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

Journal Pre-proof TonEBP-deficiency accelerates intervertebral disc degeneration underscored by matrix remodeling, cytoskeletal rearrangements, and changes in proinflammatory gene expression Steven Tessier, Victoria A. Tran, Olivia K. Ottone, Emanuel J. Novais, Alexandra Doolittle, Michael J. DiMuzio, Irving M. Shapiro, Makarand V. Risbud PII:

S0945-053X(19)30387-7

DOI:

https://doi.org/10.1016/j.matbio.2019.10.007

Reference:

MATBIO 1602

To appear in:

Matrix Biology

Received Date: 1 August 2019 Revised Date:

15 October 2019

Accepted Date: 30 October 2019

Please cite this article as: S. Tessier, V.A. Tran, O.K. Ottone, E.J. Novais, A. Doolittle, M.J. DiMuzio, I.M. Shapiro, M.V. Risbud, TonEBP-deficiency accelerates intervertebral disc degeneration underscored by matrix remodeling, cytoskeletal rearrangements, and changes in proinflammatory gene expression, Matrix Biology, https://doi.org/10.1016/j.matbio.2019.10.007. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Elsevier B.V. All rights reserved.

TonEBP-deficiency accelerates intervertebral disc degeneration underscored by matrix remodeling, cytoskeletal rearrangements, and changes in proinflammatory gene expression Steven Tessier1, Victoria A. Tran1, Olivia K. Ottone1,2, Emanuel J. Novais1,2,3,4, Alexandra Doolittle1,2, Michael J. DiMuzio1, Irving M. Shapiro1,2, Makarand V. Risbud1,2* 1

Department of Orthopaedic Surgery, Sidney Kimmel Medical College, Thomas Jefferson University, Philadelphia, PA, USA

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Graduate Program in Cell Biology and Regenerative Medicine, Jefferson College of Life Sciences, Thomas Jefferson University, Philadelphia, PA, USA

Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, Braga, Portugal 4

ICVS/3B's–PT Government Associate Laboratory, Braga, Portugal

*Corresponding author: *Makarand V. Risbud, Ph.D Department of Orthopaedic Surgery Thomas Jefferson University 1025 Walnut Street, Suite 501 College Bldg. Philadelphia PA 19107 [email protected] Fax. (215) 955-9159 Running Title: TonEBP deficiency causes intervertebral disc degeneration Disclosure Statement: Declarations of interest: None

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ABSTRACT 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 FTIRimaging 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 osmoadpatation. Keywords: Matrix remodeling, Intervertebral disc, TonEBP, Actin cytoskeleton, Inflammation Acknowledgements: We would like to thank Vedavathi Madhu for performing PCR analyses and Zariel Johnson for her contributions to the early phases of this work. Emanuel J. Novais received a PhD fellowship (PD/BD/128077/2016) from the MD/PhD Program of the University of Minho funded by the Fundação para a Ciência e a Tecnologia (FCT) during this work. Funding: This work was supported by the National Institutes of Health under grant numbers R01AR064733, R01AR055655, R01AR074813, T32AR052273.

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INTRODUCTION The intervertebral disc is a polyaxial diarthrodial joint built of three major tissues: the nucleus pulposus (NP), annulus fibrosus (AF), and hyaline cartilaginous endplates (CEPs). The NP lies at the disc’s innermost region and is characterized by its high proteoglycan to collagen content [1,2]. As glycosaminoglycan (GAG) substituents of proteoglycans are sulfated and thus negatively charged, electron neutrality within the NP compartment is achieved by an influx of positively charged ions that generate high osmotic pressure and attract water molecules into the tissue [2]. Resistance to this swelling force is provided by the circumferential AF comprised of tensile-bearing, concentric and collagenous lamellae, with interlamellar septae containing proteoglycan aggregates. Lastly, the disc is capped inferiorly and superiorly by CEPs that allow for diffusion and extrusion of water, ions, and nutrients supplied by adjacent capillary beds [3]. Altogether, these compartmental tissues coupled with osmotic pressure promote spinal stability, absorb and transmit mechanical loading, and permit the flexion-extension, rotation, and lateral bending of the vertebral column [4]. Since the matrix integrity of the disc is the primary determinant of its biomechanical function, its composition and homeostasis is tightly regulated during its maturation and aging [5–7]. While hydrophilic proteoglycan content provides the disc with the osmotic pressure to withstand mechanical forces, it also challenges resident cells with a hyperosmotic niche that fluctuates with diurnal changes in posture [8–10]. The molecular mechanism by which NP cells survive under these hyperosmotic conditions was first elucidated by Tsai et al. over a decade ago. Upon treatment with a hyperosmotic challenge, NP cells employ the osmoadaptive transcription factor, tonicity-responsive enhancer binding protein (TonEBP aka NFAT5 or OREBP), to drive the expression of genes that encode proteins and solute carriers involved in catalyzing the cytosolic accumulation of non-ionic, organic osmolytes [11]. This accumulation adjusts the osmotic balance across the cell membrane, therefore preventing deleterious changes in cell volume and harmful damage from long-term exposure to high intracellular solute concentrations [12–14]. TonEBP also stimulates transcription of heat shock proteins [15–17] and mediates expression of matrix-related genes, including aggrecan, β1, 3 glucuronosyltransferase, and collagen 2, thereby regulating the hyperosmotic niche itself [10,11,18,19]. Extracellular stimuli such as cations, integrin activation, and cytokines influence the induction of TonEBP expression and activity [20,21]. Coupling of integrin-mediated adhesion to the induction of TonEBP has been demonstrated in a carcinoma cell line wherein TonEBP was activated by α1β6 clustering upon stimulation with chemo-attractants [22]. Furthermore, impeded accumulation of intracellular osmolytes and impaired TonEBP induction by a hypertonic challenge has been shown in integrin α1-null mice and medullary collecting duct cells, respectively [23]. This mode of TonEBP regulation and our recent observation that β-actin levels are elevated in TonEBP-null NP cells during embryogenesis suggests that TonEBP may be involved in the tightly connected interactions of the actin cytoskeleton and extracellular matrix [24]. In addition, a role for TonEBP in regulating the expression of proinflammatory genes including COX2, MCP1, IL-6, IL-1β, and TNFα in cultured NP cells has been well-established [21,25,26]. This link is not surprising as TonEBP shares a Rel-like homology domain with its family members, NFAT1-4 and RelA/p65, which collectively play critical roles in immune cell function and the defense response [27]. Despite the abundance of clear evidence that the

