Growth plate chondrocyte maturation is regulated by basal intracellular calcium

Among the cellular events that are associated with the process of endochondral ossification is an incremental increase in chondrocyte basal intracellular free Ca²⁺ concentration ([Ca²⁺]_i) from 50 to 100 nM. To determine if this rise in [Ca²⁺]_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 Ca²⁺ chelator EGTA was used to deplete extracellular Ca₂₊ and lower [Ca²⁺]_i to < 50 nM or where the extracellular addition of 5 mM CaCl₂ was used to elevate [Ca²⁺]_i to > 100 nM. Although no effect on the expression of these genes was observed following treatment with 5 mM CaCl², 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 [Ca²⁺]_i. Since EGTA mimicked the effect of parathyroid hormone-related peptide (PTHrP) on GPC maturation, we examined the effect of low [Ca²⁺]_i on PTHrP expression. Suggesting that low [Ca²⁺]_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 Ca²⁺ sensitivity to the PTHrP gene. These findings underscore the importance of cellular Ca₂₊ in GPC function and suggest that PTHrP action in the growth plate is at least partially regulated by changes in basal [Ca²⁺]_i.