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.

Growth and differentiation of C2 myogenic cells are dependent on serum response factor.

In order to study to what extent and at which stage serum response factor (SRF) is indispensable for myogenesis, we stably transfected C2 myogenic cells with, successively, a glucocorticoid receptor expression vector and a construct allowing for the expression of an SRF antisense RNA under the direction of the mouse mammary tumor virus long terminal repeat. In the clones obtained, SRF synthesis is reversibly down-regulated by induction of SRF antisense RNA expression by dexamethasone, whose effect is antagonized by the anti-hormone RU486. Two kinds of proliferation and differentiation patterns have been obtained in the resulting clones. Some clones with a high level of constitutive SRF antisense RNA expression are unable to differentiate into myotubes; their growth can be blocked by further induction of SRF antisense RNA expression by dexamethasone. Other clones are able to differentiate and are able to synthesize SRF, MyoD, myogenin, and myosin heavy chain at confluency. When SRF antisense RNA expression is induced in proliferating myoblasts by dexamethasone treatment, cell growth is blocked and cyclin A concentration drops. When SRF antisense RNA synthesis is induced in arrested confluent myoblasts cultured in a differentiation medium, cell fusion is blocked and synthesis of not only SRF but also MyoD, myogenin, and myosin heavy chain is inhibited. Our results show, therefore, that SRF synthesis is indispensable for both myoblast proliferation and myogenic differentiation.

Expression and activity of serum response factor is required for expression of the muscle-determining factor MyoD in both dividing and differentiating mouse C2C12 myoblasts.

To understand the mechanism by which the serum response factor (SRF) is involved in the process of skeletal muscle differentiation, we have assessed the effect of inhibiting SRF activity or synthesis on the expression of the muscle-determining factor MyoD. Inhibition of SRF activity in mouse myogenic C2C12 cells through microinjection of either the SRE oligonucleotide (which acts by displacing SRF proteins from the endogenous SRE sequences), purified SRF-DB (a 30-kDa portion of SRF containing the DNA-binding domain of SRF, which acts as a dominant negative mutant in vivo), or purified anti-SRF antibodies rapidly prevents the expression of MyoD. Moreover, the rapid shutdown of MyoD expression after in vivo inhibition of SRF activity is observed not only in proliferating myoblasts but also in myoblasts cultured under differentiating conditions. Additionally, by using a cellular system expressing a glucocorticoid-inducible antisense-SRF (from aa 74 to 244) we have shown that blocking SRF expression by dexamethasone induction of antisense SRF results in the lack of MyoD expression as probed by both immunofluorescence and Northern blot analysis. Taken together these data demonstrate that SRF expression and activity are required for the expression of the muscle-determining factor MyoD.

The serum response factor (SRF) is needed for muscle-specific activation of CArG boxes.

CArG boxes, whose consensus sequence is CC(A/T)6GG, are involved in two very different types of transcriptional responses: response of immediate early genes to serum, mediated by so-called Serum Response Elements (SRE), and transcriptional activation of muscle-specific genes during muscle differentiation. Although previous studies have shown that the Serum Response Factor (SRF) binds to muscular CArG boxes, the role of such a binding in muscle-specific activation of CArG box-dependent genes was not directly demonstrated. Here, by transient co-transfection experiments, we demonstrate that intact SRF is required for muscle-specific transcriptional activation through CArG boxes.

Muscle-specific transcriptional activation by CArG box requires either homophilic or heterophilic interactions of the CArG box binding factors.

CArG boxes (CC(A/T)6GG sequences) are present in various promoters and are able to confer two different types of transcriptional responsiveness: serum inducibility and muscle-specific activation. Inserted upstream from the ubiquitous HSV thymidine kinase promoter, multimerized HCA1 boxes (human cardiac alpha-actin proximal CArG box) behave as strong muscle-specific activating elements. Transient expression assay was used to determine whether the muscle-specific transcriptional activation by the CArG boxes depends on the presence in the vicinity of other specific cis-acting DNA elements. Our results show that no specific association between different regulatory binding sites is required for the myogenic activity of a CArG box and that CArG elements are able to stimulate transcription in myogenic cells through either homophilic or heterophilic interactions of the CArG box binding factors.

