ABSTRACT
The enumeration of absolute cell numbers and cell proliferation in clinical samples is important for diagnostic and research purposes. Detection of cellular DNA with fluorescent dyes is the most commonly used approach for cell enumeration in cytometry. Inductively coupled plasma mass spectrometry (ICPMS) has been recently introduced to the field of protein and cell surface antigen identification via ICPMS-linked immunoassays using element-labeled affinity reagents such as gold and lanthanide-conjugated antibodies. In the present work, we describe novel methods for using metallointercalators that irreversibly bind DNA and low concentrations of rare earth metals added to cell growth media for rapid and sensitive measurement of cell numbers by mass spectrometry. We show that Ir- and Rh-containing metallointercalators are useful reagents for labeling cells and normalizing signals when studying antigen expression on different types and numbers of cells. Results are presented for solution analysis performed by conventional ICPMS and compared to measurements obtained on the novel flow cytometer mass spectrometer (FC-MS) instrument, designed to analyze multiple antigens and DNA simultaneously in single cells.
Subject(s)
Antigens/analysis , Antigens/chemistry , DNA/analysis , DNA/chemistry , Intercalating Agents/chemistry , Mass Spectrometry/methods , Antigens/immunology , Biomarkers , Cell Line , Cell Survival/drug effects , Humans , Lead/pharmacology , Molecular Structure , Palladium/pharmacology , Rhodium/pharmacologyABSTRACT
Myocyte enhancer factor 2 (MEF2) transcriptional regulatory proteins are key regulators of muscle-specific gene expression and also play a general role in the cellular response to growth factors, cytokines and environmental stressors. To identify signaling pathway components that might mediate these events, the potential role of MAP kinase and PKC signaling in the modulation of MEF2A phosphorylation and transcriptional activity were therefore studied. In transient transfection reporter assays, activated p38 MAP kinase potently increased MEF2A trans -activating potential, PKC[delta] and [epsiv] isotypes enhanced MEF2A transactivation to a lesser extent, while the ERK1/2 and JNK/SAPK pathways were without effect. A GAL4-based assay system showed that p38 MAP kinase and PKC[delta] target the MEF2A transactivation domain. We also observed an increase in p38 MAP kinase activity in congruence with the increase in MEF2A expression in differentiating primary muscle cells. COS cells overexpressing MEF2A alone or with one of the kinases were metabolically labeled with [32P]orthophosphate and MEF2A was immunoprecipitated using specific anti-MEF2A antibodies. MEF2A from cells co-transfected with activated p38 MAP kinase showed a decreased electrophoretic mobility due to phosphorylation. Subsequent phosphopeptide mapping and phosphoamino acid analysis indicated the appearance of several phoshopeptides due to p38 MAP kinase activation of MEF2A which were due to phosphorylation on serine and threonine residues. These studies position MEF2A as a nuclear target for the p38 MAP kinase signaling pathway.
Subject(s)
DNA-Binding Proteins/metabolism , Mitogen-Activated Protein Kinases , Protein Processing, Post-Translational , Signal Transduction , Transcription Factors/metabolism , Animals , COS Cells , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , DNA-Binding Proteins/genetics , HeLa Cells , Humans , MADS Domain Proteins , MEF2 Transcription Factors , Myogenic Regulatory Factors , Phosphorylation , Protein Kinase C/metabolism , Signal Transduction/genetics , Transcription Factors/genetics , Transcriptional Activation , p38 Mitogen-Activated Protein KinasesABSTRACT
Myocyte enhancer factor 2 (MEF2) has been implicated in the complex hierarchical regulation of muscle-specific gene expression and differentiation. While the MyoD family members are able to initiate the skeletal muscle differentiation program, whether MEF2 is sufficient in directing skeletal muscle differentiation is still controversial. Furthermore, how MEF2 transactivates its target genes is not fully understood. It has been suggested that the interactions of MEF2 with other factors modify its transcriptional activity. Therefore, the identification of MEF2-interacting factors may be important in understanding the mechanism by which MEF2 activates its target genes. In this study, a mitogen-activated protein kinase (MAP kinase), ERK5/BMK1 was found to interact with MEF2 in a yeast two hybrid screen. The interaction was confirmed by a glutathione S -transferase-pull down assay and a co-immunoprecipitation study indicating that endogenous ERK5 and MEF2 interact with each other in vivo . The interacting domain of MEF2 was mapped to the N-terminus which contains the highly conserved MADS and MEF2 domains. Functionally, ERK5/BMK1 was able to phosphorylate MEF2 in vitro . Furthermore, when cotransfected with ERK5/BMK1, the transactivation capacity of MEF2 was enhanced. These results suggest that the functions of MEF2 could be regulated through ERK5/BMK1.
