Association between the abnormal expression of matrix‐degrading enzymes by human osteoarthritic chondrocytes and demethylation of specific CpG sites in the promoter regions

Objective

To investigate whether the abnormal expression of matrix metalloproteinases (MMPs) 3, 9, and 13 and ADAMTS‐4 by human osteoarthritic (OA) chondrocytes is associated with epigenetic “unsilencing.”

Methods

Cartilage was obtained from the femoral heads of 16 patients with OA and 10 control patients with femoral neck fracture. Chondrocytes with abnormal enzyme expression were immunolocalized. DNA was extracted, and the methylation status of the promoter regions of MMPs 3, 9, and 13 and ADAMTS‐4 was analyzed with methylation‐sensitive restriction enzymes, followed by polymerase chain reaction amplification.

Results

Very few chondrocytes from control cartilage expressed the degrading enzymes, whereas all clonal chondrocytes from late‐stage OA cartilage were immunopositive. The overall percentage of nonmethylated sites was increased in OA patients (48.6%) compared with controls (20.1%): 20% versus 4% for MMP‐13, 81% versus 47% for MMP‐9, 57% versus 30% for MMP‐3, and 48% versus 0% for ADAMTS‐4. Not all CpG sites were equally susceptible to loss of methylation. Some sites were uniformly methylated, whereas in others, methylation was generally absent. For each enzyme, there was 1 specific CpG site where the demethylation in OA patients was significantly higher than that in controls: at −110 for MMP‐13, −36 for MMP‐9, −635 for MMP‐3, and −753 for ADAMTS‐4.

Conclusion

This study provides the first evidence that altered synthesis of cartilage‐degrading enzymes by late‐stage OA chondrocytes may have resulted from epigenetic changes in the methylation status of CpG sites in the promoter regions of these enzymes. These changes, which are clonally transmitted to daughter cells, may contribute to the development of OA.

     

Increased synovial tissue NF‐κB1 expression at sites adjacent to the cartilage–pannus junction in rheumatoid arthritis

Objective

To compare the expression of the Rel/NF‐κB subunits, NF‐κB1 (p50) and RelA (p65), in paired synovial tissue samples selected from sites adjacent to and remote from the cartilage–pannus junction (CPJ) in patients with inflammatory arthritis.

Methods

Synovial tissue was selected at arthroscopy from sites adjacent to the CPJ and from the suprapatellar pouch of patients who were referred to an early arthritis clinic. Tissue samples from patients with osteoarthritis (OA) undergoing knee arthroplasty were also studied. Rel/NF‐κB subunit activation and expression were measured by electrophoretic mobility shift assay and supershift analyses and by immunohistochemistry.

Results

Tissue samples were obtained from 10 patients with rheumatoid arthritis (RA), 7 with a seronegative arthropathy (SnA), and 6 with OA. Rel/NF‐κB was abundantly expressed in all samples. In both RA and SnA synovial tissue, the absolute number of NF‐κB1+ cells at the CPJ was significantly higher than at non‐CPJ sites (P = 0.006 and P = 0.02, respectively). The proportion of cells expressing NF‐κB1 was also significantly higher at the CPJ compared with non‐CPJ sites (P = 0.003 in RA, P = 0.009 in SnA). The numbers of RelA+ cells were consistently lower throughout. In RA synovial tissue, but not in SnA synovial tissue, both the absolute number and the proportion of RelA+ cells were significantly higher at the CPJ than at non‐CPJ sites (P = 0.003 and P = 0.01, respectively). In OA synovial tissue, the numbers of cells expressing NF‐κB1 and RelA were similar to those observed at the non‐CPJ sites in all inflammatory tissues studied.

Conclusion

In this study of early inflammatory arthritis, expression of NF‐κB1 in synovial tissue was highest at sites most likely to be associated with joint erosion. These observations are consistent with a critical role of NF‐κB1 in joint destruction, and support the rationale for specific therapeutic inhibition of NF‐κB in RA.

     

The peroxisome proliferator–activated receptor γ agonist pioglitazone reduces the development of cartilage lesions in an experimental dog model of osteoarthritis: In vivo protective effects mediated through the inhibition of key signaling and catabolic pathways

Objective

Emerging evidence indicates that peroxisome proliferator–activated receptor γ (PPARγ) may have protective effects in osteoarthritis (OA). The aim of this study was to evaluate the in vivo effect of a PPARγ agonist, pioglitazone, on the development of lesions in a canine model of OA, and to explore the influence of pioglitazone on the major signaling and metabolic pathways involved in OA pathophysiologic changes.

