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G Carnac

Publications and source records attributed to G Carnac.

At least 19 recordsLinked to original sources
PubMed2000

Protein kinase B beta/Akt2 plays a specific role in muscle differentiation.

Insulin-like growth factors positively regulate muscle differentiation through activation of the phosphatidylinositol 3-kinase/protein kinase B (PKB/Akt) signaling pathway. Here, we compare the role of the two closely related alpha (Akt1) and beta (Akt2) isoforms of PKB in muscle differentiation. During differentiation of C2.7 or L6D2 myoblasts, PKBbeta was up-regulated whereas expression of PKBalpha was unaltered. Although the two isoforms were found active in both myoblasts and myotubes, cell fractionation experiments indicated that they displayed distinct subcellular localizations in differentiated cells with only PKBbeta localized in the nuclei. In a transactivation assay, PKBbeta (either wild-type or constitutively active) was more efficient than PKBalpha in activating muscle-specific gene expression. Moreover, microinjection of specific antibodies to PKBbeta inhibited differentiation of muscle cells, whereas control or anti-PKBalpha antibodies did not. On the other hand, microinjection of the anti-PKBalpha antibodies caused a block in cell cycle progression in both non muscle and muscle cells, whereas anti-PKBbeta antibodies had no effect. Taken together, these results show that PKBbeta plays a crucial role in the commitment of myoblasts to differentiation that cannot be substituted by PKBalpha.

Cell Differentiation↗
PubMed2000

The retinoblastoma-like protein p130 is involved in the determination of reserve cells in differentiating myoblasts.

During skeletal muscle differentiation, a subset of myoblasts remains quiescent and undifferentiated but retains the capacity to self-renew and give rise to differentiating myoblasts [1] [2] [3]: this sub-population of muscle cells was recently termed 'reserve cells' [3]. In order to characterise genes that can regulate the ratio between reserve cells and differentiating myoblasts, we examined members of the retinoblastoma tumor suppressor family - Rb, p107 and p130 - an important family of negative regulators of E2F transcription factors and cell cycle progression [4]. Although pRb and p107 positively regulate muscle cell differentiation [5] [6] [7], the role of p130 in muscle cells remains unknown. We show here that p130 (protein and mRNA), but neither pRb nor p107, preferentially accumulates during muscle differentiation in reserve cells. Also, p130 is the major Rb-family protein present in E2F complexes in this sub-population of cells. Although forced expression of either p130 or pRb in mouse C2 myoblasts efficiently blocked cell cycle progression, only p130 inhibited the differentiation program. Furthermore, muscle cells overexpressing p130 had reduced levels of the muscle-promoting factor MyoD. In addition, p130 repressed the transactivation capacity of MyoD, an effect abolished by co-transfection of pRb. Thus, we propose that p130, by blocking cell cycle progression and differentiation, could be part of a specific pathway that defines a pool of reserve cells during terminal differentiation.

Animals↗
PubMed2000

A CDE/CHR-like element mediates repression of transcription of the mouse RB2 (p130) gene.

The bipartite repressor elements, termed cell cycle-dependent element (CDE)/cell cycle regulatory element (CCRE)-cell cycle homology region (CHR) control the growth-dependent transcription of the cyclin A, cdc25C, cdc2 genes. Here, we have identified a functional element displaying the signature of the CDE-CHR in the promoter of the mouse RB2 (p130) gene, encoding the retinoblastoma protein family (pRB)-related protein p130. This element locates close to the major transcription start site where it makes major groove contacts with proteins that can be detected in a cellular context using in vivo genomic footprinting techniques. Inactivation of either the CDE or CHR sequence strongly up-regulates the p130 promoter activity in exponentially growing cells, a situation where endogenous p130 gene expression is almost undetectable. Electrophoretic mobility shift assays suggest that two different protein complexes bind independently to the p130 CDE and CHR elements, and that the protein(s) bound to the CDE might be related to those bound on cyclin A and cdc2 promoters.

Animals↗
PubMed2000

The 2'-5' oligoadenylate/RNase L/RNase L inhibitor pathway regulates both MyoD mRNA stability and muscle cell differentiation.

