Techniques in embryoid body formation from human pluripotent stem cells.

Embryoid bodies (EBs) can be generated by culturing human pluripotent stem cells in ultra-low attachment culture vessels, under conditions that are adverse to pluripotency and proliferation. EBs generated in suspension cultures are capable of differentiating into cells of the ectoderm, mesoderm, and endoderm. In this chapter, we describe techniques for generation of EBs from human pluripotent stem cells.

Endogenous fluorescence signatures in living pluripotent stem cells change with loss of potency.

The therapeutic potential of stem cells is limited by the non-uniformity of their phenotypic state. Thus it would be advantageous to noninvasively monitor stem cell status. Driven by this challenge, we employed multidimensional multiphoton microscopy to quantify changes in endogenous fluorescence occurring with pluripotent stem cell differentiation.

Comparative chondrogenesis of human cell sources in 3D scaffolds.

Cartilage tissue can be engineered by starting from a diversity of cell sources, including stem-cell based and primary cell-based platforms. Selecting an appropriate cell source for the process of cartilage tissue engineering or repair is critical and challenging, due to the variety of cell options available. In this study, cellular responses of isolated human chondrocytes, human embryonic stem cells and mesenchymal stem cells (MSCs) derived from three sources, human embryonic stem cells, bone marrow and adipose tissue, were assessed for chondrogenic potential in 3D culture.

Vitamin D-binding protein influences total circulating levels of 1,25-dihydroxyvitamin D3 but does not directly modulate the bioactive levels of the hormone in vivo.

Mice deficient in the expression of vitamin D-binding protein (DBP) are normocalcemic despite undetectable levels of circulating 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)]. We used this in vivo mouse model together with cells in culture to explore the impact of DBP on the biological activity of 1,25(OH)(2)D(3). Modest changes in the basal expression of genes involved in 1,25(OH)(2)D(3) metabolism and calcium homeostasis were observed in vivo; however, these changes seemed unlikely to explain the normal calcium balance seen in DBP-null mice.

Receptor activator of nuclear factor-kappaB ligand-induced nuclear factor of activated T cells (C1) autoregulates its own expression in osteoclasts and mediates the up-regulation of tartrate-resistant acid phosphatase.

Osteoclasts are large multinucleated, bone-resorbing cells derived from hematopoietic precursors in response to receptor activator of nuclear factor-kappaB ligand (RANKL). RANKL activates a number of signal transduction pathways, which stimulate, in turn, a series of specific transcription factors that initiate the process of osteoclastogenesis. Perhaps the most important of these is nuclear factor of activated T cells cytoplasmic 1 (NFATc1), a DNA-binding protein that upon activation translocates to the nucleus where it stimulates transcription.

1,25-Dihydroxyvitamin D3 induces expression of the Wnt signaling co-regulator LRP5 via regulatory elements located significantly downstream of the gene's transcriptional start site.

Canonical Wnt signaling is essential for bone formation. Activation involves binding of secreted members of the Wnt family of proteins with a membrane receptor Frizzled on osteoblasts, an interaction that is facilitated by LRP5/LRP6 co-receptors. LRP5 is known to play a particularly important role in bone formation such that the loss of this protein results in a reduction in osteoblast number, a delay in mineralization and a reduction in peak BMD.

Perspectives on mechanisms of gene regulation by 1,25-dihydroxyvitamin D3 and its receptor.

1,25-Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) functions as a systemic signal in vertebrate organisms to control the expression of genes whose products are vital to the maintenance of calcium and phosphorus homeostasis. This regulatory capability is mediated by the vitamin D receptor (VDR) which localizes at DNA sites adjacent to the promoter regions of target genes and initiates the complex events necessary for transcriptional modulation. Recent investigations using chromatin immunoprecipitation techniques combined with various gene scanning methodologies have revealed new insights into the location, structure and function of these regulatory regions.

Enhancers located in the vitamin D receptor gene mediate transcriptional autoregulation by 1,25-dihydroxyvitamin D3.

The regulatory actions of 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) on target genes are mediated by the vitamin D receptor (VDR). Interestingly, one of the genomic targets of 1,25(OH)(2)D(3) action is the VDR gene itself; however, the mechanism underlying this regulation is unknown. We investigated VDR autoregulation by screening the mouse VDR locus from 20kb upstream of the transcriptional start site (TSS) to 10kb downstream of the last exon using chromatin immunoprecipitation (ChIP)-DNA microarray analysis (ChIP/chip).

