ISG15 Regulates RANKL-Induced Osteoclastogenic Differentiation of RAW264 Cells

Abstract

Interferon-stimulated gene 15 kDa (ISG15) is a protein upregulated by interferon-β that negatively regulates osteoclastogenesis. We investigated the role of ISG15 in receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenic differentiation of murine RAW264 cells. RANKL stimulation induced ISG15 expression in RAW264 cells at both the mRNA and protein levels. Overexpression of ISG15 in RAW264 cells resulted in suppression of cell fusion in RANKL-stimulated cells as well as the reduced expression of ATP6v0d2, a gene essential for cell fusion in osteoclastogenic differentiation. These results suggest that ISG15 suppresses RANKL-induced osteoclastogenesis, at least in part, through inhibition of ATP6v0d2 expression.Key words interferon-stimulated gene 15 kDa (ISG15); osteoclast; interferon; receptor activator of nuclear factor-κB ligand (RANKL); RAW264; ATP6v0d2Interferon-α/β is a multifunctional cytokine that exerts various biological functions in immunity, cell differentiation, and so on via the induction of downstream genes called interferonstimulated genes (ISGs).1,2) Interferon-stimulated gene 15 kDa (ISG15), a ubiquitin-like protein, is one of the proteins that are most upregulated by interferon stimulation.3,4) Upon stimulation of the cells with interferon or pathogenic materials such as lipopolysaccharides, the expression of ISG15 and several other enzymes that catalyze protein modification with ISG15 (ISGylation) is induced, followed by the ISGylation of various cellular proteins. ISGylation affects the nuclear factor-kappa B (NF-κB) signaling pathway as well as the activity of doublestranded RNA-activated protein kinase, 5,6) both of which have important roles in osteoclastogenic differentiation. 7,8) Osteoclasts are tartrate-resistant acid phosphatase (TRAP)-positive multinuclear cells derived from monocyte/macrophage lineage cells. 7,9) Receptor activator of nuclear factor-κB ligand (RANKL) is essential for the induction of osteoclastogenesis.10) The murine RAW264 cell line is a useful model of osteoclastogenesis as, upon stimulation with RANKL, RAW264 cells differentiate into TRAP-positive mononuclear cells, which subsequently undergo cell fusion to form TRAPpositive multinuclear osteoclasts. In vivo, osteoclastogenesis is tightly controlled by factors produced from cells such as osteoblasts and osteocytes, and deregulation of osteoclastogenesis results in various diseases such as osteoporosis. 7,9,11,12)

A BioID-Derived Proximity Interactome for SARS-CoV-2 Proteins

“…The top two significantly enriched terms identified for upregulated proteins were regulation of actin cytoskeleton (hsa04810; p > 10 −4 ) and RHO GTPase effectors (R-HAS-195258; p > 10 −3 ) ( Figure 3 B), suggesting mechanisms by which SARS-CoV-2 remodels cytoskeletal networks for viral entry and budding. Additionally, three subsets of protein–protein interaction networks were identified, including several proteins involved in differentiation [ 42 , 43 , 44 , 45 , 46 ]. We identified significantly enriched terms in the group of downregulated proteins, including cellular hormone metabolic process (GO:0034754; p > 10 −5 ) and maintenance of gastrointestinal epithelium ( Figure 3 C).…”

Epigenomic and Proteomic Changes in Fetal Spleens Persistently Infected with Bovine Viral Diarrhea Virus: Repercussions for the Developing Immune System, Bone, Brain, and Heart

“…In earlier studies, our group found an upregulation of IFNB , IFNG , and ISG15 in day 97 PI fetuses (21 days post maternal infection) [ 24 , 29 ]. These IFNs and ISG15 are known inhibitors of osteoclastogenesis, and their expression of these IFNs early in fetal development may not only inhibit ongoing osteoclastogenesis, but also cause the inhibition of osteoclastogenesis through hypermethylation of genes in most pathways affecting bone development and immune cell development [ 57 , 118 , 119 ]. By day 190 of gestation, Treg cells identified BVDV as self and suppressed the immune response to viral antigens; however, the epigenetic damage would have already been done.…”

Intrinsic Type 1 Interferon (IFN1) Profile of Uncultured Human Bone Marrow CD45lowCD271+ Multipotential Stromal Cells (BM-MSCs): The Impact of Donor Age, Culture Expansion and IFNα and IFNβ Stimulation

“…IFN1-tunable immunomodulatory function in vivo can be related to their control of osteoclast formation [ 55 , 56 ] as well as lymphocyte development, maturation and memory cell retention [ 41 ], with both processes known to be strongly affected by age. In the present study, we observed a strong positive correlation between BM-MSC IL7 transcript expression, involved in both lymphocyte development and memory cells retention in the BM, and the expression of several ISGs.…”

“…As documented in previous independent studies, standard BM-MSC culture expansion leads to downregulation in the expression of a large number of genes, including ISGs [24,25,56]. To get insights into the in vivo IFN1 "status" of uncultured BM-MSCs, the IFN1 profile of purified CD45 low CD271 + cells was first compared to standard cultured BM-MSCs (nonstimulated, Figure 2).…”