RESUMEN
The skeletal muscle injury triggers the inflammatory response which is crucial for damaged muscle fiber degradation and satellite cell activation. Immunodeficient mice are often used as a model to study the myogenic potential of transplanted human stem cells. Therefore, it is crucial to elucidate whether such model truly reflects processes occurring under physiological conditions. To answer this question we compared skeletal muscle regeneration of BALB/c, i.e. animals producing all types of inflammatory cells, and SCID mice. Results of our study documented that initial stages of muscles regeneration in both strains of mice were comparable. However, lower number of mononucleated cells was noticed in regenerating SCID mouse muscles. Significant differences in the number of CD14-/CD45+ and CD14+/CD45+ cells between BALB/c and SCID muscles were also observed. In addition, we found important differences in M1 and M2 macrophage levels of BALB/c and SCID mouse muscles identified by CD68 and CD163 markers. Thus, our data show that differences in inflammatory response during muscle regeneration, were not translated into significant modifications in muscle regeneration.
Asunto(s)
Inflamación/patología , Músculo Esquelético/patología , Regeneración/fisiología , Animales , Biomarcadores/metabolismo , Diferenciación Celular/fisiología , Células Cultivadas , Inflamación/metabolismo , Macrófagos/metabolismo , Macrófagos/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones SCID , Músculo Esquelético/metabolismo , Enfermedades Musculares/metabolismo , Enfermedades Musculares/patologíaRESUMEN
Changes in the expression of adhesion proteins involved in myoblast differentiation were investigated in monolayer (two-dimensional) and 3D (three-dimensional) cell cultures. The expression of integrin alpha3 subunit, integrin beta1 subunit, ADAM12 (a disintegrin and metalloproteinase 12), tetraspanins CD9 and CD81 and M-cadherin were examined in the murine myoblast cell line C2C12 and in a primary culture of rat satellite cells. Myoblasts in monolayer and 3D cultures showed significant differences in their morphology and cytoskeletal organization. All of the studied proteins participated in myoblast fusion in each culture examined, but differences in their levels of expression were observed. Satellite cell-derived myoblasts exhibited higher expression of adhesion protein mRNAs than C2C12 cells. Also, C2C12 cells from a 3D culture showed slightly higher expression of adhesion protein transcripts than the same cells cultured as a monolayer. Significantly, the levels of adhesion protein mRNAs were found to change in parallel in all cell culture types. Despite this finding, it is important that differences between satellite cell-derived myoblasts and cell line C2C12 grown in monolayer and 3D cultures are taken into account when studying processes of myoblast differentiation in vitro.
Asunto(s)
Proteínas de la Membrana/metabolismo , Mioblastos/metabolismo , Células Satélite del Músculo Esquelético/metabolismo , Proteínas ADAM/análisis , Proteínas ADAM/genética , Proteínas ADAM/metabolismo , Animales , Antígenos CD/análisis , Antígenos CD/genética , Antígenos CD/metabolismo , Cadherinas/análisis , Cadherinas/genética , Cadherinas/metabolismo , Adhesión Celular , Técnicas de Cultivo de Célula , Diferenciación Celular , Células Cultivadas , Integrina alfa3/análisis , Integrina alfa3/genética , Integrina alfa3/metabolismo , Integrina beta1/análisis , Integrina beta1/genética , Integrina beta1/metabolismo , Glicoproteínas de Membrana/análisis , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Ratones , Mioblastos/citología , ARN Mensajero/metabolismo , Ratas , Células Satélite del Músculo Esquelético/citología , Tetraspanina 28 , Tetraspanina 29RESUMEN
In this report, we focused on Pax3 and Pax7 expression in vitro during myoblast differentiation and in vivo during skeletal muscle regeneration. We showed that Pax3 and Pax7 were present in EDL (extensor digitorum longus) and Soleus muscle derived cells. These cells express in vitro a similar level of Pax3 mRNA, however, differ in the levels of mRNA encoding Pax7. Analysis of Pax3 and Pax7 proteins showed that Soleus and EDL satellite cells differ in the level of Pax3/7 proteins and also in the number of Pax3/7 positive cells. Moreover, Pax3/7 expression was restricted to undifferentiated cells, and both proteins were absent at further stages of myoblast differentiation, indicating that Pax3 and Pax7 are down-regulated during myoblast differentiation. However, we noted that the population of undifferentiated Pax3/7 positive cells was constantly present in both in vitro cultured satellite cells of EDL and Soleus. In contrast, there was no significant difference in Pax3 and Pax7 during in vivo differentiation accompanying regeneration of EDL and Soleus muscle. We demonstrated that Pax3 and Pax7, both in vitro and in vivo, participated in the differentiation and regeneration events of muscle and detected differences in the Pax7 expression pattern during in vitro differentiation of myoblasts isolated from fast and slow muscles.
