To our knowledge this is the first report describing the immunolocalisation and expression of PRG4 in cartilage in an animal model of OA. Similarly to previous reports of PRG4 immunolocalisation in normal cartilage [5
], in the present study we observed PRG4-positive cells typically in the superficial zone and not in the middle and deep zones. The lack of significant PRG4 staining in the superficial cartilage matrix in comparison with previous studies [13
] may be related to tissue processing. The tibial plateaux cartilage underwent biomechanical testing before processing for histology. Physical removal of the surface PRG4 might have occurred during the 15-minute indentation testing, which requires repeated saline lavage and swabbing of the cartilage surface. After ovine lateral meniscectomy, there was a decrease in PRG4 immunostaining with a marked loss of PRG4-positive superficial zone chondrocytes in the degenerative cartilage of the lateral compartment. Importantly, this was not associated with an appearance of PRG4-positive cells in the middle or deep zones of cartilage. Previous studies performed in late-stage human OA cartilage collected at joint replacement surgery have reported the extension of PRG4-positive chondrocytes into the deeper zones, suggesting potential adaptive responses with disease progression [7
] that were not apparent in the early stages of OA pathogenesis represented in the present study.
After ovine lateral meniscectomy, the most marked decrease in PRG4 expression was observed in the lateral compartment with the most severe histopathological alterations. However, mRNA levels were decreased in cartilage across the knee joint after meniscectomy regardless of associated degenerative changes. Darling and colleagues [16
] recently demonstrated a threefold relative abundance of PRG4 mRNA levels in the superficial zone of normal articular cartilage. Decreases in the presence and/or viability of the superficial zone cells occurring early in the present model of OA therefore probably contributed to the observed decrease in cartilage PRG4 expression. Additionally, modulation of the chondrocyte phenotype in meniscectomised cartilage by mechanical or humoral factors were likely to have been associated with the downregulation in PRG4 expression observed in the compartments not undergoing active degeneration.
Topographical variation in PRG4 expression was observed in normal ovine knee joints in the present study, with increased expression in cartilage from regions protected by a meniscus, which was consistent with the immunolocalisation of PRG4 protein. Although a recent report [16
] found no variation in PRG4 expression across distal femoral cartilage, we have previously demonstrated that topographical differences in chondrocyte metabolism were most pronounced in the tibial plateau [17
]. These differences in cartilage metabolism in the tibial plateau were probably associated with the presence of the meniscus and its effect on mechanical loading of the cartilage. In the present study we postulate that not only variation in compressive mechanical loads but also potential shearing between the meniscus and underlying cartilage may modulate PRG4 expression in this region. Wong and colleagues [18
] have previously demonstrated in chondrocyte-seeded alginate constructs that cyclic shear loading significantly upregulated PRG4 expression, whereas cyclic hydrostatic pressure was associated with a slight downregulation. Alterations in shear stress after lateral meniscectomy might therefore have contributed to the decreased cartilage PRG4 expression observed in the present study.
The mechanisms involved in regulating PRG4 expression and synthesis remain largely unknown. Increased catabolic pathways are present in OA, and the inflammatory cytokine IL-1 seems to be one of the most influential factors, demonstrating deleterious effects for cartilage in vitro
and in vivo
, acting to inhibit proteoglycan synthesis while promoting degradation of matrix components through both activation of proteases and stimulation of their production [19
]. PRG4 seems to be highly regulated by IL-1, which has been shown to inhibit its secretion in vitro
, and therefore potentially contributing to the pathogenesis of OA [2
]. Conversely, it has shown that transforming growth factor-β (TGF-β) stimulates PRG4 synthesis and may be beneficial for normal cartilage function [2
]. TGF-β has a significant role in promoting the anabolic activity of chondrocytes, and its expression has been reported to increase in early OA [25