The use of adherent monolayer cultures have produced many insights into melanoma cell growth and differentiation, but often novel therapeutics demonstrated to act on these cells are not active in vivo. It is imperative that new methods of growing melanoma cells that reflect growth in vivo are investigated. To this end, a range of human melanoma cell lines passaged as adherent cultures or induced to form melanoma spheres (melanospheres) in stem cell media have been studied to compare cellular characteristics and protein expression. Melanoma spheres and tumours grown from cell lines as mouse xenografts had increased heterogeneity when compared to adherent cells and 3D-spheroids in agar (aggregates). Furthermore, cells within the melanoma spheres and mouse xenografts each displayed a high level of reciprocal BRN2 or MITF expression, which matched more closely the pattern seen in human melanoma tumours in situ, rather than the propensity for co-expression of these important melanocytic transcription factors seen in adherent cells and 3D-spheroids. Notably, when the levels of the BRN2 and MITF proteins were each independently repressed using siRNA treatment of adherent melanoma cells, members of the NOTCH pathway responded by decreasing or increasing expression respectively. This links BRN2 as an activator and conversely MITF as a repressor of the NOTCH pathway in melanoma cells. Loss of the BRN2-MITF axis in antisense ablated cell lines decreased melanoma sphere forming capability, cell adhesion during 3D-spheroid formation, and invasion through a collagen matrix. Combined, this evidence suggests that the melanoma sphere culture system induces subpopulations of cells that may more accurately portray the in vivo disease, than growth as adherent melanoma cells.

Rev-erb␤ is an orphan nuclear receptor that selectively blocks trans-activation mediated by the retinoic acid-related orphan receptor-␣ (ROR␣). ROR␣ has been implicated in the regulation of high density lipoprotein cholesterol, lipid homeostasis, and inflammation. Reverb␤ and ROR␣ are expressed in similar tissues, including skeletal muscle; however, the pathophysiological function of Rev-erb␤ has remained obscure. We hypothesize from the similar expression patterns, target genes, and overlapping cognate sequences of these nuclear receptors that Rev-erb␤ regulates lipid metabolism in skeletal muscle. This lean tissue accounts for >30% of total body weight and 50% of energy expenditure. Moreover, this metabolically demanding tissue is a primary site of glucose disposal, fatty acid oxidation, and cholesterol efflux. Consequently, muscle has a significant role in insulin sensitivity, obesity, and the blood-lipid profile. We utilize ectopic expression in skeletal muscle cells to understand the regulatory role of Rev-erb␤ in this major mass peripheral tissue. Exogenous expression of a dominant negative version of mouse Rev-erb␤ decreases the expression of many genes involved in fatty acid/lipid absorption (including Cd36, and Fabp-3 and -4). Interestingly, we observed a robust induction (>15-fold) in mRNA expression of interleukin-6, an "exerciseinduced myokine" that regulates energy expenditure and inflammation. Furthermore, we observed the dramatic repression (>20-fold) of myostatin mRNA, another myokine that is a negative regulator of muscle hypertrophy and hyperplasia that impacts on body fat accumulation. This study implicates Rev-erb␤ in the control of lipid and energy homoeostasis in skeletal muscle. In conclusion, we speculate that selective modulators of Rev-erb␤ may have therapeutic utility in the treatment of dyslipidemia and regulation of muscle growth. Members of the nuclear receptor (NR)1 superfamily bind to specific DNA elements and function as transcriptional regulators (1, 2). In addition to the ligand-activated NRs, many members within this superfamily have no known ligand, and are referred to as "orphan NRs" (3). The orphan receptor Rev-erb␤ (NR1D2, also known as Rev-erb␣-related receptor, RVR) belongs to the family of "Reverbs" that also contain Rev-erb␣ (4, 5). The primary structure of these two receptors together with retinoic acid-related orphan receptor-␣ (ROR␣) and the Drosophila orphan receptor, E75A, is very similar especially in the DNA-binding domain and the putative ligand-binding domain (6).Two Rev-erb␤ genes have been identified; Rev-erb␤1 and Rev-erb␤2, which are alternatively spliced products of the Reverb␤ gene (7). The mRNA expression data shows that Rev-erb␤ is abundantly expressed in most tissues, although higher levels of expression are observed in skeletal muscle, brain, kidney, and liver (Refs. 4 and 8 and references therein).Rev-erb␣, Rev-erb␤, and ROR bind as monomers to the nuclear receptor half-site motif, PuGGTCA flanked 5Ј by an ATrich sequence ((A/T) 6 PuGGTCA). Although...

Nuclear hormone receptors (NRs) function as ligand dependent DNA binding proteins that translate physiological/nutritional signals into gene regulation. Dysfunctional NR signaling leads to many disorders in reproduction, inflammation, and metabolism. The opportunity to identify novel regulatory pathways in the context of human health and disease drives the challenge to unravel the biological function of the "orphan nuclear hormone receptors". For example, the Rev-erb (NR1D) subgroup (Rev-erbα/NR1D1 and Rev-erbβ/NR1D2) of orphan NRs are transcriptional silencers and negative regulators of ROR mediated trans-activation. The NR1D subgroup is highly enriched in peripheral tissues with onerous energy demands including skeletal muscle, brown and white adipose, brain, liver and kidney. This alludes to the involvement of this subgroup in metabolism. In this context, Rev-erbα-/-mice have a dyslipidemic phenotype. Recent studies in vascular smooth and skeletal muscle cells also suggest that the NR1D subgroup modulates inflammation by regulating IκBα/NFκB dependent gene expression. Rev-erbα has been identified as a critical regulator (and target) of circadian rhythm, a factor in blood pressure control and inflammation. Finally, two recent reports have demonstrated: (i) lithium mediated regulation of Rev-erbα stability and (ii) E75 (the Drosophila orthologue of human Rev-erbα) is tightly bound by heme, and functions as a "gas sensor" through interaction with CO/NO and interferes with the repression of DHR3 (the Drosophila orthologue of human RORα). In conclusion, the role of these receptors at the cross-roads of metabolism, inflammation, and circadian cycling underscores the importance of understanding the organ-specific function of the NR1D subgroup in homeostasis. The NR1D subgroup of orphan NRs, Rev-erbα (NR1D1) and Rev-erbβ (NR1D2)All members of the NR superfamily display a highly conserved structural organisation with an amino terminal AB region (that encodes activation function 1, AF-1); followed by the C-region which encodes the DNA binding domain (DBD); a linker region D and the C-terminal E region. The DE region encodes the ligand binding domain (LBD), and a transcriptional domain (in helix 12), denoted as activation function 2 (AF-2) [Gronemeyer et al., 2004].The Rev-erb/NR1D subgroup contains two members, Rev-erbα (NR1D1; [Lazar et al., 1989]) and Rev-erbβ (NR1D2;[Dumas et al., 1994;Retnakaran et al., 1994]). The Rev-erbs, and retinoid related orphan receptors (RORs; Laitinen and Staels, 2003] bind as monomers to the consensus NR half-site motif, flanked by an 6bp AT-rich sequence (A/T) 6 PuGGTCA, or as a homodimer to a direct repeat of the core motif separated by 2 bp. Although these receptors are closely related, and bind to the same motif, they function in an opposing manner. The NR1D subgroup function as dominant transcriptional silencers, and they repress trans-activation mediated by RORα [Forman et al., 1994]. Interestingly, RORα activates Rev-erbα transcription [Delerive et al., 2002;Raspe et al., 2002b]...

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