Asunto(s)
Desarrollo de Músculos , Mioblastos/fisiología , Factores de Transcripción Paired Box/metabolismo , Regeneración , Animales , Diferenciación Celular , Masculino , Fibras Musculares de Contracción Rápida/metabolismo , Fibras Musculares de Contracción Rápida/fisiología , Fibras Musculares de Contracción Lenta/metabolismo , Fibras Musculares de Contracción Lenta/fisiología , Mioblastos/citología , Mioblastos/metabolismo , Factor de Transcripción PAX3 , RatasRESUMEN
Skeletal muscles exhibit great plasticity and an ability to reconstruct in response to injury. However, the repair process is often inefficient and hindered by the development of fibrosis. We explored the possibility that during muscle repair, the different regeneration ability of the fast (extensor digitorum longus; EDL) and slow twitch (Soleus) muscles depends on the differential expression of metalloproteinases (MMP-9 and MMP-2) involved in the remodeling of the extracellular matrix. Our results show that MMP-9 and MMP-2 are present in the intact muscle and are up-regulated after crush-induced muscle injury. The expression and the activity of these two enzymes depend on the type of muscle and the phase of muscle regeneration. In the regenerating Soleus muscle, elevated levels of MMP-9 occurred during the myolysis and reconstruction phase. In contrast, regenerating EDL muscles exhibited decreased MMP-9 levels during myolysis and increased MMP-2 activity at the reconstruction phase. Moreover, satellite cells (mononuclear myoblasts) derived from Soleus and EDL muscles showed no differences in localization or activity of MMP-9 and MMP-2 during proliferation and differentiation in vitro. MMP-9 activity was present during all stages of myoblast differentiation, whereas MMP-2 activity reached its highest level during myoblast fusion. We conclude that MMPs are involved in muscle repair, and that fast and slow twitch muscles exhibit different patterns of MMP-9 and MMP-2 activity.
Asunto(s)
Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Fibras Musculares de Contracción Rápida/enzimología , Fibras Musculares de Contracción Lenta/enzimología , Músculo Esquelético/enzimología , Regeneración/fisiología , Animales , Western Blotting , Diferenciación Celular , Células Cultivadas , Masculino , Metaloproteinasa 2 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/genética , Músculo Esquelético/lesiones , Mioblastos/fisiología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
This report presents the history of the involvement of the Department of Cytology in studies of different aspects of regeneration. It can be divided into two major phases; the first focused on the regeneration of Turbellarians and the second on the regeneration of rat skeletal muscles including the differentiation of satellite cells in vitro. Regeneration of Turbellarians was investigated both at the cellular and molecular levels including the role of the protein kinase C (PKC) in this process. Studies on skeletal muscle regeneration initially focused on factors involved in regulation of signal transduction pathways. Next, we explored the influence of growth factors on the modulation of the regeneration process. Another important aspect of our studies was investigating of the distribution and function of different proteins involved in adhesion and fusion of myoblasts. Finally, we are also conducting research on the role of stem cells from other tissues in the regeneration of skeletal muscle.
Asunto(s)
Músculo Esquelético/fisiología , Planarias/fisiología , Regeneración/fisiología , Animales , Mioblastos/fisiología , Células Madre/citología , Células Madre/fisiologíaRESUMEN
Cadherins belong to a large family of membrane glycoprotein adhesion receptors that mediate homophilic, calcium-dependent cell adhesion. During myogenesis, cadherins are involved in initial cell-to-cell recognition; and it has also been suggested that they play a role in the initiation of myoblast fusion into multinuclear myotubes. One of the members of the cadherin family, M-cadherin, has been detected during embryogenesis in myogenic cells of somitic origin and in adult muscles. We investigated the distribution and function of M-cadherin and beta-catenin during differentiation of myoblasts in primary cultures of rat satellite cells. We found that M-cadherin was accumulated at the areas of contact between fusing myoblasts and that it colocalized with beta-catenin. Moreover, beta-catenin colocalized with actin in pre-fusing myoblasts. We show that myoblast differentiation is accompanied by an increase in the amounts of M-cadherin and beta-catenin both at the mRNA and the protein level. Flow cytometry analysis showed that M-cadherin expression was highest in fusing myoblasts. In addition, an antibody specific for the extracellular domain of M-cadherin inhibited the fusion of cultured myoblasts. These data suggest that regulation of the M-cadherin level plays an important role in the differentiation of satellite cells and in myoblast fusion in primary cultures.
