The role of circadian clock gene Arntl in the winter depression-like behavior in melatonin-proficient female CBA/N mice.

Seasonal affective disorder (SAD), also known as winter depression, is a subtype of depression typically manifesting in winter. Typical symptoms of SAD, such as an increased need for sleep and carbohydrate cravings associated with increased appetite and weight, are distinct from those of major depression, and the underlying mechanisms of SAD remain unclear. Although laboratory mice are generally considered non-seasonal animals, we observed depression-like behaviors in melatonin-proficient female CBA/N mice maintained under winter-mimicking conditions. Transcriptome analysis of the brains of CBA/N mice maintained under winter- and summer-mimicking conditions revealed changes in the expression of circadian clock genes, including Arntl (also known as Bmal1). We generated Arntl-deficient, melatonin-proficient CBA/N mice using the speed congenic method to examine the role of Arntl in depressive behavior. The tail suspension test in these mice revealed a depressive phenotype. These results suggested that the circadian clock gene Arntl may be involved in winter depression-like behavior.

Adolescence as a critical period for nandrolone-induced muscular strength in relation to abuse liability, alone and in conjunction with morphine, using accumbal dopamine efflux in freely moving rats.

Nandrolone, an anabolic androgenic steroid, is included in the prohibited list of the World Anti-Doping Agency. Drugs of abuse activate brain dopamine neurons and nandrolone has been suspected of inducing dependence. Accordingly, possible critical periods for the effects of nandrolone on muscular strength and dopaminergic activity have been investigated, including the effects of chronically administered nandrolone alone and on morphine-induced increases in dopamine efflux in the nucleus accumbens. Six- or 10-week-old male Sprague-Dawley rats were used. Treatment with nandrolone was initiated in adolescent (6-week-old) and young adult (10-week-old) rats. Nandrolone (5.0 mg/kg s.c.) or sesame oil vehicle was given once daily, on six consecutive days per week, for 3 weeks and then once per day for 4 consecutive days. Nandrolone enhanced the developmental increase in grip strength of 6- but not 10-week-old rats, without altering the developmental increase in body weight of either age group. Using in vivo microdialysis in freely moving 6-week-old rats given nandrolone for 4 weeks, basal accumbal dopamine efflux was unaltered, while the increase in dopamine efflux induced by acute administration of morphine (1.0 mg/kg s.c.) was reduced. The present study provides in vivo evidence that adolescence constitutes a critical period during which repeated administration of nandrolone enhances increases in muscular strength without influencing increases in body weight. Though repeated administration of nandrolone during this period of adolescence did not stimulate in vivo mesolimbic dopaminergic activity, it disrupted stimulation by an opioid, the drug class that is most commonly coabused with nandrolone.

Inhibitory Effects of Hydrolysable Tannins on Lipid Accumulation in 3T3-L1 Cells.

Obesity is currently the most common cause of metabolic diseases including type 2 diabetes and hyperlipidemia. Obesity results from excess lipid accumulation in adipose tissue. Several studies have investigated the inhibitory effects of natural plant-derived products on adipocyte differentiation and lipid accumulation. In this study, we examined the effect of hydrolysable tannins composed of gallic acid and glucose on adipocyte differentiation in 3T3-L1 cells. 1,2,3,4,6-Penta-O-galloyl-ß-D-glucose (PGG) (1), a representative gallotannin, inhibited lipid accumulation in 3T3-L1 cells, whereas ellagitannins (tellimagrandin I, eugeniin and casuarictin) did not. The expression of adipocyte differentiation-related genes, including peroxisome proliferator activator γ2 (Pparγ2), CCAAT/enhancer binding protein α (C/EBPα) and adipocyte fatty acid binding protein (aP2), was significantly suppressed in PGG (1)-treated 3T3-L1 cells beginning at day 2 after induction of differentiation. While PGG (1) did not directly reduce Pparγ2 expression, it reduced the expression of its target genes in mature adipocytes. In addition, PGG (1) treatment inhibited mitotic clonal expansion, one of earliest events of adipocyte differentiation. These findings indicate that PGG (1) has an inhibitory effect on adipocyte differentiation through the suppression of mitotic clonal expansion.

Bmal1 Regulates Prostate Growth via Cell-Cycle Modulation.

