Genomic convergence among ERRα, PROX1, and BMAL1 in the control of metabolic clock outputs.

Metabolic homeostasis and circadian rhythms are closely intertwined biological processes. Nuclear receptors, as sensors of hormonal and nutrient status, are actively implicated in maintaining this physiological relationship. Although the orphan nuclear receptor estrogen-related receptor α (ERRα, NR3B1) plays a central role in the control of energy metabolism and its expression is known to be cyclic in the liver, its role in temporal control of metabolic networks is unknown. Here we report that ERRα directly regulates all major components of the molecular clock. ERRα-null mice also display deregulated locomotor activity rhythms and circadian period lengths under free-running conditions, as well as altered circulating diurnal bile acid and lipid profiles. In addition, the ERRα-null mice exhibit time-dependent hypoglycemia and hypoinsulinemia, suggesting a role for ERRα in modulating insulin sensitivity and glucose handling during the 24-hour light/dark cycle. We also provide evidence that the newly identified ERRα corepressor PROX1 is implicated in rhythmic control of metabolic outputs. To help uncover the molecular basis of these phenotypes, we performed genome-wide location analyses of binding events by ERRα, PROX1, and BMAL1, an integral component of the molecular clock. These studies revealed the existence of transcriptional regulatory loops among ERRα, PROX1, and BMAL1, as well as extensive overlaps in their target genes, implicating these three factors in the control of clock and metabolic gene networks in the liver. Genomic convergence of ERRα, PROX1, and BMAL1 transcriptional activity thus identified a novel node in the molecular circuitry controlling the daily timing of metabolic processes.

Fibroblast apoptosis precedes cardiomyocyte mass reduction during left ventricular remodeling in hypertensive rats treated with amlodipine.

BACKGROUND: A transient induction of apoptosis accompanies the normalization of left ventricular mass index in spontaneously hypertensive rats (SHR) treated with dihydropyridine calcium-channel blockers. However, the cell type undergoing apoptosis in this model and the temporal correlation with onset cardiac remodeling remain undefined. METHODS AND RESULTS: SHR were treated either with vehicle or amlodipine (20 mg/kg per day) for 4, 7, 10, 14 or 28 days. Amlodipine stably reduced systolic blood pressure by day 2 (-26 +/- 2%) and stably reduced the left ventricular concentration of atrial natriuretic peptide (ANP) mRNA by approximately 50% as early as day 4, suggesting the early reduction of cardiomyocyte stress. Left ventricular mass index was significantly reduced by day 7 (-4.6 +/- 1.5%), in coordination with reduced DNA content (-23 +/- 2%) and non-cardiomyocyte number (-17 +/- 4%). However, the cardiomyocyte cross-sectional area was reduced only starting from day 14. Caspase-3 cleavage was significantly increased at day 7 only. Ultimately, amlodipine for 28 days induced a slight increase in capillary density without affecting total cardiomyocyte number, while reducing the total number of non-cardiomyocytes down to levels seen in untreated normotensive Wistar-Kyoto rats. Bax to Bcl-2 protein ratios were increased from day 7 to day 28. In situ double labeling by the terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) method (apoptosis) combined with rhodamine-labeled lectin binding (endothelial cell marker) revealed a significant increase (> 3-fold) in TUNEL-positive, lectin-negative non-cardiomyocytes in the interstitium between days 7 and 14. CONCLUSIONS: Left ventricular remodeling induced by amlodipine in SHR involves selective deletion of excess fibroblasts via apoptosis prior to cardiomyocyte mass reduction, but after attenuation of ANP gene expression.

Reversal of interstitial fibroblast hyperplasia via apoptosis in hypertensive rat heart with valsartan or enalapril.

OBJECTIVE: Renin-angiotensin system inhibitors transiently induce apoptosis at the onset of cardiac hypertrophy regression in spontaneously hypertensive rats (SHRs). The focus of this study is to evaluate the cell selectivity of this response. METHODS: SHRs were treated with valsartan or enalapril (30 mg kg(-1) day(-1)) or placebo for 1 to 4 weeks. Stereological and morphological data were obtained from immunohistological analyses. Apoptosis was quantified by DEVDase (caspase-3-like) activity assay and immunoblot analysis of apoptosis-regulatory proteins (Bax and Bcl-2). Identification of the apoptotic cell type was conducted using in situ TUNEL labeling, in conjunction with alpha-sarcomeric actin or lectin immunoreactivity as markers for cardiomyocytes and endothelial cells, respectively. RESULTS: Stereological analysis of the left ventricle revealed significant non-cardiomyocyte hyperplasia in placebo-treated SHRs (239+/-29x10(6) nuclei) as compared to untreated age-matched normotensive Wistar-Kyoto (WKY) rats (107+/-12x10(6)). In contrast, the number of cardiomyocyte nuclei was comparable between untreated SHRs (48+/-4x10(6)) and WKY rats. After 4 weeks of valsartan or enalapril treatment, SHRs showed significant reductions in systolic blood pressure (>28%), left ventricular hypertrophy (>9%) and cardiomyocyte cross-sectional area (>17%). Moreover, these treatments abolished non-cardiomyocyte hyperplasia in SHR left ventricle without affecting cardiomyocyte number, capillary density or number of capillary per cardiomyocyte nucleus. As a mechanism of cell deletion consistent with apoptosis induction, ventricles showed increased caspase-3 activation (>4.5-fold) as well as Bax to Bcl-2 protein ratio (>3.2-fold) within 2 weeks of valsartan or enalapril treatment. Immunohistological analysis revealed a significant increase in TUNEL-positive, lectin-negative non-cardiomyocytes, suggesting a rise in apoptotic interstitial fibroblasts in the left ventricle within 2 weeks of treatment with valsartan or enalapril (>63%), with a return to baseline (0.033+/-0.003%) at 4 weeks. Treatments did not affect right ventricular mass, apoptosis or cellularity. CONCLUSION: Cardiac apoptosis induction during regression of left ventricular hypertrophy reverses interstitial fibroblast hyperplasia in SHRs treated with inhibitors of the renin-angiotensin system.

