Circadian Clock-Deficient Mice As a Tool for Exploring Disease Etiology

Home , Hitoshi Okamura

September 2012 Biol. Pharm. Bull. 35(9) 1385–1391 (2012) 1385 Review Circadian Clock-Deﬁcient Mice as a Tool for Exploring Disease Etiology Masao Doi Department of Systems Biology, Graduate School of Pharmaceutical Sciences, Kyoto University; 46–29 Yoshida Shimoadachi, Sakyo-ku, Kyoto 606–8501, Japan. Received April 20, 2012

One of the most significant conceptual changes brought about by the analysis of circadian clock-deficient mice is that abnormalities in the circadian clock are linked not only to sleep arousal disorder but also to a wide variety of common diseases, including hypertension, diabetes, obesity, and cancer. It has recently been shown that the disruption of the two cryptochrome genes Cry1 and Cry2—core elements of the circadian clock—induces salt-dependent hypertension due to abnormally high synthesis of the mineralocorticoid aldosterone by the adrenal gland. This adrenal disorder occurs as a result of increased expression of Hsd3b6, a newly identified steroidogenic enzyme that regulates aldosterone production within the adrenal zona glomerular cells. Importantly, this enzyme is functionally conserved in humans, and the pathophysiologic condition of human idiopathic hyperaldosteronism resembles that of Cry1/2-deficient mice. This review highlights the potential utility of circadian clock-deficient mice as a tool for exploring hitherto unknown disease etiology linked to the circadian clock. Key words circadian clock; clock gene; hypertension; aldosterone

INTRODUCTION mineralocorticoid that promotes sodium and water retention by the kidney, thereby increasing vascular fluid volume and Given the increasing prevalence of sleep disorders, rotating blood pressure.7–9) Increased aldosterone production by the shift work, and irregular nocturnal lifestyles in our society adrenal gland, a symptom referred to as primary hyperal- today, it is important to elucidate how body clock disturbances dosteronism, constitutes a frequent form of secondary hyper- affect human health and disease. Hypertension (high blood tension in humans,8,9) but the underlying mechanism remains pressure) is an expanding health problem that arises from to be determined. An extensive search for the molecular path- mostly unknown causes.1) A growing number of epidemiologic way downstream of Cry in the mouse adrenal gland led to the studies indicate that shift workers, long-distance transme- identification of a previously unknown hypertension risk fac- ridian flight crews, and patients with sleep disorders show tor: the type VI 3β-hydroxyl-steroid dehydrogenase/isomerase a higher than average prevalence of hypertension-derived (Hsd3b6), an Hsd3b isoform specific to the aldosterone-pro- cardiovascular disease.2–4) Moreover, a recent clinical trial in ducing zona glomerular (ZG) cells.6) Hsd3b6 is under clock which healthy volunteers were placed in an environment that control and overexpressed in Cry-null mice. The human ho- induced rhythm abnormalities has demonstrated increased mologue of this enzyme, also expressed within the adrenal ZG blood pressure in the trial participants.5) cells, constitutes a new potential target for the development of Although the clinical and epidemiologic studies conducted antihypertensive drugs. to date have suggested a close relationship between hyper- The potential therapeutic implications raised by Cry-null tension and circadian malfunction, it remains difficult to un- mice point to the utility of circadian clock-deficient mice as derstand this relationship intuitively and confer a mechanistic a tool for exploring unknown disease etiology. This review explanation on the pathway responsible for the generation of describes an overview of the molecular mechanism of the disease. One possible straightforward approach to this prob- circadian clock and then exemplifies how pathophysiologic lem is to create clock gene-knockout mice and verify causal investigations of clockless mice may help to understand the relationships between the circadian clock and disease. Once mechanisms underlying the pathogenesis of hypertension. verified, this genetic link warrants further elucidation of the underlying mechanism, in which impaired (arrhythmic) clock- 1. CLOCK GENES controlled gene expression is expected to be a primary driver leading to the development