Cry1 Expression Research Articles

Early growth of Scots pine seedlings is affected by seed origin and light quality

Plants have evolved a suite of photoreceptors to perceive information from the surrounding light conditions. The aim of this study was to examine photomorphogenic effects of light quality on the growth of Scots pine (Pinus sylvestris L.) seedlings representing southern (60 °N) and northern (68 °N) origins in Finland. We measured the growth characteristics and the expression of light-responsive genes from seedlings grown under two LED light spectra: (1) Retarder (blue and red wavelengths in ratio 0.7) inducing compact growth, and (2) Booster (moderate in blue, green and far-red wavelengths, and high intensity of red light) promoting shoot elongation.The results show that root elongation, biomass, and branching were reduced under Retarder spectrum in the seedlings representing both origins, while inhibition in seed germination and shoot elongation was mainly detected in the seedlings of northern origin. The expression of ZTL and HY5 was related to Scots pine growth under both light spectra. Moreover, the expression of PHYN correlated with growth when exposed to Retarder, whereas CRY2 expression was associated with growth under Booster.Our data indicates that blue light and the deficiency of far-red light limit the growth of Scots pine seedlings and that northern populations are more sensitive to blue light than southern populations. Furthermore, the data analyses suggest that ZTL and HY5 broadly participate in the light-mediated growth regulation of Scots pine, whereas PHYN responses to direct sunlight and the role of CRY2 is in shade avoidance. Altogether, our study extends the knowledge of light quality and differential gene expression affecting the early growth of Scots pines representing different latitudinal origins.

SUN-LB063 Peripheral Clock System Circadian Imbalance in Cushing's Disease

Context: Glucocorticoid circulating levels in healthy individuals oscillate according to a circadian rhythm, influenced by the molecular system of clock genes. In Cushing's syndrome, there is an altered cortisol daily rhythm, which can be associated to disruption of clock genes rhythmic expression. Objective: Evaluate the expression of clock genes (CLOCK, BMAL1, CRY1, CRY2, PER1, PER2, PER3) in leukocytes of healthy individuals and in patients with Cushing's disease (CD). Design and Participants: Case-control study with female Cushing’s disease patients (n=12) and controls (n=13). Main Outcome Measures: Participants underwent salivary cortisol measurement at 0900h and 2300h. Peripheral blood samples were obtained at 0900h, 1300h, 1700h and 2300h for assessing clock genes expression by qPCR. Gene expression profiles were evaluated by Cosinor analysis method. Results: In healthy controls, there was a circadian variation of the mRNA expression of CLOCK (p<0.01), BMAL1 (p=0.02), CRY1 (p=0.02), PER2 (p<0.01), and PER3 (p<0.01), while no circadian rhythm of PER1, and CRY2 was observed. The expression of PER2 and PER3 genes in control leukocytes showed a late afternoon acrophase at 17.5h (14.9-20.1) and 19.5h (17.9-21.0), respectively, similar to CLOCK gene acrophase at 20.2h (19.0-21.4) while CRY1 showed night acrophase at 22.4h (20.1-24.6) as well as BMAL1 acrophase at 23.6h (21.7-0.49), respectively. In Cushing´s Disease patients, there was a loss of the pattern of clock genes circadian rhythmicity, in concomitance with the loss of cortisol circadian rhythm. One exception was CRY2, which presented a circadian variation (p<0.01), with acrophase during the dark phase at 0.5h (22.5-2.6). Conclusions: Our data suggest that hypercortisolism leads to dysregulation of circadian clock genes expression in Cushing's disease. CRY2 higher expression at night outlines its putative role in the cortisol circadian rhythm disruption. Although the role of clock genes dysregulation in the pathogenesis of Cushing's disease metabolic derangements is still not completely established, it is possible that it synergistically contributes to hypercortisolism to the severity of metabolic features of Cushing's disease. Unless otherwise noted, all abstracts presented at ENDO are embargoed until the date and time of presentation. For oral presentations, the abstracts are embargoed until the session begins. s presented at a news conference are embargoed until the date and time of the news conference. The Endocrine Society reserves the right to lift the embargo on specific abstracts that are selected for promotion prior to or during ENDO.

Effects of 50 Hz magnetic fields on circadian rhythm control in mice.

