ABSTRACT
Southern King Crab (SKC) represents an important fishery resource that has the potential to be a natural source of chitosan (CS) production. In tissue engineering, CS is very useful to generate biomaterials. However, CS has a lack of signaling molecules that facilitate cell-substrate interaction. Therefore, RGD (arginine-glycine-aspartic acid) peptides corresponding to the main integrin recognition site in extracellular matrix proteins have been used to improve the CS surface. The aim of this study was to evaluate in vitro cell adhesion and proliferation of CS films synthesized from SKC shell wastes functionalized with RGD peptides. The FTIR spectrum of CS isolated from SKC shells (SKC-CS) was comparable to commercial CS. Thermal properties of films showed similar endothermic peaks at 53.4 and 53.0 °C in commercial CS and SKC-CS, respectively. The purification and molecular masses of the synthesized RGD peptides were confirmed using HPLC and ESI-MS mass spectrometry, respectively. Mouse embryonic fibroblast cells showed higher adhesion on SKC-CS (1% w/v) film when it was functionalized with linear RGD peptides. In contrast, a cyclic RGD peptide showed similar adhesion to control peptide (RDG), but the highest cell proliferation was after 48 h of culture. This study shows that functionalization of SKC-CS films with linear or cyclic RGD peptides are useful to improve effects on cell adhesion or cell proliferation. Furthermore, our work contributes to knowledge of a new source of CS to synthesize constructs for tissue engineering applications.
ABSTRACT
In recent years, conditioned medium (CM) obtained from the culture of mesenchymal stromal/stem cells (MSCs) has been shown to effectively promote tissue repair and modulate the immune response in vitro and in different animal models, with potential for application in regenerative medicine. Using CM offers multiple advantages over the implantation of MSCs themselves: 1) simpler storage, transport, and preservation requirements, 2) avoidance of the inherent risks of cell transplantation, and 3) potential application as a ready-to-go biologic product. For these reasons, a large amount of MSCs research has focused on the characterization of the obtained CM, including soluble trophic factors and vesicles, preconditioning strategies for enhancing paracrine secretion, such as hypoxia, a three-dimensional (3D) environment, and biochemical stimuli, and potential clinical applications. In vitro preconditioning strategies can increase the viability, proliferation, and paracrine properties of MSCs and therefore improve the therapeutic potential of the cells and their derived products. Specifically, dynamic cultivation conditions, such as fluid flow and 3D aggregate culture, substantially impact cellular behaviour. Increased levels of growth factors and cytokines were observed in 3D cultures of MSC grown on orbital or rotatory shaking platforms, in stirred systems, such as spinner flasks or stirred tank reactors, and in microgravity bioreactors. However, only a few studies have established dynamic culture conditions and protocols for 3D aggregate cultivation of MSCs as a scalable and reproducible strategy for CM production. This review summarizes significant advances into the upstream processing, mainly the dynamic generation and cultivation of MSC aggregates, for de CM manufacture and focuses on the standardization of the soluble factor production.
ABSTRACT
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by motor neuron death. A 20% of familial ALS cases are associated with mutations in the gene coding for superoxide dismutase 1 (SOD1). The accumulation of abnormal aggregates of different proteins is a common feature in motor neurons of patients and transgenic ALS mice models, which are thought to contribute to disease pathogenesis. Developmental morphogens, such as the Wnt family, regulate numerous features of neuronal physiology in the adult brain and have been implicated in neurodegeneration. ß-catenin is a central mediator of both, Wnt signaling activity and cell-cell interactions. We previously reported that the expression of mutant SOD1 in the NSC34 motor neuron cell line decreases basal Wnt pathway activity, which correlates with cytosolic ß-catenin accumulation and impaired neuronal differentiation. In this work, we aimed a deeper characterization of ß-catenin distribution in models of ALS motor neurons. We observed extensive accumulation of ß-catenin supramolecular structures in motor neuron somas of pre-symptomatic mutant SOD1 mice. In cell-cell appositional zones of NSC34 cells expressing mutant SOD1, ß-catenin displays a reduced co-distribution with E-cadherin accompanied by an increased association with the gap junction protein Connexin-43; these findings correlate with impaired intercellular adhesion and exacerbated cell coupling. Remarkably, pharmacological inhibition of the glycogen synthase kinase-3ß (GSK3ß) in both NSC34 cell lines reverted both, ß-catenin aggregation and the adverse effects of mutant SOD1 expression on neuronal differentiation. Our findings suggest that early defects in ß-catenin distribution could be an underlying factor affecting the onset of neurodegeneration in familial ALS.