of disease. It will then be possi- In mammals, most physiologic and behavioral events are ble to conduct translational research that aims at clarifying subjected to well-controlled daily oscillations, generated by whether the molecular mechanisms in mice can be applied to an internal, self-sustained, molecular oscillator referred to as pathologies in humans. the circadian clock. The molecular framework of the circadian It has recently been shown that arrhythmic mice lack- clock is composed of transcription/translation-based autoregu- ing the core clock genes Cry1 and Cry2 (hereafter referred latory feedback loops involving a set of clock genes.10–12) One to as Cry-null mice) exhibit salt-dependent hypertension.6) of the greatest conceptual changes brought about by the dis- Cry-null mice display an adrenal disorder characterized by covery of clock genes is that circadian rhythms are generated chronic overproduction of aldosterone. Aldosterone is a potent by almost all of the cells in the body,13) and that these individ- ual rhythms are coordinated by the master clock within the The author declares no conflict of interest. hypothalamic suprachiasmatic nucleus (SCN).14–18) Previously,

Fig. 1. Autoregulatory Feedback Loops Generating Circadian Expression of Clock Genes and Output Genes Heterodimers of CLOCK and BMAL1 activate the transcription of Per and Cry genes through E-box elements, and the resulting PER and CRY proteins inhibit CLOCK:BMAL1 activity to suppress their own transcription. PER proteins are subject to CK1-catalyzed phosphorylation and subsequent proteasomal degradation via β-TrCP-mediated ubiquitination, which ensures the next initiation of E-box-mediated transcription by CLOCK:BMAL1. Assisting this core loop, DBP activates and E4BP4 inhibits the transcription of Per genes through the D-box. REV-ERBα acts on RRE to inhibit Bmal1 and E4bp4 transcription, competing with RORα. These interlocking loops generate rhythmic expression of clock genes and output genes. The extracellular signals that increase the content of Ca2+ and/or cAMP lead to the activation of ERK and CREB and thereby activate Per1 expression through a cAMP-responsive element (CRE). Besides CRE, Per1 contains glucocorticoid-responsive elements (GREs), which also activate transcription. Adopted, with modification, from Doi et al.6) it was thought that the SCN was the sole time-keeper in the The promoter regions of the core clock genes Per1 and Per2 body, but now it is recognized as a conductor governing sub- contain D-box elements through which the PAR bZip family ordinate oscillators located in the peripheral tissues.13,16) of transcriptional activators (DBP, TEF, and HLF) and the At the cellular levels, clock genes and their protein products related repressor E4BP4 exert their opposing effects on the ex- constitute multiple interlocking loops, among which the so- pression of Per genes (Fig. 1).23–27) Importantly, both Dbp and called core loop is crucial for the generation of autonomous E4bp4 are under the transcriptional control of the core loop. circadian oscillation (Fig. 1).10) In the core loop, the basic Thus, these downstream regulators of the core loop can exert helix–loop–helix-PAS domain proteins CLOCK and BMAL1 their effects on the core loop itself by acting on the D-box act as positive regulators: CLOCK forms heterodimers with elements of Per, thereby forming additional autoregulatory BMAL1 and drives the transcription of Period (Per1, Per2, limbs linked with the core loop. The RRE forms another loop, and Per3) and Cryptochrome (Cry1 and Cry2) genes through in which REV-ERBα protein indirectly regulates the expres- E-box elements (CACGT[G/T]) located in their promoter sion of Per genes by suppressing the transcription of Bmal1 regions. Once the repressor proteins PER and CRY reach a and E4bp4 through the RRE on their promoters (Fig. 1).27) critical concentration, they form complexes and feedback to The D-box and RRE-based interlocking loops are thought to inhibit CLOCK:BMAL1-mediated transcription. After a pe- enhance the stability of the core loop-dependent autonomous riod of time, PER:CRY repressor complexes are degraded via oscillation and expand circadian output pathways from the a phosphorylation-dependent posttranslational process, which core loop. allows the CLOCK:BMAL1-positive complex to reactivate E-box-mediated transcription. The