Faculty Bibliography

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-beta1 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-beta levels resulting in increased BMP signaling and increased bone deposition in adult mice.

PURPOSE OF REVIEW: Identification of patients at risk for incident disease or disease progression in osteoarthritis remains challenging, as radiography is an insensitive reflection of molecular changes that presage cartilage and bone abnormalities. Thus there is a widely appreciated need for biochemical and imaging biomarkers. We describe recent developments with such biomarkers to identify osteoarthritis patients who are at risk for disease progression. RECENT FINDINGS: The biochemical markers currently under evaluation include anabolic, catabolic, and inflammatory molecules representing diverse biological pathways. A few promising cartilage and bone degradation and synthesis biomarkers are in various stages of development, awaiting further validation in larger populations. A number of studies have shown elevated expression levels of inflammatory biomarkers, both locally (synovial fluid) and systemically (serum and plasma). These chemical biomarkers are under evaluation in combination with imaging biomarkers to predict early onset and the burden of disease. SUMMARY: Prognostic biomarkers may be used in clinical knee osteoarthritis to identify subgroups in whom the disease progresses at different rates. This could facilitate our understanding of the pathogenesis and allow us to differentiate phenotypes within a heterogeneous knee osteoarthritis population. Ultimately, such findings may help facilitate the development of disease-modifying osteoarthritis drugs (DMOADs).

OBJECTIVE.: To profile the subgingival oral microbiota abundance and diversity in never-treated, new-onset rheumatoid arthritis (NORA) patients. METHODS.: Periodontal disease (PD) status, clinical activity and sociodemographic factors were determined in patients with NORA, chronic RA (CRA) and healthy subjects. Massively parallel pyrosequencing was used to compare the composition of subgingival microbiota and establish correlations between presence/abundance of bacteria and disease phenotypes. Anti-P. gingivalis antibodies were tested to assess prior exposure. RESULTS.: The more advanced forms of periodontitis are already present at disease onset in NORA patients. The subgingival microbiota of NORA is distinct from controls. In most cases, however, these differences can be attributed to PD severity and are not inherent to RA. The presence and abundance of P. gingivalis is directly associated with PD severity as well, is not unique to RA, and does not correlate with anti-citrullinated peptide antibody (ACPA) titers. Overall exposure to P. gingivalis is similar in RA and controls, observed in 78.4% and 83.3%, respectively. Anaeroglobus geminatus correlated with ACPA/RF presence. Prevotella and Leptotrichia species are the only characteristic taxa in the NORA group irrespective of PD status. CONCLUSIONS.: NORA patients exhibit a high prevalence of PD at disease onset, despite their young age and paucity of smoking history. The subgingival microbiota of NORA patients is similar to CRA and healthy subjects of comparable PD severity. Although colonization with P. gingivalis correlates with PD severity, overall exposure is similar among groups. The role of A. geminatus and Prevotella/Leptotrichia species in this process merits further study.

OBJECTIVE: The study aimed determining whether assessment of cartilage oligomeric matrix protein (COMP) degradation products could serve as a serological disease course and therapeutic response predictor in arthritis. METHODS: We generated a panel of monoclonal antibodies against COMP fragments and developed a novel capture enzyme-linked immunosorbent assay (ELISA) for detecting COMP fragments in patients with osteoarthritis (OA) and rheumatoid arthritis (RA). This test was also used to monitor COMP fragments in surgically-induced OA, collagen-induced arthritis (CIA), and tumor necrosis factor (TNF) transgenic animal models. RESULTS: Compared with a commercial COMP ELISA kit that detected no significant difference in COMP levels between OA and control groups, a significant increase of the COMP fragments were noted in the serum of OA patients assayed by this newly established ELISA. In addition, serum COMP fragment levels were well correlated with severity in OA patients and the progression of surgically-induced OA in murine models. Furthermore, the serum levels of COMP fragments in RA patients, mice with CIA, and TNF transgenic mice were significantly higher when compared with their controls. Interestingly, treatment with TNFalpha inhibitors and methotrexate led to a significant decrease of serum COMP fragments in RA patients. Additionally, administration of Atsttrin [Tang, et al., Science 2011;332(6028):478] also resulted in a significant reduction in COMP fragments in arthritis mice models. CONCLUSION: A novel sandwich ELISA is capable of reproducibly measuring serum COMP fragments in both arthritic patients and rodent arthritis models. This test also provides a valuable means to utilize serum COMP fragments for monitoring the effects of interventions in arthritis.

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.