Mechanotransduction of bovine articular cartilage superficial zone protein by transforming growth factor beta signaling

OBJECTIVE: Mechanical signals are key determinants in tissue morphogenesis, maintenance, and restoration strategies in regenerative medicine, although molecular mechanisms of mechanotransduction remain to be elucidated. This study was undertaken to investigate the mechanotransduction process of expression of superficial zone protein (SZP), a critical joint lubricant. METHODS: Regional expression of SZP was first quantified in cartilage obtained from the femoral condyles of immature bovines, using immunoblotting, and visualized by immunohistochemistry. Contact pressure mapping in whole joints was accomplished using pressure-sensitive film and a load application system for joint testing. Friction measurements on cartilage plugs were acquired under boundary lubrication conditions using a pin-on-disk tribometer modified for reciprocating sliding. Direct mechanical stimulation by shear loading of articular cartilage explants was performed with and without inhibition of transforming growth factor beta (TGFbeta) signaling, and SZP content in media was quantified by enzyme-linked immunosorbent assay. RESULTS: An unexpected pattern of SZP localization in knee cartilage was initially identified, with anterior regions exhibiting high levels of SZP expression. Regional SZP patterns were regulated by mechanical signals and correlated with tribological behavior. Direct relationships were demonstrated between high levels of SZP expression, maximum contact pressures, and low friction coefficients. Levels of SZP expression and accumulation were increased by applying shear stress, depending on location within the knee, and were decreased to control levels with the use of a specific inhibitor of TGFbeta receptor type I kinase and subsequent phospho-Smad2/3 activity. CONCLUSION: These findings indicate a new role for TGFbeta signaling in the mechanism of cellular mechanotransduction that is especially significant for joint lubrication.     

Mechanotransduction of bovine articular cartilage superficial zone protein by transforming growth factor β signaling

Objective

Mechanical signals are key determinants in tissue morphogenesis, maintenance, and restoration strategies in regenerative medicine, although molecular mechanisms of mechanotransduction remain to be elucidated. This study was undertaken to investigate the mechanotransduction process of expression of superficial zone protein (SZP), a critical joint lubricant.

Methods

Regional expression of SZP was first quantified in cartilage obtained from the femoral condyles of immature bovines, using immunoblotting, and visualized by immunohistochemistry. Contact pressure mapping in whole joints was accomplished using pressure‐sensitive film and a load application system for joint testing. Friction measurements on cartilage plugs were acquired under boundary lubrication conditions using a pin‐on‐disk tribometer modified for reciprocating sliding. Direct mechanical stimulation by shear loading of articular cartilage explants was performed with and without inhibition of transforming growth factor β (TGFβ) signaling, and SZP content in media was quantified by enzyme‐linked immunosorbent assay.

Results

An unexpected pattern of SZP localization in knee cartilage was initially identified, with anterior regions exhibiting high levels of SZP expression. Regional SZP patterns were regulated by mechanical signals and correlated with tribological behavior. Direct relationships were demonstrated between high levels of SZP expression, maximum contact pressures, and low friction coefficients. Levels of SZP expression and accumulation were increased by applying shear stress, depending on location within the knee, and were decreased to control levels with the use of a specific inhibitor of TGFβ receptor type I kinase and subsequent phospho‐Smad2/3 activity.

Conclusion

These findings indicate a new role for TGFβ signaling in the mechanism of cellular mechanotransduction that is especially significant for joint lubrication.

     

Differential regulation of lubricin/superficial zone protein by transforming growth factor beta/bone morphogenetic protein superfamily members in articular chondrocytes and synoviocytes

OBJECTIVE: To investigate the role of transforming growth factor beta (TGFbeta)/bone morphogenetic protein (BMP) superfamily members on accumulation of superficial zone protein (SZP) in articular chondrocytes and synoviocytes. METHODS: Chondrocytes and synoviocytes were isolated from articular cartilage and synovium from calf stifle joints and cultured as monolayers in serum-free chemically defined medium. Articular chondrocytes were isolated from 3 distinct zones of the cartilage: superficial, middle, and deep. Accumulation of SZP in the culture medium in response to various members of the TGFbeta/BMP superfamily was demonstrated by immunoblotting and quantified by enzyme-linked immunosorbent assay. RESULTS: TGFbeta stimulated SZP accumulation in both superficial zone chondrocytes and synoviocytes. The 3 isoforms of TGFbeta elicited a similar dose response. Inhibition of TGFbeta receptor type I kinase by the specific inhibitor SB431542 abolished the TGFbeta-stimulated accumulation of SZP. BMPs up-regulated SZP accumulation in the superficial zone; however, the magnitude of the effects was not as great as was observed with TGFbeta. There was an additive action between TGFbeta and BMP on SZP accumulation. The response of synoviocytes to BMP was stronger than that of superficial zone chondrocytes. Activin up-regulated SZP accumulation in synoviocytes, but not in chondrocytes. CONCLUSION: TGFbeta is a critical regulator of SZP accumulation in both superficial zone articular chondrocytes and synoviocytes. TGFbeta and BMP have an additive effect. Synoviocytes are more sensitive to BMP family members and activins than are superficial zone articular chondrocytes. Thus, regulation of SZP accumulation by TGFbeta /BMP superfamily members is regulated differently in articular chondrocytes and synoviocytes.     

