Metabolic reprogramming in chondrocytes to promote mitochondrial respiration reduces downstream features of osteoarthritis.

Metabolic dysfunction in chondrocytes drives the pro-catabolic phenotype associated with osteoarthritic cartilage. In this study, substitution of galactose for glucose in culture media was used to promote a renewed dependence on mitochondrial respiration and oxidative phosphorylation. Galactose replacement alone blocked enhanced usage of the glycolysis pathway by IL1ß-activated chondrocytes as detected by real-time changes in the rates of proton acidification of the medium and changes in oxygen consumption. The change in mitochondrial activity due to galactose was visualized as a rescue of mitochondrial membrane potential but not an alteration in the number of mitochondria. Galactose-replacement reversed other markers of dysfunctional mitochondrial metabolism, including blocking the production of reactive oxygen species, nitric oxide, and the synthesis of inducible nitric oxide synthase. Of more clinical relevance, galactose-substitution blocked downstream functional features associated with osteoarthritis, including enhanced levels of MMP13 mRNA, MMP13 protein, and the degradative loss of proteoglycan from intact cartilage explants. Blocking baseline and IL1ß-enhanced MMP13 by galactose-replacement in human osteoarthritic chondrocyte cultures inversely paralleled increases in markers associated with mitochondrial recovery, phospho-AMPK, and PGC1α. Comparisons were made between galactose replacement and the glycolysis inhibitor 2-deoxyglucose. Targeting intermediary metabolism may provide a novel approach to osteoarthritis care.

Hyaluronan regulates synapse formation and function in developing neural networks.

Neurodevelopmental disorders present with synaptic alterations that disrupt the balance between excitatory and inhibitory signaling. For example, hyperexcitability of cortical neurons is associated with both epilepsy and autism spectrum disorders. However, the mechanisms that initially establish the balance between excitatory and inhibitory signaling in brain development are not well understood. Here, we sought to determine how the extracellular matrix directs synapse formation and regulates synaptic function in a model of human cortical brain development. The extracellular matrix, making up twenty percent of brain volume, is largely comprised of hyaluronan. Hyaluronan acts as both a scaffold of the extracellular matrix and a space-filling molecule. Hyaluronan is present from the onset of brain development, beginning with neural crest cell migration. Through acute perturbation of hyaluronan levels during synaptogenesis, we sought to determine how hyaluronan impacts the ratio of excitatory to inhibitory synapse formation and the resulting neural activity. We used 3-D cortical spheroids derived from human induced pluripotent stem cells to replicate this neurodevelopmental window. Our results demonstrate that hyaluronan preferentially surrounds nascent excitatory synapses. Removal of hyaluronan increases the expression of excitatory synapse markers and results in a corresponding increase in the formation of excitatory synapses, while also decreasing inhibitory synapse formation. This increased excitatory synapse formation elevates network activity, as demonstrated by microelectrode array analysis. In contrast, the addition of purified hyaluronan suppresses excitatory synapse formation. These results establish that the hyaluronan extracellular matrix surrounds developing excitatory synapses, where it critically regulates synapse formation and the resulting balance between excitatory to inhibitory signaling.

Matrix Metalloproteinase-12 Is Required for Granuloma Progression.

