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1.  Does protein kinase R mediate TNF-α- and ceramide-induced increases in expression and activation of matrix metalloproteinases in articular cartilage by a novel mechanism? 
Arthritis Research & Therapy  2003;6(1):R46-R55.
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.
PMCID: PMC400415  PMID: 14979937
articular cartilage; ceramide; matrix metalloproteinase; PKR; TNF-α
2.  The Effect of Dexamethasone and Triiodothyronine on Terminal Differentiation of Primary Bovine Chondrocytes and Chondrogenically Differentiated Mesenchymal Stem Cells 
PLoS ONE  2013;8(8):e72973.
The newly evolved field of regenerative medicine is offering solutions in the treatment of bone or cartilage loss and deficiency. Mesenchymal stem cells, as well as articular chondrocytes, are potential cells for the generation of bone or cartilage. The natural mechanism of bone formation is that of endochondral ossification, regulated, among other factors, through the hormones dexamethasone and triiodothyronine. We investigated the effects of these hormones on articular chondrocytes and chondrogenically differentiated mesenchymal stem cells, hypothesizing that these hormones would induce terminal differentiation, with chondrocytes and differentiated stem cells being similar in their response. Using a 3D-alginate cell culture model, bovine chondrocytes and chondrogenically differentiated stem cells were cultured in presence of triiodothyronine or dexamethasone, and cell proliferation and extracellular matrix production were investigated. Collagen mRNA expression was measured by real-time PCR. Col X mRNA and alkaline phosphatase were monitored as markers of terminal differentiation, a prerequisite of endochondral ossification. The alginate culture system worked well, both for the culture of chondrocytes and for the chondrogenic differentiation of mesenchymal stem cells. Dexamethasone led to an increase in glycosaminoglycan production. Triiodothyronine increased the total collagen production only in chondrocytes, where it also induced signs of terminal differentiation, increasing both collagen X mRNA and alkaline phosphatase activity. Dexamethasone induced terminal differentiation in the differentiated stem cells. The immature articular chondrocytes used in this study seem to be able to undergo terminal differentiation, pointing to their possible role in the onset of degenerative osteoarthritis, as well as their potential for a cell source in bone tissue engineering. When chondrocyte-like cells, after their differentiation, can indeed be moved on towards terminal differentiation, they can be used to generate a model of endochondral ossification, but this limitation must be kept in mind when using them in cartilage tissue engineering application.
PMCID: PMC3745539  PMID: 23977373
3.  Control of Collagen Production in Mouse Chondrocytes by Using a Combination of Bone Morphogenetic Protein-2 and Small Interfering RNA Targeting Col1a1 for Hydrogel-Based Tissue-Engineered Cartilage 
Because articular cartilage does not self-repair, tissue-engineering strategies should be considered to regenerate this tissue. Autologous chondrocyte implantation is already used for treatment of focal damage of articular cartilage. Unfortunately, this technique includes a step of cell amplification, which results in dedifferentiation of chondrocytes, with expression of type I collagen, a protein characteristic of fibrotic tissues. Therefore, the risk of producing a fibrocartilage exists. The aim of this study was to propose a new strategy for authorizing the recovery of the differentiated status of the chondrocytes after their amplification on plastic. Because the bone morphogenetic protein (BMP)-2 and the transforming growth factor (TGF)-β1 are cytokines both proposed as stimulants for cartilage repair, we undertook a detailed comparative analysis of their biological effects on chondrocytes. As a cellular model, we used mouse chondrocytes after their expansion on plastic and we tested the capability of BMP-2 or TGF-β1 to drive their redifferentiation, with special attention given to the nature of the proteins synthesized by the cells. To prevent any fibrotic character of the newly synthesized extracellular matrix, we silenced type I collagen by transfecting small interfering RNA (siRNA) into the chondrocytes, before their exposure to BMP-2 or TGF-β1. Our results showed that addition of siRNA targeting the mRNA encoded by the Col1a1 gene (Col1a1 siRNA) and BMP-2 represents the most efficient combination to control the production of cartilage-characteristic collagen proteins. To go one step further toward scaffold-based cartilage engineering, Col1a1 siRNA-transfected chondrocytes were encapsulated in agarose hydrogel and cultured in vitro for 1 week. The analysis of the chondrocyte–agarose constructs by using real-time polymerase chain reaction, Western-blotting, immunohistochemistry, and electron microscopy techniques demonstrated that the BMP-2/Col1a1 siRNA combination is effective in reinitializing correct production and assembly of the cartilage-characteristic matrix in agarose hydrogel, without production of type I collagen. Because agarose is known to favor long-term expression of the chondrocyte phenotype and agarose-based hydrogels are approved for clinical trials, this strategy appears very promising to repair hyaline cartilage.
PMCID: PMC3689937  PMID: 23311625
4.  Chondrogenic differentiation potential of osteoarthritic chondrocytes and their possible use in matrix-associated autologous chondrocyte transplantation 
Arthritis Research & Therapy  2009;11(5):R133.
Autologous chondrocyte transplantation (ACT) is a routine technique to regenerate focal cartilage lesions. However, patients with osteoarthritis (OA) are lacking an appropriate long-lasting treatment alternative, partly since it is not known if chondrocytes from OA patients have the same chondrogenic differentiation potential as chondrocytes from donors not affected by OA.
Articular chondrocytes from patients with OA undergoing total knee replacement (Mankin Score > 3, Ahlbäck Score > 2) and from patients undergoing ACT, here referred to as normal donors (ND), were isolated applying protocols used for ACT. Their chondrogenic differentiation potential was evaluated both in high-density pellet and scaffold (Hyaff-11) cultures by histological proteoglycan assessment (Bern Score) and immunohistochemistry for collagen types I and II. Chondrocytes cultured in monolayer and scaffolds were subjected to gene expression profiling using genome-wide oligonucleotide microarrays. Expression data were verified by using real-time PCR.
Chondrocytes from ND and OA donors demonstrated accumulation of comparable amounts of cartilage matrix components, including sulphated proteoglycans and collagen types I and II. The mRNA expression of cartilage markers (ACAN, COL2A1, COMP, CRTL1, SOX9) and genes involved in matrix synthesis (BGN, CILP2, COL9A2, COL11A1, TIMP4) was highly induced in 3D cultures of chondrocytes from both donor groups. Genes associated with hypertrophic or OA cartilage (ALPL, COL1A1, COL3A1, COL10A1, MMP13, POSTN, PTH1R, RUNX2) were not significantly regulated between the two groups of donors. The expression of 661 genes, including COMP, FN1, and SOX9, was differentially regulated between OA and ND chondrocytes cultured in monolayer. During scaffold culture, the differences diminished between the OA and ND chondrocytes, and only 184 genes were differentially regulated.
Only few genes were differentially expressed between OA and ND chondrocytes in Hyaff-11 culture. The risk of differentiation into hypertrophic cartilage does not seem to be increased for OA chondrocytes. Our findings suggest that the chondrogenic capacity is not significantly affected by OA, and OA chondrocytes fulfill the requirements for matrix-associated ACT.
