Plasma Membrane Of Chondrocytes Research Articles

Roles of Phosphate in Skeleton.

Phosphate is essential for skeletal mineralization, and its chronic deficiency leads to rickets and osteomalacia. Skeletal mineralization starts in matrix vesicles (MVs) derived from the plasma membrane of osteoblasts and chondrocytes. MVs contain high activity of tissue non-specific alkaline phosphatase (TNSALP), which hydrolyzes phosphoric esters such as pyrophosphates (PPi) to produce inorganic orthophosphates (Pi). Extracellular Pi in the skeleton is taken up by MVs through type III sodium/phosphate (Na+/Pi) cotransporters and forms hydroxyapatite. In addition to its roles in MV-mediated skeletal mineralization, accumulating evidence has revealed that extracellular Pi evokes signal transduction and regulates cellular function. Pi induces apoptosis of hypertrophic chondrocytes, which is a critical step for endochondral ossification. Extracellular Pi also regulates the expression of various genes including those related to proliferation, differentiation, and mineralization. In vitro cell studies have demonstrated that an elevation in extracellular Pi level leads to the activation of fibroblast growth factor receptor (FGFR), Raf/MEK (mitogen-activated protein kinase/ERK kinase)/ERK (extracellular signal-regulated kinase) pathway, where the type III Na+/Pi cotransporter PiT-1 may be involved. Responsiveness of skeletal cells to extracellular Pi suggests their ability to sense and adapt to an alteration in Pi availability in their environment. Involvement of FGFR in the Pi-evoked signal transduction is interesting because enhanced FGFR signaling in osteoblasts/osteocytes might be responsible for the overproduction of FGF23, a key molecule in phosphate homeostasis, in a mouse model for human X-linked hypophosphatemic rickets (XLH). Impaired Pi sensing may be a pathogenesis of XLH, which needs to be clarified in future.

CREB activation in hypertrophic chondrocytes is involved in the skeletal overgrowth in epiphyseal chondrodysplasia Miura type caused by activating mutations of natriuretic peptide receptor B.

Natriuretic peptide receptor B (NPRB) produces cyclic guanosine monophosphate (cGMP) when bound by C-type natriuretic peptide (CNP). Activating mutations in NPRB cause a skeletal overgrowth disorder, which has been named epiphyseal chondrodysplasia, Miura type (ECDM; OMIM #615923). Here we explored the cellular and molecular mechanisms for the skeletal overgrowth in ECDM using a mouse model in which an activating mutant NPRB is specifically expressed in chondrocytes. The mutant mice (NPRB[p.V883M]-Tg) exhibited postnatal skeletal overgrowth and increased cGMP in cartilage. Both endogenous and transgene-derived NPRB proteins were localized at the plasma membrane of hypertrophic chondrocytes. The hypertrophic zone of growth plate was thickened in NPRB[p.V883M]-Tg. An in vivo BrdU-labeling assay suggested that some of the hypertrophic chondrocytes in NPRB[p.V883M]-Tg mice continued to proliferate, although wild-type (WT) chondrocytes stopped proliferating after they became hypertrophic. In vitro cell studies revealed that NPRB activation increased the phosphorylation of cyclic AMP-responsive element binding protein (CREB) and expression of cyclin D1 in matured chondrocytes. Treatment with cell-permeable cGMP also enhanced the CREB phosphorylation. Inhibition of cyclic adenosine monophosphate (cAMP)/protein kinase A pathway had no effects on the CREB phosphorylation induced by NPRB activation. In immunostaining of the growth plates for the proliferation marker Ki67, phosphorylated CREB and cyclin D1, most signals were similarly observed in the proliferating zone in both genotypes, but some cells in the hypertrophic zone of NPRB[p.V883M]-Tg were also positively stained. These results suggest that NPRB activation evokes its signal in hypertrophic chondrocytes to induce CREB phosphorylation and make them continue to proliferate, leading to the skeletal overgrowth in ECDM.

The chondrocyte channelome: A narrative review.

