Effects Of High Glucose On Rankl-Induced Osteoclast Differentiation And Expression Of ATP6V0D2、Dc-Stamp

At present, diabetes has become a serious public health disorder, because of the prevalence and the acute or chronic complications, and osteoporosis referred to as a "silent killer" is one of the chief causes of fractures, disability, and death. The imbalance between bone formation and bone resorption could be the main cause of the increased risk of fracture in diabetes.Bone is continuously remodeled through osteoblasts and osteoclasts. Osteoclasts,the only cells capable of resorbing bone, are multinucleated giant cells derived from the monocyte/macrophage lineage. As well as osteoblasts, normal number and activity of osteoclasts is essential for normal bone turnover. Several recent studies suggests high glucose inhibits osteoclastogenesis. In many type1diabetes studies, osteoclasts show diminished number and activity. Suppression of osteoclas functions also is found in type2diabetes. So high glucose may inhibit osteoclastogenesis, tip the balance between formation of new bone and resorption of old bone, and decrease bone turnover. Currently, the specific mechanism involved in the inhibition of osteoclastogenesis remains to be elucidated. Osteoclast differentiation depends on interaction between Receptor Activator of Nuclear factor Kappa B Ligands (RANKL) and Receptor Activator of Nuclear factor Kappa B (RANK), which activate many transcription factors and downstream signaling pathways. RANKL induces osteoclast differentiation through promoting the expression of Nuclear factor Kappa B(NF-κB), c-fos, Nuclear factor of activated T cells (NFATcl), c-fos is the upstream regulatory factor of NFATcl, and cooperate with NFATcl to induce osteoclast differentiation. Fusion-mediated giant cell formation is critical for osteoclast maturation and is required to resorb bone, v-ATPase VO subunit d2(ATP6v0d2) and dendritic cell-specific transmembrane protein (DC-STAMP) expression are required for osteoclast fusion. NFATcl directly induces the expression of Atp6v0d2and DC-STAMP, which can accelerate cell-cell fusion and multinucleation process of preosteoclasts. In this study, we focused on evaluating the effects of high glucose on two essential transcription factors and the downstream molecules involved in cell-cell fusion in order to explore how high glucose affects the process of osteoclast differentiation.Part I:Effect of high glucose on RANKL-induced osteoclast differentiationObjective:Murine RAW264.7cells were treated as osteoclast precursor cells, and recombinant RANKL was used to induce osteoclast differentiation. This part intends to explore the effect of high glucose on RANKL-induced osteoclast differentiation.Methods:According to glucose concentration and RANKL intervention, cells were divided into four groups, untreated group (glucose of5.6mmol/L, no RANKL), control group (glucose of5.6 mmol/L+RANKL), high glucose group (glucose of20.2mmol/L+RANKL), osmotic control group (glucose of5.6mmol/L+mannitol of14.6mmol/L+RANKL). Raw264.7cells were seeded at a density of2×104cells/well in24-well plates cultured with RANKL and different doses of D-glucose for4days, then cells were stained using the TRAP kit. TRAP-positive multinucleated cells containing three or more nuclei were considered to be osteoclasts, and osteoclast numbers of four groups were analyzed by one-way ANOVA. RAW264.7cells were seeded in a96-well culture plate and cultured in DMEM with various concentration of D-glucose, On day4, cell viability was determined by a Cell-Counting Kit-8.Results:RANKL induced the information of TRAP positive multinucleated cells, however, the increase of TRAP-positive multinucleated cells in control cultures was inhibited by the addition of20.2mmol/L of D-glucose.(P<0.01), and cell proliferation test indicated that high glucose increased RAW264.7cells proliferation. Conclusion:high glucose significantly inhibited RANKL induced osteoclasts differentiation in RAW264.7cells.Part II:Effects of high glucose on RANKL-induced expression of ATP6v0d2、DC-STAMP and related transcription factorsObjective:Preosteoclast and osteoclast fusion was critical for multinucleation of osteoclasts, which ensured the function to resorb bone. This part focus in the effects of high glucose on gene expression of ATP6v0d2、DC-STAMP、c-fos and NFATcl to explore how high glucose inhibit osteoclastogenesis.Methods:As Part Ⅰ, RAW264.7cells were divided into four groups, untreated group (glucose of5.6mmol/L, no RANKL), control group (glucose of5.6mmol/L+RANKL), high glucose group (glucose of20.2mmol/L+RANKL), osmotic control group (glucose of5.6mmol/L+mannitol of14.6mmol/L+RANKL). RAW264.7cells were seeded at a density of1×105cells/well in6-well plates cultured with RANKL and different doses of D-glucose (5.6and20.2mmol/L D-glucose) or Mannitol for5days,then Total-RNA was extracted from cells with different doses of glucose using the TRIzol method, cDNAs were synthesized using1μg of RNA. Quantitative PCR was performed. Meanwhile, RAW264.7cells were seeded at a density of1×105cells/well in6-well plates cultured with RANKL and different doses of D-glucose (5.6and20.2mmol/L D-glucose) or Mannitol for5days, nuclear protein was extracted and the protein concentration was determined, then nuclear protein was examined by Western blot, which could quantify the protein level of c-fos and NFATcl relatively.Results:Real time RT-PCR results showed that high glucose significantly suppressed c-fos, NFATcl gene expression induced by RANKL(P<0.05, P<0.01), at the same time, high glucose significantly inhibited ATP6v0d2, DC-STAMP gene expression (P<0.01). There was no significant difference between osmotic control group and control group in gene expression of c-fos、NFATc1、 ATP6v0d2、DC-STAMP. Western blot results showed that high glucose significantly suppress c-fos, NFATcl protein expression induced by RANKL (P<0.05). Difference between osmotic control group and control group in protein expression of c-fos、NFATcl was not statistically significant.Conclusion:High glucose significantly inhibited RANKL-induced the expression of c-fos and NFATcl, at the same time, suppression on ATP6v0d2, DC-STAMP expression of high glucose was also found. Suppression on RANKL-induced cell-cell fusion of high glucose may play essential role in the decrease of osteoclasts number.