Multiple enhancer regions located at significant distances upstream of the transcriptional start site mediate RANKL gene expression in response to 1,25-dihydroxyvitamin D3.

One of the primary regulators of receptor activator of NF-kappaB ligand (RANKL) is 1,25-dihydoxyvitamin D(3) (1,25(OH)(2)D(3)). To elucidate the mechanism whereby 1,25(OH)(2)D(3) activates RANKL expression we screened some 300kb of the RANKL gene locus using a ChIP on chip analysis and identified five potential regulatory regions lying significant distances upstream of the transcription start site (TSS), the farthest over 70kb from the TSS. A direct ChIP analysis confirmed the presence of the VDR/RXR heterodimer at these sites.

Transcriptional control of receptor activator of nuclear factor-kappaB ligand by the protein kinase A activator forskolin and the transmembrane glycoprotein 130-activating cytokine, oncostatin M, is exerted through multiple distal enhancers.

Receptor activator of nuclear factor-kappaB ligand (RankL) is a potent osteoclastogenic cytokine the expression of which is regulated at the transcriptional level by 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], protein kinase A (PKA) activators such as PTH and transmembrane glycoprotein 130 (gp130)-activating cytokines such as oncostatin M. We recently identified five highly conserved chromatin domains located significant distances upstream of the RankL transcriptional start site that contribute to the ability of 1,25-(OH)2D3 and its receptor to enhance RankL gene output. We therefore screened these five common regulatory regions for their potential ability to mediate the actions of PKA- and gp130-activators using a directed chromatin immunoprecipitation approach employing antibodies to the PKA target cAMP response element-binding protein (CREB) and the gp130 target signal transducer and activator of transcription 3.

Activation of receptor activator of NF-kappaB ligand gene expression by 1,25-dihydroxyvitamin D3 is mediated through multiple long-range enhancers.

RANKL is a tumor necrosis factor (TNF)-like factor secreted by mesenchymal cells, osteoblast derivatives, and T cells that is essential for osteoclastogenesis. In osteoblasts, RANKL expression is regulated by two major calcemic hormones, 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] and parathyroid hormone (PTH), as well as by several inflammatory/osteoclastogenic cytokines; the molecular mechanisms for this regulation are unclear. To identify such mechanisms, we screened a DNA microarray which tiled across the entire mouse RankL gene locus at a 50-bp resolution using chromatin immunoprecipitation (ChIP)-derived DNA precipitated with antibodies to the vitamin D receptor (VDR) and the retinoid X receptor (RXR).

1,25-Dihydroxyvitamin D3 regulates the expression of low-density lipoprotein receptor-related protein 5 via deoxyribonucleic acid sequence elements located downstream of the start site of transcription.

The skeleton is a direct target of vitamin D action, where the hormone modulates the proliferation of osteoblast precursors, their differentiation into mature osteoblasts, and their functional activity. Some of these effects of vitamin D are reminiscent of those orchestrated by the Wnt signaling pathway wherein stimulation of the membrane receptor Frizzled and its coreceptor LRP5 leads to activation of beta-catenin and subsequent transcription-mediated changes in osteoblast biology. Indeed, LRP5 is now known to play a particularly important role in bone formation such that the loss of this component results in a reduction in osteoblast number, a delay in mineralization, and a reduction in peak bone mineral density.

The human transient receptor potential vanilloid type 6 distal promoter contains multiple vitamin D receptor binding sites that mediate activation by 1,25-dihydroxyvitamin D3 in intestinal cells.

Transient receptor potential vanilloid type 6 (TRPV6) (ECAC2, CaT1) is the major ion channel in intestinal epithelial cell membranes responsible for calcium entry. Its expression is actively regulated at the transcriptional level by 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. In this report, we identify mechanisms integral to the regulation of TRPV6 by 1,25-(OH)2D3.

Enhancers located within two introns of the vitamin D receptor gene mediate transcriptional autoregulation by 1,25-dihydroxyvitamin D3.

The biological actions of 1,25-(OH)2D3 are mediated by the vitamin D receptor (VDR), a protein that binds to target genes and alters their expression. 1,25-(OH)2D3 is also capable of inducing transcription of the VDR gene itself. In the present study, we explored both the capacity of 1,25-(OH)2D3 to induce VDR gene expression in bone cells and the mechanism instrumental to this up-regulation.

1,25-Dihydroxyvitamin D3 stimulates cyclic vitamin D receptor/retinoid X receptor DNA-binding, co-activator recruitment, and histone acetylation in intact osteoblasts.