Subject(s)
Amyotrophic Lateral Sclerosis/metabolism , Motor Neurons/metabolism , beta Catenin/metabolism , Animals , Cell Differentiation/physiology , Cells, Cultured , Disease Models, Animal , Humans , MiceABSTRACT
Ceramic and metallic nanoparticles can improve the mechanical and biological properties of polymeric scaffolds for bone tissue engineering (BTE). In this work, nanohydroxyapatite (nHAp) and nano-copper-zinc alloy (nCuZn) were added to a chitosan/gelatin (Ch/G) scaffold in order to investigate the effects on morphological, physical, and biocompatibility properties. Scaffolds were fabricated by a freeze-drying technique using different pre-freezing temperatures. Microstructure and morphology were studied by scanning electron microscopy (SEM), glass transition (Tg) was studied using differential scanning calorimetry (DSC), cell growth was estimated by MTT assay, and biocompatibility was examined in vitro and in vivo by histochemistry analyses. Scaffolds and nanocomposite scaffolds presented interconnected pores, high porosity, and pore size appropriate for BTE. Tg of Ch/G scaffolds was diminished by nanoparticle inclusion. Mouse embryonic fibroblasts (MEFs) cells loaded in the Ch/G/nHAp/nCuZn nanocomposite scaffold showed suitable behavior, based on cell adhesion, cell growth, alkaline phosphatase (ALP) activity as a marker of osteogenic differentiation, and histological in vitro cross sections. In vivo subcutaneous implant showed granulation tissue formation and new tissue infiltration into the scaffold. The favorable microstructure, coupled with the ability to integrate nanoparticles into the scaffold by freeze-drying technique and the biocompatibility, indicates the potential of this new material for applications in BTE.
ABSTRACT
Arachidonic acid (AA), a compound secreted by Sertoli cells (SC) in a FSH-dependent manner, is able to induce the release of Ca2+ from internal stores in round spermatids and pachytene spermatocytes. In this study, the possible site(s) of action of AA in round spermatids, the signalling pathways associated and the intracellular Ca2+ stores targeted by AA-induced signalling were pharmacologically characterized by measuring intracellular Ca2+ using fluorescent Ca2+ probes. Our results suggest that AA acts by interacting with a fatty acid G protein coupled receptor, initiating a G protein signalling cascade that may involve PLA2 and ERK activation, which in turn opens intracellular ryanodine-sensitive channels as well as NAADP-sensitive channels in acidic intracellular Ca2+ stores. The results presented here also suggest that AMPK and PKA modulate this AA-induced Ca2+ release from intracellular Ca2+ stores in round spermatids. We propose that unsaturated free fatty acid lipid signalling in the seminiferous tubule is a novel regulatory component of rat spermatogenesis.
