Fig 1.
PTH effects on MC3T3-E1 osteoblasts. Cells were seeded at a density of 50,000 cells/cm2 in 35 mm dishes and cultured in 10% FBS for 24 hrs prior to stimulation with 10−8 M PTH for 6 hrs in 0.1% FBS. (A) PTH stimulation of MC3T3 osteoblasts causes a significant decrease in Twist mRNA. RNA preparation and quantitative real-time RT-PCR for Twist1 was performed and normalized to GAPDH mRNA. Data represent mean ± SD expressed as fold over control. (B) PTH increases ATF4-dependent transcriptional activity in MC3T3-E1 osteoblasts. Cells were transiently transfected with p4OSE-1-luc and β-galactosidase normalization plasmid and treated for 6 hrs with 10−8M PTH in 0.1% FBS. Experiments were repeated at least three times in triplicates. *p<0.0001 (control vs PTH). (C) PTH activates the osteocalcin promoter in MC3T3-E1 osteoblasts. Left panel, cells were transiently transfected with wild-type and mutant p657mOG2-luc (mutations of both OSE2 sites which abolishes the Runx2-dependent induction of promoter activity) and β-galactosidase normalization plasmid and stimulated 48 h post transfection for 6 hrs with 10−8M PTH in 0.1% FBS. Experiments were repeated at least three times in replicates. *p<0.001 (control vs PTH). Right panel, MC3T3-E1 containing stably integrated copies of a 1.3-kb mOG2 promoter driving a firefly luciferase reporter gene were cultured in 10% FBS for 24 hrs prior to stimulation with 10−8 M PTH for 6 hrs in 0.1% FBS. Luciferase activity was normalized to total protein. Experiments were repeated at least three times in replicates. *p<0.001 (control vs PTH).