Artificial light and power frequency magnetic fields are ubiquitous in the built environment. Light is a potent zeitgeber but it is unclear whether power frequency magnetic fields can influence circadian rhythm control. To study this possibility, 8–12‐week‐old male C57BL/6J mice were exposed for 30 min starting at zeitgeber time 14 (ZT14, 2 h into the dark period of the day) to 50 Hz magnetic fields at 580 μT using a pair of Helmholtz coils and/or a blue LED light at 700 lux or neither. Our experiments revealed an acute adrenal response to blue light, in terms of increased adrenal per1 gene expression, increased serum corticosterone levels, increased time spent sleeping, and decreased locomotor activity (in all cases, P < 0.0001) compared to an unexposed control group. There appeared to be no modulating effect of the magnetic fields on the response to light, and there was also no effect of the magnetic fields alone (in both cases, P > 0.05) except for a decrease in locomotor activity (P < 0.03). Gene expression of the cryptochromes cry1 and cry2 in the adrenals, liver, and hippocampus was also not affected by exposures (in all cases, P > 0.05). In conclusion, these results suggest that 50 Hz magnetic fields do not significantly affect the acute light response to a degree that can be detected in the adrenal response. Bioelectromagnetics. 2019;9999:XX–XX. © 2019 Bioelectromagnetics Society.

Characterization of gene expression in adrenal glands of mice lacking the circadian clock protein PER1

The circadian clock regulates a variety of physiological responses throughout a 24‐hour period. At a molecular level, the clock is governed by four core transcription factors, including PER1 and CRY, which are found in every cell in the human body. All reported circadian clock gene KO mice have a blood pressure (BP) phenotype. Aldosterone, a mineralocorticoid hormone and product of the renin‐angiotensin‐aldosterone system (RAAS), is secreted by the adrenal cortex and leads to increased sodium reabsorption by the kidneys in response to low BP or low blood volume. Previously we reported that 129/sv mice lacking the circadian clock gene Per1 had reduced BP and reduced plasma aldosterone levels associated with decreased expression of HSD3B1, a key enzyme in the steroid hormone synthesis pathway. Conversely, we found that male C57BL/6 Per1 KO mice have higher BP than control mice and develop non‐dipping hypertension (a lack of dip in BP during the rest period) on a high salt diet plus mineralocorticoid treatment. Non‐dipping hypertension has been associated with an increased risk for cardiovascular disease in humans. Interestingly, female Per1 KO mice do not develop non‐dipping hypertension on a high salt diet plus mineralocorticoid treatment. The goal of this study was to determine if expression of genes involved in the aldosterone synthesis pathway is altered in C57BL/6 Per1 KO mice. These deviances could explain the BP phenotype observed at the physiological level in male mice. We obtained adrenal glands from male and female WT and Per1 KO mice at noon (midpoint of the mouse rest phase), the same time at which serum was collected for aldosterone measurements. RNA was isolated from these tissues, converted to cDNA, and mRNA levels of genes involved in circadian rhythm and aldosterone synthesis were measured by quantitative real time PCR (qPCR). Preliminary data show that Cry1 is significantly down‐regulated in female Per1 KO mice by about 50% versus WT (n=5/6 per group, p&lt;0.05). Expression of aldosterone synthesis genes CYP11B and HSD3B1 did not appear to be significantly altered in the adrenal tissue of male or female Per1 KO mice compared to WT. In contrast to what was observed with 129/sv Per1 KO, plasma aldosterone levels did not differ between C57BL/6 Per1 KO vs. WT. The observed difference in Cry1 expression suggests that RAAS and the circadian clock may work together in the maintenance of homeostatic BP. In the future we expect to perform similar experiments using male and female WT and Per1 KO mice adrenal tissue collected at a midnight time point in order to further investigate the communication occurring at the molecular level between RAAS and the circadian clock.Support or Funding InformationNIH/NIDDK 1R01DK109570; The Gatorade Trust through the UF Department of MedicineThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Impact of UV-B radiation on the photosystem II activity, pro-/antioxidant balance and expression of light-activated genes in Arabidopsis thaliana hy4 mutants grown under light of different spectral composition

