Tumor necrosis factor α induces sustained signaling and a prolonged and unremitting inflammatory response in rheumatoid arthritis synovial fibroblasts.

OBJECTIVE: The nonresolving character of synovial inflammation in rheumatoid arthritis (RA) is a conundrum. To identify the contribution of fibroblast-like synoviocytes (FLS) to the perpetuation of synovitis, we investigated the molecular mechanisms that govern the tumor necrosis factor α (TNFα)-driven inflammatory program in human FLS. METHODS: FLS obtained from the synovial tissues of patients with RA or osteoarthritis were stimulated with TNFα and assayed for gene expression and cytokine production by real-time quantitative reverse transcription-polymerase chain reaction analysis and enzyme-linked immunosorbent assay. NF-κB signaling was evaluated by Western blotting. Histone acetylation, chromatin accessibility, and NF-κB p65 and RNA polymerase II (Pol II) occupancy at the interleukin-6 (IL-6) promoter were measured by chromatin immunoprecipitation and restriction enzyme accessibility assays. RESULTS: In FLS, TNFα induced prolonged transcription of messenger RNA (mRNA) for IL-6 and progressive accumulation of IL-6 protein over 4 days. Similarly, induction of mRNA for CXCL8/IL-8, CCL5/RANTES, matrix metalloproteinase 1 (MMP-1), and MMP-3 after TNFα stimulation was sustained for several days. This contrasted with the macrophage response to TNFα, which characteristically involved a transient increase in the expression of proinflammatory genes. In FLS, TNFα induced prolonged activation of NF-κB signaling and sustained transcriptional activity, as indicated by increased histone acetylation, chromatin accessibility, and p65 and Pol II occupancy at the IL-6 promoter. Furthermore, FLS expressed low levels of the feedback inhibitors A20-binding inhibitor of NF-κB activation 3 (ABIN-3), IL-1 receptor-associated kinase M (IRAK-M), suppressor of cytokine signaling 3 (SOCS-3), and activating transcription factor 3 (ATF-3), which terminate inflammatory responses in macrophages. CONCLUSION: TNFα signaling is not effectively terminated in FLS, which leads to an uncontrolled inflammatory response. The results suggest that prolonged and sustained inflammatory responses by FLS in response to synovial TNFα contribute to the persistence of synovial inflammation in RA.

The interferon signature and STAT1 expression in rheumatoid arthritis synovial fluid macrophages are induced by tumor necrosis factor α and counter-regulated by the synovial fluid microenvironment.

OBJECTIVE: Type I interferons (IFNs) have emerged as potential activators of the IFN signature and elevated STAT-1 expression in rheumatoid arthritis (RA) synovium, but mechanisms that induce synovial IFN expression are unknown. Recently, tumor necrosis factor α (TNFα) was shown to induce a delayed IFN response in macrophages. We undertook this study to test whether TNFα, classically thought to activate inflammatory NF-κB target genes in RA, also contributes to the "IFN signature" in RA synovial macrophages. METHODS: Synovial fluid (SF) macrophages purified from 24 patients with RA and 18 patients with spondylarthritides (SpA) were lysed immediately after isolation or were cultured ex vivo in the absence or presence of blockade of endogenous type I IFN or TNFα. Expression of IFN-inducible target genes was measured by quantitative reverse transcription-polymerase chain reaction, and expression of their corresponding proteins was measured by enzyme-linked immunosorbent assay. RESULTS: Expression of an IFN signature and STAT1 in RA synovial macrophages was suppressed when type I IFNs or TNFα were blocked, whereas TNFα blockade did not affect expression of IFN response genes or STAT1 in SpA synovial macrophages. RA SF suppressed the IFN signature in RA synovial macrophages and in TNFα-, IFNα-, and IFNß-stimulated control macrophages. Type I IFNs suppressed expression of IL8 and MMP9 in RA synovial macrophages and in TNFα-stimulated control macrophages. CONCLUSION: Our findings identify a new function of TNFα in RA synovitis by implicating TNFα as a major inducer of the RA synovial IFN response. The results suggest that the expression of IFN response genes in RA synovium is regulated by interplay between TNFα and opposing homeostatic factors expressed in the synovial microenvironment.

Mitochondrial DNA mutation stimulates prostate cancer growth in bone stromal environment.

