We investigated the role of the proinflammatory cytokine TNF-α, the second messenger C2-ceramide, and protein kinase R (PKR) in bovine articular cartilage degradation. Bovine articular cartilage explants were stimulated with C2-ceramide or TNF-α for 24 hours. To inhibit the activation of PKR, 2-aminopurine was added to duplicate cultures. Matrix metalloproteinase (MMP) expression and activation in the medium were analysed by gelatin zymography, proteoglycan release by the dimethylmethylene blue assay, and cell viability by the Cytotox 96® assay. C2-ceramide treatment of cartilage explants resulted in a significant release of both pro- and active MMP-2 into the medium. Small increases were also seen with TNF-α treatment. Incubation of explants with 2-aminopurine before TNF-α or C2-ceramide treatment resulted in a marked reduction in expression and activation of both MMP-2 and MMP-9. TNF-α and C2-ceramide significantly increased proteoglycan release into the medium, which was also inhibited by cotreatment with 2-aminopurine. A loss of cell viability was observed when explants were treated with TNF-α and C2-ceramide, which was found to be regulated by PKR. We have shown that C2-ceramide and TNF-α treatment of articular cartilage result in the increased synthesis and activation of MMPs, increased release of proteoglycan, and increased cell death. These effects are abrogated by treatment with the PKR inhibitor 2-aminopurine. Collectively, these results suggest a novel role for PKR in the synthesis and activation of MMPs and support our hypothesis that PKR and its activator, PACT, are implicated in the cartilage degradation that occurs in arthritic disease.
articular cartilage; ceramide; matrix metalloproteinase; PKR; TNF-α
Recent investigations provided evidence that the sphingomyelin signal transduction pathway mediates apoptosis for tumor necrosis factor alpha (TNF-alpha) in several hematopoietic and nonhematopoietic cells. In this pathway, TNF-receptor interaction initiates sphingomyelin hydrolysis to ceramide by a sphingomyelinase. Ceramide acts as a second messenger stimulating a ceramide-activated serine/threonine protein kinase. The present studies show that ionizing radiation, like TNF, induces rapid sphingomyelin hydrolysis to ceramide and apoptosis in bovine aortic endothelial cells. Elevation of ceramide with exogenous ceramide analogues was sufficient for induction of apoptosis. Protein kinase C activation blocked both radiation-induced sphingomyelin hydrolysis and apoptosis, and apoptosis was restored by ceramide analogues added exogenously. Ionizing radiation acted directly on membrane preparations devoid of nuclei, stimulating sphingomyelin hydrolysis enzymatically through a neutral sphingomyelinase. These studies provide the first conclusive evidence that apoptotic signaling can be generated by interaction of ionizing radiation with cellular membranes and suggest an alternative to the hypothesis that direct DNA damage mediates radiation-induced cell kill.
Ceramides are signaling sphingolipids involved in cellular homeostasis but also in pathological processes such as unwanted apoptosis, growth arrest, oxidative stress, or senescence. Several enzymatic pathways are responsible for the synthesis of ceramides, which can be activated in response to exogenous stimuli such as cytokines, radiation, or oxidative stress. Endothelial cells are particularly rich in acid sphingomyelinases, which can be rapidly activated to produce ceramides, both intracellular and at the plasma membrane. In addition, neutral sphingomyelinases, the de novo pathway and the ceramide recycling pathway, may generate excessive ceramides involved in endothelial cell responses. When up-regulated, ceramides trigger signaling pathways that culminate in endothelial cell death, which in murine lungs has been linked to the development of emphysema-like disease. Furthermore, ceramides may be released paracellularly where they are believed to exert paracrine activities. Such effects, along with ceramides released by inflammatory mediators, may contribute to lung inflammation and pulmonary edema, because ceramide-challenged pulmonary endothelial cells exhibit decreased barrier function, independent of apoptosis. Reestablishing the sphingolipid homeostasis, either by modulating ceramide synthesis or by opposing its biological effects through augmentation of the prosurvival sphingosine-1 phosphate, may alleviate acute or chronic pulmonary conditions characterized by vascular endothelial cell death or dysfunction.