The effect of UV-B irradiation on the photosystem II (PSII) activity, the content of photosynthetic and UV-absorbing pigments (UAPs), activity of antioxidant enzymes such as catalase (CAT) and peroxidase (POD), as well as H2O2 content in 25-day-old wild type (WT) and the cryptochrome 1 (Cry1) mutant hy4 of Arabidopsis thaliana Col-0 plants was studied. In addition, expression of photoreceptor genes Cry1, Cry2 and UVR8, photomorphogenetic gene COP1 and transcription factors genes HY5, HYH, the gene of chlorophyll-binding protein of the PSII CAB1 as well as the flavonoid biosynthesis genes CHS, PAL and thylakoid ascorbate peroxidase gene tAPX was examined. It has been shown that UV-B leads to a decrease in the photochemical activity of PSII (FV/FM) and the PSII performance index (PIABS) of WT plants grown on white (WL) and red (RL) light and also hy4 mutants grown on WL, RL and blue light (BL). In plants grown on BL and WL, the decrease in the PSII photochemical activity was significantly greater in hy4 compared to WT. The PSII of WT plants grown in BL was resistant to UV-B. The UAPs content of hy4 grown on BL and WL was lower than that in WT. The POD and CAT activities of WT grown in BL were significantly higher than in the mutant. In WT and hy4 plants grown in RL, a noticeable difference in these enzymes activity was not found. In both types of plants grown in BL and RL, the expression of photomorphogenetic genes HYH, HY5 markedly increased after UV-B treatment but the expression of the UV-B photoreceptor gene UVR8 was reduced in hy4 grown in BL and RL. It is assumed that reduced resistance of PSII in hy4 plants grown in BL and WL can be associated with low UAPs content as well as lowered POD and CAT activities. In addition, we suggest the lowered expression of UVR8 and COP1 genes caused by Cry1 deficiency leads to a shift of balance of oxidants and antioxidants towards oxidants.

Night interruption light quality changes morphogenesis, flowering, and gene expression in Dendranthema grandiflorum

We investigated the effects of the quality of light used for interrupting the night period, termed the night interruption light (NIL), on morphogenesis, flowering, and the expression of photoperiodic genes in chrysanthemum (Dendranthema grandiflorum) cultivar ‘Gaya Yellow’, a qualitative short-day (SD) plant. Plants were raised in a closed-type plant factory under white (W) light-emitting diodes (LEDs) providing a light intensity of 180 μmol m−2 s−1 photosynthetic photon flux density, under a condition of long-day (LD, 16 h light/8 h dark), short-day (SD, 10 h light/14 h dark), or SD with a 4-h night interruption (NI) provided by 10 μmol m−2 s−1 PPF green (NI-G), blue (NI-B), red (NI-R), far-red (NI-Fr), or W (NI-W) LEDs. Plants grown in the LD condition were the tallest. The SD, NI-B, and NI-Fr conditions induced flowering. Phytochrome A (phyA) and cryptochrome 1 (cry1) were expressed at high levels in plants in NI-B, NI-Fr, and SD conditions. These results suggest that the NIL quality has significant implications on morphogenesis, flowering, and the expression of photoperiodic genes. Flowering was positively affected by the expression of phyA, cry1, and FLOWERING LOCUS T (FT), whereas it was negatively affected by the expression of phyB and anti-florigenic FT/TFL1 (AFT).

Impact of adult attention deficit hyperactivity disorder and medication status on sleep/wake behavior and molecular circadian rhythms.

Attention deficit hyperactivity disorder (ADHD) is a common neuropsychiatric condition that has been strongly associated with changes in sleep and circadian rhythms. Circadian rhythms are near 24-h cycles that are primarily generated by an endogenous circadian timekeeping system, encoded at the molecular level by a panel of clock genes. Stimulant and non-stimulant medication used in the management of ADHD has been shown to potentially impact on circadian processes and their behavioral outputs. In the current study, we have analyzed circadian rhythms in daily activity and sleep, and the circadian gene expression in a cohort of healthy controls (N = 22), ADHD participants not using ADHD-medication (N = 17), and participants with ADHD and current use of ADHD medication (N = 17). Rhythms of sleep/wake behavior were assessed via wrist-worn actigraphy, whilst rhythms of circadian gene expression were assessed ex-vivo in primary human-derived dermal fibroblast cultures. Behavioral data indicate that patients with ADHD using ADHD-medication have lower relative amplitudes of diurnal activity rhythms, lower sleep efficiency, more nocturnal activity but not more nocturnal wakenings than both controls and ADHD participants without medication. At the molecular level, there were alterations in the expression of PER2 and CRY1 between ADHD individuals with no medication compared to medicated ADHD patients or controls, whilst CLOCK expression was altered in patients with ADHD and current medication. Analysis of fibroblasts transfected with a BMAL1:luc reporter showed changes in the timing of the peak expression across the three groups. Taken together, these data support the contention that both ADHD and medication status impact on circadian processes.

