Elsevier

Laboratory Investigation

Volume 97, Issue 9, September 2017, Pages 1072-1083
Laboratory Investigation

Article
Adrenocorticotropic hormone and 1,25-dihydroxyvitamin D3 enhance human osteogenesis in vitro by synergistically accelerating the expression of bone-specific genes

https://doi.org/10.1038/labinvest.2017.62Get rights and content
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Abstract

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 D3 (1,25(OH)2D), 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)2D, 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)2D 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)2D 1α-hydroxylase (1α-hydroxylase) increased with 1,25(OH)2D, 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)2D. Inclusion of 200 nM cortisol or 10 nM ACTH in differentiation media blunted osteoblasts alkaline phosphatase response to 1,25(OH)2D and calcium. Our results point to the importance of ACTH in bone maintenance and that extra skeletal (renal) 1,25(OH)2D is required for bone mineralization despite 1α-hydroxylase expression by osteoblasts.

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Supplementary Information accompanies the paper on the Laboratory Investigation website

Adrenocorticotropic hormone and 1,25-dihydroxyvitaminD3 dramatically improve human osteoblast differentiation in vitro via synergistic mechanisms. Osteoblasts express the calcium sensing receptor and respond to calcium, but at non-physiological concentrations. Cortisol accelerates expression of some osteoblast-specific proteins, but blunts bone formation and other processes, and various hormone effects.

Supplementary information The online version of this article (doi:10.1038/labinvest.2017.62) contains supplementary material, which is available to authorized users.