PMCID: PMC2787268  PMID: 19723327
5.  Fibrin and poly(lactic-co-glycolic acid) hybrid scaffold promotes early chondrogenesis of articular chondrocytes: an in vitro study 
Synthetic- and naturally derived- biodegradable polymers have been widely used to construct scaffolds for cartilage tissue engineering. Poly(lactic-co-glycolic acid) (PLGA) are bioresorbable and biocompatible, rendering them as a promising tool for clinical application. To minimize cells lost during the seeding procedure, we used the natural polymer fibrin to immobilize cells and to provide homogenous cells distribution in PLGA scaffolds. We evaluated in vitro chondrogenesis of rabbit articular chondrocytes in PLGA scaffolds using fibrin as cell transplantation matrix.
PLGA scaffolds were soaked in chondrocytes-fibrin suspension (1 × 106cells/scaffold) and polymerized by dropping thrombin-calcium chloride (CaCl2) solution. PLGA-seeded chondrocytes was used as control. All constructs were cultured for a maximum of 21 days. Cell proliferation activity was measured at 1, 3, 7, 14 and 21 days in vitro using 3-(4,5-dimethylthiazole-2-yl)-2-, 5-diphenyltetrazolium-bromide (MTT) assay. Morphological observation, histology, immunohistochemistry (IHC), gene expression and sulphated-glycosaminoglycan (sGAG) analyses were performed at each time point of 1, 2 and 3 weeks to elucidate in vitro cartilage development and deposition of cartilage-specific extracellular matrix (ECM).
Cell proliferation activity was gradually increased from day-1 until day-14 and declined by day-21. A significant cartilaginous tissue formation was detected as early as 2-week in fibrin/PLGA hybrid construct as confirmed by the presence of cartilage-isolated cells and lacunae embedded within basophilic ECM. Cartilage formation was remarkably evidenced after 3 weeks. Presence of cartilage-specific proteoglycan and glycosaminoglycan (GAG) in fibrin/PLGA hybrid constructs were confirmed by positive Safranin O and Alcian Blue staining. Collagen type II exhibited intense immunopositivity at the pericellular matrix. Chondrogenic properties were further demonstrated by the expression of genes encoded for cartilage-specific markers, collagen type II and aggrecan core protein. Interestingly, suppression of cartilage dedifferentiation marker; collagen type I was observed after 2 and 3 weeks of in vitro culture. The sulphated-glycosaminoglycan (sGAG) production in fibrin/PLGA was significantly higher than in PLGA.
Fibrin/PLGA promotes early in vitro chondrogenesis of rabbit articular chondrocytes. This study suggests that fibrin/PLGA may serve as a potential cell delivery vehicle and a structural basis for in vitro tissue-engineered articular cartilage.
PMCID: PMC2405772  PMID: 18435862
6.  Hypoxia promotes redifferentiation and suppresses markers of hypertrophy and degeneration in both healthy and osteoarthritic chondrocytes 
Hypoxia is considered to be a positive influence on the healthy chondrocyte phenotype and cartilage matrix formation. However, hypoxia-inducible factors (HIFs) have been implicated in the pathogenesis of osteoarthritis (OA). Thus, we assessed whether healthy and OA chondrocytes have distinct responses to oxygen, particularly with regard to hypertrophy and degradation during redifferentiation.
Monolayer-expanded healthy and OA chondrocytes were redifferentiated for 14 days in pellet cultures under standard (20% oxygen) or hypoxic (2% oxygen) conditions. Cartilage matrix gene expression, matrix quality and quantity, degradative enzyme expression and HIF expression were measured.
In hypoxia, both healthy and OA chondrocytes had higher human collagen type II, α1 gene (COL2A1), and aggrecan (ACAN) expression and sulfated glycosaminoglycan (sGAG) accumulation, concomitant with lower human collagen type X, α1 gene (COL10A1), and human collagen type I, α1 gene (COL1A1), expression and collagen I extracellular accumulation. OA chondrocytes had significantly lower sGAGs/DNA than healthy chondrocytes, but only in high oxygen conditions. Hypoxia also caused significantly greater sGAG retention and hyaluronic acid synthase 2 (HAS2) expression by OA chondrocytes. Both healthy and OA chondrocytes had significantly lower expression of matrix metalloproteinases (MMPs) MMP1, MMP2, MMP3 and MMP13 in hypoxia and less active MMP2 enzyme, consistent with lower MMP14 expression. However, aggrecanase (ADAMTS4 and ADAMTS5) expression was significantly lowered by hypoxia only in healthy cells, and COL10A1 and MMP13 remained significantly higher in OA chondrocytes than in healthy chondrocytes in hypoxic conditions. HIF-1α and HIF-2α had similar expression profiles in healthy and OA cells, increasing to maximal levels early in hypoxia and decreasing over time.
Hypoxic culture of human chondrocytes has long been acknowledged to result in increased matrix accumulation, but still little is known of its effects on catabolism. We show herein that the increased expression of matrix proteins, combined with decreased expression of numerous degradative enzymes by hypoxia, minimizes but does not abolish differences between redifferentiated healthy and OA chondrocytes. Hypoxia-induced HIF expression is associated with hypertrophic marker and degradative enzyme downregulation and increased measures of redifferentiation in both healthy and OA chondrocytes. Therefore, though HIFs may be involved in the pathogenesis of OA, conditions that promote HIF expression in vitro promote matrix accumulation and decrease degradation and hypertrophy, even in cells from OA joints.
PMCID: PMC3979022  PMID: 23965235
7.  Osteoarthritic cartilage explants affect extracellular matrix production and composition in cocultured bone marrow-derived mesenchymal stem cells and articular chondrocytes 
In the present study, we established a novel in vitro coculture model to evaluate the influence of osteoarthritis (OA) cartilage explants on the composition of newly produced matrix and chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells (BMSCs) and the phenotype of OA chondrocytes. In addition, we included a “tri-culture” model, whereby a mixture of BMSCs and chondrocytes was cultured on the surface of OA cartilage explants.
Gene expression analysis, protein and glycosaminoglycan (GAG) assays, dot-blot, immunofluorescence, and biomechanical tests were used to characterize the properties of newly generated extracellular matrix (ECM) from chondrocytes and chondrogenically differentiated BMSCs and a mix thereof. We compared articular cartilage explant cocultures with BMSCs, chondrocytes, and mixed cultures (chondrocytes and BMSCs 1:1) embedded in fibrin gels with fibrin gel-embedded cells cultured without cartilage explants (monocultures).
In general, co- and tri-cultured cell regimens exhibited reduced mRNA and protein levels of collagens I, II, III, and X in comparison with monocultures, whereas no changes in GAG synthesis were observed. All co- and tri-culture regimens tended to exhibit lower Young’s and equilibrium modulus compared with monocultures. In contrast, aggregate modulus and hydraulic permeability seemed to be higher in co- and tri-cultures. Supernatants of cocultures contained significant higher levels of interleukin-1 beta (IL-1β), IL-6, and IL-8. Stimulation of monocultures with IL-1β and IL-6 reduced collagen gene expression in BMSCs and mixed cultures in general but was often upregulated in chondrocytes at late culture time points. IL-8 stimulation affected BMSCs only.