The 2'-5' oligoadenylate (2-5A)/RNase L pathway is one of the enzymatic pathways induced by interferon. RNase L is a latent endoribonuclease which is activated by 2-5A and inhibited by a specific protein known as RLI (RNase L inhibitor). This system has an important role in regulating viral infection. Additionally, variations in RNase L activity have been observed during cell growth and differentiation but the significance of the 2-5A/RNase L/RLI pathway in these latter processes is not known. To determine the roles of RNase L and RLI in muscle differentiation, C2 mouse myoblasts were transfected with sense and antisense RLI cDNA constructs. Importantly, the overexpression of RLI in C2 cells was associated with diminished RNase L activity, an increased level of MyoD mRNA, and accelerated kinetics of muscle differentiation. Inversely, transfection of the RLI antisense construct was associated with increased RNase L activity, a diminished level of MyoD mRNA, and delayed differentiation. In agreement with these data, MyoD mRNA levels were also decreased in C2 cells transfected with an inducible RNase L construct. The effect of RNase L activity on MyoD mRNA levels was relatively specific because expression of several other mRNAs was not altered in C2 transfectants. Therefore, RNase L is directly involved in myoblast differentiation, probably through its role in regulating MyoD stability. This is the first identification of a potential mRNA target for RNase L.

ATP-Binding Cassette Transporters↗
PubMed1999

cdk1- and cdk2-mediated phosphorylation of MyoD Ser200 in growing C2 myoblasts: role in modulating MyoD half-life and myogenic activity.

We have examined the role of protein phosphorylation in the modulation of the key muscle-specific transcription factor MyoD. We show that MyoD is highly phosphorylated in growing myoblasts and undergoes substantial dephosphorylation during differentiation. MyoD can be efficiently phosphorylated in vitro by either purified cdk1-cyclin B or cdk1 and cdk2 immunoprecipitated from proliferative myoblasts. Comparative two-dimensional tryptic phosphopeptide mapping combined with site-directed mutagenesis revealed that cdk1 and cdk2 phosphorylate MyoD on serine 200 in proliferative myoblasts. In addition, when the seven proline-directed sites in MyoD were individually mutated, only substitution of serine 200 to a nonphosphorylatable alanine (MyoD-Ala200) abolished the slower-migrating hyperphosphorylated form of MyoD, seen either in vitro after phosphorylation by cdk1-cyclin B or in vivo following overexpression in 10T1/2 cells. The MyoD-Ala200 mutant displayed activity threefold higher than that of wild-type MyoD in transactivation of an E-box-dependent reporter gene and promoted markedly enhanced myogenic conversion and fusion of 10T1/2 fibroblasts into muscle cells. In addition, the half-life of MyoD-Ala200 protein was longer than that of wild-type MyoD, substantiating a role of Ser200 phosphorylation in regulating MyoD turnover in proliferative myoblasts. Taken together, our data show that direct phosphorylation of MyoD Ser200 by cdk1 and cdk2 plays an integral role in compromising MyoD activity during myoblast proliferation.

Alanine↗
PubMed1998

The muscle regulatory factors MyoD and myf-5 undergo distinct cell cycle-specific expression in muscle cells.

The muscle regulators MyoD and Myf-5 control cell cycle withdrawal and induction of differentiation in skeletal muscle cells. By immunofluorescence analysis, we show that MyoD and Myf-5 expression patterns become mutually exclusive when C2 cells are induced to differentiate with Myf-5 staining present in cells which fail to differentiate. Isolation of these undifferentiated cells reveals that upon serum stimulation they reenter the cell cycle, express MyoD and downregulate Myf-5. Similar regulations of MyoD and Myf-5 were observed using cultured primary myoblasts derived from satellite cells. To further analyze these regulations of MyoD and Myf-5 expression, we synchronized proliferating myoblasts. Analysis of MyoD and Myf-5 expression during cell cycle progression revealed distinct and contrasting profiles of expression. MyoD is absent in G0, peaks in mid-G1, falls to its minimum level at G1/S and reaugments from S to M. In contrast, Myf-5 protein is high in G0, decreases during G1 and reappears at the end of G1 to remain stable until mitosis. These data demonstrate that the two myogenic factors MyoD and Myf-5 undergo specific and distinct cell cycle-dependent regulation, thus establishing a correlation between the cell cycle-specific ratios of MyoD and Myf-5 and the capacity of cells to differentiate: (a) in G1, when cells express high levels of MyoD and enter differentiation; (b) in G0, when cells express high levels of Myf-5 and fail to differentiate.

Animals↗
PubMed1998

Retinoic acid receptors and muscle b-HLH proteins: partners in retinoid-induced myogenesis.

