Lumbar instability remodels cartilage endplate to induce intervertebral disc degeneration by recruiting osteoclasts via Hippo-CCL3 signaling

Abstract

Degenerated endplate appears with cheese-like morphology and sensory innervation, contributing to low back pain and subsequently inducing intervertebral disc degeneration in the aged population.¹ However, the origin and development mechanism of the cheese-like morphology remain unclear. Here in this study, we report lumbar instability induced cartilage endplate remodeling is responsible for this pathological change. Transcriptome sequencing of the endplate chondrocytes under abnormal stress revealed that the Hippo signaling was key for this process. Activation of Hippo signaling or knockout of the key gene Yap1 in the cartilage endplate severed the cheese-like morphological change and disc degeneration after lumbar spine instability (LSI) surgery, while blocking the Hippo signaling reversed this process. Meanwhile, transcriptome sequencing data also showed osteoclast differentiation related gene set expression was up regulated in the endplate chondrocytes under abnormal mechanical stress, which was activated after the Hippo signaling. Among the discovered osteoclast differentiation gene set, CCL3 was found to be largely released from the chondrocytes under abnormal stress, which functioned to recruit and promote osteoclasts formation for cartilage endplate remodeling. Over-expression of Yap1 inhibited CCL3 transcription by blocking its promoter, which then reversed the endplate from remodeling to the cheese-like morphology. Finally, LSI-induced cartilage endplate remodeling was successfully rescued by local injection of an AAV5 wrapped Yap1 over-expression plasmid at the site. These findings suggest that the Hippo signaling induced osteoclast gene set activation in the cartilage endplate is a potential new target for the management of instability induced low back pain and lumbar degeneration.

References

[1] Ni S et al. Sensory innervation in porous endplates by Netrin-1 from osteoclasts mediates PGE2-induced spinal hypersensitivity in mice. Nat. Commun., 2019, 10

[2] Andersson GB. Epidemiological features of chronic low-back pain. Lancet, 1999, 354: 581-585

[3] Centers for Disease, C. & Prevention Prevalence and most common causes of disability among adults–United States, 2005. MMWR Morb. Mortal. Wkly Rep. 58, 421–426 (2009).

[4] Teraguchi M et al. Prevalence and distribution of intervertebral disc degeneration over the entire spine in a population-based cohort: the Wakayama Spine Study. Osteoarthr. Cartil., 2014, 22: 104-110

[5] Cheung KM et al. Prevalence and pattern of lumbar magnetic resonance imaging changes in a population study of one thousand forty-three individuals. Spine (Philos. Pa 1976), 2009, 34: 934-940

[6] Urban JP, Roberts S. Degeneration of the intervertebral disc. Arthritis Res. Ther., 2003, 5: 120-130

[7] Clouet J et al. The intervertebral disc: from pathophysiology to tissue engineering. Jt. Bone Spine, 2009, 76: 614-618

[8] Hadjipavlou AG, Tzermiadianos MN, Bogduk N, Zindrick MR. The pathophysiology of disc degeneration: a critical review. J. Bone Jt. Surg. Br., 2008, 90: 1261-1270

[9] Applebaum A, Nessim A, Cho W. Modic change: an emerging complication in the aging population. Clin. Spine Surg., 2022, 35: 12-17

[10] Desmoulin GT, Pradhan V, Milner TE. Mechanical aspects of intervertebral disc injury and implications on biomechanics. Spine (Philos. Pa 1976), 2020, 45: E457-E464

[11] Adams MA, Freeman BJ, Morrison HP, Nelson IW, Dolan P. Mechanical initiation of intervertebral disc degeneration. Spine (Philos. Pa 1976), 2000, 25: 1625-1636

[12] de Roos A, Kressel H, Spritzer C, Dalinka M. MR imaging of marrow changes adjacent to end plates in degenerative lumbar disk disease. AJR Am. J. Roentgenol., 1987, 149: 531-534

[13] Modic MT, Steinberg PM, Ross JS, Masaryk TJ, Carter JR. Degenerative disk disease: assessment of changes in vertebral body marrow with MR imaging. Radiology, 1988, 166: 193-199

[14] Ashinsky BG et al. Intervertebral disc degeneration is associated with aberrant endplate remodeling and reduced small molecule transport. J. Bone Min. Res, 2020, 35: 1572-1581

[15] Natarajan RN, Ke JH, Andersson GB. A model to study the disc degeneration process. Spine (Philos. Pa 1976), 1994, 19: 259-265

[16] Smith LJ, Nerurkar NL, Choi KS, Harfe BD, Elliott DM. Degeneration and regeneration of the intervertebral disc: lessons from development. Dis. Model Mech., 2011, 4: 31-41