sphingolipids; apoptosis; pulmonary emphysema; acute lung injury; pulmonary circulation
Alveolar cell apoptosis is involved in the pathogenesis of emphysema, a prevalent disease primarily caused by cigarette smoking. We report that ceramide, a second messenger lipid, is a critical mediator of alveolar destruction in emphysema. Inhibition of enzymes controlling de novo ceramide synthesis prevented alveolar cell apoptosis, oxidative stress and emphysema caused by blockade of the VEGF receptors in both rats and mice. Emphysema was reproduced with intra-tracheal instillation of ceramide in naïve mice. A feed-forward mechanism of ceramide synthesis due secretory acid sphingomyelinase was supported by the neutralizing effects of ceramide-specific antibody in mice and by sphingomyelinase-deficient fibroblasts. Stimulation of sphingosine-1-phosphate signaling prevented lung apoptosis, implicating that ceramide to sphingosine-1-phosphate balance is required for maintenance of alveolar septal integrity. Finally, increased lung ceramides in patients with smoking-induced emphysema position ceramide upregulation as a critical pathogenetic element and a promising target in this disease lacking effective therapies.
Human cytomegalovirus (HCMV) infection can be fatal to immunocompromised individuals. We have previously reported that gamma interferon and tumor necrosis factor alpha (TNF-α) synergistically inhibit HCMV replication in vitro. Ceramides have been described as second messengers induced by TNF-α. To investigate the mechanisms involved in the inhibition of HCMV by TNF-α, in the present study we have analyzed ceramide production by U373 MG astrocytoma cells and the effects of TNF-α versus ceramides on HCMV replication. Our results show that U373 MG cells did not produce ceramides upon incubation with TNF-α. Moreover, long-chain ceramides induced by treatment with exogenous bacterial sphingomyelinase inhibited HCMV replication in synergy with TNF-α. Surprisingly, short-chain permeant C6-ceramide increased viral replication. Our results show that the anti-HCMV activity of TNF-α is independent of ceramides. In addition, our results suggest that TNF-α and endogenous long-chain ceramides use separate pathways of cell signalling to inhibit HCMV replication, while permeant C6-ceramide appears to activate a third pathway leading to an opposite effect.
Ceramides are the main lipids in the stratum corneum and are generated during cellular stress and apoptosis by de novo synthesis or by the action of sphingomyelinase. In addition, they are lipid second messengers produced by sphingolipid metabolism and trigger important cell responses, including protein kinase C-alpha (PKC-α) activation and the stimulation of signal transduction pathways with apoptosis and stress-activated protein kinases (SAPK), such as c-jun N-terminal kinase (JNK). Thus, ceramides have anti-proliferative and apoptotic effects. This study measured the changes in the levels of epidermal ceramides and ceramide-related apoptotic signaling molecules in psoriasis patients. Samples from lesional and non-lesional epidermis were obtained from psoriasis patients. Total ceramides were fractionated using thin-layer chromatography, and the levels of PKC-α and JNK expression were measured using Western blot analysis with specific antibodies. The ceramide level was reduced significantly, and this was associated with the downregulation of apoptotic signaling molecules, such as PKC-α and JNK, in the lesional epidermis of psoriasis patients. These results suggest that the decreased level of ceramides downregulates the apoptotic pathway, leading to epidermal proliferation in psoriasis.
Apoptosis; Ceramides; JNK Mitogen-Activated Protein Kinases; Protein Kinase C-alpha; Psoriasis
Muscle atrophy associated with various pathophysiological conditions represents a major health problem, because of its contribution to the deterioration of patient status and its effect on mortality. Although the involvement of pro-inflammatory cytokines in this process is well recognized, the role of sphingolipid metabolism alterations induced by the cytokines has received little attention.
We addressed this question both in vitro using differentiated myotubes treated with TNF-α, and in vivo in a murine model of tumor-induced cachexia. Myotube atrophy induced by TNF-α was accompanied by a substantial increase in cell ceramide levels, and could be mimicked by the addition of exogenous ceramides. It could be prevented by the addition of ceramide-synthesis inhibitors that targeted either the de novo pathway (myriocin), or the sphingomyelinases (GW4869 and 3-O-methylsphingomyelin). In the presence of TNF-α, ceramide-synthesis inhibitors significantly increased protein synthesis and decreased proteolysis. In parallel, they lowered the expression of both the Atrogin-1 and LC3b genes, involved in muscle protein degradation by proteasome and in autophagic proteolysis, respectively, and increased the proportion of inactive, phosphorylated Foxo3 transcription factor. Furthermore, these inhibitors increased the expression and/or phosphorylation levels of key factors regulating protein metabolism, including phospholipase D, an activator of mammalian target of rapamycin (mTOR), and the mTOR substrates S6K1 and Akt. In vivo, C26 carcinoma implantation induced a substantial increase in muscle ceramide, together with drastic muscle atrophy. Treatment of the animals with myriocin reduced the expression of the atrogenes Foxo3 and Atrogin-1, and partially protected muscle tissue from atrophy.