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functions of TonEBP in osmoadaptation, matrix homeostasis, and inflammation are crucial to NP cell biology, the contribution of TonEBP in postnatal disc health has never been investigated in vivo, in part due to the limitation that TonEBP knockout mice evidence high embryonic lethality [28,29]. Herein, we assessed the role of TonEBP in age-dependent intervertebral disc degeneration by employing haploinsufficient mice that exhibit significant loss of TonEBP activity without compromising survival into adulthood [30]. Our studies provide strong evidence that diminished TonEBP activity causes pronounced degeneration of all three intervertebral disc compartments characterized by an increased incidence of herniation and matrix remodeling. The results presented in this study provide for the first time empirical support to the long-held hypothesis that TonEBP is essential for postnatal disc homeostasis in vivo. RESULTS TonEBP deficiency accelerates age-dependent disc degeneration of all disc compartments In the present study, we observed that TonEBP levels were abundant in NP cells of 12month-old mice and, interestingly, were reduced by 22 months (Suppl. Fig. 1). Cognizant of the important biological functions of TonEBP in disc and other tissues, we hypothesized that TonEBP-deficiency would synergize with aging and accelerate age-related disc degeneration. Mice with heterozygous deletion of exons 6 and 7 of the TonEBP DNA-binding domain (Fig. 1A) were aged to 12 or 22 months to study their spinal phenotype. Previous reports have shown that these mice are haploinsufficient and evidence significant decrease in TonEBP activity compared to wild-type controls [25,30]. Analysis comparing 22-month-old control and TonEBPdeficient mice revealed a marked reduction in taurine transporter (TauT) (Fig. 1B, B’), a wellestablished and known osmo-target of TonEBP, confirming significantly decreased TonEBP activity. Safranin-O/Fast Green/Hematoxylin staining of 12 (Fig. 1C) and 22-month-old (Fig. 1D) mice evidenced advanced disc degeneration of varying severity in comparison to agematched wild-type controls. NP tissue degeneration was characterized by increased matrix fibrosis, rearrangement of cells into a honeycomb-like pattern, and a transition from notochordal to chondrocytic cell morphology (Fig. 1C, D). Discs with severe degeneration showed loss of Safranin-O and increased Fast Green staining in the NP compartment, suggesting fibrosis of the matrix. In addition, a loss of demarcation between the NP and AF compartments was evident with clear disorganization of AF lamellae and presence of chondrocyte-like AF cells (Fig. 1C, D). These degenerative changes at both ages were apparent in average modified Thompson scores of NP and AF tissues showing higher degenerative grades in mutant mice (Fig. 1E, E’). These degenerative changes were also evident when the scores for each spinal level were plotted individually, suggesting that degeneration was widespread (Suppl. Fig. 2). Furthermore, unlike wild-type animals, numerous AF (Fig. 1D-E’) and CEP (Fig. 1F) herniations were observed in TonEBP-deficient mice with higher incidence in the lower lumbar levels (Fig. 1G). Notably, AF herniations only occurred in 22-month-old mutants, suggesting that TonEBP-deficiency indeed synergized with aging to compromise disc health. CEP herniations in mutants coincided with higher endplate cartilage scores that were indicative of cartilage disorganization and excessive proteoglycan content (Fig. 1H, Suppl. Fig. 3). Additionally, the entire subchondral space showed presence of cartilaginous tissue and lacked subchondral bone in a significantly larger percentage

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of mutant discs (Fig. 1F, I). When considering these phenotypes, it is important to note that homozygous loss of TonEBP causes developmental delay of the spine [24]. We therefore determined whether TonEBP hypomorphism also resulted in developmental delay of the spine. However, we observed comparable aspect ratios of caudal NP between wild-type and E18.5 heterozygous embryos (Suppl. Fig 4). Furthermore, previously published work by Johnson et al. has shown that there was no appreciable disc phenotype in TonEBP heterozygous mice at 4 months of age [25]. These findings suggests that the degenerative phenotype observed in aged TonEBP-deficient mice was not due to intervertebral disc developmental abnormalities. Furthermore, it should be noted that while TonEBP-deficiency promotes disc degeneration, TonEBP is not the only regulator that is required for the maintenance of disc health, and compensatory changes in other key proteins and regulators may have played an important role. Nonetheless, these histological studies clearly indicated that TonEBP-deficient mice were prone to accelerated age-dependent disc degeneration. Disc degeneration in TonEBP-deficient mice is characterized by fibrotic changes in extracellular matrix Matrix remodeling events driven by replacement of hydrophilic proteoglycans and noncollagenous proteins with a fibrocollagenous matrix is characteristic of degenerative disc disease [31–33]. As TonEBP-deficient mice showed clear evidence of degeneration, we further assessed the structure and composition of disc extracellular matrix. Total collagen content and collagen fiber orientation were visualized by Picrosirius Red staining followed by polarized light microscopy, which revealed significantly disorganized AF lamellar architecture in mutants (Fig. 2A). Furthermore, quantitative analysis of polarized images showed increased percent area of green pixels with concomitant decreased percent area of yellow pixels, suggesting increased synthesis of thin nascent fibers (green) and turnover of more mature medium-sized fibers (yellow) (Fig. 2A’) [34]. There were no apparent changes in the content of thick collagen fibers between genotypes (red). Consistent with degenerative changes seen by Safranin-O staining, 20% of mutant discs analyzed by Picrosirius Red revealed disorganized fibrillar collagen in the NP compartment (Fig. 2A, A’). We then used FTIR-imaging spectroscopy to obtain further insights into these fibrotic changes. TonEBP-deficient discs were parsed into two groups based on degree of fibrosis (nonfibrotic +/∆ vs fibrotic F+/∆) for assessment. Cluster analysis of total protein and collagen infrared spectra distinguished each disc compartment, with clear increased levels of protein and collagen in F+/∆ discs (Fig. 2B). Second derivative differentiation was applied to individual spectra of AF, NP, and CEPs of wild-type and TonEBP-deficient animals to perform quantitative analysis (Fig. 2C-C”). In accord with our Sarfanin-O and Picrosirius red analyses, the fibrotic mutant discs showed increased total protein (Fig. 2D) and collagen (Fig. 2E) content in both the CEP and NP compartments with little overall change in the AF as signified by mean absorbance of the amide I region (1660 cm-1) and collagen side chain vibration region (1338 cm-1), respectively. However, there were no significant differences between control and nonfibrotic mutants for both total protein (Fig. 2D) and collagen (Fig. 2E) content within all three disc compartments. Taken together, these results provide clear evidence that TonEBP is essential for maintaining composition and quality of matrix in the intervertebral disc. To further delineate the molecular composition and localization of matrix molecules, we performed quantitative immunohistochemistry on TonEBP mutant discs. In line with our