Synergistic interaction between enalapril, L-arginine and tetrahydrobiopterin in smooth muscle cell apoptosis and aortic remodeling induction in SHR.

Smooth muscle cell (SMC) apoptosis occurs at the onset of enalapril-induced regression of aortic hypertrophy in SHR. A potential mechanism is the correction of endothelial dysfunction (ED) leading to reduced production of reactive oxygen species and enhanced bioavailability of nitric oxide (NO), a potent apoptosis inducer. Stimulants of NO include the precursor L-arginine and the NO synthase cofactor tetrahydrobiopterin (BH(4)), which correct ED in several models. The objective was to examine the relationships between ED and the cell growth/death balance during vascular remodeling induced by enalapril in SHR. SHR, 10-week-old, received enalapril (ENA: 30 mg x kg(-1) x day(-1) p.o.) for 1 or 2 weeks, or a co-treatment of L-arginine (2.0 g x kg(-1) x day(-1) p.o.) and BH(4) (5.4 mg x kg(-1) x day(-1) i.p. twice daily) administered alone (group: LB) or in combination with enalapril (ENA+LB) for 1 week. Controls received vehicle. After 1 week, ED was completely corrected with LB but not affected significantly by ENA, whereas both treatments failed to induce SMC apoptosis or aortic remodeling. The correction of ED and the induction of SMC apoptosis (3.3-fold increase in TUNEL labeling) required 2 weeks of ENA treatment. The combination of LB with ENA for 1 week, however, was additive for the reduction of SMC proliferation, and synergistic for the induction of apoptosis and regression of vascular hypertrophy. These interactions were independent of blood pressure regulation. Our results suggest that the correction of ED is not sufficient to induce SMC apoptosis and vascular remodeling, although it facilitates these responses during enalapril treatment.

Kinin B2 receptor is not involved in enalapril-induced apoptosis and regression of hypertrophy in spontaneously hypertensive rat aorta: possible role of B1 receptor.

1. Treatment with enalapril induces smooth muscle cell apoptosis and regression of aortic hypertrophy in spontaneously hypertensive rats (SHRs), whereas combined blockade of angiotensin II AT(1) and AT(2) receptors does not. We postulated that vascular apoptosis with enalapril involves enhanced half-life of bradykinin (BK) and kinin B(2) receptor stimulation. 2. SHR, 11-weeks old, were treated for 4 weeks with enalapril (30 mg kg(-1) day(-1)), Hoe 140 (500 microg kg(-1) day(-1); B(2) receptor antagonist), alone or in combination. Controls received vehicle. 3. The half-life of hypotensive responses to intra-arterial bolus injections of BK were significantly increased in SHR anesthetized after 4 weeks of enalapril, an effect prevented by Hoe 140. The magnitude of BK-induced hypotension was significantly attenuated in all rats treated with Hoe 140. 4. As compared to placebo, enalapril treatment significantly reduced blood pressure (-34+/-2%), aortic hypertrophy (-20+/-3%), hyperplasia (-37+/-5%) and DNA synthesis (-61+/-8%), while it increased aortic DNA fragmentation by two-fold. Hoe 140 given alone or in combination with enalapril affected none of these parameters. 5. As a possible alternative mechanism, aortae isolated during the second week of enalapril treatment showed a transient upregulation of contractile responses to des-Arg(9)BK (EC(50)<1 nM), which were significantly reduced by [Leu(8)]des-Arg(9)BK (10 microM). Moreover, in vitro receptor autoradiography revealed an increase in expression of B(1) and B(2) receptor binding sites by 8-11 days of enalapril treatment. 6. Aortic apoptosis induction and hypertrophy regression with enalapril do not involve kinin B(2) receptors in SHR. Kinins acting via B(1) receptors remains a candidate mechanism.

USP2 regulates the intracellular localization of PER1 and circadian gene expression.

