Abstract
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.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Adaptor Proteins, Signal Transducing*
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Animals
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Bacterial Proteins*
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Biomarkers
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COS Cells
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Carrier Proteins / drug effects
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Carrier Proteins / genetics
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Carrier Proteins / metabolism
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Cell Differentiation / drug effects
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Cell Differentiation / genetics*
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Chondrocytes / cytology
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Chondrocytes / drug effects
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Chondrocytes / enzymology*
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Colicins / drug effects
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Colicins / genetics
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Colicins / metabolism
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Collagen Type II / drug effects
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Collagen Type II / genetics
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Collagen Type II / metabolism
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Cyclic AMP-Dependent Protein Kinase Type II
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Cyclic AMP-Dependent Protein Kinases / drug effects
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Cyclic AMP-Dependent Protein Kinases / metabolism*
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Drug Interactions / physiology
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Gene Expression Regulation, Developmental / physiology
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Growth Plate / cytology
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Growth Plate / embryology*
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Growth Plate / enzymology
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Hedgehog Proteins
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Membrane Proteins / genetics
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Membrane Proteins / metabolism
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Models, Biological
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Mutation, Missense / genetics
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Nerve Tissue Proteins*
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Osteogenesis / drug effects
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Osteogenesis / physiology*
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Parathyroid Hormone-Related Protein
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Patched Receptors
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Peptide Hormones / antagonists & inhibitors
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Peptide Hormones / genetics
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Peptide Hormones / metabolism
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Protein Structure, Tertiary / drug effects
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Protein Structure, Tertiary / physiology
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Rats
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Receptors, Cell Surface
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Recombinant Fusion Proteins
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Signal Transduction / drug effects
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Signal Transduction / physiology
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Trans-Activators / genetics
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Trans-Activators / metabolism*
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Trans-Activators / pharmacology
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Tumor Suppressor Proteins*
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Up-Regulation / drug effects
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Up-Regulation / physiology*
Substances
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Adaptor Proteins, Signal Transducing
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Bacterial Proteins
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Bin1 protein, mouse
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Biomarkers
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Carrier Proteins
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Colicins
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Collagen Type II
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Hedgehog Proteins
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Membrane Proteins
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Nerve Tissue Proteins
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Parathyroid Hormone-Related Protein
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Patched Receptors
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Peptide Hormones
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Receptors, Cell Surface
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Recombinant Fusion Proteins
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Trans-Activators
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Tumor Suppressor Proteins
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Cyclic AMP-Dependent Protein Kinase Type II
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Cyclic AMP-Dependent Protein Kinases