BMAL1 Knockdown Research Articles

The positive circadian regulators CLOCK and BMAL1 control G2/M cell cycle transition through Cyclin B1

ABSTRACTWe previously identified a tight bidirectional phase coupling between the circadian clock and the cell cycle. To understand the role of the CLOCK/BMAL1 complex, representing the main positive regulator of the circadian oscillator, we knocked down Bmal1 or Clock in NIH3T33C mouse fibroblasts (carrying fluorescent reporters for clock and cell cycle phase) and analyzed timing of cell division in individual cells and cell populations. Inactivation of Bmal1 resulted in a loss of circadian rhythmicity and a lengthening of the cell cycle, originating from delayed G2/M transition. Subsequent molecular analysis revealed reduced levels of Cyclin B1, an important G2/M regulator, upon suppression of Bmal1 gene expression. In complete agreement with these experimental observations, simulation of Bmal1 knockdown in a computational model for coupled mammalian circadian clock and cell cycle oscillators (now incorporating Cyclin B1 induction by BMAL1) revealed a lengthening of the cell cycle. Similar data were obtained upon knockdown of Clock gene expression. In conclusion, the CLOCK/BMAL1 complex controls cell cycle progression at the level of G2/M transition through regulation of Cyclin B1 expression.

Tea polyphenols direct Bmal1-driven ameliorating of the redox imbalance and mitochondrial dysfunction in hepatocytes

Circadian rhythms are intimately linked to cellular redox status homeostasis via the regulation of mitochondrial function. Tea polyphenols (TP) are nutraceuticals that possess powerful antioxidant properties, especially ameliorating oxidative stress. The objective of this study was to investigate whether circadian clock is involved in the protection effect of TP on oxidative stress cell models. TP ameliorate H2O2-triggered relatively shallow daily oscillations and phase shift of circadian clock genes transcription and protein expression. Meanwhile, TP attenuate H2O2-stimulated excessive secretions of reactive oxygen species (ROS) and restore the depletions of mitochondrial function in a Bmal1-dependent manner. Furthermore, TP treatment accelerates nuclear translocation of Nrf2 and modulates the downstream expressions of antioxidant enzymes. Intriguingly, knockdown of Bmal1 notably blocked Nrf2/ARE/HO-1 redox-sensitive transcription pathway. Our study revealed that TP, as a Bmal1-enhancing natural compound, alleviated redox imbalance via strengthening Keap1/Nrf2 antioxidant defense pathway and ameliorating mitochondrial dysfunction in a Bmal1-dependent manner.

BMAL1 deficiency promotes skeletal mandibular hypoplasia via OPG downregulation.

Skeletal mandibular hypoplasia (SMH), a common type of developmental deformities, results in impaired aesthetics of facial profile, occlusal dysfunction and poor life quality. In this study, BMAL1 deficiency leads to SMH formation, and we aim to investigate the mechanism by which BMAL1 deficiency induces SMH. Circadian rhythm-disordered mouse models were constructed by placing animals in a jet lag schedule of 6-h light advance every 7days for 4 or 8weeks. The OPG expression was evaluated by histomorphometry, immunohistochemistry and western blot analysis. The mechanism by which BMAL1 affects OPG expression was investigated by chromatin immunoprecipitation and luciferase reporter assays. The phenotypes caused by BMAL1 knockout can be rescued by exogenous supplementation with OPG. We demonstrate that the expressions of BMAL1 and OPG decreased in SMH patients. Circadian rhythm-disordered mice and Bmal1-/- mice exhibited decreased expression of OPG, reduced bone mass and bone size of mandibles. Our results revealed that BMAL1 bound directly to the Opg promoter and upregulated its expression, thus inhibiting osteoclast differentiation. BMAL1 deficiency increased osteoclast differentiation by downregulating OPG expression. In vitro, the enhancement effect of osteoclast differentiation caused by BMAL1 knockdown was significantly reversed by exogenous supplementation with OPG. Importantly, bone loss caused by BMAL1 knockout can be partially reversed by injecting OPG Intraperitoneally. These results indicate that the circadian clock plays a critical role in the growth and development of mandible by regulating OPG expression, and present a potential therapeutic strategy to prevent SMH.