Turning on the clock

Chronobiology Every day we wake and sleep under the control of the brain's master clock or suprachiasmatic nucleus (SCN). If the SCN's key cryptochrome (Cry) protein components are globally deleted, the clock is disabled. Maywood et al. have developed molecular tools to study the clock's complex transcriptional and translational feedback loops. A translational switch was designed, by expanding the genetic code, to reversibly control translation of Cry1 in the SCN. This allows an orthogonal aminoacyl–transfer RNA (tRNA) synthetase–tRNA pair to be targeted to the SCN by adeno-associated virus vectors. Cry1 is then only expressed in the SCN when a noncanonical amino acid is supplied. By using this tool, Cry1 expression can be induced in Cry-null mice to initiate circadian behavior in the otherwise arrhythmic mice. Proc. Natl. Acad. Sci. U.S.A. 115 , E12388 (2018).

Cardiomyocyte transcription is controlled by combined MR and circadian clock signalling.

We previously identified a critical pathogenic role for MR activation in cardiomyocytes that included a potential interaction between the MR and the molecular circadian clock. While glucocorticoid regulation of the circadian clock is undisputed, MR interactions with circadian clock signalling are limited. We hypothesised that the MR influences cardiac circadian clock signalling, and vice versa. 10nM aldosterone or corticosterone regulated CRY 1, PER1, PER2 and ReverbA (NR1D1) gene expression patterns in H9c2 cells over 24hr. MR-dependent regulation of circadian gene promoters containing GREs and E-box sequences was established for CLOCK, Bmal, CRY 1 and CRY2, PER1 and PER2 and transcriptional activators CLOCK and Bmal modulated MR-dependent transcription of a subset of these promoters. We also demonstrated differential regulation of MR target gene expression in hearts of mice 4hr after administration of aldosterone at 8AM versus 8PM. Our data support combined MR regulation of a subset of circadian genes and that endogenous circadian transcription factors CLOCK and Bmal modulate this response. This unsuspected relationship links MR in the heart to circadian rhythmicity at the molecular level and has important implications for the biology of MR signalling in response to aldosterone as well as cortisol. These data are consistent with MR signalling in the brain where, like the heart, it preferentially responds to cortisol. Given the undisputed requirement for diurnal cortisol release in the entrainment of peripheral clocks, the present study highlights the MR as an important mechanism for transducing the circadian actions of cortisol in addition to the GR in the heart.

Translational switching of Cry1 protein expression confers reversible control of circadian behavior in arrhythmic Cry-deficient mice

The suprachiasmatic nucleus (SCN) is the principal circadian clock of mammals, coordinating daily rhythms of physiology and behavior. Circadian timing pivots around self-sustaining transcriptional-translational negative feedback loops (TTFLs), whereby CLOCK and BMAL1 drive the expression of the negative regulators Period and Cryptochrome (Cry). Global deletion of Cry1 and Cry2 disables the TTFL, resulting in arrhythmicity in downstream behaviors. We used this highly tractable biology to further develop genetic code expansion (GCE) as a translational switch to achieve reversible control of a biologically relevant protein, Cry1, in the SCN. This employed an orthogonal aminoacyl-tRNA synthetase/tRNACUA pair delivered to the SCN by adeno-associated virus (AAV) vectors, allowing incorporation of a noncanonical amino acid (ncAA) into AAV-encoded Cry1 protein carrying an ectopic amber stop codon. Thus, translational readthrough and Cry1 expression were conditional on the supply of ncAA via culture medium or drinking water and were restricted to neurons by synapsin-dependent expression of aminoacyl tRNA-synthetase. Activation of Cry1 translation by ncAA in neurons of arrhythmic Cry-null SCN slices immediately and dose-dependently initiated TTFL circadian rhythms, which dissipated rapidly after ncAA withdrawal. Moreover, genetic activation of the TTFL in SCN neurons rapidly and reversibly initiated circadian behavior in otherwise arrhythmic Cry-null mice, with rhythm amplitude being determined by the number of transduced SCN neurons. Thus, Cry1 does not specify the development of circadian circuitry and competence but is essential for its labile and rapidly reversible activation. This demonstrates reversible control of mammalian behavior using GCE-based translational switching, a method of potentially broad neurobiological interest.