Our results suggest an inhibitory effect of OA cartilage on the production of collagens. This indicates a distinct modulatory influence that affects the collagen composition of the de novo-produced ECM from co- and tri-cultured cells and leads to impaired mechanical and biochemical properties of the matrix because of an altered fibrillar network. We suggest that soluble factors, including IL-1β and IL-6, released from OA cartilage partly mediate these effects. Thus, neighbored OA cartilage provides inhibitory signals with respect to BMSCs’ chondrogenic differentiation and matrix composition, which need to be accounted for in future cell-based OA treatment strategies.
PMCID: PMC4097830  PMID: 24916039
8.  Culture Expansion in Low-Glucose Conditions Preserves Chondrocyte Differentiation and Enhances Their Subsequent Capacity to Form Cartilage Tissue in Three-Dimensional Culture 
BioResearch Open Access  2014;3(1):9-18.
Culture conditions that preserve a stable chondrocyte phenotype are desirable in cell-based cartilage repair to maximize efficacy and clinical outcome. This study investigates whether low-glucose conditions will preserve the chondrocyte phenotype during culture expansion. Articular chondrocytes were culture-expanded in media supplemented with either low (1 mM) or high (10 mM) glucose. The metabolic phenotype, reactive oxygen species generation, and mRNA expression of markers of differentiation or catabolism were assessed by reverse-transcription quantitative polymerase chain reaction after four population doublings (PDs) and subsequent tissue formation capacity determined using pellet cultures. Continuous monolayer culture was used to determine the population doubling limit. After expansion in monolayer for four PDs, chondrocytes expanded in low-glucose conditions exhibited higher expression of the differentiation markers SOX9 and COL2A1 and reduced expression of the catabolic metalloproteinase matrix metallopeptidase 13. When chondrocytes expanded in low glucose were cultured in micropellets, they consistently generated more cartilaginous extracellular matrix than those expanded in high glucose, as evaluated by wet weight, sulfated glycosaminoglycan content, and hydroxyproline assay for collagen content. The same pattern was observed whether high or low glucose was used during the pellet culture. During expansion, chondrocytes in high-glucose generated 50% more reactive oxygen species than low-glucose conditions, despite a lower dependence on oxidative phosphorylation for energy. Furthermore low-glucose cells exhibited >30% increased population doubling limit. These data suggests that low-glucose expansion conditions better preserve the expression of differentiation markers by chondrocytes and enhance their subsequent capacity to form cartilage in vitro. Therefore, low glucose levels should be considered for the expansion of chondrocytes intended for tissue engineering applications.
PMCID: PMC3929268  PMID: 24570841
chondrocyte; Crabtree effect; differentiation markers; glucose concentration; monolayer expansion potential; regenerative potential; tissue engineering
9.  Differential gene expression associated with postnatal equine articular cartilage maturation 
Articular cartilage undergoes an important maturation process from neonate to adult that is reflected by alterations in matrix protein organization and increased heterogeneity of chondrocyte morphology. In the horse, these changes are influenced by exercise during the first five months of postnatal life. Transcriptional profiling was used to evaluate changes in articular chondrocyte gene expression during postnatal growth and development.
Total RNA was isolated from the articular cartilage of neonatal (0–10 days) and adult (4–5 years) horses, subjected to one round of linear RNA amplification, and then applied to a 9,367-element equine-specific cDNA microarray. Comparisons were made with a dye-swap experimental design. Microarray results for selected genes (COL2A1, COMP, P4HA1, TGFB1, TGFBR3, TNC) were validated by quantitative polymerase chain reaction (qPCR).
Fifty-six probe sets, which represent 45 gene products, were up-regulated (p < 0.01) in chondrocytes of neonatal articular cartilage relative to chondrocytes of adult articular cartilage. Conversely, 586 probe sets, which represent 499 gene products, were up-regulated (p < 0.01) in chondrocytes of adult articular cartilage relative to chondrocytes of neonatal articular cartilage. Collagens, matrix-modifying enzymes, and provisional matrix non-collagenous proteins were expressed at higher levels in the articular cartilage of newborn foals. Those genes with increased mRNA abundance in adult chondrocytes included leucine-rich small proteoglycans, matrix assembly, and cartilage maintenance proteins.
Differential expression of genes encoding matrix proteins and matrix-modifying enzymes between neonates and adults reflect a cellular maturation process in articular chondrocytes. Up-regulated transcripts in neonatal cartilage are consistent with growth and expansion of the articular surface. Expression patterns in mature articular cartilage indicate a transition from growth to homeostasis, and tissue function related to withstanding shear and weight-bearing stresses.
PMCID: PMC2585085  PMID: 18986532
10.  Chondrocyte source for cartilage regeneration in an immature animal: Is iliac apophysis a good alternative? 
Indian Journal of Orthopaedics  2012;46(4):402-406.
Autologous articular cartilage at present forms the main source of chondrocytes for cartilage tissue engineering. In children, iliac apophysis is a rich and readily accessible source of chondrocytes. This study compares the growth characteristics and phenotype maintenance of goat iliac apophysis growth plate chondrocytes with those sourced from goat articular cartilage, and thereby assesses their suitability for autologous chondrocyte transplantation in immature animals for growth plate and articular cartilage regeneration.
Materials and Methods:
Four sets of experiments were carried out. Cartilage samples were harvested under aseptic conditions from goat iliac apophysis and knee articular cartilage. The chondrocytes were isolated in each set and viable cells were counted and subsequently cultured as a monolayer in tissue culture flasks containing chondrogenic media at 2.5 × 103cells/cm2. The growth was periodically assessed with phase contrast microcopy and the cells were harvested on 8th and 15th days for morphology, cell yield, and phenotype assessment. Student's t-test was used for comparison of the means.
Confluence was reached in the iliac apophysis growth plate chondrocytes flasks on the 10th day and the articular cartilage chondrocytes flasks on the 14th day. Mean cell count of growth plate chondrocytes on the 8th day was 3.64 × 105 (SD = 0.601) and that of articular cartilage chondrocytes was 1.40 × 105 (SD = 0.758) per flask. The difference in the means was statistically significant (P = 0.003). On the 15th day, the mean cell number had increased to 1.35 × 106(SD = 0.20) and 1.19 × 106 (SD = 0.064) per flask, respectively. This difference was not statistically significant (P = 0.26). The population doubling time on the 8th day of cell culture was 3.18 and 6.24 days respectively, for iliac apophyseal and articular cartilage chondrocytes, which was altered to 3.59 and 3.1 days, respectively, on the 15th day. The immunocytochemistry showed 100% retention of collagen 2 positive and collagen 1 negative cells in both sets of cultures in all samples.
Iliac apophysis is a rich source of chondrocytes with a high growth rate and ability to retain phenotype when compared to articular cartilage derived chondrocytes. Further in vivo studies may determine the efficacy of physeal and articular repair in children with apophyseal chondrocytes.
PMCID: PMC3421929  PMID: 22912514
Articular cartilage; collagen 1; collagen 2; growth plate chondrocyte; immunocytochemistry; physeal bar; physis
11.  α5β1 integrin induces the expression of noncartilaginous procollagen gene expression in articular chondrocytes cultured in monolayers 
Arthritis Research & Therapy  2013;15(5):R127.
Articular chondrocytes undergo an obvious phenotypic change when cultured in monolayers. During this change, or dedifferentiation, the expression of type I and type III procollagen is induced where normal chondrocytes express little type I and type III procollagen. In this study, we attempted to determine the mechanism(s) for the induction of such procollagen expression in dedifferentiating chondrocytes.