Chondrocytes are the main cells in the extracellular matrix (ECM) of articular cartilage and possess a highly differentiated phenotype that is the hallmark of the unique physiological functions of this specialised load-bearing connective tissue. The plasma membrane of articular chondrocytes contains a rich and diverse complement of membrane proteins, known as the membranome, which defines the cell surface phenotype of the cells. The membranome is a key target of pharmacological agents and is important for chondrocyte function. It includes channels, transporters, enzymes, receptors, and anchors for intracellular, cytoskeletal and ECM proteins and other macromolecular complexes. The chondrocyte channelome is a sub-compartment of the membranome and includes a complete set of ion channels and porins expressed in these cells. Many of these are multi-functional proteins with "moonlighting" roles, serving as channels, receptors and signalling components of larger molecular assemblies. The aim of this review is to summarise our current knowledge of the fundamental aspects of the chondrocyte channelome, discuss its relevance to cartilage biology and highlight its possible role in the pathogenesis of osteoarthritis (OA). Excessive and inappropriate mechanical loads, an inflammatory micro-environment, alternative splicing of channel components or accumulation of basic calcium phosphate crystals can result in an altered chondrocyte channelome impairing its function. Alterations in Ca2+ signalling may lead to defective synthesis of ECM macromolecules and aggravated catabolic responses in chondrocytes, which is an important and relatively unexplored aspect of the complex and poorly understood mechanism of OA development.

Leptin Receptor Metabolism Disorder in Primary Chondrocytes from Adolescent Idiopathic Scoliosis Girls

To investigate the underlying mechanisms of low metabolic activity of primary chondrocytes obtained from girls with adolescent idiopathic scoliosis (AIS); AIS is a spine-deforming disease that often occurs in girls. AIS is associated with a lower bone mass than that of healthy individuals and osteopenia. Leptin was shown to play an important role in bone growth. It can also regulate the function of chondrocytes. Changes in leptin and Ob-R levels in AIS patients have been reported in several studies. The underlying mechanisms between the dysfunction of peripheral leptin signaling and abnormal chondrocytes remain unclear; The following parameters were evaluated in AIS patients and the control groups: total serum leptin levels; Ob-R expression in the plasma membrane of primary chondrocytes; JAK2 and STAT3 phosphorylation status. Then, we inhibited the lysosome and proteasome and knocked down clathrin heavy chain (CHC) expression in primary chondrocytes isolated from girls with AIS and evaluated Ob-R expression. We investigated the effects of leptin combined with a lysosome inhibitor or CHC knockdown in primary chondrocytes obtained from AIS patients; Compared with the controls, AIS patients showed similar total serum leptin levels, reduced JAK2 and STAT3 phosphorylation, and decreased cartilage matrix synthesis in the facet joint. Lower metabolic activity and lower membrane expression of Ob-R were observed in primary chondrocytes from the AIS group than in the controls. Lysosome inhibition increased the total Ob-R content but had no effect on the membrane expression of Ob-R or leptin’s effects on AIS primary chondrocytes. CHC knockdown upregulated the membrane Ob-R levels and enhanced leptin’s effects on AIS primary chondrocytes; The underlying mechanism of chondrocytes that are hyposensitive to leptin in some girls with AIS is low plasma membrane Ob-R expression that results from an imbalance between the rate of receptor endocytosis and the insertion of newly synthesized receptors into the membrane.

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.

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-1β 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.

Comparative analysis of sodium coupled vitamin C transporter 2 in human osteoarthritis grade 1 and grade 3 tissues

BackgroundNutrient levels are known to influence the development of osteoarthritis (OA), presumably by modulating levels of matrix biosynthesis and degradation. These processes may be affected by ascorbic acid (AA), an antioxidant which acts as a cofactor for numerous biochemical reactions and is essential for post-translational modifications of collagen. In this study we examined the expression of SVCT2, the only known Sodium coupled vitamin C transporter isoform present in articular cartilage, in human articular cartilage explants derived from both normal and osteoarthritis articular cartilage.MethodsOA1 and OA3 human articular cartilage was carefully dissected and macroscopically graded for degeneration via the Collins scale. The tissue samples were histologically examined by Hematoxylin and Eosin and Safranin O and Fast Green staining. SVCT2 expression analysis was performed at mRNA level by quantitative real time PCR and at a protein level by immunohistochemistry.ResultsOur quantitative real time PCR showed marked variation in the expression of SVCT2 in human osteoarthritic articular cartilage. SVCT2 expression was significantly down-regulated (p = 0.0001) in the Collins grade 3 (OA3) compared to Collins grade 1 (OA1) tissue. Furthermore, slides stained with fluorescent antibodies to SVCT2 demonstrated greatly reduced fluorescence for the SVCT2 transporter on the chondrocyte plasma membrane in the osteoarthritic tissue samples.ConclusionsThese findings demonstrate that the expression of SVCT2 transporter is significantly altered in human osteoarthritic tissues (OA3). The modulation of this transporter could therefore potentially influence the prevention, management and treatment of osteoarthritis.