Asunto(s)
Cadherinas/fisiología , Células Satélite del Músculo Esquelético/citología , Células Satélite del Músculo Esquelético/metabolismo , beta Catenina/fisiología , Animales , Adhesión Celular/fisiología , Diferenciación Celular , Masculino , Desarrollo de Músculos/fisiología , Fibras Musculares Esqueléticas/citología , Fibras Musculares Esqueléticas/metabolismo , ARN Mensajero/metabolismo , Ratas , Ratas WistarRESUMEN
OBJECTIVE: In this report, we demonstrate the participation of human cord blood (HUCB) stem cells in the skeletal muscle regeneration of SCID (severe combined immunodeficient) mice. MATERIALS AND METHODS: The HUCB cells were labeled with the PKH26 fluorescent marker or recognized by an anti-HLA-ABC or anti-beta-2-microglobulin antibody. The HUCB cells were implanted directly into the damaged mouse muscle. The regeneration process and the implanted HUCB cells were traced each day after the damage, throughout a period of 7 days, and additionally at day 30 with the use of flow cytometry and confocal microscopy. RESULTS: The PKH26-labeled cells isolated from the regenerating muscle were positive for the anti-HLA-ABC antibody. The percentage of the PKH26(+) and HLA-ABC(+) cells decreased from day 1 to day 5. In the regenerating muscle, the percentage of the HLA-ABC(+) cells increased, as measured on days 7 and 30. Moreover, myofibers containing fragments of the PKH26-labeled sarcolemma were noticed. Labeling with the anti-human beta(2)-microglobulin antibody showed the presence of positive cells and myofibers at day 7 of the regeneration, suggesting fusion of human and mouse cells. CONCLUSIONS: We suggest that the HUCB cells implanted into the damaged muscle are present there for at least 30 days and that they participate in the muscle regeneration. Moreover, our study shows that the implanted HUCB cells form human muscle precursor cells residing in the repaired mouse muscle. We suggest that the HUCB cell circulation after transplantation depends on SDF-1 (stromal-derived factor-1) expression in regenerating muscle.
Asunto(s)
Trasplante de Células Madre de Sangre del Cordón Umbilical , Células Madre Hematopoyéticas , Músculo Esquelético/lesiones , Regeneración , Heridas y Lesiones/terapia , Animales , Fusión Celular , Quimiocina CXCL12 , Quimiocinas CXC/biosíntesis , Citometría de Flujo/métodos , Regulación de la Expresión Génica , Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/patología , Humanos , Ratones , Ratones SCID , Microscopía Confocal/métodos , Músculo Esquelético/metabolismo , Miofibrillas/metabolismo , Miofibrillas/patología , Factores de Tiempo , Trasplante Heterólogo , Heridas y Lesiones/metabolismo , Heridas y Lesiones/patologíaRESUMEN
Stem cells for skeletal muscle originate from dermomyotome of the embryo. The early marker of these cells is expression of both transcription factors Pax3 and Pax7 (Pax3+/Pax7+ cells). The skeletal muscles in the adult organism have a remarkable ability to regenerate. Skeletal muscle damage induces degenerative phase, followed by activation of inflammatory and satellite cells. The satellite cells are quiescent myogenic precursor cells located between the basal membrane and the sarcolemma of myofiber and they are characterized by Pax7 expression. Activation of the satellite cells is regulated by muscle growth and chemokines. Apart from the satellite cells, a population of adult stem cells (muscle side population--mSP) exists in the skeletal muscles. Moreover, the cells trafficking from different tissues may be involved in the regeneration of damaged muscle. Trafficking of cells in the process of damaged muscle regeneration may be traced in the SCID mice.