Subject(s)
Calcium-Calmodulin-Dependent Protein Kinases/metabolism , DNA-Binding Proteins/metabolism , Mitogen-Activated Protein Kinases , Transcription Factors/metabolism , Amino Acid Sequence , Animals , Aorta/metabolism , COS Cells , Calcium-Calmodulin-Dependent Protein Kinases/genetics , Conserved Sequence , DNA-Binding Proteins/genetics , Gene Expression Regulation , Gene Library , HeLa Cells , Humans , MEF2 Transcription Factors , Mitogen-Activated Protein Kinase 7 , Molecular Sequence Data , Myogenic Regulatory Factors , Phosphorylation , Precipitin Tests , Protein Binding , Rats , Rats, Wistar , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/isolation & purification , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Transcription Factors/genetics , TransfectionABSTRACT
A subtractive hybridization and cloning strategy was used to identify genes that are up-regulated in regenerating compared with normal skeletal muscle. The gastrocnemius muscle of CD1 mice was injected with a myotoxic agent (BaCl2). A cDNA library was constructed from the regenerating muscle, and was screened with subtracted probes enriched in genes up-regulated during regeneration. Cofilin and vimentin cDNA clones were isolated. Both cofilin and vimentin were demonstrated to be overexpressed in regenerating compared with non-regenerating muscle (17-fold and 19-fold induction respectively). Cofilin and vimentin mRNAs also exhibited an increased expression in C2C12 myoblasts and a decreased expression in differentiated myotubes. Analysis of the regeneration-induced vimentin enhancer/promoter region revealed a consensus binding site for the myocyte enhancer factor 2 (MEF2) transcription factors. Electrophoretic mobility-shift assays and in vivo reporter assays revealed that MEF2 DNA-binding activity and transcriptional activation are increased in regenerating skeletal muscle, indicating that they may play a role in the activation of muscle genes during regeneration. These data suggest that both cofilin (an actin-regulatory protein) and vimentin (an intermediate filament) may be key components of the cytoskeletal reorganization that mediates muscle cell development and adult skeletal-muscle repair.
Subject(s)
Cytoskeletal Proteins/biosynthesis , DNA-Binding Proteins/metabolism , Gene Expression Regulation , Microfilament Proteins/biosynthesis , Muscle, Skeletal/metabolism , Nerve Tissue Proteins/biosynthesis , Regeneration , Transcription Factors/metabolism , Vimentin/biosynthesis , Actin Depolymerizing Factors , Animals , Base Sequence , Binding Sites , Cloning, Molecular , Consensus Sequence , Enhancer Elements, Genetic , MEF2 Transcription Factors , Mice , Myogenic Regulatory Factors , Promoter Regions, Genetic , Protein Biosynthesis , RNA, Messenger/biosynthesis , Transcription, Genetic , Vimentin/geneticsABSTRACT
A biological role for MEF2 (myocyte enhancer factor 2) activity during mammalian myogenesis has been inferred but not directly proven because of its role in the transcriptional activation of many muscle-specific genes. Therefore, our purpose was to determine whether MEF2 activity is absolutely required for mammalian myogenesis. Using a dominant-negative approach to address this question, we constructed a mutated MEF2A protein comprised of the amino-terminal DNA binding/dimerization domain of MEF2A without its trans-activation domain as a bacterial fusion protein (GST-131) or in a eukaryotic expression vector (pcDNA-131). GST-131 and the protein encoded by pcDNA-131 bind specifically to the MEF2 cis element and abrogate trans-activation of a MEF2-responsive luciferase reporter gene by wild type MEF2A, thus serving a role as trans-dominant inhibitors of MEF2 function. In congruence with their ability to interfere with wild type MEF2 function, microinjection of GST-131 or pcDNA-131 into L6E9 or C2C12 myoblasts inhibited myotube formation. Immunofluorescence analysis showed that the expression of myogenin, myosin heavy chain, and MEF2A were inhibited in the GST-131 or pcDNA-131-injected cells compared with GST or pcDNA-injected controls. We also document that this trans-dominant MEF2 inhibitor impairs the myogenic conversion of C3H10T1/2 fibroblasts by MyoD. Thus, these data provide evidence that the trans-activation function of the MEF2 proteins during mammalian myogenesis is required for muscle-specific gene expression and differentiation.
