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Fig. 4 PKA stimulators increase phosphorylation of CREB to regulate gene expression of leptin, PPARγ2 and Runx2 and the RANKL/OPG ratio. Cells were treated in AIM with IBMX (0.45 mM) after serum starvation for 48 hours. (A) PKA activity increased immediately and reached the peak at 2 hours and gradually returned to the baseline at 24 hours. (B) Cells were treated in AIM with different combinations of IBMX (0.45 mM), forskolin (100 μM), and PKI (20 μM), and PKA activities were measured at 2 hours after treatment. (C,D) The same experiment of (A). Level of CREB phosphorylation was indicated as ratio of pCREB/CREB protein level. (E) The same experiment of (B) Levels of CREB phosphorylation were measured at 2 hours after treatment. Data are presented as the mean±SD values of three separate experiments, and significance was determined by Student's t-test. (* p<0.05, ** p<0.01 versus the baseline). (F,G) Suppression of forskolin-induced modulation of gene expression of leptin, PPARγ2, Runx2, RANKL and OPG by transfection with DN CREB. Cells were transfected with control (pcDNA3) or DN CREB vector for 48 hours, followed by treatment in AIM with forskolin (100 μM) for 3 days. (F) Western blot analysis of CREB protein in the transfected cells. (G) RT-PCR analysis of gene expression in transfected cells. Transfection with DN CREB abrogated forskolin-induced block of leptin expression, initiation of PPARγ2 expression, and inhibition of Runx2 expression, and increased the ratio of RANKL/OPG gene expression.