Abstract
The biological effects of 1α,25-dihydroxyvitamin D3 (1,25 (OH)2D3) on osteoblast differentiation and function differ significantly depending upon the cellular state of maturation. To explore this phenomenon mechanistically, we examined the impact of 1,25(OH)2D3 on the transcriptomes of both pre-osteoblastic (POBs) and differentiated osteoblastic (OBs) MC3T3-E1 cells, and assessed localization of the vitamin D receptor (VDR) at sites of action on a genome-scale using ChIP sequence analysis. We observed that the 1,25(OH)2D3-induced transcriptomes of POBs and OBs were quantitatively and qualitatively different, supporting not only the altered biology observed but the potential for a change in VDR interaction at the genome as well. This idea was confirmed through discovery that VDR cistromes in POBs and OBs were also strikingly different. Depletion of VDR-binding sites in OBs, due in part to reduced VDR expression, was the likely cause of the loss of VDR-target gene interaction. Continued novel regulation by 1,25(OH)2D3, however, suggested that factors in addition to the VDR might also be involved. Accordingly, we show that transcriptomic modifications are also accompanied by changes in genome binding of the master osteoblast regulator RUNX2 and the chromatin remodeler CCAAT/enhancer-binding protein β. Importantly, genome occupancy was also highlighted by the presence of epigenetic enhancer signatures that were selectively changed in response to both differentiation and 1,25(OH)2D3. The impact of VDR, RUNX2, and C/EBPβ on osteoblast differentiation is exemplified by their actions at the Runx2 and Sp7 gene loci. We conclude that each of these mechanisms may contribute to the diverse actions of 1,25(OH)2D3 on differentiating osteoblasts.
Highlights
The biological activities of 1,25(OH)2D3 in osteoblasts are dependent upon their differentiation state
These changes are accompanied by a striking decrease in the cistromes for both RUNX2 and C/EBP, the former a master regulator of osteoblast lineage development and the latter a chromatin regulatory factor active in numerous mesenchyme-derived cells [48]
We examined whether these changes associated with differentiation were capable of altering a response to the vitamin D hormone 1,25(OH)2D3, a question sparked by previous studies, which suggested that the actions of this hormone are cell maturation-dependent both in vitro and in vivo [2]
Summary
The biological activities of 1,25(OH)2D3 in osteoblasts are dependent upon their differentiation state. The direct actions of 1,25(OH)2D3 on bone cells are complex and include both cell autonomous and paracrine mechanisms, the latter arising from the ability of the hormone to stimulate or repress the expression of growth factors and cytokines from chondrocytes, osteoblasts, and osteocytes that affect cells located nearby [2] One such paracrine factor is receptor activator of nuclear factor-B ligand that is produced in a variety of bone cell types and plays a paramount role in regulating the formation, activation, function, and survival of hematopoietic cell-derived osteoclasts [5]. 1,25(OH)2D3 plays an endocrine role in regulating the expression of fibroblast growth factor 23 (FGF23) from osteocytes; this factor acts on the distal kidney to regulate phosphate metabolism and may have independent functions in other tissues as well (6 – 8) Evidence for these homeostatic actions of 1,25(OH)2D3 derives from studies conducted both in normal rodents in vivo and in VDR- or cytochrome p450 27B1 (CYP27B1)-null mice (9 –12).
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