Subject(s)
Arachidonic Acid/pharmacology , Calcium/metabolism , Endoplasmic Reticulum/drug effects , MAP Kinase Signaling System/drug effects , Receptors, G-Protein-Coupled/agonists , Spermatids/drug effects , Animals , Cells, Cultured , Dose-Response Relationship, Drug , Endoplasmic Reticulum/metabolism , Endosomes/drug effects , Endosomes/metabolism , Kinetics , Male , Microscopy, Confocal , NADP/analogs & derivatives , NADP/metabolism , Phospholipases A2/metabolism , Rats, Sprague-Dawley , Receptors, G-Protein-Coupled/metabolism , Salicylates/pharmacology , Sesterterpenes/pharmacology , Spermatids/cytology , Spermatids/metabolism , Testis/cytology , Testis/drug effects , Testis/metabolismABSTRACT
The aim of this work was to explore the ability of free arachidonic acid, palmitic acid and the unsaturated fatty acids oleic acid and docosahexaenoic acid to modify calcium homeostasis and mitochondrial function in rat pachytene spermatocytes and round spermatids. In contrast to palmitic acid, unsaturated fatty acids produced significant increases in intracellular calcium concentrations ([Ca2+]i) in both cell types. Increases were fatty acid specific, dose-dependent and different for each cell type. The arachidonic acid effects on [Ca2+]i were higher in spermatids than in spermatocytes and persisted when residual extracellular Ca2+ was chelated by EGTA, indicating that the increase in [Ca2+]i originated from release of intracellular calcium stores. At the concentrations required for these increases, unsaturated fatty acids produced no significant changes in the plasma membrane potential of or non-specific permeability in spermatogenic cells. For the case of arachidonic acid, the [Ca2+]i increases were not caused by its metabolic conversion to eicosanoids or anandamide; thus we attribute this effect to the fatty acid itself. As estimated with fluorescent probes, unsaturated fatty acids did not affect the intracellular pH but were able to induce a progressive decrease in the mitochondrial membrane potential. The association of this decrease with reduced reactive oxygen species (ROS) production strongly suggests that unsaturated fatty acids induced mitochondrial uncoupling. This effect was stronger in spermatids than in spermatocytes. As a late event, arachidonic acid induced caspase 3 activation in a dose-dependent manner both in the absence and presence of external Ca2+. The concurrent but differential effects of unsaturated fatty acids on [Ca2+]i and mitochondrial functions are additional manifestations of the metabolic changes that germ cells undergo during their differentiation.
Subject(s)
Apoptosis , Calcium/metabolism , Fatty Acids/metabolism , Mitochondria/metabolism , Spermatids/cytology , Spermatocytes/cytology , Adenosine Triphosphate/metabolism , Animals , Arachidonic Acid/metabolism , Docosahexaenoic Acids/metabolism , Male , Membrane Potential, Mitochondrial , Rats , Rats, Sprague-Dawley , Reactive Oxygen Species/metabolism , Spermatids/metabolism , Spermatocytes/metabolismABSTRACT
Mild hypothermia condition in mammalian cell culture technology has been one of the main focuses of research for the development of breeding strategies to maximize productivity of these production systems. Despite the large number of studies that show positive effects of mild hypothermia on specific productivity of r-proteins, no experimental approach has addressed the indirect effect of lower temperatures on specific cell growth rate, nor how this condition possibly affects less specific productivity of r-proteins. To separately analyze the effects of mild hypothermia and specific growth rate on CHO cell metabolism and recombinant human tissue plasminogen activator productivity as a model system, high dilution rate (0.017 h(-1)) and low dilution rate (0.012 h(-1)) at two cultivation temperatures (37 and 33 °C) were evaluated using chemostat culture. The results showed a positive effect on the specific productivity of r-protein with decreasing specific growth rate at 33 °C. Differential effect was achieved by mild hypothermia on the specific productivity of r-protein, contrary to the evidence reported in batch culture. Interestingly, reduction of metabolism could not be associated with a decrease in culture temperature, but rather with a decrease in specific growth rate.
Subject(s)
Cell Culture Techniques/methods , Cell Proliferation/physiology , Cell Survival/physiology , Cold Temperature , Animals , CHO Cells , CricetulusABSTRACT
An accurate communication between motor neurons and skeletal muscle fibers is required for the proper assembly, growth and maintenance of neuromuscular junctions (NMJs). Several signaling and extracellular matrix molecules play stimulatory and inhibitory roles on the assembly of functional synapses. Studies in Drosophila have revealed crucial functions for early morphogens, such as members of the Wnt and Bone Morphogenetic Proteins (BMP) signaling pathways, during the assembly and maturation of the NMJ. Here, we bring together recent findings that led us to propose that BMPs also work in vertebrate organisms as diffusible cues to communicate motor neurons and skeletal muscles.
