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 1

Constitutive inactivation of Arp2/3 in disc and cartilage causes severe defects.

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Constitutive inactivation of Arp2/3 in disc and cartilage causes severe ...
(A) Generation of Arpc2-null alleles in collagen II–expressing cells. (B) Frequency of pups that survived to genotyping with an expected Mendelian distribution of 1/4 each. (C) H&E-stained ribcage of mutant at P0 compared with control (CTR). Scale bar: 100 μm. n = 1. (D and E) Images of Arp2/3 mutant mice. P6, left panel; P26, right panel; shown next to littermate controls. Red arrows indicate abnormal curvature of the spine. (F) Coronal sections of discs from a P10 animal stained by Safranin O/Fast Green/Hematoxylin. White outlines define the disc compartment. Boxes indicate higher-magnification images in bottom row, showing magnified end plates and growth plates. Yellow arrowheads show chondrocyte columns; white arrowheads show chondrocyte-like cells. Scale bars: 200 μm in top row, 50 μm in high-magnification view. n = 1; 4 discs. (G) Vertebrae at P10. Scale bar: 200 μm. n = 1; 6 vertebrae. (H) H&E-stained sections of tibia from P26 mouse (n = 1). Yellow arrowheads indicate proliferative columns, black arrowheads indicate disorganization of cells, and pink arrowheads indicate chondrocytes with hypertrophic morphology. Scale bar: 100 μm, 50 μm in higher-magnification image. (I) H&E-stained sections of embryonic tibia at E17.5. Scale bar: 100 μm, 50 μm in higher-magnification image. n = 3 embryos. (J) Ki67 IHC on E17.5 tibia. Scale bar: 100 μm. n = 1. (K) The number of proliferative columns containing 3 or more aligned cells were quantified from embryonic tibia. (L) In the same area, the number of cells in the proliferative zone were counted. (M) The heights of the proliferative zone (PZ) and hypertrophic zone (HZ) as shown in I were quantified. NP, nucleus pulposus; AF, annulus fibrosus; EP, end plate; GP, growth plate; VB, vertebral body. Quantitative measurements represent mean ± SD. Significance was determined using unpaired Student’s t test. ***P ≤ 0.01.