Articular cartilage is the lubricious, load bearing connective tissue at the end of long bones in synovial joints that normally facilitates low-friction and low-wear articulation via a number of lubrication mechanisms. Boundary lubrication is particularly important for the protection and maintenance of the articular surface, which unfortunately often becomes roughened and eroded in aging and arthritis, with the eventual development of pain and dysfunction. Synovial fluid (SF) contains molecules, such as proteoglycan 4 (PRG4) and hyaluronan (HA), that interact with and adsorb to the articular surface, and are therefore ideally positioned to contribute to boundary lubrication. This dissertation aims to contribute to the understanding of PRG4 metabolism by chondrocytes of the superficial zone of cartilage, and the mechanics of, and contributors to, boundary lubrication of articular cartilage. PRG4 metabolism by chondrocyte subpopulations in various culture systems was regulated differentially by various biochemical stimuli. The phenotype of PRG4 secretion by chondrocytes was generally maintained in that PRG4 was expressed to a much greater degree by chondrocytes from the superficial zone than by those from the middle and deep zones. PRG4 expression and secretion by chondrocytes near the articular surface was highly inhibited by IL-1a and stimulated by TGF-B1, in a dose- dependent manner, in explant cultures of cartilage. An in vitro articular cartilage-on-cartilage boundary lubrication test was implemented and extended to establish conditions where a boundary mode of lubrication was dominant. SF functioned as an effective friction-lowering boundary lubricant, even with a 3-fold decrease in constituent concentration. SF constituents, PRG4 and HA, contributed individually and in combination, both at physiological and pathophysiological concentrations, to the boundary lubrication of apposing articular cartilage surfaces. Collectively these results provide insight into the nature of PRG4 metabolism, the biomechanics of boundary lubrication of articular cartilage, and the role of PRG4, alone and in combination with other putative lubricants. The ability to modulate the dynamic regulation of PRG4, whether in homeostasis or degeneration, may ultimately be useful in prolonging the maintenance or slowing the deterioration of articular cartilage's critical mechanical functions at the end of long bones

Purpose

The objectives of this study were to assess the cartilage boundary lubricating ability of (1) nonreduced (NR) disulfide-bonded proteoglycan 4 (PRG4) multimers versus PRG4 monomers and (2) NR versus reduced and alkylated (R/A) PRG4 monomers and to assess (3) the ability of NR PRG4 multimers versus monomers to adsorb to an articular cartilage surface.

Materials and methods

PRG4 was separated into two preparations, PRG4 multimer enriched (PRG4Multi+) and PRG4 multimer deficient (PRG4Multi-), using size exclusion chromatography (SEC) and characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The cartilage boundary lubricating ability of PRG4Multi+ and PRG4Multi- was compared at a physiological concentration (450 μg/mL) and assessed over a range of concentrations (45, 150, and 450 μg/mL). R/A and NR PRG4Multi- were evaluated at 450 μg/mL. Immunohistochemistry with anti-PRG4 antibody 4D6 was performed to visualize the adsorption of PRG4 preparations to the surface of articular cartilage explants.

Results

Separation into enriched populations of PRG4Multi+ and PRG4Multi- was achieved using SEC and was confirmed by SDS-PAGE. PRG4Multi+ and PRG4Multi- both functioned as effective friction-reducing cartilage boundary lubricants at 450 μg/mL, with PRG4Multi+ being more effective than PRG4Multi-. PRG4Multi+ lubricated in a dose-dependent manner, however, PRG4Multi- did not. R/A PRG4Multi- lubricated similar to NR PRG4Multi-. PRG4-containing solutions showed 4D6 immunoreactivity at the articular surface; the immunoreactive intensity of PRG4Multi+ appeared to be similar to SF, whereas PRG4Multi- appeared to have less intensity.

Conclusions

These results demonstrate that the intermolecular disulfide-bonded multimeric structure of PRG4 is important for its ability to adsorb to a cartilage surface and function as a boundary lubricant. These findings contribute to a greater understanding of the molecular basis of cartilage boundary lubrication of PRG4. Elucidating the PRG4 structure-lubrication function relationship will further contribute to the understanding of PRG4's role in diarthrodial joint homeostasis and disease.

Objective

In gout, hyperuricemia promotes urate crystal deposition, which stimulates the NLRP3 inflammasome and interleukin-1β (IL-1β)-mediated arthritis. Incident gout without background hyperuricemia is rarely reported. To identify hyperuricemia-independent mechanisms driving gout incidence and progression, we characterized erosive urate crystalline inflammatory arthritis in a young female patient with normouricemia diagnosed as having sufficient and weighted classification criteria for gout according to the American College of Rheumatology (ACR)/EULAR gout classification criteria (the proband).

Methods

We conducted whole-genome sequencing, quantitative proteomics, whole-blood RNA-sequencing analysis using serum samples from the proband. We used a mouse model of IL-1β-induced knee synovitis to characterize proband candidate genes, biomarkers, and pathogenic mechanisms of gout.

Results

Lubricin level was attenuated in human proband serum and associated with elevated acute-phase reactants and inflammatory whole-blood transcripts and transcriptional pathways. The proband had predicted damaging gene variants of NLRP3 and of inter-α trypsin inhibitor heavy chain 3, an inhibitor of lubricin-degrading cathepsin G. Changes in the proband's serum protein interactome network supported enhanced lubricin degradation, with cathepsin G activity increased relative to its inhibitors, SERPINB6 and thrombospondin 1. Activation of Toll-like receptor 2 (TLR-2) suppressed levels of lubricin mRNA and lubricin release in cultured human synovial fibroblasts (P < 0.01). Lubricin blunted urate crystal precipitation and IL-1β induction of xanthine oxidase and urate in cultured macrophages (P < 0.001). In lubricin-deficient mice, injection of IL-1β in knees increased xanthine oxidase-positive synovial resident M1 macrophages (P < 0.05).

Conclusion

Our findings linked normouricemic erosive gout to attenuated lubricin, with impaired control of cathepsin G activity, compounded by deleterious NLRP3 variants. Lubricin suppressed monosodium urate crystallization and blunted IL-1β-induced increases in xanthine oxidase and urate in macrophages. The collective activities of articular lubricin that could limit incident and erosive gouty arthritis independently of hyperuricemia are subject to disruption by inflammation, activated cathepsin G, and synovial fibroblast TLR-2 signaling.