Biomedical subjects

B Nadal-Ginard

Publications and source records attributed to B Nadal-Ginard.

At least 19 recordsLinked to original sources

PubMed1998

Involvement of the retinoblastoma protein in brown and white adipocyte cell differentiation: functional and physical association with the adipogenic transcription factor C/EBPalpha.

We investigated the expression of the retinoblastoma protein (pRB) in adipocytes and its possible interaction with the adipogenic transcription factor CCAAT/enhancer-binding protein alpha (C/EBPalpha) in controlling the acquisition of the terminally differentiated adipocyte phenotype. The pRB was expressed (as measured by immunoblotting and/or immunofluorescence) in mice brown and white adipose tissue and in cultured adipocytes that showed lipid accumulation and expressed specific differentiation markers such as aP2 (measured using a specific cDNA probe) and in the case of brown adipocytes UCP-1 (measured using specific antibodies), but was undetectable in proliferative undifferentiated preadipocytes. Transient transfection experiments revealed a functional interaction between pRB and C/EBPalpha affecting transcription from the ucp-1 gene promoter. Thus, in immortalized brown adipocytes, co-transfection of both a C/EBPalpha and a pRB expression vectors maximally enhanced the expression of reporter chloramphenicol acetyltransferase driven by the ucp-1 promoter. Interestingly, C/EBPalpha inhibited reporter gene expression in CHO cells in an effect that was also potentiated in the presence of pRB. A positive effect of pRB on transcription from the ucp-1 promoter could be detected in C/EBPalpha-/-fibroblasts only after forced to overexpress C/EBPalpha, suggesting that the effect of pRB is dependent on its interaction with C/EBPalpha. We also found evidence that pRB and C/EBPalpha can directly bind to each other in vitro. Our results show that the expression of pRB is restricted to differentiated adipocytes, and provide evidence of a physical and functional interaction between pRB and C/EBPalpha that affects the transcriptional activity of the later on a brown adipocyte-specific gene.

3T3 Cells↗

PubMed1998

Reversal of myogenic terminal differentiation by SV40 large T antigen results in mitosis and apoptosis.

Terminally differentiated skeletal muscle myotubes are arrested in the G0 phase of the cell cycle, and this arrest is not reversed by stimulation with serum or growth factors. The myotubes have been shown to be refractory to apoptosis even under low serum conditions. When the SV40 large T antigen is induced in the C2SVTts11 myotubes, which stably harbor the T antigen gene linked to an inducible promoter, the terminally differentiated cells reenter the cell cycle to resume nuclear DNA replication representing S phase. We show here that the large T-expressing myotubes further proceeded to M phase represented by the appearance of mitotic figures with centrosomes, condensed chromosomes, and mitotic spindles. The myotubes eventually cleaved and midbodies were formed at the cleavage sites of the cytoplasm. In some cases actin filaments, reminiscent of the contractile rings, accumulated at the cleavage furrows. Thus, terminally differentiated myotubes remain able to resume at least one round of the cell cycle and consequently are considered to be capable of dedifferentiation. A subset of myotubes expressing large T did not undergo mitosis. Some of them were degenerative and contained deformed giant nuclei and pulverized nuclei. The others suffered apoptotic cell death, which was identified by morphological changes of the nuclei and the labeling with dUTP at the ends of chromatin DNA fragments. The induction of apoptosis was unlikely to be confined to a particular phase of the cell cycle. These results imply that terminally differentiated myotubes also retain a complete set of machinery for apoptosis.

Animals↗

PubMed1997

Mapping and use of a sequence that targets DNA ligase I to sites of DNA replication in vivo.

The mammalian nucleus is highly organized, and nuclear processes such as DNA replication occur in discrete nuclear foci, a phenomenon often termed "functional organization" of the nucleus. We describe the identification and characterization of a bipartite targeting sequence (amino acids 1-28 and 111-179) that is necessary and sufficient to direct DNA ligase I to nuclear replication foci during S phase. This targeting sequence is located within the regulatory, NH2-terminal domain of the protein and is dispensable for enzyme activity in vitro but is required in vivo. The targeting domain functions position independently at either the NH2 or the COOH termini of heterologous proteins. We used the targeting sequence of DNA ligase I to visualize replication foci in vivo. Chimeric proteins with DNA ligase I and the green fluorescent protein localized at replication foci in living mammalian cells and thus show that these subnuclear functional domains, previously observed in fixed cells, exist in vivo. The characteristic redistribution of these chimeric proteins makes them unique markers for cell cycle studies to directly monitor entry into S phase in living cells.

