The nuclear receptors REV-ERBα (encoded by NR1D1) and REV-ERBβ (NR1D2) have remained orphans owing to the lack of identified physiological ligands. Here we show that heme is a physiological ligand of both receptors. Heme associates with the ligand-binding domains of the REV-ERB receptors with a 1:1 stoichiometry and enhances the thermal stability of the proteins. Results from experiments of heme depletion in mammalian cells indicate that heme binding to REV-ERB causes the recruitment of the co-repressor NCoR, leading to repression of target genes including BMAL1 (official symbol ARNTL), an essential component of the circadian oscillator. Heme extends the known types of ligands used by the human nuclear receptor family beyond the endocrine hormones and dietary lipids described so far. Our results further indicate that heme regulation of REV-ERBs may link the control of metabolism and the mammalian clock.REV-ERBα was originally identified as an orphan member of the nuclear hormone receptor (NHR) family on the basis of its canonical domain structure and sequence conservation 1,2 . REV-ERBβ was subsequently identified by its homology to other NHRs and its pattern of expression, which overlaps greatly with that of REV-ERBα. Both receptors have particularly high expression in the liver, adipose tissue, skeletal muscle and brain 3-8 , where they are transcribed in a circadian manner 9-11 . The REV-ERBs are unique in the NHR superfamily in that they lack the carboxy-terminal tail (helix 12) of the ligand-binding domain (LBD), which is required for coactivator recognition 12

DEX-associated CLANs were structurally similar to those induced by mAb AP-5 and involved both increased expression and activation of αvβ3 integrins. Thus, glucocorticoid-induced CLAN formation may involve enhanced β3 integrin signaling in HTM cells, possibly by an inside-out signaling mechanism.

Steroid-induced glaucoma is an iatrogenic condition resulting from the use of glucocorticoids. Glucocorticoids such as dexamethasone (DEX) 1 raise intraocular pressure (IOP) in ϳ40% of patients in the general population, and ϳ6% of these patients will go on to develop glaucoma (1, 2). This condition is similar to primary open angle glaucoma (1-3), and is caused by a restriction in fluid outflow through the trabecular meshwork (TM), resulting in an imbalance between the amount of aqueous humor produced and the amount drained. This imbalance results in a higher IOP.It is thought that an alteration in the cytoskeletal structure or contractile properties of TM cells may result in the disruption of normal fluid flow. In support of this idea, cross-linked actin networks, referred to as CLANs, have been observed with increased frequency in the TM of glaucomatous patients and in glucocorticoid treated anterior segments as well as in TM cells in culture. CLANs are thought to alter the contractility of the TM by holding the cells in a rigid conformation, making the cells unresponsive to the change in pressure and blocking the aqueous humor outflow pathway (1,4,5). Thus, agents such as H7 and the latrunculins A and B, which disrupt the organization of the cytoskeleton, decrease IOP in porcine and monkey cultured anterior segments (6 -9).Control of the actin cytoskeleton is mediated by the Rho family of small GTPases. The Rho effector ROCK has been shown to play a part in TM contractility and modulation of IOP. Inhibition of ROCK using a dominant negative mutant or the inhibitor Y-27632 causes TM cells to "relax" by decreasing actin stress fiber formation and phosphorylation of myosin light chain (MLC) (10, 11). ROCK inhibition also decreases IOP in cultured human and porcine anterior segments (10, 11). In contrast, constitutively active RhoA (RhoA V14) increases From the ‡Departments

No abstract

The association of Lipoprotein(a) (Lp(a)) levels and increased cardiovascular risk is substantiated by human epidemiology, genetics and interventional apheresis studies. Lp(a) is a plasma lipoprotein consisting of a Low Density Lipoprotein (LDL) particle with one molecule of apolipoprotein B100 (ApoB100) covalently linked to one molecule of apolipoprotein(a). Published studies show that apo(a) synthesis and secretion from hepatoma cells is coupled to triglyceride synthesis and secretion, illustrating a point of control for Lp(a) production. The objective of this study is to compare Lp(a), apo(a) and apoB production, secretion and half-life in vivo to understand mechanisms controlling circulating levels of Lp(a). Since rodent species do not express endogenous apo(a), human apo(a) transgenic mice were created and bred with human apoB100 transgenic mice to generate Lp(a)-producing mice. The circulating level of Lp(a) in the transgenic mice is ~3.7 mg/dL that is comparable to 5.4 mg/dL measured in a healthy human subject. To inhibit triglyceride and VLDL secretion in liver, mice were treated with a microsomal triglyceride transfer protein inhibitor (MTPi), and plasma was collected in a time-course. This treatment allowed measurement of plasma Lp(a), apo(a) and apoB half-lives since secretion of nascent VLDL and LDL was blocked. MTPi caused an 81% reduction in cholesterol and 68% reduction in triglycerides after 5 days of treatment. ApoB decreased significantly within 6 hours of treatment and remained low for 5 days. The calculated half-life was 4.6 hours. By contrast, apo(a) and Lp(a) decreased significantly after 3 days of treatment with half-lives of 2.4 and 2.5 days, respectively, illustrating delayed catabolism of apo(a)/Lp(a). Hepatic Lp(a), apo(a) and apoB were decreased significantly after 5 days dosing, indicating that the apolipoproteins were likely degraded and not accumulated in the liver. Treatment of stable HepG2 cells expressing apo(a) with MTPi caused similar difference in Lp(a) and apoB half-lives. These models illustrate rapid decay of apoB, but not apo(a), when lipoprotein particle assembly is blocked, and they provide methods for future mechanistic studies of apo(a) assembly into Lp(a) and its catabolism.