All experiments were performed using primary-cultured human articular chondrocytes under approval of institutional review boards. Integrin(s) responsible for the induction of type I and type III procollagen expression were specified by RNAi experiments. The signal pathway(s) involved in the induction were determined by specific inhibitors and RNAi experiments. Adenovirus-mediated experiments were performed to identify a small GTPase regulating the activity of integrins in dedifferentiating chondrocytes. The effect of inhibition of integrins on dedifferentiation was investigated by experiments using echistatin, a potent disintegrin. The effect of echistatin was investigated first with monolayer-cultured chondrocytes, and then with pellet-cultured chondrocytes.
In dedifferentiating chondrocytes, α5β1 integrin was found to be involved in the induction of type I and type III procollagen expression. The induction was known to be mediated by v-akt murine thymoma viral oncogene homolog (AKT) signaling. Among the three AKT isoforms, AKT1 seemed to be most involved in the signaling. Elated RAS viral (r-ras) oncogene homolog (RRAS) was considered to regulate the progression of dedifferentiation by modulating the affinity and avidity of α5β1 integrin to ligands. Echistatin inhibited dedifferentiation of monolayer-cultured chondrocytes. Furthermore, the matrix formed by pellet-cultured chondrocytes more closely resembled that of normal cartilage compared with the controls.
The result of this study has shown, for the first time, that α5β1 integrin may be responsible for the induction of non-cartilaginous collagen expression in chondrocytes undergoing dedifferentiation. Again, this study has shown that the inhibition of ligand ligation to integrins may be an effective strategy to inhibit phenotypic change of cultured chondrocytes, and to improve the quality of matrix synthesized by primary cultured chondrocytes.
PMCID: PMC3978676  PMID: 24286194
12.  Induction of proliferation or hypertrophy of chondrocytes in serum-free culture: the role of insulin-like growth factor-I, insulin, or thyroxine 
The Journal of Cell Biology  1992;116(4):1035-1042.
In bone forming cartilage in vivo, cells undergo terminal differentiation, whereas most of the cells in normal articular cartilage do not. Chondrocyte hypertrophy can be induced also in vitro by diffusible signals. We have identified growth factors or hormones acting individually on 17-d chick embryo sternal chondrocytes cultured in agarose gels under strictly serum-free conditions. Insulin-like growth factor I or insulin triggered the first steps of chondrocyte maturation, i.e., cell proliferation and increased matrix deposition while the chondrocytic phenotype was maintained. However, cells did not progress to the hypertrophic stage. Proliferation and stimulated collagen production was preceded by a lag period, indicating that synthesis of other components was required before cells became responsive to insulin-like growth factor I or insulin. Very small amounts of FBS exerted effects similar to those of insulin-like growth factor I or insulin. However, FBS could act directly and elicited hypertrophy when constituting greater than 1% of the culture media. Basic FGF has been claimed to be the most potent chondrocyte mitogen, but had negligible effects under serum-free conditions. The same is true for PDGF, a major serum-mitogen. Under the direction of thyroxine, cells did not proliferate but became typical hypertrophic chondrocytes, extensively synthesizing collagen X and alkaline phosphatase.
PMCID: PMC2289336  PMID: 1734018
13.  The Effect of Chondroitin Sulphate and Hyaluronic Acid on Chondrocytes Cultured within a Fibrin-Alginate Hydrogel 
Osteoarthritis is a painful degenerative joint disease that could be better managed if tissue engineers can develop methods to create long-term engineered articular cartilage tissue substitutes. Many of the tissue engineered cartilage constructs currently available lack the chemical stimuli and cell-friendly environment that promote the matrix accumulation and cell proliferation needed for use in joint cartilage repair. The goal of this research was to test the efficacy of using a fibrin-alginate hydrogel containing hyaluronic acid (HA) and/or chondroitin sulphate (CS) supplements for chondrocyte culture. Neonatal porcine chondrocytes cultured in fibrin-alginate hydrogels retained their phenotype better than chondrocytes cultured in monolayer, as evidenced by analysis of their relative expression of type II versus type I collagen mRNA transcripts. HA or CS supplementation of the hydrogels increased matrix glycosaminoglycan (GAG) production during the first week of culture. However, the effects of these supplements on matrix accumulation were not additive and were no longer observed after two weeks of culture. Supplementation of the hydrogels with CS or a combination of both CS and HA increased the chondrocyte cell population after two weeks of culture. Statistical analysis indicated that the HA and CS treatment effects on chondrocyte numbers may be additive. This research suggests that supplementation with CS and/or HA has positive effects on cartilage matrix production and chondrocyte proliferation in three-dimensional (3D) fibrin-alginate hydrogels.
PMCID: PMC4192613  PMID: 25238548
osteoarthritis; hyaluronic acid (HA); hydrogel; tissue engineering
14.  Short-Chain Fatty Acid-Modified Hexosamine for Tissue-Engineering Osteoarthritic Cartilage 
Tissue Engineering. Part A  2013;19(17-18):2035-2044.
Inflammation and tissue degeneration play key roles in numerous rheumatic diseases, including osteoarthritis (OA). Efforts to reduce and effectively repair articular cartilage damage in an osteoarthritic environment are limited in their success due to the diseased environment. Treatment strategies focused on both reducing inflammation and increasing tissue production are necessary to effectively treat OA from a tissue-engineering perspective. In this work, we investigated the anti-inflammatory and tissue production capacity of a small molecule 3,4,6-O-tributanoylated-N-acetylglucosamine (3,4,6-O-Bu3GlcNAc) previously shown to inhibit the nuclear factor κB (NFκB) activity, a key transcription factor regulating inflammation. To mimic an inflammatory environment, chondrocytes were stimulated with interleukin-1β (IL-1β), a potent inflammatory cytokine. 3,4,6-O-Bu3GlcNAc exposure decreased the expression of NFκB target genes relevant to OA by IL-1β-stimulated chondrocytes after 24 h of exposure. The capacity of 3,4,6-O-Bu3GlcNAc to stimulate extracellular matrix (ECM) accumulation by IL-1β-stimulated chondrocytes was evaluated in vitro utilizing a three-dimensional hydrogel culturing system. After 21 days, 3,4,6-O-Bu3GlcNAc exposure induced quantifiable increases in both sulfated glycosaminoglycan and total collagen. Histological staining for proteoglycans and type II collagen confirmed these findings. The increased ECM accumulation was not due to the hydrolysis products of the small molecule, n-butyrate and N-acetylglucosamine (GlcNAc), as the isomeric 1,3,4-O-tributanoylated N-acetylglucosamine (1,3,4-O-Bu3GlcNAc) did not elicit a similar response. These findings demonstrate that a novel butanoylated GlcNAc derivative, 3,4,6-O-Bu3GlcNAc, has the potential to stimulate new tissue production and reduce inflammation in IL-1β-induced chondrocytes with utility for OA and other forms of inflammatory arthritis.
PMCID: PMC3725939  PMID: 23638885
15.  Regulation of SOX9 in normal and osteoarthritic equine articular chondrocytes by hyperosmotic loading 
Osteoarthritis and Cartilage  2010;18(11-4):1502-1508.