Methods

OA was surgically induced in dogs by sectioning of the anterior cruciate ligament. The dogs were then randomly divided into 3 treatment groups in which they were administered either placebo, 15 mg/day pioglitazone, or 30 mg/day pioglitazone orally for 8 weeks. Following treatment, the severity of cartilage lesions was scored. Cartilage specimens were processed for histologic and immunohistochemical evaluations; specific antibodies were used to study the levels of matrix metalloproteinase 1 (MMP‐1), ADAMTS‐5, and inducible nitric oxide synthase (iNOS), as well as phosphorylated MAPKs ERK‐1/2, p38, JNK, and NF‐κB p65.

Results

Pioglitazone reduced the development of cartilage lesions in a dose‐dependent manner, with the highest dosage producing a statistically significant change (P < 0.05). This decrease in lesions correlated with lower cartilage histologic scores. In addition, pioglitazone significantly reduced the synthesis of the key OA mediators MMP‐1, ADAMTS‐5, and iNOS and, at the same time, inhibited the activation of the signaling pathways for MAPKs ERK‐1/2, p38, and NF‐κB.

Conclusion

These results indicate the efficacy of pioglitazone in reducing cartilage lesions in vivo. The results also provide new and interesting insights into a therapeutic intervention for OA in which PPARγ activation can inhibit major signaling pathways of inflammation and reduce the synthesis of cartilage catabolic factors responsible for articular cartilage degradation.

     

Tumor Necrosis Factor α–Induced MicroRNA‐18a Activates Rheumatoid Arthritis Synovial Fibroblasts Through a Feedback Loop in NF‐κB Signaling

Objective

To elucidate whether the microRNA (miRNA) cluster miR‐17–92 contributes to the activated phenotype of rheumatoid arthritis synovial fibroblasts (RASFs).

Methods

RASFs were stimulated with tumor necrosis factor α (TNFα), and the expression and regulation of the miR‐17–92 cluster were studied using real‐time quantitative PCR (PCR) and promoter activity assays. RASFs were transfected with single precursor molecules of miRNAs from miR‐17–92 and the expression of matrix‐degrading enzymes and cytokines was measured by quantitative PCR and enzyme‐linked immunosorbent assay. Potential miRNA targets were identified by computational prediction and were validated using reporter gene assays and Western blotting. The activity of NF‐κB signaling was determined by reporter gene assays.

Results

We found that TNFα induces the expression of miR‐17–92 in RASFs in an NF‐κB–dependent manner. Transfection of RASFs with precursor molecules of single members of miR‐17–92 revealed significantly increased expression levels of matrix‐degrading enzymes, proinflammatory cytokines, and chemokines in precursor miR‐18a (pre‐miR‐18a)–transfected RASFs. Using reporter gene assays, we identified the NF‐κB pathway inhibitor TNFα‐induced protein 3 as a new target of miR‐18a. In addition, pre‐miR‐18a–transfected RASFs showed stronger activation of NF‐κB signaling, both constitutively and in response to TNFα stimulation.

Conclusion

Our data suggest that the miR‐17–92–derived miR‐18a contributes to cartilage destruction and chronic inflammation in the joint through a positive feedback loop in NF‐κB signaling, with concomitant up‐regulation of matrix‐degrading enzymes and mediators of inflammation in RASFs.

     

A20 suppresses inflammatory responses and bone destruction in human fibroblast‐like synoviocytes and in mice with collagen‐induced arthritis

Objective

Nuclear factor‐κB (NF‐κB) has been implicated as a therapeutic target for the treatment of rheumatoid arthritis (RA). The purpose of this study was to determine whether A20, a universal inhibitor of NF‐κB, might have antiarthritic effects.

Methods

An adenovirus containing A20 complementary DNA (AdA20) was used to deliver A20 to human rheumatoid fibroblast‐like synoviocytes (FLS) in vitro as well as to mice with collagen‐induced arthritis (CIA) in vivo via intraarticular injection into the ankle joints bilaterally.

Results

In vitro experiments demonstrated that AdA20 suppressed NF‐κB activation, chemokine production, and matrix metalloproteinase secretion induced by tumor necrosis factor α in FLS. Mice with CIA that were treated with AdA20 had a lower cumulative disease incidence and severity of arthritis, based on hind paw thickness, radiologic and histopathologic findings, and inflammatory cytokine levels, than did control virus–injected mice. The protective effects of AdA20 were mediated by the inhibition of the NF‐κB signaling pathway. The severity of arthritis was also significantly decreased in the untreated front paws, indicating a beneficial systemic effect of local suppression of NF‐κB. Surprisingly, mice treated with AdA20 after the onset of CIA had significantly decreased arthritis severity from the onset of clinical signs to the end of the study.