[17] Kushchayev SV et al. ABCs of the degenerative spine. Insights Imaging, 2018, 9: 253-274

[18] Dudli S, Fields AJ, Samartzis D, Karppinen J, Lotz JC. Pathobiology of modic changes. Eur. Spine J., 2016, 25: 3723-3734

[19] He Z, Jia M, Yu Y, Yuan C, Wang J. Roles of SDF-1/CXCR4 axis in cartilage endplate stem cells mediated promotion of nucleus pulposus cells proliferation. Biochem. Biophys. Res. Commun., 2018, 506: 94-101

[20] He Z, Pu L, Yuan C, Jia M, Wang J. Nutrition deficiency promotes apoptosis of cartilage endplate stem cells in a caspase-independent manner partially through upregulating BNIP3. Acta Biochim. Biophys. Sin. (Shanghai), 2017, 49: 25-32

[21] Wang H et al. Utilization of stem cells in alginate for nucleus pulposus tissue engineering. Tissue Eng. Part A, 2014, 20: 908-920

[22] Chen S et al. Mesenchymal stem cells protect nucleus pulposus cells from compression-induced apoptosis by inhibiting the mitochondrial pathway. Stem Cells Int., 2017, 2017

[23] Wang W et al. Transplantation of hypoxic-preconditioned bone mesenchymal stem cells retards intervertebral disc degeneration via enhancing implanted cell survival and migration in rats. Stem Cells Int., 2018, 2018

[24] Dupont S et al. Role of YAP/TAZ in mechanotransduction. Nature, 2011, 474: 179-183

[25] Kowalczyk W et al. Hippo signaling instructs ectopic but not normal organ growth. Science, 2022, 378

[26] Deng Y et al. Yap1 regulates multiple steps of chondrocyte differentiation during skeletal development and bone repair. Cell Rep., 2016, 14: 2224-2237

[27] Ariga K et al. The relationship between apoptosis of endplate chondrocytes and aging and degeneration of the intervertebral disc. Spine (Philos. Pa 1976), 2001, 26: 2414-2420

[28] Lee YH et al. Computational comparison of three posterior lumbar interbody fusion techniques by using porous titanium interbody cages with 50% porosity. Comput Biol. Med, 2016, 71: 35-45

[29] Jiang Y, Sun X, Peng X, Zhao J, Zhang K. Effect of sacral slope on the biomechanical behavior of the low lumbar spine. Exp. Ther. Med, 2017, 13: 2203-2210

[30] Yu CC et al. A new cervical artificial disc prosthesis based on physiological curvature of end plate: a finite element analysis. Spine J., 2016, 16: 1384-1391

[31] Alizadeh M et al. The use of X-shaped cross-link in posterior spinal constructs improves stability in thoracolumbar burst fracture: a finite element analysis. J. Orthop. Res., 2013, 31: 1447-1454

[32] Bleuel J, Zaucke F, Bruggemann GP, Niehoff A. Effects of cyclic tensile strain on chondrocyte metabolism: a systematic review. PLoS One, 2015, 10: e0119816

[33] Kastan N et al. Small-molecule inhibition of Lats kinases may promote Yap-dependent proliferation in postmitotic mammalian tissues. Nat. Commun., 2021, 12

[34] Huang J et al. The microRNAs miR-204 and miR-211 maintain joint homeostasis and protect against osteoarthritis progression. Nat. Commun., 2019, 10

[35] Pennicooke B, Moriguchi Y, Hussain I, Bonssar L, Hartl R. Biological treatment approaches for degenerative disc disease: a review of clinical trials and future directions. Cureus, 2016, 8: e892

[36] Sato M et al. An experimental study of the regeneration of the intervertebral disc with an allograft of cultured annulus fibrosus cells using a tissue-engineering method. Spine (Philos. Pa 1976), 2003, 28: 548-553

[37] Krut Z, Pelled G, Gazit D, Gazit Z. Stem cells and exosomes: new therapies for intervertebral disc degeneration. Cells, 2021, 10: 2241

[38] Lotz JC, Fields AJ, Liebenberg EC. The role of the vertebral end plate in low back pain. Glob. Spine J., 2013, 3: 153-164

[39] Bian Q et al. Excessive activation of TGFbeta by spinal instability causes vertebral endplate sclerosis. Sci. Rep., 2016, 6

[40] Gilbert SJ, Bonnet CS, Blain EJ. Mechanical cues: bidirectional reciprocity in the extracellular matrix drives mechano-signalling in articular cartilage. Int. J. Mol. Sci., 2021, 22: 13595