SOX9 is a transcription factor that is essential for cartilage extracellular matrix (ECM) formation. Osteoarthritis (OA) is characterised by a loss of cartilage ECM. In chondrocytes SOX9 gene expression is regulated by osmotic loading. Here we characterise SOX9 mRNA regulation through static and cyclical application of hyperosmotic conditions in normal and OA monolayer equine chondrocytes. Furthermore, we investigate whether extracellular signal-regulated protein kinase (ERK)1/2 mitogen-activated protein kinases (MAPK) pathways have a role in this regulation of SOX9.
Equine chondrocytes harvested from normal or OA joints were subjected to different osmotic loading patterns as either primary (P0) or passaged (P2) cells. The involvement of MEK–ERK signalling was demonstrated by using pharmacological inhibitors. In addition SOX9 gene stability was determined. Levels of transcripts encoding SOX9, Col2A1 and aggrecan were measured using qRT-PCR. De novo glycosaminoglycan synthesis of explants was determined with 35S sulphate during static hyperosmolar loading.
MEK–ERK signalling increases glycosaminoglycans (GAG) synthesis in explants. Static hyperosmotic conditions significantly reduced SOX9 mRNA in normal P2 and OA P0 but not normal P0 chondrocytes. SOX9 mRNA was stabilised by hyperosmotic conditions. Cyclical loading of normal P2 and OA P0 but not normal P0 cells led to an increase in SOX9 gene expression and this was prevented by MEK1/2 inhibition.
The response to osmotic loading of SOX9 mRNA is dependent on the nature of the osmotic stimulation and the chondrocyte phenotype. This variation may be important in disease progression.
PMCID: PMC3078327  PMID: 20800688
Cartilage; SOX9; ERK signalling; Osmotic loading
16.  MMP13 is a critical target gene during the progression of osteoarthritis 
Osteoarthritis (OA) is a degenerative joint disease affecting a large population of people. The mechanism of this highly prevalent disease is not fully understood. Currently there is no effective disease-modifying treatment for OA. The purpose of this study was two-fold: 1) to investigate the role of MMP13 in the development of OA; and 2) to evaluate the efficacy of the MMP13 inhibitor CL82198 as a pharmacologic treatment for preventing OA progression.
To investigate the role of the endogenous Mmp13 gene in OA development, tamoxifen was administered to two-week-old Col2CreER;Mmp13fx/fx (Mmp13Col2ER) and Cre-negative control mice for five days. OA was induced by meniscal-ligamentous injury (MLI) when the mice were 10 weeks old and MLI or sham-operated joints were harvested 4, 8, 12, or 16 weeks after surgery. To evaluate the efficacy of CL82198, MLI surgery was performed on 10-week-old wild type mice. CL82198 or saline was administered to the mice daily beginning immediately after the surgery for up to 16 weeks. The joint tissues collected from both experiments were evaluated by cartilage grading, histology/histomorphometry, immunohistochemistry (IHC), and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. The ability of CL82198 to inhibit MMP13 activity in vitro was confirmed by ELISA.
The OA progression was decelerated in Mmp13Col2ER mice 8, 12, and 16 weeks post-surgery. Cartilage grading by blinded observers confirmed decreased articular cartilage degeneration in Mmp13Col2ER mice at 8, 12 and 16 weeks compared to Cre-negative mice. Histomorphometric analysis demonstrated that Mmp13Col2ER mice had a higher articular cartilage area and thickness at 12 and 16 weeks post-surgery compared to the control mice. Results of IHC revealed greater type II collagen and proteoglycan expression in Mmp13Col2ER mice. Chondrocyte apoptosis, as determined by TUNEL staining, was higher in control mice compared to Mmp13Col2ER mice. CL82198 inhibited MMP13 activity in conditioned media from vehicle (> 85%) or bone morphogenetic protein 2 (BMP2)-treated (> 90%) primary murine sternal chondrocytes. Intraperitoneal injection of CL82198 decelerated MLI-induced OA progression, increased type II collagen and proteoglycan levels, and inhibited chondrocyte apoptosis compared to saline treatment as determined by OA grading, histology, histomorphometry, IHC, and TUNEL staining, respectively.
Mmp13 is critical for OA progression and pharmacologic inhibition of MMP13 is an effective strategy to decelerate articular cartilage loss in a murine model of injury-induced knee OA.
PMCID: PMC3672752  PMID: 23298463
17.  Damage Control Mechanisms in Articular Cartilage 
Articular chondrocytes maintain cartilage throughout life by replacing lost or damaged matrix with freshly synthesized material. Synthesis activity is regulated, rapidly increasing to well above basal levels in response to cartilage injury. Such responses suggest that synthesis activity is linked to the rate of matrix loss by endogenous "damage control" mechanisms. As a major stimulator of matrix synthesis in cartilage, insulin-like growth factor I (IGF-I) is likely to play a role in such mechanisms. Although IGF-I is nearly ubiquitous, its bioavailability in cartilage is controlled by IGF-I binding proteins (IGFBPs) secreted by chondrocytes. IGFBPs are part of a complex system, termed the IGF-I axis, that tightly regulates IGF-I activities. For the most part, IGFBPs block IGF-I activity by sequestering IGF-I from its cell surface receptor. We recently found that the expression of one binding protein, IGFBP-3, increases with chondrocyte age, paralleling an age-related decline in synthesis activity. In addition, IGFBP-3 is overexpressed in osteoarthritic cartilage, leading to metabolic disturbances that contribute to cartilage degeneration. These observations indicate that IGFBP-3 plays a crucial role in regulating matrix synthesis in cartilage, and suggest that cartilage damage control mechanisms may fail due to age-related changes in IGFBP-3 expression or distribution. Our investigation of this hypothesis began with immunolocalization studies to determine the tissue distribution of IGFBP-3 in human cartilage. We found that IGFBP-3 accumulated around chondrocytes in the pericellular/territorial matrix, where it co-localized with fibronectin, but not with the other matrix proteins tenascin-C and type VI collagen. This result suggested that the IGFBP-3 distribution is determined by binding to fibronectin. Binding studies using purified proteins demonstrated that IGFBP-3 does in fact bind to fibronectin, but not to tenascin-C or type VI collagen. Finally, we investigated the metabolic effects of fibronectin and IGFBP-3 in a chondrocyte culture system. These experiments showed that fibronectin enhanced the inhibitory effect that low concentrations of IGFBP-3 had on matrix synthesis. Taken together, these observations confirm that IGFBP-3-fibronectin interactions affect the IGF-I axis, and they indicate that IGF-I is stored in the chondrocyte territorial matrix through binding to a complex of IGFBP-3 and intact fibronectin. This arrangement may play an important role in cartilage damage control mechanisms. The local increase in matrix synthesis following injury could result from damage-induced IGF-I release from such pools. An age-related failure to organize this system may contribute to degenerative disease.
PMCID: PMC1888750  PMID: 10934618
18.  Culture of chondrocytes in alginate surrounded by fibrin gel: characteristics of the cells over a period of eight weeks 
Annals of the Rheumatic Diseases  2001;60(8):781-790.
OBJECTIVE—To produce tissue engineered cartilage by human articular chondrocytes in vitro for further use in in vivo manipulations for the treatment of cartilage defects.