Ceramide accumulation induced by TNF-α or tumor development participates in the mechanism of muscle-cell atrophy, and sphingolipid metabolism is a logical target for pharmacological or nutritional interventions aiming at preserving muscle mass in pathological situations.
15-Lipoxygenases and their metabolites have been shown to exhibit anti-inflammatory and immunomodulatory properties, but little is known regarding their expression and function in chondrocytes. The objective of this study was to evaluate the expression of 15-lipoxygenase-1 and -2 in human articular chondrocytes, and to investigate the effects of their metabolites 13(S)-hydroxy octadecadienoic and 15(S)-hydroxyeicosatetraenoic acids on IL-1β-induced matrix metalloproteinase (MMP)-1 and MMP-13 expression.
The expression levels of 15-lipoxygenase-1 and -2 were analyzed by reverse transcription PCR and Western blotting in chondrocytes, and by immunohistochemistry in cartilage. Chondrocytes or cartilage explants were stimulated with IL-1β in the absence or presence of 13(S)-hydroxy octadecadienoic and 15(S)-hydroxyeicosatetraenoic acids, and the levels of MMP-1 and MMP-13 protein production and type II collagen cleavage were evaluated using immunoassays. The role of peroxisome proliferator-activated receptor (PPAR)γ was evaluated using transient transfection experiments and the PPARγ antagonist GW9662.
Articular chondrocytes express 15-lipoxygenase-1 and -2 at the mRNA and protein levels. 13(S)-hydroxy octadecadienoic and 15(S)-hydroxyeicosatetraenoic acids dose dependently decreased IL-1β-induced MMP-1 and MMP-13 protein and mRNA expression as well as type II collagen cleavage. The effect on MMP-1 and MMP-13 expression does not require de novo protein synthesis. 13(S)-hydroxy octadecadienoic and 15(S)-hydroxyeicosatetraenoic acids activated endogenous PPARγ, and GW9662 prevented their suppressive effect on MMP-1 and MMP-13 production, suggesting the involvement of PPARγ in these effects.
This study is the first to demonstrate the expression of 15-lipoxygenase-1 and -2 in articular chondrocytes. Their respective metabolites, namely 13(S)-hydroxy octadecadienoic and 15(S)-hydroxyeicosatetraenoic acids, suppressed IL-1β-induced MMP-1 and MMP-13 expression in a PPARγ-dependent pathway. These data suggest that 15-lipoxygenases may have chondroprotective properties by reducing MMP-1 and MMP-13 expression.
Articular cartilage is physiologically exposed to repeated loads. The mechanical properties of cartilage are due to its extracellular matrix, and homeostasis is maintained by the sole cell type found in cartilage, the chondrocyte. Although mechanical forces clearly control the functions of articular chondrocytes, the biochemical pathways that mediate cellular responses to mechanical stress have not been fully characterised. The aim of our study was to examine early molecular events triggered by dynamic compression in chondrocytes. We used an experimental system consisting of primary mouse chondrocytes embedded within an agarose hydrogel; embedded cells were pre-cultured for one week and subjected to short-term compression experiments. Using Western blots, we demonstrated that chondrocytes maintain a differentiated phenotype in this model system and reproduce typical chondrocyte-cartilage matrix interactions. We investigated the impact of dynamic compression on the phosphorylation state of signalling molecules and genome-wide gene expression. After 15 min of dynamic compression, we observed transient activation of ERK1/2 and p38 (members of the mitogen-activated protein kinase (MAPK) pathways) and Smad2/3 (members of the canonical transforming growth factor (TGF)-β pathways). A microarray analysis performed on chondrocytes compressed for 30 min revealed that only 20 transcripts were modulated more than 2-fold. A less conservative list of 325 modulated genes included genes related to the MAPK and TGF-β pathways and/or known to be mechanosensitive in other biological contexts. Of these candidate mechanosensitive genes, 85% were down-regulated. Down-regulation may therefore represent a general control mechanism for a rapid response to dynamic compression. Furthermore, modulation of transcripts corresponding to different aspects of cellular physiology was observed, such as non-coding RNAs or primary cilium. This study provides new insight into how chondrocytes respond to mechanical forces.
The mechanisms involved in hypoxic pulmonary vasoconstriction (HPV) are not yet fully defined. The aim of the study was to determine the role of protein kinase C ζ (PKCζ) and neutral sphingomyelinase (nSMase) in HPV.