The Clock Protein Bmal1 Regulates Circadian Expression and Activity of Sulfotransferase 1a1 in Mice.

Sulfotransferase 1a1 (Sult1a1) is a phase II enzyme that contributes extensively to metabolism and detoxification of various drugs and chemicals. Here we aimed to investigate a potential role of the clock protein Bmal1 (brain and muscle Arnt-like protein-1) in circadian regulation of Sult1a1 in mice. The regulatory effects of Bmal1 on Sult1a1 were assessed both in vivo (using Bmal1- deficient mice) and in vitro (using both normal and serum-shocked Hepa-1c1c7 cells). The relative mRNA and protein levels of Sult1a1 in the cells or mouse livers were measured by RT-qPCR and Western blotting, respectively. Sulfation activities of two Sult1a1 substrates (i.e., p-nitrophenol and galangin) were determined using mouse liver S9 fractions. Transcriptional regulation of Sult1a1 by Bmal1 was investigated using luciferase reporter, electrophoretic mobility shift (EMSA), and chromatin immunoprecipitation (ChIP) assays. We first showed that hepatic Sult1a1 was rhythmically expressed at both mRNA and protein levels (higher expressions during the night than the daytime). Consistently, the liver sulfation activities toward two Sult1a1 substrates were circadian time dependent with a higher activity at ZT14 than at ZT2. Furthermore, deletion of Bmal1 in mice blunted the circadian rhythmicity of hepatic Sult1a1 (with reduced expression levels). Likewise, Bmal1 positively regulated Sult1a1 expression in conventionally cultured Hepa-1c1c7 cells, and Bmal1 knockdown blunted expression rhythmicity of Sult1a1 in serum-shocked Hepa-1c1c7 cells. A combination of promoter analysis, EMSA and ChIP assays revealed that Bmal1 stimulated Sult1a1 transcription through its specific binding to the-571- to -554-bp region (an E-box element) in the promoter. In conclusion, Bmal1 activated the transcription of Sult1a1 and controlled circadian expression and activity of the enzyme.

Proliferative Glioblastoma Cancer Cells Exhibit Persisting Temporal Control of Metabolism and Display Differential Temporal Drug Susceptibility in Chemotherapy.

Even in immortalized cell lines, circadian clocks regulate physiological processes in a time-dependent manner, driving transcriptional and metabolic rhythms, the latter being able to persist without transcription. Circadian rhythm disruptions in modern life (shiftwork, jetlag, etc.) may lead to higher cancer risk. Here, we investigated whether the human glioblastoma T98G cells maintained quiescent or under proliferation keep a functional clock and whether cells display differential time responses to bortezomib chemotherapy. In arrested cultures, mRNAs for clock (Per1, Rev-erbα) and glycerophospholipid (GPL)-synthesizing enzyme genes, 32P-GPL labeling, and enzyme activities exhibited circadian rhythmicity; oscillations were also found in the redox state/peroxiredoxin oxidation. In proliferating cells, rhythms of gene expression were lost or their periodicity shortened whereas the redox and GPL metabolisms continued to fluctuate with a similar periodicity as under arrest. Cell viability significantly changed over time after bortezomib treatment; however, this rhythmicity and the redox cycles were altered after Bmal1 knock-down, indicating cross-talk between the transcriptional and the metabolic oscillators. An intrinsic metabolic clock continues to function in proliferating cells, controlling diverse metabolisms and highlighting differential states of tumor suitability for more efficient, time-dependent chemotherapy when the redox state is high and GPL metabolism low.

Bmal1 knockdown suppresses wake and increases immobility without altering orexin A, corticotrophin-releasing hormone, or glutamate decarboxylase.

