Objective. To measure cartilage pH in patients with osteoarthritis (OA) and to analyze the presence of cathepsin K, the recently discovered acidic endoproteinase, in phenotypically altered chondrocytes.Methods. Intraoperative measurements of the pH of clinically normal, fibrillated, superficially fissured, and deeply fissured cartilage surfaces (grades 0-3, respectively) in OA patients undergoing primary hip replacement surgery were performed with the use of a sting electrode sterilized with microbicidic plasma. Fluorescent pH probes were used for in situ assessment of cartilage matrix pH. Cathepsin K was assessed using quantitative reverse transcriptase-polymerase chain reaction and immunohistochemistry methods.Results. The pH of grade 0 cartilage surfaces was 7.1 ؎ 0.4 (mean ؎ SD), compared with 6.2 ؎ 0.9 (P < 0.05), 5.7 ؎ 1.0 (P < 0.001), and 5.5 ؎ 1.0 (P < 0.001) for grades 1-3 cartilage surfaces, respectively. Fluorescent pH probes and acid-dependent autocatalytic conversion of cathepsin K into its active, low molecular weight form in cartilage confirmed these findings. Cathepsin K messenger RNA levels increased in relation to the severity of OA, and the number of cathepsin K-containing chondrocytes increased from a mean ؎ SD of 12 ؎ 3 in grade 0 cartilage surfaces to 47 ؎ 7, 50 ؎ 6, and 100 ؎ 12 in grades 1-3 cartilage surfaces, respectively (P < 0.001 for all comparisons).Conclusion. Acid-activated, but pharmacologically inhibitable, cathepsin K is induced in phenotypically altered chondrocytes in OA. The findings suggest that cathepsin K, rather than neutral matrix metalloproteinases, degrades the superficial gliding surfaces of the articular hyaline cartilage in OA.Osteoarthritis (OA) is a common and crippling degenerative joint disease that affects the elderly population. In adult cartilage, chondrocytes occupy Ͻ5% of the total tissue volume. They reside in an avascular, anoxic environment and depend on anaerobic metabolism. Articular hyaline cartilage consists of a hydrated, proteoglycan-rich matrix in which a network of type II collagen-rich fibers is embedded (1,2). The hydrated proteoglycan matrix has a high swelling pressure, which is counteracted by the stiff collagen network (2). This gives unique biomechanical properties to the articular hyaline cartilage.While depletion of proteoglycans alone is considered to be reversible, once the collagen network is destroyed, the damage is permanent, because the articular hyaline cartilage has an almost nonexistent capacity to regenerate (3-5). Therefore, degradation of type II collagen is the crucial event in the pathogenesis of OA.OA has been considered to be strictly a wearand-tear disease, in which the destruction of the collagen fiber network is mechanically induced. However, it is now widely accepted that OA is a biochemically mediSupported by clinical EVO research grants (TYH 0056, TYH 0215, TYH 0341, and TYH 8307), an Invalid Foundation 9750/2 grant, and by the Academy of Finland/Centre for Technological Advancement (TEKES)/Ministry of Education (...
Normal bone remodeling and pathological bone destruction have been considered to be osteoclast-driven. Osteoclasts are able to attach to bare bone surface and produce an acidic subcellular space. This leads to acid dissolution of hydroxyapatite, allowing cathepsin K to degrade the organic type I collagen-rich osteoid matrix under the acidic condition prevailing in Howship lacunae. Using a sting pH electrode, the interface membrane around a loosened total hip replacement prosthesis was found to be acidic.
In the differentiation of osteoclasts the differentiation factor (RANKL) interacts with the receptor activator of NF-kappaB (RANK) in a direct cell-to-cell contact between osteoblast and (pre)osteoclast. This is inhibited by soluble osteoprotegerin (OPG). The mRNA levels of both RANKL (p < 0.01) and RANK (p < 0.05) were high in peri-implant tissue and RANKL+ and RANK+ cells were found in such tissue. Double labelling also disclosed soluble RANKL bound to RANK+ cells. We were unable to stimulate fibroblasts to express RANKL in vitro, but monocyte activation with LPS gave a fivefold increase in RANK mRNA levels. In contrast to RANKL and RANK expression in peri-implant tissue, expression of OPG was restricted to vascular endothelium. Endothelial cell OPG mRNA levels were regulated by TNF-alpha and VEGF, but not by hypoxia. It is concluded that activated cells in the interface tissue overproduce both RANKL and RANK and they can interact without interference by OPG.
Podosomes and invadopodia are actin-based structures at the ventral cell membrane, which have a role in cell adhesion, migration and invasion. Little is known about the differences and dynamics underlying these structures. We studied podosome-like structures of oral squamous carcinoma cells and invadopodia of their invasive variant that has undergone a spontaneous epithelial-mesenchymal transition (EMT). In 3D imaging, podosomes were relatively large structures that enlarged in time, whereas invadopodia of invasive cells remained small, but were more numerous, degraded more extracellular matrix (ECM) and were morphologically strikingly different from podosomes. In live-cell imaging, highly dynamic, invadopodia-embedded actin tails were frequently released and rocketed through the cytoplasm. Resembling invadopodia, we found new club-ended cell extensions in EMT-experienced cells, which contained actin, cortactin, vinculin and MT1-matrix metalloproteinase. These dynamic cell extensions degraded ECM and, in field emission scanning electron microscopy, protruded from the dorsal cell membrane. Plectin, αII-spectrin, talin and focal adhesion kinase immunoreactivities were detected in podosome rings, whereas they were absent from invadopodia. Tensin potentially replaced talin in invadopodia. Integrin α3β1 surrounded both podosomes and invadopodia, whereas integrin αvβ5 localized only to invadopodia heads. Pacsin 2, in conjunction with filamin A, was detected early in podosomes, whereas pacsin 2 was not found in invadopodia and filamin A showed delayed accumulation. Fluorescence recovery after photobleaching indicated faster reorganization of actin, cortactin and filamin A in podosomes compared to invadopodia. In conclusion, EMT affects the invasion machinery of oral squamous carcinoma cells. Non-invasive squamous carcinoma cells constitutively organize podosomes, whereas invasive cells form invadopodia. The club-ended cell extensions, or externalized invadopodia, are involved in ECM degradation and maintenance of contact to adhesion substrate and surrounding cells during invasion.
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