BACKGROUND CONTEXT Destruction of extracellular matrix (ECM) leads to intervertebral disc degeneration (IDD), which underlies many spine-related disorders. Matrix metalloproteinases (MMPs), and disintegrins and metalloproteinases with thrombospondin motifs (ADAMTSs) are believed to be the major proteolytic enzymes responsible for ECM degradation in the intervertebral disc (IVD). PURPOSE To summarize the current literature on gene expression and regulation of MMPs, ADAMTSs, and tissue inhibitors of metalloproteinases (TIMPs) in IVD aging and IDD. METHODS A comprehensive literature review of gene expression of MMP, ADAMTS, and TIMP in human IDD and reported studies on regulatory factors controlling their expressions and activities in both human and animal model systems. RESULTS Upregulation of specific MMPs (MMP-1, -2, -3, -7, -8, -10, and -13) and ADAMTS (ADAMTS-1, -4, and -15) were reported in human degenerated IVDs. However, it is still unclear from conflicting published studies whether the expression of ADAMTS-5, the predominant aggrecanase, is increased with IDD. Tissue inhibitors of metalloproteinase-3 is downregulated, whereas TIMP-1 is upregulated in human degenerated IVDs relative to nondegenerated IVDs. Numerous studies indicate that the expression levels of MMP and ADAMTS are modulated by a combination of many factors, including mechanical, inflammatory, and oxidative stress, some of which are mediated in part through the p38 mitogen-activated protein kinase pathway. Genetic predisposition also plays an important role in determining gene expression of MMP-1, -2, -3, and -9. CONCLUSIONS Upregulation of MMP and ADAMTS expression and enzymatic activity is implicated in disc ECM destruction, leading to the development of IDD. Future IDD therapeutics depends on identifying specific MMPs and ADAMTSs whose dysregulation result in pathological proteolysis of disc ECM.

Selective interference of mTORC1/RAPTOR protects against inflammation-induced apoptosis, senescence, and matrix catabolism possibly through Akt and autophagy induction in human disc cells.

IntroductionThe longitudinal degradation mechanism of extracellular matrix (ECM) in the interbertebral disc remains unclear. Our objective was to elucidate catabolic and anabolic gene expression profiles and their balances in intervertebral disc degeneration using a static compression model.MethodsForty-eight 12-week-old male Sprague-Dawley rat tails were instrumented with an Ilizarov-type device with springs and loaded statically at 1.3 MPa for up to 56 days. Experimental loaded and distal-unloaded control discs were harvested and analyzed by real-time reverse transcription-polymerase chain reaction (PCR) messenger RNA quantification for catabolic genes [matrix metalloproteinase (MMP)-1a, MMP-2, MMP-3, MMP-7, MMP-9, MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4, and ADAMTS-5], anti-catabolic genes [tissue inhibitor of metalloproteinases (TIMP)-1, TIMP-2, and TIMP-3], ECM genes [aggrecan-1, collagen type 1-α1, and collagen type 2-α1], and pro-inflammatory cytokine genes [tumor necrosis factor (TNF)-α, interleukin (IL)-1α, IL-1β, and IL-6]. Immunohistochemistry for MMP-3, ADAMTS-4, ADAMTS-5, TIMP-1, TIMP-2, and TIMP-3 was performed to assess their protein expression level and distribution. The presence of MMP- and aggrecanase-cleaved aggrecan neoepitopes was similarly investigated to evaluate aggrecanolytic activity.ResultsQuantitative PCR demonstrated up-regulation of all MMPs and ADAMTS-4 but not ADAMTS-5. TIMP-1 and TIMP-2 were almost unchanged while TIMP-3 was down-regulated. Down-regulation of aggrecan-1 and collagen type 2-α1 and up-regulation of collagen type 1-α1 were observed. Despite TNF-α elevation, ILs developed little to no up-regulation. Immunohistochemistry showed, in the nucleus pulposus, the percentage of immunopositive cells of MMP-cleaved aggrecan neoepitope increased from 7 through 56 days with increased MMP-3 and decreased TIMP-1 and TIMP-2 immunopositivity. The percentage of immunopositive cells of aggrecanase-cleaved aggrecan neoepitope increased at 7 and 28 days only with decreased TIMP-3 immunopositivity. In the annulus fibrosus, MMP-cleaved aggrecan neoepitope presented much the same expression pattern. Aggrecanase-cleaved aggrecan neoepitope increased at 7 and 28 days only with increased ADAMTS-4 and ADAMTS-5 immunopositivity.ConclusionsThis rat tail sustained static compression model mimics ECM metabolic imbalances of MMPs, aggrecanases, and TIMPs in human degenerative discs. A dominant imbalance of MMP-3/TIMP-1 and TIMP-2 relative to ADAMTS-4 and ADAMTS-5/TIMP-3 signifies an advanced stage of intervertebral disc degeneration.

Objective: The mammalian target of rapamycin (mTOR) is a serine/threonine kinase that integrates nutrients to execute cell growth. We hypothesized that mTOR is influential in the intervertebral discdlargest avascular, low-nutrient organ. Our objective was to identify the optimal mTOR inhibitor for treating human degenerative disc disease. Design: mTOR complex 1 (mTORC1) regulates p70/ribosomal S6 kinase (p70/S6K), negatively regulates autophagy, and is controlled by Akt. Akt is controlled by phosphatidylinositol 3-kinase (PI3K) and mTOR complex 2 (mTORC2). mTORC1 inhibitorsdrapamycin, temsirolimus, everolimus, and curcumin, mTORC1&mTORC2 inhibitordINK-128, PI3K&mTOR inhibitordNVP-BEZ235, and Akt inhibitordMK-2206dwere applied to human disc nucleus pulposus (NP) cells. mTOR signaling, autophagy, apoptosis, senescence, and matrix metabolism were evaluated. Results: mTORC1 inhibitors decreased p70/S6K but increased Akt phosphorylation, promoted autophagy with light chain 3 (LC3)-II increases and p62/sequestosome 1 (p62/SQSTM1) decreases, and suppressed pro-inflammatory interleukin-1 beta (IL-1b)-induced apoptotic terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) positivity (versus rapamycin, 95% confidence interval (CI) À0.431 to À0.194; temsirolimus, 95% CI À0.529 to À0.292; everolimus, 95% CI À0.477 to À0.241; curcumin, 95% CI À0.248 to À0.011) and poly (ADP-ribose) polymerase (PARP) and caspase-9 cleavage, senescent senescence-associated beta-galactosidase (SA-b-gal) positivity (versus rapamycin, 95% CI À0.437 to À0.230; temsirolimus, 95% CI À0.534 to À0.327; everolimus, 95% CI À0.485 to À0.278; curcumin, 95% CI À0.210 to À0.003) and p16/INK4A expression, and catabolic matrix metalloproteinase (MMP) release and activation. Meanwhile, dual mTOR inhibitors decreased p70/S6K and Akt phosphorylation without enhanced autophagy and suppressed apoptosis, senescence, and matrix catabolism. MK-2206 counteracted protective effects of temsirolimus. Additional disc-tissue analysis found relevance of mTOR signaling to degeneration grades. Conclusion: mTORC1 inhibitorsdnotably temsirolimus with an improved water solubilitydbut not dual mTOR inhibitors protect against inflammation-induced apoptosis, senescence, and matrix catabolism in human disc cells, which depends on Akt and autophagy induction.