To determine the effect of Bmal1 knockdown (KD) on sleep, activity, immobility, hypothalamic levels of orexin, corticotrophin-releasing hormone (CRH), and GABAergic glutamate decarboxylase (GAD). We used Bmal1 siRNA, or control siRNA intracerebroventricular (ICV) injection to knock down Bmal1 in C57BL/6 mice. Sleep polysomnography, wheel-running activity, and tail suspension test were performed. Polysomnographic (PSG) recordings in both groups were preceded by ICV injection made during both the light phase and the dark phase. We also measured brain orexin A and CRH using an ELISA and measured GAD using immunoblotting. Compared with control group, Bmal1 KD group had reduced wheel activity and increased immobility. Compared with control, the Bmal1 KD group had reduced wheel activity and increased immobility. During the first 24hours after treatment, we observed that control siRNA induced a much greater increase in sleep during the dark phase, which was associated with lower orexin levels. However, beginning 24hours after treatment, we observed an increase in sleep and a decrease in time spent awake during the dark phase in the Bmal1 KD group. These changes were not associated with changes in brain levels of orexin A, CRH, or GAD. Bmal1 KD led to reduced activity, increased immobility, and dramatic reduction in time spent awake as well as an increase in sleep during the dark phase. Early after injection, there was a slight change in sleep but brain levels of orexin, CRH, and GAD remain unchanged. Control siRNA also affected sleep associated with changes in orexin levels.

High dietary sodium causes dyssynchrony of the renal molecular clock in rats.

Speed JS, Hyndman KA, Roth K, Heimlich JB, Kasztan M, Fox BM, Johnston JG, Becker BK, Jin C, Gamble KL, Young ME, Pollock JS, Pollock DM. High dietary sodium causes dyssynchrony of the renal molecular clock in rats. Am J Physiol Renal Physiol 314: F89-F98, 2018. First published September 27, 2017; doi:10.1152/ajprenal.00028.2017.-Dyssynchrony of circadian rhythms is associated with various disorders, including cardiovascular and metabolic diseases. The cell autonomous molecular clock maintains circadian control; however, environmental factors that may cause circadian dyssynchrony either within or between organ systems are poorly understood. Our laboratory recently reported that the endothelin (ET-1) B (ETB) receptor functions to facilitate Na+ excretion in a time of day-dependent manner. Therefore, the present study was designed to determine whether high salt (HS) intake leads to circadian dyssynchrony within the kidney and whether the renal endothelin system contributes to control of the renal molecular clock. We observed that HS feeding led to region-specific alterations in circadian clock components within the kidney. For instance, HS caused a significant 5.5-h phase delay in the peak expression of Bmal1 and suppressed Cry1 and Per2 expression in the renal inner medulla, but not the renal cortex, of control rats. The phase delay in Bmal1 expression appears to be mediated by ET-1 because this phenomenon was not observed in the ETB-deficient rat. In cultured inner medullary collecting duct cells, ET-1 suppressed Bmal1 mRNA expression. Furthermore, Bmal1 knockdown in these cells reduced epithelial Na+ channel expression. These data reveal that HS feeding leads to intrarenal circadian dyssynchrony mediated, in part, through activation of ETB receptors within the renal inner medulla.

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 β (PDGFRβ), a protein necessary for maintaining BBB integrity. Knockdown of Bmal1 downregulated PDGFRβ 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.

Bmal1 interference impairs hormone synthesis and promotes apoptosis in porcine granulosa cells

In mammals, granulosa cell proliferation, differentiation, luteinization, apoptosis, and hormone synthesis are tightly related to oocyte maturation, follicular development and ovarian function. In current study, we investigated the role of the key circadian clock gene, brain and muscle arnt-like protein-1 (Bmal1), on porcine granulosa cell hormone secretion and apoptosis. The transcription levels of circadian clock genes, including Bmal1 and period circadian clock 2 (Per2), were detected by RT-qPCR. We found that the circadian clock genes exhibited rhythmic change and were further enhanced by dexamethasone synchronization in granulosa cells. Bmal1 knockdown reduced transcriptional levels of hormone receptor genes, including follicle stimulating hormone receptor (Fshr), luteinizing hormone/choriogonadotropin receptor (Lhcgr) and estrogen receptor 2 (Esr2), and decreased the mRNA and protein levels of cytochrome P450 family 11 subfamily A member 1 (Cyp11a1), cytochrome P450 family 19 subfamily A member 1 (Cyp19a1) and steroidogenic acute regulatory protein (Star), which are the key enzymes involved in hormone synthesis. Synthesis of progesterone and estradiol were also inhibited by Bmal1 siRNA treatment in granulosa cells. Moreover, flow cytometry analysis demonstrated suppressing Bmal1 promoted granulosa cells apoptosis. Western blot analysis showed that Bmal1 interference inactivated the PI3K/Akt/mTOR signaling pathway. In conclusion, Bmal1 plays a critical role in secretion of hormone and apoptosis of porcine granulosa cells via the PI3K/Akt/mTOR signaling pathway.

