Ihh enhances differentiation of CFK-2 chondrocytic cells and antagonizes PTHrP-mediated activation of PKA.
Indian Hedgehog (Ihh), a member of the hedgehog (HH) family of secreted morphogens, and parathyroid hormone-related peptide (PTHrP) are key regulators of cartilage cell (chondrocyte) differentiation. We have investigated, in vitro, the actions of HH signalling and its possible interplay with PTHrP using rat CFK-2 chondrocytic cells. Markers of chondrocyte differentiation [alkaline phosphatase (ALP) activity, and type II (Col2a1) and type X collagen (Col10a1) expression] were enhanced by overexpression of Ihh or its N-terminal domain (N-Ihh), effects mimicked by exogenous administration of recombinant N-terminal HH peptide. Moreover, a missense mutation mapping to the N-terminal domain of Ihh (W160G) reduces the capacity of N-Ihh to induce differentiation. Prolonged exposure of CFK-2 cells to exogenous N-Shh (5x10(-9) M) in the presence of PTHrP (10(-8) M) or forskolin (10(-7) M) resulted in perturbation of HH-mediated differentiation. In addition, overexpression of a constitutively active form of the PTHrP receptor (PTHR1 H223R) inhibited Ihh-mediated differentiation, implicating activation of protein kinase A (PKA) by PTHR1 as a probable mediator of the antagonistic effects of PTHrP. Conversely, overexpression of Ihh/N-Ihh or exogenous treatment with N-Shh led to dampening of PTHrP-mediated activation of PKA. Taken together, our data suggest that Ihh harbors the capacity to induce rather than inhibit chondrogenic differentiation, that PTHrP antagonizes HH-mediated differentiation through a PKA-dependent mechanism and that HH signalling, in turn, modulates PTHrP action through functional inhibition of signalling by PTHR1 to PKA.
Deckelbaum
Ron A
RA
Department of Medicine and Lady Davis Institute for Medical Research, Sir Mortimer B. Davis-Jewish General Hospital, McGill University, Montreal, QC, Canada H3T 1E2.
Chan
George
G
Miao
Dengshun
D
Goltzman
David
D
Karaplis
Andrew C
AC
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D001426:Bacterial Proteins; D015415:Biomarkers; D019556:COS Cells; D002352:Carrier Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects / Q000235:genetics*; D019902:Chondrocytes / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D003087:Colicins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D024043:Collagen Type II / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D054746:Cyclic AMP-Dependent Protein Kinase Type II; D017868:Cyclic AMP-Dependent Protein Kinases / Q000187:drug effects / Q000378:metabolism*; D004347:Drug Interactions / Q000502:physiology; D018507:Gene Expression Regulation, Developmental / Q000502:physiology; D006132:Growth Plate / Q000166:cytology / Q000196:embryology* / Q000201:enzymology; D053823:Hedgehog Proteins; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008954:Models, Biological; D020125:Mutation, Missense / Q000235:genetics; D009419:Nerve Tissue Proteins; D010012:Osteogenesis / Q000187:drug effects / Q000502:physiology*; D044162:Parathyroid Hormone-Related Protein; D000072079:Patched Receptors; D036361:Peptide Hormones / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000187:drug effects / Q000502:physiology; D051381:Rats; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D025521:Tumor Suppressor Proteins; D015854:Up-Regulation / Q000187:drug effects / Q000502:physiology*
D048868:Adaptor Proteins, Signal Transducing; D001426:Bacterial Proteins; C109221:Bin1 protein, mouse; D015415:Biomarkers; D002352:Carrier Proteins; D003087:Colicins; D024043:Collagen Type II; D053823:Hedgehog Proteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D044162:Parathyroid Hormone-Related Protein; D000072079:Patched Receptors; D036361:Peptide Hormones; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; D025521:Tumor Suppressor Proteins; D054746:Cyclic AMP-Dependent Protein Kinase Type II; D017868:Cyclic AMP-Dependent Protein Kinases