Metabolic Labeling Reveals a Critical Role for Decorin in the Assembly and Turnover of Cartilage Matrix

Introduction: The development of effective cartilage regeneration is challenged by our incomplete understanding of molecular activities that regulate the assembly, turnover and degradation of its extracellular matrix (ECM) [1], a hydrated composite of collagen II/IX/XI fibrils and aggrecan macromolecules [2]. Our recent study identified a critical role for decorin, a small leucine-rich proteoglycan (SLRP), in regulating cartilage ECM integrity in both health and osteoarthritis (OA). In decorin-null (Dcn‒/‒) mice, articular cartilage exhibits reduced aggrecan levels in both the pericellular matrix (PCM) and territorial/interterritorial (T/IT)-ECM, as well as perturbed chondrocyte mechanotransduction [3]. This role was attributed to a novel “physical linkage” function of decorin, acting to enhance aggrecan-aggrecan and aggrecan-collagen fibril molecular adhesion [4]. In cartilage ECM, aggrecan undergoes active turnover [5], with a higher turnover rate in the PCM [6]. Given the role of decorin in aggrecan retention, we investigated how decorin mediates the turnover of newly synthesized proteins and glycosaminoglycans (GAGs) using a bio-orthogonal click-labeling method [7] and elucidated how decorin regulates the collagen-aggrecan integration by AFM-nanoindentation on cartilage following enzymatic GAG removal and re-infiltration of exogenous decorin.

Materials and

Methods: Femoral head cartilage explants were harvested from 3-week-old Dcn‒/‒ and wild-type (WT) C57BL/6 mice. Following a 2-day pre-culture, explants were cultured for 3 days with either 30 mM N-azidoacetylgalactosamine-tetraacylated (GAL) (1086, ClickChemistryTools) or L-azidohomoalanine (AHA) (1066, ClickChemistryTools) in chondrogenic DMEM [8] to metabolically label nascent GAGs and proteins [7], respectively. Then, explants were tagged with 30 mM AZDye 488 DBCO (1278, ClickChemistryTools) and cultured for 6 days in chondrogenic DMEM, with or without IL-1β (10 ng/mL) to elicit catabolism [9]. Nascent GAL-labeled GAGs and AHA-labeled proteins released into the supernatant were collected every other day for quantification. On day 6, tissues were digested with 2% papain to quantify the nascent protein/GAG retained in the explants. Confocal images (Zeiss LSM700) were taken to assess the spatial distribution of nascent proteins and GAGs. Following our established procedure [4], AFM-nanoindentation was applied to quantify the modulus, Eind, of 3-week-old Dcn‒/‒ and WT femoral condyle cartilage using a microspherical tip (R ≈ 5 μm, k ≈ 5.4 N/m) in PBS. We tested untreated cartilage, cartilage depleted of sulfated GAGs by 0.5 U/ml chondroitinase ABC and 500 U/ml hyaluronidase digestion for 24 hrs each, and GAG-removed cartilage reinfiltrated with exogenous decorin (D8428, Sigma, 20 μg/mL for 24 hours). Repeated measure two-way ANOVA was applied to study the effects of genotype and IL-1β on GAG/protein release, and two-way ANOVA was applied to study the effects of genotype and treatment on Eind, followed by Tukey-Kramer multiple comparison at α = 0.05.

Results, Conclusions, and Discussions: This study highlights a crucial role for decorin in regulating the assembly of newly synthesized aggrecan in normal and degenerative cartilage. Fluorescence images showed clear differences in the distributions of nascent GAGs versus proteins (Fig. 1a,b). Specifically, we observed a higher concentration of nascent GAGs surrounding the cells with or without IL-1β stimulation, indicating their preferred localization in the PCM. As most GAGs constitute the side chains of aggrecan [10], the accelerated release of nascent GAGs (Fig. 2a) and their reduced fluorescent signal (Fig. 1a) from Dcn‒/‒ cartilage supports the role of decorin in increasing the retention of newly synthesized aggrecan (Fig. 1a). In contrast, nascent proteins were distributed nearly homogeneously throughout the intercellular space and loss of decorin did not impact their release (Fig. 2b).

We also demonstrate that decorin increases the retention of aggrecan through strengthening the collagen network. Under AFM-nanoindentation, sGAG removal significantly reduced tissue modulus, which was partially restored by decorin infiltration for WT cartilage. This suggests that decorin strengthens the collagen fibrillar network, likely by providing physical linkages through its hydrophobic core protein to connect adjacent collagen II fibrils. This decorin-strengthened collagen network thus provides higher resistance to limit the diffusive loss of aggrecan entrapped therein, which is driven by the osmotic pressure from the fixed negative charges on sGAGs [11]. In contrast, for Dcn‒/‒ cartilage, the modulus of untreated tissue was lower than that of WT, while neither sGAG removal nor decorin infiltration had a significant impact (Fig. 3). This absence of exogenous decorin rescue effect on Dcn‒/‒ cartilage is possibly due to the altered collagen fibril structure resulted from decorin loss, i.e., higher fibril heterogeneity [4], which renders exogenous decorin unable to effectively establish physical linkages with this altered network.

Our results highlight a critical role for decorin in increasing the retention of newly synthesized aggrecan in both normal and degenerative cartilage. One mechanism is through its strengthening effect of the collagen fibril network. Results thus provide the path for developing decorin-based strategies for improving cartilage regeneration and OA intervention.

Acknowledgements (Optional): This work was supported by NIH R01AR074490.

References: [1] Huey+ 2012. [2] Han+ 2011. [3] Chery+ 2021. [4] Han+ 2019. [5] Lohmander+ 1999. [6] Quinn+ 1999. [7] Porter+ 2022. [8] Loebel+ 2020. [9] Li+ 2020. [10] Poole+ 1996. [11] Warren+ 2022.