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
Parathyroid hormone (PTH; 10(-7) to 10(-15) M) decreased terminal chondrogenesis in the avian sterna. During the first half of an 8-day culture, 100 nM PTH (1-34) significantly increased sternal length and downregulated the deposition of type X collagen and its mRNA expression. However, it remains unclear how PTH increased cartilaginous growth. In this study, we examined growth by both cell proliferation and analysis of cyclin d1 and collagen mRNA. Types II, IX, and X collagens and cyclin d1 mRNA were quantified through real-time RT-PCR, while Ki-67 was used as an immunohistochemical proliferation marker. Extracellular matrix content was measured through mRNA quantification of types II, IX, and X collagen and observing deposition of the same collagens. PTH significantly increased the proliferation marker Ki-67 in the sternal cephalic region. There was less type II and X collagen in PTH-treated sterna with concomitant decreases in mRNA production, suggesting that proliferation was the major contributor to cartilage growth in the presence of PTH/PTH-related peptide receptor activation. In conclusion, these experiments demonstrated that PTH increased cartilage growth by upregulating cell proliferation or other extracellular matrix components.
Subject(s)
Cell Proliferation , Chondrocytes/metabolism , Chondrogenesis , Hyaline Cartilage/growth & development , Parathyroid Hormone-Related Protein/metabolism , Peptide Fragments/metabolism , Sternum/growth & development , Teriparatide/analogs & derivatives , Animals , Cell Proliferation/drug effects , Chick Embryo , Chondrocytes/drug effects , Chondrogenesis/drug effects , Collagen/genetics , Collagen/metabolism , Cyclin D1/genetics , Cyclin D1/metabolism , Gene Expression Regulation, Developmental , Hyaline Cartilage/drug effects , Hyaline Cartilage/embryology , Hyaline Cartilage/metabolism , Immunohistochemistry , Ki-67 Antigen/metabolism , Organ Culture Techniques , Parathyroid Hormone/metabolism , Peptide Fragments/pharmacology , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Sternum/drug effects , Sternum/embryology , Sternum/metabolism , Teriparatide/metabolism , Teriparatide/pharmacology , Time FactorsABSTRACT
Parathyroid hormone (PTH) regulates calcium and phosphate homeostasis through the endocrine system. Parathyroid hormone-related peptide (PTHrP) is a heterogeneous polypeptide with sequence homology to PTH in its first 13 amino acid residues. Both bind and activate a common receptor, the type 1 PTH/PTHrP receptor (PTH1R). Activation of this G-protein-coupled receptor by PTHrP has been shown to regulate chondrogenesis in a manner that attenuates chondrocyte hypertrophy. Here, we report the dose-response (10(-7) to 10(-15) M) effects of PTH on chondrogenesis using an avian sternal organ culture model. PTH increased cartilaginous tissue length and downregulated the deposition of type X collagen and its mRNA expression. In addition, PTH increased chondrocyte cell diameter in prehypertrophic and proliferative regions while decreasing chondrocyte apoptosis in the hypertrophic zone. In conclusion, these experiments demonstrate that PTH regulates cartilage growth, chondrocytic apoptosis, deposition of type X collagen protein, and expression of type X collagen mRNA. Type X collagen mRNA expression was downregulated by PTH in this organ culture model, but cell size, another marker for terminal differentiation, increased.