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previous work which has shown that aggrecan is a target of TonEBP [11], the results indicated lower levels of aggrecan as evidenced by decreased staining of the NP and AF compartments (Fig. 3A, A’). There was also decreased levels of aggrecan neoepitope ARGxx in AF indicating lower turnover by ADAMTS4/5 or of lower aggrecan levels (Fig. 3B, B’). Interestingly, however, chondroitin sulfate staining did not mirror decreased aggrecan levels (Fig. 3 C, C’), suggesting that the osmotic pressure required for withstanding spinal forces was maintained by increased substitution of aggrecan, by production of other CS-bearing proteoglycans, or by other functional modifications to hyaluronan properties [35]. Indeed, we observed an increase in levels of CS-rich β domain-containing versican (Fig. 3E, E’). The matrix changes in TonEBP-deficient discs were also associated with decreased levels of fibromodulin in the outer AF (Fig. 3 F, F’), which is a small leucin-rich proteoglycan (SLRP) known to enhance collagen fibril formation [36]. Additionally, while no change was observed in cartilage oligomeric matrix protein (COMP) (Fig. 3 G, G’) or collagen 1 (COLI) (Fig. 3 H, H’), the AF evidenced a significant reduction in collagen 2 (COLII) (Fig. 3I, I’), and the NP showed increased deposition of the hypertrophic marker collagen 10 (COLX). In agreement with changes in these collagen isoforms, TonEBPdeficient discs showed increased levels of matrix metalloproteinase 13 (MMP13), which were proportional to the degree of degeneration (Fig. 3K, K’). Matrix remodeling, actin cytoskeleton rearrangements, and altered expression of proinflammatory genes coincide with fibrotic changes in TonEBP-deficient mice To gain further mechanistic insights into the disc phenotype of TonEBP-deficient mice, we performed microarray analysis on isolated NP and AF tissues from lumbar discs (L1-S1) of 1-year-old mutant and wild-type animals (Fig. 4A). Three-dimensional principle component analysis (PCA) of transcriptomic profiles clearly showed that samples with the same genotype clustered together (Fig. 4B, B’). At p ≤ 0.05, a total of 3,814 and 1,383 genes were differentially expressed in AF and NP cells, respectively. Heatmaps of these differentially expressed genes between TonEBP-deficient and wild-type animals in both AF (Fig. 4D) and NP (Fig. 4E) tissue are shown. Volcano plots were also generated to show the relationship between fold change (2fold threshold) and p-value (–log10) for both AF (Fig. 4D’) and NP (Fig. 4E’) samples. Differentially regulated genes with greater than 2-fold change at p ≤ 0.05 were analyzed for gene ontology using DAVID functional annotation bioinformatics. This analysis identified changes in extracellular matrix, cell adhesion, and the actin cytoskeleton among the most enriched in the AF (Fig. 4F), with broad changes in chromatin dynamics, transcription, and immune-related genes as the most prominent in NP (Fig. 4F’). Comparison of differentially expressed genes with 2-fold change between TonEBP-deficient AF and NP tissues revealed an overlap of 104 genes (6.3%). These common genes constituted pathways regarding glycoproteins, transcriptional regulation, cell adhesion, and the immune response (Fig. 4G, G’). All annotated gene sets and clusters are shown in Suppl. Table 1-3. Interestingly, these analyses also showed that a greater number of genes were differentially expressed in TonEBP-deficient AF tissue than NP tissue at the transcriptomic level. Consistent with our histological and FTIR analyses, a number of downregulated extracellular matrix genes in the AF corresponded to collagen assembly and organization. These genes included ABI family member 3 binding protein (Abi3bp), cartilage intermediate layer protein (Cilp, Cilp2), procollagen c-endopeptidase enhancer 2 (Pcolce2), fibromodulin (Fmod),

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asporin (Aspn), fibronectin 1 (Fn1), tenascin X (Tnxb), and fibulin 1 (Fbln1). Furthermore, the AF showed downregulation of genes that encode crucial proteoglycans such as lubricin (Prg4), aggrecan (Acan), and syndecan 4 (Sdc4) (Fig. 5A). In accordance with these changes, both collagen 1α1 and 1α2 (Col1a1, Col1a2) were upregulated, along with genes expressed in dense connective tissues such as tenacin C (Tnc), bone sialoprotein 2 (Ibsp), and osteoblast specific factor (Postn) (Fig. 5A). Interestingly, the results also showed upregulation of the SLRP lumican (Lum), consistent with previous studies which showed that lumican levels are increased in fibromodulin-deficient mice [37]. Additionally, the analysis showed robust elevation in Mmp13 expression in AF tissue (Fig. 5B), consistent with our immunohistochemistry analysis (Fig. 3K, K’). Additional upregulated genes involved in matrix degradation included Mmp2, Mmp8, Mmp9, Mmp16, a disintegrin and metalloproteinase with thrombospondin motifs 10 (Adamts10), fibroblast activation protein α (Fap), and cathepsin K (Ctsk) (Fig. 5B). Mmp2, Mmp8, and Mmp9 have been strongly correlated with disc degeneration in humans [38,39]. Collectively, these matrix changes portray hallmark characteristics of degenerative disc disease. It is well-known that matrix composition of the intervertebral disc is paramount for its proper biomechanical function, wherein transformation from gelatinous to fibrocartilaginous matrix is strongly associated with increased tissue stiffness [40]. It is also known that feedback of stiffer extracellular matrix on cells induces adhesion complexes and cytoskeleton adjustments [41]. Interestingly, perturbations in AF matrix composition of TonEBP-deficient mice were associated with increased expression of key adhesion molecules, including integrin subunit β-2 (Igtb2), integrin β-2-like precursor (Igtb2l), integrin subunit α-M (Intam), tensin 3 (Tns3), and vinculin (Vcl) (Fig. 5C). Of the important proteins that are involved in linking integrins to cytoplasmic actin networks, vasodilator-stimulated phosphoprotein (Vasp), filamin α (flna), and talin 1 (Tln1) were also significantly upregulated, with a fold induction of 1.76, 1.7, and 1.64fold, respectively. In agreement with the observed fibrotic changes and upregulation of adhesion molecules in TonEBP-deficient AF tissue, numerous actin-related genes were increased, including small GTPases of the Rho family Rac1 and Rac2 (Rac1, Rac2), major components of the actin-branching Arp2/3 complex (Actr2, Actr3, Arpc1b, Arpc4), profilin 1 (Pfn1), tropomyosin 1α (Tpm1), coronin 1A, 1C, and 7 (Coro1a, Coro1c, Coro7), and several additional genes shown in Fig. 5D. Changes in expression of select genes in AF (Abi3bp, Cilp, Tnc, Mmp13, Arpc1, Vcl) observed on microarray were confirmed by qPCR (Fig. 5F-K). Taken together, these results suggest that AF cells in TonEBP-deficient discs augmented their actinbased adhesion pathways to mediate their interaction with the surrounding fibrotic and disorganized extracellular matrix. Gene ontology analysis using DAVID functional annotation bioinformatics yielded nucleus, DNA binding, and immune-related pathways among the most enriched in the NP (Fig. 4F’). While prominent downregulation of histone expression and alterations in epigenetic mechanisms (methylation, acetylation) of gene regulation emerged, as well as changes in solute carrier transporters and downregulation of Aqp1 in NP cells (Suppl. Fig. 5), we focused on immune-related changes in NP cells due to the importance of inflammation in the context of intervertebral disc degeneration. Nearly all immune-related genes that were differentially expressed were downregulated, consistent with the role of TonEBP as a transcriptional enhancer. Compared to controls, TonEBP-deficient NP cells expressed lower levels of proteoglycan 2 (Prg2), myeloperoxidase (Myo), eosinophil cationic protein 1, 2, 3, 6, 7, and 12 (Ear1, 2, 3, 6, 7,