BACKGROUND AND OBJECTIVES: Mitochondrial DNA (mtDNA) mutations, inherited and somatically acquired, are common in clinical prostate cancer. We have developed model systems designed to study specific mtDNA mutations in controlled experiments. Because prostate cancer frequently metastasizes to bone we tested the hypothesis that mtDNA mutations enhance prostate cancer growth and survival in the bone microenvironment. METHODS: The pathogenic nucleotide position (np) 8993 mDNA mutation was introduced into PC3 prostate cancer cells by cybrid formation. Wild-type and mutant cybrids were grown as nude mouse subcutaneous xenografts with or without bone stromal cell co-inoculation. Cybrids were also grown in the intratibial space. Tumor growth was assayed by direct tumor measurement and luciferase chemiluminescence. Gene expression was assayed using cDNA microarrays confirmed by real time PCR, western blot analysis and immunohistochemistry. RESULTS: Cybrids with the 8,993 mtDNA mutation grew faster than wild-type cybrids. Further growth acceleration was demonstrated in the bone microenvironment. A 37 gene molecular signature characterized the growth advantage conferred by the mtDNA mutation and bone microenvironment. Two genes of known importance in clinical prostate cancer, FGF1 and FAK, were found to be substantially upregulated only when both mtDNA mutation and bone stromal cell were present. CONCLUSIONS: The ATP6 np 8,993 mtDNA mutation confers a growth advantage to human prostate cancer that is most fully manifest in the bone microenvironment. The identification of specific molecular alterations associated with mtDNA mutation and growth in bone may allow new understanding of prostate cancer bone metastasis.

Anti-Mullerian hormone may be a useful adjunct in the diagnosis of polycystic ovary syndrome in nonobese adolescents.

OBJECTIVES: This study aimed to [1] confirm that nonobese adolescents with polycystic ovary syndrome (PCOS) have higher anti-Mullerian hormone (AMH) than controls; [2] examine the relationship of AMH with PCOS features and hormonal profile; and [3] approximate an AMH value that discriminates between adolescents with PCOS and controls. DESIGN: Case-control study. SETTING: Subspecialty ambulatory clinic. PATIENTS: Thirty-one nonobese adolescent girls (age 13-21 years), 15 with PCOS diagnosed using the National Institutes of Health (NIH) criteria and 16 healthy control subjects. Subjects and controls were comparable for body mass index z-score, age and ethnicity. MAIN OUTCOME MEASURE(S): AMH in PCOS subjects and control groups, correlation of AMH with hormonal parameters. RESULTS: AMH was higher in PCOS subjects (4.4±3.4 ng/mL) than in controls (2.4±1.3 ng/mL), when adjusted for menstrual age. In the entire group (PCOS and controls), AMH correlated with androgens, ovarian size and the presence of polycystic ovary (PCO) appearance. There was no difference in average ovarian size between PCOS (7.1±2.6 cm³) and controls (6.7±1.8 cm³). PCOS subjects were 1.49 times more likely to have AMH >3.4 ng/mL (confidence interval 0.98-2.26 ng/mL). CONCLUSIONS: Our data suggest that AMH may be a useful adjunct in the diagnosis of PCOS in adolescents.

Differential roles for caspase-mediated and calpain-mediated cell death in 1- and 3-week-old rat cortical cultures.

Necrosis and apoptosis are well established as two primary cell death pathways. Mixed neuroglial cultures are commonly used to study cell death mechanisms in neural cells. However, the ages of these cultures vary across studies and little attention has been paid to how cell death processes may change as the cultures mature. To clarify whether neuroglial culture age affects cell death mechanisms, we treated 1- and 3-week-old neuroglial cultures with either the excitotoxic stimulus, N-methyl-D-aspartate (NMDA), or with the oxidative stressor, hydrogen peroxide (H2O2). Although NMDA is known to be toxic only in cultures that are at least 2 weeks old, H2O2 is toxic in cultures of all ages. Here, we confirm that, in 1-week-old neuroglial cultures, NMDA does not induce toxicity, whereas H2O2 induces both calpain-mediated and caspase-mediated neuronal death. In 3-week-old cultures, both NMDA and H2O2 trigger calpain-mediated, but not caspase-mediated, neuronal death. Further, we observed a decrease in caspase-3 levels and an increase in calpain levels in untreated neuroglial cultures as they aged. The findings presented here show that neuronal cell death mechanisms vary with culture age and highlight the necessity of considering culture age when interpreting neural cell culture data.