Expression of Keratinocyte Growth Factor and Its Receptor in Rat Tracheal Cartilage: Possible Involvement in Wound Healing of the Damaged Cartilage

Keratinocyte growth factor (KGF) is involved in the development and regeneration of a variety of tissues. To clarify the role of KGF in cartilage wound healing, we examined the expression of KGF and its receptor (KGFR) immunohistochemically in the wound healing area of rat tracheal cartilage, and the direct effect of recombinant KGF on the proliferation and differentiation of primary cultures of rat chondrocytes. KGF was found in the cytoplasm of both chondrocytes and perichondrial cells. On the other hand, KGFR was detected only in the plasma membrane of chondrocytes. Although the expression of KGF was similar in the cartilage and perichondrial area before and after injury, KGFR expression was induced after injury and limited to proliferating chondrocytes. The staining pattern of KGF and KGFR was same in the mature and the immature rat tracheal cartilage. Moreover, in vitro experiments using primary cultured chondrocytes revealed that KGF at 200 ng/ml significantly increased the number of chondrocytes (~1.5-fold), and significantly reduced acid mucopolysaccharide production. These results indicate that KGF stimulates chondrocyte proliferation, suggesting that KGF could therapeutically modulate the wound healing process in the tracheal cartilage.

Bone-Targeted Replacement Therapy for Hypophosphatasia

Bone-Targeted Replacement Therapy for Hypophosphatasia

Effect of a glucosamine derivative on impact-induced chondrocyte apoptosis in vitro. A preliminary report

Here, we report that a lipophilic derivative of glucosamine, Glu5, is able to prevent impact-induced chondrocyte death by the putative mechanism of reducing mitochondrial depolarisation following a single impact load in vitro.

Effects of vibration and hyaluronic acid on activation of three‐dimensional cultured chondrocytes

To investigate the effects of vibration (Vib) and hyaluronic acid (HA) on 3-dimensional cultured cartilage. Chondrocytes were obtained from metatarsophalangeal joints of freshly killed 6-month-old pigs. Twenty-four-well plates containing type I collagen sponge disks were used to culture samples. The frequency and the amplitude of the vibration of the well plate were 100 Hz and 0.5 nm, respectively. We produced 3-dimensional cartilage tissue using HA and vibration with collagen sponge as a carrier. Four different culture conditions were examined: a control HA-Vib- group, an HA-Vib+ group, an HA+Vib- group, and an HA+Vib+ group. Each group was cultured for 2 weeks. After culture days 3, 7, 10, and 14 (every 3.5 days), the levels of chondroitin 4-sulfate (C4S) and chondroitin 6-sulfate (C6S) isomers synthesized in each culture medium were measured. Histologic analysis, immunohistochemical analysis, and electron microscopic examination were performed. Mean C4S and C6S synthesis had increased rapidly after 7 days of culture and continued to increase thereafter. There were significant differences among the 4 groups (P < 0.01). Synthesis of both C4S and C6S was most abundant in the HA+Vib+ group and the lowest in the HA-Vib- group. After 1 and 2 weeks of culture, the chondrocytes had formed stratified structures on the collagen sponges in all groups, although the thickest structure was observed in the HA+Vib+ group and the thinnest in the HA-Vib- group. Under immunofluorescence, the HA+Vib+ group exhibited the strongest chromatic features. Under electron microscopy, the chondrocytes in the HA+Vib+ group exhibited many long and slender prominences on their surface, and extracellular substance could be observed associated with the cells. Our results indicate that the combination of vibration and HA activates the production of proteoglycan in 3-dimensional cultured chondrocytes and stimulates MAPK and beta-catenin. This suggests that some mechanoreceptors for vibration exist on the plasma membrane of chondrocytes and activate the intracellular signal transduction system.

