Fig. 5.
Enhanced osteoblastogenic differentiation and runx2 transcriptional activity of MC3T3-E1 cells overexpressing wild-type ANK or the F376del mutant form of ANK. (A) Intracellular PPi in COS cells transfected with empty expression vector (Vector) or vector containing wild-type ank (ANK-WT) or F376del ank (ANK-Fdel) cDNA. Intracellular PPi concentration was assayed after 3 days of transfection. The intracellular PPi concentration of COS cells transfected with empty vector was set as 100%. Data were obtained from three different experiments and are expressed as means ± SD (ap < .01 versus empty vector–transfected COS cells; bp < .01 versus F376del-transfected COS cells). (B) Expression of ANK protein in MC3T3-E1 cells transfected with empty expression vector (Vector) or expression vector containing wild-type ank cDNA (ANK-WT) or F376del ank cDNA (ANK-Fdel) was analyzed by immunoblotting with an antibody specific for ANK. Immunoblotting with an antibody specific for actin was performed to demonstrate equal loading of the gel and to show that transfection of MC3T3-E1 cells affects only ANK expression and is not toxic to the cells. (C) mRNA levels of APase, bone sialoprotein (BSP), osteocalcin (OC), type I collagen (Col1A1), and (D) osterix and runx2 in MC3T3-E1 cells transfected with empty vector (Vector), vector containing ank cDNA (ANK-WT), or vector containing F376del ank cDNA (ANK-Fdel) were determined by real-time PCR and SYBR Green and normalized to 18S RNA levels. Data were obtained from triplicate PCRs using RNA from three different cultures, and values are presented as means ± SD (ap < .01 versus cells transfected with empty vector; bp < .01 versus cells transfected with vector containing wild-type ank cDNA). (E) MC3T3-E1 cells were cotransfected with a firefly luciferase reporter construct containing six runx2 DNA-binding elements (pOSE2-luc), a Renilla luciferase reporter construct, and either empty vector (Vector), vector containing ank cDNA (ANK-WT), or vector containing F376del ank cDNA (ANK-Fdel) and cultured for 48 hours after transfection. Cell lysates were analyzed for firefly luciferase activity and normalized to Renilla luciferase activity. Data were obtained from three different experiments, and values are presented as means ± SD (ap < 0.01 versus cells transfected with empty vector; bp < .01 versus cells transfected with vector containing wild-type ank cDNA).