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C Pinset

Publications and source records attributed to C Pinset.

At least 37 records · Page 2Linked to original sources
PubMed1996

Long-term secretion of therapeutic proteins from genetically modified skeletal muscles.

Protein delivery from genetically modified skeletal muscle has been reported previously. However, a stable and prolonged secretion was obtained in immunocompromised or newborn animals only. To evaluate the clinical relevance of this approach, we have transduced myoblasts from an adult beta-glucuronidase-deficient (MPS VII) mouse with retroviral vectors carrying either the human beta-glucuronidase cDNA or the murine erythropoietin (Epo) cDNA. The cells were then grafted into the tibialis anterior muscle of adult immunocompetent MPS VII recipients. Protein expression was controlled either by ubiquitous or muscle-specific transcriptional regulatory elements. Animals were analyzed over an 8-month period. The in situ detection of beta-glucuronidase activity revealed up to 60% of genetically modified myofibers in the recipient muscles. The human desmin promoter and enhancer showed the highest in vivo expression. Secretion of beta-glucuronidase induced a disappearance of lysosomal storage lesions in the liver and spleen of recipient animals. Delivery of Epo led to a permanent increase of hematocrit values over 3 months. These results showed that the transplantation of genetically modified myoblasts allowed a sustained secretion of recombinant proteins at therapeutic levels in immunocompetent adult mice. They suggest that the approach may be considered for human applications.

Animals↗
PubMed1996

Autonomous differentiation in the mouse myogenic cell line, C2, involves a mutual positive control between insulin-like growth factor II and MyoD, operating as early as at the myoblast stage.

We have studied the contribution of the endogenous production of insulin-like growth factor II (IGFII) and of the muscle regulatory factor, MyoD, to the autonomy of differentiation in isolated skeletal myoblasts. Inhibition of MyoD and IGFII gene expression in myoblasts of the mouse myogenic cell line, C2, was achieved by transfection and selection of stably transfected cells (anti-MyoD and anti-IGFII cells) with vectors producing MyoD or IGFII antisense RNA. We observed that inhibiting either MyoD or IGFII has multiple and similar consequences. In addition to the inhibition of the target gene, expression of MyoD transcripts in anti-IGFII myoblasts and expression of IGFII in anti-MyoD myoblasts were also abolished, whereas accumulation of transcripts for the muscle regulatory factor, Myf5, was markedly increased in both cell types. However, despite this Myf5 up-regulation, both anti-IGFII and anti-MyoD myoblasts lost the ability to undergo autonomous differentiation (differentiation in the absence of added IGF), further indicating that Myf5 and MyoD are not strictly interchangeable. Additional evidence of a link between MyoD and IGFII was obtained: (1) forced expression of the MyoD cDNA stimulated IGFII gene expression, and (2) treatment of C2 myoblasts with fibroblast growth factor, not only diminished MyoD expression and compromised differentiation as previously shown by others, but also abolished IGFII expression. These experiments showing loss or gain of function argue in favor of a mutual positive control between IGFII and MyoD operating as early as the myoblast stage.

Animals↗
PubMed1994

Myf5, MyoD, myogenin and MRF4 myogenic derivatives of the embryonic mesenchymal cell line C3H10T1/2 exhibit the same adult muscle phenotype.

Cells of the embryonic mesenchymal cell line C3H10T1/2 have revealed the potential that the four regulatory factors belonging to the MyoD family have to activate myogenesis. In the present study we have further investigated the myogenic phenotype of C3H10T1/2 cells stably transfected with either Myf5, MyoD, myogenin or MRF4 cDNAs. We have studied the influence of each transfected cDNA on expression of the four endogenous muscle regulatory genes and on the ability of these embryonic myogenic derivatives to express adult muscle genes. No trace of endogenous transcripts distinct from the exogenous one was found in any of the four converted populations at the myoblast stage. This indicates that cross-activation within the MyoD family does not occur at the myoblast stage in these cells. Similarly, evidence was obtained that auto- or cross-activation of the Myf5 gene occurs neither at the myoblast stage nor at the myotube stage and that no autoactivation of the MRF4 gene occurs. Our results together with previous observations indicate that in C3H10T1/2 myogenic derivatives: (1) Autoactivation at the myoblast stage is restricted to MyoD (2) Expression from each cDNA alone is sufficient to establish and maintain the myoblast phenotype (3) The endogenous Myf5 gene is not mobilized. We have also observed that endogenous transcripts for MyoD and myogenin begin to accumulate at the onset of differentiation in the four myogenic derivatives, whereas accumulation of endogenous MRF4 transcripts starts after myotubes have formed and occurs at a much lower level (100- to 500-fold lower) than in differentiated cultures of myosatellite cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Activation of gene expression via CArG boxes during myogenic differentiation.