The circadian clock system exists in most organs and regulates diverse physiological processes, including growth. Here, we used a prostate-specific Bmal1-knockout mouse model (pBmal1 KO: PbsnCre+; Bmal1fx/fx) and immortalized human prostate cells (RWPE-1 and WPMY-1) to elucidate the role of the peripheral prostate clock on prostate growth. Bmal1 KO resulted in significantly decreased ventral and dorsolateral lobes with less Ki-67-positive epithelial cells than the controls. Next, the cap analysis of gene expression revealed that genes associated with cell cycles were differentially expressed in the pBmal1 KO prostate. Cdkn1a (coding p21) was diurnally expressed in the control mouse prostate, a rhythm which was disturbed in pBmal1 KO. Meanwhile, the knockdown of BMAL1 in epithelial RWPE-1 and stromal WPMY-1 cell lines decreased proliferation. Furthermore, RWPE-1 BMAL1 knockdown increased G0/G1-phase cell numbers but reduced S-phase numbers. These findings indicate that core clock gene Bmal1 is involved in prostate growth via the modulation of the cell cycle and provide a rationale for further research to link the pathogenesis of benign prostatic hyperplasia or cancer with the circadian clock.

Disruption of Bmal1 Impairs Blood-Brain Barrier Integrity via Pericyte Dysfunction.

Circadian rhythm disturbances are well established in neurological diseases. However, how these disruptions cause homeostatic imbalances remains poorly understood. Brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein 1 (Bmal1) is a major circadian clock transcriptional activator, and Bmal1 deficiency in male Bmal1nestin-/- mice induced marked astroglial activation without affecting the number of astrocytes in the brain and spinal cord. Bmal1 deletion caused blood-brain barrier (BBB) hyperpermeability with an age-dependent loss of pericyte coverage of blood vessels in the brain. Using Nestin-green fluorescent protein (GFP) transgenic mice, we determined that pericytes are Nestin-GFP+ in the adult brain. Bmal1 deletion caused Nestin-GFP+ pericyte dysfunction, including the downregulation of platelet-derived growth factor receptor ß (PDGFRß), a protein necessary for maintaining BBB integrity. Knockdown of Bmal1 downregulated PDGFRß transcription in the brain pericyte cell line. Thus, the circadian clock component Bmal1 maintains BBB integrity via regulating pericytes.SIGNIFICANCE STATEMENT Circadian rhythm disturbances may play a role in neurodegenerative disorders, such as Alzheimer's disease. Our results revealed that one of the circadian clock components maintains the integrity of the blood-brain barrier (BBB) by regulating vascular-embedded pericytes. These cells were recently identified as a vital component for the control of BBB permeability and cerebral blood flow. Our present study demonstrates the involvement of circadian clock component Bmal1 in BBB homeostasis and highlights the role of Bmal1 dysfunction in multiple neurological diseases.

Deletion of Bmal1 Prevents Diet-Induced Ectopic Fat Accumulation by Controlling Oxidative Capacity in the Skeletal Muscle.

Brain and muscle arnt-like protein 1 (BMAL1), is a transcription factor known to regulate circadian rhythm. BMAL1 was originally characterized by its high expression in the skeletal muscle. Since the skeletal muscle is the dominant organ system in energy metabolism, the possible functions of BMAL1 in the skeletal muscle include the control of metabolism. Here, we established that its involvement in the regulation of oxidative capacity in the skeletal muscle. Muscle-specific Bmal1 KO mice (MKO mice) displayed several physiological hallmarks for the increase of oxidative capacity. This included increased energy expenditure and oxygen consumption, high running endurance and resistance to obesity with improved metabolic profiles. Also, the phosphorylation status of AMP-activated protein kinase and its downstream signaling substrate acetyl-CoA carboxylase in the MKO mice were substantially higher than those in the Bmal1flox/flox mice. In addition, biochemical and histological studies confirmed the substantial activation of oxidative fibers in the skeletal muscle of the MKO mice. The mechanism includes the regulation of Cacna1s expression, followed by the activation of calcium-nuclear factor of activated T cells (NFAT) axis. We thus conclude that BMAL1 is a critical regulator of the muscular fatty acid level under nutrition overloading and that the mechanism involves the control of oxidative capacity.

Aryl Hydrocarbon Receptor Plays Protective Roles against High Fat Diet (HFD)-induced Hepatic Steatosis and the Subsequent Lipotoxicity via Direct Transcriptional Regulation of Socs3 Gene Expression.

Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor regulating the expression of genes involved in xenobiotic response. Recent studies have suggested that AhR plays essential roles not only in xenobiotic detoxification but also energy metabolism. Thus, in this study, we studied the roles of AhR in lipid metabolism. Under high fat diet (HFD) challenge, liver-specific AhR knock-out (AhR LKO) mice exhibited severe steatosis, inflammation, and injury in the liver. Gene expression analysis and biochemical study revealed thatde novolipogenesis activity was significantly increased in AhR LKO mice. In contrast, induction of suppressor of cytokine signal 3 (Socs3) expression by HFD was attenuated in the livers of AhR LKO mice. Rescue of theSocs3gene in the liver of AhR LKO mice cancelled the HFD-induced hepatic lipotoxicities. Promoter analysis established Socs3 as novel transcriptional target of AhR. These results indicated that AhR plays a protective role against HFD-induced hepatic steatosis and the subsequent lipotoxicity effects, such as inflammation, and that the mechanism of protection involves the direct transcriptional regulation ofSocs3expression by AhR.

Different Roles of Negative and Positive Components of the Circadian Clock in Oncogene-induced Neoplastic Transformation.

In mammals, circadian rhythms in physiological function are generated by a molecular oscillator driven by transcriptional-translational feedback loop consisting of negative and positive regulators. Disruption of this circadian clock machinery is thought to increase the risk of cancer development, but the potential contributions of each component of circadian clock to oncogenesis have been little explored. Here we reported that negative and positive transcriptional regulators of circadian feedback loop had different roles in oncogene-induced neoplastic transformation. Mouse embryonic fibroblasts prepared from animals deficient in negative circadian clock regulators, Period2 (Per2) or Cryptochrome1/2 (Cry1/2), were prone to transformation induced by co-expression of H-ras(V12) and SV40 large T antigen (SV40LT). In contrast, mouse embryonic fibroblasts prepared from mice deficient in positive circadian clock regulators, Bmal1 or Clock, showed resistance to oncogene-induced transformation. In Per2 mutant and Cry1/2-null cells, the introduction of oncogenes induced expression of ATF4, a potent repressor of cell senescence-associated proteins p16INK4a and p19ARF. Elevated levels of ATF4 were sufficient to suppress expression of these proteins and drive oncogenic transformation. Conversely, in Bmal1-null and Clock mutant cells, the expression of ATF4 was not induced by oncogene introduction, which allowed constitutive expression of p16INK4a and p19ARF triggering cellular senescence. Although genetic ablation of either negative or positive transcriptional regulators of the circadian clock leads to disrupted rhythms in physiological functions, our findings define their different contributions to neoplastic cellular transformation.

The intrinsic microglial clock system regulates interleukin-6 expression.

Similar to neurons, microglia have an intrinsic molecular clock. The master clock oscillator Bmal1 modulates interleukin-6 upregulation in microglial cells exposed to lipopolysaccharide. Bmal1 can play a role in microglial inflammatory responses. We previously demonstrated that gliotransmitter ATP induces transient expression of the clock gene Period1 via P2X7 purinergic receptors in cultured microglia. In this study, we further investigated mechanisms underlying the regulation of pro-inflammatory cytokine production by clock molecules in microglial cells. Several clock gene transcripts exhibited oscillatory diurnal rhythmicity in microglial BV-2 cells. Real-time luciferase monitoring also showed diurnal oscillatory luciferase activity in cultured microglia from Per1::Luciferase transgenic mice. Lipopolysaccharide (LPS) strongly induced the expression of pro-inflammatory cytokines in BV-2 cells, whereas an siRNA targeting Brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein 1 (Bmal1), a core positive component of the microglial molecular clock, selectively inhibited LPS-induced interleukin-6 (IL-6) expression. In addition, LPS-induced IL-6 expression was attenuated in microglia from Bmal1-deficient mice. This phenotype was recapitulated by pharmacological disruption of oscillatory diurnal rhythmicity using the synthetic Rev-Erb agonist SR9011. Promoter analysis of the Il6 gene revealed that Bmal1 is required for LPS-induced IL-6 expression in microglia. Mice conditionally Bmal1 deficient in cells expressing CD11b, including microglia, exhibited less potent upregulation of Il6 expression following middle cerebral artery occlusion compared with that in control mice, with a significant attenuation of neuronal damage. These results suggest that the intrinsic microglial clock modulates the inflammatory response, including the positive regulation of IL-6 expression in a particular pathological situation in the brain, GLIA 2016. GLIA 2017;65:198-208.

Effect of a dosing-time on quetiapine-induced acute hyperglycemia in mice.

