Osteocytes are derived from osteoblast lineage cells that become progressively embedded in mineralized bone. Development of the osteocytogenic cell line IDG-SW3 has enabled a temporal and mechanistic investigation of this process. Through RNA-sequencing analyses, we show that although substantial changes in gene expression occur during the osteoblast to osteocyte transition, the majority of the transcriptome remains qualitatively osteoblast like. Genes either up-regulated or expressed uniquely in the osteocyte include local and systemic factors such as Sost and Fgf23 as well as genes implicated in neuronal, muscle, vascular, or regulatory function. As assessed by chromatin immunoprecipitation coupled to high-throughput sequencing, numerous changes in epigenetic histone modifications also occur during osteocytogenesis; these are largely qualitative rather than quantitative. Specific epigenetic changes correlate with altered gene expression patterns that are observed during the transition. These genomic changes likely influence the highly restricted transcriptomic response to 1,25(OH)(2)D(3) that occurs during differentiation. VDR binding in osteocytes revealed an extensive cistrome co-occupied by retinoid X receptor and located predominantly at sites distal to regulated genes. Although sites of VDR binding were apparent near many 1,25(OH)(2)D(3)-regulated genes, the expression of others adjacent to VDR-binding sites were unaffected; lack of VDR binding was particularly prevalent at down-regulated genes. Interestingly, 1,25(OH)(2)D(3) was found to induce the Boc and Cdon coreceptors that are active in hedgehog signaling in osteocytes. We conclude that osteocytogenesis is accompanied by changes in gene expression that may be driven by both genetic and epigenetic components. These changes are likely responsible for the osteocyte phenotype and may contribute to reduced sensitivity to 1,25(OH)(2)D(3).

Virtually all of the known biological actions of the hormonal ligand 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) are mediated by the vitamin D receptor (VDR). Following binding and activation by the ligand, the VDR localizes in the nucleus to the regulatory regions of target genes and recruits chromatin-active coregulatory complexes which, in turn, modulate transcriptional output. The failure of the VDR to function due to crippling mutations results in total hereditary resistance to 1,25(OH)(2)D(3) in both mice and humans. In this review, we summarize the structural and functional properties of the VDR and the role of 1,25(OH)(2)D(3) in receptor activation, and then describe the results of recent studies using genome-wide analyses that define the overarching principles through which the VDR modulates genes expression. We also focus on the recent analysis of a specific 1,25(OH)(2)D(3) regulated gene that provides confirmation of the principles identified through these genome-wide methodologies. Taken together, these studies suggest an unanticipated increase in the complexity of the molecular processes that govern gene regulation by hormones and other factors.

Fat mass may be modulated by the number of brown-like adipocytes in white adipose tissue (WAT) in humans and rodents. Bone remodeling is dependent on systemic energy metabolism and, with age, bone remodeling becomes uncoupled and brown adipose tissue (BAT) function declines. To test the interaction between BAT and bone, we employed Misty (m/m) mice, which were reported be deficient in BAT. We found that Misty mice have accelerated age-related trabecular bone loss and impaired brown fat function (including reduced temperature, lower expression of Pgc1a and less sympathetic innervation compared to wildtype (+/+)). Despite reduced BAT function, Misty mice had normal core body temperature, suggesting heat is produced from other sources. Indeed, upon acute cold exposure (4°C for 6 hr), inguinal WAT from Misty mice compensated for BAT dysfunction by increasing expression of Acadl, Pgc1a, Dio2 and other thermogenic genes. Interestingly, acute cold exposure also decreased Runx2 and increased Rankl expression in Misty bone, but only Runx2 was decreased in wildtype. Browning of WAT is under the control of the sympathetic nervous system (SNS) and, if present at room temperature, could impact bone metabolism. To test whether SNS activity could be responsible for accelerated trabecular bone loss, we treated wildtype and Misty mice with the β-blocker, propranolol. As predicted, propranolol slowed trabecular BV/TV loss in the distal femur of Misty mice without affecting wildtype. Finally, the Misty mutation (a truncation of DOCK7) also has a significant cell-autonomous role. We found DOCK7 expression in whole bone and osteoblasts. Primary osteoblast differentiation from Misty calvaria was impaired, demonstrating a novel role for DOCK7 in bone remodeling. Despite the multifaceted effects of the Misty mutation, we have shown that impaired brown fat function leads to altered SNS activity and bone loss, and for the first time that cold exposure negatively affects bone remodeling.

The biological actions of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) are mediated by the vitamin D receptor (VDR), which is expressed in numerous target tissues in a cell type-selective manner. Recent studies using genomic analyses and recombineered bacterial artificial chromosomes (BACs) have defined the specific features of mouse and human VDR gene loci in vitro. In the current study, we introduced recombineered mouse and human VDR BACs as transgenes into mice and explored their expression capabilities in vivo. Individual transgenic mouse strains selectively expressed BAC-derived mouse or human VDR proteins in appropriate vitamin D target tissues, thereby recapitulating the tissue-specific expression of endogenous mouse VDR. The mouse VDR transgene was also regulated by 1,25(OH)2D3 and dibutyryl-cAMP. When crossed into a VDR-null mouse background, both transgenes restored wild-type basal as well as 1,25(OH)2D3-inducible gene expression patterns in the appropriate tissues. This maneuver resulted in the complete rescue of the aberrant phenotype noted in the VDR-null mouse, including systemic features associated with altered calcium and phosphorus homeostasis and disrupted production of parathyroid hormone and fibroblast growth factor 23, and abnormalities associated with the skeleton, kidney, parathyroid gland, and the skin. This study suggests that both mouse and human VDR transgenes are capable of recapitulating basal and regulated expression of the VDR in the appropriate mouse tissues and restore 1,25(OH)2D3 function. These results provide a baseline for further dissection of mechanisms integral to mouse and human VDR gene expression and offer the potential to explore the consequence of selective mutations in VDR proteins in vivo.

Receptor activator of NF-B ligand (Rankl) is a TNF-like factor that induces the formation of osteoclasts responsible for bone resorption. Although T cell activation up-regulates this gene, the molecular mechanism of its transcriptional control remains unknown. We used ChIP-chip analysis in mouse primary T cells and a T cell hybridoma to define the regulatory enhancers responsible for this up-regulation and to characterize their properties. Elevated H3/H4 acetylation and increased RNA polymerase II density were evident at mRL-D5, a known enhancer located 76 kb upstream of the TSS, as well as at a cluster of regulatory sites located even further upstream between ؊123 to ؊156 kb, termed the T cell control region (TCCR). Based upon the ability of calcium signaling and MAPK inhibitors to block Rankl expression, we conducted further ChIP-chip analysis of the transcriptional mediators c-Fos, NF-B, and Nfat. T cell activation induced c-Fos binding at the mRL-D5 enhancer and within the TCCR. The interaction of NF-B wasobserved at the transcriptional start site and at mRL-D5. Both mRL-D5 and segments of the TCCR exhibited robust transcriptional activity in reporter assays, and site-specific mutagenesis of c-Fos and Nfat elements abrogated reporter activity, suggesting a role for both factors in the control of enhancer-mediated Rankl transcription. Finally, chromosome conformation capture analysis confirmed that mRL-D5 and segments of the TCCR were located in proximity to the Rankl gene promoter and thus potentially able to influence directly Rankl gene promoter activity. We conclude that both mRL-D5 and the TCCR represent control segments that play an integral role in Rankl expression in T cells.