Asunto(s)
Músculo Esquelético/citología , Músculo Esquelético/fisiología , Regeneración/fisiología , Células Madre/citología , Células Madre/fisiología , AnimalesRESUMEN
Mesenchymal stem cells derived from bone marrow or several fetal tissues can be expanded and differentiated into other cell lines. The fetal liver is the source of early hematopoietic cells and also, as a fetal tissue, may be considered as a source of pluripotent stem cells. The differentiation potential of fetal rat liver cells have been examined. Freshly isolated liver cells from 14-d fetuses were cultured in Dulbecco medium supplemented with 10% FCS. The plastic-adherent cells were then passaged up to 10 times. Freshly isolated cells and cells from every passage were cultured in hematopoiesis-promoting environment that consists of methylcelulose supplemented with FCS, rat IL-3, human IL-6 and Epo. Parallely these cells were incubated in co-culture with rat muscle satellite cells (Dulbecco medium with 10% FCS and 10% HS) to examine their myogenic potential. Culture in methylcelulose resulted in a high number of GM and Mix colonies in case of freshly isolated liver cells and the number of colonies decreased according to the number of passages. In case of cells from 4th passage, there ware no hematopoietic colonies in culture. In contrast--freshly isolated cells were not able to fuse with rat satellite cells and form the myotubes. This ability appeared in plastic-adherent cells just from the second passage and increases to 5th passage. The cells from every next passage up to 10th when co-cultured with satellite cells participated in myotube formation at the same high level. This result may suggest that in the 14-d rat liver there exist at least two subpopulations of cells: the non-adherent hematopoietic cell population, and the population of plastic-adherent cells capable of differentiating into myotubes. Since the attempts to redifferentiate hematopoietic subpopulation into myopoiesis, or myopoietic subpopulation into hematopoiesis failed, it may be concluded that at least under our experimental conditions the fetal liver cells do not reveal the "plasticity" features.
Asunto(s)
Diferenciación Celular/fisiología , Hígado/citología , Hígado/embriología , Células Madre/citología , Células Madre/fisiología , Animales , Humanos , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/fisiología , RatasRESUMEN
Pluripotent stem cells are a potential source of various cell types for use in regenerative medicine. Despite accumulating knowledge, there is currently no efficient and reproducible protocol that does not require genetic manipulation for generation of myogenic cells from pluripotent stem cells. Here, we examined whether mouse embryonic stem (ES) cells are able to undergo myogenic differentiation and fusion in response to signals released by differentiating myoblasts. Using ES cells expressing the histone 2B-green fluorescent fusion protein, we were able to detect hybrid myotubes formed by ES cells and differentiating myoblasts. ES cells that fused with myoblasts downregulated the expression of pluripotency markers and induced the expression of myogenic markers, while unfused ES cells did not exhibit this expression pattern. Thus, the signals released by myoblasts were not sufficient to induce myogenic differentiation of ES cells. Although ES cells synthesize many proteins involved in myoblast adhesion and fusion, we did not observe any myotubes formed exclusively by ES cells. We found that ES cells lacked M-cadherin and vascular cell adhesion molecule-1, which may account for the low frequency of hybrid myotube formation in ES cell-myoblast co-cultures and the inability of ES cells alone to form myotubes.
Asunto(s)
Diferenciación Celular , Células Madre Embrionarias/metabolismo , Desarrollo de Músculos , Mioblastos/metabolismo , Animales , Antígenos de Diferenciación/metabolismo , Fusión Celular , Línea Celular , Células Madre Embrionarias/citología , Ratones , Mioblastos/citologíaRESUMEN
Different sources of stem cells are considered as a potential source of precursor cells that could improve skeletal muscle regeneration. Under physiological conditions muscle regeneration is based on the satellite cells, i.e. adult muscle precursor cells that are localized between muscle fiber and surrounding basal lamina. These cells remain quiescent but after skeletal muscle injury activate, proliferate, differentiate, and fuse either to form new muscle fibers or reconstruct the damaged ones. As it was shown in many studies few populations of stem cells other than satellite cells are able to support skeletal muscle regeneration. Among them are mesenchymal stem cells (MSCs) that are present in many niches within adult organism and also in fetal tissues, such as human umbilical cord blood (HUCB) or umbilical cord connective tissue, i.e. Wharton's jelly. Thus, MSCs are intensively tested to prove that they are able to differentiate into various cell types, including skeletal myoblasts, and therefore could be useful in regenerative medicine. In our previous study we showed that MSCs isolated from Wharton's jelly expressed pluripotency as well as myogenic markers and were able to undergo myogenic differentiation both in vitro and in vivo. We also analyzed the potential of HUCB cells population which contains not only MSCs but also hematopoietic precursors. Our analyses of whole population of HUCB cells showed that these cells express myogenic regulatory factors, i.e. MyoD, and are able to contribute to skeletal muscle regeneration. In the present study we document that adherent fraction of HUCB cells, i.e. the cells that constitute the subpopulation enriched in MSCs, expresses pluripotency and myogenic markers, and have a positive impact at the regeneration of injured mouse skeletal muscle.