Background: Sarcoidosis is a chronic inflammatory disease of unknown cause characterized by granuloma formation. Mechanisms for chronic persistence of granulomas are unknown. Matrix Metalloproteinase-12 (MMP12) degrades extracellular matrix elastin and enables infiltration of immune cells responsible for inflammation and granuloma formation. Previous studies report increased MMP12 in sarcoidosis patients and association between MMP12 expression and disease severity. We also observed elevated MMP12 in our multiwall carbon nanotube (MWCNT) murine model of granulomatous inflammation. Here we hypothesized that MMP12 is important to acute and late phases of granuloma pathogenesis. To test this hypothesis, we analyzed granulomatous and inflammatory responses of Mmp12 knock-out (KO) mice at 10 (acute) and 60 days (late) after MWCNT instillation. Methods: C57BL/6 (wildtype) and Mmp12 KO mice underwent oropharyngeal instillation of MWCNT. Lungs were harvested at 3, 10, 20, and 60 days post instillation for evaluation of MMP12 expression and granulomatous changes. Bronchoalveolar lavage (BAL) cells were analyzed 60 days after MWCNT instillation for expression of mediators thought to play a role in sarcoid granulomatosis: peroxisome proliferator-activated receptor-gamma (PPARγ), interferon-gamma (IFN-γ), and CCL2 (MCP-1). Results: Pulmonary granuloma appearance at 10 days after MWCNT instillation showed no differences between wildtype and Mmp12 KO mice. In contrast, by 60 days after MWCNT instillation, Mmp12 KO mice revealed markedly attenuated granuloma formation together with elevated PPARγ and reduced IFNγ expression in BAL cells compared to wildtype. Unexpectedly, Mmp12 KO mice further demonstrated increased alveolar macrophages with increased CCL2 at 60 days. Conclusions: The striking reduction of granuloma formation at day 60 in Mmp12 KO mice suggests that MMP12 is required to maintain chronic granuloma pathophysiology. The increased PPARγ and decreased IFNγ findings suggest that these mediators also may be involved since previous studies have shown that PPARγ suppresses IFNγ and PPARγ deficiency amplifies granuloma formation. Interestingly, a role of MMP12 in granuloma resolution is also suggested by increases in both macrophage influx and CCL2. Overall, our results strongly implicate MMP12 as a key factor in granuloma persistence and as a possible therapeutic target in chronic pulmonary sarcoidosis.

Suppression of murine osteoarthritis by 4-methylumbelliferone.

Using in vitro models, we previously reported that 4-methylumbelliferone (4-MU) blocked many of the pro-catabolic features of activated chondrocytes. 4-MU also blocked safranin O loss from human cartilage explants exposed to interleukin 1ß (IL1ß) in vitro. However, the mechanism for this chondroprotective effect was independent of the action of 4-MU as a hyaluronan (HA) inhibitor. Interestingly, overexpression of HA synthase 2 (HAS2) also blocked the same pro-catabolic features of activated chondrocytes as 4-MU via a mechanism independent of extracellular HA accumulation. Data suggest that altering UDP-sugars may be behind these changes in chondrocyte metabolism. However, all of our previous experiments with 4-MU or HAS2 overexpression were performed in vitro. The purpose of this study was to confirm whether 4-MU was effective at limiting the effects of osteoarthritis (OA) on articular cartilage in vivo. The progression of OA was evaluated after destabilization of the medial meniscus (DMM) surgery on C57BL/6 mice in the presence or absence of 4-MU-containing chow. Mice fed 4-MU after DMM surgery exhibited significant suppression of OA starting from an early stage in vivo. Mice fed 4-MU exhibited lower OARSI scores after DMM; reduced osteophyte formation and reduced MMP3 and MMP13 immunostaining. 4-MU also exerted pronounced chondroprotective effects on murine joint cartilage exposed to IL1ß in vitro and, blocked IL1ß-enhanced lactate production in cartilage explants. Therefore, 4-MU is effective at significantly reducing the loss of proteoglycan and reducing MMP production both in vitro and in vivo as well as cartilage damage and osteophyte formation in vivo after DMM. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res. 38:1122-1131, 2020.

Inhibition of CD44 intracellular domain production suppresses bovine articular chondrocyte de-differentiation induced by excessive mechanical stress loading.

CD44 fragmentation is enhanced in chondrocytes of osteoarthritis (OA) patients. We hypothesized that mechanical stress-induced enhancement of CD44-intracellular domain (CD44-ICD) production plays an important role in the de-differentiation of chondrocytes and OA. This study aimed to assess the relationship between CD44-ICD and chondrocyte gene expression. Monolayer cultured primary bovine articular chondrocytes (BACs) were subjected to cyclic tensile strain (CTS) loading. ADAM10 inhibitor (GI254023X) and γ-secretase inhibitor (DAPT) were used to inhibit CD44 cleavage. In overexpression experiments, BACs were electroporated with a plasmid encoding CD44-ICD. CTS loading increased the expression of ADAM10 and subsequent CD44 cleavage, while decreasing the expression of SOX9, aggrecan, and type 2 collagen (COL2). Overexpression of CD44-ICD also resulted in decreased expression of these chondrocyte genes. Both GI254023X and DAPT reduced the production of CD44-ICD upon CTS loading, and significantly rescued the reduction of SOX9 expression by CTS loading. Chemical inhibition of CD44-ICD production also rescued aggrecan and COL2 expression following CTS loading. Our findings suggest that CD44-ICD is closely associated with the de-differentiation of chondrocytes. Excessive mechanical stress loading promoted the de-differentiation of BACs by enhancing CD44 cleavage and CD44-ICD production. Suppression of CD44 cleavage has potential as a novel treatment strategy for OA.