Stimulation of dna synthesis by ascorbate in cultures of articular chondrocytes

The addition of 0.2 mM Na L-ascorbate increased the incorporation of ³H-thymidine by rabbit articular chondrocytes in cell and organ culture. The stimulatory response of explants to ascorbate was potentiated by pretreatment of the cartilage with 0.2% clostridial collagenase (type 1) or trypsin for 15–30 minutes. In explants there was a latent period of 3 to 4 days before increased labeling of the nuclei could be detected. The effect was transient and declined after 8 days of culture. It was more evident in organ cultures of immature (3-month-old) than 2- to 3-year-old rabbits. Age differences were not detected in cell cultures. Explants of adult human articular cartilage were stimulated by ascorbate when the medium was supplemented with 10% fresh human serum but not by fetal bovine serum. The findings indicated that synthesis of DNA by articular chondrocytes in situ is regulated by responsiveness of the cells proper to compounds such as vitamin C, by properties of the extracellular matrix, and by factors in the serum. Ascorbate was cytotoxic at concentrations >0.2 mM in the presence of certain batches of serum and when added to monolayers before the cells attached to the surface of the flask.     

Differential regulation of lubricin/superficial zone protein by transforming growth factor β/bone morphogenetic protein superfamily members in articular chondrocytes and synoviocytes

Objective

To investigate the role of transforming growth factor β (TGFβ)/bone morphogenetic protein (BMP) superfamily members on accumulation of superficial zone protein (SZP) in articular chondrocytes and synoviocytes.

Methods

Chondrocytes and synoviocytes were isolated from articular cartilage and synovium from calf stifle joints and cultured as monolayers in serum‐free chemically defined medium. Articular chondrocytes were isolated from 3 distinct zones of the cartilage: superficial, middle, and deep. Accumulation of SZP in the culture medium in response to various members of the TGFβ/BMP superfamily was demonstrated by immunoblotting and quantified by enzyme‐linked immunosorbent assay.

Results

TGFβ stimulated SZP accumulation in both superficial zone chondrocytes and synoviocytes. The 3 isoforms of TGFβ elicited a similar dose response. Inhibition of TGFβ receptor type I kinase by the specific inhibitor SB431542 abolished the TGFβ‐stimulated accumulation of SZP. BMPs up‐regulated SZP accumulation in the superficial zone; however, the magnitude of the effects was not as great as was observed with TGFβ. There was an additive action between TGFβ and BMP on SZP accumulation. The response of synoviocytes to BMP was stronger than that of superficial zone chondrocytes. Activin up‐regulated SZP accumulation in synoviocytes, but not in chondrocytes.

Conclusion

TGFβ is a critical regulator of SZP accumulation in both superficial zone articular chondrocytes and synoviocytes. TGFβ and BMP have an additive effect. Synoviocytes are more sensitive to BMP family members and activins than are superficial zone articular chondrocytes. Thus, regulation of SZP accumulation by TGFβ /BMP superfamily members is regulated differently in articular chondrocytes and synoviocytes.