Intake of an Obesogenic Cafeteria Diet Affects Body Weight, Feeding Behavior, and Glucose and Lipid Metabolism in a Photoperiod-Dependent Manner in F344 Rats.

We previously demonstrated that chronic exposure to different photoperiods induced marked variations in several glucose and lipid metabolism-related parameters in normoweight Fischer 344 (F344) rats. Here, we examined the effects of the combination of an obesogenic cafeteria diet (CAF) and the chronic exposure to three different day lengths (L12, 12 h light/day; L18, 18 h light/day; and L6, 6 h light/day) in this rat strain. Although no changes were observed during the first 4 weeks of adaptation to the different photoperiods in which animals were fed a standard diet, the addition of the CAF for the subsequent 7 weeks triggered profound physiologic and metabolic alterations in a photoperiod-dependent manner. Compared with L12 rats, both L6 and L18 animals displayed lower body weight gain and cumulative food intake in addition to decreased energy expenditure and locomotor activity. These changes were accompanied by differences in food preferences and by a sharp upregulation of the orexigenic genes Npy and Ghsr in the hypothalamus, which could be understood as a homeostatic mechanism for increasing food consumption to restore body weight control. L18 rats also exhibited higher glycemia than the L6 group, which could be partly attributed to the decreased pAkt2 levels in the soleus muscle and the downregulation of Irs1 mRNA levels in the gastrocnemius muscle. Furthermore, L6 animals displayed lower whole-body lipid utilization than the L18 group, which could be related to the lower lipid intake and to the decreased mRNA levels of the fatty acid transporter gene Fatp1 observed in the soleus muscle. The profound differences observed between L6 and L18 rats could be related with hepatic and muscular changes in the expression of circadian rhythm-related genes Cry1, Bmal1, Per2, and Nr1d1. Although further research is needed to elucidate the pathophysiologic relevance of these findings, our study could contribute to emphasize the impact of the consumption of highly palatable and energy dense foods regularly consumed by humans on the physiological and metabolic adaptations that occur in response to seasonal variations of day length, especially in diseases associated with changes in food intake and preference such as obesity and seasonal affective disorder.

The adrenal gland circadian clock exhibits a distinct phase advance in spontaneously hypertensive rats.

The circadian clock influences a multitude of cellular and biological processes, including blood pressure control. Spontaneously hypertensive rats (SHR) exhibit aberrant circadian rhythms affecting cardiovascular parameters, and they also have abnormal clock gene expression profiles in several organs. Given the important role of the adrenal gland in orchestrating circadian oscillations, we investigated the adrenal gland circadian clock in SHR and control Wistar-Kyoto rats maintained under a 12-hour light-dark cycle. Adrenal glands, livers, and serum samples were collected every 4 h and mRNA was extracted for analysis of clock gene expression. Serum levels of corticosterone and aldosterone were also analyzed. Overall, the circadian profiles of Bmal1, Per2, Per3, Cry1, RevErba, Revervb, and Dbp gene expression in SHR adrenal glands were phase-advanced relative to controls. The expression profile of StAR (a representative gene under circadian control in the adrenal gland), as well as the circadian rhythms of serum concentrations of corticosteroid and aldosterone were also phase advanced. E4bp4 gene expression was significantly higher during the dark period, yet the expression of its transcriptional activator, Rora, was significantly lower throughout the 24 h period in SHR adrenal glands than in controls. This paradoxical high E4bp4 gene expression was, however, not observed in the liver. In addition, Per1, Per2, Per3, Reverba, and Reverbb mRNA tended to be lower in SHR adrenal glands than in controls. Thus, we conclude that SHR possess an abnormal adrenal circadian clock, which may affect the transcriptional regulation of clock-controlled genes, and steroid hormone secretion by the adrenal gland.