Although rare, second-generation antipsychotic drugs cause severe hyperglycemia within several days after the initiation of therapy. Because glucose tolerance exhibits circadian rhythmicity, we evaluated an effect of a dosing-time on quetiapine-induced acute hyperglycemia in mice. A single intraperitoneal dose of quetiapine dosing-time-independently induced insulin resistance in fasted C57BL/6J mice. However, acute hyperglycemic effect was detected only after dosing of the drug at the beginning of an active phase. Under the conditions in which hepatic glucose production was stimulated by pyruvate administration, hyperglycemic effect of quetiapine was dosing-time-independently observed. In addition, the dosing-time-dependent hyperglycemic effect of quetiapine disappeared in the liver-specific circadian clock-disrupted mice in which circadian rhythmicity in hepatic glucose production is deranged. Furthermore, the dosing-time had little impact on the pharmacokinetics of quetiapine in normal mice. These results suggest that quetiapine acutely causes hyperglycemia only when hepatic glucose production elevates. Therefore, quetiapine therapy with once daily dosing at a rest phase might be safer than that at an active phase. Further studies are needed to confirm the hypothesis.

Neuroprotective effect of S-allyl-l-cysteine derivatives against endoplasmic reticulum stress-induced cytotoxicity is independent of calpain inhibition.

S-allyl-l-cysteine (SAC) is known to have neuroprotective properties. We synthesized various SAC derivatives and tested their effects on endoplasmic reticulum stress-induced neurotoxicity in cultured hippocampal neurons (HPNs). Among the compounds tested, S-propyl-l-cysteine (SPC) exhibited the strongest neuroprotective activity in HPNs, followed by S-ethyl-l-cysteine (SEC) and S-methyl-l-cysteine (SMC). Unlike SAC and SMC, SPC and SEC did not have inhibitory activity on µ-calpain, suggesting that the mechanism underlying the protective activity of SPC and SEC differs from that of SAC.

Sympathetic activation induces skeletal Fgf23 expression in a circadian rhythm-dependent manner.

The circadian clock network is well known to link food intake and metabolic outputs. Phosphorus is a pivotal nutritional factor involved in energy and skeletal metabolisms and possesses a circadian profile in the circulation; however, the precise mechanisms whereby phosphate metabolism is regulated by the circadian clock network remain largely unknown. Because sympathetic tone, which displays a circadian profile, is activated by food intake, we tested the hypothesis that phosphate metabolism was regulated by the circadian clock network through the modification of food intake-associated sympathetic activation. Skeletal Fgf23 expression showed higher expression during the dark phase (DP) associated with elevated circulating FGF23 levels and enhanced phosphate excretion in the urine. The peaks in skeletal Fgf23 expression and urine epinephrine levels, a marker for sympathetic tone, shifted from DP to the light phase (LP) when mice were fed during LP. Interestingly, ß-adrenergic agonist, isoproterenol (ISO), induced skeletal Fgf23 expression when administered at ZT12, but this was not observed in Bmal1-deficient mice. In vitro reporter assays revealed that ISO trans-activated Fgf23 promoter through a cAMP responsive element in osteoblastic UMR-106 cells. The mechanism of circadian regulation of Fgf23 induction by ISO in vivo was partly explained by the suppressive effect of Cryptochrome1 (Cry1) on ISO signaling. These results indicate that the regulation of skeletal Fgf23 expression by sympathetic activity is dependent on the circadian clock system and may shed light on new regulatory networks of FGF23 that could be important for understanding the physiology of phosphate metabolism.

FASTER: an unsupervised fully automated sleep staging method for mice.

Identifying the stages of sleep, or sleep staging, is an unavoidable step in sleep research and typically requires visual inspection of electroencephalography (EEG) and electromyography (EMG) data. Currently, scoring is slow, biased and prone to error by humans and thus is the most important bottleneck for large-scale sleep research in animals. We have developed an unsupervised, fully automated sleep staging method for mice that allows less subjective and high-throughput evaluation of sleep. Fully Automated Sleep sTaging method via EEG/EMG Recordings (FASTER) is based on nonparametric density estimation clustering of comprehensive EEG/EMG power spectra. FASTER can accurately identify sleep patterns in mice that have been perturbed by drugs or by genetic modification of a clock gene. The overall accuracy is over 90% in every group. 24-h data are staged by a laptop computer in 10 min, which is faster than an experienced human rater. Dramatically improving the sleep staging process in both quality and throughput FASTER will open the door to quantitative and comprehensive animal sleep research.