     

Inhibition of beta-catenin signaling in articular chondrocytes results in articular cartilage destruction

OBJECTIVE: Osteoarthritis is a degenerative joint disease whose molecular mechanism is currently unknown. Wnt/beta-catenin signaling has been demonstrated to play a critical role in the development and function of articular chondrocytes. To determine the role of beta-catenin signaling in articular chondrocyte function, we generated Col2a1-ICAT-transgenic mice to inhibit beta-catenin signaling in chondrocytes. METHODS: The expression of the ICAT transgene was determined by immunostaining and Western blot analysis. Histologic analyses were performed to determine changes in articular cartilage structure and morphology. Cell apoptosis was determined by TUNEL staining and the immunostaining of cleaved caspase 3 and poly(ADP-ribose) polymerase (PARP) proteins. Expression of Bcl-2, Bcl-x(L), and Bax proteins and caspase 9 and caspase 3/7 activities were examined in primary sternal chondrocytes isolated from 3-day-old neonatal Col2a1-ICAT-transgenic mice and their wild-type littermates and in primary chicken and porcine articular chondrocytes. RESULTS: Expression of the ICAT transgene was detected in articular chondrocytes of the transgenic mice. Associated with this, age-dependent articular cartilage destruction was observed in Col2a1-ICAT-transgenic mice. A significant increase in cell apoptosis in articular chondrocytes was identified by TUNEL staining and the immunostaining of cleaved caspase 3 and PARP proteins in these transgenic mice. Consistent with this, Bcl-2 and Bcl-x(L) expression were decreased and caspase 9 and caspase 3/7 activity were increased, suggesting that increased cell apoptosis may contribute significantly to the articular cartilage destruction observed in Col2a1-ICAT-transgenic mice. CONCLUSION: Inhibition of beta-catenin signaling in articular chondrocytes causes increased cell apoptosis and articular cartilage destruction in Col2a1-ICAT- transgenic mice.     

The accumulation of intracellular ITEGE and DIPEN neoepitopes in bovine articular chondrocytes is mediated by CD44 internalization of hyaluronan

OBJECTIVE: A dramatic loss of aggrecan proteoglycan from cartilage is associated with osteoarthritis. The fate of residual G1 domains of aggrecan is unknown, but inefficient turnover of these domains may impede subsequent repair and retention of newly synthesized aggrecan. Thus, the objective of this study was to determine whether ITEGE- and DIPEN-containing G1 domains, generated in situ, are internalized by articular chondrocytes, and whether these events are dependent on hyaluronan (HA) and its receptor, CD44. METHODS: ITEGE and DIPEN neoepitopes were detected by immunofluorescence staining of bovine articular cartilage chondrocytes treated with or without interleukin-1alpha (IL-1alpha). Additionally, purified ITEGE- or DIPEN-containing G1 domains were aggregated with HA and then added to articular chondrocytes, articular chondrocytes transfected with CD44delta67, or COS-7 cells transfected with or without full-length CD44. Internalized epitopes were distinguished by their resistance to extensive trypsinization of the cell surface. RESULTS: Both ITEGE and DIPEN were visualized within the extracellular cell-associated matrix of chondrocytes as well as within intracellular vesicles. Following trypsinization, the intracellular accumulation of both epitopes was clearly visible. IL-1 treatment increased extracellular as well as intracellular ITEGE epitope accumulation. Once internalized, the ITEGE neoepitope became localized within the nucleus and displayed little colocalization with HA, DIPEN, or other G1 domain epitopes. The internalization of both ITEGE and DIPEN G1 domains was dependent on the presence of HA and CD44. CONCLUSION: One important mechanism for the elimination of residual G1 domains following extracellular degradation of aggrecan is CD44-mediated co-internalization with HA.     

Sphingomyelinase decreases type II collagen expression in bovine articular cartilage chondrocytes via the ERK signaling pathway

OBJECTIVE: Ceramide, a mediator of proinflammatory cytokine signaling, induces cartilage degradation and reduces type II collagen synthesis in articular cartilage. The accumulation of ceramide is associated with arthritis in Farber's disease. The aim of this study was to investigate the mechanism of ceramide-induced down-regulation of type II collagen. METHODS: Bovine articular chondrocytes were stimulated with sphingomyelinase (SMase) to increase levels of endogenous ceramide. Components of the ERK pathway were inhibited by Raf-1 kinase inhibitor and the MEK inhibitor, PD98059. Cell extracts were analyzed by Western blotting for ERK-1/2, SOX9, c-Fos, and type II collagen, and the level of c-fos messenger RNA (mRNA) was analyzed by quantitative polymerase chain reaction. Localization of ERK-1/2, SOX9, and c-Fos was assessed by immunocytochemistry and confocal microscopy. RESULTS: SMase treatment of chondrocytes caused sustained phosphorylation of ERK-1/2 throughout the cytoplasm and nucleus that was reduced by inhibitors of Raf-1 kinase and MEK-1/2. SMase treatment of chondrocytes also induced translocation of c-Fos to the nucleus and phospho-SOX9 to the cytoplasm and increased expression of c-fos mRNA. Type II collagen expression, which was down-regulated by SMase treatment, was restored by the MEK-1/2 inhibitor, PD98059. CONCLUSION: SMase down-regulates type II collagen in articular chondrocytes via activation of the ERK signaling cascade, redistribution of SOX9, and recruitment of c-Fos. This new mechanism for cartilage degradation provides potential targets for future treatment of arthritic disease.     