Chondrocytes Utilize a Cholesterol-Dependent Lipid Translocator To Externalize Phosphatidylserine

During endochondral ossification, growth plate chondrocytes release plasma membrane (PM) derived matrix vesicles (MV), which are the site of initial hydroxyapatite crystal formation. MV constituents which facilitate the mineralization process include the integral membrane ectoenzymes alkaline phosphatase (ALPase) and nucleotide pyrophosphatase phosphodiesterase (NPP1/PC-1), along with a phosphatidylserine- (PS-) rich membrane surface that binds annexins and calcium, resulting in enhanced calcium entry into MV. In this study, we determined that chick growth plate MV were highly enriched in membrane raft microdomains containing high levels of cholesterol, glycophosphatidylinositol- (GPI-) anchored ALPase, and phosphatidylserine (PS) localized to the external leaflet of the bilayer. To determine how such membrane microdomains arise during chondrocyte maturation, we explored the role of PM cholesterol-dependent lipid assemblies in regulating the activities of lipid translocators involved in the externalization of PS. We first isolated and determined the composition of detergent-resistant membranes (DRMs) from chondrocyte PM. DRMs isolated from chondrocyte PM were enhanced in ganglioside 1 (GM1) and cholesterol as well as GPI-anchored ALPase. Furthermore, these membrane domains were enriched in PS (localized to the external leaflet of the bilayer) and had significantly higher ALPase activity than non-cholesterol-enriched domains. To understand the role of cholesterol-dependent lipid assemblies in the externalization of PS, we measured the activities of two lipid transporters involved in PS externalization, aminophospholipid translocase (APLT) and phospholipid scramblase (PLSCR1), during maturation of a murine chondrocytic cell line, N1511. In this report, we provide the first evidence that maturing chondrocytes express PLSCR1 and have scramblase activity. We propose that redistribution of PS is dependent on an increase in phospholipid scramblase activity and a decrease in APLT activity. Lastly, we show that translocator activity is most likely to be modulated by membrane cholesterol levels through a membrane raft microdomain.

Phosphodiesterase Activity of Alkaline Phosphatase in ATP-initiated Ca2+ and Phosphate Deposition in Isolated Chicken Matrix Vesicles

Inorganic pyrophosphate is a potent inhibitor of bone mineralization by preventing the seeding of calcium-phosphate complexes. Plasma cell membrane glycoprotein-1 and tissue nonspecific alkaline phosphatase were reported to be antagonistic regulators of mineralization toward inorganic pyrophosphate formation (by plasma cell membrane glycoprotein-1) and degradation (by tissue nonspecific alkaline phosphatase) under physiological conditions. In addition, they possess broad overlapping enzymatic functions. Therefore, we examined the roles of tissue nonspecific alkaline phosphatase within matrix vesicles isolated from femurs of 17-day-old chick embryos, under conditions where these both antagonistic and overlapping functions could be evidenced. Addition of 25 microM ATP significantly increased duration of mineralization process mediated by matrix vesicles, while supplementation of mineralization medium with levamisole, an alkaline phosphatase inhibitor, reduces the ATP-induced retardation of mineral formation. Phosphodiesterase activity of tissue nonspecific alkaline phosphatase for bis-p-nitrophenyl phosphate was confirmed, the rate of this phosphodiesterase activity is in the same range as that of phosphomonoesterase activity for p-nitrophenyl phosphate under physiological pH. In addition, tissue nonspecific alkaline phosphatase at pH 7.4 can hydrolyze ADPR. On the basis of these observations, it can be concluded that tissue nonspecific alkaline phosphatase, acting as a phosphomonoesterase, could hydrolyze free phosphate esters such as pyrophosphate and ATP, while as phosphodiesterase could contribute, together with plasma cell membrane glycoprotein-1, in the production of pyrophosphate from ATP.