Chondroprotective effects of 4-methylumbelliferone and hyaluronan synthase-2 overexpression involve changes in chondrocyte energy metabolism.

Hyaluronan is a critical component of articular cartilage and partially helps retain aggrecan within the extracellular matrix of this tissue. During osteoarthritis, hyaluronan and aggrecan loss are an early sign of tissue damage. However, our recent attempts to mimic hyaluronan loss with the hyaluronan inhibitor 4-methylumbelliferone (4MU) did not exacerbate arthritis-like features of in vitro models of arthritis, but surprisingly, caused the reverse (i.e. provided potent chondroprotection). Moreover, the protective effects of 4MU did not depend on its role as a hyaluronan inhibitor. To understand the molecular mechanism in 4MU-mediated chondroprotection, we considered recent studies suggesting that shifts in intracellular UDP-hexose pools promote changes in metabolism. To determine whether such metabolic shifts are associated with the mechanism of 4MU-mediated pro-catabolic inhibition, using molecular and metabolomics approaches, we examined whether bovine and human chondrocytes exhibit changes in the contribution of glycolysis and mitochondrial respiration to ATP production rates as well as in other factors that respond to or might drive these changes. Overexpression of either HA synthase-2 or 4MU effectively reduced dependence on glycolysis in chondrocytes, especially enhancing glycolysis use by interleukin-1ß (IL1ß)-activated chondrocytes. The reduction in glycolysis secondarily enhanced mitochondrial respiration in chondrocytes, which, in turn, rescued phospho-AMP-activated protein kinase (AMPK) levels in the activated chondrocytes. Other glycolysis inhibitors, unrelated to hyaluronan biosynthesis, namely 2-deoxyglucose and dichloroacetate, caused metabolic changes in chondrocytes equivalent to those elicited by 4MU and similarly protected both chondrocytes and cartilage explants. These results suggest that fluxes in UDP-hexoses alter metabolic energy pathways in cartilage.

Hyaluronan synthase 2 (HAS2) overexpression diminishes the procatabolic activity of chondrocytes by a mechanism independent of extracellular hyaluronan.

Osteoarthritis (OA) is a progressive degenerative disease of the joints caused in part by a change in the phenotype of resident chondrocytes within affected joints. This altered phenotype, often termed proinflammatory or procatabolic, features enhanced production of endoproteinases and matrix metallo-proteinases (MMPs) as well as secretion of endogenous inflammatory mediators. Degradation and reduced retention of the proteoglycan aggrecan is an early event in OA. Enhanced turnover of hyaluronan (HA) is closely associated with changes in aggrecan. Here, to determine whether experimentally increased HA production promotes aggrecan retention and generates a positive feedback response, we overexpressed HA synthase-2 (HAS2) in chondrocytes via an inducible adenovirus construct (HA synthase-2 viral overexpression; HAS2-OE). HAS2-OE incrementally increased high-molecular-mass HA >100-fold within the cell-associated and growth medium pools. More importantly, our results indicated that the HAS2-OE expression system inhibits MMP3, MMP13, and other markers of the procatabolic phenotype (such as TNF-stimulated gene 6 protein (TSG6)) and also enhances aggrecan retention. These markers were inhibited in OA-associated chondrocytes and in chondrocytes activated by interleukin-1ß (IL1ß), but also chondrocytes activated by lipopolysaccharide (LPS), tumor necrosis factor α (TNFα), or HA oligosaccharides. However, the enhanced extracellular HA resulting from HAS2-OE did not reduce the procatabolic phenotype of neighboring nontransduced chondrocytes as we had expected. Rather, HA-mediated inhibition of the phenotype occurred only in transduced cells. In addition, high HA biosynthesis rates, especially in transduced procatabolic chondrocytes, resulted in marked changes in chondrocyte dependence on glycolysis versus oxidative phosphorylation for their metabolic energy needs.

Simvastatin promotes restoration of chondrocyte morphology and phenotype.