The results reported here indicate that retinoic acid (RA) induces growth arrest and differentiation only in MyoD-expressing muscle cells. Transient transfection assays reveal a functional interaction between MyoD, a key myogenic regulator and RA-receptors, principal mediators of RA actions. Interestingly, we demonstrate that RXR-MyoD-containing complexes are recruited at specific MyoD DNA-binding sites in muscle cells. Furthermore, we also demonstrate that RA-receptors and the muscle basic helix-loop-helix (b-HLH) proteins interact physically. Mutational analysis suggests that this interaction occurs via the basic region of muscle b-HLH proteins and the DNA-binding domain of RA-receptors and is important for functional interactions between these two families of transcription factors. In conclusion, these results highlight novel interactions between two distinct groups of regulatory proteins that influence cell growth and differentiation.

Binding Sites↗
PubMed1998

Functional specificity of the two retinoic acid receptor RAR and RXR families in myogenesis.

In C2 myoblasts, retinoic acid (RA) is an efficient inducer of both growth arrest and differentiation. These RA effects are mediated through at least two classes of retinoic acid receptors (RARs and RXRs), which belong to the nuclear receptor superfamily. To determine the role played by each RAR or RXR family in this model system, we have analysed the effects of RA in C2 myoblasts expressing a dominant negative RAR (dnRAR) or a dominant negative RXR (dnRXR). The stable expression of dnRAR or dnRXR in C2 cells delays the RA-induced growth arrest and differentiation, an effect which is more pronounced in C2-dnRXR myoblasts. Furthermore, the RA-inducible expression of MyoD gene is lost in C2-dnRXR but not in C2-dnRAR cells, indicating that each family of retinoid receptors RAR and RXR may regulate distinct subsets of RA-responsive genes. Finally, using C2 cell lines with different retinoid responsiveness, we provided evidence for a link between the RXR and MyoD families in the process of myogenic differentiation. These results illustrate a critical role for RA-receptors in RA-control of C2 myogenesis and provide tools for studying the function of RA and its receptors during vertebrate development.

Cell Differentiation↗
PubMed1998

RhoA GTPase and serum response factor control selectively the expression of MyoD without affecting Myf5 in mouse myoblasts.

MyoD and Myf5 belong to the family of basic helix-loop-helix transcription factors that are key operators in skeletal muscle differentiation. MyoD and Myf5 genes are selectively activated during development in a time and region-specific manner and in response to different stimuli. However, molecules that specifically regulate the expression of these two genes and the pathways involved remain to be determined. We have recently shown that the serum response factor (SRF), a transcription factor involved in activation of both mitogenic response and muscle differentiation, is required for MyoD gene expression. We have investigated here whether SRF is also involved in the control of Myf5 gene expression, and the potential role of upstream regulators of SRF activity, the Rho family G-proteins including Rho, Rac, and CDC42, in the regulation of MyoD and Myf5. We show that inactivation of SRF does not alter Myf5 gene expression, whereas it causes a rapid extinction of MyoD gene expression. Furthermore, we show that RhoA, but not Rac or CDC42, is also required for the expression of MyoD. Indeed, blocking the activity of G-proteins using the general inhibitor lovastatin, or more specific antagonists of Rho proteins such as C3-transferase or dominant negative RhoA protein, resulted in a dramatic decrease of MyoD protein levels and promoter activity without any effects on Myf5 expression. We further show that RhoA-dependent transcriptional activation required functional SRF in C2 muscle cells. These data illustrate that MyoD and Myf5 are regulated by different upstream activation pathways in which MyoD expression is specifically modulated by a RhoA/SRF signaling cascade. In addition, our results establish the first link between RhoA protein activity and the expression of a key muscle regulator.

3T3 Cells↗
PubMed1997

The community effect in Xenopus myogenesis is promoted by dorsalizing factors.

The community effect describes a process required for Xenopus muscle progenitor cells to progress to the expression of myogenic genes. Past work has suggested that this effect is dependent on secreted factors released by dorsolateral or dorsal lip cells. We show here that known dorsalizing molecules as well as other natural dorsal lip factors contribute to, but do not wholly account for, the community effect. We conclude that, in addition to dorsalizing molecules, the community effect requires factors or conditions peculiar to the dorsolateral mesoderm, a region of the embryo that contains muscle progenitor cells.

Animals↗
PubMed1996

RXR alpha is essential for mediating the all-trans retinoic acid-induced growth arrest of C2 myogenic cells.

In C2 muscle cells, retinoic acid (RA) induces growth arrest associated with terminal differentiation. These RA actions are presumed to be mediated through nuclear receptors (RARs and RXRs) that belong to the superfamily of ligand-dependent transcription factors. In this study, we have characterized a myogenic C2 subclone, that unlike parental cells, is resistant to growth inhibition and differentiation by RA. Examination of these RA-sensitive and resistant C2 cells for the expression of retinoid acid receptors revealed a lack of RXR alpha expression at the myoblast stage in resistant C2 cells. To determine the functions of RXR alpha, we introduced an RXR alpha expression vector into RA-resistant C2 cells by transient or stable transfections. Our results show that RXR alpha restores the response to RA in this subclone with respect to AP1 inhibition and growth arrest. These observations indicate that RXR alpha plays a crucial role in mediating RA induced growth arrest of C2 myogenic cells.

