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Figure 2. Inhibition of the cAMP/PKA signaling pathway did not inhibit PTHrP-induced TRAP⁺ cell formation in co-cultures. (A) Co-cultures were pre-treated with or without Rp-cAMP (100 µM), H89 (1 µM), or PKI (1 µM) for 30 min, followed by incubation with either PTHrP (10^–8 M) or PGE₂ (10^–8 M), together with Dex (10^–7 M), for 10 days. TRAP⁺ cells were counted and expressed as number of TRAP⁺ cells per culture well (values are mean ± SEM, n = 3). Similar results were obtained in 3 independent experiments. *P < 0.01 vs. PGE₂-treated cultures. (B) Microscopic view of TRAP⁺ cells. Bar = 100 µm. (C) Human PDL cells were pre-treated with PKI (1 µM) for 30 min, followed by incubation with PTHrP (10^–8 M) for 72 hrs. Total RNA was isolated from PDL cells, and expression levels of RANKL, OPG, and GAPDH mRNA were measured by RT-PCR analysis. Numbers below the gels represent n-fold increase in intensity of RANKL or OPG relative to corresponding GAPDH mRNA signals. Similar results were obtained in 3 independent experiments. (D) Human PDL cells were pre-treated with or without PKI (0.1, 1 µM) for 30 min, followed by incubation with PTHrP (10^–8 M) or PGE₂ (10^–8 M) for 20 min together with Dex. Total cell lysates were used for PKA assay, with the PepTag PKA assay kit. Similar results were obtained in 3 independent experiments. (E) PKA activity was measured by densitometry. Each column indicates the relative value of phosphorylation by PKA vs. the intensity of the phosphorylation signal without PTHrP. *P < 0.01 vs. PTHrP or PGE₂-treated cultures. (F) Co-cultures of mouse bone marrow cells (1 x 10⁵ cells/well) and mouse calvaria cells (1 x 10⁵ cells/well) were pre-treated with or without H89 (1 µM) or PKI (1 µM) for 30 min, followed by incubation with PTHrP (10^–8 M) for 5 days. TRAP⁺ multinucleated cells (MNCs) were counted. Data shown are the number of TRAP⁺ MNCs per culture well (values are mean ± SEM, n = 3). Similar results were obtained in 3 independent experiments. (G) Microscopic view of TRAP⁺ cells and MNCs. Bar = 100 µM.

J DENT RES, Vol. 84, No. 4, 329-334 (2005)
DOI: 10.1177/154405910508400407

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