In this study we examined whether the action of simvastatin affects re-differentiation of passaged chondrocytes and if so, whether this was mediated via changes in cholesterol or cholesterol intermediates. Bovine articular chondrocytes, of varying passage number, human knee chondrocytes and rat chondrosarcoma chondrocytes were treated with simvastatin and examined for changes in mRNA and protein expression of markers of the chondrocyte phenotype as well as changes in cell shape, proliferation and proteoglycan production. In all three models, while still in monolayer culture, simvastatin treatment alone promoted changes in phenotype and morphology indicative of re-differentiation most prominent being an increase in SOX9 mRNA and protein expression. In passaged bovine chondrocytes, simvastatin stimulated the expression of SOX9, ACAN, BMP2 and inhibited the expression of COL1 and α-smooth muscle actin. Co-treatment of chondrocytes with simvastatin plus exogenous cholesterol-conditions that had previously reversed the inhibition on CD44 shedding, did not alter the effects of simvastatin on re-differentiation. However, the co-treatment of chondrocytes with simvastatin together with other pathway intermediates, mevalonate, geranylgeranylpyrophosphate and to a lesser extent, farnesylpyrophosphate, blocked the pro-differentiation effects of simvastatin. Treatment with simvastatin stimulated expression of SOX9 and COL2a and enhanced SOX9 protein in human OA chondrocytes. The co-treatment of OA chondrocytes with mevalonate or geranylgeranylpyrophosphate, but not cholesterol, blocked the simvastatin effects. These results lead us to conclude that the blocking of critical protein prenylation events is required for the positive effects of simvastatin on the re-differentiation of chondrocytes.

The pericellular hyaluronan of articular chondrocytes.

The story of hyaluronan in articular cartilage, pericellular hyaluronan in particular, essentially is also the story of aggrecan. Without properly tethered aggrecan, the load bearing function of cartilage is compromised. The anchorage of aggrecan to the cell surface only occurs due to the binding of aggrecan to hyaluronan-with hyaluronan tethered either to a hyaluronan synthase or by multivalent binding to CD44. In this review, details of hyaluronan synthesis are discussed including how HAS2 production of hyaluronan is necessary for normal chondrocyte development and matrix assembly, how an abundance or deficit of pericellular hyaluronan alters chondrocyte metabolism, and whether hyaluronan size matters or changes with aging or disease. The biomechanical role and matrix assembly function of hyaluronan in addition to the functions of hyaluronidases are discussed. The turnover of hyaluronan is considered including mechanisms by which its turnover, at least in part, is mediated by endocytosis by chondrocytes and regulated by aggrecan degradation. Differences between turnover and clearance of newly synthesized hyaluronan and aggrecan versus the half-life of hyaluronan remaining within the inter-territorial matrix of cartilage are discussed. The release of neutral pH-acting hyaluronidase activity remains one unanswered question concerning the loss of cartilage hyaluronan in osteoarthritis. Signaling events driven by changes in hyaluronan-chondrocyte interactions may involve a chaperone function of CD44 with other receptors/cofactors as well as the changes in hyaluronan production functioning as a metabolic rheostat.

Simvastatin inhibits CD44 fragmentation in chondrocytes.

In human osteoarthritic chondrocytes, the hyaluronan receptor CD44 undergoes proteolytic cleavage at the cell surface. CD44 cleavage is thought to require transit of CD44 into cholesterol-rich lipid rafts. The purpose of this study was to investigate whether statins exert a protective effect on articular chondrocytes due to diminution of cholesterol. Three model systems of chondrocytes were examined including human HCS-2/8 chondrosarcoma cells, human osteoarthritic chondrocytes and normal bovine articular chondrocytes. Treatment with IL-1ß + Oncostatin M resulted in a substantial increase in CD44 fragmentation in each of the three chondrocyte models. Pre-incubation with simvastatin prior to treatment with IL-1ß + Oncostatin M decreased the level of CD44 fragmentation, decreased the proportion of CD44 that transits into the lipid raft fractions, decreased ADAM10 activity and diminished the interaction between CD44 and ADAM10. In HCS-2/8 cells and bovine articular chondrocytes, fragmentation of CD44 was blocked by the knockdown of ADAM10. Inhibition of CD44 fragmentation by simvastatin also resulted in improved retention of pericellular matrix. Addition of cholesterol and farnesyl-pyrophosphate reversed the protective effects of simvastatin. Thus, the addition of simvastatin exerts positive effects on chondrocytes including reduced CD44 fragmentation and enhanced the retention of pericellular matrix.

