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D Paulin

Publications and source records attributed to D Paulin.

At least 55 records · Page 3Linked to original sources
PubMed1997

Desmin is essential for the tensile strength and integrity of myofibrils but not for myogenic commitment, differentiation, and fusion of skeletal muscle.

A null mutation was introduced into the mouse desmin gene by homologous recombination. The desmin knockout mice (Des -/-) develop normally and are fertile. However, defects were observed after birth in skeletal, smooth, and cardiac muscles (Li, Z., E. Colucci-Guyon, M. Pincon-Raymond, M. Mericskay, S. Pournin, D. Paulin, and C. Babinet. 1996. Dev. Biol. 175:362-366; Milner, D.J., G. Weitzer, D. Tran, A. Bradley, and Y. Capetanaki. 1996. J. Cell Biol. 134:1255- 1270). In the present study we have carried out a detailed analysis of somitogenesis, muscle formation, maturation, degeneration, and regeneration in Des -/- mice. Our results demonstrate that all early stages of muscle differentiation and cell fusion occur normally. However, after birth, modifications were observed essentially in weight-bearing muscles such as the soleus or continually used muscles such as the diaphragm and the heart. In the absence of desmin, mice were weaker and fatigued more easily. The lack of desmin renders these fibers more susceptible to damage during contraction. We observed a process of degeneration of myofibers, accompanied by macrophage infiltration, and followed by a process of regeneration. These cycles of degeneration and regeneration resulted in a relative increase in slow myosin heavy chain (MHC) and decrease in fast MHC. Interestingly, this second wave of myofibrillogenesis during regeneration was often aberrant and showed signs of disorganization. Subsarcolemmal accumulation of mitochondria were also observed in these muscles. The lack of desmin was not compensated by an upregulation of vimentin in these mice either during development or regeneration. Absence of desmin filaments within the sarcomere does not interfere with primary muscle formation or regeneration. However, myofibrillogenesis in regenerating fibers is often abortive, indicating that desmin may be implicated in this repair process. The results presented here show that desmin is essential to maintain the structural integrity of highly solicited skeletal muscle.

Adenosine Triphosphatases↗
PubMed1997

Increase in the proliferative capacity of human myoblasts by using the T antigen under the vimentin promoter control.

Normal myoblasts have a strictly limited growth potential and senesce after a defined number of population doubling. The objective of this study was to determine whether the proliferative capacity of human myoblasts could be extended without inhibiting myogenic differentiation. We have established a stable transfected human myoblast cell line that expresses the SV 40 large T antigen under the control of the human vimentin promoter. We show that these cells have an increased proliferative capacity compared with that of normal myoblasts. Indeed, the final proliferative capacity was increased to 19 passages (5 for normal myoblasts). Moreover, they retained their capacity to differentiate fully, as indicated by their morphology and electrophysiological properties as well as by the expression of different markers of differentiation. The generation of human myogenic cell lines with the ability to proliferate for a longer period of time than primary myoblasts and while retaining the capacity to differentiate into myotubes could provide a valuable tool for the derivation of cell lines from human diseased muscle cells.

Antigens, Viral, Tumor↗
PubMed1997

Null mutation in the desmin gene gives rise to a cardiomyopathy.

A null mutation in the desmin gene has been introduced into the germ line of mice. Such mice develop and reproduce normally proving that desmin is not needed either for the formation of the heart or the alignment of functioning myofibrils. However, cardiovascular lesions and a skeletal myopathy were observed in growing and adult mice. In the present study we have carried out a detailed analysis of these cardiac lesions. Homozygous mutant mice, which were confirmed to lack expression of desmin mRNA and desmin protein in the heart, were revealed by electron microscopy to contain degenerating cardiomyocytes as early as 5 days post-partum. At 10 days post-partum and onwards the degeneration of cardiomyocytes gave rise to areas with an accumulation of macrophages, fibrosis and calcification preferentially in the inter-ventricular septum and the free wall of the right ventricle. The localization of the lesions mainly to these sites suggested that it is not the work load and contractions per se which were the pathogenic events leading to the cardiomyopathy. It might be that stress related to lengthening of the myocytes occur more in the right ventricle than in the left. At the ultrastructural level changes in the intercalated discs, disruption of the sarcolemma and supercontraction of myofibrils seemed to be the key events leading to cardiomyocyte death. Thus, the intermediate filaments are required to maintain the basic integrity of cardiomyocytes and especially the link between the intermediate filaments and the sarcolemma appear more important than previously realized.