ABSTRACT
Different pathways activated by morphogens of the early embryonic development, such as the Wnt and the Bone Morphogenetic Protein (BMP) ligands, are involved in diverse physiological and pathological conditions of the nervous system, including neurodegeneration. In this work, we have analyzed the endogenous activity of the canonical Wnt/ß-catenin and BMP/Smad-dependent pathways in an in vitro model of amyotrophic lateral sclerosis (ALS), given by motor neuron-like NSC34 cells stably expressing wild-type or G93A mutated forms of human Cu/Zn superoxide dismutase-1 (SOD1). As ALS-derived motor neurons, NSC34 cells expressing mutated hSOD1 show a decreased proliferation rate, are more susceptible to oxidation-induced cell death and display Golgi fragmentation. In addition, they display an impaired ability to induce the expression of the motor neuronal marker Hb9 and, consistently, to morphologically differentiate into a motor neuronal phenotype. Regarding signaling, our data show that the transcriptional activity associated to the Wnt/ß-catenin pathway is decreased, a finding possibly associated to the cytosolic aggregation of ß-catenin. In turn, the BMP-dependent phosphorylation of Smad1 and the transcriptional activation of the BMP/Smad pathway is increased in the pathologic model. Together, these findings suggest that Wnt/ß-catenin and the BMP-dependent pathways could play relevant roles in the neurodegeneration of motor neurons in the context of ALS.
ABSTRACT
Vitamin C plays key roles in cell homeostasis, acting as a potent antioxidant as well as a positive modulator of cell differentiation. In skeletal muscle, the vitamin C/sodium co-transporter SVCT2 is preferentially expressed in oxidative slow fibers. Besides, SVCT2 is up-regulated upon the early fusion of primary myoblasts. However, our knowledge of the postnatal expression profile of SVCT2 remains scarce. Here we have analyzed the expression of SVCT2 during postnatal development of the chicken slow anterior and fast posterior latissimus dorsi muscles, ranging from day 7 to adulthood. SVCT2 expression is consistently higher in the slow than in the fast muscle at all stages. After hatching, SVCT2 expression is significantly down-regulated in the anterior latissimus dorsi, which nevertheless maintains a robust slow phenotype. Taking advantage of the C2C12 cell line to recapitulate myogenesis, we confirmed that SVCT2 is expressed in a biphasic fashion, reaching maximal levels upon early myoblasts fusion and decreasing during myotube growth. Together, these findings suggest that the dynamic expression levels of SVCT2 could be relevant for different features of skeletal muscle physiology, such as muscle cell formation, growth and activity.
Subject(s)
Muscle Fibers, Slow-Twitch/metabolism , Sodium-Coupled Vitamin C Transporters/metabolism , Animals , Cell Line , Chickens , Down-Regulation , Growth and Development/physiologyABSTRACT
Reactive oxygen species (ROS) and reactive nitrogen species (RNS) like superoxide and nitric oxide are produced by testis and spermatogenic cells in response to heat stress. However, the magnitude and mechanisms of this production in spermatogenic cells have not been described. In this work, we evaluated ROS/RNS production, its pharmacology, mitochondrial oxidative metabolism, membrane potential and antioxidant capacity at different temperatures in isolated rat pachytene spermatocytes and round spermatids. Our results showed an increment in ROS/RNS production by pachytene spermatocytes when increasing the temperature to 40â°C. Instead, ROS/RNS production by round spermatids did not change at temperatures higher than 33â°C. ROS/RNS production was sensitive to NADPH oxidase inhibitor diphenylene iodonium or the mitochondrial complex I inhibitor rotenone. No additive effects were observed for these two compounds. Our results suggest an important mitochondrial ROS/RNS production in spermatogenic cells. Oligomycin-insensitive oxygen consumption (uncoupled oxygen consumption) increased with temperature and was significantly larger in round spermatids than pachytene spermatocytes, indicating a likely round spermatid mitochondrial uncoupling at high temperatures. A similar conclusion can be reached by measuring the mitochondrial membrane potential using rhodamine 123 fluorescence in permeabilized cells or JC-1 fluorescence in intact cells. The antioxidant capacity was higher in round spermatids than pachytene spermatocytes at 40â°C. Our results strongly suggest that at high temperatures (40â°C) pachytene spermatocytes are more susceptible to oxidative stress, but round spermatids are more protected because of a temperature-induced mitochondrial uncoupling together with a larger antioxidant capacity.