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12), elastase (Elane), neutrophilic cytosolic factor 1 and 4 (Ncf1, 4), and a number of others shown in Fig. 5I. Changes in Prg2 expression was confirmed by qPCR (Fig. 5L). These findings are in accord with the ability of TonEBP to act on pro-inflammatory stimuli such as microbial products and prompt anti-pathogenic responses, especially in immune cells. Interestingly, microarray analysis of AF also suggested a change in about 50 genes related to the immune system process (Fig 4. F); however, the directionality of these changes did not follow a clear trend of reduced expression as was observed in the NP tissue profile. Additionally, quantitative immunohistochemistry was performed on 22-month-old mutant and wild-type control discs for localization of select immune markers. Results clearly showed a marked reduction in IL-6, Cox2, and Mcp1 levels in NP cells of mutant mice supporting microarray results (Fig. 6A-C’) and consistent with the proinflammatory role of TonEBP. Vertebral bodies of TonEBP-deficient mice show sparse distribution of trabecular bone Previous work has shown that TonEBP-deficient mice evidence retarded growth and delayed development of the spinal column [24,29]. However, a detailed analysis of postnatal vertebral bone in TonEBP-deficient mice has not been conducted. We therefore performed micro-computed tomography (MicroCT) analysis on lumbar vertebrae (L1-4) of TonEBPdeficient mice at 22-months of age (Fig. 7A). The mutant vertebrae were significantly shorter than controls (Fig. 7B) with a reduced disc height and disc height index (Fig. 7C, D). Notably, 3D reconstruction of trabecular bone showed a reduction in the trabecular network (Fig. 7E), marked by decreased bone volume fraction (BV/TV) (Fig. 7F) without significant changes in trabecular thickness (Fig. 7G). This reduction in BV/TV was associated with decreased trabecular number (Fig. 7H) and corresponding increase in trabecular spacing (Fig. 7I). On the other hand, there were no prominent changes in the appearance of cortical bone as seen from 3D reconstruction (Fig. 7J). Correspondingly, cortical bone volume (BV) (Fig. 7K), mean total cross-sectional bone area (Fig. 7L), cortical cross-sectional thickness (Fig. 7M), and mean polar moment of inertia (Fig. 7N) showed little differences between genotypes. Taken together, these results suggested that TonEBP plays an important role in the homeostasis of vertebral trabecular bone, and interestingly, may explain why less subchondral bone was observed in the endplates of TonEBP-deficient animals. DISCUSSION Our previous work has unveiled a dichotomous yet primarily homeostatic role for TonEBP in NP cells. Results of these studies have clearly shown that TonEBP plays an important role in osmoadaptation and extracellular matrix synthesis, allowing NP cell survival under hyperosmotic conditions [11,17,18,20,42]. More recent work has shown that this transcription factor is critical in expression of proinflammatory molecules as a likely adaptation to osmotic loading [21,25,26]. These in vitro studies provided a strong rationale for the notion that TonEBP is essential for disc homeostasis in vivo. For the first time, the work presented here characterized the spinal phenotype of aging TonEBP-deficient mice with an aim to test this longheld hypothesis. Spontaneous herniation of the endplate and annulus fibrosus was an interesting consequence of TonEBP-deficiency in aging discs. Under healthy conditions, endplates act as

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physical barriers to the NP compartment, distributing intradiscal pressures and preventing NP tissue from protruding into the adjacent vertebral centrum. Under diseased states, resultant damage can cause weakened focal points within the endplate making it more prone to failure [3,43]. This pathology is consistent with the endplate phenotype of TonEBP-deficient mice as they presented with significant cartilage disorganization and increased proteoglycan content. Furthermore, as increased deposition of proteoglycans and collagen have been shown to reduce the size of endplate pores [44], the observed endplate changes in mutants may have impeded nutrient diffusion into the avascular NP compartment and thus contributed to the degeneration cascade, especially in fibrotic discs. Similarly, increased susceptibility to annular herniation seen in TonEBP mutants was associated with compromised AF matrix structure and composition, as reflected by our histological and microarray analyses. On histology, the variation observed in the degenerative phenotype of TonEBP mutants was likely caused by intrinsic intra- and interanimal variation. Such variation is commonly seen in disc tissue because the character and extent of biomechanical stress differs significantly along the spinal column [45]. For instance, flexionextension range of motion has been shown to be greater in the caudal-most lumbar motion segments in humans, especially at L5-S1, whereas lateral bending was greater at the center of the lumbar spine with L2-3, L3-4, and L4-5 [46]. In agreement with this phenomenon, level-by-level assessment of TonEBP mutant discs showed higher modified Thompson grades in the caudalmost lumbar discs (Suppl. Fig. 2). Furthermore, human twin studies have described that genetic and environmental influences differ substantially for upper versus lower lumbar levels. [47]. While it is possible that poor vertebral bone quality in TonEBP-deficient mice also altered the biomechanics of the spine and thus contributed to the disc phenotype, recent work in mice has shown that alterations in adjacent vertebral bone morphology due to elevated systemic inflammation can increase the incidence of herniations but does not exert degenerative effects on the disc [48]. Interestingly, on the other hand, it has also been suggested that the fibrotic status of intervertebral discs can induce bone remodeling responses in adjacent vertebrae as a result of load redistribution [49]. The matrix composition of the intervertebral disc is a major determinant of its biomechanical properties. Our results suggest that TonEBP-deficiency drives matrix remodeling events characteristic of fibrosis, a condition known to result in increased tissue stiffness [31,50]. Morphologically, this transformation is characterized by an increased presence of chondrocytelike disc cells, matrix fibrosis, and lamellar disorganization. At the molecular level, we also observed a downregulation of collagen assembly/organizing proteins (i.e. FMOD, Abi3bp, Cilp) and water-imbibing matrix molecules (i.e. ACAN, COLII, Prg4, Sdc4), as well as an upregulation of matrix catabolic enzymes (i.e. MMP13, Mmp2, Mmp9) and matrix constituents of dense connective tissues (i.e. Col1a1, Col1a2, Tnc, Ibsp, Postn). Further, elevation of lumican may in part explain the increased percentage of thin fibers in the AF of TonEBP mutants as lumican has been implicated in formation of thin collagen fibrils [51]. As feedback from stiffer extracellular matrix on cells is known to induce adhesion complexes and cytoskeleton adjustments [41], it is not unreasonable to assume that these fibrotic changes prompted AF cells to enhance their actin-based adhesion pathways to mediate their interaction with the fibrotic matrix of TonEBP-deficient discs. Evidence for this phenomenon is provided by our microarray results on AF tissue which showed an upregulation of genes involved in cell adhesion (i.e. Igtb2, Tns3, Vcl, Vasp, flna, Tln1) and actin dynamics (i.e. Rac1, Rac2, Actr2, Actr3, Pfn1, Tpm1, Coro1a). A relationship between many of these integrin-associated and actin-binding proteins