Animals↗
PubMed1997

Desmin-lacZ transgene expression and regeneration within skeletal muscle transplants.

The purpose of this study was to investigate the initiation and time course of the regeneration process in fragments of skeletal muscle transplants as a function of muscle tissue age at implantation. The appearance of desmin occurs at the very beginning of myogenesis. The transgenic desmin nls lacZ mice used in the study bear a transgene in which the 1 kb DNA 5' regulatory sequence of the desmin gene is linked to a reporter gene coding for Escherichia coli beta-galactosidase. The desmin lacZ transgene labels muscle cells in which the desmin synthesis programme has commenced. We implanted pectoralis muscle fragments from fetal transgenic embryos and mature and old transgenic mice into mature non-transgenic mice. Early events of myogenesis occurring during regeneration started sooner in transplants from 4-month-old (day 3 post-implantation) muscle than in those from 24-month-old (day 5-6 post-implantation) muscle, and they lasted longer in those from young (day 17 post-implantation) than in those from old (day 14 post-implantation) muscle fragments. In adult muscle, transgene activation proceeded from the periphery toward the centre of the transplant. In transplants from fetal 18-day-old pectoralis, myotubes with transgene activity were observed from day 1 to day 19. Desmin immunoreactivity, which appeared about one day after transgene activation, was followed by myosin expression. In adult transplants, the continuity of laminin labelling was disrupted around degenerative fibres, illustrating alteration of the extracellular matrix. Our data suggest that satellite cells from old muscle tissue have lower proliferative capacity and/or less access to trophic substances released by the host (damaged fibres, vascularization) than those from fetal or young adult muscle.

Animals↗
PubMed1997

Characterization of a newly established human bone marrow endothelial cell line: distinct adhesive properties for hematopoietic progenitors compared with human umbilical vein endothelial cells.

Human bone marrow endothelial cells (HBMEC) are intimately involved in the homing of hematopoietic progenitor cells (HPC) to the bone marrow and in the regulation of proliferation and differentiation of these cells. Because availability of primary HBMEC and their capacity to be cultured in vitro are limited, we used isolated HBMEC to establish a cloned cell line by microinjection of a recombinant plasmid expressing simian virus 40 early genes under the control of a deletion mutant of the human vimentin promoter. Serum requirements for growth of a transformed HBMEC line (TrHBMEC) were markedly decreased compared with those of primary cells, and added growth factors were not required for proliferation. Cells took up acetylated low-density lipoprotein normally, bound to Ulex europaeus lectin, and stained positively for von Willebrand factor, P-selectin, CD31, CD34, CD44, very late antigen-5, and intercellular adhesion molecule-2 (ICAM-2). After treatment with TNF-alpha or lipopolysaccharide, TrHBMEC increased surface expression of E-selectin, vascular cell adhesion molecule-1 (VCAM-1), and ICAM-1 in a manner similar to primary HBMEC. In contrast, IL-1 beta elicited much less up-regulation of these adhesion molecules than in primary cells. In previous work, we reported that, in a flow adhesion model, rolling of peripheral blood CD34+ cells on primary HBMEC was E-selectin-dependent, whereas VCAM-1 and ICAM-1 contributed to firm adhesion. In the present study, we show that HPC adhere in a similar way to TrHBMEC. A less-pronounced role for VCAM-1 and ICAM-1 was found in the adhesion of HPC to human umbilical vein endothelial cells. Furthermore, significantly more CD34+ cells adhered to TNF-alpha-stimulated HBMEC and TrHBMEC than to similarly stimulated human umbilical vein endothelial cells. These data emphasize the importance of using microvessel HBMEC for studying the homing of HPC to the bone marrow, and indicate the usefulness of the above-described bone marrow endothelial cell line.