We have studied gene activation via CArG boxes in the context of myogenesis. The proximal CArG box of the human cardiac actin gene (HCA1) stimulates transcription from the herpes simplex virus thymidine kinase (TK) promoter in a tissue-specific fashion. Thus in transient transfection assays, when the expression of chloramphenicol acetyltransferase (CAT) from p(HCA1)4 TKCAT is compared to that derived from p(M1)4 TKCAT which contains an inactive mutated version (M1) of the HCA1 element, high levels of expression are seen in C2 mouse myoblasts and myotubes, and in the T4 myoblast cell line derived from the C3H10T1/2 cell line by 5-azacytidine treatment, whereas only low levels of expression are seen in the mouse L fibroblast cell line. The parental C3H10T1/2 cell line shows intermediate levels of expression. A similar situation is seen in stably transfected cell lines. Gene activation via CArG boxes was also analyzed in the course of myogenic conversion of C3H10T1/2 cells treated with 5-azacytidine. Our results indicate that activation of the CAT gene from the HCA1 element is slightly posterior to the appearance of the first MyoD1 and myogenin transcripts, concomitant with the appearance of cardiac alpha-actin transcripts, but clearly precedes the accumulation of myosin light-chain 1a transcripts and the appearance of troponin T-positive cells. These results further establish that CArG boxes can be seen as muscle-specific cis-acting regulatory element prior to terminal differentiation.

Actins↗
PubMed1993

Muscle differentiation: insulin-like growth factors as positive modulators of myogenic regulatory genes?

The contribution of autocrine production of insulin-like growth factor II (IGFII) to myogenic differentiation was studied in the mouse myogenic C2 cells. Permissive C2 cells were stably transfected with a vector generating antisense RNA complementary to IGF RNAs. Our results show that: (1) accumulation of IGFII mRNA is dramatically decreased (this mRNA is undetectable by RNA blotting); (2) accumulation of MyoD mRNA also becomes undetectable by RNA blot analysis; (3) the ability of the cells to differentiate, including the activation of myogenin and myosin genes, is severely compromised; (4) expression of the MyoD genes and the ability to differentiate are restored following the addition of insulin or IGF to the cells. Our observations indicate that the autonomous differentiation of myogenic cells requires autocrine production of IGF and raise the possibility that IGFII positively regulates expression of the MyoD gene, one of the members of the myogenic regulatory factor family.

Animals↗
PubMed1992

Preferential binding of insulin-like growth factor-II (IGF-II) to a putative alpha 2 beta 2 IGF-II receptor type in C2 myoblasts.

