Figure 2

cAMP/PKA signaling induces Hif-1α expression. (a) Levels of Hif-1α protein were measured in wild-type osteoblasts following 4 h of treatment with 10 nmol·L⁻¹ PTH or vehicle (NT), preceded by 30 min of treatment with 100 μg·mL⁻¹ cycloheximide (Chx). (b) Levels of Akt (S473) and p70 S6 kinase protein phosphorylation were measured in wild-type osteoblasts following 1 h of treatment with 10 nmol·L⁻¹ PTH. (c) Hif-1α protein expression was measured in control and mTOR-deficient osteoblasts following treatment with 10 nmol·L⁻¹ PTH for 4 h. (d) Densitometric quantitation of Hif-1α protein as shown in a and c. (e and g) Akt and p70 S6 kinase phosphorylation (e) and Hif-1α protein expression (g) were measured in wild-type osteoblasts following 30 min (e) or 6 hours (g) of treatment with Forskolin at the indicated concentrations. (f) Quanitification of Akt phosphorylation shown in e in Forskolin-treated osteoblasts relative to untreated. (h) Relative expression of Hif-1α protein as shown in g. (i and l) Levels of Akt and p70 S6 kinase phosphorylation (i) and Hif-1α protein expression (l) were measured following 30 min (i) or 6 h (l) of treatment with 10 nmol·L⁻¹ PTH, preceded by 30 min of treatment with 10 μmol·L⁻¹ H-89. (j and k) Quantification of Akt (j) and p70 S6 kinase (k) phosphorylation shown in i in PTH- and/or H-89-treated osteoblasts relative to untreated. (m) Relative expression of Hif-1α protein as shown in l. *P≤0.05.