CRISPR/Cas9 knockout of HAS2 in rat chondrosarcoma chondrocytes demonstrates the requirement of hyaluronan for aggrecan retention.

Hyaluronan (HA) plays an essential role in cartilage where it functions to retain aggrecan. Previous studies have suggested that aggrecan is anchored indirectly to the plasma membrane of chondrocytes via its binding to cell-associated HA. However, reagents used to test these observations such as hyaluronidase and HA oligosaccharides are short term and may have side activities that complicate interpretation. Using the CRISPR/Cas9 gene editing approach, a model system was developed by generating HA-deficient chondrocyte cell lines. HA synthase-2 (Has2)-specific single guide RNA was introduced into two different variant lines of rat chondrosarcoma chondrocytes; knockout clones were isolated and characterized. Two other members of the HA synthase gene family were expressed at very low relative copy number but showed no compensatory response in the Has2 knockouts. Wild type chondrocytes of both variants exhibited large pericellular matrices or coats extending from the plasma membrane. Addition of purified aggrecan monomer expanded the size of these coats as the proteoglycan became retained within the pericellular matrix. Has2 knockout chondrocytes lost all capacity to assemble a particle-excluding pericellular matrix and more importantly, no matrices formed around the knockout cells following the addition of purified aggrecan. When grown as pellet cultures so as to generate a bioengineered neocartilage tissue, the Has2 knockout chondrocytes assumed a tightly-compacted morphology as compared to the wild type cells. When knockout chondrocytes were transduced with Adeno-ZsGreen1-mycHas2, the cell-associated pericellular matrices were restored including the capacity to bind and incorporate additional exogenous aggrecan into the matrix. These results suggest that HA is essential for aggrecan retention and maintaining cell separation during tissue formation.

4-Methylumbelliferone Diminishes Catabolically Activated Articular Chondrocytes and Cartilage Explants via a Mechanism Independent of Hyaluronan Inhibition.

Depletion of the cartilage proteoglycan aggrecan is one of the earliest events that occurs in association with osteoarthritis. This loss is often accompanied by a coordinate loss in another glycosaminoglycan, hyaluronan. Chondrocytes experimentally depleted of cell-associated hyaluronan respond by switching to a pro-catabolic metabolism that includes enhanced production of endogenous inflammatory mediators and increased synthesis of matrix metalloproteinases. Hyaluronan turnover is also increased. Together, such a response provides for possible establishment of a self-perpetuating spiral of events that maintains or prolongs the pro-catabolic state. Chondrocytes or cartilage can also be activated by treatment with pro-inflammatory cytokines and mediators such as IL-1ß, TNFα, LPS, fibronectin fragments, and hyaluronan oligosaccharides. To determine the mechanism of chondrocyte activation due to hyaluronan loss, a depletion method was required that did not include degrading the hyaluronan. In recent years, several laboratories have used the coumarin derivative, 4-methylumbelliferone, as a potent inhibitor of hyaluronan biosynthesis, due in part to its ability to sequester intracellular UDP-glucuronic acid and inhibition of hyaluronan synthase transcription. However, contrary to our expectation, although 4-methylumbelliferone was indeed an inhibitor of hyaluronan biosynthesis, this depletion did not give rise to an activation of chondrocytes or cartilage. Rather, 4-methylumbelliferone directly and selectively blocked gene products associated with the pro-catabolic metabolic state of chondrocytes and did so through a mechanism preceding and independent of hyaluronan inhibition. These data suggest that 4-methylumbelliferone has additional useful applications to block pro-inflammatory cell activation events but complicates how it is used for defining functions related to hyaluronan.

Hypoxia-inducible factor-2α induces expression of type X collagen and matrix metalloproteinases 13 in osteoarthritic meniscal cells.

