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Osteoblasts produce the receptor activator of nuclear factor-kappa B ligand (RANKL) and osteoprotegerin, the inducer and the suppressor of osteoclast differentiation and activation. We previously proposed that the degradation of osteoprotegerin by lysine-specific gingipain of Porphyromonas gingivalis and neutrophil elastase is one of the mechanisms of bone resorption associated with infection and inflammation. In the present study, we found that cathepsin K (CTSK) also degraded osteoprotegerin in an acidic milieu and the buffer with a pH of 7.4. The 37 k fragment of osteoprotegerin produced by the reaction with CTSK was further degraded into low molecular weight fragments, including a 13 k fragment, depending on the reaction time. The N-terminal amino acid sequence of the 37 k fragment matched that of the intact osteoprotegerin, indicating that CTSK preferentially hydrolyzes the death domain-like region of osteoprotegerin, not its RANKL-binding region. The 13 k fragment of osteoprotegerin was the C-terminal 13 k portion within the RANKL-binding region of the 37 k fragment. Finally, CTSK restored RANKL-dependent osteoclast differentiation that was suppressed by the addition of osteoprotegerin. Collectively, CTSK is a possible positive regulator of osteoclastogenesis., (© 2023. The Society for In Vitro Biology.)

The articular cartilage is an avascular tissue, and oxygen tensions in its superficial and deeper zones are estimated to be 6% and 1%. Degeneration of the articular cartilage begins from the surface zone in osteoarthritis. We previously reported that monocarboxylate transporter-1, a transmembrane transporter for monocarboxylates, played an essential role in the interleukin-1β-induced expression of NADPH oxidase-2, a reactive oxygen species-producing enzyme, and reactive oxygen species-dependent death of mouse chondrogenic ATDC5 cells cultured in a normal condition (20% oxygen). Here, we investigated the effect of oxygen tension on interleukin-1β-induced events described above in ATDC5 cells. Interleukin-1β induced the death of ATDC5 cells under 20% and 6% oxygen but did not under 2% and 1% oxygen. Interleukin-1β induced Mct1 (monocarboxylate transporter-1 gene) and Nox2 (NADPH oxidase-2 gene) mRNAs' expression under 20% oxygen in 24 h, respectively, but not under 2% oxygen. On the other hand, a 24-h incubation with interleukin-1β upregulated the expression of Nos2 (inducible nitric oxide synthase gene) mRNA irrespective of oxygen tension. Furthermore, inhibition of I-κB kinase suppressed the interleukin-1β-induced expression of Mct1 mRNA in the cells cultured under 20% and 2% oxygen, indicating NF-κB plays an essential role in the induction of the Mct1 gene expression. The results suggest that interleukin-1β induces monocarboxylate transporter-1 in an oxygen tension-dependent manner required for cell death in ATDC5 cells. These results might explain some part of the degenerative process of the articular cartilage, which begins from its superficial zone in the pathogenesis of osteoarthritis., (© 2022. The Society for In Vitro Biology.)

Down's syndrome is one of the most common human congenital genetic diseases and affected patients have increased risk of periodontal disease. To examine involvement of the disease with periodontal disease development, we established immortalized periodontal ligament cells obtained from a Down's syndrome patient by use of SV40T-Ag and hTERT gene transfection. Expressions of SV40T-Ag and hTERT were observed in periodontal ligament cell-derived immortalized cells established from healthy (STPDL) and Down's syndrome patient (STPDLDS) samples. Primary cultured periodontal ligament cells obtained from a healthy subject (pPDL) had a limited number of population doublings (< 40), while STPDL and STPDLDS cells continued to grow with more than 80 population doublings. Primary cultured periodontal ligament cells obtained from the patient showed a chromosome pattern characteristic of Down's syndrome with trisomy 21, whereas STPDLDS samples showed a large number of abnormal chromosomes in those results. Gene expression analysis revealed that expression of DSCR-1 in STPDLDS is greater than that in STPDL. These results suggest that the newly established STPDLDS cell line may be a useful tool for study of periodontal disease in Down's syndrome patients., (© 2021. The Author(s).)