Subject(s)
Cold Temperature , Hot Temperature , Pachytene Stage/physiology , Reactive Nitrogen Species/metabolism , Reactive Oxygen Species/metabolism , Spermatids/metabolism , Spermatocytes/metabolism , Animals , Antioxidants/metabolism , Body Temperature/physiology , Cells, Cultured , Heat-Shock Response/physiology , Male , Rats , Rats, Sprague-Dawley , Spermatids/physiology , Spermatocytes/physiology , Spermatogenesis/physiologyABSTRACT
Bone morphogenetic proteins (BMPs) regulate several aspects of neuronal behavior. For instance, BMP-2 has the ability to modulate, either positively or negatively, the outgrowth of neuronal processes in diverse cell types. In Drosophila motor neurons, the BMP type II receptor (BMPRII) homolog wishful thinking plays crucial roles on neuromuscular synaptogenesis signaling through Smad-dependent and Smad-independent pathways. However, a role for BMP signaling at the vertebrate neuromuscular junction has not been addressed. Herein, we have analyzed the expression of BMPRII and the effect of BMP-2 during the morphological differentiation of motor neuron-like NSC-34 cells. Our data indicate that BMPRII is up-regulated and becomes accumulated in somas and growth cones upon motor neuronal differentiation. BMP-2 inhibits the differentiation of NSC-34 cells, an effect that correlates with activation of a Smad-dependent pathway, induction of the inhibitory Id1 transcription factor, and down-regulation of the neurogenic factor Mash1. BMP-2 also activates effectors of Smad-independent pathways. Remarkably, BMP-2 treatment significantly increases the expression of BMPRII. Our findings provide the first evidence to suggest a role for BMP pathways on the differentiation of motor neurons leading to successful assembly and/or regeneration of the vertebrate neuromuscular synapse.
Subject(s)
Bone Morphogenetic Protein 2/pharmacology , Bone Morphogenetic Protein Receptors, Type II/metabolism , Motor Neurons/cytology , Neurites/drug effects , Up-Regulation/drug effects , Animals , Basic Helix-Loop-Helix Transcription Factors/metabolism , Bone Morphogenetic Protein Receptors, Type II/genetics , Cell Differentiation , Hybrid Cells , Lim Kinases/metabolism , Luciferases, Renilla/metabolism , Mice , Neurofilament Proteins/metabolism , Phosphorylation/drug effects , RNA, Messenger/metabolism , Signal Transduction/drug effects , Time Factors , Transcription Factors/genetics , Transcription Factors/metabolism , Transfection , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
The neuromuscular junction has been extensively employed in order to identify crucial determinants of synaptogenesis. At the vertebrate neuromuscular synapse, extracellular matrix and signaling proteins play stimulatory and inhibitory roles on the assembly of functional synapses. Studies in invertebrate species have revealed crucial functions of early morphogens during the assembly and maturation of the neuromuscular junction. Here, we discuss growing evidence addressing the function of Wnt and Bone morphogenetic protein (BMP) signaling pathways at the vertebrate neuromuscular synapse. We focus on the emerging role of Wnt proteins as positive and negative regulators of postsynaptic differentiation. We also address the possible involvement of BMP pathways on motor neuron behavior for the assembly and/or regeneration of the neuromuscular junction.
Subject(s)
Bone Morphogenetic Proteins/metabolism , Neurogenesis , Neuromuscular Junction/metabolism , Wnt Proteins/metabolism , Wnt Signaling Pathway , Animals , Bone Morphogenetic Proteins/genetics , Extracellular Matrix/metabolism , Neuromuscular Junction/growth & development , Wnt Proteins/geneticsABSTRACT
Maitotoxin (MTX), a potent polyether marine biotoxin, induces Ca(2+) entry in different mammalian cells by activation of Ca(2+) channels. The identity and modulation of the MTX-activated Ca(2+) entry pathway is not known. In this work, we show, for the first time, that glucose and lactate can modulate the excitability of spermatogenic cell MTX-activated Ca(2+) channels. Physiological and pharmacological evidences indicate that glucose and lactate differentially affect MTX-activated Ca(2+) entry mainly through changes that these substrates induce on intracellular Ca(2+) stores and the concentration of intracellular Ca(2+) ([Ca(2+)](i)) in spermatogenic cells. Our findings strongly suggest that MTX-activated Ca(2+) channels in spermatogenic cells can be regulated by a Ca(2+)-CaM-dependent protein kinase.
