Identification of BMAL1-Regulated circadian genes in mouse liver and their potential association with hepatocellular carcinoma: Gys2 and Upp2 as promising candidates.

Identification and functional analysis of key genes regulated by the circadian clock system will provide a comprehensive understanding of the underlying mechanisms through which circadian clock disruption impairs the health of living organisms. The initial phase involved bioinformatics analysis, drawing insights from three RNA-seq datasets (GSE184303, GSE114400, and GSE199061) derived from wild-type mouse liver tissues, which encompassed six distinct time points across a day. As expected, 536 overlapping genes exhibiting rhythmic expression patterns were identified. By intersecting these genes with differentially expressed genes (DEGs) originating from liver RNA-seq data at two representative time points (circadian time, CT: CT2 and CT14) in global Bmal1 knockout mice (Bmal1-/-), hepatocyte-specific Bmal1 knockout mice (L-Bmal1-/-), and their corresponding control groups, 80 genes potentially regulated by BMAL1 (referred to as BMAL1-regulated genes, BRGs) were identified. These genes were significantly enriched in glycolipid metabolism, immune response, and tumorigenesis pathways. Eight BRGs (Nr1d1, Cry1, Gys2, Homer2, Serpina6, Slc2a2, Nmrk1, and Upp2) were selected to validate their expression patterns in both control and L-Bmal1-/- mice livers over 24 h. Real-time quantitative polymerase chain reaction results demonstrated a comprehensive loss of rhythmic expression patterns in the eight selected BRGs in L-Bmal1-/- mice, in contrast to the discernible rhythmic patterns observed in the livers of control mice. Additionally, significant reductions in the expression levels of these selected BRGs, excluding Cry1, were also observed in L-Bmal1-/- mice livers. Chromatin immunoprecipitation (ChIP)-seq (GSE13505 and GSE39860) and JASPAR analyses validated the rhythmic binding of BMAL1 to the promoter and intron regions of these genes. Moreover, the progression of conditions, from basic steatosis to non-alcoholic fatty liver disease, and eventual malignancy, demonstrated a continuous gradual decline in Bmal1 transcripts in the human liver. Combining the aforementioned BRGs with DEGs derived from human liver cancer datasets identified Gys2 and Upp2 as potential node genes bridging the circadian clock system and hepatocellular carcinoma (HCC). In addition, CCK8 and wound healing assays demonstrated that the overexpression of human GYS2 and UPP2 proteins inhibited the proliferation and migration of HepG2 cells, accompanied by elevated expression of p53, a tumor suppressor protein. In summary, this study systematically identified rhythmic genes in the mouse liver, and a subset of circadian genes potentially regulated by BMAL1. Two circadian genes, Gys2 and Upp2, have been proposed and validated as potential candidates for advancing the prevention and treatment of HCC.

Hypoxia activation attenuates progesterone synthesis in goat trophoblast cells via NR1D1 inhibition of StAR expression†.

Trophoblast plays a crucial role in gestation maintenance and embryo implantation, partly due to the synthesis of progesterone. It has been demonstrated that hypoxia regulates invasion, proliferation, and differentiation of trophoblast cells. Additionally, human trophoblasts display rhythmic expression of circadian clock genes. However, it remains unclear if the circadian clock system is present in goat trophoblast cells (GTCs), and its involvement in hypoxia regulation of steroid hormone synthesis remains elusive. In this study, immunofluorescence staining revealed that both BMAL1 and NR1D1 (two circadian clock components) were highly expressed in GTCs. Quantitative real-time PCR analysis showed that several circadian clock genes were rhythmically expressed in forskolin-synchronized GTCs. To mimic hypoxia, GTCs were treated with hypoxia-inducing reagents (CoCl2 or DMOG). Quantitative real-time PCR results demonstrated that hypoxia perturbed the mRNA expression of circadian clock genes and StAR. Notably, the increased expression of NR1D1 and the reduction of StAR expression in hypoxic GTCs were also detected by western blotting. In addition, progesterone secretion exhibited a notable decline in hypoxic GTCs. SR9009, an NR1D1 agonist, significantly decreased StAR expression at both the mRNA and protein levels and markedly inhibited progesterone secretion in GTCs. Moreover, SR8278, an NR1D1 antagonist, partially reversed the inhibitory effect of CoCl2 on mRNA and protein expression levels of StAR and progesterone synthesis in GTCs. Our results demonstrate that hypoxia reduces StAR expression via the activation of NR1D1 signaling in GTCs, thus inhibiting progesterone synthesis. These findings provide new insights into the NR1D1 regulation of progesterone synthesis in GTCs under hypoxic conditions.

