Identification of kaempferol-regulated proteins in rat calvarial osteoblasts during mineralization by proteomics

Kaempferol, a flavonoid, promotes osteoblast mineralization in vitro and bone formation in vivo; however, its mechanism of action is yet unknown. We adopted proteomic approach to identify the differential effect of kaempferol on rat primary calvarial osteoblasts during mineralization. The primary rat calvarial osteoblasts were treated with kaempferol (5.0 microM) for 9 days under mineralizing condition that resulted in significant increase in alkaline phosphatase activity and mineralization of the cells. Further, 2-D analysis of the kaempferol-treated osteoblast lysates revealed 18 differentially expressed proteins (nine upregulated and nine downregulated) on the basis of >/<2.0-fold as cut-off (p<0.01) that were then identified by MALDI-TOF MS. These included cytoskeletal proteins, intracellular signaling protein, chaperone, extracellular matrix protein, and proteins involved in glycolysis and cell-matrix interactions. Proteomics data were confirmed by Western blotting and quantitative real-time PCR by randomly selecting two upregulated and two downregulated proteins. Western blot analysis confirmed upregulation of HSP-70 and cytokeratin-14 levels, and downregulation of aldose reductase and caldesmon expression. We further demonstrated that kaempferol treatment inhibits aldose reductase activity in osteoblasts indicating an altered cellular metabolism by decelerating polyol pathway that was associated with the kaempferol-induced osteoblast mineralization. In conclusion, this is a first comprehensive study on the differential regulation of proteins by kaempferol in primary osteoblast, which would further help to elucidate the role of the identified proteins in the process of osteoblast mineralization.