Modulation of H+ Transport Mechanisms by Interleukin-1 in Isolated Bovine Articular Chondrocytes

The proinflammatory cytokine interleukin-1 (IL-1) promotes the degradation of articular cartilage by inhibiting matrix synthesis and stimulating degradative enzyme activity. Generation of nitric oxide (NO) in response to IL-1 is implicated in these actions. The catabolic actions of IL-1 can be inhibited by manoeuvres which are predicted to dissipate H⁺ gradients across the chondrocyte plasma membrane. In the present study, the effects of IL-1 on H⁺ extrusion from bovine articular chondrocytes were investigated. pH was measured using the H⁺-sensitive fluorescent dye BCECF. Cells were acidified by ammonium rebound and the contribution of the Na⁺-H⁺ exchanger (NHE) and of the vacuolar H⁺-ATPase to acid extrusion was characterised by ion substitution and inhibitor studies. Overnight (18h) exposure to IL-1 stimulated acid extrusion in a dose-dependent fashion. This effect represented stimulation of both NHE and the ATPase. Characterisation of the timecourse of this response indicated that, while stimulation of acid extrusion was rapid, effects on the ATPase were only apparent after greater than 8h incubation with the cytokine. In keeping with this observation, the protein synthesis inhibitor cycloheximide abolished the stimulatory effect of IL-1 on ATPase-mediated extrusion. The upregulation of ATPase activity by IL-1 was inhibited by the NOS inhibitor L-NAME and by the NO scavenger PTIO. In cells which had not been exposed to IL-1, treatment with the NO donor SNAP also stimulated acid extrusion by the ATPase. In contrast, NHE activity was not altered by any of these compounds. Taken together, these results imply that IL-1 can stimulate acid extrusion in chondrocytes and that this reflects rapid upregulation of NHE with slower induction of H⁺-ATPase activity which requires elevated levels of NO. While ATPase induction involves protein synthesis, this process may not constitute synthesis of ATPase proteins per se, but rather of some associated regulatory process.

Aquaporin water channels AQP1 and AQP3, are expressed in equine articular chondrocytes

Chondrocytes exist in an unusual and highly variable ionic and osmotic environment in the extracellular matrix of articular cartilage. Alterations to the ionic and osmotic environment of chondrocytes influence the volume and ionic content of the cells, which, in turn, modifies the rate at which extracellular matrix macromolecules are synthesized and degraded. Thus, regulation of the water and solute content of chondrocytes will profoundly affect their anabolic and catabolic functions. The water content of cells is effectively influenced by the abundance of aquaporin (AQP) water channels. Recent studies have shown that several AQP water channel isoforms are expressed in chondrocytes from Meckel’s cartilage, developing teeth and other orofacial tissues. The aim of the present investigation was to determine if chondrocytes from equine articular cartilage express AQP water channels. Polyclonal antibodies to AQP1, AQP2 and AQP3 were used in conjunction with immunohistochemistry, immunoblotting and quantitative flow cytometry to determine if AQP1, AQP2 and AQP3 are expressed in equine articular chondrocytes. Our studies show that AQP1 and AQP3 are expressed by chondrocytes in articular cartilage in situ and in isolated chondrocytes. We found no evidence for expression of AQP2, the vasopressin-regulated water channel in chondrocytes. AQP1 and AQP3 may be involved in the transport of water and small solutes and osmotically active metabolites across the chondrocyte plasma membrane during volume regulatory behaviour. AQP1 may be involved in transporting metabolic water. AQP3 may participate in the transport of glycerol and structurally related molecules.

Latrunculin and cytochalasin decrease chondrocyte matrix retention.

