Arp2/3 inactivation causes intervertebral disc and cartilage degeneration with dysregulated TonEBP-mediated osmoadaptation
Steven Tessier, Alexandra C. Doolittle, Kimheak Sao, Jeremy D. Rotty, James E. Bear, Veronica Ulici, Richard F. Loeser, Irving M. Shapiro, Brian O. Diekman, Makarand V. Risbud
Steven Tessier, Alexandra C. Doolittle, Kimheak Sao, Jeremy D. Rotty, James E. Bear, Veronica Ulici, Richard F. Loeser, Irving M. Shapiro, Brian O. Diekman, Makarand V. Risbud
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Research Article Bone biology Cell biology

Arp2/3 inactivation causes intervertebral disc and cartilage degeneration with dysregulated TonEBP-mediated osmoadaptation

Abstract

Extracellular matrix and osmolarity influence the development and homeostasis of skeletal tissues through Rho GTPase–mediated alteration of the actin cytoskeleton. This study investigated whether the actin-branching Arp2/3 complex, a downstream effector of the Rho GTPases Cdc42 and Rac1, plays a critical role in maintaining the health of matrix-rich and osmotically loaded intervertebral discs and cartilage. Mice with constitutive intervertebral disc– and cartilage-specific deletion of the critical Arp2/3 subunit Arpc2 (Col2-Cre; Arpc2fl/fl) developed chondrodysplasia and spinal defects. Since these mice did not survive to adulthood, we generated mice with inducible Arpc2 deletion in disc and cartilage (Acan-CreERT2; Arpc2fl/fl). Inactivation of Arp2/3 at skeletal maturity resulted in growth plate closure, loss of proteoglycan content in articular cartilage, and degenerative changes in the intervertebral disc at 1 year of age. Chondrocytes with Arpc2 deletion showed compromised cell spreading on both collagen and fibronectin. Pharmacological inhibition of Cdc42 and Arp2/3 prevented the osmoadaptive transcription factor TonEBP/NFAT5 from recruiting cofactors in response to a hyperosmolarity challenge. Together, these findings suggest that Arp2/3 plays a critical role in cartilaginous tissues through the regulation of cell–extracellular matrix interactions and modulation of TonEBP-mediated osmoadaptation.

Authors

Steven Tessier, Alexandra C. Doolittle, Kimheak Sao, Jeremy D. Rotty, James E. Bear, Veronica Ulici, Richard F. Loeser, Irving M. Shapiro, Brian O. Diekman, Makarand V. Risbud

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Figure 3

Postnatal inactivation of Arp2/3 causes growth plate closure and articular cartilage defects.

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Postnatal inactivation of Arp2/3 causes growth plate closure and articul...
(A) Acan-CreERT2 mice were injected with tamoxifen at 4 months and evaluated at 6 and 12 months of age. (B) H&E-stained tibia sections of mice at 6 months and 1 year. White outline defines the growth plate (GP) in control animals; white arrows show reminiscent growth plate in mutant mice. Scale bar: 100 μm. n ≥ 6. (C) Growth plate area was quantified by tracing in ImageJ. (D) Safranin O/Fast Green staining of the articular cartilage in 1-year-old mice after tamoxifen-induced loss of Arp2/3 at 4 months as compared with controls. Scale bar: 50 μm. n ≥ 6. (E and F) Stained slides were graded for proteoglycan loss (E) and articular cartilage structure (OARSI) (F). In both measures, scores are summed from each of 4 cartilage surfaces graded on a 12-point scale, where higher scores indicate worse OA. (G and I) High-magnification images of growth plate (G) and articular chondrocytes (I) isolated at P19 after induction of Arp2/3 loss at P12. Scale bar: 25 μm. (H and J) Spread cell area of growth plate (H) and articular chondrocytes (J) were quantified to determine the extent of interaction with the underlying collagen I (COL1) or fibronectin (FN). Quantitative measurements represent mean cell area ± SD; n ≥ 33 cells/group. Significance for growth plate areas and articular cartilage grades was determined by 1-way ANOVA and Kruskal-Wallis, respectively. Significance between cell spreading was determined using Mann-Whitney U test; *P ≤ 0.05; ***P ≤ 0.001; ****P ≤ 0.0001.