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Ihh enhances differentiation of CFK-2 chondrocytic cells and antagonizes PTHrP-mediated activation of PKA

¹ Department of Medicine and Lady Davis Institute for Medical Research, Sir Mortimer B. Davis-Jewish General Hospital, McGill University, Montreal, QC, Canada H3T 1E2
² Calcium Research Laboratory, Department of Medicine, McGill University Health Centre and McGill University, Montreal, QC, Canada H3A 1A1

View larger version (31K): [in a new window]   Fig. 1. Expression of Ihh variants in COS-1 and CFK-2 cells. (A) Schematic representation of the cDNAs for Ihh, its myc-tagged variant, and its N-terminal domain (N-Ihh), used by insertion into the pcDNA3 mammalian expression vector. Indicated are the internal signal sequence and the conserved cleavage site between Gly241 and Cys242. (B) Western blot analysis of lysates (lanes 1-4; 9-10) or conditioned media (lanes 5-8; 11-12) from COS-1 cells transiently transfected with pcDNA3 (lanes 1,5,9,11), Ihh (lanes 2,6), Ihh-myc (lanes 3,7,10,12) or N-Ihh (lanes 4,8). The detection of a 46 kDa and 19 kDa species in lysates from Ihh or Ihh-myc transfectants was facilitated by the use of -Ihh-N antibody specific to the N-terminal domain of Ihh (lanes 1-8). The 26 kDa C-terminal domain of Ihh was identified with the use of an anti-myc antibody (lanes 9-12). (C) Western blot analysis of COS-1 cell lysates following transient transfection with pcDNA3, N-Ihh-pcDNA3 or N-Ihh W160G-pcDNA3. The 19 kDa peptide species attributed to the N-terminal domain of Ihh in its normal and mutant forms was detected by the -Ihh-N antibody.

View larger version (24K): [in a new window]   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.

View larger version (35K): [in a new window]   Fig. 2. Induction of Ptc receptor expression by Ihh, N-Ihh, N-Ihh W160G or exogenous N-Shh in CFK-2 cells. (A) Expression of Ptc in stably transfected CFK-2 cells. CFK-2 cells that were stably transfected with pcDNA3, Ihh-pCDNA3, N-Ihh-pcDNA3 or N-Ihh W160G-pcDNA3 were subjected to postconfluent growth conditions for a period of 16 days and total RNA was extracted intermittently at the indicated times. Ptc mRNA expression was assessed by northern blot analysis and compared to the respective transgene level. (B) A graphic depiction of the results shown in A, comparing Ptc expression with the levels of GAPDH expression where bars represent average levels at the three time points (*P<0.01). (C) Comparison of Ptc expression to transgene levels in Ihh-, N-Ihh- and N-Ihh W160G-transfected cells after these were normalized to GAPDH mRNA. (D) Dose response induction of Ptc expression by exogenous N-Shh. CFK-2 cells were subjected to exogenous treatment with increasing concentrations of recombinant N-Shh or vehicle for a period of 6 days in postconfluent culture, after which total cellular RNA was extracted and subjected to northern blot analysis.

View larger version (40K): [in a new window]   Fig. 3. Induction of chondrogenic markers in CFK-2 cells by Ihh is mimicked by recombinant N-Shh. (A) Alkaline phosphatase (ALP) activity is enhanced by Ihh or N-Ihh, but not by N-Ihh W160G. CFK-2 cells stably transfected with pcDNA3, Ihh or N-Ihh underwent postconfluent growth and were analyzed for ALP enzymatic activity during the indicated time periods (left). ALP activity was also compared in stably transfected CFK-2 cell populations expressing N-Ihh or its mutant variant form, N-Ihh W160G (right). (B) Induction of Col2a1 and Col10a1 collagens by Ihh or N-Ihh. Stably transfected CFK-2 cell populations expressing Ihh, N-Ihh, N-Ihh W160G, or vector alone were subjected to postconfluent growth conditions and total RNA was extracted at the indicated time periods. Northern blot analysis was used to evaluate expression levels of the chondrogenic markers Col2a1 and Col10a1. (C) Dose-response induction of ALP, Col2a1 and Col10a1 by exogenous N-Shh. Naive CFK-2 cells grown to postconfluence were subjected to treatment by vehicle or exogenous N-Shh peptide at the indicated concentrations. After 6 days of treatment, ALP enzymatic activity in cell extracts was measured (left) and total RNA was analyzed for Col2a1 and Col10a1 expression (right). A maximal response was observed at a concentration of 10-8 M N-Shh.

View larger version (27K): [in a new window]   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 Cß 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).

View larger version (15K): [in a new window]   Fig. 6. Proposed model for interaction between the PTHrP and HH signalling pathways. Signalling by all mammalian HH proteins is mediated by the Gli family of transcription factors that consist of three members: Gli1, Gli2 and Gli3. Although all Gli proteins harbour consensus PKA phosphorylation sites, only Gli2 and Gli3 are known to be functionally converted to transcriptional repressors following phosphorylation by PKA. A plausible scenario for PTHrP to antagonize HH would be through activation of PKA via PTHR1. PKA may differentially phosphorylate Gli2 or Gli3, consequently converting them to repressors and causing downregulation of some HH target genes (ALP, Col2a1, Col10a1, PTHR1) while not affecting others (Ptc). HH, in turn, may antagonize PKA activation, possibly through functional inhibition of PTHR1, thereby ensuring its own continued signalling capability.

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