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 6

Arp2/3 and Cdc42 control TonEBP activity.

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Arp2/3 and Cdc42 control TonEBP activity. (A–D) Quantitative immunohisto...
(A–D) Quantitative immunohistostaining of TonEBP (A and B) and TauT (C and D) in NP cells of 1-year-old lumbar discs. Scale bar: 100 μm. n = 10 discs; 5 mice. (E and F) Western blot and densitometric analysis showing reduced TonEBP induction with hypertonic stimulation (n = 6 independent experiments). (G) Depiction of the binary TonTAD-GAL4 system used to measure TonTAD activity. (H) NaCl-induced activation of TonTAD with inhibition of Arp2/3 (CK666), Cdc42 (ML-141), and Rac1-GEF (NSC23766) in primary NP cells. (I–K) Luciferase assays measuring TauT (I), Hsp70 (J), and AR (K) promoter activities with NaCl treatment and Arp2/3 or Cdc42 inhibition (n = 3, 3 replicates/experiment). (L and M) Luciferase assay measuring TauT (L) and Hsp70 (M) mutant promoter activity with NaCl treatment and Arp2/3 or Cdc42 inhibition (n = 3, 3 replicates/experiment). (N–P) Quantitative PCR measuring mRNA levels of TauT (N), SMIT (O), and AR (P) from primary NP cells treated with NaCl and Arp2/3 or Cdc42 inhibitors (n ≥ 5 independent experiments). (Q) Mechanistic model depicting Arp2/3-mediated regulation of TonEBP. Quantitative measurements represent mean ± SD. Significance was determined using 1-way ANOVA or Kruskal-Wallis, when making multiple comparisons between groups, or unpaired Student’s t test, when making comparisons between only 2 groups. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001.