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has been established by a vast body of literature [41], as well as their dynamic reciprocity with extracellular matrix components. For instance, loss of Sdc-4 in endothelial cells has been shown to dramatically alter actin cytoskeleton dynamics and β1 integrin localization at focal adhesions, as well as decouple vinculin from actin filaments [52]. Increased actin-related and adhesion molecules may have impacted the tensile-sensing, cordlike outer AF cells that form an interconnected network along the longitudinal length of the lamella, and the inner AF cells comprising extensive sinuous processes. As these processes are rich with actin and have been suggested to function in mechanical sensing and cell-matrix adhesion, it is possible that dysregulation of actin networks and matrix interactions in these cells contributed to a homoeostatic imbalance which favored a degenerative phenotype [53,54]. Due to the degenerative status of mutant discs, it should be noted that it is difficult to infer whether TonEBP-deficiency played a direct role in these cytoskeleton and adhesion changes or whether it was consequence of cellular responses to the changing matrix composition. Importantly, in addition to these changes, expression of mRNAs encoding several transport proteins including Aqp1 was altered in both NP and AF compartment of mutant mice, suggesting that the osmotic environment was altered. Similar changes in transporters are noted by Zhang et al. in a mouse model of spontaneous disc degeneration [32]. Notably, a link between cytoskeletal rearrangements and osmosensing has been previously described [55,56] and there is a wellestablished connection between integrin-mediated adhesion and TonEBP transcriptional activity [22,23], suggesting that some of these changes may well reflect a compensatory change in response to TonEBP-deficiency. A shift from the production of anabolic to catabolic factors by resident cells of the intervertebral disc is a hallmark of degenerative disc disease. This pathological state is widely believed to be driven by the upregulation of pro-inflammatory molecules, particularly TNF-α and IL-1β, which are elevated in degenerated and herniated human discs [57,58]. Interestingly, our group recently reported that the discs of polyarthritic Tg197 mice that overexpress human TNFα show abundant GAG-rich matrix, increased NP cell number and a resultant expansion of the NP cell band, a striking contrast to the hypothesis that chronic systemic inflammation would result in increased matrix degradation and fibrosis [48]. Additionally, IL-1α/β double knockout mice showed a more degenerative phenotype rather than having a protective effect against age-related disc degeneration [59]. These findings support the idea first suggested by Johnson and colleagues that pro-inflammatory genes may serve a homeostatic role in disc cells [25]. Our findings that TonEBP-deficient NP cells express reduced levels of immune-related and pro-inflammatory genes (i.e. IL-6, MCP1, COX2, Prg2, Myo, Ear1, Elane, Ncf1) are in agreement with this hypothesis, contributing to a growing body of literature which suggests that the effects of inflammation on disc homeostasis are more complex than previously thought. This may be due to the fact that the majority of disc tissue is avascular and immune privileged, and therefore shielded from the effects of the immune system unless there is a breach caused by herniation. In corroboration of this view, TonEBP-deficiency has been shown to have a protective effect against immune-driven inflammatory arthritis whereas, as we show here, TonEBP-deficiency in the disc leads to pronounced degeneration, a stark contrast in phenotypes analogous to what is observed in Tg197 mice [48,60,61]. While it is well-established that pro-inflammatory cytokines activate matrix-degrading enzymes, the notion that TonEBP mutant discs showed fibrotic changes of the matrix without an augmentation in pro-inflammatory genes lends credence to the

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possibility that these genes have an important function in disc maintenance when expressed at physiological levels. In summary, our studies show for the first time that TonEBP-deficiency causes pronounced degeneration of all three intervertebral disc compartments with high incidence of herniations. The disc phenotype is marked by extracellular matrix remodeling, actin cytoskeleton rearrangements, and suppressed proinflammatory gene expression, advancing our understanding of the diverse contributions of TonEBP in intervertebral disc homeostasis and disease. METHODS Mice All mouse experiments were performed under protocols approved by the Institutional Animal Care and Use Committee (IACUC) of Thomas Jefferson University in accordance with the IACUC’s relevant guidelines and regulations. The initial characterization of TonEBP haploinsufficient mice used in this study has been provided by Go et al. [30]. Mice were on a C57BL/6J background and of mixed sex for morphological analyses. Histological Analysis Lumbar spines were fixed in 4% PFA for 2 days at 4 °C and then decalcified in 20% EDTA at 4°C for 14 days. Spines were embedded in paraffin for sectioning in the coronal plane. Sevenmicron sections were stained with 1% Safranin-O, 0.05% Fast Green, and 1% Hematoxylin and then visualized by light microscopy (Axio Imager 2, Carl Zeiss) using 5×/0.15 N-Achroplan or 20×/0,5 EC Plan-Neofluar (Carl Zeiss) objectives. Imaging of sections was conducted with the Axiocam 105 color camera (Carl Zeiss) using Zen2™ software (Carl Zeiss). Disc degeneration was evaluated by 5 blinded observers using a modified Thompson grading scale and endplate cartilage score criteria shown in Suppl. Fig 3 [31,62]. Presence of subchondral bone within the endplate was manually quantified as a binary variable. All lumbar levels from five 12-month-old (n=5 mice/genotype) and six 22-month-old mutants (n=6 mice/genotype) were scored representing a total of 66 lumbar discs per genotype. Picrosirius RedTM staining of lumbar discs (n = 5 animals/genotype; 2 discs/animal; total 10 discs/genotype) was visualized using a polarized microscope (Eclipse LV100 POL, Nikon). The AF compartments of stained sections were delineated and quantified by percent green, yellow, or red pixels. Aspect ratio was measured using ImageJ 1.52a (http://rsb. info.nih.gov/ij/), where the major axis was divided by the minor axis determined by Fit Ellipse measurements. Aspect ratio data were collected from three E18.5 embryos per genotype with three discs per embryo (9 discs/genotype). Immunohistochemistry Coronal sections of the lumbar intervertebral disc were de-paraffinized in histoclear and rehydrated in a series of ethanol solutions (100%-70%). De-paraffinized sections were incubated in boiled citrate-based unmasking solution (Vector Laboratories, H-3301) for 20 minutes and then cooled to room temperature for 30 minutes. Next, the sections were incubated for 1 hour in the appropriate blocking solution (either 5-10% Normal Goat Serum, 10% Fetal Bovine Serum, or reagent from M.O.M.™ Immunodetection Kit; vector Laboratories, BMK-2202) and subsequently incubated overnight at 4°C with primary antibody against taurine transporter

