FIGURE 5.

Osteopontin mRNA knockdown and constitutively active PTH/ PTHrP receptor (H223R) synergistically enhanced cAMP-response element activity in MC3T3-E1 osteoblastic cells. MC3T3-E1 osteoblastic cells were plated on 24-well plates at a density of 10⁴ cells per well, and small interference RNAs for OPN and H223R were cotransfected the following day. CRE activity was evaluated by the luciferase assay, and OPN mRNA expression was measured by quantitative real-time PCR after 48 h. OPN mRNA expression was efficiently knocked down by siRNA in this experiment (a). As a reference, control siRNA were used. b, cAMP-response element activity after cotransfection of OPN siRNA and H223R. H223R increased CRE activity by 32.4-fold. Cotransfection of OPN siRNA and H223R synergistically enhanced CRE activity by over 350-fold (*, p < 0.05; **, p < 0.01). Data represent three independent experiments.