Antigens, CD↗
PubMed1997

The importance of intermediate filaments in the adaptation of tissues to mechanical stress: evidence from gene knockout studies.

Research over the past few years on the function of intermediate filaments in cells in culture has not produced convincing results, because the key role of intermediate filaments is within tissues and at certain periods of development. Only recently the technique of gene knockout has been used to examine intermediate filaments in mice and has provided the first evidence that intermediate filaments are directly involved in cell resilience and the maintenance of tissue integrity. Knockout of the gene encoding keratin K8 is lethal in the embryo, and results in hepatic or intestinal lesions, while knockout of the K14 or K10 genes leads to rupture of stratified epithelia. Knockout of the gene encoding desmin causes the rupture of skeletal and cardiac muscle, and collapse of blood vessel walls. Knockout of the gene coding for GFAP leads to a loss of cerebral white matter, and knockout of the gene coding for vimentin causes degeneration of the cerebellar Purkinje cells. The results reveal the lack of compensation by another intermediate filament. Tissues without intermediate filaments fall apart; they are mechanically unstable, unable to resist physical stress, and this leads to cell degeneration. By maintaining the shape and plasticity of the cell, the intermediate filament network acts as an integrator within the cell space. The state of mechanical force imposed on a tissue or a cell can alter the shape of certain elements of the cytoskeleton and thus participate to the control of cell functions.

Adaptation, Physiological↗
PubMed1996

PEA3 transactivates vimentin promoter in mammary epithelial and tumor cells.

We have used differential display RT-PCR method to detect the genes specifically activated or repressed between mammary tumor and normal mammary epithelial cells. One of the genes identified is vimentin. The vimentin gene is abundantly expressed in both human and mouse mammary tumor cells and its expression decreased dramatically in normal mammary epithelial cells. The expression of vimentin gene correlates with the expression of transcription factor PEA3. Since the promoters of human and mouse vimentin genes contain one PEA3 binding site we investigated the ability of PEA3 to transactivate the vimentin promoter in mouse mammary epithelial cell CLS1, mouse mammary tumor MMT and human mammary tumor cell lines MCF7 and MDA231. Our results suggest that PEA3 specifically transactivates vimentin promoter through PEA3 site. Among members of the ETS transcription factor family only Erg showed ability to transactivate vimentin promoter besides PEA3. Our results also suggest that NFkB site on the vimentin promoter may act as a positive regulatory element for the transcription of vimentin. In metastatic mammary tumors derived from mice carrying the polyoma middle T or neu transgene, PEA3 is overexpressed and vimentin has been shown to play a key role in the motility of cells. Our results suggest that one of the roles of PEA3 in mammary tumor is to participate the activation of vimentin gene whose gene product in turn contributes to the metastatic potential of mammary tumors.

Animals↗
PubMed1996

Cardiovascular lesions and skeletal myopathy in mice lacking desmin.

In order to further our understanding of the biological role of desmin, the muscle-specific intermediate filament protein, a null mutation in the desmin gene was introduced into the germ line of mice. Despite the complete lack of desmin, these mice developed and reproduced. Since we show that skeletal, cardiac, and smooth muscles form in the Des-/- mice, it is reasonable to propose that desmin is not essential for myogenic commitment or for myoblast fusion or differentiation in vivo. However, morphological abnormalities were observed in the diaphragm of adult mice; these were demonstrated by disorganized, distended, and nonaligned fibers. The heart presented areas of hemorrhaging in which fibrosis and ischemia were observed. We have also shown that the absence of desmin produces specific defects in smooth muscles. In conclusion, our results have demonstrated that desmin is not required for the differentiation of skeletal, cardiac, and smooth muscles but is essential to strengthen and maintain the integrity of these tissues.

Animals↗
PubMed1996

A 28-bp negative element with multiple factor-binding activity controls expression of the vimentin-encoding gene.