Subject(s)
DNA-Binding Proteins/antagonists & inhibitors , Muscle Development , Transcription Factors/antagonists & inhibitors , DNA/metabolism , Glutathione Transferase/genetics , Glutathione Transferase/metabolism , HeLa Cells , Humans , MADS Domain Proteins , MEF2 Transcription Factors , Microinjections , MyoD Protein/antagonists & inhibitors , Myogenic Regulatory Factors , Myogenin/pharmacology , Recombinant Fusion Proteins/administration & dosage , Recombinant Fusion Proteins/pharmacology , Transcriptional ActivationABSTRACT
Tissue-specific gene expression can be mediated by complex transcriptional regulatory mechanisms. Based on the dichotomy of the ubiquitous distribution of the myocyte enhancer factor 2 (MEF2) gene mRNAs compared to their cell type-restricted activity, we investigated the basis for their tissue specificity. Electrophoretic mobility shift assays using the muscle creatine kinase MEF2 DNA binding site as a probe showed that HeLa, Schneider, L6E9 muscle, and C2C12 muscle cells have a functional MEF2 binding activity that is indistinguishable based on competition analysis. Interestingly, chloramphenicol acetyltransferase reporter assays showed MEF2 site-dependent trans-activation in myogenic C2C12 cells but no trans-activation by the endogenous MEF2 proteins in HeLa cells. By immunofluorescence, we detected abundant nuclear localized MEF2A and MEF2D protein expression in HeLa cells and C2C12 muscle cells. Using immuno-gel shift analysis and also co-immunoprecipitation studies, we show that the predominant MEF2 DNA binding complex bound to MEF2 sites from either the muscle creatine kinase or c-jun regulatory regions in C2C12 muscle cells is comprised of a MEF2A homodimer, whereas in HeLa cells, it is a MEF2A:MEF2D heterodimer. Thus, the presence of MEF2 DNA binding complexes is not necessarily coupled with trans-activation of target genes. The ability of the MEF2 proteins to activate transcription in vivo correlates with the specific dimer composition of the DNA binding complex and the cellular context.
