Restoring the dampened expression of the core clock molecule BMAL1 protects against compression-induced intervertebral disc degeneration

Abstract

The circadian clock participates in maintaining homeostasis in peripheral tissues, including intervertebral discs (IVDs). Abnormal mechanical loading is a known risk factor for intervertebral disc degeneration (IDD). Based on the rhythmic daily loading pattern of rest and activity, we hypothesized that abnormal mechanical loading could dampen the IVD clock, contributing to IDD. Here, we investigated the effects of abnormal loading on the IVD clock and aimed to inhibit compression-induced IDD by targeting the core clock molecule brain and muscle Arnt-like protein-1 (BMAL1). In this study, we showed that BMAL1 KO mice exhibit radiographic features similar to those of human IDD and that BMAL1 expression was negatively correlated with IDD severity by systematic analysis based on 149 human IVD samples. The intrinsic circadian clock in the IVD was dampened by excessive loading, and BMAL1 overexpression by lentivirus attenuated compression-induced IDD. Inhibition of the RhoA/ROCK pathway by Y-27632 or melatonin attenuated the compression-induced decrease in BMAL1 expression. Finally, the two drugs partially restored BMAL1 expression and alleviated IDD in a diurnal compression model. Our results first show that excessive loading dampens the circadian clock of nucleus pulposus tissues via the RhoA/ROCK pathway, the inhibition of which potentially protects against compression-induced IDD by preserving BMAL1 expression. These findings underline the importance of the circadian clock for IVD homeostasis and provide a potentially effective therapeutic strategy for IDD.

References

[1] Patke A, Young MW, Axelrod S. Molecular mechanisms and physiological importance of circadian rhythms. Nat. Rev. Mol. Cell Biol., 2020, 21: 67-84

[2] Dudek M, Meng QJ. Running on time: the role of circadian clocks in the musculoskeletal system. Biochem J., 2014, 463: 1-8

[3] Dibner C, Schibler U, Albrecht U. The mammalian circadian timing system: organization and coordination of central and peripheral clocks. Annu. Rev. Physiol., 2010, 72: 517-549

[4] Dudek M et al. The chondrocyte clock gene Bmal1 controls cartilage homeostasis and integrity. J. Clin. Invest., 2016, 126: 365-376

[5] McDearmon EL et al. Dissecting the functions of the mammalian clock protein BMAL1 by tissue-specific rescue in mice. Science, 2006, 314: 1304-1308

[6] Firsov D, Bonny O. Circadian rhythms and the kidney. Nat. Rev. Nephrol., 2018, 14: 626-635

[7] Asher G et al. SIRT1 regulates circadian clock gene expression through PER2 deacetylation. Cell, 2008, 134: 317-328

[8] Yang G et al. Timing of expression of the core clock gene Bmal1 influences its effects on aging and survival. Sci. Transl. Med., 2016, 8: 324ra316

[9] Lutshumba J et al. Deletion of BMAL1 in Smooth Muscle Cells Protects Mice From Abdominal Aortic Aneurysms. Arterioscler Thromb. Vasc. Biol., 2018, 38: 1063-1075

[10] Zhou B et al. CLOCK/BMAL1 regulates circadian change of mouse hepatic insulin sensitivity by SIRT1. Hepatology, 2014, 59: 2196-2206

[11] Wang Y et al. Disruption of the circadian clock alters antioxidative defense via the SIRT1-BMAL1 pathway in 6-OHDA-induced models of Parkinson’s disease. Oxid. Med. Cell Longev., 2018, 2018: 4854732

[12] Kc R et al. Environmental disruption of circadian rhythm predisposes mice to osteoarthritis-like changes in knee joint. J. Cell Physiol., 2015, 230: 2174-2183

[13] Kelleher FC, Rao A, Maguire A. Circadian molecular clocks and cancer. Cancer Lett., 2014, 342: 9-18

[14] Thummadi NB, Jagota A. Aging renders desynchronization between clock and immune genes in male Wistar rat kidney: chronobiotic role of curcumin. Biogerontology, 2019, 20: 515-532

[15] Clark S, Horton R. Low back pain: a major global challenge. Lancet, 2018, 391: 2302

[16] Cieza A et al. Global estimates of the need for rehabilitation based on the Global Burden of Disease study 2019: a systematic analysis for the Global Burden of Disease Study 2019. Lancet, 2021, 396: 2006-2017

[17] Diseases GBD, Injuries C. Global burden of 369 diseases and injuries in 204 countries and territories, 1990-2019: a systematic analysis for the Global Burden of Disease Study 2019. Lancet, 2020, 396: 1204-1222

[18] 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

[19] Bian Q et al. Mechanosignaling activation of TGFbeta maintains intervertebral disc homeostasis. Bone Res., 2017, 5: 17008

[20] Zheng L et al. Ciliary parathyroid hormone signaling activates transforming growth factor-beta to maintain intervertebral disc homeostasis during aging. Bone Res., 2018, 6: 21

[21] He, R. et al. HIF1A Alleviates compression-induced apoptosis of nucleus pulposus derived stem cells via upregulating autophagy. Autophagy 17, 3338–3360 (2021).

