Tokyo Dental College

Tartrate-resistant acid phosphatase (TRAP) staining is widely used to stain osteoclasts in histological bone sections. The red dye formed by the conventional TRAP enzymatic reaction using naphthol AS-MX (or AS-BI) phosphate and fast red-violet (or garnet) chromogens is readily soluble in alcohol or xylene and requires air-drying prior to cover slipping or the use of an aqueous mounting medium. However, the use of an aqueous mounting medium makes it difficult to store stained specimens for a long time. In this modified method, a new fuchsin (NewFuchsin) was used as a chromogen, which enabled dehydration and clearing after staining and the use of a non-aqueous organic solvent-based mounting medium. Samples prepared using this modified TRAP activity staining method (NewFuchsin TRAP staining) have the following advantages over conventional TRAP staining:•The staining of sections provides a clear histological image and allows for long-term preservation.•The red dye formed by NewFuchsin TRAP staining can be detected not only in the bright field, but also in the fluorescent field.•Combined with tissue clearing using ethyl cinnamate, osteoclasts are observed using three-dimensional imaging.

Hypervitaminosis D is induced iatrogenically or endogenously. We previously reported that the vitamin D receptor (VDR) in osteoblast lineage cells mediates bone resorption and soft-tissue calcification in hypervitaminosis D. However, bone formation in hypervitaminosis D remains understudied. Here, we show that abundant 1α,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] suppresses bone formation through VDR in osteoblast lineage cells. High-dose 1,25(OH)₂D₃ suppressed bone formation and increased serum sclerostin, a bone formation inhibitor, in Control but not osteoblast lineage-specific VDR-cKO [Osterix (Osx)-VDR-cKO] mice. However, Sost mRNA expression in bone was downregulated by 1,25(OH)₂D₃ in Control but not Osx-VDR-cKO mice. Meanwhile, mRNA expression of β-1,4-N-acetyl-galactosaminyltransferase 3 (B4GALNT3), whose function is reported to decrease circulating sclerostin, was suppressed by 1,25(OH)₂D₃ in bone in Control but not Osx-VDR-cKO mice. Overexpressed B4galnt3 in rodent osteoblast-lineage cell lines increased GalNAcβ1→4GlcNAc- (LDN-) glycosylated sclerostin, suggesting that this modification can explain the discordance between serum sclerostin levels and mRNA in bone. Although excessive 1,25(OH)₂D₃ increased mRNA levels of Fibroblast growth factor 23 (Fgf23), another osteotropic factor, by 10-fold through VDR in osteoblast lineage cells, it was previously shown to increase serum FGF23 levels by several hundred-fold. 1,25(OH)₂D₃-induced changes of FGF23-degradation regulators, such as furin, polypeptide N-acetylgalactosaminyltransferase 3 (GALNT3), and family with sequence similarity member 20 C (FAM20C), did not match the markedly high FGF23 levels, suggesting the existence of other regulators of FGF23. These findings suggest that VDR plays pivotal roles in the suppression of bone formation in hypervitaminosis D, possibly by increasing circulations of sclerostin and FGF23 through post-translational or post-transcriptional mechanisms.

Translocator protein (TSPO) is a mitochondrial outer membrane protein expressed on a variety of immune cells, including macrophages, dendritic cells, and T cells, in addition to neurons and steroid-producing cells. Previous studies of TSPO ligands have suggested that TSPO is involved in multiple cellular functions, including steroidogenesis, immunomodulation, and cell proliferation. Currently, there are limited reports on the effects of TSPO or TSPO ligands on T cell-mediated immune responses. Here, we investigated the involvement of TSPO/TSPO ligand in T cell responses using a 2,4-dinitro-1-fluorobenzene (DNFB)-induced contact hypersensitivity (CH) model. Treatment with Ro5-4864, a TSPO ligand, during DNFB sensitization reduced the number and activation status of CD4+ and CD8+ T cells in draining lymph nodes and alleviated skin inflammation after DNFB challenge. Adoptive transfer of Ro5-4864-treated mouse-derived DNFB-sensitized T cells to naive mice inhibited CH responses after DNFB challenge. Ro5-4864-treated sensitized T cells showed lower proliferative responses when stimulated with DNFB-pulsed antigen-presenting cells compared to control-treated sensitized T cells. Ro5-4864 also suppressed cell proliferation, as well as adenosine triphosphate and lactate production, during T cell activation. Moreover, the inhibitory effects of Ro5-4864 on T cell responses were conserved in TSPO-deficient cells. Our results suggest that Ro5-4864 inhibits CH responses by suppressing energy metabolism, at least via glycolysis, to reduce the T cell primary response in a TSPO-independent manner.