Adrenocorticotropic hormone and 1,25-dihydroxyvitamin D 3 enhance human osteogenesis in vitro by synergistically accelerating the expression of bone-specific genes.

To improve definition of the physical and hormonal support of bone formation, we studied differentiation of human osteoblasts in vitro at varying combinations of ACTH, 1α,25-dihydroxyvitamin D ₃ (1,25(OH) ₂ D), and extracellular calcium, with and without added cortisol. Bone mineralization, alkaline phosphatase activity, and osteoblast-specific markers RunX2, osterix, and collagen I increased with 10 pM ACTH, 10 nM 1,25(OH) ₂ D, or at 2 mM calcium with important synergistic activity of combinations of any of these stimuli. Signals induced by ACTH at 10-30 min included cAMP, TGF-β, and Erk1/2 phosphorylation. Affymetrix gene expression analysis showed that 2 h treatment of ACTH or 1,25(OH) ₂ D increased the expression of bone regulating and structural mRNAs, including collagen I, biglycan, the vitamin D receptor, and TGF-β. Accelerating expression of these bone-specific genes was confirmed by quantitative PCR. Expression of 1,25(OH) ₂ D 1α-hydroxylase (1α-hydroxylase) increased with 1,25(OH) ₂ D, ACTH, and extracellular calcium from 0.5 to 2 mM. Unlike renal 1α-hydroxylase, in osteoblasts, 1α-hydroxylase activity is independent of parathyroid hormone. In keeping with calcium responsivity, calcium-sensing receptor RNA and protein increased with 10 nM ACTH or 1,25(OH) ₂ D. Inclusion of 200 nM cortisol or 10 nM ACTH in differentiation media blunted osteoblasts alkaline phosphatase response to 1,25(OH) ₂ D and calcium. Our results point to the importance of ACTH in bone maintenance and that extra skeletal (renal) 1,25(OH) ₂ D is required for bone mineralization despite 1α-hydroxylase expression by osteoblasts.