[22] Kang L et al. The mitochondria-targeted anti-oxidant MitoQ protects against intervertebral disc degeneration by ameliorating mitochondrial dysfunction and redox imbalance. Cell Prolif., 2020, 53: e12779

[23] Ke W et al. The distinct roles of myosin IIA and IIB under compression stress in nucleus pulposus cells. Cell Prolif., 2021, 54: e12987

[24] Wang Y et al. SIRT1 alleviates high-magnitude compression-induced senescence in nucleus pulposus cells via PINK1-dependent mitophagy. Aging, 2020, 12: 16126-16141

[25] Phillips KL, Jordan-Mahy N, Nicklin MJ, Le Maitre CL. Interleukin-1 receptor antagonist deficient mice provide insights into pathogenesis of human intervertebral disc degeneration. Ann. Rheum. Dis., 2013, 72: 1860-1867

[26] Malko JA, Hutton WC, Fajman WA. An in vivo magnetic resonance imaging study of changes in the volume (and fluid content) of the lumbar intervertebral discs during a simulated diurnal load cycle. Spine (Philos. Pa 1976), 1999, 24: 1015-1022

[27] Morris H, Goncalves CF, Dudek M, Hoyland J, Meng QJ. Tissue physiology revolving around the clock: circadian rhythms as exemplified by the intervertebral disc. Ann. Rheum. Dis., 2021, 80: 828-839

[28] Kaila-Kangas L et al. Sleep disturbances as predictors of hospitalization for back disorders-a 28-year follow-up of industrial employees. Spine (Philos. Pa 1976), 2006, 31: 51-56

[29] Zhao I, Bogossian F, Turner C. The effects of shift work and interaction between shift work and overweight/obesity on low back pain in nurses: results from a longitudinal study. J. Occup. Environ. Med, 2012, 54: 820-825

[30] Dudek M et al. The intervertebral disc contains intrinsic circadian clocks that are regulated by age and cytokines and linked to degeneration. Ann. Rheum. Dis., 2017, 76: 576-584

[31] Kim, Y. K., Kang, D., Lee, I. & Kim, S. Y. Differences in the incidence of symptomatic cervical and lumbar disc herniation according to age, sex and national health insurance eligibility: a pilot study on the disease’s association with work. Int. J. Environ. Res. Public Health 15, 2094 (2018).

[32] Yang S, Zhang F, Ma J, Ding W. Intervertebral disc ageing and degeneration: the antiapoptotic effect of oestrogen. Ageing Res. Rev., 2020, 57: 100978

[33] Burridge K, Monaghan-Benson E, Graham DM. Mechanotransduction: from the cell surface to the nucleus via RhoA. Philos. Trans. R. Soc. Lond. B Biol. Sci., 2019, 374: 20180229

[34] Lessey EC, Guilluy C, Burridge K. From mechanical force to RhoA activation. Biochemistry, 2012, 51: 7420-7432

[35] Appleton CT, Usmani SE, Mort JS, Beier F. Rho/ROCK and MEK/ERK activation by transforming growth factor-alpha induces articular cartilage degradation. Lab. Invest, 2010, 90: 20-30

[36] Liang J et al. Leptin-mediated cytoskeletal remodeling in chondrocytes occurs via the RhoA/ROCK pathway. J. Orthop. Res., 2011, 29: 369-374

[37] Vriend J, Reiter RJ. Melatonin feedback on clock genes: a theory involving the proteasome. J. Pineal Res, 2015, 58: 1-11

[38] Xu Y et al. Melatonin attenuates choroidal neovascularization by regulating macrophage/microglia polarization via inhibition of RhoA/ROCK signaling pathway. J. Pineal Res., 2020, 69: e12660

[39] Balsalobre A et al. Resetting of circadian time in peripheral tissues by glucocorticoid signaling. Science, 2000, 289: 2344-2347

[40] Gossan N et al. The circadian clock in murine chondrocytes regulates genes controlling key aspects of cartilage homeostasis. Arthritis Rheum., 2013, 65: 2334-2345

[41] Chen Y et al. Age-related BMAL1 change affects mouse bone marrow stromal cell proliferation and osteo-differentiation potential. Arch. Med. Sci., 2012, 8: 30-38

[42] An K et al. A circadian rhythm-gated subcortical pathway for nighttime-light-induced depressive-like behaviors in mice. Nat. Neurosci., 2020, 23: 869-880

[43] Hayter EA et al. Distinct circadian mechanisms govern cardiac rhythms and susceptibility to arrhythmia. Nat. Commun., 2021, 12

[44] Maury E, Navez B, Brichard SM. Circadian clock dysfunction in human omental fat links obesity to metabolic inflammation. Nat. Commun., 2021, 12

[45] Yang W et al. Clock gene Bmal1 modulates human cartilage gene expression by crosstalk with Sirt1. Endocrinology, 2016, 157: 3096-3107