Methods and results
Ceramide content was measured by immunocytochemistry and voltage-gated potassium channel (KV) currents were recorded by the patch clamp technique in isolated rat pulmonary artery smooth muscle cells (PASMC). Contractile responses were analysed in rat pulmonary arteries mounted in a wire myograph. Pulmonary pressure was recorded in anesthetized open-chest rats. Protein and mRNA expression were measured by western blot and RT–PCR, respectively. We found that hypoxia increased ceramide content in PASMC which was abrogated by inhibition of nSMase, but not acid sphingomyelinase (aSMase). The hypoxia-induced vasoconstrictor response in isolated pulmonary arteries and the inhibition of KV currents were strongly reduced by inhibition of PKCζ or nSMase but not aSMase. The nSMase inhibitor GW4869 prevented HPV in vivo. The vasoconstrictor response to hypoxia was mimicked by exogenous addition of bacterial Smase and ceramide. nSMase2 mRNA expression was ∼10-fold higher in pulmonary compared with mesenteric arteries. In mesenteric arteries, hypoxia failed to increase ceramide but exogenous SMase induced a contractile response.
nSMase-derived ceramide production and the activation of PKCζ are early and necessary events in the signalling cascade of acute HPV.
Hypoxic pulmonary vasoconstriction; Neutral sphingomyelinase; Protein kinase C ζ; Pulmonary arteries
Cyclooxygenase-2 (COX-2) is known to modulate bone metabolism, including bone formation and resorption. Because cartilage serves as a template for endochondral bone formation and because cartilage development is initiated by the differentiation of mesenchymal cells into chondrocytes (Ahrens et al., 1977; Sandell and Adler, 1999; Solursh, 1989), it is of interest to know whether COX-2 expression affect chondrocyte differentiation. Therefore, we investigated the effects of COX-2 protein on differentiation in rabbit articular chondrocyte and chick limb bud mesenchymal cells. Overexpression of COX-2 protein was induced by the COX-2 cDNA transfection. Ectopic expression of COX-2 was sufficient to causes dedifferentiation in articular chondrocytes as determined by the expression of type II collagen via Alcian blue staining and Western blot. Also, COX-2 overexpression caused suppression of SOX-9 expression, a major transcription factor that regulates type II collagen expression, as indicated by the Western blot and RT-PCR. We further examined ectopic expression of COX-2 in chondrifying mesenchymal cells. As expected, COX-2 cDNA transfection blocked cartilage nodule formation as determined by Alcian blue staining. Our results collectively suggest that COX-2 overexpression causes dedifferentiation in articular chondrocytes and inhibits chondrogenic differentiation of mesenchymal cells.
chondrocytes; cyclooxygenase 2; cell dedifferentiation; cell differentiation; collagen type II; mesenchymal cells
During in vivo development, articular cartilage is exposed to several different forms of stress. This study examined the effects of radial confinement and passive axial compression-induced vertical confinement, on the biomechanical, biochemical, and histological properties of self-assembled chondrocyte constructs. The self-assembled constructs, engineered without the use of an exogenous scaffold, exhibited significant increases in stiffness in the direction orthogonal to that of the confinement surface. With radial confinement, the significantly increased aggregate modulus was accompanied by increased collagen organization in the direction perpendicular to the articular surface, with no change in collagen or glycosaminoglycan (GAG) content. Additionally, radial confinement was most beneficial when applied before t=2 wks. With passive axial compression, the significantly increased Young’s modulus and ultimate tensile strength were accompanied by a significant increase in collagen production. This study is the first to demonstrate the beneficial effects of confinement on tissue engineered constructs in the direction orthogonal to that of the confinement surface.
OBJECTIVE—To investigate the effect of insulin-like growth factor 1 (IGF1) on the release of collagen, and the production and expression of matrix metalloproteinases (MMPs) induced by the proinflammatory cytokine interleukin 1α (IL1α) in combination with oncostatin M (OSM) from bovine nasal cartilage and primary human articular chondrocytes.
METHODS—Human articular chondrocytes and bovine nasal cartilage were cultured with and without IGF1 in the presence of IL1α or IL1α + OSM. The release of collagen was measured by an assay for hydroxyproline. Collagenase activity was determined with the diffuse fibril assay using 3H acetylated collagen. The expression of MMP-1, MMP-3, MMP-8, MMP-13, and tissue inhibitor of metalloproteinase 1 (TIMP-1) mRNA was analysed by northern blot.
