Effects of knockout of the receptor for advanced glycation end-products on bone mineral density and synovitis in mice with intra-articular fractures.

Our group employed the mouse closed intra-articular fracture (IAF) model to test the hypothesis that the innate immune system plays a role in initiating synovitis and post-traumatic osteoarthritis (PTOA) in fractured joints. A transgenic strategy featuring knockout of the receptor for advanced glycation end-products (RAGE -/- ) was pursued. The 42 and 84 mJ impacts used to create fractures were in the range previously reported to cause PTOA at 60 days post-fracture. MicroCT (µCT) was used to assess fracture patterns and epiphyseal and metaphyseal bone loss at 30 and 60 days post-fracture. Cartilage degeneration, synovitis, and matrix metalloproteinase (MMP-3, -13) expression were evaluated by histologic analyses. In wild-type mice, µCT imaging showed that 84 mJ impacts led to significant bone loss at 30 days (p < 0.05), but recovered to normal at 60 days. Bone losses did not occur in RAGE-/- mice. Synovitis was significantly elevated in 84 mJ impact wild-type mice at both endpoints (30 day, p = 0.001; 60 day, p = 0.05), whereas in RAGE-/- mice synovitis was elevated only at 30 days (p = 0.02). Mankin scores were slightly elevated in both mouse strains at 30 days, but not at 60 days. Immunohistochemistry revealed significant fracture-related increases in MMP-3 and -13 expression at 30 days (p < 0.05), with no significant difference between genotypes. These findings indicated that while RAGE -/- accelerated recovery from fracture and diminished synovitis, arthritic changes were temporary and too modest to detect an effect on the pathogenesis of PTOA. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2439-2449, 2018.

Induction of an altered CD40 signaling complex by an antagonistic human monoclonal antibody to CD40.

Blocking the interaction of CD40 with its ligand CD154 is a desirable goal of therapies for preventing and/or ameliorating autoimmune diseases and transplant rejection. CD154-blocking mAbs used in human clinical trials resulted in unanticipated vascular complications, leading to heightened interest in the therapeutic potential of antagonist mAbs specific for human CD40. Abs that do not require physical competition with CD154 to inhibit CD40 signaling have particular therapeutic promise. In this study, we demonstrate that the antagonist anti-human CD40 mAb PG102 fails to trigger CD40-mediated activation, as well as impairs CD154-mediated CD40 activation, via a distinct nonstimulatory CD40 signaling mechanism. PG102 did not induce early CD40-induced signaling events, and it inhibited early kinase and transcription factor activation by CD154 or agonist anti-CD40 mAbs. However, PG102 stimulated normal CD40-mediated TNFR-associated factor (TRAF)2 and TRAF3 degradation. PG102 induced the formation of a CD40 signaling complex that contained decreased amounts of both TRAF2 and TRAF3 and TRAF2-associated signaling proteins. Additionally, PG102-induced CD40 signaling complexes failed to recruit TRAF6 to detergent-insoluble membrane fractions. Fab fragments of PG102, while retaining CD40 binding, did not induce TRAF degradation, nor could they inhibit CD154-stimulated B cell signaling, indicating that CD40 aggregation is required for the signaling inhibition induced by PG102. The antagonistic impact of PG102 on CD40 signaling reveals that the manner of CD40 ligation can determine sharply different outcomes for CD40 signaling and suggests that such information can be used to therapeutically manipulate these outcomes.

Follistatin-like protein 1 regulates chondrocyte proliferation and chondrogenic differentiation of mesenchymal stem cells.

