Fig. 5. HH-signalling upregulates PTHR1 expression while dampening PTHrP-mediated cAMP-dependent protein kinase A (PKA) activity. (A) Northern blot analysis for PTHR1 mRNA expression in CFK-2 cells treated with vehicle, PTHrP (10^-8 M) or N-Shh peptide (10^-9 M) for 7 days during postconfluent growth (left). This experiment was also repeated with CFK-2 cells undergoing treatment with N-Shh in the absence or presence of PTHrP (10^-8 M) or forskolin (10^-7 M) (right). (B) Response to PTHrP (1-34) was assessed by measuring PKA activity in stable populations of CFK-2 cells following a 24-hour period of serum starvation. Cells were treated with 300 µM isobutylmethylxanthine (IBMX) in the presence or absence of 10^-8 M PTHrP (1-34) for a period of 20 minutes. Lysates were then used to measure PKA activity and results are depicted as pmol of phosphate transferred to the kemptide substrate per unit time per mg of protein in the lysate. Activity in PTHrP-treated cells was also measured in the presence of a specific inhibitor to the catalytic subunit of PKA as control (PTHrP + PKI). Results from three independent experiments where samples were assayed in triplicates are depicted (^*P<0.05; ^**P<0.001; left panel). Duplicate protein samples from cell lysates used for PKA activity measurement were subjected to western blot analysis for the detection of the catalytic subunit of PKA. Two catalytic subunits representing the C and C_ß forms of PKA are shown (right). (C) Naive CFK-2 cells were treated for 7 days during postconfluent growth with the indicated concentrations of recombinant N-Shh. Following 24 hours of serum starvation cells were then transiently treated with PTHrP (10^-8 M) and PKA activity was measured (^*P<0.01; ^**P<0.001).