Unlabelled: 1,25(OH)2D3 induces gene expression through the VDR. We used chromatin immunoprecipitation techniques to explore this 1,25(OH)2D3-induced process on the 25-hydroxyvitamin D3-24-hydroxylase (Cyp24) and Opn gene promoters in intact osteoblasts. Our studies show that 1,25(OH)2D3-induced transactivation is a dynamic process that involves promoter-specific localization of VDR and RXR, recruitment of histone acetyltransferase complexes, and in the case of the Cyp24 gene, modification of histone 4.

2-Methylene-19-nor-(20S)-1,25-dihydroxyvitamin D3 potently stimulates gene-specific DNA binding of the vitamin D receptor in osteoblasts.

2-Methylene-19-nor-(20S)-1,25-dihydroxyvitamin D3 (2MD) is a highly potent analog of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) whose actions are mediated through the vitamin D receptor (VDR). In this report, we have replicated this increased potency of 2MD in vitro using osteoblastic cells and explored its underlying molecular mechanism. 2MD stimulates the expression of several vitamin D-sensitive genes including 25-hydroxyvitamin D3-24 hydroxylase (Cyp24), osteopontin and receptor activator of NF kappa B ligand and suppresses osteoprotegerin at concentrations two logs lower than that for 1,25(OH)2D3.

Synthetic LXXLL peptide antagonize 1,25-dihydroxyvitamin D3-dependent transcription.

The vitamin D receptor (VDR) is known to mediate the biological actions of 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) through its ability to regulate cellular programs of gene expression. We identified VDR- and retinoid X receptor (RXR)-interacting LXXLL peptides using a mammalian two-hybrid system and examined whether these molecules could block vitamin D and 9-cis retinoic acid (9-cis RA) response. Peptides were identified that were reactive to RXR alone as well as to both VDR and RXR.

Inhibition of 1,25-dihydroxyvitamin D3-dependent transcription by synthetic LXXLL peptide antagonists that target the activation domains of the vitamin D and retinoid X receptors.

The vitamin D receptor (VDR) is known to mediate the biological actions of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] through its ability to regulate cellular programs of gene expression. Although RXR appears to participate as a heterodimeric partner with the VDR, absolute evidence for its role remains equivocal in vivo. To test this role and to investigate the requirement for comodulator interaction, we identified VDR- and retinoid X receptor (RXR)-interacting LXXLL peptides and examined whether these molecules could block vitamin D and 9-cis retinoic acid (9-cis RA) response.

A potent analog of 1alpha,25-dihydroxyvitamin D3 selectively induces bone formation.

1,25-Dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] is a principal regulator of calcium and phosphorus homeostasis through actions on intestine, kidney, and bone. 1,25(OH)(2)D(3) is not considered to play a significant role in bone formation, except for its role in supporting mineralization. We report here on the properties of 2-methylene-19-nor-(20S)-1alpha,25(OH)(2)D(3) (2MD), a highly potent analog of 1,25(OH)(2)D(3) that induces bone formation both in vitro and in vivo.

Vitamin D receptor-mediated gene regulation mechanisms and current concepts of vitamin D analog selectivity.

Lipophilic hormones of steroidal origin such as the sex hormones and 1,25-dihydroxy vitamin D(3) (1,25[OH](2)D(3)) function by regulating patterns of gene expression in cells. The mediators of such actions are nuclear receptors that recognize these ligands with high affinity and selectivity and function through several mechanisms as gene specific transcription factors. As a result of the mechanistic complexity of nuclear receptor action, recent studies have revealed that both synthetic analogs as well as novel mimetics of a receptor's natural hormonal ligand are capable of modulating functional responses in both cell- and gene-selective manners.

Distinct molecular mechanism for initiating TRAF6 signalling.

Tumour-necrosis factor (TNF) receptor-associated factor 6 (TRAF6) is the only TRAF family member that participates in signal transduction of both the TNF receptor (TNFR) superfamily and the interleukin-1 receptor (IL-1R)/Toll-like receptor (TLR) superfamily; it is important for adaptive immunity, innate immunity and bone homeostasis. Here we report crystal structures of TRAF6, alone and in complex with TRAF6-binding peptides from CD40 and TRANCE-R (also known as RANK), members of the TNFR superfamily, to gain insight into the mechanism by which TRAF6 mediates several signalling cascades. A 40 degrees difference in the directions of the bound peptides in TRAF6 and TRAF2 shows that there are marked structural differences between receptor recognition by TRAF6 and other TRAFs.