Cryptochrome 1 promotes osteogenic differentiation of human osteoblastic cells via Wnt/β-Catenin signaling

AimsThe exact mechanism underlying osteoblast differentiation and proliferation remains to be further elucidated. The circadian clock has been universally acknowledged controls behavioral activities and biological process in mammals. Cryptochrome 1 (Cry1), one of the core circadian genes, is associated with bone metabolism. However, the exact role and potential mechanism of Cry1 in regulating osteogenesis are still unclear. Main methodsWestern blotting and qRT-PCR were applied to detect Cry1 expression levels, molecules in osteogenesis related signaling pathways and osteogenic transcriptional markers. The ALP staining and Alizarin red S staining were performed to weigh osteogenic state, while CCK8 assay was used to detect cell growth rates. Osteogenic capability of osteoblasts was determined using an ectopic bone formation assay. Key findingsCry1 was upregulated in the process of osteoblast differentiation, along with osteogenic transcriptional factors. Then, Cry1 upregulation and knockdown cell lines were established and we found Cry1 overexpression promoted osteogenesis and proliferation of osteoblasts both in vitro and in vivo. Besides, the canonical Wnt/β-Catenin signaling was increasingly activated by Cry1 overexpression, whereas inhibition of β-Catenin restrained enhanced osteogenic capability of Cry1 upregulated osteoblasts. SignificanceIn conclusion, these results suggest that Cry1 promotes osteogenic differentiation of human osteoblasts through the canonical Wnt/β-Catenin signaling.

Rhythms of Core Clock Genes and Spontaneous Locomotor Activity in Post-Status Epilepticus Model of Mesial Temporal Lobe Epilepsy.

The interaction of Mesial Temporal Lobe Epilepsy (mTLE) with the circadian system control is apparent from an oscillatory pattern of limbic seizures, daytime's effect on seizure onset and the efficacy of antiepileptic drugs. Moreover, seizures per se can interfere with the biological rhythm output, including circadian oscillation of body temperature, locomotor activity, EEG pattern as well as the transcriptome. However, the molecular mechanisms underlying this cross-talk remain unclear. In this study, we systematically evaluated the temporal expression of seven core circadian transcripts (Bmal1, Clock, Cry1, Cry2, Per1, Per2, and Per3) and the spontaneous locomotor activity (SLA) in post-status epilepticus (SE) model of mTLE. Twenty-four hour oscillating SLA remained intact in post-SE groups although the circadian phase and the amount and intensity of activity were changed in early post-SE and epileptic phases. The acrophase of the SLA rhythm was delayed during epileptogenesis, a fragmented 24 h rhythmicity and extended active phase length appeared in the epileptic phase. The temporal expression of circadian transcripts Bmal1, Cry1, Cry2, Per1, Per2, and Per3 was also substantially altered. The oscillatory expression of Bmal1 was maintained in rats imperiled to SE, but with lower amplitude (A = 0.2) and an advanced acrophase in the epileptic phase. The diurnal rhythm of Cry1 and Cry2 was absent in the early post-SE but was recovered in the epileptic phase. Per1 and Per2 rhythmic expression were disrupted in post-SE groups while Per3 presented an arrhythmic profile in the epileptic phase, only. The expression of Clock did not display rhythmic pattern in any condition. These oscillating patterns of core clock genes may contribute to hippocampal 24 h cycling and, consequently to seizure periodicity. Furthermore, by using a pool of samples collected at 6 different Zeitgeber Times (ZT), we found that all clock transcripts were significantly dysregulated after SE induction, except Per3 and Per2. Collectively, altered SLA rhythm in early post-SE and epileptic phases implies a possible role for seizure as a nonphotic cue, which is likely linked to activation of hippocampal–accumbens pathway. On the other hand, altered temporal expression of the clock genes after SE suggests their involvement in the MTLE.

Chronic treatment with dexamethasone alters clock gene expression and melatonin synthesis in rat pineal gland at night.