Amino Acid Sequence↗

PubMed1997

Identification of a cis-acting regulatory element conferring inducibility of the atrial natriuretic factor gene in acute pressure overload.

To identify the cis-acting regulatory element(s) which control the induction of the atrial natriuretic factor (ANF) gene in acute pressure overload, DNA constructs consisting of promoter elements linked to a reporter gene were injected into the myocardium of dogs, which underwent aortic banding or were sham-operated. Expression of a reporter gene construct harboring the ANF promoter (-3400ANF) was induced 6-12-fold after 7 d of pressure overload. An internal deletion of 556 bp (nucleotide sequence -693 to -137) completely abrogated the inducibility of the ANF reporter gene construct. An activator protein-1 (AP1)-like site (-496 to -489) and a cAMP regulatory element (CRE) (-602 to -596) are located within the deleted sequence. Site-directed mutagenesis of the AP1-like site but not the CRE completely prevented the induction of this construct to acute pressure overload. Further, the AP1-like site was able to confer inducibility of a heterologous promoter (beta-myosin heavy chain) to higher values than controls. Gel mobility shift assay (GMSA) supershift analysis was performed using a radiolabeled probe of the ANF promoter (-506/-483) that included the AP1-like site (ATGAATCA) sequence, as well as a probe converted to contain an AP1 consensus sequence (ATGACTCA). GMSA analysis demonstrated that the ANF AP1-like element could bind both a constitutively expressed factor and the AP1 proteins, and conversion to a true AP1 site increased its affinity for AP1. However, 7 d after the onset of pressure overload, the AP1 proteins were present only at low levels, and the major complex formed by the ANF AP1-like probe was not supershifted by a jun antibody. Using a large animal model of pressure overload, we have demonstrated that a unique cis-acting element was primarily responsible for the overload induction of the ANF gene.

Animals↗

PubMed1997

Myocyte-specific enhancer factor 2 and thyroid hormone receptor associate and synergistically activate the alpha-cardiac myosin heavy-chain gene.

The muscle-specific regulatory region of the alpha-cardiac myosin heavy-chain (MHC) gene contains the thyroid hormone response element (TRE) and two A/T-rich DNA sequences, designated A/T1 and A/T2, the putative myocyte-specific enhancer factor 2 (MEF2) binding sites. We investigated the roles of the TRE and MEF2 binding sites and the potential interaction between thyroid hormone receptor (TR) and MEF2 proteins regulating the alpha-MHC promoter. Deletion mutation analysis indicated that both the A/T2 motif and TRE were required for muscle-specific expression of the alpha-MHC gene. The alpha-MHC enhancer containing both the A/T2 motif and TRE was synergistically activated by coexpression of MEF2 and TR in nonmuscle cells, whereas neither factor by itself activated the alpha-MHC reporters. The reporter construct containing the A/T2 sequence and the TRE linked to a heterologous promoter also showed synergistic activation by coexpression of MEF2 and TR in nonmuscle cells. Moreover, protein binding assays demonstrated that MEF2 and TR specifically bound to one another in vitro and in vivo. The MADS domain of MEF2 and the DNA-binding domain of TR were necessary and sufficient to mediate their physical interaction. Our results suggest that the members of the MADS family (MEF2) and steroid receptor superfamily (TR) interact with one another to synergistically activate the alpha-cardiac MHC gene expression.

Animals↗

PubMed1996

E2F1 inhibition of transcription activation by myogenic basic helix-loop-helix regulators.

Cellular transcription factor E2F1 is thought to regulate the expression of genes important for cell cycle progression and cell proliferation. Deregulated E2F1 expression induces S-phase entry in quiescent cells and inhibits myogenic differentiation. We show here that E2F1 inhibits the activation of gene transcription by myogenic basic helix-loop-helix proteins myoD and myogenin. Transfection assay using different deletion constructs indicates that both the DNA binding and the transactivation domains of E2F1 are required for its inhibition of myoD transcription activation. However, the retinoblastoma protein (RB) binding domain is not required. Furthermore, co-transfection with the RB, which inhibits the transcription activity of E2F1, can also repress E2F1 inhibition of myoD transactivation. These results suggest an essential role of E2F1-mediated transcription in its inhibition of myogenesis.

Animals↗

PubMed1996

Functional and physical interactions between mammalian achaete-scute homolog 1 and myocyte enhancer factor 2A.

