Faculty Bibliography

OBJECTIVE: To address the role of the nuclear receptor 4A (NR4A) family of orphan nuclear receptors in synoviocyte transformation, hyperplasia, and regulation of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in models of inflammatory arthritis. METHODS: NR4A messenger RNA levels in synovial tissue and primary synoviocytes were measured by quantitative reverse transcription-polymerase chain reaction (RT-PCR). NR4A2 was stably overexpressed in normal synoviocytes, and cell proliferation, survival, anchorage-independent growth, migration, and invasion were monitored in vitro. MMP and TIMP expression levels were analyzed by quantitative RT-PCR, and MMP-13 promoter activity was measured using reporter assays. Stable depletion of endogenous NR4A levels was achieved by lentiviral transduction of NR4A short hairpin RNA (shRNA), and the effects on proliferation, migration, and MMP-13 expression were analyzed. RESULTS: NR4A2 was expressed at elevated levels in normal, OA, and RA synovial tissue and in primary RA synoviocytes. Tumor necrosis factor alpha (TNFalpha) rapidly and selectively induced expression of NR4A2 in synoviocytes. Ectopic expression of NR4A2 in normal synoviocytes significantly increased proliferation and survival, promoted anchorage-independent growth, and induced migration and invasion. MMP-13 gene expression was synergistically induced by NR4A2 and TNFalpha, while expression of TIMP-2 was antagonized. NR4A2 directly transactivated the proximal MMP-13 promoter, and a point mutation in the DNA binding domain of NR4A2 abolished transcriptional activation. Depletion of endogenous NR4A receptors with shRNA reduced synoviocyte proliferation, migration, and MMP-13 expression. CONCLUSION: The orphan nuclear receptor NR4A2 is a downstream mediator of TNFalpha signaling in synovial tissue. NR4A2 transcriptional activity contributes to the hyperplastic and invasive phenotype of synoviocytes that leads to cartilage destruction, suggesting that this receptor may show promise as a therapeutic target in inflammatory arthritis.

OBJECTIVE: Mutations in LMNA encoding the A-type lamins cause several diseases, including those with features of premature aging and skeletal abnormalities. The aim of this study was to examine the expression of lamin A in cartilage from patients with osteoarthritis (OA) and the effects of its overexpression on chondrocyte senescence and apoptosis. METHODS: Human chondrocyte-like cells (SW-1353) were used. RNA isolated from human OA and non-OA cartilage was used for profiling messenger RNA expression, using Affymetrix microarray analysis. The effects of lamin A overexpression on mitochondrial function and apoptosis were examined by assessing mitochondrial membrane potential, ATP levels, and cytochrome c release, and with a TUNEL assay. Western blotting was performed to determine protein expression. RESULTS: Lamin A expression was markedly elevated in OA cartilage samples compared with non-OA control samples. Western blot analysis confirmed increased expression of lamin A in OA compared with non-OA cartilage. Interleukin-1beta treatment inhibited lamin A accumulation, whereas treatment with prostaglandin E(2) (PGE(2) ) caused a marked increase in lamin A accumulation. These effects of exogenous PGE(2) on lamin A expression were mediated via the EP(2) /EP(4) receptors. Transfected chondrocytes that expressed lamin A displayed markers of early senescence/apoptosis. CONCLUSION: The results of this study suggest that lamin A is up-regulated in OA chondrocytes, and that increased nuclear accumulation of lamin A in response to catabolic stress may account for the premature aging phenotype and apoptosis of OA chondrocytes.

