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Suppression of Sost/Sclerostin and Dickkopf-1 Augment Intervertebral Disc Structure in Mice.

ABSTRACT: Intervertebral disc (IVD) degeneration is a leading cause of low back pain, characterized by accelerated extracellular matrix breakdown and IVD height loss, but there is no approved pharmacological therapeutic. Deletion of Wnt ligand competitor Lrp5 induces IVD degeneration, suggesting that Wnt signaling is essential for IVD homeostasis. Therefore, the IVD may respond to neutralization of Wnt ligand competitors sost(gene)/sclerostin(protein) and/or dickkopf-1 (dkk1). Anti-sclerostin antibody (scl-Ab) is an FDA-approved bone therapeutic that activates Wnt signaling. We aimed to (i) determine if pharmacological neutralization of sclerostin, dkk1, or their combination would stimulate Wnt signaling and augment IVD structure and (ii) determine the prolonged adaptation of the IVD to global, persistent deletion of sost. Nine-week-old C57Bl/6J female mice (n = 6-7/group) were subcutaneously injected 2×/week for 5.5 weeks with scl-Ab (25 mg/kg), dkk1-Ab (25 mg/kg), 3:1 scl-Ab/dkk1-Ab (18.75:6.25 mg/kg), or vehicle (veh). Separately, IVD of sost KO and wild-type (WT) mice (n = 8/group) were harvested at 16 weeks of age. First, compared with vehicle, injection of scl-Ab, dkk1-Ab, and 3:1 scl-Ab/dkk1-Ab similarly increased lumbar IVD height and β-catenin gene expression. Despite these similarities, only injection of scl-Ab alone strengthened IVD mechanical properties and decreased heat shock protein gene expressions. Genetically and compared with WT, sost KO enlarged IVD height, increased proteoglycan staining, and imbibed IVD hydration. Notably, persistent deletion of sost was compensated by upregulation of dkk1, which consequently reduced the cell nuclear fraction for Wnt signaling co-transcription factor β-catenin in the IVD. Lastly, RNA-sequencing pathway analysis confirmed the compensatory suppression of Wnt signaling and revealed a reduction of cellular stress-related pathways. Together, suppression of sost/sclerostin or dkk1 each augmented IVD structure by stimulating Wnt signaling, but scl-Ab outperformed dkk1-Ab in strengthening the IVD. Ultimately, postmenopausal women prescribed scl-Ab injections to prevent vertebral fracture may also benefit from a restoration of IVD height and health. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

SUBMITTER: Kroon T

PROVIDER: S-EPMC9320845 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The expression of Interleukin-1ß (IL-1ß) and its antagonist and Interleukin-1 receptor antagonist (IL-1Ra) are correlated with greater human intervertebral disc (IVD) degeneration, suggesting that elevated IL-1β activity promotes disc degeneration. Many in vitro studies support such a mechanistic relationship, whereas few in vivo investigations have been reported. The present study tests the effect of increased IL-1β activity on intervertebral disc in mice with an IL-1Ra gene deletion. IL-1Ra-/- mice and wild-type (WT) C57Bl6J mice were examined at 3 and 12 months of age. Caudal IVD segments were evaluated for disc degeneration by histopathology, functional testing, and inflammatory gene expression relevant to IL-1β pathways. To test differences in injury response, pinprick annular puncture was performed on IL-1Ra-/- and WT mice and evaluated similarly. IL-1Ra-/- IVDs had significantly worse histopathology at 3 months compared to WT controls, but not at 12 months. IL-1Ra-/- IVDs exhibited significantly more viscous mechanical properties than WT IVDs. qPCR revealed downregulation of inflammatory genes at 3 and 12 months in IL-1Ra-/- IVDs, with concomitant downregulation of anabolic and catabolic genes. Annular puncture yielded no appreciable differences between 2-week and 6-week post-injured WT and IL1-Ra-/- IVDs in histopathology or biomechanics, but inflammatory gene expression was sharply downregulated in IL-1Ra-/- mice at 2 weeks, returning by 6 weeks post injury. In the present study, IL-1Ra deletion resulted in increased IVD histopathology, inferior biomechanics, and transiently decreased pro-inflammatory cytokine gene expression. The histopathology of IL-1Ra-/- IVDs on a C57BL/6J background is less severe than a previous report of IL1Ra-/- on a BALB/c background, yet both strains exhibit IVD degeneration, reinforcing a mechanistic role of IL-1β signaling in IVD pathobiology. Despite a pro-inflammatory environment, the annular puncture was no worse in IL-1Ra-/- mice, suggesting that response to injury involves pathways other than inflammation. Overall, this study supports the hypothesis that IL-1β-driven inflammation is important in IVD degeneration.

Project description:Patients with anterior cruciate ligament (ACL) rupture are two times as likely to develop posttraumatic osteoarthritis (PTOA). Annually, there are ?900,000 knee injuries in the United States, which account for ?12% of all osteoarthritis (OA) cases. PTOA leads to reduced physical activity, deconditioning of the musculoskeletal system, and in severe cases requires joint replacement to restore function. Therefore, treatments that would prevent cartilage degradation post-injury would provide attractive alternatives to surgery. Sclerostin (Sost), a Wnt antagonist and a potent negative regulator of bone formation, has recently been implicated in regulating chondrocyte function in OA. To determine whether elevated levels of Sost play a protective role in PTOA, we examined the progression of OA using a noninvasive tibial compression overload model in SOST transgenic (SOSTTG ) and knockout (Sost-/- ) mice. Here we report that SOSTTG mice develop moderate OA and display significantly less advanced PTOA phenotype at 16 weeks post-injury compared with wild-type (WT) controls and Sost-/- . In addition, SOSTTG built ?50% and ?65% less osteophyte volume than WT and Sost-/- , respectively. Quantification of metalloproteinase (MMP) activity showed that SOSTTG had ?2-fold less MMP activation than WT or Sost-/- , and this was supported by a significant reduction in MMP2/3 protein levels, suggesting that elevated levels of SOST inhibit the activity of proteolytic enzymes known to degrade articular cartilage matrix. Furthermore, intra-articular administration of recombinant Sost protein, immediately post-injury, also significantly decreased MMP activity levels relative to PBS-treated controls, and Sost activation in response to injury was TNF? and NF-?B dependent. These results provide in vivo evidence that sclerostin functions as a protective molecule immediately after joint injury to prevent cartilage degradation. © 2018 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals Inc.

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Suppression of Sost/Sclerostin and Dickkopf-1 Augment Intervertebral Disc Structure in Mice.

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