The promoter of the human vimentin-encoding gene (VIM) contains two enhancers separated by a negative region. The distal and proximal enhancers bind the transcription factors, AP-1 and NK-kappaB, respectively, which contribute to serum induction of Vim synthesis. We were interested in looking for particular regulatory elements that might be responsible for tissue-specific extinction and culture-dependent activation of human VIM. We have identified a 48-bp sequence in the distal enhancer which had not been reported before. This sequence includes a negative element, NE2, which confers transcriptional repression in transfection experiments and binds at least two factors in vitro. NE2 may participate in the differentiation-stage-specific control of VIM expression which involves multiple regulatory sequences and several positive and negative trans-acting factors.

Base Composition↗
PubMed1996

Relationships between intermediate filaments and cell-specific functions in renal cell lines derived from transgenic mice harboring the temperature-sensitive T antigen.

Four renal cell lines were derived from glomeruli, proximal, distal, and cortical collecting tubules microdissected from the kidneys of transgenic mice carrying the temperature-sensitive mutant of the simian virus 40 large T antigen under the control of the vimentin promoter. All four cell lines contained large T antigen in their nuclei, grew rapidly, and contained vimentin filaments when grown in serum-enriched medium at the permissive temperature of 33 degrees C. The glomerular cell line formed multiple layers of cells and contained smooth muscle actin and desmin filaments, features of mesangial cells. The three tubule cell lines formed monolayers of polarized cuboid cells separated by tight junctions and having a patchy distribution of cytokeratins K8-K18. A shift from 33 degrees C to the restrictive temperature (39.5 degrees C) stopped cell growth in all cell lines and caused profound changes in the content of intermediate filaments. Vimentin was still present in mesangial-like cells, but the proximal, distal, and collecting tubule cells contained uniform networks of cytokeratins K8-K18 and desmoplakin I and II around the cell peripheries. Potassium transport, mediated by Na+-K+ ATPase pumps and specific cAMP hormonal sensitivities, significantly increased in proximal, distal, and collecting tubule cells when shifted from 33 degrees C to 39.5 degrees C. Thus, the temperature-dependent inactivation of large T antigen, responsible for the arrest of cell growth, did not affect the phenotype of mesangial-like glomerular cells but induced some changes in the expression of intermediate filaments and restored, at least partially, the main parental cell-specific functions in proximal, distal, and collecting tubule cultured cells.

Animals↗
PubMed1996

Human desmin gene: cDNA sequence, regional localization and exclusion of the locus in a familial desmin-related myopathy.

Desmin is a muscle-specific intermediate filament that is encoded by a gene assigned to human chromosome 2q35. Desmin-related myopathies are inherited disorders characterized by an intrasarcoplasmic accumulation of desmin. Recently, the knockout of the desmin gene was shown to generate a myopathic syndrome in transgenic mice, suggesting that functional abnormality of desmin may generate similar clinical symptoms in mouse and human. To determine the potential role of the desmin gene in a well-defined desmin-related myopathy (autosomal dominant form of Fardeau), human desmin cDNAs obtained from affected and unaffected individuals were cloned, sequenced and compared. No obvious mutation was detected. A BssHII restriction fragment length polymorphism (RFLP) was identified in exon 6 of the desmin gene. This RFLP was associated with a previously identified EcoRV RFLP in exon 4 to generate a tetra-allelic system, which was tested for linkage to the desmin-related myopathy in three families. The human desmin gene was localized within an 11-cM interval on chromosome 2q using a panel of radiation hybrids. This 11-cM region was clearly excluded by linkage analysis in the three desmin-related myopathy families using a set of highly polymorphic microsatellite markers. These results suggest that the desmin gene is not primarily involved in this disease.

Alternative Splicing↗
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

Protective effect of platelet activating factor antagonists on cultured endothelial cell lysis induced by elastase or activated neutrophils.