The compartment-specific lipid changes in femoral-tibial bone of healthy controls and mild osteoarthritis (OA) patients were quantified at 3.0 T. Healthy volunteers [Kellgren-Lawrence (KL) grade = 0; n = 15, 4 females, 11 males, mean age 39 +/- 16 years, age range = 24-78 years] and mild OA patients (KL = 1, 2; n = 26, 12 females, 14 males, mean age 61 +/- 14 years, age range = 27-80 years) were scanned on a 3 T scanner. Clinical proton density (PD)-weighted fast spin echo (FSE) images in the sagittal (without fat-saturation), axial and coronal (fat-saturation) planes were acquired for cartilage Whole-Organ MR Imaging Score (WORMS) grading. A voxel of 10 x 10 x 10 mm(3) was positioned in the medial and lateral compartments of the tibia [medial tibial (MT) and lateral tibial (LT)] and femur [medial femoral (MF) and lateral femoral (LF)] for MRS measurements using the single voxel-stimulated echo acquisition mode (STEAM) pulse sequence. All MRS data were processed with Java-based Magnetic Resonance User Interface (JMRUI). Wilcoxon's rank sum test and mixed model two-way analysis of variance (anova) were performed to determine significant differences between different compartments as well as examine the effect of OA grade and compartment, and their interactions. Generally, the MF compartment index of unsaturation was increased in healthy subjects compared with OA subjects (whether graded by KL or WORMS score). Differences between MF at KL0 and all other compartments at KL1 except LF approached statistical significance (p < 0.05). Differences in saturated lipids signals could be observed predominantly in the 2.03 p.p.m. frequency shift. Healthy controls in the MF compartment had the lowest saturated lipid signals, and mild OA patients with KL2 and WORMS5-6 in the MF compartment had the highest saturated lipid signals compared with other compartments at 2.03 p.p.m. (p < 0.05).

Purpose: To determine whether inhibition of inducible nitric oxide synthase (iNOS) can slow progression of knee osteoarthritis (OA), using the rate of joint space narrowing (JSN) in the medial tibiofemoral compartment as the primary outcome measure. Methods: This was a 2-year multinational, multicenter, double-blind, parallel group trial enrolling subjects with symptomatic knee OA in which subjects were randomly assigned to receive once daily 50 mg or 200 mg of the selective iNOS inhibitor, SD-6010, or placebo. Subjects were required to have a body mass index (BMI) >=25 and <= 40 kg/m² and Kellgren and Lawrence Grade (KLG) 2 or 3 in the study knee. Randomization was stratified by KLG. Radiographs were acquired using the modified Lyon-schuss protocol, at baseline, 48 and 96 weeks for measuring JSN. Clinical benefit was recorded at 12-week then every 24 weeks; use of acetaminophen, NSAIDs and/or weak opioids was permitted throughout the trial. The primary analysis of the rate of JSN used a continuous time random coefficients MMRM model. The slope over the entire 96 week period was used to assess the rate of change in joint space width (JSW). Results: Of 1457 randomized subjects (SD-6010 50mg, n=485; SD-6010 200mg, n=486; placebo, n=486), 1048 (71.9%) completed the study. Subjects were predominantly female (76.5%) with mean age 61. 0 years and mean BMI of 31.8 kg/m². Fifty-six percent had KLG3. The primary analysis did not demonstrate the superiority of SD-6010 in either treatment group over placebo. In an exploratory discrete time MMRM analysis of KLG2 subjects, the loss of JSW after 48 weeks was significantly smaller with SD-6010 (50 mg or 200 mg) than placebo. Least squared (LS) mean +/- standard error (SE) losses in JSW for SD-6010 50mg (-0.048 +/- 0.028 mm) and 200mg (-0.062 +/- 0.028 mm) were 59.9% (95% CI: 6.8%, 106.9%) and 48.7% (95% CI: -8.4%, 93.9%) of placebo (-0.120 +/- 0.028 mm; P=0.032 and P=0.081, respectively). After 96 weeks, the LS mean losses in JSW for SD-6010 50mg (-0.132 +/- 0.036 mm) and 200mg (-0.156 +/- 0.037 mm) were 20.9% (95% CI: -50.5%, 64.0%) and 6.8% (95% CI: -72.7%, 51.7%) of the LS-mean JSW loss in the placebo group (-0.167+/-0.036 mm) (P=0.460 and P=0.812, respectively). In a similar analysis of KLG3 subjects, no improvement in JSN was observed. Although SD-6010 showed no efficacy with respect to improvement in joint pain or function, it was generally safe and well tolerated in this population. Conclusions: During the first 48 weeks of treatment, subjects with mild OA who were treated with an iNOS inhibitor had a lower rate of JSN; however, this improvement was not sustained at 96 weeks. iNOS inhibition did not slow OA progression in subjects with more severe radiographic OA. The observed early effect on JSN in subjects with mild OA supports the role of iNOS in OA pathogenetic mechanisms. However, the loss of efficacy over time and the lack of effect in subjects with more severe disease suggest that alternative biochemical catabolic pathways overcame the effects of nitric oxide inhibition alone or that in more severe radiographic OA, biomechanical factors may not be amenable to iNOS inhibition