Clock gene Bmal1 is dispensable for intrinsic properties of murine hematopoietic stem cells.

BACKGROUND: Circadian rhythms are known to influence a variety of biological phenomena such as cell cycle, sleep-wake rhythm, hormone release and other important physiological functions. Given that cell cycle entry of hibernating hematopoietic stem cells (HSCs) plays a critical role in controlling hematopoiesis, we asked functional significance of the clock gene Bmal1, which plays a central role in regulating circadian rhythms as a transcription factor. Here we investigated the necessity of Bmal1 for HSC functions using Bmal1 deficient (Bmal1⁻/⁻) mice. FINDINGS: Using colony-forming assays in vitro, we found that the frequency of mixed colony formation between Bmal1⁺/⁺ and Bmal1⁻/⁻ CD34-KSL cells does not differ significantly. Competitive bone marrow assays also revealed that Bmal1⁻/⁻ bone marrow cells competed normally with wild-type cells and displayed long-term multi-hematopoietic lineage reconstitution. In addition, there were no significant differences in the frequencies and hibernation state of bone marrow HSCs between Bmal1⁺/⁺ and Bmal1⁻/⁻ mice, suggesting that they are independent of circadian rhythms. CONCLUSIONS: This paper discusses the necessity of circadian rhythms for HSC functions. Our data clearly shows that a key circadian clock gene Bmal1 is dispensable for intrinsic functions of HSCs, such as differentiation, proliferation and repopulating ability.

Aryl hydrocarbon receptor modulates NADPH oxidase activity via direct transcriptional regulation of p40phox expression.

A member of the NADPH oxidase subunits, p40(phox) plays an important role in the regulation of NADPH oxidase activity and the subsequent production of reactive oxygen species (ROS). In this study, we show that mouse p40(phox) is a novel transcriptional target of the aryl hydrocarbon receptor (AhR), known as a dioxin receptor or xenobiotic receptor, in the liver. Treatment of mice with 3-methylcholanthrene (3MC) increased p40(phox) gene expression in the liver, but this induction of p40(phox) gene expression was diminished by the deletion of the AhR gene in the liver. Consistent with the in vivo results, the expression of the p40(phox) gene was increased in 3MC-treated Hepa1c1c7 cells in an AhR-dependent manner. In addition, promoter analysis established p40(phox) as a transcriptional target of AhR. Studies using the RNA-interference technique revealed that p40(phox) is involved in the increase of NADPH oxidase activity and the subsequent ROS production in AhR-activated Hepa1c1c7 cells. Consequently, the results obtained here may provide a novel molecular mechanism for ROS production after exposure to dioxins.

Clock genes influence gene expression in growth plate and endochondral ossification in mice.

We have previously shown transient promotion by parathyroid hormone of Period-1 (Per1) expression in cultured chondrocytes. Here we show the modulation by clock genes of chondrogenic differentiation through gene transactivation of the master regulator of chondrogenesis Indian hedgehog (IHH) in chondrocytes of the growth plate. Several clock genes were expressed with oscillatory rhythmicity in cultured chondrocytes and rib growth plate in mice, whereas chondrogenesis was markedly inhibited in stable transfectants of Per1 in chondrocytic ATDC5 cells and in rib growth plate chondrocytes from mice deficient of brain and muscle aryl hydrocarbon receptor nuclear translocator-like (BMAL1). Ihh promoter activity was regulated by different clock gene products, with clear circadian rhythmicity in expression profiles of Ihh in the growth plate. In BMAL1-null mice, a predominant decrease was seen in Ihh expression in the growth plate with a smaller body size than in wild-type mice. BMAL1 deficit led to disruption of the rhythmic expression profiles of both Per1 and Ihh in the growth plate. A clear rhythmicity was seen with Ihh expression in ATDC5 cells exposed to dexamethasone. In young mice defective of BMAL1 exclusively in chondrocytes, similar abnormalities were found in bone growth and Ihh expression. These results suggest that endochondral ossification is under the regulation of particular clock gene products expressed in chondrocytes during postnatal skeletogenesis through a mechanism relevant to the rhythmic Ihh expression.

[Dyslipidemia induced disorder of circadian rhythm].