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.

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.

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).

Transferrin gene expression in choroid plexus of the adult rat brain.

Transferrin immunoreactivity and transferrin messenger RNA (mRNA) were recently found to be present in oligodendrocytes of the adult rat brain by using immunohistochemistry and in situ hybridization procedure. The present study demonstrates, in the same way, that epithelial cells of the choroid plexus also contain transferrin together with transferrin mRNA. Choroid plexus of the lateral and the third ventricle are rich in transferrin mRNA, while choroid plexus of the fourth ventricle contain few if any transferrin mRNA. These results demonstrate that epithelial cells of the choroid plexus as well as oligodendrocytes express the transferrin gene in the adult rat brain.

In vivo developmental modifications of the expression of genes encoding muscle-specific enzymes in rat.

cDNA clones for rat muscle-type creatine kinase and glycogen phosphorylase and aldolase A were isolated from a rat muscle cDNA library. An additional clone recognizing an unidentified 2.7-kilobase pair mRNA species was also isolated. These cDNA clones were used as probes to investigate the expression of the corresponding mRNAs during muscle development. Two aldolase A mRNA species were detected, one of 1650 bases expressed in non-muscle tissues, fetal muscle, and adult slow-twitch muscle, the other of 1550 bases was highly specific of adult fast-twitch skeletal muscle differentiation. These aldolase A mRNAs were shown by primer extension to differ by their 5' ends. The accumulation of muscle-type phosphorylase and creatine kinase and muscle-specific aldolase A mRNA accumulation during muscle development seems to be a coordinate process occurring progressively from the 17th day of intrauterine life up to the 30th day after birth. In contrast, the 2.7-kilobase pair RNA species is maximally expressed at the 1st week after birth as is the neonatal form of myosin heavy chain mRNA.

Transferrin gene expression visualized in oligodendrocytes of the rat brain by using in situ hybridization and immunohistochemistry.

The presence and production of transferrin in the adult rat brain have been investigated using both immunohistochemistry and in situ hybridization in tissue sections. Indirect immunofluorescence with four distinct antisera against rat and human transferrin and one monoclonal antibody against human transferrin demonstrated labeling of the cytoplasm of oligodendrocytes (a category of glial cells) in most parts of the brain, especially in the white matter. In situ hybridization using rat transferrin 32P-labeled cDNA as a probe revealed the presence of transferrin mRNA in glial cells whose appearance, distribution, and organization exactly matched those of the cells decorated with the transferrin antibodies. These results provide evidence that the transferrin gene is expressed in the central nervous system and that transferrin is synthesized by and stored within oligodendrocytes in the adult rat brain. These data suggest that this molecule could have a specific function in nervous system activity.

Transient transcriptional inhibition of the transferrin gene by cyclic AMP.

Transferrin mRNA content and gene transcription rate were measured in the liver of rats submitted to iron overload or depletion, castration, treatment with sexual steroid hormones, glucagon and cyclic AMP. The influence of puberty in males and females and of pregnancy was also analysed. Glucagon and cyclic AMP reduced mRNA level by about 50% at the 12th hour of treatment and transferrin gene transcription by as much as 95% at the 30th minute of drug infusion, with a secondary increase of the transcription rate for a protracted treatment. None of the other hormones tested had any detectable effect on transferrin gene expression, the same being true for iron overload or depletion.

Expression of the transferrin gene during development of non-hepatic tissues: high level of transferrin mRNA in fetal muscle and adult brain.

Using a cloned rat transferrin cDNA probe, we looked for transferrin mRNA in the various rat tissues during development. In all the cases the mRNA detected seemed to be the same and to be product of a single gene. The transferrin gene is early expressed at a high level during liver differentiation. In the muscle and other non-hepatic and non-nervous tissues, the gene expression is maximal just before birth (19-20th day of gestational age), then markedly decreases during the postnatal development, the mRNA level being very low in the adult tissues. In brain, by contrast, transferrin mRNA level is very low before birth, then gradually increases during the postnatal development and reaches a plateau in the adult. Maximal mRNA concentration in fetal muscle (2 days before birth) and adult brain is about 1:7 to 1:10 of that obtained in adult liver. These results are analyzed in the light of the evidence that transferrin is not only an iron-binding protein, but also a factor involved in cell proliferation and differentiation, and particularly in nerve control of muscle differentiation.

