Type VI Collagen Regulates Pericellular Matrix Properties, Chondrocyte Swelling, and Mechanotransduction in Mouse Articular Cartilage

Abstract

Objective: Mechanical factors play a critical role in the physiology and pathology of articular cartilage, although the mechanisms of mechanical signal transduction are not fully understood. We undertook this study to test the hypothesis that type VI collagen is necessary for mechanotransduction in articular cartilage by determining the effects of type VI collagen knockout on the activation of the mechano-osmosensitive, calcium-permeable channel TRPV4 (transient receptor potential vanilloid channel 4) as well as on osmotically induced chondrocyte swelling and pericellular matrix (PCM) mechanical properties.

Methods: Confocal laser scanning microscopy was used to image TRPV4-mediated calcium signaling and osmotically induced cell swelling in intact femora from 2- and 9-month-old wild-type (WT) and type VI collagen-deficient (Col6a1(-/-)) mice. Immunofluorescence-guided atomic force microscopy was used to map PCM mechanical properties based on the presence of perlecan.

Results: Hypo-osmotic stress-induced TRPV4-mediated calcium signaling was increased in Col6a1(-/-) mice relative to WT controls at 2 months. Col6a1(-/-) mice exhibited significantly increased osmotically induced cell swelling and decreased PCM moduli relative to WT controls at both ages.

Conclusion: In contrast to our original hypothesis, type VI collagen was not required for TRPV4-mediated Ca(2+) signaling; however, knockout of type VI collagen altered the mechanical properties of the PCM, which in turn increased the extent of cell swelling and osmotically induced TRPV4 signaling in an age-dependent manner. These findings emphasize the role of the PCM as a transducer of mechanical and physicochemical signals, and they suggest that alterations in PCM properties, as may occur with aging or osteoarthritis, can influence mechanotransduction via TRPV4 or other ion channels.

Figure 1. Schematic of in situ Ca²⁺ imaging configuration

A. Experimental system for fluorescence imaging of Ca²⁺ in chondrocytes within intact murine femoral condyle on a confocal laser scanning microscope. Media was exchanged with a peristaltic pump while fluorescence ratio imaging was performed. B. Representative frame of fluorescence imaging with Fura Red (red) and Fluo-4 (green). C. Representative fluorescence recording of single signaling cell over time. Red and green lines show Fura Red and Fluo-4 intensity (respectively), and black shows ratio of green to red. Stars mark significant increases in intracellular Ca²⁺ concentration.

Figure 2. Immunohistochemistry for type VI collagen and perlecan in mouse cartilage

A. Immunohistochemistry shows the localized pericellular presence of type VI collagen in WT but not Col6a1^−/− mice at both 2 and 9 month ages. B. Immunolabeling shows clear presence of perlecan in the pericellular region of WT as well as Col6a1^−/− mice at both 2 and 9 month time points.

Figure 3. Osmotically-induced Ca²⁺ signaling in chondrocytes in situ

The percentage of cells responding with a Ca²⁺ signal was significantly higher in response to −100mOsm change in osmolarity than to iso-osmotic control, and this hypo-osmotic signaling was significantly greater in Col6a1^−/− mice than in WT for 2 month old mice. All signaling was significantly inhibited with the TRPV4 antagonist GSK205, indicating that the response was mediated by TRPV4. *: Significantly greater than iso-osmotic control, #: Significantly less than −100mOsm treatment, %: Significant difference between WT and Col6a1^−/−, & Significantly greater than 9 month group. All analyses performed with a chi-squared test (p<0.05).

Figure 4. In situ measurements of chondrocyte volume and osmotically-induced swelling

A. Representative images of one z-stack slice for volume reconstructions obtained with Calcein-AM intracellular label before (300mOsm) and after hypotonic fluid exchange (200mOsm). B. Rendering of 3-D reconstruction of a representative cell before (left) and after (right) hypotonic fluid exchange. C. Percent increase in chondrocyte volume in intact femoral condyle in response to hypotonic media change. WT chondrocytes showed a significantly smaller volume increase with hypo-osmotic stress than Col6a1^−/− chondrocytes, regardless of age (2-way ANOVA, genotype p<0.0085, age p=0.12, interaction p=0.93). Bars show mean±SEM.

Figure 5. Immunofluorescence-guided AFM for mapping ECM and PCM moduli

A. Sample AFM force maps show matrix stiffness around cells in cartilage from 2 month and 9 month old mice. Perlecan labeling was used to define the boundaries of the PCM around each cell. B. ECM elastic moduli were greater in 2 month mice than 9 month mice. There was a trend towards WT having a greater ECM modulus than Col6a1^−/− (2-way ANOVA on log transformed data, age p=0.016, genotype p=0.087, interaction p=0.23). C. PCM moduli of Col6a1^−/− mice were significantly lower than those of WT mice regardless of age (2-way ANOVA on log transformed data, genotype p=0.0013, age p=0.67, interaction p=0.21). Bars show mean±SEM.