Osteosarcoma (OS) is the most common primary malignant bone tumor. However, the therapeutic results of the advanced cases at the first visit were still extremely poor. Therefore, more effective therapeutic options based on molecular profiling of OS are needed. In this study, we investigated the functions of endoplasmic reticulum (ER) stress activities in OS and elucidated whether ER stress inhibitors could exert antitumor effects. The expression of 84 key genes associated with unfolded protein response (UPR) was assessed in four OS cells (143B, MG63, U2OS and KHOS) by RT2 Profiler PCR Arrays. Based on results, we performed both siRNA and inhibitor assays focusing on IRE1α-XBP1 and PERK pathways. All OS cell lines showed resistance to PERK inhibitors. Furthermore, ATF4 and EIF2A inhibition by siRNA did not affect the survival of OS cell lines. On the other hand, IRE1α-XBP1 inhibition by toyocamycin suppressed OS cell growth (IC50: < 0.075 μM) and cell viability was suppressed in all OS cell lines by silencing XBP1 expression. The expression of XBP1s and XBP1u in OS cell lines and OS surgical samples were confirmed using qPCR. In MG63 and U2OS, toyocamycin decreased the expression level of XBP1s induced by tunicamycin. On the other hand, in 143B and KHOS, stimulation by toyocamycin did not clearly change the expression level of XBP1s induced by tunicamycin. However, morphological apoptotic changes and caspase activation were observed in these two cell lines. Inhibition of the IRE1α-XBP1s pathway is expected to be a promising new target for OS., (© 2021. The Author(s).)

Environmental contaminations of 129 I were continuously monitored in various sample preparation rooms for accelerator mass spectrometry at the University of Tsukuba. Monitoring of 129 I was performed in the rooms used for the treatment of samples in the past, in order to compare with the results obtained in the sample preparation rooms. Ambient levels of atmospheric 129 I in each room were estimated from the measured concentrations in the alkali trap solutions. This article reports the results of one year of monitoring the temporal changes of stable iodine ( 127 I) and 129 I contamination rates in the alkali trap solutions. It was found that 129 I contamination rates were lower than approximately 10 4 atoms cm -2 day -1 in the rooms where ether no samples or only samples with environmental background levels of 129 I were handled. Values from 10 4 to 10 5 atoms cm -2 day -1 were recorded in another room where environmental samples, such as the samples derived from nuclear power plant accidents, were treated. Higher levels of 129 I, ranging from 10 6 to 10 7 atoms cm -2 day -1 , were recorded in rooms used for treating neutron-activated iodine. The experimental results show that the 129 I level depended on the 129 I sample-preparation histories for the respective rooms. It is possible to estimate the 129 I contamination risk from the atmosphere to the samples by knowing the 129 I level in the preparation room.

Osteocytes regulate bone remodeling, especially in response to mechanical loading and unloading of bone, with nitric oxide reported to play an important role in that process. In the present study, we found that 8-nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP), a second messenger of nitric oxide in various types of cells, was produced by osteocytes in bone tissue as well as cultured osteocytic Ocy454 cells. The amount of 8-nitro-cGMP in Ocy454 cells increased during incubation with parathyroid hormone or prostaglandin E 2 , both of which are known to upregulate receptor activator of nuclear factor-κB ligand (RANKL) mRNA expression in osteocytes. On the other hand, exogenous 8-nitro-cGMP did not have effects on either the presence or absence of these bioactive substances. Furthermore, neither an inhibitor of nitric oxide synthase nor 8-bromo-cGMP, a cell-permeable analog of cGMP, showed remarkable effects on mRNA expression of sclerostin or RANKL. These results indicate that neither nitric oxide nor its downstream compounds, including 8-nitro-cGMP, alone are sufficient for induction of functional changes in osteocytes.