OBJECTIVES: To evaluate whether Hypoxia-inducible factor-2α (HIF-2α) regulates expression of endochondral ossification-related molecules in human OA meniscus. METHODS: Expressions of HIF-2α, type X collagen (COL10), matrix metalloproteinase (MMP)-13, and vascular endothelial growth factor (VEGF) in non-OA and OA menisci were analyzed by real-time RT-PCR and immunohistochemistry (IHC). Meniscal cells from OA patients were treated with interleukin-1ß (IL-1ß) and gene expression was analyzed. After knockdown of HIF-2α in OA meniscal cells, COL10 and MMP-13 expression were analyzed by RT-PCR, western blotting, immunofluorescence and ELISA. RESULT: Histological analysis demonstrated weak staining of the superficial layer and large round cells in OA meniscus. RT-PCR analysis showed that HIF-2α, COL10, MMP-13, and VEGF mRNA expressions were higher in OA than non-OA meniscal cells. IHC showed a coordinated staining pattern of HIF-2α, COL10, and MMP-13 in OA meniscus. IL-1ß treatment increased HIF-2α, COL10, and MMP-13 expressions in OA meniscal cells, and knockdown of HIF-2α suppressed IL-1ß-mediated increase in COL10 and MMP-13 expression. CONCLUSIONS: These results suggested that HIF-2α may cause meniscal matrix degradation by transactivation of MMP-13. HIF-2α may be a therapeutic target for modulating matrix degradation in both articular cartilage and meniscus during knee OA progression.

CD44 knock-down in bovine and human chondrocytes results in release of bound HYAL2.

CD44 shedding occurs in osteoarthritic chondrocytes. Previous work of others has suggested that the hyaluronidase isoform HYAL2 has the capacity to bind to CD44, a binding that may itself induce CD44 cleavage. Experiments were developed to elucidate whether chondrocyte HYAL2: (1) was exposed on the extracellular plasma membrane of chondrocytes, (2) bound to CD44, (3) underwent shedding together with CD44 and lastly, (4) exhibited hyaluronidase activity within a near-neutral pH range. Enhancing CD44 shedding by IL-1ß resulted in a proportional increase in HYAL2 released from human and bovine chondrocytes into the medium. CD44 knockdown by siRNA also resulted in increased accumulation of HYAL2 in the media of chondrocytes. By hyaluronan zymography only activity at pH3.7 was observed and this activity was reduced by pre-treatment of chondrocytes with trypsin. CD44 and HYAL2 were found to co-immunoprecipitate, and to co-localize within intracellular vesicles and at the plasma membrane. Degradation of hyaluronan was visualized by agarose gel electrophoresis. With this approach, hyaluronidase activity could be observed at pH4.8 under assay conditions in which CD44 and HYAL2 binding remained intact; additionally, weak hyaluronidase activity could be observed at pH6.8 under these conditions. This study suggests that CD44 and HYAL2 are bound at the surface of chondrocytes. The release of HYAL2 when CD44 is shed could provide a mechanism for weak hyaluronidase activity to occur within the more distant extracellular matrix of cartilage.

Extracellular processing of the cartilage proteoglycan aggregate and its effect on CD44-mediated internalization of hyaluronan.

In many cells hyaluronan receptor CD44 mediates the endocytosis of hyaluronan and its delivery to endosomes/lysosomes. The regulation of this process remains largely unknown. In most extracellular matrices hyaluronan is not present as a free polysaccharide but often is found in complex with other small proteins and macromolecules such as proteoglycans. This is especially true in cartilage, where hyaluronan assembles into an aggregate structure with the large proteoglycan termed aggrecan. In this study when purified aggrecan was added to FITC-conjugated hyaluronan, no internalization of hyaluronan was detected. This suggested that the overall size of the aggregate prevented hyaluronan endocytosis and furthermore that proteolysis of the aggrecan was a required prerequisite for local, cell-based turnover of hyaluronan. To test this hypothesis, limited C-terminal digestion of aggrecan was performed to determine whether a size range of aggrecan exists that permits hyaluronan endocytosis. Our data demonstrate that only limited degradation of the aggrecan monomer was required to allow for hyaluronan internalization. When hyaluronan was combined with partially degraded, dansyl chloride-labeled aggrecan, blue fluorescent aggrecan was also visualized within intracellular vesicles. It was also determined that sonicated hyaluronan of smaller molecular size was internalized more readily than high molecular mass hyaluronan. However, the addition of intact aggrecan to hyaluronan chains sonicated for 5 and 10 s reblocked their endocytosis, whereas aggregates containing 15-s sonicated hyaluronan were internalized. These data suggest that hyaluronan endocytosis is regulated in large part by the extracellular proteolytic processing of hyaluronan-bound proteoglycan.

