F-spondin deficient mice have a high bone mass phenotype.

F-spondin is a pericellular matrix protein upregulated in developing growth plate cartilage and articular cartilage during osteoarthritis. To address its function in bone and cartilage in vivo, we generated mice that were deficient for the F-spondin gene, Spon1. Spon1-/- mice were viable and developed normally to adulthood with no major skeletal abnormalities. At 6 months, femurs and tibiae of Spon1-/- mice exhibited increased bone mass, evidenced by histological staining and micro CT analyses, which persisted up to 12 months. In contrast, no major abnormalities were observed in articular cartilage at any age group. Immunohistochemical staining of femurs and tibiae revealed increased levels of periostin, alkaline phosphate and tartrate resistant acid phosphatase (TRAP) activity in the growth plate region of Spon1-/- mice, suggesting elevated bone synthesis and turnover. However, there were no differences in serum levels of TRAP, the bone resorption marker, CTX-1, or osteoclast differentiation potential between genotypes. Knockout mice also exhibited reduced levels of TGF-ß1 in serum and cultured costal chondrocytes relative to wild type. This was accompanied by increased levels of the BMP-regulatory SMADs, P-SMAD1/5 in tibiae and chondrocytes. Our findings indicate a previously unrecognized role for Spon1 as a negative regulator of bone mass. We speculate that Spon1 deletion leads to a local and systemic reduction of TGF-ß levels resulting in increased BMP signaling and increased bone deposition in adult mice.

Orphan nuclear receptor NR4A2 induces synoviocyte proliferation, invasion, and matrix metalloproteinase 13 transcription.

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 α (TNFα) 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 TNFα, 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 TNFα 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.

F-spondin regulates chondrocyte terminal differentiation and endochondral bone formation.

This study examines the role of F-spondin, an extracellular matrix protein of osteoarthritic cartilage, during chondrocyte maturation in embryonic growth plate cartilage. In chick tibia, F-spondin expression localized to the hypertrophic and calcified zones of the growth plate. Functional studies using tibial organ cultures indicated that F-spondin inhibited (∼35%, p = 0.02), and antibodies to F-spondin increased (∼30%, p < 0.1) longitudinal limb growth relative to untreated controls. In cell cultures, induction of chondrocyte maturation, by retinoic acid (RA) or transforming growth factor (TGF)-ß treatment led to a significant upregulation of F-spondin (p < 0.05). F-spondin transfection increased mineral deposition, alkaline phosphatase (AP) and matrix metalloproteinase (MMP)-13 mRNA levels (p < 0.05), and AP activity following RA stimulation, compared to mock transfected controls. Using AP as a differentiation marker we then investigated the mechanism of F-spondin promaturation effects. Blocking endogenous F-spondin via its thrombospondin (TSR) domain inhibited RA induced AP activity 40% compared to controls (p < 0.05). The stimulatory effect of F-spondin on AP expression was also inhibited following depletion of TGF-ß from culture supernatants. Our findings indicate that F-spondin is expressed in embryonic cartilage, where it has the capacity to enhance chondrocyte terminal differentiation and mineralization via interactions in its TSR domain and TGF-ß dependent pathways.

F-spondin, a neuroregulatory protein, is up-regulated in osteoarthritis and regulates cartilage metabolism via TGF-beta activation.

In osteoarthritis (OA) articular chondrocytes undergo phenotypic changes culminating in the progressive loss of cartilage from the joint surface. The molecular mechanisms underlying these changes are poorly understood. Here we report enhanced (approximately 7-fold) expression of F-spondin, a neuronal extracellular matrix glycoprotein, in human OA cartilage (P<0.005). OA-specific up-regulation of F-spondin was also demonstrated in rat knee cartilage following surgical menisectomy. F-spondin treatment of OA cartilage explants caused a 2-fold increase in levels of the active form of TGF-beta1 (P<0.01) and a 10-fold induction of PGE2 (P<0.005) in culture supernatants. PGE2 induction was found to be dependent on TGF-beta and the thrombospondin domain of the F-spondin molecule. F-spondin addition to cartilage explant cultures also caused a 4-fold increase in collagen degradation (P<0.05) and a modest reduction in proteoglycan synthesis (approximately 20%; P<0.05), which were both TGF-beta and PGE2 dependent. F-spondin treatment also led to increased secretion and activation of MMP-13 (P<0.05). Together these studies identify F-spondin as a novel protein in OA cartilage, where it may act in situ at lesional areas to activate latent TGF-beta and induce cartilage degradation via pathways that involve production of PGE2.

Transcriptional repression of matrix metalloproteinase gene expression by the orphan nuclear receptor NURR1 in cartilage.