Asunto(s)
Sangre Fetal/citología , Células Madre Mesenquimatosas/citología , Desarrollo de Músculos/fisiología , Músculo Esquelético/fisiología , Medicina Regenerativa , Adulto , Animales , Diferenciación Celular , Células Cultivadas , Femenino , Sangre Fetal/fisiología , Humanos , Técnicas para Inmunoenzimas , Células Madre Mesenquimatosas/fisiología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Músculo Esquelético/citología , Factores Reguladores Miogénicos/genética , Factores Reguladores Miogénicos/metabolismo , Embarazo , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Formation of mammalian skeletal muscle myofibers, that takes place during embryogenesis, muscle growth or regeneration, requires precise regulation of myoblast adhesion and fusion. There are few evidences showing that adhesion proteins play important role in both processes. To follow the function of these molecules in myoblast differentiation we analysed integrin alpha3, integrin beta1, ADAM12, CD9, CD81, M-cadherin, and VCAM-1 during muscle regeneration. We showed that increase in the expression of these proteins accompanies myoblast fusion and myotube formation in vivo. We also showed that during myoblast fusion in vitro integrin alpha3 associates with integrin beta1 and ADAM12, and also CD9 and CD81, but not with M-cadherin or VCAM-1. Moreover, we documented that experimental modification in the expression of integrin alpha3 lead to the modification of myoblast fusion in vitro. Underexpression of integrin alpha3 decreased myoblasts' ability to fuse. This phenomenon was not related to the modifications in the expression of other adhesion proteins, i.e. integrin beta1, CD9, CD81, ADAM12, M-cadherin, or VCAM-1. Apparently, aberrant expression only of one partner of multiprotein adhesion complexes necessary for myoblast fusion, in this case integrin alpha3, prevents its proper function. Summarizing, we demonstrated the importance of analysed adhesion proteins in myoblast fusion both in vivo and in vitro.
Asunto(s)
Diferenciación Celular , Integrina alfa3/metabolismo , Ratas/fisiología , Células Satélite del Músculo Esquelético/fisiología , Animales , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Fusión Celular , Células Cultivadas , Técnicas de Cocultivo , Expresión Génica , Integrina alfa3/genética , Masculino , Ratones , Músculo Esquelético/citología , Músculo Esquelético/fisiología , Cultivo Primario de Células , RegeneraciónRESUMEN
Nonhematopoietic cord blood cells and mesenchymal cells of umbilical cord Wharton's jelly have been shown to be able to differentiate into various cell types. Thus, as they are readily available and do not raise any ethical issues, these cells are considered to be a potential source of material that can be used in regenerative medicine. In our previous study, we tested the potential of whole mononucleated fraction of human umbilical cord blood cells and showed that they are able to participate in the regeneration of injured mouse skeletal muscle. In the current study, we focused at the umbilical cord mesenchymal stromal cells isolated from Wharton's jelly. We documented that limited fraction of these cells express markers of pluripotent and myogenic cells. Moreover, they are able to undergo myogenic differentiation in vitro, as proved by coculture with C2C12 myoblasts. They also colonize injured skeletal muscle and, with low frequency, participate in the formation of new muscle fibers. Pretreatment of Wharton's jelly mesenchymal stromal cells with SDF-1 has no impact on their incorporation into regenerating muscle fibers but significantly increased muscle mass. As a result, transplantation of mesenchymal stromal cells enhances the skeletal muscle regeneration.
Asunto(s)
Células Madre Mesenquimatosas/citología , Cordón Umbilical/citología , Animales , Diferenciación Celular , Células Cultivadas , Quimiocina CXCL12/farmacología , Técnicas de Cocultivo , Humanos , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/efectos de los fármacos , Ratones , Ratones Endogámicos NOD , Fibras Musculares Esqueléticas/metabolismo , Mioblastos/citología , Receptores CXCR4/metabolismo , Regeneración/efectos de los fármacos , Gelatina de Wharton/citologíaRESUMEN
Satellite cells are myogenic precursor cells, participating in growth, and regeneration of skeletal muscles. The proteins that play a role in myogenesis are integrins. In this report, we show that the integrin alpha3 subunit is expressed in quiescent satellite cells and activated myoblasts. We also find that in myoblasts the integrin alpha3 subunit is localized at cell-cell and cell-extracellular matrix contacts. We notice that increase in protein and mRNA encoding the integrin alpha3 subunit accompanies myoblast differentiation. Using double immunofluorescence and immunoprecipitation experiments, we demonstrate that the integrin alpha3 subunit co-localizes with actin, and binds the integrin beta1 subunit and ADAM12, suggesting that the complex alpha3beta1/ADAM12 is probably involved in myoblast fusion. Importantly, overexpression of the full-length integrin alpha3 subunit increases myoblast fusion whereas an antibody against its extracellular domain inhibits fusion. These data demonstrate that the integrin alpha3 subunit may contribute to satellite cell activation and then myoblast adhesion and fusion.