[46] Yang N et al. Cellular mechano-environment regulates the mammary circadian clock. Nat. Commun., 2017, 8

[47] Allada R, Bass J. Circadian mechanisms in medicine. N. Engl. J. Med., 2021, 384: 550-561

[48] Hermida RC, Fernandez JR, Mojon A, Hygia Project I. Chronotherapy of hypertension, asleep ambulatory blood pressure, and glaucoma. Eur. Heart J., 2020, 41: 1605

[49] Ruan W, Yuan X, Eltzschig HK. Circadian rhythm as a therapeutic target. Nat. Rev. Drug Disco., 2021, 20: 287-307

[50] Sancar, A. & Van Gelder, R. N. Clocks, cancer, and chronochemotherapy. Science 371, eabb0738 (2021).

[51] Saran AR, Dave S, Zarrinpar A. Circadian rhythms in the pathogenesis and treatment of fatty liver disease. Gastroenterology, 2020, 158: 1948-1966 e1941

[52] Chen F et al. Melatonin alleviates intervertebral disc degeneration by disrupting the IL-1beta/NF-kappaB-NLRP3 inflammasome positive feedback loop. Bone Res., 2020, 8: 10

[53] Dominguez-Rodriguez A, Abreu-Gonzalez P, Sanchez-Sanchez JJ, Kaski JC, Reiter RJ. Melatonin and circadian biology in human cardiovascular disease. J. Pineal Res., 2010, 49: 14-22

[54] Fang J et al. Melatonin prevents senescence of canine adipose-derived mesenchymal stem cells through activating NRF2 and inhibiting ER stress. Aging (Albany NY), 2018, 10: 2954-2972

[55] Heo JI et al. Melatonin improves insulin resistance and hepatic steatosis through attenuation of alpha-2-HS-glycoprotein. J. Pineal Res., 2018, 65: e12493

[56] Yoo SH et al. PERIOD2::LUCIFERASE real-time reporting of circadian dynamics reveals persistent circadian oscillations in mouse peripheral tissues. Proc. Natl. Acad. Sci. USA, 2004, 101: 5339-5346

[57] Zheng, Y. et al. Characterization of Cre recombinase mouse lines enabling cell type-specific targeting of postnatal intervertebral discs. J. Cell Physiol., https://doi.org/10.1002/jcp.28166 (2019). Epub ahead of print.

[58] Wang D et al. Quercetin suppresses apoptosis and attenuates intervertebral disc degeneration via the SIRT1-autophagy pathway. Front. Cell Dev. Biol., 2020, 8: 613006

[59] Xu X et al. Progerin accumulation in nucleus pulposus cells impairs mitochondrial function and induces intervertebral disc degeneration and therapeutic effects of sulforaphane. Theranostics, 2019, 9: 2252-2267

[60] Stannard JT et al. Development of a whole organ culture model for intervertebral disc disease. J. Orthop. Transl., 2016, 5: 1-8

[61] Liu, J. W. et al. An in vitro organ culture model of the murine intervertebral disc. J. Vis. Exp. 122, 55437 (2017).

[62] Sun Z et al. CK8 phosphorylation induced by compressive loads underlies the downregulation of CK8 in human disc degeneration by activating protein kinase C. Lab. Invest., 2013, 93: 1323-1330

[63] Sun Z et al. Notochordal-cell-derived exosomes induced by compressive load inhibit angiogenesis via the miR-140-5p/Wnt/beta-catenin axis. Mol. Ther. Nucleic Acids, 2020, 22: 1092-1106

[64] Sun Z et al. Adipose-derived stromal cells protect intervertebral disc cells in compression: implications for stem cell regenerative disc therapy. Int. J. Biol. Sci., 2015, 11: 133-143

[65] Hirata H et al. A rat tail temporary static compression model reproduces different stages of intervertebral disc degeneration with decreased notochordal cell phenotype. J. Orthop. Res., 2014, 32: 455-463

[66] Iatridis JC, Mente PL, Stokes IA, Aronsson DD, Alini M. Compression-induced changes in intervertebral disc properties in a rat tail model. Spine (Philos. Pa 1976), 1999, 24: 996-1002

[67] Yan Z et al. Static compression induces ECM remodeling and integrin alpha2beta1 expression and signaling in a rat tail caudal intervertebral disc degeneration model. Spine (Philos. Pa 1976), 2017, 42: E448-E458

[68] Yurube T et al. Rat tail static compression model mimics extracellular matrix metabolic imbalances of matrix metalloproteinases, aggrecanases, and tissue inhibitors of metalloproteinases in intervertebral disc degeneration. Arthritis Res. Ther., 2012, 14: R51

[69] Han B et al. A simple disc degeneration model induced by percutaneous needle puncture in the rat tail. Spine (Philos. Pa 1976), 2008, 33: 1925-1934

Funding

National Natural Science Foundation of China (National Science Foundation of China)(81730065)

The Medical Research Council (UK) MR/T016744/1 and MR/P010709/1 and the Versus Arthritis Senior Research Fellowship Award 20875.