promoter regions of Per1 2. CLOCK-CONTROLLED GENES and Per2 undergo circadian fluctuation in histone H3 acetylation, which coincides with the recruitment of cAMP response Functional cellular clocks are present not only in the SCN element binding protein (CREB)-binding protein and CLOCK but also in virtually all cells in the body and contribute to the to the E-box elements.19,20) Importantly, both proteins possess circadian regulation of physiologies in local tissues and or- histone acetyltransferase activity,21) indicating that the control gans. Gene array studies have demonstrated that at least 10% of chromatin remodeling constitutes a key regulatory step of the cellular genes are under the transcriptional control of governing the cellular clock machinery.11) the clock.28–30) Furthermore, it was shown that the clock con- Besides the E-box, the core loop regulates at least two addi- trols the genes corresponding to rate-limiting steps of various tional cis-elements, called the D-box (the DBP/E4BP4-binding cellular pathways,28) demonstrating that the physiologic roles element) and the ROR/REV-ERB-binding element (RRE).22) of the clock are selective and efficient. Given the pervasive September 2012 1387

Fig. 2. Role of Circadian Clock-Regulated ZG-Specific 3β-HSD Subtype in Causing Aldosterone Disorder (A) Immunocytochemistry reveals ZG-specific expression of Hsd3b6 is in the mouse adrenal gland. ZF, zona fasciculata; ZR, zona reticularis; M, medulla. Bar, 100 µm. (B) Zone-specific steroidogenesis in the rodent adrenal gland. Aldosterone is produced specifically in the ZG, where Cyp11b2 (aldosterone synthase) is expressed. Thus, the site specificity of Hsd3b6 indicates that the major role of this enzyme is devoted to aldosterone synthesis. (C) Schematic model of abnormal regulation of Hsd3b6 in Cry-null mice. In wild-type mice, Hsd3b6 is rhythmically controlled by the circadian regulators DBP and E4BP4. In Cry-null mice, however, constitutive expression of DBP disrupts the equilibrium, leading to imbalanced overexpression of Hsd3b6. Continuously enhanced 3β-HSD activity within the ZG cells results in increased PAC and the development of salt-sensitive hypertension. Adopted, with modification, from Doi et al.6) influence of the clock on physiology, it may not be surprising new genetic links between circadian clock malfunction and that the disruption of this system can lead to the pathogenesis the pathogenesis of various lifestyle-related diseases, such as of various diseases. metabolic syndrome, cancer, and cardiovascular complaints. The molecular mechanisms by which the clock controls the Recent pathophysiologic studies using arrhythmic clock transcription of output genes are essentially the same as those gene-mutant mice have provided novel lines of evidence, for the regulation of clock genes. Mainly, three output routes linking the Period gene to cancer37) and Clock and Bmal1 to have been identified, dependent on the regulatory cis-elements metabolic syndrome (diabetes and obesity).38–41) The absence involved: E-box; D-box; and RRE (Fig. 1). For example, the of Cry1 and Cry2 induces salt-sensitive hypertension6) and expression of vasopressin in the SCN is regulated by an E-box exacerbates arthritis.42) site on its promoter, which is activated by CLOCK:BMAL1 Interestingly, the pathophysiologic consequences of circa- in a circadian time-dependent manner.31) In the liver, albumin dian clock-deficient mice are diverse, depending on the gene and Cyp2A5 are regulated via D-box sites, positively by DBP that has been deleted. The phenotypic differences between and negatively by E4BP4.32) Bmal1 expression, as well as the genotypes appear to reflect differential states of circadian many clock-controlled genes expressed during circadian night, output genes. For example, E-box-mediated output genes in is regulated via RRE sites by the opposite actions of REV- Cry-null mice are constitutively upregulated (de-repressed) ERBα and RORs.33) It is conceivable that these three differ- as a result of impaired circadian repression.43–45) This con- ential cis-elements diversify output pathways and expand the trasts sharply with Clock- and Bmal1-mutant mice that exhibit ways by which the circadian clock contributes to physiology continuously decreased (inactivated) expression of E-box- and pathophysiology. mediated genes.31,46,47) In this context, it is