The mammalian achaete-scute homolog 1 (MASH1) protein is required for the early development of the nervous system. However, the molecular and biochemical mechanism by which MASH1 acts to determine neurogenesis are still unknown. The myocyte enhancer factor 2A (MEF2A) is a MADS transcription factor that is essential for the specification and differentiation of the muscle lineage. Here we show that MEF2A and MASH1 are coordinately induced during the differentiation of the teratocarcinoma cell line P19 along a neuronal lineage and that in transient transfection assays, MEF2A and MASH1 cooperatively activate gene expression. This cooperativity appears to be due to a specific physical interaction between MEF2A and MASH1. Taken together, these findings suggest that MASH1 via a cooperative interaction with MEF2A may regulate the expression of specific genes that are critical for neuronal differentiation.

Animals↗

PubMed1996

Cloning and characterization of an olfactory cyclic nucleotide-gated channel expressed in mouse heart.

Regulation of ionic currents in the heart is partly achieved by signaling cascades which alter intracellular levels of cyclic nucleotides. Changes in cyclic nucleotide levels can regulate channels either directly, like the direct binding of cAMP to the i(f) channel in pacemaker tissues, or indirectly through phosphorylation of channels by cAMP-dependent, or cGMP-dependent protein kinases. These types of regulation generally alter the voltage sensitivities of channels. A class of voltage-insensitive channels, first discovered in retinal rods and olfactory neurons, were recently identified in the heart. These channels are opened by the direct binding of cyclic nucleotides, providing a means of regulating ionic currents outside the influence of membrane voltage. Since different isoforms have different affinities for cAMP and cGMP, it is important to determine which isoforms are expressed in heart in order to predict their roles in heart function. We have cloned the olfactory channel from mouse heart, and find that although the message is very rare, Western blot analysis indicates the olfactory channel protein is stable in heart sarcolemma. Our data also suggest the olfactory channel protein forms homomeric channels in the heart since other isoforms or splice variants were not detected either by PCR amplification or by RNase protection. In addition, we have isolated and sequenced the mouse olfactory cyclic nucleotide-gated channel gene, and show the genomic organization is remarkably similar to that found in the human retinal channel gene. Part of this work was presented in abstract form.

Amino Acid Sequence↗

PubMed1996

Early expression of the different isoforms of the myocyte enhancer factor-2 (MEF2) protein in myogenic as well as non-myogenic cell lineages during mouse embryogenesis.

MEF2 proteins are a family of transcription factors that have muscle-specific DNA binding activity and bind to conserved A/T rich elements in the regulatory regions of numerous muscle-specific genes. They are thought have an important role in the development and differentiation of skeletal muscle. Recent in situ hybridization studies using mouse MEF2 probes have shown that MEF2 gene transcripts are detected very early in development at high levels in the myogenic cells of the myotome and embryonic heart. However, MEF2A and MEF2D transcripts have been detected in many adult tissues where they are not translated or the corresponding proteins are rapidly degraded. Therefore, it is important to establish the temporal and spatial correlation between MEF2 RNA and protein expression. In the present study we have performed in situ immunohistochemistry of whole mount mouse embryos at different stages of development using polyclonal antibodies specific for the MEF2A, MEF2C and MEF2D isoforms. At day 8.5 of development, all three MEF2 isoforms are expressed in the heart. MEF2A and C are detected in a few cells in the rostral-most somite by day 9 of development. Their expression then proceeds in a rostro-caudal direction concurrent with somite maturation. The pattern of expression of the MEF2D isoform is similar to that of MEF2C but the amount detected is much lower. Interestingly, MEF2A is also detected as early as day 8.5 p.c. in cells of the embryonic vasculature and non-myogenic cells. These results demonstrate that MEF2 proteins are detected early in development in the somites and heart, thus supporting their importance in the early stages of the hierarchical cascade of myogenesis. Their presence in non-muscle cells further suggests they could also play a role in the determination of other mesodermal derivatives, including cells of the vasculature.

Age Factors↗

PubMed1995

Cardiac myocyte terminal differentiation. Potential for cardiac regeneration.