Subject(s)
DNA-Binding Proteins/genetics , DNA/metabolism , Muscles/metabolism , Transcription Factors/genetics , Transcription, Genetic , Biopolymers , Cell Nucleus/metabolism , DNA-Binding Proteins/metabolism , HeLa Cells , Humans , MADS Domain Proteins , MEF2 Transcription Factors , Muscles/cytology , Myogenic Regulatory Factors , Phosphorylation , Transcription Factors/metabolismABSTRACT
Dystrophin is a subsarcolemmal protein that interacts with cytoskeletal actin and a glycoprotein complex in the plasma membrane. One potential function of dystrophin is its ability to stabilize the sarcolemmal membrane during muscle contraction. We hypothesized 1) that chronic muscle use and disuse would alter the expression of dystrophin as a compensatory mechanism designed to prevent muscle damage, and 2) that other subsarcolemmal cytoskeletal proteins (vinculin, M-vinculin, aciculin 60/63 kDa) that colocalize with dystrophin in muscle adherens junctions would be changed in parallel. Chronic muscle use induced by voluntary running or 10-Hz chronic stimulation did not alter dystrophin levels in rat muscle. In contrast, muscle disuse induced by 6 d of microgravity, or 7 and 21 d of denervation, increased dystrophin levels by 1.8-, 1.9- and 3.2-fold, respectively. Thus, this increase in dystrophin levels appears to be dependent on the duration of muscle disuse, independent of the presence of the nerve. Denervation also induced 3.3-fold increases in vinculin and aciculin 60 kDa, in parallel with dystrophin. However, in contrast to its effects on dystrophin, chronic stimulation increased the levels of vinculin and aciculin 60 kDa by 3.4- and 6.4-fold, respectively. Thus, both the removal and the augmentation of muscle activity resulted in increases of these two cytoskeletal proteins. The data indicate that the concentrations of these proteins are independently regulated. They further indicate that chronic muscle use is not a stimulus for the induction of dystrophin levels, suggesting that normal levels are sufficient for the protective effect on the sarcolemma that dystrophin may confer. The results reveal an interesting area of muscle plasticity, and the adaptation observed may have profound implications for the structure and function of skeletal muscle responding to changes in contractile activity.
Subject(s)
Cytoskeletal Proteins/analysis , Dystrophin/analysis , Muscle, Skeletal/chemistry , Phosphoglucomutase , Vinculin/analysis , Adaptation, Physiological , Animals , Male , Muscle Contraction/physiology , Rats , Rats, Sprague-DawleyABSTRACT
We evaluated mitochondrial adaptations in the hearts of euthyroid and hypothyroid rats subject to aortic constriction for 2, 4, 7, 14, 21, and 28 d to induce a pressure-overload (PO), compared to sham-operated (SH) controls. PO animals attained higher arterial pressures than SH animals, by 55% in the euthyroid group, but only 14% in hypothyroid rats after 28 d. The left ventricle/body weight ratio was increased 44% by PO in the euthyroid group, and 26% in the hypothyroid group. PO attenuated the decline in cardiac growth in the hypothyroid group. Thus, hypothyroidism reduces the magnitude of the PO, but not the potential for hypertrophy in response to PO. Cytochrome c oxidase activity (CYTOX) was unchanged by PO in the euthyroid animals, indicating that the synthesis of mitochondria paralleled adaptive growth. However, CYTOX activity decreased up to 20% in the hypothyroid groups (P < 0.05) and was unaltered by PO. Thus, PO prevented the decline in growth, but not the decline in mitochondrial enzymes due to hypothyroidism. The lack of effect of PO on mitochondria was partly due to pretranslational changes since CYTOX subunit VIc mRNA was reduced by PO in the hypothyroid animals, but not in the euthyroid group. Levels of the chaperones HSP60 and GRP75, as well as HSP60 mRNA were unaffected by hypothyroidism, but paralleled adaptive growth induced by PO. Hypothyroidism changes the pattern of gene expression within the heart leading to altered mitochondrial composition. This cannot be compensated for by conditions of increased physiological demand.