We have studied insulin-like-growth-factor (IGF) binding in two subclones of the C2 myogenic cell line. In the permissive parental subclone, myoblasts differentiate spontaneously into myotubes in medium supplemented with fetal calf serum. Unlike permissive myoblasts, inducible myoblasts require high concentrations of insulin (1.6 microM) or lower concentrations of IGF-I (25 nM) to differentiate, and expression of MyoD1 is not constitutive. IGF receptors were studied in microsomal membranes of proliferating and quiescent myoblasts and myotubes. IGF-II binding was also studied in inducible myoblasts transfected with the MyoD1 cDNA (clone EP5). Both inducible and permissive cells exhibited a single class of binding sites with similar affinity for IGF-I (Kd 0.8-1.2 nM). Affinity cross-linking of [125I]IGF-I to microsomal membranes, under reducing conditions, revealed a binding moiety with an apparent molecular mass of 130 kDa in permissive cells and 140 kDa in inducible cells, which corresponded to the alpha subunit of the IGF-I receptor. In permissive quiescent myoblasts, linear Scatchard plots suggested that [125I]IGF-II bound to a single class of binding sites (Kd 0.6 nM) compatible with binding to the IGF-II/M6P receptor. This was confirmed by affinity cross-linking experiments showing a labeled complex with an apparent molecular mass of 260 kDa and 220 kDa when studied under reducing and non-reducing conditions, respectively. In contrast, competitive inhibition of [125I]IGF-II binding to inducible quiescent myoblasts generated curvilinear Scatchard plots which could be resolved into two single classes of binding sites. One of them corresponded to the IGF-II/M6P receptor (Kd 0.2 nM) as evidenced by cross-linking experiments. The second was the binding site of highest affinity (Kd 0.04 nM) which was less inhibited by IGF-I than by IGF-II and was not inhibited by insulin. It migrated in SDS/PAGE at a position equivalent a molecular mass of 140 kDa, under reducing conditions, and at approximately 300 kDa, under non-reducing conditions. The labeling of this atypical binding moiety was not inhibited by anti(IGF-II/M6P-receptor) immunoglobulin. It was also observed in permissive and inducible myoblasts at proliferating stage. It was absent for permissive quiescent myoblasts and from permissive and inducible myotubes. Forced expression of MyoD1 in inducible cells (EP5 cells) dramatically reduced [125I]IGF-II binding to this atypical receptor. It emerges from these experiments that C2 cells express a putative alpha 2 beta 2 IGF-II receptor structurally related to the insulin/IGF-I receptor family. It is present in myoblasts but not in myotubes.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1992

3,5,3'-Triiodothyronine positively regulates both MyoD1 gene transcription and terminal differentiation in C2 myoblasts.

Thyroid hormones are among the positive regulators of muscle development in vivo, but little is known about the way they work. We demonstrate here that MyoD1, one of the master genes controlling myogenesis, is a target of T3. After proliferating C2 myoblasts have been treated with T3 for 15 h, we observed a rise in MyoD1 expression at both the mRNA and protein levels. This is the first positive hormonal control of MyoD1 gene expression reported so far. We also provide data which suggest that T3 nuclear receptor(s) have a direct role on MyoD1 gene transcription: 1) C2 cells express the alpha 1 form of T3 nuclear receptors; 2) T3 up-regulates MyoD1 gene transcription and does not affect MyoD1 mRNA stability, as demonstrated by run-on and actinomycin D chase experiments, respectively; and 3) this transcriptional activation does not need the synthesis of intermediate protein(s) since it is not abolished by simultaneous treatment with cycloheximide. Moreover, in presence of T3, the increase of MyoD1 transcripts is associated with a faster terminal differentiation. Indeed we observed an earlier expression of various markers of myogenesis including myogenin (a regulatory gene of the MyoD1 family mainly involved in the triggering of terminal differentiation), myosin light chain 1A, and troponin T in T3-treated cells vs. untreated cells. We suggest that the regulation of a pivotal myogenic gene could be an important step in the control exerted by T3 on muscle development in vivo.

Animals↗
PubMed1991

Functional adult acetylcholine receptor develops independently of motor innervation in Sol 8 mouse muscle cell line.