BackgroundMelatonin is a neuroendocrine hormone that regulates many functions involving energy metabolism and behavior in mammals throughout the light/dark cycle. It is considered an output signal of the central circadian clock, located in the suprachiasmatic nucleus of the hypothalamus. Melatonin synthesis can be influenced by other hormones, such as insulin and glucocorticoids in pathological conditions or during stress. Furthermore, glucocorticoids appear to modulate circadian clock genes in peripheral tissues and are associated with the onset of metabolic diseases. In the pineal gland, the modulation of melatonin synthesis by clock genes has already been demonstrated. However, few studies have shown the effects of glucocorticoids on clock genes expression in the pineal gland.ResultsWe verified that rats treated with dexamethasone (2 mg/kg body weight, intraperitoneal) for 10 consecutive days, showed hyperglycemia and pronounced hyperinsulinemia during the dark phase. Insulin sensitivity, glucose tolerance, melatonin synthesis, and enzymatic activity of arylalkylamine N-acetyltransferase, the key enzyme of melatonin synthesis, were reduced. Furthermore, we observed an increase in the expression of Bmal1, Per1, Per2, Cry1, and Cry2 in pineal glands of rats treated with dexamethasone.ConclusionThese results show that chronic treatment with dexamethasone can modulate both melatonin synthesis and circadian clock expression during the dark phase.

Altered circadian genes expression in breast cancer tissue according to the clinical characteristics.

Breast cancer has a multifactorial etiology. One of the supposed and novel mechanisms is an alteration of circadian gene expression. Circadian genes play a crucial role in many physiological processes. These processes, such as genomic stability, DNA repair mechanism and apoptosis, are frequently disrupted in breast tumors. To assess the significance of circadian gene expression in breast cancer, we carried out an analysis of CLOCK, BMAL1, NPAS2, PER1, PER2, PER3 and CRY1, CRY2, TIMELESS, CSNK1E expression by the use of the quantitative Real-Time PCR technique in tumor tissue and non-tumor adjacent normal tissue sampled from 107 women with a newly diagnosed disease. The obtained data were compared to the clinical and histopathological features. PER1, PER2, PER3, CRY2 were found to be significantly down-expressed, while CLOCK, TIMELESS were over-expressed in the studied tumor samples compared to the non-tumor samples. Only gene expression of CRY1 was significantly down-regulated with progression according to the TNM classification. We found significantly decreased expression of CRY2, PER1, PER2 genes in the ER/PR negative breast tumors compared to the ER/PR positive tumors. Additionally, expression of CRY2, NPAS2 genes had a decreased level in the poorly differentiated tumors in comparison with the well and moderately differentiated ones. Our results indicate that circadian gene expression is altered in breast cancer tissue, which confirms previous observations from various animal and in vitro studies.

Electroconvulsive Seizure Alters the Expression and Daily Oscillation of Circadian Genes in the Rat Frontal Cortex.

ObjectiveElectroconvulsive therapy (ECT) is the most effective treatment for mood disorders. Accumulating evidence has suggested the important role of circadian genes in mood disorders. However, the effects of ECT on circadian genes have not been systemically investigated. MethodsWe examined the expression and daily oscillation of major circadian genes in the rat frontal cortex after electroconvulsive seizure (ECS). ResultsFirstly, mRNA and protein level were investigated at 24 hr after single ECS (E1X) and repeated ECS treatements for 10 days (E10X), which showed more remarkable changes after E10X than E1X. mRNA expression of Rorα, Bmal1, Clock, Per1, and Cry1 was decreased, while Rev-erbα expression was increased at 24 hr after E10X compared to sham. The proteins showed similar pattern of changes. Next, the effects on oscillation and rhythm properties (mesor, amplitude, and acrophase) were examined, which also showed more prominent changes after E10X than E1X. After E10X, mesor of Rorα, Bmal1, and Cry1 was reduced, and that of Rev-erbα was increased. Five genes, Rev-erbα, Bmal1, Per1, Per2, and Cry2, showed earlier acrophase after E10X. Conclusion The findings suggest that repeated ECS induces reduced expression and phase advance of major circadian genes in the in vivo rat frontal cortex.

Cryptochrome 2 (CRY2) Suppresses Proliferation and Migration and Regulates Clock Gene Network in Osteosarcoma Cells.

