Faculty Bibliography

Postmenopausal osteoporosis is characterized by an imbalance of bone resorption exceeding bone formation, resulting in a net loss of bone mineral density (BMD). Estrogen deficiency is known to promote bone resorption. However, the causative factors that impair bone formation have not been identified. Women after menopause experience not only estrogen deficiency but also iron accumulation as a result of cessation of menstruation. In this study we investigated whether increased iron plays a role in osteoporosis. By growing primary mouse osteoclast and osteoblast progenitor cells as well as immortalized cell lines in the presence of iron, we found that increased iron had minimal effects on osteoclast cell differentiation. Interestingly, iron, particularly in its inorganic form, and to a lesser extent ferritin and transferrin all suppressed alkaline phosphatase (ALP) activities in osteoblasts. Moreover, iron downregulated mRNA levels of several other osteoblastogenic markers such as Runx2, osterix, osteopontin, and osteocalcin. To further show that this in vitro finding is relevant to the in vivo condition, we demonstrated that iron-accumulated mice with intact ovaries exhibited a significant decrease in BMD. Although iron inhibited preosteoblast cell differentiation, it did enhance preosteoblast cell proliferation, as evidenced by increased cell growth and expression of cell cycle regulator genes such as CDK4, CDK6, cyclin D1, and cyclin D3 and G(2) /M phase cell population. Taken together, our results suggest that increased iron could be a factor that slows down bone formation in postmenopausal women. (c) 2011 American Society for Bone and Mineral Research

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 ( approximately 35%, p = 0.02), and antibodies to F-spondin increased ( approximately 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)-beta 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-beta 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-beta dependent pathways.

MicroRNAs (miRNAs) represent a class of non-coding RNA molecules playing pivotal roles in cellular and developmental processes. miRNAs modulate the expression of multiple target genes at the post-transcriptional level and are predicted to affect up to one-third of all human protein-encoding genes. Recently, miRNA involvement in the adaptive and innate immune systems has been recognized. Rheumatoid arthritis serves an example of a chronic inflammatory disorder in which miRNAs modulate the inflammatory process in the joints, with the potential to serve as biomarkers for both the inflammatory process and the potential for therapeutic response. This review discusses the investigations that led to miRNA discovery, miRNA biogenesis and mode of action, and the diverse roles of miRNAs in modulating the immune and inflammatory responses. We conclude with a discussion of the implications of miRNA biology in rheumatoid arthritis and other autoimmune disorders

Osteoarthritis (OA) is often a progressive and disabling disease, which occurs in the setting of a variety of risk factors--such as advancing age, obesity and trauma--that collude to incite a cascade of pathophysiological events within joint tissues. An important emerging theme in OA is a broadening of focus from a disease of cartilage to one of the 'whole joint.' The synovium, bone and cartilage are each involved in pathological processes that lead to progressive joint degeneration. Additional themes that have emerged over the past decade are novel mechanisms of cartilage degradation and repair, the relationship between biomechanics and biochemical pathways, the importance of inflammation and the role of genetics. In this article, we review the molecular, clinical and imaging evidence that synovitis is not an 'incidental finding of OA', but plays a significant role in disease pathogenesis, and could therefore represent a target for future treatments

T cell receptor (TCR)-dependent regulatory T cell (Treg) activity controls effector T cell (Teff) function and is inhibited by the inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha). Protein kinase C-theta (PKC-theta) recruitment to the immunological synapse is required for full Teff activation. In contrast, PKC-theta was sequestered away from the Treg immunological synapse. Furthermore, PKC-theta blockade enhanced Treg function, demonstrating PKC-theta inhibits Treg-mediated suppression. Inhibition of PKC-theta protected Treg from inactivation by TNF-alpha, restored activity of defective Treg from rheumatoid arthritis patients, and enhanced protection of mice from inflammatory colitis. Treg freed of PKC-theta-mediated inhibition can function in the presence of inflammatory cytokines and thus have therapeutic potential in control of inflammatory diseases

