ADAMTS5 Deficiency Protects Mice From Chronic Tobacco Smoking-induced Intervertebral Disc Degeneration

Abstract

Study Design ADAMTS5-deficient and WT mice were chronically exposed to tobacco smoke to investigate effects on intervertebral disc degeneration (IDD). Objective To demonstrate a role for ADAMTS5 in mediating tobacco smoking-induced IDD Summary of Background Data We previously demonstrated that chronic tobacco smoking causes IDD in mice due, in part, to proteolytic destruction of disc aggrecan. However, it was unknown which matrix proteinase(s) drive these detrimental effects. Methods Three-month old Wt (C57BL/6) and ADAMTS5-/- mice were chronically exposed to tobacco smoke (4 cigarettes/day, 5 day/week for 6 months). ADAMTS-mediated cleavage of disc aggrecan was analyzed by Western blot. Disc total glycosaminoglycan (GAG) content was assessed by dimethyl methylene blue assay and safranin O/fast green histology. Vertebral osteoporosity was measured by micro-computed tomography. Human nucleus pulposus (hNP) cell cultures were also exposed directly to tobacco smoke extract (TSE), a condensate containing the water-soluble compounds inhaled by smokers, to measure ADAMTS5 expression and ADAMTS-mediated cleavage of aggrecan. Activation of NF-κB, a family of transcription factors essential for modulating the cellular response to stress, was measured by immunofluorescence assay. Results Genetic depletion of ADAMTS5 prevented vertebral bone loss, substantially reduced loss of disc GAG content, and completely obviated ADAMTS-mediated proteolysis of disc aggrecan within its interglobular domain (IGD) in mice following exposure to tobacco smoke. hNP cell cultures exposed to TSE also resulted in upregulation of ADAMTS5 protein expression and a concomitant increase in ADAMTS-mediated cleavage within aggrecan IGD. Activation of NF-κB, known to be required for ADAMTS5 gene expression, was observed in both TSE-treated hNP cell cultures and disc tissue of tobacco smoke-exposed mice. Discussion The findings demonstrate that ADAMTS5 is the primary aggrecanase mediating smoking-induced disc aggrecanolysis and IDD. Mouse models of chronic tobacco smoking are important and useful for probing the mechanisms of disc aggrecan catabolism and IDD.

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“…No. 31460, Thermo Fisher; Ngo, Pohl et al, ). Expression of p53 protein was examined by extracting protein from spine discs using T‐PER Tissue Protein Extraction Reagent with proteinase inhibitor cocktail as per the manufacturer's instructions (Cat.…”

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“…Activation of other degrading enzymes may have been involved in the decrease of these matrix proteins. Recently, Ngo et al demonstrated that ADAMTS5 is the primary aggrecanase mediating smoking-induced IVD degeneration in mouse models of chronic tobacco smoking using ADAMTS5 -deficient mice [32]. Thus, ADAMTS5 may also be the enzyme responsible for the degradation in the current study.…”

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