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(1:100; Abcam, ab196821), aggrecan (1:50; MilliporeSigma, AB1031), ARGxx (1:200; Abcam, ab3773), chondroitin sulfate (1:300; Abcam, ab11570), versican (1:200; MilliporeSigma, Ab1033), fibromodulin (1:250; Abcam, ab81443), cartilage oligomeric matrix protein (1:200; Abcam, ab231977), collagen I (1:100; Abcam, ab34710), collagen II (1:400; Fitzgerald, 70RCR008), collagen X (1:500; Abcam, ab58632), matrix metalloproteinase 13 (1:200; Abcam, ab39012), interleukin-6 (1:50; Novus, NB600-1131), cyclooxygenase 2 (1:200; Cell Signaling, #12282), and monocyte chemoattractant protein-1 (1: 150; Abcam ab25124). The ARGxx antibody recognizes the ARG neoepitope generated after aggrecanase (ADAMTS-1,-4,-5) cleavage between amino acids EGE and ARG within aggrecan’s interglobular domain. After washing with PBS, sections were incubated for 1 hour at room temperature with Alexa Fluor®594 secondary antibody (1:700, Jackson ImmunoResearch Lab, Inc.). The sections were washed with PBS before mounting with ProLong® Gold Antifade Mountant containing DAPI (Thermo Fisher Scientific, P36934), and visualized by fluorescence microscopy (Axio Imager 2, Carl Zeiss) using the 5×/0.15 N-Achroplan or 20×/0,5 EC Plan-Neofluar (Carl Zeiss) objectives. The stained sections were imaged using X-Cite® 120Q Excitation Light Source (Excelitas), the AxioCam MRm camera (Carl Zeiss), and Zen2™ software (Carl Zeiss). Five animals per timepoint and genotype were used for analysis. Staining was performed on two representative discs per animal (5 animals/genotype; 2 discs/animal; total 10 discs/stain). Digital Image Analysis All imaged sections stained by immunohistochemistry were analyzed using ImageJ 1.52a (http://rsb. info.nih.gov/ij/) in the grayscale. The boundaries of the NP and AF were digitally traced using the Freehand Tool. These images were then thresholded to create binary images. Designated regions of interest (ROI) were analyzed using the Area Fraction measurement for each section. Area Fraction represents the percentage of positive pixels normalized to ROI, therefore representing protein expression within a given area. Fourier Transform Infrared (FTIR) Imaging Spectroscopy 5 µm-thick sections of decalcified lumbar discs were collected from control and TonEBP hypomorphic animals (n = 5 animals/genotype; 2 discs/animal; total 10 discs/genotype) and used to acquire infrared (IR) spectral imaging data. Data were collected using a Spectrum Spotlight 400 FT-IR Imaging system (Perkin Elmer, Shelton, CT), operating in the mid-IR region of 4,000-850 cm-1 with a spectral resolution of 8 cm-1 and spatial resolution of 25 µm. To reduce section quality-based variation, data were collected and averaged across three consecutive sections per disc. Second derivative differentiation with 9-point Savitzky-Golay smoothing was applied to the spectral absorbance data to enhance the separation of overlapping peaks. The resultant spectra were multiplied by a factor of negative one for positive visualization of the spectra. Mean absorbances in the amide I region (1660 cm-1) and collagen side chain vibration region (1338 cm1) were quantified and compared across the control, non-firbotic and fibrotic TonEBP-deficient discs in the AF, NP, and EP compartments. Significant differences in parameters were assessed using a one-way ANOVA and post-hoc Tukey test, where relevant, with p < 0.05 considered significant. Tissue RNA extraction NP and AF tissue was dissected separately from levels L1-S1 of 1-year-old male mice and immediately placed in TRIzol® Reagent (Life Technologies). Four mice per genotype were