Clock Regulation of Metabolites Reveals Coupling between Transcription and Metabolism

The intricate connection between the circadian clock and metabolism remains poorly understood. We used high temporal resolution metabolite profiling to explore clock regulation of mouse liver and cell-autonomous metabolism. In liver, ∼50% of metabolites were circadian, with enrichment of nucleotide, amino acid, and methylation pathways. In U2 OS cells, 28% were circadian, including amino acids and NAD biosynthesis metabolites. Eighteen metabolites oscillated in both systems and a subset of these in primary hepatocytes. These 18 metabolites were enriched in methylation and amino acid pathways. To assess clock dependence of these rhythms, we used genetic perturbation. BMAL1 knockdown diminished metabolite rhythms, while CRY1 or CRY2 perturbation generally shortened or lengthened rhythms, respectively. Surprisingly, CRY1 knockdown induced 8hr rhythms in amino acid, methylation, and vitamin metabolites, decoupling metabolite from transcriptional rhythms, with potential impact on nutrient sensing invivo. These results provide the first comprehensive views of circadian liver and cell-autonomous metabolism.

Clock Gene Bmal1 Modulates Human Cartilage Gene Expression by Crosstalk With Sirt1.

The critical regulation of the peripheral circadian gene implicated in osteoarthritis (OA) has been recently recognized; however, the causative role and clinical potential of the peripheral circadian rhythm attributable to such effects remain elusive. The purpose of this study was to elucidate the role of a circadian gene Bmal1 in human cartilage and pathophysiology of osteoarthritis. In our present study, the mRNA and protein levels of circadian rhythm genes, including nicotinamide adenine dinucleotide oxidase (NAD(+)) and sirtuin 1 (Sirt1), in human knee articular cartilage were determined. In OA cartilage, the levels of both Bmal1 and NAD(+) decreased significantly, which resulted in the inhibition of nicotinamide phosphoribosyltransferase activity and Sirt1 expression. Furthermore, the knockdown of Bmal1 was sufficient to decrease the level of NAD(+) and aggravate OA-like gene expression changes under the stimulation of IL-1β. The overexpression of Bmal1 relieved the alteration induced by IL-1β, which was consistent with the effect of the inhibition of Rev-Erbα (known as NR1D1, nuclear receptor subfamily 1, group D). On the other hand, the transfection of Sirt1 small interfering RNA not only resulted in a reduction of the protein expression of Bmal1 and a moderate increase of period 2 (per2) and Rev-Erbα but also further exacerbated the survival of cells and the expression of cartilage matrix-degrading enzymes induced by IL-1β. Overexpression of Sirt1 restored the metabolic imbalance of chondrocytes caused by IL-1β. These observations suggest that Bmal1 is a key clock gene to involve in cartilage homeostasis mediated through sirt1 and that manipulating circadian rhythm gene expression implicates an innovative strategy to develop novel therapeutic agents against cartilage diseases.

CLOCK and BMAL1 Regulate Muscle Insulin Sensitivity via SIRT1 in Male Mice.