CD44 and hyaluronan promote the bone morphogenetic protein 7 signaling response in murine chondrocytes.

OBJECTIVE: Cell-matrix interactions promote cartilage homeostasis. We previously found that Smad1, the transcriptional modulator of the canonical bone morphogenetic protein 7 (BMP-7) pathway, interacted with the cytoplasmic domain of CD44, the principal hyaluronan receptor on chondrocytes. To elucidate the physiologic function of CD44-Smad1 interactions, as well as the role of hyaluronan, we studied the response of chondrocytes isolated from CD44(-/-) and BALB/c (wild-type [WT]) mice to stimulation with BMP-7. METHODS: In primary murine chondrocytes, CD44 expression was decreased by small interfering RNA (siRNA) transfection or was enhanced by plasmid transfection. Pericellular hyaluronan was removed by hyaluronidase treatment, or its endogenous synthesis was inhibited. Changes in response to BMP-7 stimulation were evaluated by Western blotting of Smad1 phosphorylation and aggrecan messenger RNA (mRNA) expression. RESULTS: Chondrocytes from CD44(-/-) mice and WT mice transfected with CD44 siRNA were less responsive than untransfected chondrocytes from WT mice to BMP-7. CD44(-/-) mouse chondrocytes transfected with pCD44 showed increased sensitivity to BMP-7. Significant increases in aggrecan mRNA were observed in WT mouse chondrocytes in response to 10 ng/ml of BMP-7, whereas at least 100 ng/ml of BMP-7 was required for CD44(-/-) mouse chondrocytes. However, in chondrocytes from CD44(-/-) and WT mice, hyaluronidase treatment decreased cellular responses to BMP-7. Treatment of both bovine and murine chondrocytes with 4-methylumbelliferone to reduce the synthesis of endogenous hyaluronan confirmed that hyaluronan promoted BMP-7 signaling. CONCLUSION: Taken together, these investigations into the mechanisms underlying BMP-7 signaling in chondrocytes revealed that while hyaluronan-dependent pericellular matrix is critical for BMP-7 signaling, the expression of CD44 promotes the cellular response to lower concentrations of BMP-7.

Chondroprotective Effect of Kartogenin on CD44-Mediated Functions in Articular Cartilage and Chondrocytes.

OBJECTIVE: A recent report identified the small molecule kartogenin as a chondrogenic and chondroprotective agent. Since changes in hyaluronan metabolism occur during cartilage degeneration in osteoarthritis, we began studies to determine whether there was a connection between extracellular hyaluronan, CD44-hyaluronan interactions and the effects of kartogenin on articular chondrocytes. METHODS: Chondrocytes cultured in monolayers, bioengineered neocartilages, or cartilage explants were treated with kartogenin with or without stimulation by IL-1ß. Accumulation of matrix was visualized by a particle exclusion assay or by safranin O staining and release of sulfated glycosaminoglycans was determined. Production of aggrecanases and aggrecan G1-ITEGE neoepitope, fragmentation of CD44 and the SMAD1/5/8 signaling pathway were evaluated by western blotting. RESULTS: Kartogenin treatment enhanced chondrocyte pericellular matrix assembly and retention in the presence of IL-1ß. The chondroprotective effects of kartogenin on IL-1ß-induced release of sulfated glycosaminoglycans from articular cartilage explants, reduction in safranin O staining of neocartilage discs as well as a reduction in aggrecan G1-ITEGE neoepitope in chondrocyte and explant cartilage cultures were observed. Kartogenin partially blocked the IL-1ß-induced increased expression of ADAMTS-5. Additionally, kartogenin-treated articular chondrocytes exhibited a decrease in CD44 proteolytic fragmentation. However, kartogenin treatment did not enhance proteoglycan in control, non-IL-1ß-treated cultures. Similarly, kartogenin enhanced the SMAD1 phosphorylation but only following pretreatment with IL-1ß. CONCLUSION: These studies provide novel information on the chondroprotective function of kartogenin in adult articular cartilage. The effects of kartogenin are significant after activation of chondrocytic chondrolysis, which may occur following disruption of homeostasis maintained by hyaluronan-CD44 interactions.

