Osteoprotegerin (OPG) serum levels are associated with liver fibrogenesis and have been proposed as a biomarker for diagnosis. However, the source and role of OPG in liver fibrosis are unknown, as is the question whether OPG expression responds to treatment. Therefore, we aimed to elucidate the regulation of OPG production and its biological activity in human and mouse livers. OPG levels were significantly higher in lysates of human cirrhotic and mouse fibrotic livers compared to healthy livers. Hepatic OPG expression localized in cirrhotic collagenous bands in and around myofibroblasts. Single cell sequencing of murine liver cells showed hepatic stellate cells (HSC) to be the main producers of OPG in healthy livers. Using mouse precision-cut liver slices, we found OPG production induced by transforming growth factor β1 (TGFβ1) stimulation. Moreover, OPG itself stimulated expression of genes associated with fibrogenesis in liver slices through TGFβ1, suggesting profibrotic activity of OPG. Resolution of fibrosis in mice was associated with significantly lower OPG levels in livers as compared to their fibrotic counterparts.OPG stimulates fibrogenesis through TGFβ1 and is closely associated with the degree of fibrogenesis. It may therefore be a novel drug target for liver fibrosis or be used as a biomarker for treatment success of novel antifibrotics.

Oncostatin M (OSM), an inflammatory cytokine belonging to interleukin-6 superfamily, is recognized as an essential factor secreted by osteal macrophages to orchestrate bone remodelling. Osteoprotegerin (OPG) produced by osteoblasts regulates osteoclastogenesis. We previously demonstrated that bone morphogenetic protein-4 (BMP-4) stimulates OPG synthesis in osteoblast-like MC3T3-E1 cells, and that SMAD1/5/8(9), p38 mitogen-activated protein kinase (MAPK), and p70 S6 kinase positively regulates the OPG synthesis. The present study is aimed to investigate the effect of OSM on BMP-4-stimulated OPG synthesis in osteoblasts. OSM suppressed the release and the mRNA expression of OPG upregulated by BMP-4 in MC3T3-E1 cells.  Neither the BMP-4-induced phosphorylation of SMAD1/5/9 nor that of p38 MAPK affected by OSM. On the other hand, the BMP-4-stimulated phosphorylation of p70 S6 kinase was considerably suppressed by OSM. These results strongly suggest that OSM suppresses the BMP-4-stimulated OPG synthesis via inhibition of the p70 S6 kinase-mediated pathway in osteoblasts.

Butyric acid as a histone deacetylase (HDAC) inhibitor was produced by a number of periodontal and root canal microorganisms (such as Porphyromonas, Fusobacterium etc.). Butyric acid may affect the biological activities of periodontal/periapical cells such as osteoblasts, periodontal ligament cells etc., and thus affect periodontal/periapical tissue destruction and healing. The purposes of this study were to study the toxic effects of butyrate on matrix and mineralization markers’ expression of MG-63 osteoblasts. Cell viability and proliferation were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Cellular apoptosis and necrosis were analyzed by propidium iodide/Annexin V flow cytometry. Protein and mRNA expression of OPG, and RANKL were analyzed by western blotting and RT-PCR. OPG, soluble RANKL (sRANKL), 8-isoprostane, pro-collagen I, MMP-2, osteonectin (SPARC), osteocalcin and osteopontin secretion into culture medium were measured by enzyme-linked immunosorbant assay. Histone H3 acetylation levels were evaluated by immunofluorescent staining (IF) and western blot. We found that butyrate induced morphologic changes of growing MG-63 cells, with bigger and flattened in appearance. Butyrate activated histone H3 acetylation of MG-63 cells. Exposure of MG-63 cells to butyrate partly decreased cell number with no marked increase in apoptosis and necrosis. Butyrate stimulated RANKL protein expression, whereas it inhibited OPG protein expression. Butyrate also inhibited the secretion of OPG in MG-63 cells, whereas sRANKL level was below detection limit. Butyrate stimulated 8-isoprostane, MMP-2 and osteopontin secretion, but not procollagen I, osteonectin, osteocalcin in MG-63 cells. In conclusion, butyric acid generated by periodontal and root canal microorganisms may potentially induce bony destruction and impair bone repair by alteration of OPG/RANKL expression/secretion, 8-isoprostane, MMP-2, and osteopontin secretion, and affect cell proliferation. These effects are possibly related to increased histone acetylation. These events are important in the pathogenesis of periodontal and periapical destruction.

Background: Osteoclastic bone resorption in the compression zone of periodontal ligament (PDL) plays a role in orthodontic tooth movement, and is regulated by the balance of RANKL and OPG. Compression downregulates OPG, conversely, tension upregulates OPG in PDL cells. However, the regulatory mechanism of OPG expression in PDL cells under different mechanical stresses remains unclear. Methods: To study microRNA (miRNA) expression profiles, compression (2g/cm²) or tension (15%-elongation) was applied to immortalized human PDL (HPL) cells, and miRNA was extracted. The miRNA expression was analyzed using a human miRNA microarray, and the changes of the miRNA expression were confirmed by real-time RT-PCR. In addition, miR-3198-mimic and -inhibitor were transfected into HPL cells to understand the resulting OPG expression and production. Results: Certain miRNAs were expressed differentially under compression and tension. Some miRNAs including miR-3198 were upregulated only by compression. Real-time RT-PCR confirmed that compression induced miR-3198, but tension reduced it, in HPL cells. miR-3198-inhibitor upregulated and miR-3198-mimic reduced OPG in HPL cells. miR-3198-inhibitor rescued the compression-mediated downregulation of OPG. On the other hand, miR-3198-mimic reduced OPG expression under tension. Conclusion: We conclude that miR-3198 is upregulated by compression and is downregulated by tension, suggesting that miR-3198 downregulates OPG in response to mechanical stress.