interesting to note that whereas both Bmal1-null mice and Cry-null mice are 3. CLOCK GENES AND DISEASE behaviorally arrhythmic, they are distinct from each other in blood pressure phenotype: Bmal1-null mice are hypotensive,48) 3.1. Genetic Evidence Linking the Molecular Clock a phenotype opposite to that of Cry-null mice. and Disease In general, sleep arousal disorders are thought 3.2. Aldosterone-Dependent Salt-Sensitive Hyperten- to be the primary (or sole) example of circadian clock-related sion in Cry-Null Mice Aldosterone, secreted by the adrenal diseases.34–36) However, the discovery of clock genes and gland, is a key steroid hormone that controls epithelial Na+ the subsequent clarification of the molecular feedback loops channel activity in the aldosterone-sensitive distal nephron, challenged this view and opened up opportunities to define thereby controlling sodium balance, blood volume, and blood 1388 Vol. 35, No. 9 pressure.7–9) In wild-type mice, both plasma aldosterone to aldosterone production. The increment of Hsd3b6 within concentration (PAC) and blood pressure undergo circadian the ZG cells of the Cry-null adrenal gland was confirmed at fluctuations, peaking almost synchronously at night (when the protein and enzymatic activity levels.6) Furthermore, phar- nocturnal animals are active). By contrast, in Cry-null mice, macologic inhibition of 3β-HSD enzymatic activity in vivo by these parameters are not circadian,6) reflecting the loss of the treating mice with trilostane (2α-cyano-4α,5α-epoxy-17β-olan- endogenous circadian clock system.49–51) Moreover, we found drostane-3-one)53–56) leads to the reduction of PAC in Cry-null that in Cry-null mice PAC is markedly and constitutively mice. These observations suggest that Hsd3b6 is a rate-lim- elevated and that this elevation renders blood pressure salt iting enzyme for aldosterone production and that the chronic sensitive.6) Normally, blood pressure is resilient to changes in elevation of its activity instigates abnormally high synthesis of the amount of dietary salt intake. However, Cry-null mice ex- aldosterone in the adrenal gland. perience a drastic rise in blood pressure when switched from The molecular mechanism underlying the abnormal regula- a normal-salt to high-salt diet. Importantly, this elevation was tion of Hsd3b6 is also worthy of investigation to understand reversed by treatment with an aldosterone blocker, eplerenone, the etiology behind the adrenal-autonomous circadian disor- demonstrating that the salt-dependent hypertension of Cry- der. ZG cells have an endogenous clock that governs thou- null mice is aldosterone dependent.6) sands of clock-controlled genes. The Hsd3b6 promoter bears 3.3. Enhanced Baroreflex Sensitivity in Cry-Null Mice two functional D-box cis-elements,6) allowing the circadian Despite the increased PAC, Cry-null mice are not hypertensive regulator DBP to activate transcription and E4BP4 to suppress when fed a normal-salt diet.6) This suggests that compensa- DBP-mediated transactivation (Fig. 2). In the Cry-null adre- tory processes minimalize the effect of increased PAC on nal gland, the expression levels of E4bp4 are constantly low, blood pressure in Cry-null mice. The baroreflex appears to whereas those of Dbp are constitutively high because of the be among such compensatory processes, since Cry-null mice loss of CRY-mediated transcriptional inhibition (Fig. 1).25,43) present increased baroreflex sensitivity as compared to wild- Thus, the DBP-dependent constitutive activation is likely to be type mice.52) The baroreflex, or baroreceptor reflex, is a short- a primary driver leading to the imbalanced superexpression of term negative feedback mechanism by which an increase in Hsd3b6 (see model in Fig. 2). This model is consistent with a blood pressure reflexively (thus acutely) causes the heart rate recent independent study showing that the genetic inactivation to decrease, thereby suppressing the elevation of blood pres- of PAR bZip proteins (Dbp−/− Tef −/− Hlf −/−) leads, conversely, sure. Thus, enhanced baroreflex sensitivity in Cry-null mice to reduced aldosterone production in the adrenal gland.57) is likely to counterbalance the aldosterone-dependent elevation of blood pressure. 