Asunto(s)
Integrina alfa3/fisiología , Mioblastos Esqueléticos/citología , Células Satélite del Músculo Esquelético/citología , Proteínas ADAM/metabolismo , Proteína ADAM12 , Actinas/metabolismo , Animales , Anticuerpos/farmacología , Adhesión Celular , Fusión Celular , Células Cultivadas , Integrina alfa3/análisis , Integrina alfa3/genética , Integrina beta1/metabolismo , Masculino , Proteínas de la Membrana/metabolismo , Mioblastos Esqueléticos/efectos de los fármacos , Mioblastos Esqueléticos/metabolismo , ARN Mensajero/análisis , Ratas , Ratas Wistar , Células Satélite del Músculo Esquelético/metabolismoRESUMEN
It was shown that syndecans have a potential role in muscle development. We focused this study on the role of syndecan-4 distribution and phosphorylation during the differentiation of satellite cells isolated from Soleus muscle. Syndecans are cell surface heparan sulfate proteoglycans (HSPGs) that bind numerous ligands through their HS glycosaminoglycan chains (GAG). They play a role in cell-extracellular matrix and cell-cell adhesion, signal transduction and the targeting of growth factors and other molecules to the cell surface. Syndecan-4 acts as a co-receptor or, along with integrins, is localized to the cell membrane of focal contacts. Syndecan-4 participates in the organization of the structure of focal contacts reacting with extracellular matrix molecules. The interaction of syndecan-4 with protein kinase C (PKC) isoforms is the main mechanism regulating its distribution in cells. Our current study focused on the role of the distribution of syndecan-4, and its interactions with PKC isoforms during the differentiation of activated satellite cells. We used the PKC activator TPA (12-O-tetradecanoyl phorbol 13-acetate) and the PKC inhibitor Calphostin C (Cal C). We concluded that syndecan-4 was important not only in the activation of satellite cells, but also in myoblast differentiation. During our research, we observed the presence of syndecan-4 and changes in its location over the course of that process. We also showed that TPA and Cal C treatment had an influence on the subcellular distribution of syndecan-4, but there was no influence on myoblast differentiation. We speculated that the reason for changes after TPA treatment was the interactions with activated PKC alpha, which provoked syndecan-4/PKC alpha complex translocation to integrins. We also supposed that Cal C treatment inhibited PKC delta activity and probably induced PKC lpha association to syndecan-4, and syndecan-4 translocation to integrins.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Glicoproteínas de Membrana/efectos de los fármacos , Músculo Esquelético/efectos de los fármacos , Naftalenos/farmacología , Proteoglicanos/efectos de los fármacos , Células Satélite del Músculo Esquelético/efectos de los fármacos , Acetato de Tetradecanoilforbol/farmacología , Animales , Western Blotting , Técnica del Anticuerpo Fluorescente , Masculino , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/efectos de los fármacos , Ratas , Ratas Wistar , Sindecano-4RESUMEN
Satellite cells (myogenic stem cells) dissociated from adult muscle tissue proliferate, fuse and form multinucleate myotubes when placed in culture. This study focused on the role of talin distribution during the differentiation of satellite cells. Talin plays a key role in anchoring actin filaments to integrins as well as to the plasma membrane in focal contacts. We demonstrated that there is a colocalization of talin and phosphoserine residues during the differentiation of satellite cells, and that it changes after TPA (a protein kinase C activator) treatment, and showed that talin existing in the cell-extracellular matrix and cell-cell contact area was not phosphorylated. In the presence of TPA (24 and 48 h exposure) the level of colocalization of both talin and phosphoserine residues was the same in the treated cells and in the control cells, but the level of talin phosphorylation was higher in the treated cells. We found that in myotubes from TPA treated cultures (144 h exposure to TPA), talin had localized near the cell membrane in the absence of phosphoserine residues, and that the level of talin phosphorylation was lower than in the control cells. We also demonstrated that the expression of talin during satellite cell differentiation was constant in both the control and TPA-treated cells.