Cell Differentiation↗
PubMed1996

The homeobox gene Siamois is a target of the Wnt dorsalisation pathway and triggers organiser activity in the absence of mesoderm.

Siamois, a Xenopus zygotic homeobox gene with strong dorsalising activity, is expressed in the dorsal-vegetal organiser known as the Nieuwkoop centre. We show that, in contrast to Spemann organiser genes such as goosecoid, chordin and noggin, Siamois gene expression is not induced following overexpression of mesoderm inducers in ectodermal (animal cap) cells. However, Siamois is induced by overexpressing a dorsalising Wnt molecule. Furthermore, like Wnt, Siamois can dorsalise ventral mesoderm and cooperate with Xbrachyury to generate dorsal mesoderm. These results suggest that Siamois is a mediator of the Wnt-signalling pathway and that the synergy between the Wnt and mesoderm induction pathways occurs downstream of the early target genes of these two pathways. Overexpression of Siamois in animal cap cells reveals that this gene can act in a non vegetal or mesodermal context. We show the following. (1) Animal cap cells overexpressing Siamois secrete a factor able to dorsalise ventral gastrula mesoderm in tissue combination experiments. (2) The Spemann organiser-specific genes goosecoid, Xnr-3 and chordin, but not Xlim.1, are activated in these caps while the ventralising gene Bmp-4 is repressed. However, the dorsalising activity of Siamois-expressing animal caps is significantly different from that of noggin- or chordin-expressing animal caps, suggesting the existence of other dorsalising signals in the embryo. (3) Ectodermal cells overexpressing Siamois secrete a neuralising signal and can differentiate into cement gland and, to a lesser extent, into neural tissue. Hence, in the absence of mesoderm induction, overexpression of Siamois is sufficient to confer organiser properties on embryonic cells.

Activins↗
PubMed1996

The Xenopus T-box gene, Antipodean, encodes a vegetally localised maternal mRNA and can trigger mesoderm formation.

We have used differential display to identify genes inducible by activin and isolated a novel member of the T-box gene family that includes the Xenopus genes Xbrachyury and Eomesodermin. Here we show that this novel gene is unique within the T-box family because it is maternally expressed at a high level. Furthermore, it belongs to a rare class of maternal mRNAs in Xenopus that are localised to the vegetal hemisphere of the egg and we have therefore named it Antipodean. We show here that low amounts of Antipodean injected into ectoderm (animal cap cells) strongly induce pan mesodermal genes such as Xbrachyury and ventral mesodermal genes such as Xwnt-8. Overexpression of Antipodean generates mesoderm of ventral character, and induces muscle only weakly. This property is consistent with the observed late zygotic Antipodean mRNA expression in the posterior paraxial mesoderm and ventral blastopore, and its exclusion from the most dorsal mesodermal structure, the notochord. Antipodean is induced by several molecules of the TGF-beta class, but in contrast to Xbrachyury, not by bFGF. This result suggests that the expression of these T-box genes may be under the control of different regulatory pathways. Finally, we demonstrate that Antipodean and Eomesodermin induce each other and both are able to induce Xbrachyury. The early zygotic expression of Antipodean is not induced by Xbrachyury, though later it is to some extent. Considering its maternal content, Antipodean could initiate a cascade of T-box gene activations. The expression of these genes may, in turn, sustain each other's expression to define and maintain the mesoderm identity in Xenopus.

Amino Acid Sequence↗
PubMed1994

Nuclear import of the myogenic factor MyoD requires cAMP-dependent protein kinase activity but not the direct phosphorylation of MyoD.

MyoD is a nuclear phosphoprotein that belongs to the family of myogenic regulatory factors and acts in the transcriptional activation of muscle-specific genes. We have investigated the role of cAMP-dependent protein kinase (A-kinase) in modulating the nuclear locale of MyoD. Purified MyoD protein microinjected into the cytoplasm of rat embryo fibroblasts is rapidly translocated into the nucleus. Inhibition of A-kinase activity through injection of the specific inhibitory peptide PKI prevents this nuclear localisation. This inhibition of nuclear location is specifically reversed by injection of purified A-kinase catalytic subunit, showing the requirement for A-kinase in the nuclear import of MyoD. Site-directed mutagenesis of all the putative sites for A-kinase-dependent phosphorylation on MyoD, substituting serine or threonine residues for the non-phosphorylatable amino acid alanine, had no effect on nuclear import of mutated MyoD. These data exclude the possibility that the effect of A-kinase on the nuclear translocation of MyoD is mediated by direct phosphorylation of MyoD and imply that A-kinase operates through phosphorylation of components involved in the nuclear transport of MyoD.

