TRPV4-mediated mechanotransduction regulates the metabolic response of chondrocytes to dynamic loading

Abstract

Mechanical loading of joints plays a critical role in maintaining the health and function of articular cartilage. The mechanism(s) of chondrocyte mechanotransduction are not fully understood, but could provide important insights into new physical or pharmacologic therapies for joint diseases. Transient receptor potential vanilloid 4 (TRPV4), a Ca(2+)-permeable osmomechano-TRP channel, is highly expressed in articular chondrocytes, and loss of TRPV4 function is associated with joint arthropathy and osteoarthritis. The goal of this study was to examine the hypothesis that TRPV4 transduces dynamic compressive loading in articular chondrocytes. We first confirmed the presence of physically induced, TRPV4-dependent intracellular Ca(2+) signaling in agarose-embedded chondrocytes, and then used this model system to study the role of TRPV4 in regulating the response of chondrocytes to dynamic compression. Inhibition of TRPV4 during dynamic loading prevented acute, mechanically mediated regulation of proanabolic and anticatabolic genes, and furthermore, blocked the loading-induced enhancement of matrix accumulation and mechanical properties. Furthermore, chemical activation of TRPV4 by the agonist GSK1016790A in the absence of mechanical loading similarly enhanced anabolic and suppressed catabolic gene expression, and potently increased matrix biosynthesis and construct mechanical properties. These findings support the hypothesis that TRPV4-mediated Ca(2+) signaling plays a central role in the transduction of mechanical signals to support cartilage extracellular matrix maintenance and joint health. Moreover, these insights raise the possibility of therapeutically targeting TRPV4-mediated mechanotransduction for the treatment of diseases such as osteoarthritis, as well as to enhance matrix formation and functional properties of tissue-engineered cartilage as an alternative to bioreactor-based mechanical stimulation.

Keywords: TGF-beta; calcium signaling; ion channel; mechanobiology; tissue engineering.

Fig. 1.

TRPV4 function in agarose-embedded chondrocytes. (A) Positive labeling of TRPV4 in agarose-embedded chondrocytes (i) and no labeling in the no primary control (ii); scale bar, 10 μm. (B) Confocal images of a chondrocyte signaling in response to GSK101 (arrow), indicated by the increased ratio of green:red fluorescence post-GSK101 incubation; scale bar, 15 μm. (C) Representative Ca²⁺ traces for conditions that demonstrate significant [Ca²⁺]_i signaling compared with the isoosmotic control. (D) Percentage of cells responding to the below osmotic and chemical conditions. Data not sharing a common superscript letter indicate a significant difference (P < 0.05). Bars do not have error bars because the percent responding metric does not have an error associated with it. Numbers inside the bars are the total of number of cells in each group. (E) Average number of peaks elicited by a signaling cell during the imaging period. *P < 0.05 vs. other groups, n = 5–6, mean ± SEM.

Fig. 2.

TRPV4-dependent gene expression in 2-wk precultured chondrocyte-laden agarose constructs 24 and 72 h after mechanical loading. No change in (A) ACAN or (E) SOX9 gene expression was observed. Mechanical loading enhanced TGF-β3 (F) and suppressed ADAMTS5 gene expression (D) in a TRPV4-dependent manner. Inhibition of TRPV4 during mechanical loading also enhanced NOS2 (C) and suppressed COL2α1 (B) gene expression. *greater than all other groups (P < 0.05); ^#smaller than all other groups (P < 0.05); **less than control and GSK205, greater than Loaded+GSK205 (P < 0.05), n = 4–6, mean ± SEM.

Fig. 3.

TRPV4 antagonist GSK205 blocks the biosynthetic response of chondrocyte-laden constructs to dynamic mechanical loading. (A) Wet weight, (B) DNA content, (C) total s-GAG, (D) total collagen, (E) Young’s modulus, and (F) dynamic modulus of constructs following 4 wk of mechanical loading. *greater than all other groups (P < 0.05); **greater than GSK205 (P < 0.05); ***smaller than all other groups (P < 0.05), n = 3–4, mean ± SEM.

Fig. 4.

TRPV4-dependent gene expression in 2-wk precultured chondrocyte-laden agarose constructs 24 and 72 h following TRPV4 agonist GSK101 treatment. GSK101 enhanced (B) COL2α1 and (F) TGF-β3 and decreased (C) NOS2 (D) and ADAMTS5 gene expression, with no effect on (A) ACAN and (E) SOX9 expression. *greater than control (P < 0.05), n = 4–6, mean ± SEM.

Fig. 5.

TRPV4 activation via daily osmotic loading or GSK101 enhances extracellular matrix accumulation. (A) Wet weight, (B) DNA content, (C) total s-GAG, (D) s-GAG/DNA, (E) total collagen, (F) total collagen–DNA, (G) Young’s modulus, and (H) dynamic modulus of constructs following 2 and 4 wk of stimulation. (I) Gross morphology, Safranin-O, and collagen type II immunostaining after 4 wk of culture; scale bar, 2 mm. Data not sharing a common superscript letter indicate a significant difference (P < 0.05), n = 6–8, mean ± SEM.