Circadian misalignment induces insulin resistance in both human and animal models, and skeletal muscle is the largest organ response to insulin. However, how circadian clock regulates muscle insulin sensitivity and the underlying molecular mechanisms are still largely unknown. Here we show circadian locomotor output cycles kaput (CLOCK) and brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein (BMAL)-1, two core circadian transcription factors, are down-regulated in insulin-resistant C2C12 myotubes and mouse skeletal muscle. Furthermore, insulin signaling is attenuated in the skeletal muscle of Clock(Δ19/Δ19) mice, and knockdown of CLOCK or BMAL1 by small interfering RNAs induces insulin resistance in C2C12 myotubes. Consistently, ectopic expression of CLOCK and BMAL1 improves insulin sensitivity in C2C12 myotubes. Moreover, CLOCK and BMAL1 regulate the expression of sirtuin 1 (SIRT1), an important regulator of insulin sensitivity, in C2C12 myotubes and mouse skeletal muscle, and two E-box elements in Sirt1 promoter are responsible for its CLOCK- and BMAL1-dependent transcription in muscle cells. Further studies show that CLOCK and BMAL1 regulate muscle insulin sensitivity through SIRT1. In addition, we find that BMAL1 and SIRT1 are decreased in the muscle of mice maintained in constant darkness, and resveratrol supplementation activates SIRT1 and improves insulin sensitivity. All these data demonstrate that CLOCK and BMAL1 regulate muscle insulin sensitivity via SIRT1, and activation of SIRT1 might be a potential valuable strategy to attenuate muscle insulin resistance related to circadian misalignment.

The chondrocyte-intrinsic circadian clock is disrupted in human osteoarthritis

ABSTRACTPeripheral clocks are essential for driving cell differentiation. In osteoarthritis, loss of the normal differentiated chondrocyte (cartilage cell) phenotype is causative of disease. We investigated whether clock gene expression differed in osteoarthritic compared to “healthy” chondrocytes and used RNAi to determine whether the differences observed could affect chondrocyte phenotype. Following serum shock, PER2 expression was significantly higher, whereas BMAL1 expression was significantly lower, in osteoarthritic chondrocytes. Knockdown of BMAL1 in “healthy” chondrocytes was associated with higher cell proliferation and MMP13 expression, features characteristic of the osteoarthritic chondrocyte phenotype. Chondrocyte-intrinsic clock disruption may be a critical early step in osteoarthritis development.

Regulation of transforming growth factor-beta1 (TGF-β1)-induced pro-fibrotic activities by circadian clock gene BMAL1.

BackgroundBMAL1 is a transcriptional activator of the molecular clock feedback network. Besides its role in generating circadian rhythms, it has also been shown to be involved in the modulation of cell proliferation, autophagy and cancer cell invasion. However, the role of BMAL1 in pulmonary fibrogenesis is still largely unknown. In this study, we investigated the crosstalk between BMAL1 and the signaling transduction and cellular activities of TGF-β1, a key player in lung fibrogenesis.MethodsLungs from wild type and TGF-β1-adenovirus-infected mice were harvested and homogenized for isolation of RNA and protein. RT-PCR and Western Blotting were employed to measure the expression level of clock genes and TGF-β1-induced downstream target genes. siRNA against human BMAL1 gene was transfected by using lipofectamine RNAiMAX to knockdown the endogenous BMAL1 in both lung epithelial cells and fibroblasts.ResultsOur results showed that TGF-β1 is able to up-regulate BMAL1 expression in both lung epithelial cells and normal lung fibroblasts. In animal models of pulmonary fibrosis, BMAL1 expression was also significantly higher in adenovirus-TGF-β1-infected mice than in the control group. Interestingly, BMAL1 was mostly found in a deacetylated form in the presence of TGF-β1. Importantly, siRNA–mediated knockdown of BMAL1 significantly attenuated the canonical TGF-β1 signaling pathway and altered TGF-β1-induced epithelial-mesenchymal transition and MMP9 production in lung epithelial cells. In addition, BMAL1 knockdown inhibited the fibroblast to myofibroblast differentiation of normal human lung fibroblasts.ConclusionsOur results indicate that activation of TGF-β1 promotes the transcriptional induction of BMAL1. Furthermore, BMAL1 is required for the TGF-β1-induced signaling transduction and pro-fibrotic activities in the lung.