The proteoglycan-rich extracellular matrix (ECM) directly associated with the cells of articular cartilage is anchored to the chondrocyte plasma membrane via interaction with the hyaluronan receptor CD44. The cytoplasmic tail of CD44 interacts with the cortical cytoskeleton. The objective of this study was to determine the role of the actin cytoskeleton in CD44-mediated matrix assembly by chondrocytes and cartilage matrix retention and homeostasis. Adult bovine articular cartilage tissue slices and isolated chondrocytes were treated with latrunculin or cytochalasin. Tissues were processed for histology and chondrocytes were examined for CD44 expression and pericellular matrix assembly. Treatments that disrupt the actin cytoskeleton reduced chondrocyte pericellular matrix assembly and the retention of proteoglycan within cartilage explants. There was enhanced detection of a neoepitope resulting from proteolysis of aggrecan. Cytoskeletal disruption did not reduce CD44 expression, as monitored by flow cytometry, but detergent extraction of CD44 was enhanced and hyaluronan binding was decreased. Thus, disruption of the cytoskeleton reduces the anchorage of CD44 in the chondrocyte membrane and the capacity of CD44 to bind its ligand. The results suggest that cytoskeletal disruption within cartilage uncouples chondrocytes from the matrix, resulting in altered metabolism and deleterious changes in matrix structure.

Ectopic mineralization of articular cartilage in the bullfrog Rana catesbeiana and its possible involvement in bone closure.

Mineralization of the articular cartilage is a pathological condition associated with age and certain joint diseases in humans and other mammals. In this work, we describe a physiological process of articular cartilage mineralization in bullfrogs. Articular cartilage of the proximal and distal ends of the femur and of the proximal end of the tibia-fibula was studied in animals of different ages. Mineralization of the articular cartilage was detected in animals at 1 month post-transformation. This mineralization, which appeared before the hypertrophic cartilage showed any calcium deposition, began at a restricted site in the lateral expansion of the cartilage and then progressed to other areas of the epiphyseal cartilage. Mineralized structures were identified by von Kossa's staining and by in vivo incorporation of calcein green. Element analysis showed that calcium crystals consisted of poorly crystalline hydroxyapatite. Mineralized matrix was initially spherical structures that generally coalesced after a certain size to occupy larger areas of the cartilage. Alkaline phosphatase activity was detected at the plasma membrane of nearby chondrocytes and in extracellular matrix. Apoptosis was detected by the TUNEL (TDT-mediated dUTP-biotin nick end-labeling) reaction in some articular chondrocytes from mineralized areas. The area occupied by calcium crystals increased significantly in older animals, especially in areas under compression. Ultrastructural analyses showed clusters of needle-like crystals in the extracellular matrix around the chondrocytes and large blocks of mineralized matrix. In 4-year-old animals, some lamellar bone (containing bone marrow) occurred in the same area as articular cartilage mineralization. These results show that the articular cartilage of R. catesbeiana undergoes precocious and progressive mineralization that is apparently stimulated by compressive forces. We suggest that this mineralization is involved in the closure of bone extremities, since mineralization appears to precede the formation of a rudimentary secondary center of ossification in older animals.

Temporal expression of CD44 during embryonic chick limb development and modulation of its expression with retinoic acid.

Hyaluronan-cell interactions are initiated co-ordinately with mesenchymal condensation during chondrogenic differentiation in the limb bud. Hyaluronan is responsible for the retention and organization of proteoglycan within the cartilage matrix. Hyaluronan-CD44 binding also retains proteoglycan aggregates to the chondrocyte plasma membrane. A sequence for CD44 protein in chick has recently been reported, but never evaluated in chick chondrocytes. Total RNA was isolated from embryonic chick limb buds, stages 18, 19, 24, 25 and 30. Using semi-quantitative RT-PCR, expression of aggrecan, this chick CD44 orthologue and GAPDH mRNA was analyzed. Aggrecan expression was detected at all stages, but was increased at stage 30. CD44 mRNA was detected at extremely low levels at stage 18 to higher levels in the latter stages. Thus, the temporal expression of CD44 mRNA correlated with the onset of pre-cartilage condensation. The full-length chick chondrocyte CD44 cDNA was obtained following RT-PCR using RNA derived from tibial chondrocytes from stage 37 chick embryos. The nucleotide sequence was used to generate an amino acid sequence and analyses revealed homologies of 44.4% with mouse, 47.8% with bovine and 46.3% with human CD44. Tibial chondrocytes were cultured in the presence or absence of retinoic acid for 36 or 72 h. By RT-PCR, expression of aggrecan and the CD44 mRNA by chick chondrocytes was decreased after retinoic acid treatment, while GAPDH expression showed no change. As expected, control chondrocytes exhibited a round morphology while retinoic acid-treated chondrocytes were elongated. The retinoic acid-treated chondrocytes also exhibited reduced hyaluronan binding. This functional assay indicates a role for a CD44 receptor in matrix retention by chick chondrocytes.