We have defined culture conditions, using a feeder layer of cells from the embryonic mesenchymal cell line, 10T1/2 and a serum-free medium, which allow cells from the mouse myogenic cell line Sol 8 to form contracting myotubes for two weeks. Under these culture conditions, Sol 8 myotubes undergo a maturation process characterized by a sequential expression of two phenotypes. An early phenotype is typified by the expression of the nicotinic acetylcholine receptor (AChR) gamma-subunit transcripts and the presence of low conductance ACh-activated channels, typical of embryonic AChR. A late phenotype is characterized by the expression of AChR epsilon-subunit transcripts, the decreased accumulation of gamma-subunit transcripts and the appearance of high conductance ACh-activated channels, typical of adult AChR. These results indicate that the expression of functional adult type AChR does not require the presence of the motor nerve and therefore represents an intrinsic feature of the Sol 8 muscle cells. Chronic exposure of the cells to the voltage-sensitive Na+ channel blocking agent tetrodotoxin does not affect the appearance of the AChR epsilon-subunit transcripts but prevents the reduction of the steady-state level of the AChR gamma-subunit transcripts and yields a reduced proportion of the adult type channels. Thus, activity seems to facilitate the switch from the embryonic to the adult phenotype of the AChR protein. The Sol 8 cell system might be useful to analyse further the genetic and epigenetic regulation of muscle fibre maturation in mammals.

Animals↗
PubMed1991

Developmental patterns in the expression of Myf5, MyoD, myogenin, and MRF4 during myogenesis.

By using the polymerase chain reaction to amplify specific regions of the respective cDNAs, we have studied the expression of genes encoding the myogenic regulatory factors Myf5, MyoD, Myogenin, and MRF4 (Myf6, herculin) in cultured mouse muscle cells (inducible and permissive C2 cells and Sol8 cells). These cell lines may represent distinct stages in the progression of determined, or committed, muscle cells toward terminal differentiation. Transcripts for Myf5 were detected at the myoblast stage in all the committed muscle cells tested. Expression of the gene for MyoD appeared to be optional at the myoblast stage (MyoD is present in permissive myoblasts and absent from inducible myoblasts) but, like Myogenin, was found to accompany terminal differentiation. Furthermore, forced expression of MyoD converted inducible C2 cells into permissive cells. Expression of MRF4 was found to follow expression of the three other factors and to occur after the onset of terminal differentiation. Of particular interest was the finding that expression of MRF4 was temporally correlated with expression of the gene for the acetylcholine receptor epsilon-subunit that is characteristic of the adult receptor. In vivo, the only transcripts for myogenic regulatory factors to be detected in 8-day mouse embryos were those for Myf5, while expression of MRF4 followed expression of Myf5, MyoD, and Myogenin in developing limbs. Temporal and phenotypic differences related to the expression of Myf5, MyoD, Myogenin, and MRF4 suggest that these factors fulfil distinct roles in the control of myogenesis.

Animals↗
PubMed1990

CC Ar GG boxes, cis-acting elements with a dual specificity. Muscle-specific transcriptional activation and serum responsiveness.

The influence of different CC Ar GG boxes derived from either muscle-specific or serum-responsive genes, on the specificity of different promoters has been investigated. Inserted upstream from an 85 base-pair long minimal promoter of the human cardiac alpha-actin gene, a single copy of both the cognate CC Ar GG element (HCA1) and the c-fos gene serum response element (SRE) stimulate transcription four- to fivefold more efficiently in C2 myogenic cells than in L fibroblastic cells, SRE being two- to threefold more active than HCA1. Inserted upstream from the ubiquitous Herpes simplex thymidine kinase (HSV-tk) promoter, multimerized CC Ar GG boxes behave as strong muscle-specific activating elements, about 20-fold more active in myogenic C2 cells than in L fibroblasts and hepatoma HepG2 cells. They also confer serum responsiveness on the HSV-tk promoter. Efficiency of HCA1 and SRE tetramers in conferring both muscle specificity and serum responsiveness is roughly similar. It appears, therefore, that regardless of their origin (either muscle-specific or serum-responsive genes) CC Ar GG boxes behave by themselves as both muscle-specific activating and serum-responsive elements.

Actins↗
PubMed1990

Quantitative estimation of minor mRNAs by cDNA-polymerase chain reaction. Application to dystrophin mRNA in cultured myogenic and brain cells.