The circadian clock gene Bmal1 acts as a potential anti-oncogene in pancreatic cancer by activating the p53 tumor suppressor pathway

Disruption of the circadian clock has been shown to be associated with tumor development. This study aimed to investigate the role of the core circadian gene Bmal1 in pancreatic cancer (PC). We first found that the levels of Bmal1 were downregulated in PC samples and were closely correlated with the clinicopathological features of patients. To dissect the underlying mechanism, we performed a RNA-seq assay followed by systematic gene function and pathway enrichment analyses. We detected an anti-apoptotic and pro-proliferative transcriptome profile after Bmal1 knockdown in PC cells. Further in vitro and in vivo studies confirmed that Bmal1 overexpression significantly inhibited cell proliferation and invasion and induced G2/M cell cycle arrest, whereas Bmal1 knockdown promoted PC growth, as demonstrated in Bmal1-manipulated AsPC-1 and BxPC-3 cell lines. Our mechanistic studies indicated that Bmal1 could directly bind to the p53 gene promoter and thereby transcriptionally activate the downstream tumor suppressor pathway in a p53-dependent manner. In sum, our findings suggest that Bmal1 acts as an anti-oncogene in PC and represents a potential biomarker for its diagnosis.

Circadian Modulation of 8-Oxoguanine DNA Damage Repair.

The DNA base excision repair pathway is the main system involved in the removal of oxidative damage to DNA such as 8-Oxoguanine (8-oxoG) primarily via the 8-Oxoguanine DNA glycosylase (OGG1). Our goal was to investigate whether the repair of 8-oxoG DNA damage follow a circadian rhythm. In a group of 15 healthy volunteers, we found a daily variation of Ogg1 expression and activity with higher levels in the morning compared to the evening hours. Consistent with this, we also found lower levels of 8-oxoG in morning hours compared to those in the evening hours. Lymphocytes exposed to oxidative damage to DNA at 8:00 AM display lower accumulation of 8-oxoG than lymphocytes exposed at 8:00 PM. Furthermore, altered levels of Ogg1 expression were also observed in a group of shift workers experiencing a deregulation of circadian clock genes compared to a control group. Moreover, BMAL1 knockdown fibroblasts with a deregulated molecular clock showed an abolishment of circadian variation of Ogg1 expression and an increase of OGG1 activity. Our results suggest that the circadian modulation of 8-oxoG DNA damage repair, according to a variation of Ogg1 expression, could render humans less susceptible to accumulate 8-oxoG DNA damage in the morning hours.

The Peripheral Clock Regulates Human Pigmentation

Although the regulation of pigmentation is well characterized, it remains unclear whether cell-autonomous controls regulate the cyclic on-off switching of pigmentation in the hair follicle (HF). As human HFs and epidermal melanocytes express clock genes and proteins, and given that core clock genes (PER1, BMAL1) modulate human HF cycling, we investigated whether peripheral clock activity influences human HF pigmentation. We found that silencing BMAL1 or PER1 in human HFs increased HF melanin content. Furthermore, tyrosinase expression and activity, as well as TYRP1 and TYRP2 mRNA levels, gp100 protein expression, melanocyte dendricity, and the number gp100+ HF melanocytes, were all significantly increased in BMAL1 and/or PER1-silenced HFs. BMAL1 or PER1 silencing also increased epidermal melanin content, gp100 protein expression, and tyrosinase activity in human skin. These effects reflect direct modulation of melanocytes, as BMAL1 and/or PER1 silencing in isolated melanocytes increased tyrosinase activity and TYRP1/2 expression. Mechanistically, BMAL1 knockdown reduces PER1 transcription, and PER1 silencing induces phosphorylation of the master regulator of melanogenesis, microphthalmia-associated transcription factor, thus stimulating human melanogenesis and melanocyte activity in situ and in vitro. Therefore, the molecular clock operates as a cell-autonomous modulator of human pigmentation and may be targeted for future therapeutic strategies.

