title: Growth plate chondrocyte maturation is regulated by basal intracellular calcium.
abstract: Among the cellular events that are associated with the process of endochondral ossification is an incremental increase in chondrocyte basal intracellular free Ca(2+) concentration ([Ca(2+)](i)) from 50 to 100 nM. To determine if this rise in [Ca(2+)](i) functionally participates in the maturational process of growth plate chondrocytes (GPCs), we examined its effect on several markers of hypertrophy, including annexin V, bone morphogenetic protein-6, type X collagen, and indian hedgehog. Expression of these genes was determined under conditions either where the Ca(2+) chelator EGTA was used to deplete extracellular Ca(2+) and lower [Ca(2+)](i) to < 50 nM or where the extracellular addition of 5 mM CaCl(2) was used to elevate [Ca(2+)](i) to > 100 nM. Although no effect on the expression of these genes was observed following treatment with 5 mM CaCl(2), 4 mM EGTA significantly inhibited their expression. This effect was recapitulated in sternal chondrocytes and was reversed following withdrawal of EGTA. Based on these findings, we hypothesized that the EGTA-induced suppression of these genes was mediated by a factor whose expression is responsive to changes in basal [Ca(2+)](i). Since EGTA mimicked the effect of parathyroid hormone-related peptide (PTHrP) on GPC maturation, we examined the effect of low [Ca(2+)](i) on PTHrP expression. Suggesting that low [Ca(2+)](i) suppression of hypertrophy was PTHrP-dependent in GPCs, (a) treatment with 4 mM EGTA increased PTHrP expression, (b) the EGTA effect was rescued by blocking PTHrP binding to its receptor with the competitive antagonist TIP(7-39), and (c) EGTA could mimic the PTHrP stimulation of AP-1 binding to DNA. Additionally, PTHrP promoter analysis identified a domain (-1498 to -862, relative to the start codon) involved with conferring Ca(2+) sensitivity to the PTHrP gene. These findings underscore the importance of cellular Ca(2+) in GPC function and suggest that PTHrP action in the growth plate is at least partially regulated by changes in basal [Ca(2+)](i).
authors.affiliation: Center for Musculoskeletal Research, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642, USA. michael_zuscik@urmc.rochester.edu
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Zuscik
authors.forename: Mary
authors.initials: M
authors.lastname: D'Souza
authors.forename: Andreia M
authors.initials: AM
authors.lastname: Ionescu
authors.forename: Karlene K
authors.initials: KK
authors.lastname: Gunter
authors.forename: Thomas E
authors.initials: TE
authors.lastname: Gunter
authors.forename: Regis J
authors.initials: RJ
authors.lastname: O'Keefe
authors.forename: Edward M
authors.initials: EM
authors.lastname: Schwarz
authors.forename: J Edward
authors.initials: JE
authors.lastname: Puzas
authors.forename: Randy N
authors.initials: RN
authors.lastname: Rosier
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D017304:Annexin A5 / Q000235:genetics / Q000378:metabolism; D055418:Bone Morphogenetic Protein 6; D019485:Bone Morphogenetic Proteins / Q000235:genetics / Q000378:metabolism; D002118:Calcium / Q000172:deficiency*; D020013:Calcium Signaling / Q000187:drug effects / Q000235:genetics*; D002454:Cell Differentiation / Q000187:drug effects / Q000235:genetics*; D002478:Cells, Cultured; D002614:Chelating Agents; D002642:Chick Embryo; D002645:Chickens; D019902:Chondrocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D024121:Collagen Type X / Q000235:genetics / Q000378:metabolism; D005110:Extracellular Space / Q000187:drug effects / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects / Q000235:genetics*; D006132:Growth Plate / Q000196:embryology* / Q000254:growth & development / Q000378:metabolism; D053823:Hedgehog Proteins; D006984:Hypertrophy / Q000235:genetics / Q000378:metabolism; D007424:Intracellular Fluid / Q000378:metabolism*; D010012:Osteogenesis / Q000235:genetics*; D044162:Parathyroid Hormone-Related Protein; D011506:Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism; D018808:Transcription Factor AP-1 / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D015854:Up-Regulation / Q000187:drug effects / Q000235:genetics
chemical_list: D017304:Annexin A5; D055418:Bone Morphogenetic Protein 6; D019485:Bone Morphogenetic Proteins; D002614:Chelating Agents; D024121:Collagen Type X; D053823:Hedgehog Proteins; D044162:Parathyroid Hormone-Related Protein; D011506:Proteins; D015534:Trans-Activators; D018808:Transcription Factor AP-1; D002118:Calcium