Conclusion

These results suggest that using A20 to block the NF‐κB pathway in rheumatoid joints reduces both the inflammatory response and the tissue destruction. The development of an immunoregulatory strategy based on A20 may therefore have therapeutic potential in the treatment of RA.

     

RhoA‐mediated,tumor necrosis factor α–induced activation of NF‐κB in rheumatoid synoviocytes: Inhibitory effect of simvastatin

Objective

Increasing evidence indicates that RhoA may play a central role in the inflammatory response. This study was conducted to examine the role of RhoA in mediating the activation of NF‐κB in tumor necrosis factor α (TNFα)–stimulated rheumatoid synoviocytes, and to evaluate the modulatory effects of statins on the TNFα‐induced activation of RhoA and NF‐κB and the secretion of proinflammatory cytokines by rheumatoid synoviocytes.

Methods

Rheumatoid synoviocytes obtained from patients with active rheumatoid arthritis were stimulated with TNFα and incubated with simvastatin (SMV) (1 μM). RhoA activity was assessed by a pull‐down assay. NF‐κB DNA binding activity and nuclear translocation of NF‐κB were measured by a sensitive multiwell colorimetric assay and confocal fluorescence microscopy, respectively.

Results

TNFα stimulation elicited a robust increase in RhoA activity in a dose‐dependent manner, and SMV mitigated this increase. TNFα also hastened NF‐κB nuclear translocation of subunit p65 and increased DNA binding activity, luciferase reporter gene expression, degradation of IκB, and secretion of interleukin‐1β (IL‐1β) and IL‐6. SMV prevented the increase in NF‐κB activation and rise in IL‐1β and IL‐6 levels induced by TNFα, whereas mevalonate and geranylgeranyl pyrophosphate reversed the inhibitory effects of SMV on activation of NF‐κB and RhoA. Furthermore, cotransfection with a dominant‐negative mutant of RhoA demonstrated that the TNFα‐induced signaling pathway involved sequential activation of RhoA, leading to NF‐κB activation and, ultimately, to secretion of cytokines.

Conclusion

This study identifies RhoA as the key regulator of TNFα‐induced NF‐κB activation, which ultimately results in the secretion of proinflammatory cytokines in rheumatoid synoviocytes. The findings provide a new rationale for the antiinflammatory effects of statins in inflammatory arthritis.

     

NF‐κB protects Behçet's disease T cells against CD95‐induced apoptosis up‐regulating antiapoptotic proteins

Objective

To determine whether prolongation of the inflammatory reaction in patients with Behçet's disease (BD) is related to apoptosis resistance and is associated with the up‐regulation of antiapoptotic factors.

Methods

The percentage of cell death was evaluated by flow cytometry in peripheral blood mononuclear cells from 35 patients with BD and 30 healthy volunteers. The expression levels of antiapoptotic factors and NF‐κB regulatory proteins were measured using Western blotting and immunohistochemical analyses. To down‐regulate NF‐κB nuclear translocation, BD T lymphocytes were exposed in vitro to thalidomide and subjected to transfection with NF‐κB small interfering RNA.

Results

Although CD95 is highly expressed in BD T cells, the absence of sensitivity to CD95‐induced apoptosis observed may be attributable to the inhibitory action of antiapoptotic genes. Immunoblot analysis for major antiapoptotic proteins showed considerable up‐regulation of the short form of cellular FLIP (cFLIP) and Bcl‐x_L in BD activated T cells, while levels of Bcl‐2, caspase 3, and caspase 8 in activated T cells from patients with BD were comparable with those in activated T cells from normal donors. Moreover, expression of IKK and IκB was up‐regulated, whereas NF‐κB translocated to the nucleus in BD T cells, suggesting that NF‐κB activation may modulate the expression of antiapoptotic genes. Interestingly, thalidomide and NF‐κB small interfering RNA down‐regulated cFLIP and Bcl‐x_L expression levels and sensitized BD activated T cells to CD95‐induced apoptosis.

Conclusion

Taken together, these results indicate that NF‐κB contributes to the regulation of the apoptosis‐related factors and death receptors leading to apoptosis resistance in BD T cell subsets. Our results suggest that NF‐κB plays a crucial role in the pathogenesis of BD, and that its pharmacologic control could represent a key strategy in modulating specific immune‐mediated disease.