4. TRANSLATIONS TO HUMAN ALDOSTERONISM 3.4. Primary Hyperaldosteronemia in Cry-Null Mice The biosynthesis of aldosterone takes place exclusively in the 4.1. Cry-Null Mice Show Pathophysiology Similar to adrenal ZG cells, which constitute the outermost layer of the Idiopathic Hyperaldosteronism Primary hyperaldostero- adrenal cortex (Fig. 2). The activity of aldosterone production nism in humans can be classified into two distinct diagnos- in the ZG cells is strictly controlled by the renin-angioten sin- tic categories: aldosterone-producing adenoma (APA); and aldosterone system.1) When blood pressure is low, aldosterone idiopathic hyperaldosteronism (IHA).8,9) Although both APA is released from the ZG in response to renin-induced angi- and IHA exhibit salt-sensitive hypertension, characterized otensin II production. By contrast, when blood pressure is by increased PAC and decreased PRA, the pathologies of the high, aldosterone production is lowered following the reduc- two are distinct. Normally, APA is a unilateral benign tumor tion of plasma renin activity (PRA). Importantly, PRA in Cry- that can be corrected by surgery. By contrast, IHA is nontu- null mice is markedly reduced throughout the day, indicating morigenic and characterized by a bilateral hyperplasia of ZG that the enhanced PAC in Cry-null mice is not a secondary cells, a condition similar to that of Cry-null mice. IHA has result of enhanced PRA. Rather, the primary cause seems been called “idiopathic” because the underlying mechanism is intrinsic to the adrenal gland. Histologically, Cry-null mice not known. Even worse, IHA does not allow for surgical cor- show bilateral hyperplasia of ZG cells. Moreover, aldosterone rection by adrenalectomy because of its bilateral nature. Thus, secretion from ex vivo slice cultures of the Cry-null adrenal the identification of Cry-null mice as a new animal model of gland is autonomously enhanced relative to that of the wild- IHA would provide opportunities to understand the etiology type adrenal gland. These results indicate that the abnormality of IHA and conduct drug screening for its treatment. of Cry-null mice exists within the adrenal gland. 4.2. Identification of a Human Orthologue of Hsd3b6 3.5. Identification of Hsd3b6 as a New Hypertension as a Key Target for Translational Research Human Risk Factor Linked to the Circadian Clock To identify HSD3B1 is a functional counterpart of mouse Hsd3b6 (note the primary cause of the adrenal disorder in Cry-null mice, that the gene numbers do not reflect functional similarity as DNA microarray-based comparative analysis was performed the members of 3β-HSD protein family have been chrono- on the adrenal glands of wild-type and Cry-null mice.6) logically designated according to their order of identification Among dozens of steroid synthetic enzymes, Hsd3b6, a sub- in each species).6) Subtype-selective Taqman qPCR analysis type of 3β-hydroxyl-steroid dehydrogenase (3β-HSD, a group with laser-microdissected human adrenal specimens revealed of enzymes that catalyze the conversion of pregnenolone to that the expression of HSD3B1 is specific to ZG cells, whereas progesterone, an enzymatic reaction known to be essential human HSD3B2 (a counterpart of mouse Hsd3b1) is enriched for the biosynthesis of all sex steroids, glucocorticoids, and in a different cortical layer, termed the zona fasciculata (ZF). mineralocorticoids) was found to be under clock control and Interestingly, human HSD3B1 is expressed not only in the overexpressed in Cry-null mice (Fig. 2). Notably, Hsd3b6 ex- adrenal ZG but also in the skin and placenta, in a pattern sim- hibits ZG-specific expression, indicating its major contribution ilar to that of mouse Hsd3b6.58) These observations support September 2012 1389 the notion that human HSD3B1, but not HSD3B2, represents a REFERENCES functional counterpart of mouse Hsd3b6. Human HSD3B1 may be a critical target for translational re- 1) Lifton RP, Gharavi AG, Geller DS. Molecular mechanisms of search. It has recently been shown that single-nucleotide poly- human hypertension. Cell, 104, 545–556 (2001). morphisms (SNPs) of HSD3B1 are associated with elevated 2) Furlan R, Barbic F, Piazza S, Tinelli M, Seghizzi P, Malliani A. mean systolic and diastolic blood pressures59) and PAC,60) Modifications of cardiac autonomic profile associated with a shift schedule of work. Circulation, 102, 1912–1916 (2000). suggesting a functional contribution of HSD3B1 to aldosterone 3) Bradley TD, Floras JS. 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