METHODS—Human articular chondrocytes were cultured in 0.5%, 1.0%, and 2.0% of alginate for up to four weeks. The optimal concentration of an alginate matrix for cell replication and for aggrecan synthesis by chondrocytes was determined. DNA content in the different culture conditions was measured after two and four weeks. Aggrecan synthesis rates and accumulation in the surrounding extracellular matrix were assessed by [35S]sulphate incorporation after the same periods of culture. To follow the outgrowth of chondrocytes from the alginate beads, chondrocytes were cultured for four weeks in 0.5 or 1.0% alginate surrounded by 0.25 or 0.5% fibrin gel. DNA content of each culture was measured after different culture periods. Finally, human chondrocytes in 1.0% alginate beads were embedded in 0.5% fibrin gel for eight weeks. Immunohistochemical analysis for aggrecan, type I and II collagen was performed weekly.
RESULTS—At two weeks the DNA content in each culture significantly increased in 0.5 and 1.0% alginate cultures in comparison with baseline values. This increase continued until week 4 at the three alginate concentrations. Aggrecan synthesis at two weeks was highest in 0.5 and 1.0% alginate cell cultures. At four weeks aggrecan synthesis rates decreased independently of the alginate concentrations. Aggrecan mainly accumulated in the interterritorial matrix. Proliferation of chondrocytes in alginate and outgrowth of these cells in the surrounding fibrin gel were evident throughout the culture period. The accumulation of aggrecan and type II collagen around the cells, in alginate as well as in fibrin gel, gradually increased over the culture period. Type I collagen appeared after six weeks in alginate and in the surrounding fibrin.
CONCLUSION—Human chondrocytes proliferate in this culture system, show an outgrowth into the surrounding fibrin, and synthesise a cartilage-like matrix for up to eight weeks.

PMCID: PMC1753804  PMID: 11454643
19.  Subtype-Specific Translocation of the δ Subtype of Protein Kinase C and Its Activation by Tyrosine Phosphorylation Induced by Ceramide in HeLa Cells 
Molecular and Cellular Biology  2001;21(5):1769-1783.
We investigated the functional roles of ceramide, an intracellular lipid mediator, in cell signaling pathways by monitoring the intracellular movement of protein kinase C (PKC) subtypes fused to green fluorescent protein (GFP) in HeLa living cells. C2-ceramide but not C2-dihydroceramide induced translocation of δPKC-GFP to the Golgi complex, while αPKC- and ζPKC-GFP did not respond to ceramide. The Golgi-associated δPKC-GFP induced by ceramide was further translocated to the plasma membrane by phorbol ester treatment. Ceramide itself accumulated to the Golgi complex where δPKC was translocated by ceramide. Gamma interferon also induced the δPKC-specific translocation from the cytoplasm to the Golgi complex via the activation of Janus kinase and Mg2+-dependent neutral sphingomyelinase. Photobleaching studies showed that ceramide does not evoke tight binding of δPKC-GFP to the Golgi complex but induces the continuous association and dissociation of δPKC with the Golgi complex. Ceramide inhibited the kinase activity of δPKC-GFP in the presence of phosphatidylserine and diolein in vitro, while the kinase activity of δPKC-GFP immunoprecipitated from ceramide-treated cells was increased. The immunoprecipitated δPKC-GFP was tyrosine phosphorylated after ceramide treatment. Tyrosine kinase inhibitor abolished the ceramide-induced activation and tyrosine phosphorylation of δPKC-GFP. These results suggested that gamma interferon stimulation followed by ceramide generation through Mg2+-dependent sphingomyelinase induced δPKC-specific translocation to the Golgi complex and that translocation results in δPKC activation through tyrosine phosphorylation of the enzyme.
PMCID: PMC86731  PMID: 11238914
20.  Prostaglandin PGE2 at very low concentrations suppresses collagen cleavage in cultured human osteoarthritic articular cartilage: this involves a decrease in expression of proinflammatory genes, collagenases and COL10A1, a gene linked to chondrocyte hypertrophy 
Suppression of type II collagen (COL2A1) cleavage by transforming growth factor (TGF)-β2 in cultured human osteoarthritic cartilage has been shown to be associated with decreased expression of collagenases, cytokines, genes associated with chondrocyte hypertrophy, and upregulation of prostaglandin (PG)E2 production. This results in a normalization of chondrocyte phenotypic expression. Here we tested the hypothesis that PGE2 is associated with the suppressive effects of TGF-β2 in osteoarthritic (OA) cartilage and is itself capable of downregulating collagen cleavage and hypertrophy in human OA articular cartilage. Full-depth explants of human OA knee articular cartilage from arthroplasty were cultured with a wide range of concentrations of exogenous PGE2 (1 pg/ml to 10 ng/ml). COL2A1 cleavage was measured by ELISA. Proteoglycan content was determined by a colorimetric assay. Gene expression studies were performed with real-time PCR. In explants from patients with OA, collagenase-mediated COL2A1 cleavage was frequently downregulated at 10 pg/ml (in the range 1 pg/ml to 10 ng/ml) by PGE2 as well as by 5 ng/ml TGF-β2. In control OA cultures (no additions) there was an inverse relationship between PGE2 concentration (range 0 to 70 pg/ml) and collagen cleavage. None of these concentrations of added PGE2 inhibited the degradation of proteoglycan (aggrecan). Real-time PCR analysis of articular cartilage from five patients with OA revealed that PGE2 at 10 pg/ml suppressed the expression of matrix metalloproteinase (MMP)-13 and to a smaller extent MMP-1, as well as the proinflammatory cytokines IL-1β and TNF-α and type X collagen (COL10A1), the last of these being a marker of chondrocyte hypertrophy. These studies show that PGE2 at concentrations much lower than those generated in inflammation is often chondroprotective in that it is frequently capable of selectively suppressing the excessive collagenase-mediated COL2A1 cleavage found in OA cartilage. The results also show that chondrocyte hypertrophy in OA articular cartilage is functionally linked to this increased cleavage and is often suppressed by these low concentrations of added PGE2. Together these initial observations reveal the importance of very low concentrations of PGE2 in maintaining a more normal chondrocyte phenotype.
PMCID: PMC2206385  PMID: 17683641
21.  Targeted Induction of Endoplasmic Reticulum Stress Induces Cartilage Pathology 
PLoS Genetics  2009;5(10):e1000691.
Pathologies caused by mutations in extracellular matrix proteins are generally considered to result from the synthesis of extracellular matrices that are defective. Mutations in type X collagen cause metaphyseal chondrodysplasia type Schmid (MCDS), a disorder characterised by dwarfism and an expanded growth plate hypertrophic zone. We generated a knock-in mouse model of an MCDS–causing mutation (COL10A1 p.Asn617Lys) to investigate pathogenic mechanisms linking genotype and phenotype. Mice expressing the collagen X mutation had shortened limbs and an expanded hypertrophic zone. Chondrocytes in the hypertrophic zone exhibited endoplasmic reticulum (ER) stress and a robust unfolded protein response (UPR) due to intracellular retention of mutant protein. Hypertrophic chondrocyte differentiation and osteoclast recruitment were significantly reduced indicating that the hypertrophic zone was expanded due to a decreased rate of VEGF–mediated vascular invasion of the growth plate. To test directly the role of ER stress and UPR in generating the MCDS phenotype, we produced transgenic mouse lines that used the collagen X promoter to drive expression of an ER stress–inducing protein (the cog mutant of thyroglobulin) in hypertrophic chondrocytes. The hypertrophic chondrocytes in this mouse exhibited ER stress with a characteristic UPR response. In addition, the hypertrophic zone was expanded, gene expression patterns were disrupted, osteoclast recruitment to the vascular invasion front was reduced, and long bone growth decreased. Our data demonstrate that triggering ER stress per se in hypertrophic chondrocytes is sufficient to induce the essential features of the cartilage pathology associated with MCDS and confirm that ER stress is a central pathogenic factor in the disease mechanism. These findings support the contention that ER stress may play a direct role in the pathogenesis of many connective tissue disorders associated with the expression of mutant extracellular matrix proteins.