Endogenous 24-h rhythms in physiology are driven by a network of circadian clocks located in most tissues. The molecular clock mechanism is based on feedback loops involving clock genes and their protein products. Posttranslational modifications, including ubiquitination, are important for regulating the clock feedback mechanism. Recently, we showed that the deubiquitinating enzyme ubiquitin-specific peptidase 2 (USP2) associates with clock proteins and deubiquitinates PERIOD1 (PER1) but does not affect its overall stability. Mice devoid of USP2 display defects in clock function. Here, we show that USP2 regulates nucleocytoplasmic shuttling and nuclear retention of PER1 and its repressive role on the clock transcription factors CLOCK and BMAL1. The rhythm of nuclear entry of PER1 in Usp2 knockout mouse embryonic fibroblasts (MEFs) was advanced but with reduced nuclear accumulation of PER1. Although Per1 mRNA expression rhythm remained intact in the Usp2 KO MEFs, the expression profiles of other core clock genes were altered. This was also true for the expression of clock-controlled genes (e.g., Dbp, Tef, Hlf, E4bp4). A similar phase advance of PER1 nuclear localization rhythm and alteration of clock gene expression profiles were also observed in livers of Usp2 KO mice. Taken together, our results demonstrate a novel function of USP2 in the molecular clock in which it regulates PER1 function by gating its nuclear entry and accumulation.

Regulation of behavioral circadian rhythms and clock protein PER1 by the deubiquitinating enzyme USP2.

Endogenous 24-hour rhythms are generated by circadian clocks located in most tissues. The molecular clock mechanism is based on feedback loops involving clock genes and their protein products. Post-translational modifications, including ubiquitination, are important for regulating the clock feedback mechanism. Previous work has focused on the role of ubiquitin ligases in the clock mechanism. Here we show a role for the rhythmically-expressed deubiquitinating enzyme ubiquitin specific peptidase 2 (USP2) in clock function. Mice with a deletion of the Usp2 gene (Usp2 KO) display a longer free-running period of locomotor activity rhythms and altered responses of the clock to light. This was associated with altered expression of clock genes in synchronized Usp2 KO mouse embryonic fibroblasts and increased levels of clock protein PERIOD1 (PER1). USP2 can be coimmunoprecipitated with several clock proteins but directly interacts specifically with PER1 and deubiquitinates it. Interestingly, this deubiquitination does not alter PER1 stability. Taken together, our results identify USP2 as a new core component of the clock machinery and demonstrate a role for deubiquitination in the regulation of the circadian clock, both at the level of the core pacemaker and its response to external cues.

Dynein light chain Tctex-type 1 modulates orexin signaling through its interaction with orexin 1 receptor.

Orexins (OX-A, OX-B) are neuropeptides involved in the regulation of the sleep-wake cycle, feeding and reward, via activation of orexin receptors 1 and 2 (OX1R, OX2R). The loss of orexin peptides or functional OX2R has been shown to cause the sleep disorder, narcolepsy. Since the regulation of orexin receptors remains largely undefined, we searched for novel protein partners of the intracellular tail of orexin receptors. Using a yeast two-hybrid screening strategy in combination with co-immunoprecipitation experiments, we found interactions between OX1R and the dynein light chains Tctex-type 1 and 3 (Dynlt1, Dynlt3). These interactions were mapped to the C-terminal region of the dynein light chains and to specific residues within the last 10 amino acids of OX1R. Hence, we hypothesized that dynein light chains could regulate orexin signaling. In HEK293 cells expressing OX1R, stimulation with OX-A produced a less sustained extracellular signal-regulated kinases 1/2 (ERK1/2) activation when Dynlt1 was co-expressed, while it was prolonged under reduced Dynlt1 expression. The amount of OX1R located at the plasma membrane as well as the kinetics and extent of OX-A-induced internalization of OX1R (disappearance from membrane) were not altered by Dynlt1. However, Dynlt1 reduced the localization of OX1R in early endosomes following initial internalization. Taken together, these data suggest that Dynlt1 modulates orexin signaling by regulating OX1R, namely its intracellular localization following ligand-induced internalization.

The crosstalk between physiology and circadian clock proteins.

In mammals, many physiological processes present diurnal variations, and most of these rhythms persist even in absence of environmental timing cues. These endogenous circadian rhythms are generated by intracellular timing mechanisms termed circadian clocks. In mammals, the master clock is located in the suprachiasmatic nuclei (SCN), but other brain regions and most peripheral tissues contain circadian clocks. These clocks are responsive to environmental cues, in particular light/dark and feeding/fasting cycles. In the last few years, tissue-specific knock-out and transgenic mouse models have helped to define the physiological roles of specific clocks. Recent reports indicate that the clock-physiology connection is bi-directional, and physiological cues, in particular the energetic status of the cell, can feed into the clockwork. This effect was discovered unexpectedly in molecular analyses of clock protein modifications. Beyond the positive and negative transcription/translation feedback loops of the molecular oscillator lies another level of complexity. Post-translational modifications of clock proteins are both critical for the timing of the clock feedback mechanism and to provide regulatory fine-tuning. This review summarizes recent advances in our understanding of the roles of peripheral clocks and of post-translational modifications occurring on clock proteins. These two matters are at the intersection of physiology, metabolism, and the circadian system.