OBJECTIVES: Chondrocytes, the only cells in the articular cartilage, play a pivotal role in osteoarthritis (OA) because they are responsible for maintenance of the extracellular matrix (ECM). Follistatin-like protein 1 (FSTL1) is a secreted protein found in mesenchymal stem cells (MSCs) and cartilage but whose function is unclear. FSTL1 has been shown to modify cell growth and survival. In this work, we sought to determine whether FSTL1 could regulate chondrogenesis and chondrogenic differentiation of MSCs. METHODS: To study the role of FSTL1 in chondrogenesis, we used FSTL1 knockout (KO) mice generated in our laboratory. Proliferative capacity of MSCs, obtained from skulls of E18.5 embryos, was analysed by flow cytometry. Chondrogenic differentiation of MSCs was carried out in a pellet culture system. Gene expression differences were assessed by microarray analysis and real-time PCR. Phosphorylation of Smad3, p38 MAPK and Akt was analysed by western blotting. RESULTS: The homozygous FSTL1 KO embryos showed extensive skeletal defects and decreased cellularity in the vertebral cartilage. Cell proliferation of FSTL1-deficient MSCs was reduced. Gene expression analysis in FSTL1 KO MSCs revealed dysregulation of multiple genes important for chondrogenesis. Production of ECM proteoglycans and collagen II expression were decreased in FSTL1-deficient MSCs differentiated into chondrocytes. Transforming growth factor ß signalling in FSTL1 KO cells was significantly suppressed. CONCLUSIONS: FSTL1 is a potent regulator of chondrocyte proliferation, differentiation and expression of ECM molecules. Our findings may lead to the development of novel strategies for cartilage repair and provide new disease-modifying treatments for OA.

Enhanced Toll-like receptor (TLR) responses of TNFR-associated factor 3 (TRAF3)-deficient B lymphocytes.

The key role of TRAF6 in TLR signaling pathways is well known. More recent evidence has implicated TRAF3 as another TRAF family member important to certain TLR responses of myeloid cells. Previous studies demonstrate that TRAF3 functions are highly context-dependent, displaying receptor and cell-type specificity. We thus examined the TLR responses of TRAF3(-/-)mouse B lymphocytes to test the hypothesis that TRAF3 plays distinct roles in such responses, depending on cell type. TRAF3(-/-) DC are known to have a defect in type 1 IFN production and here, showed diminished production of TNF and IL-10 and unaltered IL-6. In marked contrast, TRAF3(-/-) B cells made elevated amounts of TNF and IL-6 protein, as well as IL-10 and IP-10 mRNA, in response to TLR ligands. Also, in contrast to TRAF3(-/-) DC, the type 1 IFN pathway was elevated in TRAF3(-/-) B cells. Increased early responses of TRAF3(-/-) B cells to TLR signals were independent of cell survival or proliferation but associated with elevated canonical NF-κB activation. Additionally, TRAF3(-/-) B cells displayed enhanced TLR-mediated expression of AID and Ig isotype switching. Thus, TRAF3 plays varied and cell type-specific, biological roles in TLR responses.

The signal from the initiation of meiotic recombination to the first division of meiosis.

Two of the unique events that occur in meiosis are high levels of genetic recombination and the reductional division. Our previous work demonstrated that the REC102, REC104, REC114, and RAD50 genes, required to initiate meiotic recombination in Saccharomyces cerevisiae, are needed for the proper timing of the first meiotic (MI) division. If these genes are absent, the MI division actually begins at an earlier time. This paper demonstrates that the meiotic recombination genes MER2/REC107, SPO11, and MRE2 and the synaptonemal complex genes HOP1 and RED1 are also required for the normal delay of the MI division. A rec103/ski8 mutant starts the MI division at the same time as in wild-type cells. Our data indicate no obvious correlation between the timing of premeiotic S phase and the timing of the first division in Rec- mutants. Cells with rec102 or rec104 mutations form MI spindles before wild-type cells, suggesting that the initiation signal acts prior to spindle formation. Neither RAD9 nor RAD24 is needed to transduce the signal, which delays the first division. The timing of the MI division in RAD24 mutants indicates that the pachytene checkpoint is not active in Rec+ cells and suggests that the coordination between recombination and the MI division in wild-type cells may occur primarily due to the initiation signal. Finally, at least one of the targets of the recombination initiation signal is the NDT80 gene, a transcriptional regulator of middle meiotic gene expression required for the first division.