Fig. 4. PTHrP antagonizes hedgehog-induced chondrogenic differentiation through a PKA-dependent mechanism. (A) PTHrP and forskolin impede HH-induced ALP activity. Naive CFK-2 cells grown to confluence were subjected to treatment with vehicle or N-Shh (5x10^-9 M) in the absence or presence of PTHrP 1-34 (10^-8 M) or forskolin (10^-7 M) for a period of 8 days. Cell extracts were used to measure ALP enzymatic activity. (B) Selective inhibition of HH-induced gene expression by PTHrP. Northern blot analysis for the assessment of Col2a1, Col10a1 and Ptc expression was performed on total RNA extracted from postconfluent CFK-2 cells that were subjected to 8-day treatment with vehicle or N-Shh (5x10^-9 M) in the absence or presence of PTHrP 1-34 (10^-8 M) or forskolin (10^-7 M). (C) Constitutive activation of PKA via PTHR1 H223R interferes with HH-induced ALP activity. CFK-2 cell populations were generated following double stable transfection with Ihh-pcDNA3/pcDNA3.1, Ihh-pcDNA3/PTHrP-pcDNA3.1 or Ihh-pcDNA3/PTHR1 (H223R)-pcDNA3.1. ALP specific activity was measured following 10 days of postconfluent culture.