The NR4A orphan receptors (Nur77, NURR1, and NOR-1) are emerging as key regulators of cytokine and growth factor action in chronic inflammatory diseases. In this study, we address the role of these receptors in cartilage homeostasis during inflammatory joint disease. We document for the first time expression of the NR4A receptors in osteoarthritic cartilage. Relative to Nur77 and NOR-1, NURR1 is expressed at the highest level and correlates with cyclooxygenase-2 levels in cartilage. Consistent with this observation, cyclooxygenase-2-derived prostaglandin E(2) (PGE(2)) rapidly and potently induces NURR1 expression in chondrocytes, suggesting that this receptor may regulate PGE(2)-mediated processes in cartilage. We demonstrate that PGE(2) represses interleukin-1beta-induced matrix metalloproteinase (MMP)-1 and that transient overexpression of NURR1 is sufficient to antagonize expression of this gene. Furthermore, MMP-1 promoter activity is potently suppressed by NURR1, resulting in a significant reduction in endogenous MMP-1 mRNA and secreted pro-MMP-1 protein. In addition, NURR1 selectively antagonizes cytokine-induced MMP-3 and -9 expression with minimal effects on MMP-2 and -13 and tissue inhibitor of matrix metalloproteinases-1 and -2. To explore the molecular mechanisms of NURR1 transrepression, we reveal that this receptor targets a critical region of the MMP-1 promoter (-1772 to -1546 bp) and that repression does not require consensus binding sites for NURR1. We confirm that NURR1 targets a 40-bp promoter sequence that is also positively regulated by ETS transcription factors. Finally, functional studies indicate that transcriptional antagonism exists between NURR1 and ETS1 on the MMP-1 promoter. We propose a protective function for NURR1 in cartilage homeostasis by selectively repressing MMP gene expression during inflammation.

Functional genomics approaches in arthritis.

The post-genomic era of functional genomics and target validation will allow us to narrow the bridge between clinically correlative data and causative data for complex diseases, such as arthritis, for which the etiological agent remains elusive. The availability of human and other annotated genome sequences, and parallel developments of new technologies that allow analysis of minute amounts of human and animal cells (peripheral blood cells and infiltrating cells) and tissues (synovium and cartilage) under different pathophysiological conditions, has facilitated high-throughput gene mining approaches that can generate vast amounts of clinically correlative data. Characterizing some of the correlative/causative genes will require reverting to the hypothesis-driven, low throughput method of complementary experimental biology using genomic approaches as a tool. This will include in silico gene expression arrays, genome-wide scans, comparative genomics using various animal models (such as rodents and zebrafish), bioinformatics and a team of well trained translational scientists and physicians. For the first time, the "genomic tools" will allow us to analyze small amounts of surgical samples (such as needle biopsies) and clinical samples in the context of the whole genome. Preliminary genomic analysis in osteoarthritis has already resurrected the debate on the semantic issues in the definition of inflammation. Further analyses will not only facilitate the development of unbiased hypotheses at the molecular level, but also assist us in the identification and characterization of novel targets and disease markers for pharmacological intervention, gene therapy, and diagnosis.

Functional genomic analysis of type II IL-1beta decoy receptor: potential for gene therapy in human arthritis and inflammation.

Gene expression arrays show that human epithelial cells and human arthritis-affected cartilage lack detectable amounts of mRNA for IL-1 antagonizing molecules: IL-1Ra and IL-1RII, but constitutively express IL-1. Functional genomic analysis was performed by reconstituting human IL-1RII expression in various IL-1RII-deficient cell types to examine its antagonist role using gene therapy approaches. Adenovirus-expressing IL-1RII when transduced into human and bovine chondrocytes, human and rabbit synovial cells, human epithelial cells, and rodent fibroblasts expressed membrane IL-1RII and spontaneously released functional soluble IL-1RII. The IL-1RII(+) (but not IL-1RII(-)) cells were resistant to IL-1beta-induced, NO, PGE(2), IL-6, and IL-8 production or decreased proteoglycan synthesis. IL-1RII inhibited the function of IL-1 in chondrocytes and IL-1- and TNF-alpha-induced inflammatory mediators in human synovial and epithelial cells. IL-1RII(+) chondrocytes were more resistant to induction of NO and PGE(2) by IL-1beta compared with IL-1RII(-) cells incubated with a 10-fold (weight) excess of soluble type II IL-1R (sIL-1RII) protein. In cocultures, IL-1RII(+) synovial cells released sIL-1RII, which in a paracrine fashion protected chondrocytes from the effects of IL-1beta. Furthermore, IL-1RII(+) (but not IL-1RII(-)) chondrocytes when transplanted onto human osteoarthritis-affected cartilage in vitro, which showed spontaneous release of sIL-1RII for 20 days, inhibited the spontaneous production of NO and PGE(2) in cartilage in ex vivo. In summary, reconstitution of IL-1RII in IL-1RII(-) cells using gene therapy approaches significantly protects cells against the autocrine and paracrine effects of IL-1 at the signaling and transcriptional levels.