RESULTS—IGF1 can partially inhibit the release of collagen induced by IL1α or IL1α + OSM from bovine nasal cartilage. This was accompanied by a reduced secretion and activation of collagenase by bovine nasal cartilage. IGF1 can also down regulate IL1α or IL1α + OSM induced MMP-1, MMP-3, MMP-8, and MMP-13 mRNA expression in human articular chondrocytes and bovine chondrocytes. It had no significant effect on the production and expression of TIMP-1 mRNA in chondrocytes.
CONCLUSION—This study shows for the first time that IGF1 can partially block the release of collagen from cartilage and suggests that down regulation of collagenases by IGF1 may be an important mechanism in preventing cartilage resorption initiated by proinflammatory cytokines.
Accumulation of advanced glycation end products (AGEs) in joints is important in the development of cartilage destruction and damage in age-related osteoarthritis (OA). The aim of this study was to investigate the roles of peroxisome proliferator-activated receptor γ (PPARγ), toll-like receptor 4 (TLR4), and receptor for AGEs (RAGE) in AGEs-induced inflammatory signalings in human OA chondrocytes. Human articular chondrocytes were isolated and cultured. The productions of metalloproteinase-13 and interleukin-6 were quantified using the specific ELISA kits. The expressions of related signaling proteins were determined by Western blotting. Our results showed that AGEs enhanced the productions of interleukin-6 and metalloproteinase-13 and the expressions of cyclooxygenase-2 and high-mobility group protein B1 and resulted in the reduction of collagen II expression in human OA chondrocytes. AGEs could also activate nuclear factor (NF)-κB activation. Stimulation of human OA chondrocytes with AGEs significantly induced the up-regulation of TLR4 and RAGE expressions and the down-regulation of PPARγ expression in a time- and concentration-dependent manner. Neutralizing antibodies of TLR4 and RAGE effectively reversed the AGEs-induced inflammatory signalings and PPARγ down-regulation. PPARγ agonist pioglitazone could also reverse the AGEs-increased inflammatory signalings. Specific inhibitors for p38 mitogen-activated protein kinases, c-Jun N-terminal kinase and NF-κB suppressed AGEs-induced PPARγ down-regulation and reduction of collagen II expression. Taken together, these findings suggest that AGEs induce PPARγ down-regulation-mediated inflammatory signalings and reduction of collagen II expression in human OA chondrocytes via TLR4 and RAGE, which may play a crucial role in the development of osteoarthritis pathogenesis induced by AGEs accumulation.
Pulsed low intensity ultrasound (PLIUS) has been used successfully for bone fracture repair and has therefore been suggested for cartilage regeneration. However, previous in vitro studies with chondrocytes show conflicting results as to the effect of PLIUS on the elaboration of extracellular matrix. This study tests the hypothesis that PLIUS, applied for 20 min/day, stimulates the synthesis of sulphated glycosaminoglycan (sGAG) by adult bovine articular chondrocytes cultured in either monolayer or agarose constructs. For both culture models, PLIUS at either 30 or 100 mW/cm2 intensity had no net effect on the total sGAG content. Although PLIUS at 100 mW/cm2 did induce a 20% increase in sGAG content at day 2 of culture in agarose, this response was lost by day 5. Intensities of 200 and 300 mW/cm2 resulted in cell death probably due to heating from the ultrasound transducers. The lack of a sustained up-regulation of sGAG synthesis may reflect the suggestion that PLIUS only induces a stimulatory effect in the presence of a tissue injury response. These results suggest that PLIUS has a limited potential to provide an effective method of stimulating matrix production as part of a tissue engineering strategy for cartilage repair.
LIPUS; PLIUS; Cartilage; Glycosaminoglycan; Ultrasound
Small hyaluronan (HA) oligosaccharides serve as competitive receptor antagonists to displace HA from the cell surface and induce cell signaling events. In articular chondrocytes this cell signaling is mediated by the HA receptor CD44 and induces stimulation of genes involved in matrix degradation such as matrix metalloproteinases as well as matrix repair genes including collagen type II, aggrecan and HA synthase-2. The objective of this study was to determine changes in the expression and function of aggrecanases after disruption of chondrocyte CD44-HA interactions.