Intracellular domain fragment of CD44 alters CD44 function in chondrocytes.

The hyaluronan receptor CD44 undergoes sequential proteolytic cleavage at the cell surface. The initial cleavage of the CD44 extracellular domain is followed by a second intramembranous cleavage of the residual CD44 fragment, liberating the C-terminal cytoplasmic tail of CD44. In this study conditions that promote CD44 cleavage resulted in a diminished capacity to assemble and retain pericellular matrices even though sufficient non-degraded full-length CD44 remained. Using stable and transient overexpression of the cytoplasmic domain of CD44, we determined that the intracellular domain interfered with anchoring of the full-length CD44 to the cytoskeleton and disrupted the ability of the cells to bind hyaluronan and assemble a pericellular matrix. Co-immunoprecipitation assays were used to determine whether the mechanism of this interference was due to competition with actin adaptor proteins. CD44 of control chondrocytes was found to interact and co-immunoprecipitate with both the 65- and 130-kDa isoforms of ankyrin-3. Moreover, this interaction with ankyrin-3 proteins was diminished in cells overexpressing the CD44 intracellular domain. Mutating the putative ankyrin binding site of the transiently transfected CD44 intracellular domain diminished the inhibitory effects of this protein on matrix retention. Although CD44 in other cells types has been shown to interact with members of the ezrin/radixin/moesin (ERM) family of adaptor proteins, only modest interactions between CD44 and moesin could be demonstrated in chondrocytes. The data suggest that release of the CD44 intracellular domain into the cytoplasm of cells such as chondrocytes exerts a competitive or dominant-negative effect on the function of full-length CD44.

Chondrogenic capacity and alterations in hyaluronan synthesis of cultured human osteoarthritic chondrocytes.

During osteoarthritis there is a disruption and loss of the extracellular matrix of joint cartilage, composed primarily of type II collagen, aggrecan and hyaluronan. In young patients, autologous chondrocyte implantation can be used to repair cartilage defects. However, for more elderly patients with osteoarthritis, such a repair approach is contraindicated because the procedure requires a large expansion of autologous chondrocytes in vitro leading a rapid, perhaps irreversible, loss of the chondrocyte phenotype. This study investigates whether osteoarthritic chondrocytes obtained from older patients can be expanded in vitro and moreover, induced to re-activate their chondrocyte phenotype. A decrease in chondrocyte phenotype markers, collagen II, aggrecan and SOX9 mRNA was observed with successive expansion of cells in monolayer culture. However, chondrogenic induction in three-dimensional pellet culture successfully rescued the expression of all three marker genes to native levels, even with 4th passage cells-cells representing an approximate 625-fold expansion in cell number. This data supports the use of osteoarthritic cells for autologous implantation repair. In addition, another set of gene products were explored as useful markers of the chondrocyte phenotype. Differentiated primary chondrocytes exhibited a common pattern of hyaluronan synthase isoforms that changed upon cell expansion in vitro and, reverted back to the original pattern following pellet culture. Moreover, the change in isoform pattern correlated with changes in the molecular size of synthesized hyaluronan.

Matrix metalloproteinase-9 in a unique proteoglycan form in avian embryonic growth plate cartilage.

The appearance of a high molecular weight gelatinolytic enzyme (230 kDa) correlated with cartilage collagen loss in chick embryonic tibias cultured with lipopolysaccharide. This 230 kDa enzyme was purified and its activity was measured on synthetic and natural substrates. The enzyme was activated by aminophenylmercuric acetate and inhibited by ethylenediaminetetraacetic acid, phenanthroline, marimastat or tissue inhibitors of metalloproteinases. Amino acid sequences of peptides derived from the purified enzyme showed identity with avian MMP-9. Digestion of the intact enzyme with chondroitinase decreased the size of the molecule to 80 kDa on SDS-PAGE. When chick embryonic tibia cultures were radiolabeled with (35)S-sulfate, the radiolabel co-purified with the 230 kDa gelatinase. Chondroitinase treated 230 kDa gelatinase also reacted with specific anti-chondroitin sulfate antibodies and FACE analysis revealed a predominance of chondroitin-4-sulfate. These results demonstrate this avian matrix metalloproteinase contained glycosaminoglycan chains. To our knowledge, this is the first report of a matrix metalloproteinase in a proteoglycan form.