Cell Type-Specific Functions of Period Genes Revealed by Novel Adipocyte and Hepatocyte Circadian Clock Models

In animals, circadian rhythms in physiology and behavior result from coherent rhythmic interactions between clocks in the brain and those throughout the body. Despite the many tissue specific clocks, most understanding of the molecular core clock mechanism comes from studies of the suprachiasmatic nuclei (SCN) of the hypothalamus and a few other cell types. Here we report establishment and genetic characterization of three cell-autonomous mouse clock models: 3T3 fibroblasts, 3T3-L1 adipocytes, and MMH-D3 hepatocytes. Each model is genetically tractable and has an integrated luciferase reporter that allows for longitudinal luminescence recording of rhythmic clock gene expression using an inexpensive off-the-shelf microplate reader. To test these cellular models, we generated a library of short hairpin RNAs (shRNAs) against a panel of known clock genes and evaluated their impact on circadian rhythms. Knockdown of Bmal1, Clock, Cry1, and Cry2 each resulted in similar phenotypes in all three models, consistent with previous studies. However, we observed cell type-specific knockdown phenotypes for the Period and Rev-Erb families of clock genes. In particular, Per1 and Per2, which have strong behavioral effects in knockout mice, appear to play different roles in regulating period length and amplitude in these peripheral systems. Per3, which has relatively modest behavioral effects in knockout mice, substantially affects period length in the three cellular models and in dissociated SCN neurons. In summary, this study establishes new cell-autonomous clock models that are of particular relevance to metabolism and suitable for screening for clock modifiers, and reveals previously under-appreciated cell type-specific functions of clock genes.

A Meeting of Two Chronobiological Systems: Circadian Proteins Period1 and BMAL1 Modulate the Human Hair Cycle Clock

The hair follicle (HF) is a continuously remodeled mini organ that cycles between growth (anagen), regression (catagen), and relative quiescence (telogen). As the anagen-to-catagen transformation of microdissected human scalp HFs can be observed in organ culture, it permits the study of the unknown controls of autonomous, rhythmic tissue remodeling of the HF, which intersects developmental, chronobiological, and growth-regulatory mechanisms. The hypothesis that the peripheral clock system is involved in hair cycle control, i.e., the anagen-to-catagen transformation, was tested. Here we show that in the absence of central clock influences, isolated, organ-cultured human HFs show circadian changes in the gene and protein expression of core clock genes (CLOCK, BMAL1, and Period1) and clock-controlled genes (c-Myc, NR1D1, and CDKN1A), with Period1 expression being hair cycle dependent. Knockdown of either BMAL1 or Period1 in human anagen HFs significantly prolonged anagen. This provides evidence that peripheral core clock genes modulate human HF cycling and are an integral component of the human hair cycle clock. Specifically, our study identifies BMAL1 and Period1 as potential therapeutic targets for modulating human hair growth.

REV-ERBα Inhibits the <i>PTGS2</i> Expression in Bovine Uterus Endometrium Stromal and Epithelial Cells Exposed to Ovarian Steroids

The nuclear receptor REV-ERBα (encoded by NR1D1) has a critical role in metabolism and physiology as well as circadian rhythm. Here, we investigated the possible contribution of clock genes including NR1D1 to the secretion of prostaglandin F2α (PGF2α) from bovine uterine stromal (USCs) and epithelial cells (UECs) by modulating the expression of PTGS2. The circadian oscillation of clock genes in the cells was weak compared with that reported in rodents, but the expression of BMAL1, PER1, and NR1D1 was changed temporally by treatment with ovarian steroids. Significant expression of clock genes including NR1D1 was detected in USCs exposed to progesterone. NR1D1 was also significantly expressed in UECs exposed to estradiol. The expression of PTGS2 was suppressed in USCs exposed to progesterone, while the expression was initially suppressed in UECs exposed to estradiol and then increased after long-term exposure to estradiol. BMAL1 knockdown with specific siRNA caused a significant decrease in the transcript levels of NR1D1 and PTGS2 in USCs, but not in UECs. The production of PGF2α also decreased in USCs after BMAL1 knockdown, while its level did not significantly change in UECs. The transcript level of PTGS2 was increased by treatment with the antagonist of REV-ERBα in both cell types, but the agonist was ineffective. In these two cell types treated with the agonist or antagonist, the PGF2α production coincided well with the PTGS2 expression. Collectively, these results indicate that REV-ERBα plays an inhibitory role in the expression of PTGS2 in both bovine USCs and UECs treated with ovarian steroids.