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sacrificed for tissue RNA isolation. Isolated tissues were homogenized with a Pellet Pestle Motor (Sigma Aldrich, Z359971) for 1 min. Total RNA was extracted from the tissue lysates using Direct-zol® MiniPrep Plus kit (Zymo Research). Microarray Analysis and Enriched Pathways RNA from biological triplicates (n=3 animals/genotype; L1-S1, 6 discs/animal) were used for analysis. Fragmented biotin labeled cDNA was synthesized using the GeneChip WT Plus kit according to ABI protocol (Thermo Fisher Scientific). Gene chips (Mouse Clariom S) were hybridized with fragmented and biotin-labeled cDNA. Arrays were washed and stained with GeneChip hybridization wash & stain kit and scanned on an Affymetrix Gene Chip Scanner 3000 7G, using Command Console Software. Quality Control of the experiment was performed by Expression Console Software v 1.4.1. Chp files were generated by sst-rma normalization from Affymetrix cel file using Expression Console Software. Experimental group was compared with control group by using Transcriptome array console 4.0 software. Detection above background higher than 50% was used for Significance Analysis of Microarrays (SAM), 2-Fold-change and p-value < 0.05. DAVID tool was used to compute enriched pathways. Functional Categories Keywords, Gene ontology (GO) terms for biological process (BP), molecular function (MF) and cell compartment (CC) as well as KEGG pathways were examined. The genes that were included in the SAM analysis served as the background gene set. Analyses and visualizations were done in Transcriptome array console 4.0 software. Real-Time qRT-PCR DNA-free tissue RNA was converted into cDNA using EcoDry premix (Clontech). PCR reactions using gene-specific primers (IDT, IA) and SYBR Green master mix (Applied Biosystems) were measured by the Step-One Plus System (Applied Biosystems). Experiments were conducted with four biological replicates (n=3-4 animals/genotype; L1-S1, 6 discs/animal). Micro-computed tomography analysis Lumbar spines fixed in 4% PFA and stored in 70% EtOH were scanned using the Bruker Skyscan 1275 microCT system at 50kV and 200µA with an exposure time of 85ms, generating a resolution of 15µm. An aluminum filter was used for each scan. Images were reconstructed using the nRecon (Bruker) program and analysis was conducted using CTan (Bruker). Height of the intervertebral discs and vertebral bones were measured at the dorsal, midline, and ventral regions along the sagittal plane and averaged. Disc height index (DHI) was calculated as previously described [63]. Transverse cross-sectional images were analyzed to evaluate trabecular and cortical bone morphology. For trabecular analysis, a region of interest (ROI) was selected by contouring the boundary between trabecular and cortical bone throughout the vertebral body. 3D assessment of trabecular bone obtained values for bone volume percent (BV/TV), trabecular thickness (Tb.Th), number (Tb.N) and separation (Tb.Sp). 2D analysis of cortical bone assessed bone volume (BV), cross-sectional thickness (Cs.Th), mean total cross-sectional bone area (B.Ar), and mean polar moment of inertia (MMI). Lumbar levels L1-4 were used to obtain vertebral heights required for DHI calculations whereas level L1-3 were used for bone morphology analysis. Both 22-month-old wild-type and TonEBP-deficient mice (n ≥ 5 mice; 15 vertebrae/genotype; 18 discs/genotype) were analyzed. Statistical Analysis

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Data is presented as mean ± SD. Differences between genotypes were analyzed using the Student’s t test when only two groups presented on graph or one-way ANOVA with a Sidak’s multiple comparison test between groups. Significance between data presented as contingency graphs was determined using χ2 test. All statistical analyses were done using Prism7 (GraphPad Software). p ≤ 0.05 was the threshold for statistical significance. Author contributions Study design: MVR, ST. Study conduct: MVR, IMS. Data collection: ST, VAT, OKO, AD, MJD. Data analysis: MVR, ST, EJN. Data interpretation: MVR, ST. Drafting manuscript: MVR, ST. Revising manuscript content: MVR, IMS, ST. Approving final version of manuscript: MVR, IMS, ST, VAT, OKO, EJN, AD, MJD. Acknowledgements This work was supported by the National Institutes of Health under grant numbers R01AR064733, R01AR055655, R01AR074813, T32AR052273. The authors thank Zariel I. Johnson for contributions to the early phases of this work.

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Figure Legends Figure 1: TonEBP deficiency accelerates age-related disc degeneration. (A) Schematic showing targeting of exons 6 and 7 of the Nfat5 gene and resultant mRNA product. (B, B’) Evaluation of taurine transporter (TauT) levels in NP cells by quantitative immunohistochemistry. 5x scale = 200 µm; 20x scale = 100 µm. Coronal sections of discs from (C) 12-month-old and (D) 22month old animals stained by Safranin-O/Fast Green/Hematoxylin. White arrowheads show chondrocyte-like disc cells; black arrowheads show loss of NP/AF demarcation. 5x scale = 200 µm; 20x scale = 50 µm. (E) Average modified Thompson score for 12-month (n=5 mice/genotype; L1-S1, 6 discs/animal; total 30 discs/genotype) and (E) 22-month-old animals (n=6 mice/genotype; L1-S1, 6 discs/animal; total 36 discs/genotype), where higher scores indicate worsening changes. (E’) Distribution of histological grades for 12-month and 22-monthold animals. (F) High magnification image of CEPs stained by Safranin-O/Fast Green/Hematoxylin. Black arrowheads show subchondral bone; White arrowheads show CEP; black arrow shows CEP herniation. 5x scale = 100 µm; 20x scale = 50 µm. (G) Level-by-level mapping of AF and CEP herniation events. (H) Endplate cartilage score for combined timepoints and (I) percentage of discs with present subchondral bone within the endplate or lack thereof representing combined timepoints (n = 11 mice/genotype; L1-S1, 6 discs/animal; total 66 discs/genotype). NP: Nucleus Pulposus; AF: Annulus fibrosus; CEP: cartilaginous end plate; GP: Growth plate; VB: Vertebral body. Quantitative measurements represent mean ± SD. Significance was determined using either unpaired t-test or χ2 test where graphs represent contingency plots. n.s. = not significant; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001. Figure 2: Disc degeneration in TonEBP-deficient mice is characterized by fibrocartilaginous changes. (A) Picrosirius Red staining of 22-month-old lumbar discs showing collagen content in AF (Left) and NP (right) tissue visualized by bright field and polarized microscopy (n=5 mice; 2 discs/mouse; 10 discs/genotype). 20x scale = 50 µm. (A’) Quantification of collagen fiber thickness distribution. (A”) Percentage of fibrotic NP compartments with Picrosirius Redpositive staining among analyzed discs. (B) Cluster analysis images of infrared spectra of total protein (Prot) and collagen (Col) content (left panels) with respect to corresponding grey-scale scans (right column). +/∆ represents non-fibrotic TonEBP hypomorphic discs whereas F+/∆ indicates the fibrotic population. (C-C”) Representative graphs of average second derivative spectra from AF, CEP, and NP clusters, inverted for positive visualization of the spectra. (D) Total protein content represented by mean absorbance peak values measured at 1660 cm-1. (E) Total collagen content represented by mean absorbance peak values measured at 1156 cm-1. AU: arbitrary units. Quantitative measurements represent mean ± SD. Significance was determined using unpaired t-test when only two groups were compared, χ2 test for the contingency plot, and one-way ANOVA for multiple comparisons. n.s. = not significant; *, p ≤ 0.05; **, p ≤ 0.01; ****, p ≤ 0.0001. Figure 3: TonEBP-deficient discs show matrix composition characteristic of degeneration. Quantitative immunohistochemistry performed on 22-month-old lumbar discs stained by the following: (A ,A’) aggrecan (ACAN); (B, B’) the aggrecanase-generated neoepitope, ARGxx; (C, C’) chondroitin sulfate (CS); (E, E’) versican β domain (VCAN); (F, F’) fibromodulin (FMOD); (G, G’) cartilage oligomeric matrix protein (COMP); (H, H’) collagen 1 (COLI); (I, I’) collagen 2 (COLII); (J, J’) collagen 10 (COLX); (K, K’) matrix metalloproteinase 13 (MMP13).

