24,25-(OH)₂D₃ regulates cartilage and bone via autocrine and endocrine mechanisms

The purpose of this paper is to summarize recent advances in our understanding of the physiological role of 24(R),25(OH)₂D₃ in bone and cartilage and its mechanism of action. With the identification of a target cell, the growth plate resting zone (RC) chondrocyte, we have been able to use cell biology methodology to investigate specific functions of 24(R),25(OH)₂D₃ and to determine how 24(R),25(OH)₂D₃ elicits its effects. These studies indicate that there are specific membrane-associated signal transduction pathways that mediate both rapid, nongenomic and genomic responses of RC cells to 24(R),25(OH)₂D₃. 24(R),25(OH)₂D₃ binds RC chondrocyte membranes with high specificity, resulting in an increase in protein kinase C (PKC) activity. The effect is stereospecific; 24R,25(OH)₂D₃, but not 24S,25-(OH)₂D₃, causes the increase, indicating a receptor-mediated response. Phospholipase D-2 (PLD2) activity is increased, resulting in increased production of diacylglycerol (DAG), which in turn activates PKC. 24(R),25(OH)₂D₃ does not cause translocation of PKC to the plasma membrane, but activates existing PKCα. There is a rapid decrease in Ca²⁺ efflux, and influx is stimulated. 24(R),25(OH)₂D₃ also reduces arachidonic acid release by decreasing phospholipase A₂ (PLA₂) activity, thereby decreasing available substrate for prostaglandin production via the action of cyclooxygenase-1. PGE₂ that is produced acts on the EP1 and EP2 receptors expressed by RC cells to downregulate PKC via protein kinase A, but the reduction in PGE₂ decreases this negative feedback mechanism. Both pathways converge on MAP kinase, leading to new gene expression. One consequence of this is production of new matrix vesicles containing PKCα and PKCζ and an increase in PKC activity. The chondrocytes also produce 24(R),25(OH)₂D₃, and the secreted metabolite acts directly on the matrix vesicle membrane. Only PKCζ is directly affected by 24(R),25(OH)₂D₃ in the matrix vesicles, and activity of this isoform is inhibited. This effect may be involved in the control of matrix maturation and turnover. 24(R),25(OH)₂D₃ causes RC cells to mature along the endochondral developmental pathway, where they become responsive to 1α,25(OH)₂D₃ and lose responsiveness to 24(R),25(OH)₂D₃, a characteristic of more mature growth zone (GC) chondrocytes. 1α,25(OH)₂D₃ elicits its effects on GC through different signal transduction pathways than those used by 24(R),25(OH)₂D₃. These studies indicate that 24(R),25(OH)₂D₃ plays an important role in endochondral ossification by regulating less mature chondrocytes and promoting their maturation in the endochondral lineage.