The exact mechanism of terminal differentiation in cardiac myocytes is currently unknown. Studies in the skeletal muscle system provided a model where muscle lineage termination gene directly interacts with Rb to produce and maintain the terminally differentiated state. This interaction provided the critical components for the lock in cell cycle arrest in skeletal muscle cell. Cardiac muscle appears on the surface very similar to skeletal muscle especially since they share large numbers of structural and contractile proteins. However, it is clear that cardiac muscle cells are distinct biologically at the regulatory level. First and foremost, differentiation and capacity for hyperplasia (mitosis) is not mutually exclusive, in that the heart being the first functional organ embryologically is able to grow via cell division until shortly after birth. Thereafter further growth is provided by hypertrophy. In skeletal muscle, these two processes, differentiation and ability to undergo mitosis, appear to be mutually exclusive. Second, cardiac muscles have not been shown to express any of the skeletal muscle determination basic helix loop helix factors like myoD or any proteins that are functionally similar. Third, heterokaryons of cardiac myocytes and fibroblasts reveal a lack of dominance of the cardiac muscle phenotype. This is distinctly different in skeletal muscle, whose phenotype is dominant which provided a platform to identify the skeletal muscle determination gene, myoD. Although various basic helix loop helix proteins and homeobox genes have been identified in cardiac myocytes, their function remains to be elucidated. At this time no cardiac determination gene has been identified. Despite these differences, we have shown that the biology of pocket proteins Rb and P107 is similar in skeletal and cardiac myocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

Regulation of cyclins and p34CDC2 expression during terminal differentiation of C2C12 myocytes.

Little is known about the expression of cell cycle regulatory genes upon terminal differentiation of skeletal muscle cells. In this report, we demonstrate that the expressions of cyclin A, cyclin D1 and p34cdc2 are downregulated upon C2C12 myocytes differentiation and are not inducible in differentiated myotubes. SV40 large T antigen can induce cell cycle entry of myotubes through its induction of these genes' expressions and pRB phosphorylation as well as its suppression of Rb expression. These results provide the first direct evidence that the irreversible downregulation of cyclins and cyclin-dependent kinases is one mechanism for the permanent cell cycle withdrawal of myotubes.

Animals↗

PubMed1995

Molecular cardiomyoplasty: potential cardiac gene therapy for chronic heart failure.

In this study, we evaluated the feasibility of converting cardiac fibroblasts into skeletal muscle cells by forced expression of the MyoD gene, one of the basic helix-loop-helix myogenic factors. Primary cardiac fibroblasts, isolated from newborn rats, were infected with retrovirus-carrying sense or antisense MyoD gene. Ten days after infection, expression of MyoD protein was demonstrated in 95% of cells infected with sense MyoD virus by intense nuclear immunostaining with a MyoD polyclonal antibody. In contrast, none of the cells infected with antisense MyoD virus showed staining. On withdrawal of serum, 95% of MyoD positive cells became elongated and, in the presence of appropriate cell density, fused to form multinucleated myotubes, morphologically similar to striated muscle cell. Expression of downstream myogenic differentiation markers, myosin heavy chain and myocyte-specific enhancer factor 2, in 95% of these myotubes were detected by intense cytoplasmic and nuclear immunostaining, respectively, with specific antibodies. In contrast, no detectable staining was noted in MyoD negative cells. Spontaneous contractile movements were noted in a few clusters of myotubes. In summary, cardiac fibroblasts were able to be converted into bonafide potentially functional skeletal myocytes as shown by definitive morphologic and biochemical changes. Further studies with in vivo models are needed to explore this unique molecular strategy to treat patients with chronic heart failure.

Animals↗

PubMed1995

Overexpression of Gs alpha protein in the hearts of transgenic mice.

Alterations in beta-adrenergic receptor-Gs-adenylyl cyclase coupling underlie the reduced catecholamine responsiveness that is a hallmark of human and animal models of heart failure. To study the effect of altered expression of Gs alpha, we overexpressed the short isoform of Gs alpha in the hearts of transgenic mice, using a rat alpha-myosin heavy chain promoter. Gs alpha mRNA levels were increased selectively in the hearts of transgenic mice, with a level 38 times the control. Despite this marked increase in mRNA, Western blotting identified only a 2.8-fold increase in the content of the Gs alpha short isoform, whereas Gs activity was increased by 88%. The discrepancy between Gs alpha mRNA and Gs alpha protein levels suggests that the membrane content of Gs alpha is posttranscriptionally regulated. The steady-state adenylyl cyclase catalytic activity was not altered under either basal or stimulated conditions (GTP + isoproterenol, GTP gamma S, NaF, or forskolin). However, progress curve studies did show a significant decrease in the lag period necessary for GppNHp to stimulate adenylyl cyclase activity. Furthermore, the relative number of beta-adrenergic receptors binding agonist with high affinity was significantly increased. Our data demonstrate that a relatively small increase in the amount of the coupling protein Gs alpha can modify the rate of catalyst activation and the formation of agonist high affinity receptors.

Adenylyl Cyclases↗

PubMed1995

Inhibition of in vitro myogenic differentiation by cellular transcription factor E2F1.

