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

Abstract

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.

Ameloblastin modulates osteoclastogenesis through the integrin/ERK pathway

“…The human Keratin 14 (K14) promoter was chosen to drive the Ambn transgene because K14 was expressed in HERS, ERM and bone [25], and the K14 promoter has resulted in substantial overexpression yields in our laboratory [26]. For our studies, two transgenic constructs were generated using a modified pSKII-trans vector in which the K14 promoter, the polyA signal (a generous gift from Dr. Elaine Fuchs, Rockefeller University), the β- Globulin intron, the mouse Ambn coding region, or the LacZ gene were inserted.…”

Nanotechnology towards prevention of anaemia and osteoporosis: from concept to market

“…The bone micro-architecture was well maintained up to 30 days after withdrawal of formulated kaempferol. However, deterioration started within 15 days in the osteoporosis rats administered with free kaempferol [47]. It is a novel formulation to increase the bioavailability of flavanoids for the stimulation of osteoblasts and maintenance of skeletal health.…”

Identification of GRP78 as a molecular target of medicarpin in osteoblast cells by proteomics

“…SDS-PAGE was performed at constant current (10 mA/gel) using running buffer (25 mM tris-HCl, 192 mM glycine, and 0.1 % SDS). The proteins were visualized using silver staining as reported previously [16]. Equal amount of protein was loaded in each experiment and similar procedure for staining was used to minimize the variations arising due to sample loading, staining procedures, and run-to-run variability.…”

“…The sample for MS was prepared using standard procedure [16]. Briefly, differentially expressed silver-stained spots were excised, cut into pieces, and washed with water with agitation on a vortex mixer.…”