BACKGROUND: A lack of biomarkers that identify patients at risk for severe osteoarthritis (OA) complicates development of disease-modifying OA drugs. OBJECTIVE: To determine whether inflammatory genetic markers could stratify patients with knee OA into high and low risk for destructive disease. METHODS: Genotype associations with knee OA severity were assessed in two Caucasian populations. Fifteen single nucleotide polymorphisms (SNPs) in six inflammatory genes were evaluated for association with radiographic severity and with synovial fluid mediators in a subset of the patients. RESULTS: Interleukin 1 receptor antagonist (IL1RN) SNPs (rs419598, rs315952 and rs9005) predicted Kellgren-Lawrence scores independently in each population. One IL1RN haplotype was associated with lower odds of radiographic severity (OR=0.15; 95% CI 0.065 to 0.349; p<0.0001), greater joint space width and lower synovial fluid cytokine levels. Carriage of the IL1RN haplotype influenced the age relationship with severity. CONCLUSION: IL1RN polymorphisms reproducibly contribute to disease severity in knee OA and may be useful biomarkers for patient selection in disease-modifying OA drug trials

Purpose: Stem cell-based therapies aimed at introducing progenitor cells into cartilage lesions hold great promise for the restoration of damaged articular surfaces following joint injury or osteoarthritis. Key to the generation of a functional repair tissue is the controlled differentiation into the desired phenotype. To this end microRNAs (miRNAs) may be important molecules that regulate this process. By acting as transcriptional repressors, their modulation during differentiation may enable commitment to a specific lineage by suppressing the expression of other lineage markers. In this study we profiled MSCs for miRNA expression following induction into the chondrocyte (C), osteoblast (O) and smooth muscle (SM) lineages. Results: Human bone marrow-derived Mesenchymal Stem Cells (MSCs) were obtained from NIH, or from the discarded hips of patients undergoing joint replacement surgery. SM differentiation was induced by treating monolayer cultures with 1 mM thromboxane-A2 [DP1] in the presence of 0.25% serum. C differentiation was induced by seeding MSCs in aggregate cultures in the presence of dexamethasone and TGF-b1 (10 ng/ml). O differentiation was induced by treatment of monolayer cultures with dexamethasone, ascorbate and beta-glycerolphosphate. Profiling of miRNAs by microarray (Agilent) or QPCR (SA Biosciences) revealed differential regulation of miR-7 and miR-130b, among 376 probes. Following SM and C differentiation, miR-7 expression was down-regulated up to 6.9-fold and 3-fold respectively. Conversely, during O differentiation, its expression was induced approximately 7-fold. Analysis of theoretical mRNA targets using TargetScan online software (www.targetscan.org) identified conserved sites in several genes associated with chondrocyte and myoblast lineages. Putative chondrogenic targets were found to include COL2A1, IGFR1, and GDF5, while potential smooth muscle modulators included EGFR1, PIK3CD, IRS1/IRS2, KLF4, CNN3 and IGF1R. Following a similar trend to miR-7, miR-130b was down-regulated up to 3.2-fold and 3.1-fold in C and SM differentiation respectively, while O differentiation induced its expression 2-fold. TargetScan analysis identified putative chondrogenic targets, TGF-BRII, Sox5, BMP-2 and IGF1; Potential smooth muscle regulators included ESR1, TGF-BRII, MBLN1, TGFBR1 and IGF2BP1. Together these observations suggest that miR-7 and miR-130b act to negatively regulate myogenic and chondrogenic cell fates via regulation of lineage specific genes. Conclusion: Our findings suggest that miR-7 and miR-130b, via the targeting of lineage specific molecules, regulate cell fate in adult human MSCs by inhibiting smooth muscle and chondrocyte differentiation, thereby promoting 'default' differentiation into the osteoblast lineage. [DP1]0.25% FBS 24 hours prior to addition of 1.0mM of the TxA2 chemical analog U46619