Changes in cell surface antigen expression on human articular chondrocytes induced by gamma-interferon

Ia antigens (class II HLA molecules) have been detected on cells eluted from affected human cartilage in certain disease states, but not on normal cartilage cells. Because the presence of Ia antigens on chondrocytes may play an important role in rheumatic diseases, we investigated the induction of these molecules by γ-interferon (γ-IFN), a potent Ia-inducing lymphokine. Human articular chondrocytes were incubated with recombinant γ-IFN, and the expression of Ia antigens was studied by cell sorter analysis, using a panel of reagents that detect monomorphic and polymorphic specificities of the DR and DQ Ia antigen families. While the induction of DR antigens, including polymorphic DR specificities, was readily obtained with γ-IFN (50–95% positive cells), DQ antigens were negative or were displayed only on a lower percentage of chondrocytes (5–60%). In addition, incubation with γ-IFN led to an increased expression of HLA class I antigens. The expression of various other surface markers either remained unchanged (as in 4F2 and BA-2) or showed tendencies toward decreased percentages (as in 83c2) or increased percentages (as in MøR-17). No apparent change in cell morphology or growth pattern was observed.     

Vulnerability of the superficial zone of immature articular cartilage to compressive injury

Objective

The zonal composition and functioning of adult articular cartilage causes depth‐dependent responses to compressive injury. In immature cartilage, shear and compressive moduli as well as collagen and sulfated glycosaminoglycan (sGAG) content also vary with depth. However, there is little understanding of the depth‐dependent damage caused by injury. Since injury to immature knee joints most often causes articular cartilage lesions, this study was undertaken to characterize the zonal dependence of biomechanical, biochemical, and matrix‐associated changes caused by compressive injury.

Methods

Disks from the superficial and deeper zones of bovine calves were biomechanically characterized. Injury to the disks was achieved by applying a final strain of 50% compression at 100%/second, followed by biomechanical recharacterization. Tissue compaction upon injury as well as sGAG density, sGAG loss, and biosynthesis were measured. Collagen fiber orientation and matrix damage were assessed using histology, diffraction‐enhanced x‐ray imaging, and texture analysis.

Results

Injured superficial zone disks showed surface disruption, tissue compaction by 20.3 ± 4.3% (mean ± SEM), and immediate biomechanical impairment that was revealed by a mean ± SEM decrease in dynamic stiffness to 7.1 ± 3.3% of the value before injury and equilibrium moduli that were below the level of detection. Tissue areas that appeared intact on histology showed clear textural alterations. Injured deeper zone disks showed collagen crimping but remained undamaged and biomechanically intact. Superficial zone disks did not lose sGAG immediately after injury, but lost 17.8 ± 1.4% of sGAG after 48 hours; deeper zone disks lost only 2.8 ± 0.3% of sGAG content. Biomechanical impairment was associated primarily with structural damage.

Conclusion

The soft superficial zone of immature cartilage is vulnerable to compressive injury, causing superficial matrix disruption, extensive compaction, and textural alteration, which results in immediate loss of biomechanical function. In conjunction with delayed superficial sGAG loss, these changes may predispose the articular surface to further softening and tissue damage, thus increasing the risk of development of secondary osteoarthritis.

     

Sulfate incorporation by articular chondrocytes in monolayer culture

Articular chondrocytes in primary monolayers or subcultures synthesize sulfated macromolecules, presumably mucopolysaccharides. Intracellular ³⁵SO₄ incorporation by postnatal rabbit chondrocytes reached a steady level by 24 hr of incubation; the nondialyzable extracellular (centrifuged medium + trypsin digest) counts increased over 72 hr, when they were up to 100 times the intracellular value. Cell for cell, radiosulfate counts in each fraction were higher in chondrocytes than fibroblasts. Data from a 3-year-old rabbit were comparable to those from recently weaned animals. Secretion of sulfated mucopolysaccharide was also found in cultures of adult human and bovine articular chondrocytes. Although Ham's F12 medium is advantageous in establishing a primary culture of chondrocytes, sulfate incorporation was greater when Dulbecco-Eagle medium, with or without supplemental L-proline, was used for subcultures. Ascorbic acid (5 mg/100 ml) added to an already confluent monolayer had no consistent effect.     

