No abstract

CTLA4‐Ig (cytotoxic T‐lymphocyte antigen 4‐immunoglobulin; Abatacept) is a biologic drug for rheumatoid arthritis. CTLA4 binds to the CD80/86 complex of antigen‐presenting cells and blocks the activation of T cells. Although previous reports showed that CTLA4‐Ig directly inhibited osteoclast differentiation, the whole inhibitory mechanism of CTLA4‐Ig for osteoclast differentiation is unclear. Bone marrow macrophages (BMMs) from WT mice were cultured with M‐CSF and RANKL with or without the recombinant mouse chimera CTLA4‐Ig. Intracellular calcium oscillations of BMMs with RANKL were detected by staining with calcium indicator fura‐2 immediately after administration of CTLA4‐Ig or after one day of treatment. Calcium oscillations were analyzed using Fc receptor gamma‐ (FcRγ‐) deficient BMMs. CTLA4‐Ig inhibited osteoclast differentiation and reduced the expression of the nuclear factor of activated T cells NFATc1 in BMMs in vitro. Calcium oscillations in BMMs were suppressed by CTLA4‐Ig both immediately after administration and after one day of treatment. CTLA4‐Ig did not affect osteoclastogenesis and did not cause remarkable changes in calcium oscillations in FcRγ‐deficient BMMs. Finally, to analyze the effect of CTLA4‐Ig in vivo, we used an LPS‐induced osteolysis model. CTLA4‐Ig suppressed LPS‐induced bone resorption in WT mice, not in FcRγ‐deficient mice. In conclusion, CTLA4‐Ig inhibits intracellular calcium oscillations depending on FcRγ and downregulates NFATc1 expression in BMMs. © 2019 American Society for Bone and Mineral Research.

Lubricin encoded by the proteoglycan 4 (Prg4) gene is produced from superficial zone (SFZ) cells of articular cartilage and synoviocytes, which is indispensable for lubrication of joint surfaces. Loss‐of‐function of human and mouse Prg4 results in early‐onset arthropathy accompanied by lost SFZ cells and hyperplastic synovium. Here, we focused on increases in the thickness of articular cartilage in Prg4‐knockout joints and analyzed the underlying mechanisms. In the late stage of articular cartilage development, the articular cartilage was thickened at 2 to 4 weeks and the SFZ disappeared at 8 weeks in Prg4‐knockout mice. Similar changes were observed in cultured Prg4‐knockout femoral heads. Cell tracking showed that Prg4‐knockout SFZ cells at 1 week of age expanded to deep layers after 1 week. In in vitro experiments, overexpression of Prg4 lacking a mucin‐like domain suppressed differentiation of ATDC5 cells markedly, whereas pellets of Prg4‐knockout SFZ cells showed enhanced differentiation. RNA sequencing identified matrix metalloproteinase 9 (Mmp9) as the top upregulated gene by Prg4 knockout. Mmp9 expressed in the SFZ was further induced in Prg4‐knockout mice. The increased expression of Mmp9 by Prg4 knockout was canceled by IκB kinase (IKK) inhibitor treatment. Phosphorylation of Smad2 was also enhanced in Prg4‐knockout cell pellets, which was canceled by the IKK inhibitor. Expression of Mmp9 and phosphorylated Smad2 during articular cartilage development was enhanced in Prg4‐knockout joints. Lubricin contributes to homeostasis of articular cartilage by suppressing differentiation of SFZ cells, and the nuclear factor‐kappa B‐Mmp9‐TGF‐β pathway is probably responsible for the downstream action of lubricin. © 2020 American Society for Bone and Mineral Research (ASBMR).

Ectopic endochondral ossification in the tendon/ligament is caused by repetitive mechanical overload or inflammation. Tendon stem/progenitor cells (TSPCs) contribute to tissue repair, and some express lubricin [proteoglycan 4 (PRG4)]. However, the mechanisms of ectopic ossification and association of TSPCs are not yet known. Here, we investigated the characteristics of Prg4-positive ( + ) cells and identified that R-spondin 2 (RSPO2), a WNT activator, is specifically expressed in a distinct Prg4 + TSPC cluster. The Rspo2 + cluster was characterized as mostly undifferentiated, and RSPO2 overexpression suppressed ectopic ossification in a mouse Achilles tendon puncture model via chondrogenic differentiation suppression. RSPO2 expression levels in patients with ossification of the posterior longitudinal ligament were lower than those in spondylosis patients, and RSPO2 protein suppressed chondrogenic differentiation of human ligament cells. RSPO2 was induced by inflammatory stimulation and mechanical loading via nuclear factor κB. Rspo2 + cells may contribute to tendon/ligament homeostasis under pathogenic conditions.

The Runt-related transcription factor (Runx) family plays various roles in the homeostasis of cartilage. Here, we examined the role of Runx2 and Runx3 for osteoarthritis development in vivo and in vitro. Runx3-knockout mice exhibited accelerated osteoarthritis following surgical induction, accompanied by decreased expression of lubricin and aggrecan. Meanwhile, Runx2 conditional knockout mice showed biphasic phenotypes: heterozygous knockout inhibited osteoarthritis and decreased matrix metallopeptidase 13 (Mmp13) expression, while homozygous knockout of Runx2 accelerated osteoarthritis and reduced type II collagen (Col2a1) expression. Comprehensive transcriptional analyses revealed lubricin and aggrecan as transcriptional target genes of Runx3, and indicated that Runx2 sustained Col2a1 expression through an intron 6 enhancer when Sox9 was decreased. Intra-articular administration of Runx3 adenovirus ameliorated development of surgically induced osteoarthritis. Runx3 protects adult articular cartilage through extracellular matrix protein production under normal conditions, while Runx2 exerts both catabolic and anabolic effects under the inflammatory condition.