Diphenoloxidases in various forms of myopathy which are transmitted by different genetic mechanisms.

In a previous article (Demos et al. 1981), we reported a significant and specific reduction of the activity index (AI) of the diphenoloxidases (DPox) in patients and heterozygotes with progressive Duchenne muscular dystrophy (DMD), which is transmitted genetically by female subjects by a sex-linked recessive mechanism (SLR). This same anomaly was detected in patients suffering from other types of dystrophy: Becker, limbgirdle, fascio-scapulo-humeral, and in heterozygotes of either sex in diseases transmitted by an obviously recessive autosomic mechanism. These anomalies were detected using blood spots collected on absorbent paper and stored at 4 degrees C for different periods. They were of the same type as had previously been detected using blood platelets (Demos 1973).

Diphenoloxidases in X-linked recessive (Duchenne) muscular dystrophy.

In extracts derived from whole blood, a high molecular weight fraction of the diphenoloxidase enzymes has a significantly diminished specific activity in patients and definite carriers (heterozygotes) of the X-linked, recessive (Duchenne) form of muscular dystrophy. This anomaly was studied using spots of blood which had been collected on absorbent paper and stored at 4 degrees C for variable periods of time. Fractions enriched in the enzymes were obtained by subjecting aqueous extracts of the spots to treatment with an anion exchange resin (DEAE Sephadex A 50) followed by gel filtration on Sephadex G-25. It is of interest that this anomaly was observed in some definite carriers of the mutant gene who had on several occasions a serum creatine kinase level in the normal range. The significance of these observations is discussed.

Purification and characterization of a novel human red cell enzyme with diphenol oxidase activity.

A new enzyme protein, diphenol oxidase, has been isolated and purified from human red cells and catalyzes the in vitro formation of adrenochrome from epinephrine and of melanin from dihydroxyphenylalanine. Some immunological and physicochemical properties of this protein have been studied.

The pattern of urinary catecholamines and their metabolites in Duchenne myopathy, in relation to disease evolution.

In this report we have tried to determine whether or not catecholamines are involved in the progressive muscular dystrophy. Catecholamines and their metabolites were studied in urines of children with Duchenne disease or other forms of myopathy (limb-girdle and facio-scapulo humeral myopathies). Catecholamine deaminated metabolites were normal in either form of myopathy; in contrast, Duchenne patients, contrarily to other children, eliminated excessive amounts of most amines (catecholamines and methoxylated amines) in relation to age and degree of disease evolution. Our results indicate that catecholamines are not the primary factors involved in the pathogenesis of Duchenne myopathy, but are rather secondary to some disease effects. It is suggested that the high excretion of catecholamines and their methoxylated amine metabolites observed in severely affected Duchenne boys might be related to thermoregulatory process or/and to alterations in some enzymatic systems.

[Diphenoloxydase (DPOx) in red cell membranes]. / Etude des diphénoloxydases (DPOx) dans les membranes de globules rouges.

Two diphenoloxydases (DPOx) with similar molecular weights (158 000) are found in normal human red cell hemolysates. One of these enzymes appears to be tightly bound to the membranes while the other is not, or at most only weakly bound.

Progressive muscular dystrophy. Functional improvement after a renal allograft.

From his first years a child showed signs of a primary and rapidly developing muscular dystrophy. The diagnosis was established by an increased serum CK level and by electromyography and muscle biopsies. Afterwards this child developed a severe renal deficiency which needed binephrectomy and the graft of a normal kidney. During the few months just after the graft, the disability increased and the patient could not stand upright by himself. Later on, he gradually became able to walk on his own and without bracing. He could climb stairs and stand up from the floor. The CK activity returned to normal. At present, 4 years after the graft (the patient is 16 years), the improvement of his functional abilities is constant, although the CK activity has increased again. In this article we give evidence that this patient suffers from a primary muscular dystrophy. We discuss the type of dystrophy concerned. We believe that it is the graft of a normal kidney which was responsible for the improvement observed, and not the physiotherapy or the drugs administered after the graft.