Subject(s)
Calcium/physiology , Animals , Calcium/pharmacology , Cytoplasm/metabolism , Germ Cells/metabolism , Glucose , Ions/metabolism , Lactic Acid , Male , Marine Toxins , Oxocins , Rats , Rats, Sprague-DawleyABSTRACT
In rat seminiferous tubules (ST), cells that contain polar and neutral lipids with long-chain polyenoic fatty acids (PUFA) and sphingomyelins (SM) and ceramides (Cer) with very long chain (VLC) PUFA of the n-6 series coexist. In this study, pachytene spermatocytes and round spermatids were isolated to determine how these lipids change during spermatogenesis. As the amount per cell of PUFA-rich glycerophospholipids (GPL) decreased with cell size, the 22:5/20:4 ratio increased with cell differentiation. The elovl2 and elovl5 genes, required for 22:5 formation, were expressed (mRNA) in both cell types. Residual bodies- particles with compacted organelles and materials discarded from late spermatids-concentrated cholesterol, 22:5-rich triacylglycerols, and GPL, including plasmalogens and phosphatidylserine. Species of SM and Cer with nonhydroxylated (n-) VLCPUFA (28:4, 30:5, and 32:5) predominated in pachytene spermatocytes, whereas species with the corresponding 2-hydroxy (2-OH) VLCPUFA prevailed in round spermatids. Thus, a dramatic increase in the 2-OH/n-VLCPUFA ratio in SM and Cer was a hallmark of differentiation. A substantial decrease of 2-OH SM occurred between spermatids and mature spermatozoa and 2-OH SM species were collected in residual bodies "en route" to Sertoli cells. Notably, spermatids and spermatozoa gained a significant amount of ceramides devoid of n-VLCPUFA but having 2-OH VLCPUFA as their main fatty acids.
Subject(s)
Cell Differentiation , Fatty Acids, Unsaturated/metabolism , Spermatids/chemistry , Spermatids/metabolism , Spermatocytes/metabolism , Acetyltransferases/genetics , Acetyltransferases/metabolism , Animals , Ceramides/metabolism , Fatty Acid Elongases , Male , Rats , Rats, Wistar , Spermatids/cytology , Spermatocytes/cytology , Spermatogenesis , Sphingomyelins/metabolismABSTRACT
BACKGROUND: The conversion of one cell type into another has been suggested to be, at the molecular level, the consequence of change(s) in the expression level of key developmental genes. Myoblasts have the ability to differentiate either to skeletal muscle or osteogenic lineage depending of external stimuli. Extracellular matrix (ECM) has been shown to be essential for skeletal muscle differentiation, through its direct interaction with myoblasts' cell receptors. We attempt to address if ECM also plays a role in the osteogenic differentiation of skeletal muscle cells. RESULTS: Inhibition of proteoglycan sulfation by sodium chlorate in myoblast cultures strongly affects ECM synthesis and deposition and induces the expression of the osteogenic lineage markers alkaline phosphatase (ALP) and osteocalcin in mononuclear cells. Induction of ALP by sodium chlorate does not affect the expression of specific muscle determination transcription factors, such as MyoD and Myf-5, in the same cells. The osteogenic transcription factor Cbfa-1 expression is also unaffected. Induction of ALP is not inhibited by a soluble form of BMP receptor IA. This suggests that the deviation of the myogenic pathway of C2C12 myoblasts into the osteogenic lineage by inhibitors of proteoglycan sulfation is BMP-2 independent. The increase of osteogenic markers expression can be totally prevented by an exogenous ECM. Interestingly, a similar BMP-2-independent ALP activity induction can be observed in myoblasts cultured on an ECM previously synthesized by BMP-2 treated myoblasts. Under in vivo conditions of increased ECM turn-over and deposition, as in the mdx dystrophic muscle and during skeletal muscle regeneration, an induction and relocalization of ALP is observed in a subpopulation of skeletal muscle fibers, whereas in normal skeletal muscle, ALP expression is restricted to blood vessels and some endomysial mononuclear cells. CONCLUSION: These results suggest that signals arising from the ECM induce the expression of osteogenic markers in muscle cells by a mechanism independent of BMP-2 and without affecting the expression of key muscle or osteogenic determination genes. An induction and relocalization of ALP is also observed in mdx and regenerating skeletal muscles, in vivo conditions of increased muscle ECM deposition or turnover.