Structure and promoter analysis of the mouse membrane-bound transferrin-like protein (MTf) gene.

Recently, we purified membrane-bound transferrin-like protein (MTf) from the plasma membrane of rabbit chondrocytes and showed that the expression levels of MTf protein and mRNA were much higher in cartilage than in other tissues [Kawamoto T, Pan, H., Yan, W., Ishida, H., Usui, E., Oda, R., Nakamasu, K., Noshiro, M., Kawashima-Ohya, Y., Fujii, M., Shintani, H., Okada, Y. & Kato, Y. (1998) Eur. J. Biochem. 256, 503--509]. In this study, we isolated the MTf gene from a constructed mouse genomic library. The mouse MTf gene was encoded by a single-copy gene spanning approximately 26 kb and consisting of 16 exons. The transcription-initiation site was located 157 bp upstream from the translation-start codon, and a TATA box was not found in the 5' flanking region. The mouse MTf gene was mapped on the B3 band of chromosome 16 by fluorescence in situ hybridization. Using primary chondrocytes, SK-MEL-28 (melanoma cell line), ATDC5 (chondrogenic cell line) and NIH3T3 (fibroblast cell line) cells, we carried out transient expression studies on various lengths of the 5' flanking region of the MTf gene fused to the luciferase reporter gene. Luciferase activity in SK-MEL-28 cells was higher than in primary chondrocytes. Although no luciferase activity was detectable in NIH3T3 cells, it was higher in chondrocytes than in ATDC5 chondrogenic cells. These findings were consistent with the levels of expression of MTf mRNA in these cells cultured under similar conditions. The patterns of increase and decrease in the luciferase activity in chondrocytes transfected with various 5' deleted constructs of the MTf promoter were similar to that in ATDC5 cells, but differed from that in SK-MEL-28 cells. The findings obtained with primary chondrocytes suggest that the regions between -693 and -444 and between -1635 and -1213 contain positive and negative cis-acting elements, respectively. The chondrocyte-specific expression of the MTf gene could be regulated via these regulatory elements in the promoter region.

Sodium transport systems in human chondrocytes. I. Morphological and functional expression of the Na+,K(+)-ATPase alpha and beta subunit isoforms in healthy and arthritic chondrocytes.

The chondrocyte is the cell responsible for the maintenance of the articular cartilage matrix. The negative charges of proteoglycans of the matrix draw cations, principally Na+, into the matrix to balance the negative charge distribution. The Na+,K(+)-ATPase is the plasma membrane enzyme that maintains the intracellular Na+ and K+ concentrations. The enzyme is composed of an alpha and a beta subunit, so far, 4 alpha and 3 beta isoforms have been identified in mammals. Chondrocytes are sensitive to their ionic and osmotic environment and are capable of adaptive responses to ionic environmental perturbations particularly changes to extracellular [Na+]. In this article we show that human fetal and adult chondrocytes express three alpha (alpha 1, alpha 2 and the neural form of alpha 3) and the three beta isoforms (beta 1, beta 2 and beta 3) of the Na+,K(+)-ATPase. The presence of multiple Na+,K(+)-ATPase isoforms in the plasma membrane of chondrocytes suggests a variety of kinetic properties that reflects a cartilage specific and very fine specialization in order to maintain the Na+/K+ gradients. Changes in the ionic and osmotic environment of chondrocytes occur in osteoarthritis and rheumatoid arthritis as result of tissue hydration and proteoglycan loss leading to a fall in tissue Na+ and K+ content. Although the expression levels and cellular distribution of the proteins tested do not vary, we detect changes in p-nitrophenylphosphatase activity "in situ" between control and pathological samples. This change in the sodium pump enzymatic activity suggests that the chondrocyte responds to these cationic environmental changes with a variation of the active isozyme types present in the plasma membrane.