Bovine articular chondrocytes or bovine cartilage tissue were pre-treated with a variety of inhibitors of major signaling pathways prior to the addition of HA oligosaccharides. Changes in aggrecanase were monitored by real time reverse transcriptase-polymerase chain reaction and western blot analysis of ADAMTS4, ADAMTS5 and aggrecan proteolytic fragments. To test the interactions between ADAMTS4 and MT4-MMP, protein lysates purified from stimulated chondrocytes were subjected to co-immunoprecipitation.
Disruption of chondrocyte CD44-HA interactions with HA oligosaccharides induced the transcription of ADAMTS4 and ADAMTS5 in time- and dose-dependent manner. The association of GPI-anchored MT4-MMP with ADAMTS4 was also induced in articular chondrocytes by HA oligosaccharides. Inhibition of the NF-κB pathway blocked HA oligosaccharides-mediated stimulation of aggrecanases.
Disruptive changes in chondrocyte-matrix interactions by HA oligosaccharides induce matrix degradation and elevate aggrecanases via the activation of the NF-κB signaling pathway.
Hyaluronan; Hyaluronan oligosaccharides; CD44; ADAMTS4; ADAMTS5; MT4-MMP
Inflammation accompanied by severe oxidative stress plays a vital role in the orchestration and progression of neurodegeneration prevalent in chronic and acute CNS pathologies as well as in aging. The proinflammatory cytokine tumor necrosis factor alpha (TNFα) elicits the formation of the bioactive ceramide by stimulating the hydrolysis of the membrane lipid sphingomyelin by sphingomyelinase activities. Ceramide stimulates the formation of reactive oxygen species (ROS) and apoptotic mechanisms in both neurons and non-neuronal cells establishing a link between sphingolipid metabolism and oxidative stress. We demonstrated in SH-SY5Y human neuroblastoma cells and primary cortical neurons, that TNFα is a potent stimulator of Mg2+-dependent neutral sphingomyelinase (Mg2+-nSMase) activity and sphingomyelin hydrolysis rather than de novo synthesis, was the predominant source of ceramide increases. Mg2+-nSMase activity preceded an accumulation of ROS by a neuronal NADPH oxidase (NOX). Notably, TNFα provoked a NOX-dependent oxidative damage to sphingosine kinase-1, which generates sphingosine-1-phosphate, a ceramide metabolite associated with neurite outgrowth. Indeed, ceramide and ROS inhibited neurite outgrowth of DRG neurons by disrupting growth cone motility. Blunting ceramide and ROS formation both rescued sphingosine kinase-1 activity and neurite outgrowth. Our studies suggest that TNFα-mediated activation of Mg2+-nSMase and NOX in neuronal cells not only produced the neurotoxic intermediates ceramide and ROS but also directly antagonized neuronal survival mechanisms thus accelerating neurodegeneration.
sphingomyelinase; ceramide; NADPH oxidase; sphingosine kinase; neuroinflammation
Increases in ceramide levels have been implicated in the pathogenesis of both acute or chronic lung injury models. However, the role of individual ceramide species, or of the enzymes that are responsible for their synthesis, in lung health and disease has not been clarified. We now show that C24- and C16-ceramides are the most abundant lung ceramide species, paralleled by high expression of their synthetic enzymes, ceramide synthase 2 (CerS2) and CerS5, respectively. Furthermore, the ceramide species synthesis in the lung is homeostatically regulated, since mice lacking very long acyl chain C24-ceramides due to genetic deficiency of CerS2 displayed a ten-fold increase in C16-ceramides and C16-dihydroceramides along with elevation of acid sphingomyelinase and CerS5 activities. Despite relatively preserved total lung ceramide levels, inhibition of de novo sphingolipid synthesis at the level of CerS2 was associated with significant airflow obstruction, airway inflammation, and increased lung volumes. Our results suggest that ceramide species homeostasis is crucial for lung health and that CerS2 dysfunction may predispose to inflammatory airway and airspace diseases.
Chondrocytes isolated from a variety of sources, including auricular (AU) and articular (AR) cartilage, can differ in cell behavior, growth, and extracellular matrix (ECM) production, which can impact neocartilage properties in tissue engineering approaches. This behavior is also affected by the surrounding microenvironment, including soluble factors, biomaterials, and mechanical loading. The objective of this study was to investigate differences in juvenile AU and AR chondrocyte behavior when encapsulated in radically polymerized hyaluronic acid hydrogels. When implanted in vivo, differences in macroscopic appearance, mechanical properties, glycosaminoglycan content, and collagen content were observed depending on the chondrocyte type encapsulated. Specifically, AU constructs exhibited construct growth and neocartilage formation with increases in aggregate modulus and ECM accumulation with culture, whereas AR constructs retained their construct size and remained translucent with only a minimal increase in the compressive modulus. When cultured in vitro, both cell types remained viable and differences in gene expression were observed for type I and II collagens. Likewise, differences in gene expression were noted after dynamic mechanical loading, where stimulated AR constructs exhibited 2.3- and 1.5-fold increases in type II collagen and aggrecan over free-swelling controls, while AU samples exhibited smaller fold increases of 1.4- and 1.3-fold, respectively. Thus, these data indicate that the specific cell source, cell/material interactions, and loading environment are important in the final properties of tissue-engineered products.