1. The mechanism(s) responsible for injury of endothelial cells induced by human leukocyte elastase (HLE) was investigated in an immortalized venous human endothelial cell line (IVEC). 2. First, the proteinase concentrations and incubation delays necessary to trigger a significant IVEC cytotoxicity were determined by chromium assays. Thus, exposure of IVEC for 6 h to 10 micrograms ml-1 HLE resulted in 22 +/- 2.8% lysis and 36.4 +/- 5.4% detachment (mean +/- s.e. mean; n = 4; P < 0.05). 3. WEB 2086, a specific platelet-activating factor (PAF) receptor antagonist, induced a significant concentration-dependent decrease of such a lysis (39.6 +/- 7.7% protection at 100 microM; n = 4). This potential role for PAF was confirmed with two other antagonists of this lipid mediator, i.e., BN 52021 and RP 48740. 4. Finally, we demonstrated that pretreatment of IVEC with WEB 2086 protected significantly against cell lysis induced by stimulated human neutrophils, an experimental model in which HLE participates.

Azepines↗
PubMed1996

The SM 22 promoter directs tissue-specific expression in arterial but not in venous or visceral smooth muscle cells in transgenic mice.

The transcriptional signals underlying smooth muscle differentiation are currently unknown. We report here the complete sequence and characterization of the single mouse gene for the smooth muscle-specific protein SM 22 and the transcriptional activity of its promoter in cultured smooth muscle cells in vitro and in transgenic mice. In the transgenic animals, promoter constructs ranging in length from 445 to 2126 bp directed reporter expression initially in the heart and the somites of embryos and subsequently in the arteries of the vascular system, but in none of the visceral muscles, nor in the veins. Expression in the heart was spatially restricted to the presumptive right ventricle and outflow tract and disappeared in the adult. Likewise, expression in the somites was only transitory and was not observed after about 14.5 days post coitum in the embryo. In the adult mouse, SM 22 promoter activity persisted in the smooth muscle cells of the arteries and was still notably absent from other smooth muscles, despite the ubiquitous presence of the endogenous SM 22 protein. These findings on the transcriptional activity of a smooth muscle promoter in vivo reveal the existence of different differentiation programmes for smooth muscle cells in the veins and the arteries and raise the expectation of a further subdivision of programmes among the visceral muscles.

Animals↗
PubMed1996

Targeting widespread sites of damage in dystrophic muscle: engrafted macrophages as potential shuttles.

Inherited muscle diseases are characterized by widespread muscle damage in the body. This limits the clinical relevance of cell or gene therapy based upon direct injections into muscles. One way to circumvent this obstacle would be to use circulating cells, capable of homing naturally to the sites of lesion, to deliver therapeutic substances. Certain muscular dystrophies present successive cycles of degeneration-regeneration. These sporadic necrotic lesions trigger local inflammations with subsequent infiltration of blood-borne mononuclear cells. We have, therefore, tested the possibility that homing monocytes and macrophages could be appropriate shuttles for delivering a therapeutic agent to disseminated pathogenic sites, their targeting being triggered by the pathogeny itself. First, fluorescently labeled immortalized monocytes were intravenously injected into mice which had previously undergone freeze-damaging of individual muscles. In agreement with our hypothesis, intense labelling was observed in the muscle, specifically in damaged regions. Second, the technique was adapted to meet the needs of chronic diseases with characteristic continuous, widespread degeneration of muscle fibers, by creating a reservoir of genetically engineered monocytes, via bone marrow transplantation. Mdx mice received bone marrow from transgenic mice expressing the lacZ reporter gene, under the control of the vimentin promoter, which is active in monocytes and macrophages. Histological and molecular analyses demonstrated the homing of engineered macrophages at the sites of muscle damage, for periods as long as 2 months. Bone marrow progenitor cells, appropriately engineered to elicit the synthesis, in macrophages, of therapeutically relevant substances, may be of clinical value in various pathologies involving an inflammatory phase.

Animals↗
PubMed1995

Myotube driven myogenic recruitment of cells during in vitro myogenesis.

