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

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.

TonEBP-deficiency accelerates intervertebral disc degeneration underscored by matrix remodeling, cytoskeletal rearrangements, and changes in proinflammatory gene expression.

The tonicity-responsive enhancer binding protein (TonEBP) plays an important role in intervertebral disc and axial skeleton embryogenesis. However, the contribution of this osmoregulatory transcription factor in postnatal intervertebral disc homeostasis is not known in vivo. Here, we show for the first time that TonEBP-deficient mice have pronounced age-related degeneration of the intervertebral disc with annular and endplate herniations. Using FTIR-imaging spectroscopy, quantitative immunohistochemistry, and tissue-specific transcriptomic analysis, we provide morphological and molecular evidence that the overall phenotype is driven by a replacement of water-binding proteoglycans with fibrocartilaginous matrix. Whereas TonEBP deficiency in the AF compartment caused tissue fibrosis associated with alterations in actin cytoskeleton and adhesion molecules, predominant changes in pro-inflammatory pathways were seen in the NP compartment of mutants, underscoring disc compartment-specific effects. Additionally, TonEBP-deficient mice presented with compromised trabecular bone properties of vertebrae. These results provide the first in vivo support to the long-held hypothesis that TonEBP is crucial for postnatal homeostasis of the spine and controls a multitude of functions in addition to cellular osmoadaptation.

COX-2 expression mediated by calcium-TonEBP signaling axis under hyperosmotic conditions serves osmoprotective function in nucleus pulposus cells.

The nucleus pulposus (NP) of intervertebral discs experiences dynamic changes in tissue osmolarity because of diurnal loading of the spine. TonEBP/NFAT5 is a transcription factor that is critical in osmoregulation as well as survival of NP cells in the hyperosmotic milieu. The goal of this study was to investigate whether cyclooxygenase-2 (COX-2) expression is osmoresponsive and dependent on TonEBP, and whether it serves an osmoprotective role. NP cells up-regulated COX-2 expression in hyperosmotic media. The induction of COX-2 depended on elevation of intracellular calcium levels and p38 MAPK pathway, but independent of calcineurin signaling as well as MEK/ERK and JNK pathways. Under hyperosmotic conditions, both COX-2 mRNA stability and its proximal promoter activity were increased. The proximal COX-2 promoter (-1840/+123 bp) contained predicted binding sites for TonEBP, AP-1, NF-κB, and C/EBP-ß. While COX-2 promoter activity was positively regulated by both AP-1 and NF-κB, AP-1 had no effect and NF-κB negatively regulated COX-2 protein levels under hyperosmotic conditions. On the other hand, TonEBP was necessary for both COX-2 promoter activity and protein up-regulation in response to hyperosmotic stimuli. Ex vivo disc organ culture studies using hypomorphic TonEBP+/- mice confirmed that TonEBP is required for hyperosmotic induction of COX-2. Importantly, the inhibition of COX-2 activity under hyperosmotic conditions resulted in decreased cell viability, suggesting that COX-2 plays a cytoprotective and homeostatic role in NP cells for their adaptation to dynamically loaded hyperosmotic niches.

TNF-α promotes nuclear enrichment of the transcription factor TonEBP/NFAT5 to selectively control inflammatory but not osmoregulatory responses in nucleus pulposus cells.

Intervertebral disc degeneration (IDD) causes chronic back pain and is linked to production of proinflammatory molecules by nucleus pulposus (NP) and other disc cells. Activation of tonicity-responsive enhancer-binding protein (TonEBP)/NFAT5 by non-osmotic stimuli, including proinflammatory molecules, occurs in cells involved in immune response. However, whether inflammatory stimuli activate TonEBP in NP cells and whether TonEBP controls inflammation during IDD is unknown. We show that TNF-α, but not IL-1ß or LPS, promoted nuclear enrichment of TonEBP protein. However, TNF-α-mediated activation of TonEBP did not cause induction of osmoregulatory genes. RNA sequencing showed that 8.5% of TNF-α transcriptional responses were TonEBP-dependent and identified genes regulated by both TNF-α and TonEBP. These genes were over-enriched in pathways and diseases related to inflammatory response and inhibition of matrix metalloproteases. Based on RNA-sequencing results, we further investigated regulation of novel TonEBP targets CXCL1, CXCL2, and CXCL3 TonEBP acted synergistically with TNF-α and LPS to induce CXCL1-proximal promoter activity. Interestingly, this regulation required a highly conserved NF-κB-binding site but not a predicted TonE, suggesting cross-talk between these two members of the Rel family. Finally, analysis of human NP tissue showed that TonEBP expression correlated with canonical osmoregulatory targets TauT/SLC6A6, SMIT/SLC5A3, and AR/AKR1B1, supporting in vitro findings that the inflammatory milieu during IDD does not interfere with TonEBP osmoregulation. In summary, whereas TonEBP participates in the proinflammatory response to TNF-α, therapeutic strategies targeting this transcription factor for treatment of disc disease must spare osmoprotective, prosurvival, and matrix homeostatic activities.