Amplification of the mRNA polymerase chain reaction is a very sensitive technique to detect low-abundance transcripts. We describe in this paper conditions necessary to make this technique quantitative. Quantification is performed in the exponential phase of the amplification process and the results are standardized with respect to those obtained with an exogenous mRNA which is co-reverse-transcribed and co-amplified in the same reaction as the analyzed transcripts. The primers are chosen in different exons to distinguish the amplification of mRNA fragments from the amplification of contaminating DNA. Analysis of the kinetics of amplification and parameters influencing this kinetics shows that: (a) in the exponential phase of amplification, the amount of amplified fragments is proportional to the initial amount of transcripts; (b) in a certain range of length fragment, the yield of amplification is inversely proportional to the length of the amplified fragments. Using this method we have demonstrated that the dystrophin gene is already activated at the myoblastic stage. A quantitative estimation of the transcript showed that the expression of this gene increases strongly in the course of in vitro myogenesis. In primary culture of mouse brain cells, the dystrophin gene was found to be more expressed in neuronal than in glial cells.

Animals↗
PubMed1989

Expression of MyoD1 coincides with terminal differentiation in determined but inducible muscle cells.

We have examined the expression of MyoD1, a potential determination factor of myogenic cells, in permissive and inducible C2 myoblasts. These two types of myoblasts exhibit distinct requirements to undergo terminal differentiation. Unlike permissive cells, inducible cells fail to differentiate in the presence of growth medium plus fetal calf serum and require insulin to undergo terminal differentiation. We show that while expression of MyoD1 is constitutive in permissive cells, no trace of MyoD1 transcripts is found in inducible cells at the myoblast stage. In these cells, however, expression of MyoD1 accompanies differentiation. This indicates that MyoD1 may not be required for the maintenance of the myoblast phenotype, and could act as an effector of terminal differentiation in already determined muscle cells. Our results provide new evidence that permissive and inducible cells represent two distinct stages of the progression of determined muscle cells toward terminal differentiation.

Autoradiography↗
PubMed1989

Murine muscle-specific enolase: cDNA cloning, sequence, and developmental expression.

In vertebrates, the glycolytic enzyme enolase (EC 4.2.1.11) is present as homodimers and heterodimers formed from three distinct subunits of identical molecular weight, alpha, beta, and gamma. We report the cloning and sequencing of a cDNA encoding the beta subunit of murine muscle-specific enolase. The corresponding amino acid sequence shows greater than 80% homology with the beta subunit from chicken obtained by protein sequencing and with alpha and gamma subunits from rat and mouse deduced from cloned cDNAs. In contrast, there is no homology between the 3' untranslated regions of mouse alpha, beta, and gamma enolase mRNAs, which also differ greatly in length. The short 3' untranslated region of beta enolase mRNA accounts for its distinct length, 1600 bases. It is known that a progressive transition from alpha alpha to beta beta enolase occurs in developing skeletal muscle. We show that this transition mainly results from a differential regulation of alpha and beta mRNA levels. Analysis of myogenic cell lines shows that beta enolase gene is expressed at the myoblast stage. Moreover, transfection of premyogenic C3H10T1/2 cells with MyoD1 cDNA shows that the initial expression of beta transcripts occurs during the very first steps of the myogenic pathway, suggesting that it could be a marker event of myogenic lineage determination.

Actins↗
PubMed1988

High level of c-fos mRNA accumulation is not obligatory for renewed cell proliferation.

We have examined the expression of the c-fos gene and the formation of inositol phosphates with respect to the reentry of inducible C2 myoblasts into the cell cycle. GI arrested myoblasts were stimulated to proliferate by addition of fresh medium containing either 20% foetal calf serum (FCS) or 1.6 10(-6) M insulin and 7 microM Na+ vanadate. Our results show that renewed proliferation, which occurred in the presence of insulin + vanadate, was neither preceded by increased inositol phosphate formation, nor by high level of c-fos mRNA accumulation, while, as classically observed, FCS induced proliferation was. These results suggest that increased inositol phospholipids breakdown and transient accumulation of c-fos mRNA at a high level, are not obligatory for renewed cell proliferation.

Animals↗
PubMed1988

The 'CC.Ar.GG' box. A protein-binding site common to transcription-regulatory regions of the cardiac actin, c-fos and interleukin-2 receptor genes.