Several epidemiological studies have suggested that the perturbation of circadian rhythm has adverse metabolic consequences (e.g., dyslipidemia) in humans. At the molecular level, circadian rhythms are encoded by an autoregulatory loop composed of a set of transcription activators (BMAL1/CLOCK) that induce expression of repressors (PER/CRY). The mammalian molecular clock is not only expressed within the master suprachiasmatic nucleus pacemaker neurons, but also within nearly all cells. In addition to this core loop, BMAL1/CLOCK also induce expression of the orphan nuclear hormone receptor, which modulates Bmal1 transcription. Disruption of clock genes results in metabolic deregulation in mice. In this article, the roles of clock genes in the regulation of metabolism were summarized based on the phenotypes of the knockout mice.

[The roles of clock genes in obesity].

Several epidemiological studies have suggested that the perturbation of circadian rhythm has adverse metabolic consequences (e.g., obesity) in humans. At the molecular level, circadian rhythms are encoded by an autoregulatory loop composed of a set of transcription activators (BMAL1/CLOCK) that induce expression of repressors (PER/CRY). The mammalian molecular clock is not only expressed within the master suprachiasmatic nucleus pacemaker neurons, but also within nearly all cells. In addition to this core loop, BMAL1/CLOCK also induce expression of the orphan nuclear hormone receptor, which modulates Bmal1 transcription. Disruption of clock genes results in metabolic dysregulation in mice. In this article, the roles of clock genes in the regulation of metabolism were summarized based on the phenotypes of the knockout mice.

Arntl deficiency in myeloid cells reduces neutrophil recruitment and delays skeletal muscle repair.

After a muscle injury, a process comprising inflammation, repair, and regeneration must occur in a time-sensitive manner for skeletal muscle to be adequately repaired and regenerated. This complex process is assumed to be controlled by various myeloid cell types, including monocytes and macrophages, though the mechanism is not fully understood. Aryl hydrocarbon receptor nuclear translocator-like (Arntl or Bmal1) is a transcription factor that controls the circadian rhythm and has been implicated in regulating myeloid cell functions. In the present study, we generated myeloid cell-specific Arntl conditional knockout (cKO) mice to assess the role of Arntl expressed in myeloid cell populations during the repair process after muscle injury. Myeloid cell-specific Arntl deletion impaired muscle regeneration after cardiotoxin injection. Flow cytometric analyses revealed that, in cKO mice, the numbers of infiltrating neutrophils and Ly6Chi monocytes within the injured site were reduced on days 1 and 2, respectively, after muscle injury. Moreover, neutrophil migration and the numbers of circulating monocytes were significantly reduced in cKO mice, which suggests these effects may account, at least in part, for the impaired regeneration. These findings suggest that Arntl, expressed in the myeloid lineage regulates neutrophil and monocyte recruitment and is therefore required for skeletal muscle regeneration.

Hairless modulates ligand-dependent activation of the vitamin D receptor-retinoid X receptor heterodimer.

The active form of vitamin D, 1α,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], binds to the vitamin D receptor (VDR) and regulates various physiological and pharmacological processes. Secondary bile acids, such as lithocholic acid (LCA), also act as endogenous VDR ligands. The molecular basis of ligand-selective VDR action remains largely unknown. Hairless (HR) acts as a coregulator of VDR through a direct interaction. HR mutations confer an alopecia phenotype similar to VDR mutations in mice and humans, but the underlying molecular mechanisms have not been elucidated. We examined the effect of HR on VDR activation induced by 1,25(OH)(2)D(3) and LCA. HR repressed VDR transactivation induced by both 1,25(OH)(2)D(3) and LCA. HR also repressed transactivation of VDR E269A and R391A mutants, but less effectively than that of wild-type VDR. These residues are involved in retinoid X receptor (RXR) heterodimer allosteric communication, through which information from ligands is transmitted to dimer and coactivator interfaces. In the presence of HR cotransfection, LCA activated these VDR mutants more effectively than wild-type VDR. In mammalian two-hybrid assays, HR enhanced the association of VDR with a corepressor, nuclear receptor corepressor. These findings indicate that HR affects VDR-RXR heterodimer allosteric communication and corepressor complex formation. Interestingly, HR knockdown in keratinocyte-derived HaCaT cells increased ligand-induced cytochrome P450, family 24, subfamily A, polypeptide 1 (CYP24A1) expression but suppressed expression of cathelicidin antimicrobial peptide, indicating that HR acts not only as a corepressor but also as a coactivator. HR may be a VDR modulator that affects the RXR allosteric communication network in order to regulate transcription in a gene-selective manner.