Subject(s)
Aorta, Abdominal/physiopathology , Electron Transport Complex IV/metabolism , Hypothyroidism/metabolism , Mitochondria, Heart/metabolism , Animals , Animals, Newborn , Chaperonin 60 , Constriction, Pathologic , Pressure , RNA, Messenger , RatsABSTRACT
Molecular chaperones and cytosolic stress proteins are actively involved in the stabilization, import and refolding of precursor proteins into mitochondria. The purpose of the present study was to evaluate the relationship between mitochondrial content under steady-state conditions, and during the induction of organelle biogenesis, with the expression of stress proteins and mitochondrial chaperonins. A comparison of steady-state levels of mitochondrial enzyme activity [cytochrome c oxidase (CYTOX)] with chaperonin levels [the heat-shock protein HSP60, the glucose-regulated protein GRP75 (mtHSP70)] in striated muscles possessing a wide range of oxidative capacities revealed a proportional expression between the two. This relationship was disrupted by chronic contractile activity brought about by 10 days of 10 Hz stimulation of the tibialis anterior (TA) muscle, which induced 2.4-fold increases in CYTOX activity, but 3.2- and 9.3-fold increases in HSP60 and GRP75 respectively. The inducible stress protein HSP70i was detected at low levels in control TA muscle, and was increased 9.6-fold by chronic contractile activity, to values comparable with those found in the unstressed soleus muscle. This increase occurred in the absence of changes in type I MHC levels, indicating independent regulation of these genes. Despite the increases in HSP60 and HSP70i proteins, contractile activity did not alter their respective mRNA levels, illustrating post-transcriptional mechanisms of gene regulation during contractile activity. In contrast, the mRNA levels encoding the co-chaperonin CPN10 were increased 3.3-fold by contractile activity. Thus, the expression of individual mitochondrial chaperonins is independently regulated and uncoordinated. The extent of the induction of these stress proteins and chaperonins by contractile activity exceeded that of membrane enzymes (e.g. CYTOX). It remains to be determined whether this marked induction of proteins comprising part of the protein import machinery is beneficial for the translocation of enzyme precursors into the mitochondria during conditions of accelerated biogenesis.
Subject(s)
Chaperonin 60/metabolism , HSP70 Heat-Shock Proteins/metabolism , Membrane Proteins/metabolism , Mitochondria/metabolism , Muscle, Skeletal/metabolism , Animals , Chaperonin 10/genetics , Chaperonin 10/metabolism , Chaperonin 60/genetics , Electric Stimulation , Electron Transport Complex IV/metabolism , HSP70 Heat-Shock Proteins/genetics , Male , Membrane Proteins/genetics , Muscle Contraction , Muscle, Skeletal/ultrastructure , RNA, Messenger/metabolism , Rats , Rats, Sprague-DawleyABSTRACT
Existing literature provides an equivocal picture of the behavior of mitochondrial synthesis during the time course of cardiac hypertrophy. Therefore, we examined the effect of cardiac hypertrophy on mitochondrial cytochrome c oxidase (CYTOX) activity, the content of CYTOX subunit VIc mRNA, and the expression of molecular chaperones. Adult male Sprague-Dawley rats were subjected to either abdominal aortic constriction to induce pressure overload (PO) or a sham operation (SH). Animals were studied 2, 4, 7, 14, 21, or 28 days after surgery. Aortic constriction resulted in a significant evaluation in arterial pressure by 4 days after surgery. Significant (p < 0.05) hypertrophy was attained by 4 days and was stabilized at 37% between 7 and 28 days. CYTOX activity (U/g) did not differ significantly between PO and SH animals at either early (< 7 days) or later time points, indicating that mitochondrial content increased in proportion to adaptive cellular hypertrophic growth. The concentration of the molecular chaperones HSP60 and GRP75 involved in mitochondrial protein import did not change with PO treatment. The levels of mRNAs encoding both CYTOX subunit VIc and HSP60 remained constant, in proportion to cardiac growth. This suggests that the accelerated synthesis of CYTOX and HSP60 during cardiac hypertrophy is regulated transcriptionally. The data help to resolve the controversy in the literature regarding mitochondrial biogenesis during moderate, stable cardiac hypertrophy, and they indirectly indicate that proportional mitochondrial synthesis relative to cellular hypertrophy is regulated at the transcriptional level.
