ABSTRACT
Type X collagen (Col-X) deposition is a marker of terminal differentiation during chondrogenesis, in addition to appositional growth and apoptosis. The parathyroid hormone/parathyroid hormone related peptide (PTH/PTHrP) receptor, or PPR, is a G-Protein coupled receptor (GPCR), which activates several downstream pathways, moderating chondrocyte differentiation, including suppression of Col-X deposition. An Avian sterna model was used to analyze the PPR GPCR downstream kinase role in growth rate and extracellular matrix (ECM) including Col-II, IX, and X. Phosphatidylinositol kinase (PI3K), mitogen activating protein kinase (MAPK) and protein kinase A (PKA) were inhibited with specific established inhibitors LY294002, PD98059, and H89, respectively to test the hypothesis that they could reverse/inhibit the PTH/PTHrP pathway. Excised E14 chick sterna were PTH treated with or without an inhibitor and compared to controls. Sternal length was measured every 24 hr. Cultured sterna were immuno-stained using specific antibodies for Col-II, IX, or X and examined via confocal microscopy. Increased growth in PTH-treated sterna was MAPK, PI3K, and PKA dose dependent, suggesting growth was regulated through multiple pathways. Col-X deposition was rescued in PTH-treated sterna in the presence of PI3K or MAPK inhibitors, but not with the PKA inhibitor. All three inhibitors moderately disrupted Col-II and Col-IX deposition. These results suggest that PTH can activate multiple pathways during chondrocyte differentiation.
Subject(s)
Cell Proliferation , Chondrocytes/enzymology , Collagen Type X/metabolism , Mitogen-Activated Protein Kinases/metabolism , Parathyroid Hormone/metabolism , Peptide Fragments/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Receptors, Parathyroid Hormone/metabolism , Sternum/enzymology , Animals , Cattle , Cell Proliferation/drug effects , Chick Embryo , Chickens , Chondrocytes/drug effects , Collagen Type II/metabolism , Collagen Type IX/metabolism , Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors , Cyclic AMP-Dependent Protein Kinases/metabolism , Immunohistochemistry , Microscopy, Confocal , Mitogen-Activated Protein Kinases/antagonists & inhibitors , Phosphoinositide-3 Kinase Inhibitors , Protein Kinase Inhibitors/pharmacology , Signal Transduction , Sternum/drug effects , Sternum/embryology , Time Factors , Tissue Culture TechniquesABSTRACT
Crude preparations of lysyl hydroxylase were extracted from chick-embryo tendons synthesizing exclusively type I collagen, chick-embryo sterna synthesizing exclusively type II collagen and HT-1080 sarcoma cells synthesizing exclusively type IV collagen. No differences were found in the Km values for Fe2+, 2-oxoglutarate and ascorbate between these three enzymes preparations. Similarly no differences were found in the Km values for type I and type II protocollagens and the rate at which type IV protocollagen is hydroxylated between these enzyme preparations. The extent to which type I protocollagen could be hydroxylated by the three enzymes was likewise identical. These data strongly argue against the existence of collagen-type-specific lysyl hydroxylase isoenzymes.