Amino Acid Sequence↗
PubMed1993

Serum-induced inhibition of myogenesis is differentially relieved by retinoic acid and triiodothyronine in C2 murine muscle cells.

We recently reported that triiodothyronine (T3) enhances MyoD gene expression and accelerates terminal differentiation in murine C2 myoblasts. In this paper, we are interested in the effects of other hormones acting through related nuclear receptors. Retinoic acid (RA), but not estradiol or dexamethasone, is also able to enhance MyoD gene expression (about threefold). However, the effects of RA and T3 on myogenesis are quite distinct, with a much more potent RA action. Indeed, although T3 and RA positively regulate myogenesis with similar efficiency in poorly mitogenic conditions, in presence of high serum concentrations T3 can no longer trigger terminal differentiation whereas RA still remains efficient. Thus, serum concentration is a crucial parameter in discriminating between the effects of T3 and RA on myogenesis. The differential effects between these two hormone are likely to be related to the ability of RA-activated endogenous retinoic acid receptors (RARs) to induce C2 myoblasts growth-arrest and to extinguish AP1 activity (thought to act as an inhibitor of myogenesis) whereas T3-activated endogenous thyroid hormones receptors (THRs) are relatively inefficient. We propose that the much higher level of RARs in C2 cells versus THRs could to some extent account for the differential ability of T3 and RA to antagonize serum-regulated mitogenic pathways in myogenic cells. This study provides clear evidence for an important role of RA on MyoD gene expression and myogenesis and suggests that T3 and RA could play overlapping, but distinct, roles on muscle development.

Animals↗
PubMed1993

9-cis-retinoic acid regulates the expression of the muscle determination gene Myf5.

Myf5 is a member of the MyoD family, a set of four helix-loop-helix transcription factors that controls myogenic differentiation. The Myf5 gene has both in vivo and in vitro expression patterns consistent with an involvement in the first events of myogenesis, such as acquisition and/or maintenance of myogenic "determined" phenotype. To date, very little is known about the mechanism underlying the tight regulation of Myf5 expression. We report here that retinoic acid (RA) reduces the level of Myf5 message in both mouse C2 and rat L6 cell lines, probably at the transcriptional level, because Myf5 mRNA stability is not affected by RA. This repression is dose dependent, starting at 0.1 microM of all-trans RA, and is not abrogated by cycloheximide, suggesting a direct involvement of RA receptors in the control of Myf5 expression. Furthermore, we compared the efficiency of natural (all-trans RA and 9-cis RA) or synthetic (TTNPB) retinoids that differentially activate the two families of RA receptors, RA receptors and retinoid X-receptors (9-cis RA). As 9-cis RA is about 10 times more efficient than all-trans RA in repressing Myf5, whereas TTNPB, which preferentially activates RA receptors, is far less potent, our data provide evidence for an important role of ligand-bound retinoid X-receptors in the mediation of this inhibition.

Animals↗
PubMed1993

Overexpression of c-erbA proto-oncogene enhances myogenic differentiation.

Triiodothyronine (T3) positively regulates both the expression of the MyoD gene, a key myogenic regulator, and C2 muscle cell differentiation. To directly examine the role of its nuclear receptors in the control of myogenesis, we introduced a c-erbA expression vector into C2 muscle cells by transient or stable transfection. Our results show that c-erbA can play a potent role in the triggering of muscle terminal differentiation since its overexpression leads to: (1) a complete abrogation of the activity of the myogenesis inhibitor AP-1 (fos/jun) transcription factor; (2) an enhanced induction of MyoD expression upon T3 treatment; (3) the acquisition by T3 of the ability to trigger both growth arrest and terminal differentiation in the presence of large amounts of serum mitogens, a property that is otherwise specific to retinoic acid (RA). Thus, c-erbA is one of the two protooncogenes (with c-ski) that acts as positive regulator of muscle differentiation. Furthermore, the fact that c-erbA overexpression allows T3 to largely mimic the RA effects indicates that their biological differences in the modulation of myogenic program primarily rely on the differential expression of their receptors in C2 muscle cells rather than on an intrinsic specificity of their target genes.

Animals↗