title: Effects of erythropoietin on osteoblast proliferation and function.
abstract: The purposes of this study were to investigate the effects of erythropoietin (EPO) on the proliferation and function of human osteoblast cells (hFOB 1.19) cultured in vitro and to explore the underlying molecular mechanisms to provide a theoretical foundation for clinical applications of EPO in oral implant and restoration therapies. Cultured hFOB 1.19 cells were treated with high and low doses of EPO. Changes in cell viability after 24 and 48 h of treatment were evaluated with the Mosmann tetrazolium assay. Changes in cell proliferation after 48 h of EPO treatment were measured by bromodeoxyuridine (BrdU) labeling, and changes in alkaline phosphatase (ALP) activity were determined by a specific assay. The effects of EPO on osteocalcin secretion were determined with the enzyme-linked immunosorbent assay, and changes in the protein expression of osteoprotegerin (OPG), osteopontin (OPN) and receptor activator of NF-κB ligand (RANKL) were assayed by western blot. The effects of EPO treatment on the levels of the EPO receptor (EPOR), phosphorylated Jak2 (P-Jak2) and phosphorylated Stat3 (P-Stat3) in hFOB 1.19 cells were evaluated in conjunction with a Jak2/Stat3 inhibitor. After 24 h of EPO treatment, hFOB 1.19 cells showed increased cell viability compared with the blank control group (p < 0.05). After 48 h, cell viability and growth were further improved relative to controls, with a significant increase observed for viability (p < 0.05). A significant increase in the proportion of BrdU-labeled proliferating cells was observed in the high-dose EPO group (p < 0.05), and EPO-treated cells also showed enhanced ALP activity (p < 0.05). There were no statistically significant differences in osteocalcin secretion between groups after 48 h of EPO treatment (p > 0.05); however, increased secretion was observed in EPO-treated cells after 96 h of treatment (p < 0.05). EPO treatment significantly promoted OPG and OPN expression (p < 0.05) while significantly inhibiting RANKL expression (p < 0.01). EPO treatment also significantly upregulated the levels of EPOR, P-Jak2 and P-Stat3 in hFOB 1.19 cells (p < 0.01); these effects were abrogated by co-treatment with a Jak2/Stat3 inhibitor (AG490) (p < 0.01). EPO significantly stimulated osteoblast proliferation and differentiation. The underlying molecular mechanism is associated with the ability of EPO to promote ALP activity, osteocalcin secretion and OPG and OPN expression and to inhibit RANKL expression in osteoblasts. This mechanism appears to be mediated by the Jak2/Stat3 pathway.
authors.affiliation: The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) and Key Laboratory of Oral Biomedicine Ministry of Education, School and Hospital of Stomatology, Wuhan University, 237 Luoyu Road, Wuhan, 430079, People's Republic of China.
authors.forename: Lvhua
authors.initials: L
authors.lastname: Guo
authors.forename: Tao
authors.initials: T
authors.lastname: Luo
authors.forename: Ying
authors.initials: Y
authors.lastname: Fang
authors.forename: Lan
authors.initials: L
authors.lastname: Yang
authors.forename: Liping
authors.initials: L
authors.lastname: Wang
authors.forename: Jingwen
authors.initials: J
authors.lastname: Liu
authors.forename: Bin
authors.initials: B
authors.lastname: Shi
mesh_terms: D000469:Alkaline Phosphatase / Q000378:metabolism; D015153:Blotting, Western; D049109:Cell Proliferation / Q000187:drug effects*; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D004797:Enzyme-Linked Immunosorbent Assay; D004921:Erythropoietin / Q000378:metabolism*; D006801:Humans; D010006:Osteoblasts / Q000187:drug effects* / Q000502:physiology*; D015675:Osteocalcin / Q000378:metabolism; D053495:Osteopontin / Q000378:metabolism; D053244:Osteoprotegerin / Q000378:metabolism; D053245:RANK Ligand / Q000378:metabolism
chemical_list: C554270:EPO protein, human; D053244:Osteoprotegerin; D053245:RANK Ligand; C505688:TNFSF11 protein, human; D015675:Osteocalcin; D053495:Osteopontin; D004921:Erythropoietin; D000469:Alkaline Phosphatase