LEF1‐mediated MMP13 gene expression is repressed by SIRT1 in human chondrocytes

Abstract

Reduced SIRT1 activity and levels during osteoarthritis (OA) promote gradual loss of cartilage. Loss of cartilage matrix is accompanied by an increase in matrix metalloproteinase (MMP) 13, partially because of enhanced LEF1 transcriptional activity. In this study, we assessed the role of SIRT1 in LEF1-mediated MMP13 gene expression in human OA chondrocytes. Results showed that MMP13 protein levels and enzymatic activity decreased significantly during SIRT1 overexpression or activation by resveratrol. Conversely, MMP13 gene expression was reduced in chondrocytes transfected with SIRT1 siRNA or treated with nicotinamide (NAM), a sirtuin inhibitor. Chondrocytes challenged with IL-1b, a cytokine involved in OA pathogenesis, enhanced LEF1 protein levels and gene expression, resulting in increased MMP13 gene expression; however, overexpression of SIRT1 during IL-1b challenge impeded LEF1 levels and MMP13 gene expression. Previous reports showed that LEF1 binds to the MMP13 promoter and transactivates its expression, but we observed that SIRT1 repressed LEF1 protein and mRNA expression, ultimately reducing LEF1 transcriptional activity, as judged by luciferase assay. Finally, mouse articular cartilage from Sirt1 2/2 presented increased LEF1 and MMP13 protein levels, similar to human OA cartilage. Thus, demonstrating for the first time that SIRT1 represses MMP13 in human OA chondrocytes, which appears to be mediated, at least in part, through repression of the transcription factor LEF1, a known modulator of MMP13 gene expression.-Elayyan, J., Lee, E.-J., Gabay, O., Smith, C. A., Qiq, O., Reich, E., Mobasheri, A., Henrotin, Y., Kimber, S. J., Dvir-Ginzberg, M. LEF1-mediated MMP13 gene expression is repressed by SIRT1 in human chondrocytes. FASEB J. 31, 3116-3125 (2017). www.fasebj.orgArticular cartilage undergoes age-related degenerative changes leading to the joint disease osteoarthritis (OA). Gradual loss of hyaline joint cartilage during OA is evoked through chronic synovitis, which involves the accumulation of proinflammatory cytokines, as IL1b, TNF-a, and IL6 within the joint (1-5), subsequently leading to a steady increase in matrix metalloproteinases (MMPs) and a disintegrin and MMP with thrombospondin motifs (ADAMTS) proteins, which further promote cartilage destruction (6-12). Recent experimental attempts ABBREVIATIONS: ACAN, aggrecan gene; ADAMTS, a disintegrin and metalloproteinase with thrombospondin-like motifs; BMP, bone morphogenic protein; ChIP, chromatin immunoprecipitation; COL2A1, collagen-2 a-1 gene; FBS, fetal bovine serum; FGF, fibroblast growth factor; GDF, growth differentiation factor; GSK3b, glycogen synthase kinase-3b; hESC, human embryonic stem cell; KO, knockout; LEF, lymphoid enhancer factor; MMP, matrix metalloproteinase; NAM, nicotinamide adenine mononucleotide; OA, osteoarthritis; Res, resveratrol; qPCR, quantitative PCR; siRNA, small interfering RNA; SIRT1, silent mating type information regulation 2 homolog 1 (sirtuin-1); SOX9, sex-determining-region-on-the-Y-chromosome-relate...

A predicted unstructured C-terminal loop domain in SIRT1 is required for cathepsin B cleavage

“…5C,D). However, in contrast to cells overexpressing flSIRT1 or ΔSIRT1, cells overexpressing 75SIRT1 demonstrated a 20% increase in H3 acetylation and a twofold increase in p65 acetylation, indicating that 75SIRT1 is enzymatically inactive, as previously reported for chondrocytes and cartilage (Elayyan et al, 2017;Oppenheimer et al, 2012). Of note, pcDNA (expression vector) controls possess similar substrate acetylation states to those presented for flSIRT1 and ΔSIRT1, which might be related to possible saturation of the deacetylation activity beyond substrate availability.…”

“…Why the enzymatic activity of SIRT1 is disrupted during aging is, as yet, unclear, although several (not mutually exclusive) factors have been hypothesized to affect this activity; most notably, the availability of NAD in the cell and various translational and posttranslational modifications, such as alternative splicing, SIRT1 phosphorylation, ubiquitination or SUMOylation (Lynch et al, 2010;Sasaki et al, 2008;Peng et al, 2015;Yang et al, 2007). We previously found that, in human cartilage samples and mouse models of osteoarthritis (OA; an age-associated disease), inflammation induces reduced SIRT1 activity via its truncation by the lysosomal cysteine protease cathepsin B (Dvir-Ginzberg et al, 2011;Elayyan et al, 2017). Although cathepsins are typically bound to the acidic lumen of the lysosome (where they participate in protein recycling), exposure to pro-inflammatory mediators, such as TNFα, can drive lysosomal permeability (Guicciardi et al, 2000) and the subsequent release of cathepsins to the cytoplasm and nucleus, where they target cellular proteins and alter various cellular circuits, including epigenetic reprogramming and apoptosis (Dvir-Ginzberg et al, 2011;Duncan et al, 2008;Droga-Mazovec et al, 2008;Oppenheimer et al, 2012;Cirman et al, 2004).…”

Homocysteine causes dysfunction of chondrocytes and oxidative stress through repression of SIRT1/AMPK pathway: A possible link between hyperhomocysteinemia and osteoarthritis

“…Also, the aberrant SIRT1/AMPK/PGC-1α signaling was found in OA chondrocytes [11] . It has been shown that the diminished SIRT1 in OA [12] was critical for chondrocyte survival via downregulation of a potent pro-apoptotic protein, PTP1B [13] , or suppression of cartilage-degrading enzymes through modulation of the NF-κB pathway [14] , [15] . Given that Hcy is involved in mitochondrial dysfunction and downregulation of SIRT1/AMPK/PGC-1α may lead to OA, we sought to investigate whether elevation of Hcy caused mitochondrial dysfunction in cartilage via inhibition of SIRT1.…”

Osteoarthritis year in review 2017: biology

“…Later stages of OA with loss of expression of chondrocyte SIRT1 corresponded to higher levels of p53 and p53 acetylation, relative to age-matched controls (trauma patients without OA). In addition, Elayyan and colleagues provided evidence for a mechanistic link between SIRT1 and control of MMP-13 gene expression mediated by the LEF1 transcription factor in human OA chondrocytes 42 . Overexpression of SIRT1 resulted in suppression of MMP-13 and LEF1 as well as their induction by IL-1b.…”