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(n = 5 mice; 10 discs/genotype). 5x scale = 200 µm; 20x scale = 100 µm; 40x scale = 50 µm. Quantitative measurements represent mean ± SD. Significance was determined using unpaired ttest. n.s. = not significant; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001. Figure 4: Microarray analyses of AF and NP tissue from TonEBP-deficient discs. (A) Experimental design for microarray analysis wherein NP and AF tissues were separated and pooled upon dissection of lumbar spinal levels (L1-S1) of individual mice. (B, B’) Clustering of biological triplicates within genotypes assessed by three-dimensional principle component analysis (PCA). (C, C’) Total differential expression of genes at p ≤ 0.05 in (C) AF and (C’) NP tissues. Heatmaps of differentially expressed genes from (D) AF and (E) NP samples comparing triplicates of each genotype. Volcano plots showing the relationship between fold change (>2fold) and p-value (–log10) for AF (D’) and (E’) NP samples. Representative annotation plots of gene sets among highest enrichment scores in (F) AF and (G) NP samples. (H) Venn diagram comparing genes differentially expressed at p ≤ 0.05 and >2-fold of AF and NP tissue from TonEBP-deficient mice. (H’) Representative annotation plots of gene sets among highest enrichment scores from the common genes identified in H. (n = 3 mice/genotype; L1-S1, 6 discs/animal). Figure 5: Differential regulation of genes associated with extracellular matrix remodeling, cell adhesion, the actin cytoskeleton, and proinflammatory signaling in TonEBP-deficient discs. Representative expression changes from AF samples greater than 2-fold at p ≤ 0.05 of genes involved in (A) extracellular matrix composition, (B) matrix degradation, (C) cell adhesion, and (D) the actin cytoskeleton (n = 3 mice/genotype; L1-S1, 6 discs/animal). (E) Representative immune-related genes expressed in NP cells with a differential greater than 2-fold at p ≤ 0.05. (n = 3 mice/genotype). Changes in the mRNA expression levels of (F) Abi3bp, (G) Cilp, (H) Tnc, (I) Mmp13, (J) Arpc1, (K) Vcl, and (L) Prg2 (n = 3-4 mice/genotype; L1-S1, 6 discs/animal). Differential expression levels on microarray graphs are normalized to wild-type expression levels. Quantitative measurements represent mean ± SD. Significance was determined using unpaired t-test. *, p ≤ 0.05; **, p ≤ 0.01; ****, p ≤ 0.0001. Figure 6: TonEBP-deficient NP cells show reduced levels of pro-inflammatory molecules. Quantitative immunohistochemistry performed on 22-month-old lumbar discs stained with (A, A’) interleukin-6 (IL-6), (B’ B’) cyclooxygenase-2 (COX2), and (C, C’) monocyte chemoattractant protein-1 (MCP1). (n = 5 mice; 10 discs/genotype). 10x scale = 200 µm. Quantitative measurements represent mean ± SD. Significance was determined using unpaired ttest. *, p ≤ 0.05; ****, p ≤ 0.0001. Figure 7: Vertebral bodies of TonEBP-deficient mice show compromised trabecular bone. (A) Representative MicroCT scans of 22-month-old wild-type and mutant lumbar spines. The vertebral levels labeled in yellow indicate levels analyzed. White arrowheads indicate the disc space analyzed. (B) Vertebral height, (C) disc height, and (D) disc height index measurements are shown. (E) Coronal (Top) and transverse (Bottom) views of trabecular bone 3D reconstructions. (F) Bone volume fraction (BV/TV), (G) trabecular thickness (Tb.Th), (H) trabecular number (Tb.N) and trabecular spacing (Tb.Sp) measurements are shown. (J) Coronal (Top) and transverse (Bottom) views of cortical bone 3D reconstructions. (K) Cortical bone volume (BV), mean total cross-sectional bone area (B.Ar), cortical cross-sectional thickness

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(Cs.Th), and mean polar moment of inertia (MMI) measurements are shown. (n ≥ 5 mice; 15 vertebrae/genotype; 18 discs/genotype). Scale = 1 mm. Quantitative measurements represent mean ± SD. Significance was determined using unpaired t-test. n.s. = not significant; *, p ≤ 0.05; **, p ≤ 0.01. Supplemental Figure 1: TonEBP abundance in NP cells decreases with aging. Quantitative immunohistochemistry performed on (A, A’,C) 12-month and (B, B’,C) 22-month-old lumbar wild-type discs stained with TonEBP (n = 5 mice; 2 discs/animal; 10 discs/genotype). 5x scale = 200 µm; 20x scale = 100 µm. Quantitative measurements represent mean ± SD. Significance was determined using unpaired t-test. **, p ≤ 0.01. Supplemental Figure 2: Level-by-level scores of lumbar discs using a modified Thompson scale of (A) 1-year-old and (B) 22-month-old animals. Supplemental Figure 3: Criteria use to score endplate cartilage. Cartilaginous endplates are outlined in black and degenerative features are indicated by white arrows. Supplemental Figure 4: Safranin-O/Fast Green/Hematoxylin staining of (A) lumbar and (B) caudal intervertebral discs at E18.5 (20x scale = 50 µm). (B’) Quantitative analysis of caudal NP aspect ratios. (n = 3 embryos/genotype; 3 discs/embryo). Quantitative measurements represent mean ± SD. Significance was determined using unpaired t-test. n.s. = not significant. Supplemental Figure 5: Representative (A) histone-related and (B, B’) transporter genes expressed in NP and AF cells with a differential greater than 2-fold at p ≤ 0.05. Differential expression levels on microarray graphs are normalized to wild-type expression levels. (n = 3). Supplemental Table 1: Annotated gene sets and clusters of AF samples (>2-fold at p ≤ 0.05). (n = 3 mice/genotype). Supplemental Table 2: Annotated gene sets and clusters of NP samples (>2-fold at p ≤ 0.05). (n = 3 mice/genotype). Supplemental Table 3: Annotated common gene sets and clusters between AF and NP samples of TonEBP-deficient mice (>2-fold at p ≤ 0.05). (n = 3 mice/genotype).

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Highlights • TonEBP-deficiency causes pronounced age-dependent intervertebral disc degeneration • Fibrocartilaginous matrix changes are associated with actin rearrangements and altered expression of adhesion molecules • TonEBP-deficient mice show suppressed expression of immune-related and proinflammatory genes • Vertebral bodies of TonEBP-deficient mice show compromised trabecular bone