[41] Peng B et al. Possible pathogenesis of painful intervertebral disc degeneration. Spine (Philos. Pa 1976), 2006, 31: 560-566

[42] Saito T et al. Transcriptional regulation of endochondral ossification by HIF-2alpha during skeletal growth and osteoarthritis development. Nat. Med, 2010, 16: 678-686

[43] Yang S et al. Hypoxia-inducible factor-2alpha is a catabolic regulator of osteoarthritic cartilage destruction. Nat. Med., 2010, 16: 687-693

[44] Burleigh A et al. Joint immobilization prevents murine osteoarthritis and reveals the highly mechanosensitive nature of protease expression in vivo. Arthritis Rheum., 2012, 64: 2278-2288

[45] Gruber J et al. Induction of interleukin-1 in articular cartilage by explantation and cutting. Arthritis Rheum., 2004, 50: 2539-2546

[46] Gilbert SJ et al. Inflammatory and degenerative phases resulting from anterior cruciate rupture in a non-invasive murine model of post-traumatic osteoarthritis. J. Orthop. Res, 2018, 36: 2118-2127

[47] Furman BD et al. Targeting pro-inflammatory cytokines following joint injury: acute intra-articular inhibition of interleukin-1 following knee injury prevents post-traumatic arthritis. Arthritis Res. Ther., 2014, 16: R134

[48] Hu K et al. Pathogenesis of osteoarthritis-like changes in the joints of mice deficient in type IX collagen. Arthritis Rheum., 2006, 54: 2891-2900

[49] Pan D. The hippo signaling pathway in development and cancer. Dev. Cell, 2010, 19: 491-505

[50] Deng Y et al. Reciprocal inhibition of YAP/TAZ and NF-kappaB regulates osteoarthritic cartilage degradation. Nat. Commun., 2018, 9

[51] Koch AE et al. Macrophage inflammatory protein-1 alpha. A novel chemotactic cytokine for macrophages in rheumatoid arthritis. J. Clin. Invest., 1994, 93: 921-928

[52] Collins FL et al. CCL3 and MMP-9 are induced by TL1A during death receptor 3 (TNFRSF25)-dependent osteoclast function and systemic bone loss. Bone, 2017, 97: 94-104

[53] Jordan LA et al. Inhibition of CCL3 abrogated precursor cell fusion and bone erosions in human osteoclast cultures and murine collagen-induced arthritis. Rheumatol. (Oxf.), 2018, 57: 2042-2052

[54] Khoury M et al. Adeno-associated virus type 5-mediated intraarticular administration of tumor necrosis factor small interfering RNA improves collagen-induced arthritis. Arthritis Rheum., 2010, 62: 765-770

[55] Moya IM, Halder G. Hippo-YAP/TAZ signalling in organ regeneration and regenerative medicine. Nat. Rev. Mol. Cell Biol., 2019, 20: 211-226

[56] Zanconato F, Cordenonsi M, Piccolo S. YAP/TAZ at the roots of cancer. Cancer Cell, 2016, 29: 783-803

[57] Harvey KF, Zhang X, Thomas DM. The Hippo pathway and human cancer. Nat. Rev. Cancer, 2013, 13: 246-257

[58] Li H et al. Nanoscaled bionic periosteum orchestrating the osteogenic microenvironment for sequential bone regeneration. ACS Appl. Mater. Interfaces, 2020, 12: 36823-36836

[59] Bian Q et al. Mechanosignaling activation of TGFbeta maintains intervertebral disc homeostasis. Bone Res, 2017, 5

[60] Fan F et al. Pharmacological targeting of kinases MST1 and MST2 augments tissue repair and regeneration. Sci. Transl. Med, 2016, 8: 352ra108

[61] Boos N et al. Classification of age-related changes in lumbar intervertebral discs: 2002 Volvo Award in basic science. Spine (Philos. Pa 1976), 2002, 27: 2631-2644

[62] Masuda K et al. A novel rabbit model of mild, reproducible disc degeneration by an anulus needle puncture: correlation between the degree of disc injury and radiological and histological appearances of disc degeneration. Spine (Philos. Pa 1976), 2005, 30: 5-14

[63] Pan W et al. Verteporfin can reverse the paclitaxel resistance induced by YAP over-expression in HCT-8/T cells without photoactivation through inhibiting YAP expression. Cell Physiol. Biochem., 2016, 39: 481-490

[64] Wang H et al. Moderate mechanical stimulation antagonizes inflammation of annulus fibrosus cells through YAP-mediated suppression of NF-κB signaling. J. Orthop. Res, 2023, 41: 2667-2684

Funding

National Natural Science Foundation of China (National Science Foundation of China)(82172468, 82372436 and 32301416)