Author Summary
Mutations in genes for extracellular matrix proteins are generally thought to exert their pathogenic effects because of resulting defects in extracellular matrix. However, it is becoming increasingly clear that such mutations can also have significant effects inside the cell due to the induction of ER stress. Mutations in type X collagen cause a dwarfism called metaphyseal chondrodysplasia type Schmid. A gene targeted mouse model expressing mutant type X collagen exhibited an expanded hypertrophic zone of the growth plate and significant increases in cellular ER stress, as noted previously. VEGF expression was disrupted leading to decreases in the rate of vascular invasion. To directly assess the role of elevated ER stress in disease pathogenesis, transgenic mouse lines expressing an exogenous, ER stress–inducing protein (cog mutant of thyroglobulin—Tgcog) targeted to hypertrophic chondrocytes were generated. Mice expressing Tgcog protein showed elevated ER stress, an expanded hypertrophic zone, and reduced bone growth demonstrating that elevated ER stress and the resultant UPR is the principal pathogenic mechanism causing this cartilage pathology. It is possible that therapeutic strategies aimed at alleviating ER stress may be beneficial in this and other connective tissue diseases caused by mutant extracellular matrix genes.
PMCID: PMC2757901  PMID: 19834559
22.  In vivo model of cartilage degradation--effects of a matrix metalloproteinase inhibitor. 
Annals of the Rheumatic Diseases  1995;54(8):662-669.
OBJECTIVES--To develop a model of cartilage degradation that (i) enables the testing of synthetic, small molecular weight matrix metalloproteinase (MMP) inhibitors as agents to prevent cartilage erosion, (ii) permits the direct assay of the principal constituents of the extracellular matrix (collagen and proteoglycan) in both the non-calcified articular cartilage and the calcified cartilage compartments, and (iii) is mediated by a chronic, granulomatous tissue that closely apposes intact articular cartilage, and in this respect resembles the pannus-cartilage junction of rheumatoid arthritis. METHODS--Femoral head cartilage was obtained from donor rats, wrapped in cotton and implanted subcutaneously into recipient animals. After a two stage papain digestion procedure, the proteoglycan and collagen contents were measured by assaying for glycosaminoglycans and hydroxyproline, respectively, in both the non-calcified cartilage that comprises the articular surface layer and the calcified cartilage compartment. The incorporation in vitro of [35S]-sulphate into glycosaminoglycans was assayed as a measure of proteoglycan biosynthesis. An osmotic minipump was cannulated to the implanted femoral head cartilage and synthetic MMP inhibitors (MI-1 and MI-2) were infused continuously over a 14 day period. RESULTS--The implanted, cotton wrapped femoral head cartilages provoked a granulomatous response that resulted in the removal of collagen and proteoglycan from the cartilage matrix. The removal of proteoglycan and collagen was exclusively from the non-calcified articular cartilage, whereas the proteoglycan and collagen content of the calcified compartment increased during the experiments. MI-1 reproducibly reduced the degradation of proteoglycan and collagen in implanted femoral head cartilage. CONCLUSIONS--We have described an in vivo model of cartilage degradation that permits the measurement of proteoglycan and collagen in both non-calcified articular cartilage and calcified cartilage compartments. The model can be used to test the effects of agents of unknown systemic bioavailability and pharmacokinetic profile by infusing them directly to the site of cartilage degradation. The removal of cartilage extracellular matrix by granulomatous tissue was inhibited by an MMP inhibitor, thus proving the involvement of this family of proteinases in cartilage catabolism in this model.
PMCID: PMC1009964  PMID: 7677443
23.  Bone morphogenetic protein-2-induced Wnt/β-catenin signaling pathway activation through enhanced low-density-lipoprotein receptor-related protein 5 catabolic activity contributes to hypertrophy in osteoarthritic chondrocytes 
Events normally taking place in the terminal chondrocyte differentiation in the growth plate are also observed during osteoarthritis (OA) development, suggesting that molecules, such as Wnts and bone morphogenetic proteins (BMPs) regulating chondrocyte activity in the growth plate, may play a key role in osteoarthritis pathogenesis. The aim of the study was to investigate the possible cross-talk between BMP-2 and Wnt/β-catenin pathways in OA progression.
Low-density-lipoprotein receptor-related protein 5 (LRP-5) and 6, BMP-2, -4, and -7, bone morphogenetic protein receptor-IA and IB (BMPR-IA and BMPR-IA), lymphoid enhancer factor-1 (LEF-1), and transcription factor 4 (TCF-4) expression levels were investigated in normal and osteoarthritic chondrocytes. LRP-5, β-catenin (phospho and active form), matrix metalloproteinases (MMPs) 7, 9, 13, 14, ADAMTS-4, 5, as well as collagen X (COL10A1) expression levels were evaluated after LRP-5 silencing in BMP-2-treated chondrocytes. The investigation of Smad1/5/8 binding to LRP-5 promoter was assessed with chromatin immunoprecipitation (ChIP). Furthermore, we evaluated the effect of experimental activation of the Wnt/β-catenin pathway with LiCl and LEF-1 silencing, in LiCl-treated chondrocytes, on matrix metalloproteinases (MMPs) 7, 9, 13, 14, ADAMTS-4, 5, and collagen X (COL10A1) expression, as well as possible interactions between LEF-1 and MMPs and COL10A1 promoters by using a ChIP assay.
LRP-5, BMP-2, BMP-4, BMPR-IA, and LEF-1 mRNA and protein expression levels were found to be significantly upregulated in osteoarthritic chondrocytes compared with normal. We showed that treatment of cultured chondrocytes with BMP-2 resulted in increased β-catenin nuclear translocation and LRP-5 expression and that the BMP-2-induced LRP-5 upregulation is mediated through Smad1/5/8 binding on LRP-5 promoter. LRP-5 silencing reduced nuclear β-catenin protein levels, MMPs and collagen X expression, whereas increased phospho-β-catenin protein levels in BMP-2-treated chondrocyte. Furthermore, we demonstrated that activation of the Wnt/β-catenin signaling pathway by LiCl and LEF-1 downregulation by using siRNA regulates MMP-9, 13, 14, ADAMTS-5, and COL10A1 expression, evidenced by the observed strong binding of LEF-1 to MMP-9, 13, 14, ADAMTS-5 and COL10A promoters.
Our findings suggest, for the first time to our knowledge, that BMP-2-induced Wnt/β-catenin signaling activation through LRP-5 may contribute to chondrocyte hypertrophy and cartilage degradation in osteoarthritis.