Subject(s)
Cardiomegaly/genetics , Cardiomegaly/metabolism , Electron Transport Complex IV/biosynthesis , Mitochondria/metabolism , Animals , Aorta/physiopathology , Hypertension/metabolism , Immunoblotting , Male , RNA, Messenger/biosynthesis , Rats , Rats, Sprague-Dawley , Time FactorsABSTRACT
A new protein was found at the site of interaction of cytoskeletal filaments and the plasma membrane in desmosomes of human cardiac muscle intercalated discs. As revealed by the indirect immunofluorescence method, monoclonal antibody XVE2 was able to stain intercalated discs of cardiac muscle and desmosomes of human skin epidermal cells, whereas it did not react with sections from human uterine smooth muscle, vascular tissue and liver. Western immunoblot analysis of extracts of total human heart and uterus demonstrated that a doublet of 65 kDa and 70 kDa polypeptides were the major proteins recognized by the monoclonal antibody XVE2. These 65 kDa and 70 kDa proteins are immunologically distinct from other known intercalated disc proteins such as vinculin, meta-vinculin, filamin, talin, alpha-actinin, desmin and desmoplakins. The distribution of the XVE2 monoclonal antibody antigens raises the possibility that these polypeptides are involved in linking intermediate filaments to the dense plaque of desmosomes of cardiac muscle intercalated discs.
Subject(s)
Muscle Proteins/metabolism , Myocardium/metabolism , Antibodies, Monoclonal , Antigens , Cell Membrane/metabolism , Cytoskeleton/metabolism , Desmosomes/metabolism , Female , Humans , Immunohistochemistry , Skin/metabolismABSTRACT
Vinculin- and caldesmon-immunoreactive forms and actin isoform patterns were studied in samples of normal and atherosclerotic human aorta. After removal of adventitia and endothelium, the remaining tissue was divided into three layers: media, muscular-elastic (adjacent to media) intima, and subendothelial (juxtaluminal) intima. In media of normal aorta, meta-vinculin accounted for 41.0 +/- 0.9% (mean +/- SEM) of total immunoreactive vinculin (meta-vinculin + vinculin); 150-kDa caldesmon accounted for 78.2 +/- 5.1% of immunoreactive caldesmon (150-kDa + 70-kDa); the fractional contents of alpha-smooth muscle actin, beta-nonmuscle, and gamma-isoactins were 49.0 +/- 0.6%, 30.4 +/- 0.6%, and 20.8 +/- 0.8%, respectively. Muscular-elastic intima was very similar to media by these criteria. In subendothelial intima, the fractional content of meta-vinculin and 150-kDa caldesmon was significantly lower (6.9 +/- 1.5% and 32.7 +/- 7.0%, respectively) than in muscular-elastic intima and media, whereas the isoactin pattern was identical to that in adjacent layers, demonstrating the smooth muscle origin of subendothelial intima cells. In atherosclerotic fibrous plaque, the fractional content of alpha-actin was decreased in subendothelial intima, rather than in media and muscular-elastic intima. Additionally, the proportion of subendothelial intima cells [i.e., the cells that express low amounts of smooth muscle phenotype markers (meta-vinculin, 150-kDa caldesmon, and alpha-actin)] in the total intima cell population increased dramatically in atherosclerotic fibrous plaque. The results suggest that changes in the relative content of meta-vinculin and 150-kDa caldesmon as well as alpha-actin in human aortic intima are associated with atherosclerosis although, in subendothelial intima of normal aorta, a certain smooth muscle cell population exists that expresses reduced amounts of "contractile" phenotype markers, even in the absence of the disease.