Muscular dysgenesis (mdg) is a recessive lethal mutation in the mouse which drastically affects skeletal muscle development during embryonic life. Physiologically, the disease is characterized by a complete paralysis resulting from a lack of excitation-contraction coupling. Existing electrophysiological, biochemical, and genetic evidence shows that mdg/mdg mice express a basic alteration of L-type voltage-sensitive Ca2+ channels in skeletal muscle. Studies on mdg/mdg myotubes in primary culture have shown that +/+ fibroblasts or +/+ Schwann cells may fuse with them and correct their functional deficiency by genetic complementation. As the spontaneous formation of heterocaryons is thought to be an exclusive property of myoblasts, we asked whether fibroblasts may have changed their properties before fusion occurred. We used primary cells issued from sciatic nerves dissected from newborn transgenic mice carrying the pHuDes1-nls-LacZ transgene (Des-LacZ cells) as non-muscle cells. These cells were mainly fibroblasts (80%) positive for Thy1.1 and Schwann cells positive for S100. The cultures were negative for myogenic markers (desmin, troponin T), did not form myotubes long-term, and did not display significant activation of the muscle reporter gene (pHuDes1-nls-LacZ). After a few days in coculture with dysgenic or normal myotubes, the muscle reporter gene (beta-galactosidase) was detected both within dysgenic myotubes, correlating with the restoration of normal contractile activity, and normal myotubes. As well as confirming that fusion takes place, this shows that Des-LacZ cells nuclei incorporated into recipient myotubes express their own myogenic genes. Moreover, individual mononucleated Des-LacZ cells expressing beta-galactosidase were observed, indicating that myogenic genes were being expressed before fusion. This suggests a mechanism of myotube driven myogenic recruitment of cells during the in vitro myogenesis. Analysis of the distribution of the induced Des-LacZ cells (positive for beta-galactosidase) in compartmentalized muscle cocultures showed that in the presence of dysgenic myotubes, these cells were equally distributed in both myotube free and enriched areas, whereas in the presence of normal myotubes, the positive cells remained in close vicinity of the myotubes. This difference could be explained by the fact that the dysgenic phenotype might include release of the induction process from its normal controls. Our results are consistent with the idea of a transcellular mechanism triggering myogenic differentiation in non-muscle cells, and that myotubes themselves are able to drive myogenic recruitment of cells during the in vitro myogenesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Migration of myogenic cells from the somites to the fore-limb buds of developing mouse embryos.

In this study, we have isolated newly formed somites from the caudal regions of 8.5 day mouse embryos and transplanted them orthotopically into correspondingly staged hosts at the level of the prospective limb-forming region. The experimental embryos were then cultured intact for 32-36 hr. The donor somites used were pre-labelled with DiI, a fluorescent lipophilic dye, or were obtained from transgenic embryos that carried a 1 kb 5' regulatory sequence of the desmin gene linked to the gene encoding Escherichia coli beta-galactosidase. The transgene is specifically expressed in skeletal muscles (Li et al. [1993] Development 117:947-959). The aim of these experiments was to show definitively that the musculature of the mammalian limb is derived from the somites. The results demonstrated that DiI-labelled cells from the implanted somites were able to invade the proximal region of the fore-limb bud during the course of development. The use of transgenic somites as grafts confirmed that some of the somitic cells found in the limbs were myogenic cells. To determine whether the displacement of somitic cells is an active or passive process, somatopleure obtained from the prospective limb-forming regions of day 8.5 day embryos was implanted into 8.5 day hosts. We did not detect the presence of DiI-labelled somatopleural cells in the fore-limb after 32-36 hr of culture. This suggests that somitic cells reached the limb bud via active locomotion rather than as a result of being passively dragged there, as the limb elongates during development. In addition, we injected latex beads into the somites, as probes, to determine whether extracellular matrix-driven translocation plays a role in driving the somitic cells to the limb bud. In a majority of the specimens examined, we could not detect the presence of these beads in the limb bud. However, in the trunk of these embryos, the beads were found dispersed throughout the ventral neural crest pathway.

Animals↗