PMCID: PMC3446456  PMID: 22513174
24.  Lung Injury and Lung Cancer Caused by Cigarette Smoke-Induced Oxidative Stress: Molecular Mechanisms and Therapeutic Opportunities Involving the Ceramide-Generating Machinery and Epidermal Growth Factor Receptor 
Antioxidants & Redox Signaling  2014;21(15):2149-2174.
Chronic obstructive pulmonary disease (COPD) and lung cancer are frequently caused by tobacco smoking. However, these diseases present opposite phenotypes involving redox signaling at the cellular level. While COPD is characterized by excessive airway epithelial cell death and lung injury, lung cancer is caused by uncontrolled epithelial cell proliferation. Notably, epidemiological studies have demonstrated that lung cancer incidence is significantly higher in patients who have preexisting emphysema/lung injury. However, the molecular link and common cell signaling events underlying lung injury diseases and lung cancer are poorly understood. This review focuses on studies of molecular mechanism(s) underlying smoking-related lung injury (COPD) and lung cancer. Specifically, the role of the ceramide-generating machinery during cigarette smoke-induced oxidative stress leading to both apoptosis and proliferation of lung epithelial cells is emphasized. Over recent years, it has been established that ceramide is a sphingolipid playing a major role in lung epithelia structure/function leading to lung injury in chronic pulmonary diseases. However, new and unexpected findings draw attention to its potential role in lung development, cell proliferation, and tumorigenesis. To address this dichotomy in detail, evidence is presented regarding several protein targets, including Src, p38 mitogen-activated protein kinase, and neutral sphingomyelinase 2, the major sphingomyelinase that controls ceramide generation during oxidative stress. Furthermore, their roles are presented not only in apoptosis and lung injury but also in enhancing cell proliferation, lung cancer development, and resistance to epidermal growth factor receptor-targeted therapy for treating lung cancer. Antioxid. Redox Signal. 21, 2149–2174.
I. Introduction
II. Oxidative Stress and Pulmonary Disease
A. Smoking, oxidative stress, and inflammation
B. Smoking and emphysema/lung injury: the role of apoptosis in the disease development
III. The Role of Ceramide in Oxidative Stress-Induced Lung Epithelial Apoptosis
A. Neutral sphingomyelinase 2-induced ceramide generation as a specific target in CS-induced lung injury
B. The surprising role of Src in controlling nSMase2/ceramide generation
IV. Oxidative Stress and Lung Cancer
A. Stress-driven endocytosis of tyrosine-phosphorylated EGFR leads to tumorigenesis: the critical role of oxidative stress
1. Canonical EGFR activation, intracellular trafficking, and degradation
2. CS produces H2O2-induced oxidative stress that aberrantly activates EGFR
3. The lack of c-Cbl binding to EGFR causes prolonged proliferation signaling under oxidative stress
4. EGFR perinuclear sorting under oxidative stress
5. An aberrant activated conformation of EGFR under oxidative stress underlies lung cancer resistance to targeted therapy
V. Smoking and Lung Diseases: The Enigmatic Association Between Lung Injury and Lung Cancer
VI. The Dichotomous Response of Airway Epithelial Cells to CS Oxidants: A Critical Role for Src
VII. The Unexpected Role of Ceramide in Cell Proliferation and Tumorigenesis
A. Cell membrane ceramide-enriched signaling platform and stabilization of aberrantly activated EGFR
B. Membrane ordered lipid domains
C. Ceramide selectively displaces cholesterol from ordered lipid domains (rafts)
D. Ceramide/cholesterol ratio affects EGFR and Src
E. Ceramide, EGFR, and Src
F. The link between ceramide and Src underlies their dual roles in apoptosis and proliferation
VIII. Propagation of Oncogenes and miRNA via nSMase2-Dependent Ceramide Generation and Exosome Secretion
IX. Updates on Novel Unresolved Complexities in the Ceramide-Generating Machinery
X. Therapeutic Perspectives
XI. Concluding Remarks
PMCID: PMC4215561  PMID: 24684526
25.  Fibroblast growth factor receptor 1 is principally responsible for fibroblast growth factor 2-induced catabolic activities in human articular chondrocytes 
Arthritis Research & Therapy  2011;13(4):R130.
Cartilage degeneration driven by catabolic stimuli is a critical pathophysiological process in osteoarthritis (OA). We have defined fibroblast growth factor 2 (FGF-2) as a degenerative mediator in adult human articular chondrocytes. Biological effects mediated by FGF-2 include inhibition of proteoglycan production, up-regulation of matrix metalloproteinase-13 (MMP-13), and stimulation of other catabolic factors. In this study, we identified the specific receptor responsible for the catabolic functions of FGF-2, and established a pathophysiological connection between the FGF-2 receptor and OA.
Primary human articular chondrocytes were cultured in monolayer (24 hours) or alginate beads (21 days), and stimulated with FGF-2 or FGF18, in the presence or absence of FGFR1 (FGF receptor 1) inhibitor. Proteoglycan accumulation and chondrocyte proliferation were assessed by dimethylmethylene blue (DMMB) assay and DNA assay, respectively. Expression of FGFRs (FGFR1 to FGFR4) was assessed by flow cytometry, immunoblotting, and quantitative real-time PCR (qPCR). The distinctive roles of FGFR1 and FGFR3 after stimulation with FGF-2 were evaluated using either pharmacological inhibitors or FGFR small interfering RNA (siRNA). Luciferase reporter gene assays were used to quantify the effects of FGF-2 and FGFR1 inhibitor on MMP-13 promoter activity.
Chondrocyte proliferation was significantly enhanced in the presence of FGF-2 stimulation, which was inhibited by the pharmacological inhibitor of FGFR1. Proteoglycan accumulation was reduced by 50% in the presence of FGF-2, and this reduction was successfully rescued by FGFR1 inhibitor. FGFR1 inhibitors also fully reversed the up-regulation of MMP-13 expression and promoter activity stimulated by FGF-2. Blockade of FGFR1 signaling by either chemical inhibitors or siRNA targeting FGFR1 rather than FGFR3 abrogated the up-regulation of matrix metalloproteinases 13 (MMP-13) and a disintegrin and metalloproteinase with a thrombospondin type 1 motif 5 (ADAMTS5), as well as down-regulation of aggrecan after FGF-2 stimulation. Flow cytometry, qPCR and immunoblotting analyses suggested that FGFR1 and FGFR3 were the major FGFR isoforms expressed in human articular chondrocytes. FGFR1 was activated more potently than FGFR3 upon FGF-2 stimulation. In osteoarthritic chondrocytes, FGFR3 was significantly down regulated (P < 0.05) with a concomitant increase in the FGFR1 to FGFR3 expression ratio (P < 0.05), compared to normal chondrocytes. Our results also demonstrate that FGFR3 was negatively regulated by FGF-2 at the transcriptional level through the FGFR1-ERK (extracellular signal-regulated kinase) signaling pathway in human articular chondrocytes.
FGFR1 is the major mediator with the degenerative potential in the presence of FGF-2 in human adult articular chondrocytes. FGFR1 activation by FGF-2 promotes catabolism and impedes anabolism. Disruption of the balance between FGFR1 and FGFR3 signaling ratio may contribute to the pathophysiology of OA.
PMCID: PMC3239372  PMID: 21835001

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