Subject(s)
Actins/analysis , Calmodulin-Binding Proteins/analysis , Muscle Proteins/analysis , Muscle, Smooth, Vascular/drug effects , Adult , Arteriosclerosis/metabolism , Humans , Middle Aged , Molecular Weight , Phenotype , VinculinABSTRACT
Meta-vinculin, a vinculin-related protein, has been isolated from human uterus smooth muscle. Specific antibodies to meta-vinculin, which distinguish between meta-vinculin and vinculin, were prepared by absorption of anti-meta-vinculin serum on vinculin coupled to nitrocellulose. Meta-vinculin specific antibody demonstrates only smooth and cardiac muscle specificity and is able to cross-react with a small 21-kD fragment of the meta-vinculin polypeptide chain. This antibody does not interact with protease resistant 95-kD core shared by vinculin and meta-vinculin. Meta-vinculin specific antibody was used for the localization of meta-vinculin in smooth and cardiac muscles by the indirect immunofluorescence method. At the light microscopy resolution level it was found that meta-vinculin and vinculin are localized in the same cellular adhesive structures. Meta-vinculin is present in membrane-associated microfilament-bound plaques of smooth muscle, in intercalated discs and costameres of cardiac muscle. In primary culture of smooth muscle cells from human aorta, meta-vinculin and vinculin were found to be present in focal contacts of the cells. During the cultivation of smooth muscle cells, the quantity of meta-vinculin decreased progressively and finally meta-vinculin completely disappeared from the focal contacts. The data show that in smooth and cardiac muscles meta-vinculin could be a structural component of microfilament-membrane attachment sites, defined earlier by the localization of vinculin.
Subject(s)
Membrane Proteins/analysis , Muscle Proteins/analysis , Muscle, Smooth/analysis , Myocardium/analysis , Antibody Specificity , Aorta , Cross Reactions , Electrophoresis, Polyacrylamide Gel , Female , Fluorescent Antibody Technique , Humans , Immunoassay , Immunohistochemistry , Isoelectric Focusing , Muscle Proteins/immunology , Muscle, Smooth, Vascular/analysis , Muscles/analysis , Uterus , VinculinABSTRACT
Microheterogeneity of different vinculin and meta-vinculin isoforms in adult human tissues and cultured cells was studied by two-dimensional gel electrophoresis and immunoblotting technique. Four isoforms of vinculin (alpha, alpha', beta, and gamma) and two isoforms of meta-vinculin (alpha and beta) were resolved. alpha-, alpha'-, and beta-isoforms of vinculin were found in all cell types and tissue samples analyzed in the present study. gamma-Isoform of vinculin and both alpha- and beta-isoforms of meta-vinculin were found in smooth (aorta wall and myometrium) and cardiac muscle, rather than in skeletal muscle, liver, foreskin fibroblasts, and macrophages. In the primary culture of human aorta smooth muscle cells, the fractional content of gamma-isoform of vinculin and meta-vinculin was dramatically reduced, and, by the onset of intensive cell division, the proteins could hardly be detected. Subcultured human aorta smooth muscle cells did not contain gamma-vinculin and meta-vinculin. We analyzed the microheterogeneity of vinculin and meta-vinculin in three smooth muscle layers of human aorta wall--media, muscular-elastic (adjacent to media) intima, and subendothelial (juxtaluminal) intima. It was shown that in media the fractional content of gamma-isoform of vinculin was 45% and meta-vinculin, 42%; in muscular-elastic intima the fractional content of gamma-vinculin was 42% and meta-vinculin, 36%. However, in subendothelial intima, the share of these proteins was significantly lower than in adjacent muscular-elastic intima and media. Isoactin pattern that is characteristic of smooth muscle was identical in all aortic layers, thus proving the smooth muscle origin of subendothelial intima cells. These findings demonstrate that human aortic smooth muscle cells in vivo and in vitro undergo coordinated differential expression of smooth muscle specific variants of vinculin, i.e. gamma-vinculin and meta-vinculin.
Subject(s)
Aorta/metabolism , Genetic Variation , Muscle Proteins/genetics , Muscle, Smooth, Vascular/metabolism , Adult , Cells, Cultured , Humans , Immunoassay , Isoelectric Focusing , Muscle Proteins/isolation & purification , VinculinABSTRACT
150 kDa caldesmon was shown to be characteristic of vascular smooth muscle cells in normal tissue rather than in subculture. Subcultured smooth muscle cells from human aorta contained only the 70 kDa immunoreactive form of caldesmon. During the course of primary culture the amount of 150 kDa caldesmon as well as metavinculin decreased significantly whilst 70 kDa caldesmon became the predominant form, and by the onset of cell division the 150 kDa form was practically substituted by 70 kDa caldesmon. The data show that the predominance of 150 kDa caldesmon is characteristic of contractile smooth muscle cells, while in proliferating cells 70 kDa caldesmon is expressed.