We previously showed that degradation of cellular sphingomyelin (SM) by SMase C results in a greater stimulation of cholesterol translocation to endoplasmic reticulum, compared to its degradation by SMase D. Here we investigated the hypothesis that the effect of SMase C is partly due to the generation of ceramide, rather than due to depletion of SM alone. Inhibition of hydroxymethylglutaryl CoA reductase (HMGCR) activity was used as a measure of cholesterol translocation. Treatment of fibroblasts with SMase C resulted in a 90% inhibition of HMGCR, whereas SMase D treatment inhibited it by 29%. Treatment with exogenous ceramides, or increasing the endogenous ceramide levels also inhibited HMGCR by 60–80%. Phosphorylation of HMGCR was stimulated by SMase C or exogenous ceramide. The effects of ceramide and SMase D were additive, indicating the independent effects of SM depletion and ceramide generation. These results show that ceramide regulates sterol trafficking independent of cellular SM levels.
Sphingomyelin; Ceramide; ACAT; HMG CoA reductase; Sterol trafficking
While designing poly(ethylene glycol) hydrogels with high moduli suitable for in situ placement is attractive for cartilage regeneration, the impact of a tighter crosslinked structure on the organization and deposition of matrix is not fully understood. The objectives for this study were to characterize the composition and spatial organization of neo-matrix as a function of gel crosslinking and study its impact on chondrocytes via anabolic and catabolic gene expressions and catabolic activity. Bovine articular chondrocytes were encapsulated in hydrogels of three crosslinking densities (compressive moduli were 60, 320 and 590 kPa) and cultured for 25 days. Glycosaminoglycan production increased with culture time and was greatest in gels with lowest crosslinking. Collagens II and VI, aggrecan, link protein, and decorin were localized to pericellular regions in all gels, but their presence decreased with increases in gel crosslinking. Collagen II and aggrecan expressions were initially up-regulated in gels with higher crosslinking, but increased similarly up to day 15. Matrix metalloproteinases (MMP)-1 and -13 expressions were elevated (~25-fold) in gels with higher crosslinking throughout the study, while MMP-3 was not affected by gel crosslinking. The presence of aggecan and collagen degradation products confirmed MMP activity. These findings indicate that chondrocytes synthesize the major cartilage components within PEG hydrogels, however, gel structure strongly impacts the composition and spatial organization of the neo-tissue and impacts how chondrocytes respond to their environment, particularly with respect to their catabolic expressions.
hydrogels; cartilage; chondrocytes; extracellular matrix; pericellular matrix
Interleukin-1β (IL-1β) is overproduced in human and rodent epileptogenic tissue and it exacerbates seizures upon brain application in rodents. Moreover, pharmacological prevention of IL-1β endogenous synthesis, or IL-1 receptor blockade, mediates powerful anticonvulsive actions indicating a significant role of this cytokine in ictogenesis. The molecular mechanisms of the proconvulsive actions of IL-1β are not known. We show here that EEG seizures induced by intrahippocampal injection of kainic acid in C57BL6 adult mice were increased by 2-fold on average by pre-exposure to IL-1β and this effect was blocked by 3-O-methylsphingomyelin (3-O-MS), a selective inhibitor of the ceramide-producing enzyme sphingomyelinase. C2-ceramide, a cell permeable analog of ceramide, mimicked IL-1β action suggesting that ceramide may be the second messenger of the proconvulsive effect of IL-1β. The seizure exacerbating effects of either IL-1β or C2-ceramide were dependent on activation of the Src family of tyrosine kinases since they were prevented by CGP76030, an inhibitor of this enzyme family. The proconvulsive IL-1β effect was associated with increased Tyr418 phosphorylation of Src-family of kinases indicative of its activation, and Tyr1472 phosphorylation of one of its substrate, the NR2B subunit of the N-methyl-d-aspartate receptor, which were prevented by 3-O-MS and CGP76030. Finally, the proconvulsive effect of IL-1β was blocked by ifenprodil, a selective NR2B receptor antagonist. These results indicate that the proconvulsive actions of IL-1β depend on the activation of a sphingomyelinase- and Src-family of kinases-dependent pathway in the hippocampus which leads to the phosphorylation of the NR2B subunit, thus highlighting a novel, non-transcriptional mechanism underlying seizure exacerbation in inflammatory conditions.