Terminal differentiation of cultured myocytes requires withdrawal of the cells from the cell cycle. Constitutive overexpression of several oncogenes in myoblasts can inhibit in vitro myogenesis. Here we studied the role of the cellular transcription factor E2F1 on myogenic differentiation. E2F1 expression is irreversibly down-regulated during differentiation of C2C12 myocytes. Furthermore, deregulated E2F1 expression in C2C12 cells prevented myogenic differentiation. This inhibition of myogenesis was associated with the repression of myogenin expression and an elevated cyclin D1 expression. Moreover, E2F1-overexpressing myocytes failed to exit the cell cycle under differentiation conditions. These results are consistent with the notion that E2F1 can function as an oncogene and further suggest that E2F1 down-regulation is required for myogenic differentiation.

Animals↗

PubMed1994

Activation of the myogenic lineage by MEF2A, a factor that induces and cooperates with MyoD.

Muscle enhancer factor-2A (MEF2A), a member of the MADS family, induced myogenic development when ectopically expressed in clones of nonmuscle cells of human clones, a function previously limited to the muscle basic helix-loop-helix (bHLH) proteins. During myogenesis, MEF2A and bHLH proteins cooperatively activate skeletal muscle genes and physically interact through the MADS domain of MEF2A and the three myogenic amino acids of the muscle bHLH proteins. Thus, skeletal myogenesis is mediated by two distinct families of mutually inducible and interactive muscle transcription factors, either of which can initiate the developmental cascade.

Animals↗

PubMed1994

Smooth muscle-specific switching of alpha-tropomyosin mutually exclusive exon selection by specific inhibition of the strong default exon.

Exons 2 and 3 of alpha-tropomyosin are spliced in a strict mutually exclusive manner. Exon 3 is a default choice, being selected in almost all cell types where the gene is expressed. The default selection arises from a competition between the two exons, in which the stronger branch point/pyrimidine tract elements of exon 3 win. Exon 2 is selected predominantly or exclusively only in smooth muscle cells. We show here that the basis for the smooth muscle-specific switching of exon selection is inhibition of exon 3. Exon 3 is still skipped with smooth muscle specificity, even in the absence of exon 2. We have defined two conserved sequence elements, one in each of the introns flanking exon 3, that are essential for this regulation. Mutation of either element severely impairs regulated suppression of exon 3. No other exon or intron sequences appear to be necessary for regulation. We have also demonstrated skipping of exon 3 that is dependent upon both regulatory elements in an in vitro splicing assay. We further show that both splice sites of exon 3 must be inhibited in a concerted fashion to switch to selection of exon 2. This may relate to the requirement for negative elements on both sides of the exon.

Alternative Splicing↗

PubMed1994

Specific protein-protein interactions between the essential mammalian spliceosome-associated proteins SAP 61 and SAP 114.

Spliceosome-associated proteins (SAPs) 61, 62, and 114 can be UV-crosslinked to pre-mRNA in purified spliceosomal complexes and are associated with U2 small nuclear ribonucleoproteins (snRNP). These proteins also compose the essential heterotrimeric splicing factor SF3a, and products of yeast pre-mRNA processing genes PRP9, PRP11, and PRP21 are their likely yeast counterparts. We report the isolation of a cDNA encoding SAP 61 and find that it is 30% identical in amino acid sequence to PRP9. A C-terminal Cys2His2 zinc-finger-like motif, which could be involved in the pre-mRNA binding, is the most highly conserved region of the protein. We also demonstrate specific protein-protein interactions between SAPs 61 and 114 and show that the N terminus of SAP 61 is required for this interaction. Significantly, the corresponding proteins are also known to interact in yeast: PRP9 interacts with PRP21, and the N-terminal portion of PRP9 is required. Previous work showed that direct interactions also occur between SAPs 62 and 114 and between the corresponding PRPs 11 and 21. These observations indicate that the specific protein-protein interactions that occur between the three prespliceosomal factors have been conserved between yeast and mammals.

Amino Acid Sequence↗

PubMed1994

Reversal of terminal differentiation mediated by p107 in Rb-/- muscle cells.

The terminal differentiation of mammalian muscle cells requires the tumor suppressor retinoblastoma protein (Rb). Unlike their wild-type counterparts, multinucleated myotubes from mouse cells deficient in Rb (Rb-/-) were induced by serum to re-enter the cell cycle. Development of the myogenic phenotype in Rb-/- cells correlated with increased expression of p107, which interacted with myogenic transcription factors. Serum-induced cell cycle reentry, on the other hand, correlated with decreased p107 expression. Thus, although p107 could substitute for Rb as a cofactor for differentiation, it could not maintain the terminally differentiated state in Rb-/- myotubes.

Animals↗