Subject(s)
Calmodulin-Binding Proteins/analysis , Muscle, Smooth, Vascular/analysis , Aorta , Cells, Cultured , Contractile Proteins/analysis , Filamins , Humans , Immunosorbent Techniques , Microfilament Proteins/analysis , Muscle Proteins/analysis , VinculinABSTRACT
We have developed a monoclonal antibody that specifically interacts with a surface antigen of human fibroblasts and smooth muscle cells. The antibody (antibody IIG10) recognizes a polypeptide of molecular mass 330,000, revealed by immunoblotting in fibroblast and smooth muscle cell extract, but not in vascular endothelial cells, peritoneal macrophages, peripheral blood lymphocytes nor hepatocytes. In tissue sections the antibody stained smooth muscle cells of myometrium, aorta and smaller blood vessels, and fibroblasts of connective tissue. Specificity of the antibody was further confirmed by double staining of aorta sections. Antibody IIG10 was used to identify smooth muscle-derived foam cells in the atherosclerotic plaque of human aorta.
Subject(s)
Aorta/pathology , Arteriosclerosis/pathology , Muscle, Smooth, Vascular/pathology , Antibodies, Monoclonal , Fluorescent Antibody Technique , Humans , Organ Culture TechniquesABSTRACT
Meta-vinculin distribution in adult human tissue was studied by immunoblotting technique. Meta-vinculin was found in smooth (aorta wall and myometrium) and cardiac muscle, rather than in skeletal muscle, liver, kidney and cultured cells - macrophages, foreskin fibroblasts, peripheral blood lymphocytes and vascular endothelial cells. In the primary culture of smooth muscle cells from human aorta the meta-vinculin/vinculin ratio was reduced, and on the onset of cell division meta-vinculin could hardly be detected. Subcultured smooth muscle cells from human aorta did not contain meta-vinculin. The data show that the presence of meta-vinculin is characteristic of 'contractile' smooth muscle cells rather than of proliferating in vitro.
Subject(s)
Muscle Proteins/analysis , Aorta/analysis , Cells, Cultured , Humans , Muscle, Smooth/analysis , Myocardium/analysis , VinculinABSTRACT
Monoclonal antibody discriminating between endothelial and smooth muscle cells is suggested to be used as a vector for directed transport of drugs to injured (denuded) areas of blood vessel wall. An in vitro model system was used in the studies: vascular smooth muscle or endothelial cells grown on plastic surface were treated with specific mouse monoclonal antibody recognizing an antigen localized on the surface of smooth muscle rather than endothelial cells; then erythrocytes coated with secondary (rabbit antimouse) antibodies were added. The results were analyzed spectrophotometrically or with scanning electron microscopy. Under the experimental conditions, erythrocytes, possible 'containers' for carrying the drugs, were found to bind only to smooth muscle cells. The data show that antibody provides absolute discrimination between endothelial and smooth muscle cells and, thus, may be used as a vector for drug targeting.
Subject(s)
Antibodies, Monoclonal/administration & dosage , Erythrocytes/immunology , Muscle, Smooth, Vascular/immunology , Pharmaceutical Vehicles , Animals , Endothelium/immunology , Humans , Mice , Microscopy, Electron, ScanningABSTRACT
A monoclonal antibody has been generated that interacts with the surface of cultured human aorta smooth muscle cells and does not bind to the endothelial cells from aorta and umbilical vein. An antigen recognized by the antibody has a molecular mass of 330 kDa as determined by electrophoresis of immunoprecipitate in SDS-polyacrylamide gel. The same antigen appeared to be present on the fibroblast surface while neither immunofluorescence, flow cytofluorimetry nor immunoprecipitation reveal it on the endothelial cell surface or in the Triton X-100 extract.