARY The external cell layer that surrounds the fish primary myotome provides the myogenic precursors necessary for muscle growth, suggesting that this epithelium is equivalent to the amniote dermomyotome. In this study we report the identification of a trout orthologue of the dermal marker Dermo-1, and show that trout somitic external cells, which are all potentially myogenic as indicated by the transcription of Pax7 gene, express Dermo-1. This finding and our previous observation that external cells express collagen I show that these cells have dermis-related characteristics in addition to exhibiting myogenic features. In an effort to identify novel genes expressed in the external cell epithelium we performed an in situ hybridisation screen and found both collectin sub-family member 12, a transmembrane C-type lectin, and Seraf, an EGF-like repeat autocrine factor. In situ hybridisation of staged trout embryos revealed that the expression of Dermo-1, collectin sub-family member 12 and Seraf within the external cell layer epithelium was preceded by a complex temporal and spatial expression pattern in the early somite.

Expression patterns of collagen I (α1) encoding gene and muscle‐specific genes reveal that the lateral domain of the fish somite forms a connective tissue surrounding the myotome

Somites are repeated, epithelial structures that are derived from the unsegmented paraxial mesoderm located lateral to the notochord. In higher vertebrates, somites differentiate into a sclerotome that subsequently forms the vertebrae and the ribs and into a dermomyotome that gives rise to a myotome, from which arises the skeletal muscle, and to a dermatome, from which arises the dermis. Fish somites have been shown to produce a sclerotome and a myotome, but very little is known regarding their participation in the formation of connective tissues, especially at the junction between the epidermis and the myotome. To investigate the formation of connective tissues in fish somites, we have examined the expression pattern of the collagen I (α1) chain. As somitogenesis proceeds rostrocaudally, collagen I (α1) expression marks the sclerotomal cells and delineates the formation of the vertebrae. Surprisingly, after the completion of the segmentation, transcript for the collagen I (α1) chain appeared in a distinct epithelial‐like monolayer situated at the periphery of the developing somite facing the surface epidermis. This epithelial monolayer of somitic cells that covered the superficial slow muscle cells, did not express the myogenic transcriptional regulator myogenin and was devoid of contractile filament. As the somite increased in size, these collagen‐expressing epithelial cells flattened, forming a thin cellular layer underlying the epidermis and recovering the lateral surface of the myotome. In conclusion, the lateral domain of the fish somite forms a distinct epithelial cell layer sharing many characteristics with amniote dermatome. Developmental Dynamics 233:605–611, 2005.

Gene expression profiling of the hyperplastic growth zones of the late trout embryo myotome using laser capture microdissection and microarray analysis

BackgroundA unique feature of fish is that new muscle fibres continue to be produced throughout much of the life cycle; a process termed muscle hyperplasia. In trout, this process begins in the late embryo stage and occurs in both a discrete, continuous layer at the surface of the primary myotome (stratified hyperplasia) and between existing muscle fibres throughout the myotome (mosaic hyperplasia). In post-larval stages, muscle hyperplasia is only of the mosaic type and persists until 40% of the maximum body length is reached. To characterise the genetic basis of myotube neoformation in trout, we combined laser capture microdissection and microarray analysis to compare the transcriptome of hyperplastic regions of the late embryo myotome with that of adult myotomal muscle, which displays only limited hyperplasia.ResultsGene expression was analysed using Agilent trout oligo microarrays. Our analysis identified more than 6800 transcripts that were significantly up-regulated in the superficial hyperplastic zones of the late embryonic myotome compared to adult myotomal muscle. In addition to Pax3, Pax7 and the fundamental myogenic basic helix-loop-helix regulators, we identified a large set of up-regulated transcriptional factors, including Myc paralogs, members of Hes family and many homeobox-containing transcriptional regulators. Other cell-autonomous regulators overexpressed in hyperplastic zones included a large set of cell surface proteins belonging to the Ig superfamily. Among the secreted molecules found to be overexpressed in hyperplastic areas, we noted growth factors as well as signalling molecules. A novel finding in our study is that many genes that regulate planar cell polarity (PCP) were overexpressed in superficial hyperplastic zones, suggesting that the PCP pathway is involved in the oriented elongation of the neofibres.ConclusionThe results obtained in this study provide a valuable resource for further analysis of novel genes potentially involved in hyperplastic muscle growth in fish. Ultimately, this study could yield insights into particular genes, pathways or cellular processes that may stimulate muscle regeneration in other vertebrates.