BackgroundCircadian disruption is a potential cancer risk factor in humans. However, the role of the clock gene, cryptochrome 2 (CRY2), in osteosarcoma (OS) is still not clear.Material/MethodsTo evaluate the potential role of CRY2 in HOS osteosarcoma cells, CRY2-silenced cell lines were established. Furthermore, we investigated the effect of CRY2 knockdown on HOS cells by CCK-8, colony formation, migration assay, and flow cytometry, in vitro.ResultsCRY2 knockdown promoted HOS OS cell proliferation and migration. We used a cell cycle assay to show that CRY2 knockdown increased the S phase cell population and reduced the G1 phase cell population. Western blot analyses showed that CRY2 knockdown decreased P53 expression and increased expression of c-myc and cyclin D1. Simultaneously, CRY2 knockdown increased the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2, but did not change the phosphorylation of c-Jun N terminal kinase (JNK) and P38. CRY2 knockdown also increased the expression of matrix metalloproteinase (MMP)-2 and β-catenin, and increased OS cell proliferation and migration by inducing cell cycle progression and promoting mitogen-activated protein kinase (MAPK) and Wnt/β-catenin signaling pathways. Although it has previously been unclear whether the expression of CRY2 affects the expression of other clock genes in the clock gene network, our results show that knockdown of CRY2 significantly increased the mRNA expression of CRY1, Period (PER) 1, PER2, BMAL1, and CLOCK.ConclusionsOur results suggest that CRY2 may be an anti-oncogene in OS, whose functions involve both downstream genes and other circadian genes.

Melatonin Attenuates Dysregulation of the Circadian Clock Pathway in Mice With CCl4-Induced Fibrosis and Human Hepatic Stellate Cells.

Dysregulation of the circadian clock machinery is a critical mechanism in the pathogenesis of fibrosis. This study aimed to investigate whether the antifibrotic effect of melatonin is associated with attenuation of circadian clock pathway disturbances in mice treated with carbon tetrachloride (CCl4) and in human hepatic stellate cells line LX2. Mice received CCl4 5 μL/g body weight i.p. twice a week for 4 or 6 weeks. Melatonin was given at 5 or 10 mg/kg/day i.p., beginning 2 weeks after the start of CCl4 administration. Treatment with CCl4 resulted in fibrosis evidenced by the staining of α-smooth muscle actin (α-SMA) positive cells and a significant decrease of peroxisome proliferator-activated receptor (PPARα) expression. CCl4 led to a lower expression of brain and muscle Arnt-like protein 1 (BMAL1), circadian locomotor output cycles kaput (CLOCK), period 1–3 (PER1, 2, and 3), cryptochrome 1 and 2 (CRY1 and 2) and the retinoic acid receptor-related orphan receptor (RORα). The expression of the nuclear receptor REV-ERBα showed a significant increase. Melatonin significantly prevented all these changes. We also found that melatonin (100 or 500 μM) potentiated the inhibitory effect of REV-ERB ligand SR9009 on α-SMA and collagen1 expression and increased the expression of PPARα in LX2 cells. Analysis of circadian clock machinery revealed that melatonin or SR9009 exposure upregulated BMAL1, CLOCK, PER2, CRY1, and RORα expression, with a higher effect of combined treatment. Findings from this study give new insight into molecular pathways accounting for the protective effect of melatonin in liver fibrosis.

Reciprocal Regulation among miR-181d/CRY2/FBXL3/c-myc Signaling Axis Modulates Metabolism in Colorectal Cancer

Patients with CRC (colorectal cancer) usually have a poor prognosis and the cure rate of CRC remained unsatisfied due to unfavorable curative effect. It is well known that microRNAs (miRNAs) and energy metabolism have pivotal roles in CRC progression. In a recent article in Cell Death & Disease by Xiaofeng Guo. et al. 2017, we have reported an oncogenic role of miR-181d in CRC by promoting glycolysis, and its underlying molecular mechanism about a new feedback loop among miR-181d/CRY2/FBXL3/c-myc signaling axis. Among these, we have identified the level of miR-181d was upregulated in CRC and the inhibition of miR-181d decreased glycolysis in CRC cells. We also found that c-myc played a central role in regulating cell glycolysis, which is required for the metabolic shift induced by miR-181d. Besides, we have demonstrated FBXL3 and CRY2 were direct targets of miR-181d and c-myc promoted miR-181d upregulation while inhibiting the expression of CRY2 and FBXL3 in CRC cells. The data from our recent article strongly suggest a new light onto the oncogenic function of the miR-181d in CRC. Furthermore, these findings represent a novel potential approach for silencing miR-181d/c-myc signaling pathway in CRC treatment.