experimental epilepsy; glia activation; cytokines; NMDA receptor; inflammation
The de novo pathway of ceramide synthesis has been implicated in the pathogenesis of excessive lung apoptosis and murine emphysema. Intracellular and paracellular-generated ceramides may trigger apoptosis and propagate the death signals to neighboring cells, respectively. In this study we compared the sphingolipid signaling pathways triggered by the paracellular- versus intracellular-generated ceramides as they induce lung endothelial cell apoptosis, a process important in emphysema development. Intermediate–chain length (C8:0) extracellular ceramides, used as a surrogate of paracellular ceramides, triggered caspase-3 activation in primary mouse lung endothelial cells, similar to TNF-α–generated endogenous ceramides. Inhibitory siRNA against serine palmitoyl transferase subunit 1 but not acid sphingomyelinase inhibited both C8:0 ceramide– and TNF-α (plus cycloheximide)–induced apoptosis, consistent with the requirement for activation of the de novo pathway of sphingolipid synthesis. Tandem mass spectrometry analysis detected increases in both relative and absolute levels of C16:0 ceramide in response to C8:0 and TNF-α treatments. These results implicate the de novo pathway of ceramide synthesis in the apoptotic effects of both paracellular ceramides and TNF-α–stimulated intracellular ceramides in primary lung endothelial cells. The serine palmitoyl synthase-regulated ceramides synthesis may contribute to the amplification of pulmonary vascular injury induced by excessive ceramides.
apoptosis; lung; cytokines; signaling; sphingolipids
The in vitro phenotype of bovine articular chondrocytes is described. Chondrocytes plated at high density in roller-bottle and dish cultures were maintained in vitro. The major matrix macromolecules, collagen and proteoglycan, synthesized by these cells were characterized during the course of the culture period. The chondrocytes synthesized mainly Type II collagen, which was found predominantly in the cell-associated matrix. The media contained a mixture of Type II and Type III collagens. Type I collagen was detectable in neither the medium nor the cell-associated matrix. The proteoglycan monomers found in media and cell-associated matrix had the same hydrodynamic sizes as monomers synthesized by cartilage slices or those extracted from adult articular cartilage. The majority of proteoglycans synthesized by the cells were found in high molecular weight aggregates which were readily recovered from the media and were extractable from cell-associated matrix with low ionic strength buffers. The results demonstrate the long-term in vitro phenotypic stability of the bovine articular chondrocytes. The advantages of the in vitro system as a model for studying the effects of external agents, such as drugs and vitamins, are discussed.
Osteoarthritis (OA), the commonest form of arthritis and a major cause of morbidity, is characterized by progressive degeneration of the articular cartilage. Along with increased production and activation of degradative enzymes, altered synthesis of cartilage matrix molecules and growth factors by resident chondrocytes is believed to play a central role in this pathological process. We used an ovine meniscectomy model of OA to evaluate changes in chondrocyte expression of types I, II and III collagen; aggrecan; the small leucine-rich proteoglycans (SLRPs) biglycan, decorin, lumican and fibromodulin; transforming growth factor-β; and connective tissue growth factor. Changes were evaluated separately in the medial and lateral tibial plateaux, and were confirmed for selected molecules using immunohistochemistry and Western blotting. Significant changes in mRNA levels were confined to the lateral compartment, where active cartilage degeneration was observed. In this region there was significant upregulation in expession of types I, II and III collagen, aggrecan, biglycan and lumican, concomitant with downregulation of decorin and connective tissue growth factor. The increases in type I and III collagen mRNA were accompanied by increased immunostaining for these proteins in cartilage. The upregulated lumican expression in degenerative cartilage was associated with increased lumican core protein deficient in keratan sulphate side-chains. Furthermore, there was evidence of significant fragmentation of SLRPs in both normal and arthritic tissue, with specific catabolites of biglycan and fibromodulin identified only in the cartilage from meniscectomized joints. This study highlights the focal nature of the degenerative changes that occur in OA cartilage and suggests that altered synthesis and proteolysis of SLRPs may play an important role in cartilage destruction in arthritis.