We have previously suggested that a repeated sequence motif in the upstream region of the human cardiac actin gene 'CC.Ar.GG', where Ar is an (A + T)-rich six-base-pair-sequence, may be important in the muscle-specific expression of this gene [Minty, A. & Kedes, L. (1986) Mol. Cell Biol. 6, 2125-2136]. Here we show that this sequence binds a nuclear protein, and that binding is abolished by mutating either the CC and GG dinucleotides or the (A + T)-rich centre. Mutation of the CC and GG nucleotides also abolishes the transcription-stimulating activity of this sequence on the cardiac actin promoter. A similar sequence has been implicated in the serum-response of the c-fos gene [Treisman, R. (1986) Cell 46, 567-574]. We show that this c-fos 'CC.Ar.GG' sequence competes with the cardiac actin sequence for factor binding. Our results suggest that the minimum sequence requirements for binding of the serum response factor may correspond to the 'CC.Ar.GG' box sequence. Using this criterion, we predict and confirm the existence of such a binding site in a regulatory region of the interleukin-2 receptor gene. It appears therefore that interactions between 'CC.Ar.GG' boxes and similar proteic factors could be involved in the control of different genes responding to different stimuli, e.g. muscle differentiation (cardiac actin gene) or growth stimulation (c-fos, cytoskeletal actin or interleukin-2 receptor genes).

Actins↗
PubMed1988

Functional activity of the two promoters of the myosin alkali light chain gene in primary muscle cell cultures: comparison with other muscle gene promoters and other culture systems.

Proximal upstream flanking sequences of the mouse myosin alkali light chain gene encoding MLC1F and MLC3F, the mouse alpha-cardiac actin gene and the chicken gene for the alpha-subunit of the acetylcholine receptor were linked to the bacterial chloramphenicol acetyl transferase (CAT) gene and transfected into primary cultures derived from mouse skeletal muscle or into myogenic cell lines. We demonstrate that the mouse MLC1F/MLC3F gene has two functional promoters. In primary muscle cultures, a 1200 bp sequence flanking exon 1 (MLC1F) and a 438 bp sequence flanking exon 2 (MLC3F) direct CAT activity in myotubes, but not in myoblasts or in non myogenic 3T6 and CV1 cells. Developmentally regulated expression is also seen with the alpha-cardiac actin (320 bp) and acetylcholine receptor alpha-subunit (850 bp) upstream sequences in the primary culture system. Transfection experiments with myogenic cell lines show different results with a given promoter construct, reflecting possible differences in the levels of regulatory factors between lines. Different muscle gene promoters behave differently in a given cell line, suggesting different regulatory factor requirements between these promoters.

Actins↗
PubMed1988

Calcitonin gene-related peptide enhances the rate of desensitization of the nicotinic acetylcholine receptor in cultured mouse muscle cells.

Calcitonin gene-related peptide (CGRP) is a neuropeptide that coexists with acetylcholine in spinal cord motoneurons. The effects of CGRP on the functional properties of the nicotinic acetylcholine receptor (AcChoR) were examined by electrophysiological methods. Using the whole-cell patch-clamp technique and a mouse cell line derived from soleus muscle, we found that CGRP produces a progressive and reversible enhancement of the rapid-decay phase of AcChoR desensitization. Single-channel data further show that CGRP decreases acetylcholine-activated channel opening frequency. This decrease occurs when CGRP and acetylcholine are applied on different cell-surface areas and thus is likely mediated by a second-messenger system. CGRP is also shown to increase cAMP accumulation in this cell line. The effects of CGRP on macroscopic acetylcholine-activated currents are mimicked by external application of forskolin (10 microM) or by internal perfusion of the cell with cAMP (1 microM). In both these cases, further application of CGRP produces no additional enhancement of AcChoR desensitization. These results suggest that, on mouse muscle cells, CGRP regulates AcChoR desensitization by a mechanism that involves, at least in part, cAMP-dependent phosphorylation of the AcChoR.

Animals↗