Subject(s)
Alkaline Phosphatase/metabolism , Bone Morphogenetic Protein 2/metabolism , Extracellular Matrix/metabolism , Muscle Fibers, Skeletal/metabolism , Osteocalcin/metabolism , Animals , Cell Line , Chlorates/chemistry , Chlorates/pharmacology , Core Binding Factor Alpha 1 Subunit/metabolism , Mice , Muscle Fibers, Skeletal/cytology , Myoblasts/metabolism , Myogenic Regulatory Factors/metabolism , Proteoglycans/antagonists & inhibitors , Proteoglycans/metabolismABSTRACT
Proteoglycans have been identified within the extracellular matrices (ECM) of bone and are known to play a role in ECM assembly, mineralization, and bone formation. Bone morphogenetic protein-2 (BMP-2) specifically converts the differentiation pathway of C2C12 myoblasts into that of osteoblast lineage cells. Microarray analyses of the mouse myoblast cell line C2C12 and its differentiation into osteoblastic cells in response to BMP-2 have suggested the up-regulation of several proteoglycan species, although there is a lack of biochemical evidence for this response. In this study we have biochemically analyzed and characterized the proteoglycan populations that are induced in C2C12 cells upon osteoblastic differentiation produced by BMP-2. An important and specific increase in the synthesis of secreted decorin was observed in BMP-2-treated cells, as compared to untreated myoblasts and myoblasts induced to differentiate into myotubes. Decorin was seen to contain larger glycosaminoglycan (GAG) chains in induced than in non-induced cells. BMP-2 also produced an augment in the synthesis of different heparan sulfate proteoglycans such syndecan-2, - 3, glypican, and perlecan in detergent-soluble and non-soluble cellular fractions. We also examined whether the evident changes induced by BMP-2 in secreted decorin could have a functional role. BMP-2 signaling dependent as well as induction of alkaline phosphatase (ALP) activity was diminished in decorin null myoblasts compared to wild type myoblats although cell surface level of BPM-2 receptors was unchanged. These results are the first biochemical evidence and analysis for the effect of BMP-2 on the synthesis of proteoglycan during osteogenic conversion of myoblasts and suggest a role for decorin in cell response to BMP-2.
Subject(s)
Bone Morphogenetic Proteins/metabolism , Cell Differentiation/physiology , Extracellular Matrix Proteins/metabolism , Myoblasts/metabolism , Osteoblasts/metabolism , Proteoglycans/biosynthesis , Transforming Growth Factor beta/metabolism , Alkaline Phosphatase/metabolism , Animals , Bone Morphogenetic Protein 2 , Cell Line , Culture Media, Conditioned , Decorin , Extracellular Matrix/chemistry , Extracellular Matrix/metabolism , Extracellular Matrix Proteins/chemistry , Extracellular Matrix Proteins/genetics , Inhibitor of Differentiation Protein 1/genetics , Inhibitor of Differentiation Protein 1/metabolism , Mice , Myoblasts/cytology , Osteoblasts/cytology , Proteoglycans/chemistry , Proteoglycans/genetics , Proteoglycans/metabolism , Signal Transduction/physiologyABSTRACT
Transcription of specific skeletal muscle genes requires the expression of the muscle regulatory factor myogenin. To assess the role of the extracellular matrix (ECM) in skeletal muscle differentiation, the specific inhibitors of proteoglycan synthesis, sodium chlorate and beta-D-xyloside, were used. Treatment of cultured skeletal muscle cells with each inhibitor substantially abolished the expression of creatine kinase and alpha-dystroglycan. This inhibition was totally reversed by the addition of exogenous ECM. Myoblast treatment with each inhibitor affected the deposition and assembly of the ECM constituents glypican, fibronectin, and laminin. These treatments did not affect MyoD, MEF2A, and myogenin expression and nuclear localization. Differentiated myoblast treatment with RGDS peptides completely inhibited myogenesis without affecting the expression or nuclear localization of myogenin. Integrin-mediated signaling of focal adhesion kinase was partially inhibited by chlorate and beta-D-xyloside, an effect reversed by the addition of exogenous ECM gel. These results suggested that the expression of myogenin is not sufficient to successfully drive skeletal muscle formation and that ECM is required to complete the skeletal muscle differentiation process.