Transglutaminase activity in aging articular chondrocytes and articular cartilage vesicles

Objective. Transglutaminases (TGases) (E.G. 2.3.2.13) catalyze a posttranslational modification of proteins and are associated with biomineralization in growth plate cartilage. Type II TGase participates in the activation of latent transforming growth factor β (TGFß), a crucial factor for both normal cartilage mineralization and the pathologic mineralization that results in calcium pyrophosphate dihydrate (CPPD) crystal formation in aging articular cartilage. To explore a possible association between TGase levels and CPPD crystal formation in mature articular cartilage, TGase activity in articular chondrocytes from old and young pigs and in the articular cartilage vesicle (ACV) fraction of porcine articular cartilage was examined. In addition, the effects of TGase inhibitors on the production of inorganic pyrophosphate (PPi), a process necessary for CPPD crystallogenesis, were determined. Methods. TGase activity was measured with a radiometric assay in cultured articular chondrocytes from the knee joints of old (3–5 years old) and young (2–6 weeks old) pigs and in the ACVs. PPi levels were measured in chondrocyte-conditioned media in the presence of TGase inhibitors or control compounds. Results. Levels of TGase activity in the cytosolic fraction of old chondrocytes were 7-fold higher than those in identically cultured young chondrocytes. The mean ± SD activity level in the membrane fraction of lysed chondrocytes was 6.0 ± 0.6 units/mg protein in old articular chondrocytes and was undetectable in young chondrocytes. In ACVs, the mean ± SD TGase activity level was 1.23 ± 0.1 units/mg protein. Type II TGase protein was present in chondrocyte cytosol and in ACVs. TGase activity was increased by TGFß to 120% of control values (P < 0.01), and decreased by insulin-like growth factor 1 to 80% of control values (P < 0.01). TGase inhibitors blocked media accumulation of PPi, an essential precursor of CPPD crystal formation, and a sensitive marker of TGFß effect. Conclusion. These data suggest a potential link between TGase activity and processes of pathologic biomineralization that result in CPPD crystal formation in aging articular cartilage.     

Effects of sulfasalazine and tofacitinib on the protein profile of articular chondrocytes

Abstract

Objective. Sulfasalazine (SSZ) and tofacitinib are effective for treating rheumatoid arthritis, however, their effects on chondrocytes have not been fully understood. We here tried to elucidate their effects on chondrocyte proteins.

Methods. We treated chondrocytes from five osteoarthritis patients with IL-1β, IL-1β+ SSZ, IL-1β+ tofacitinib, SSZ alone, and tofacitinib alone. Then, we compared protein profiles of the chondrocytes using two-dimensional differential gel electrophoresis. Further, we identified altered proteins by mass spectrometry.

Results. Out of 892 detected protein spots, the IL-1β stimulation changed intensity of 43 spots more than 1.3-fold or less than 1/1.3-fold significantly. SSZ suppressed the IL-1β-induced intensity alteration in 16 (37%) out of the 43 protein spots. Tofacitinib suppressed the IL-1β-induced alteration in 4 (9.3%) out of the 43 spots. The production of AMP deaminase 2 and procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 were increased by IL-1β and the increase was suppressed by SSZ and by tofacitinib. SSZ alone altered intensity of 273 (31%) out of the 852 spots significantly, whereas tofacitinib alone altered intensity of only 24 (2.7%) out of them.

Conclusion. SSZ and, to lesser extent, tofacitinib suppress the effects of IL-1β on the protein profiles of chondrocytes. Our data would promote understanding of effects of the drugs on chondrocytes.     

Inorganic pyrophosphate release by rabbit articular chondrocytes in vitro

Release of inorganic pyrophosphate (PPi) by rabbit articular chondrocytes in vitro was measured by a newly developed assay which utilizes radioactive orthophosphate (32Pi) labeling and anion exchange high performance liquid chromatography. Chondrocytes in monolayer and high density culture failed to release PPi. Explants (cartilage fragments), however, released newly formed PPi into the culture medium. Trypsin treatment of cartilage fragments almost completely blocked the PPi extrusion. Collagenase treatment had no effect on PPi extrusion. There was no clear correlation between proteoglycan synthesis, measured by 35SO4 incorporation, and PPi release. Suppression of proteoglycan synthesis with tunicamycin did not influence the PPi release of the explants.