Foxp3(+)CD4(+)CD25(high) regulatory T cell (Treg) suppression of inflammation depends on T-cell receptor-mediated Nuclear Factor of Activated T cells c1 (NFATc1) activation with reduced Akt activity. We investigated the role of the scaffold protein Disc large homolog 1 (Dlgh1) in linking the T-cell receptor to this unique signaling outcome. The Treg immunological synapse (IS) recruited fourfold more Dlgh1 than conventional CD4(+) T-cell IS. Tregs isolated from patients with active rheumatoid arthritis, or treated with tumor necrosis factor-alpha, displayed reduced function and diminished Dlgh1 recruitment to the IS. Furthermore, Dlgh1 silencing abrogated Treg function, impaired NFATc1 activation, reduced phosphatase and tensin homolog levels, and increased Akt activation. Dlgh1 operates independently of the negative feedback pathway mediated by the related adapter protein Carma1 and thus presents an array of unique targets to selectively manipulate Treg function.

Objective: Non-steroidal anti-inflammatory drugs (NSAIDs) that are prescribed for treatment of osteoarthritis (OA) symptoms including pain and inflammation target the production eicosanoids which exhibit numerous functions in various cell types. In these studies, we have (a) identified the diverse eicosanoid pathways that are activated in human chondrocytes of normal and OA cartilage, (b) delineated the modulation of eicosanoids in the presence of NSAIDS and selective COX-2 inhibitors, and (c) characterized eicosanoid products and various transcripts modulated by various inhibitors of eicosanoids in human OA cartilage by gene expression arrays. Methods: Immunoassay analysis of culture supernatants were utilized to determine the spectrum of eicosanoids derived from both the cyclooxygenase (COX) and lipoxygenase (LOX) pathways of normal and human OA cartilage in ex-vivo conditions. Human OA cartilage was incubated in ex-vivo conditions to examine spontaneous or IL-1 induced production of eicosanoids in the presence of various COX inhibitors. Gene expression analysis was performed to analyze the expression of mRNA in the presence and absence of COX-2 inhibitors in OA cartilage in ex-vivo conditions. Results: Normal and OA human cartilage explants produced multiple eicosanoids of the COX and LOX pathways. PGF1alpha, PGF2alpha, PGE2 > TXB2, PGD2, and LTB4 were spontaneously generated by normal and OA cartilage. Among these, elevated levels of PGE2 and LTB4 were generated in OA as compared to normal cartilage. IL-1 treatment further enhanced these eicosanoids production. Treatment of OA cartilage explants with cyclooxygenase inhibitors (celecoxib & indomethacin) augmented LTB4 accumulation by 2- to 4-fold. A follow-up pharmacogenomic analysis identified approximately 90 cytokine and growth factor related transcripts that were modulated following selective COX-2 inhibition. Conclusion: These studies for the first time demonstrate that normal and OA cartilage generates multiple and differential eicosanoid products. Inhibition of the COX- pathway in human OA cartilage caused accumulation of end products (LTB4) of the 5LO pathway. Furthermore, celecoxib, a selective COX-2 inhibitor, regulated numerous genes in cartilage, which are linked to the NFkB and AP-1 pathways at the mRNA level. In conclusion, these experiments demonstrate the complex and pleotropic role of eicosanoids in human cartilage homeostasis and pathophysiology of OA.