Autophagy activation attenuates the circadian clock oscillators in U2OS cells via the ATG5 pathway.

The circadian clock and autophagy are essential biological mechanisms involved in regulating many physiological processes. Accumulating evidence has revealed that autophagic activity is regulated by the circadian clock system. However, whether autophagy regulates the circadian clock system remains unclear. In this study, rapamycin and AICAR, two classical activators of autophagy, were used to create autophagy activation models in BMAL1-dLuc U2OS cell line. The results showed that the mRNA expression of MAP1LC3B and ATG5 were significantly upregulated after autophagy activation, whereas the mRNA expression of circadian clock genes (BMAL1, PER2, REV-ERBα, and DBP) were significantly decreased. Consistent with these data, the relative ratio of LC3-II/LC3-I and the protein level of ATG5 were increased after rapamycin or AICAR treatment. In contrast, BMAL1 and REV-ERBα levels were decreased. Notably, the mRNA expression of circadian clock genes (BMAL1, PER2, REV-ERBα, and DBP) and autophagy-related genes (MAP1LC3B and ATG5) showed rhythmic expression patterns in both untreated and rapamycin/AICAR-treated U2OS cells. Moreover, the autophagy inhibitor 3-methyladenine partially reversed the inhibitory effects of autophagy on circadian clock genes expression and BMAL1-Luc oscillations. Another critical finding was that ATG5 knockout alleviates the inhibitory effect of rapamycin-mediated autophagy activation on the circadian clock oscillators in U2OS cells. Collectively, our data indicate that autophagy activation attenuates the circadian clock oscillators in U2OS cells via the ATG5 pathway.

Transcriptional Feedback Loops in the Caprine Circadian Clock System.

The circadian clock system is based on interlocked positive and negative transcriptional and translational feedback loops of core clock genes and their encoded proteins. The mammalian circadian clock system has been extensively investigated using mouse models, but has been poorly investigated in diurnal ruminants. In this study, goat embryonic fibroblasts (GEFs) were isolated and used as a cell model to elucidate the caprine circadian clock system. Real-time quantitative PCR analysis showed that several clock genes and clock-controlled genes were rhythmically expressed in GEFs over a 24 h period after dexamethasone stimulation. Immunofluorescence revealed that gBMAL1 and gNR1D1 proteins were expressed in GEFs, and western blotting analysis further verified that the proteins were expressed with circadian rhythmic changes. Diurnal changes in clock and clock-controlled gene expression at the mRNA and protein levels were also observed in goat liver and kidney tissues at two representative time points in vivo. Amino acid sequences and tertiary structures of goat BMAL1 and CLOCK proteins were found to be highly homologous to those in mice and humans. In addition, a set of goat representative clock gene orthologs and the promoter regions of two clock genes of goats and mice were cloned. Dual-luciferase reporter assays showed that gRORα could activate the promoter activity of the goat BMAL1, while gNR1D1 repressed it. The elevated pGL4.10-gNR1D1-Promoter-driven luciferase activity induced by mBMAL1/mCLOCK was much higher than that induced by gBMAL1/gCLOCK, and the addition of gCRY2 or mPER2 repressed it. Real-time bioluminescence assays revealed that the transcriptional activity of BMAL1 and NR1D1 in goats and mice exhibited rhythmic changes over a period of approximately 24 h in NIH3T3 cells or GEFs. Notably, the amplitudes of gBMAL1 and gNR1D1 promoter-driven luciferase oscillations in NIH3T3 cells were higher than those in GEFs, while mBMAL1 and mNR1D1 promoter-driven luciferase oscillations in NIH3T3 cells had the highest amplitude. In sum, transcriptional and translational loops of the mammalian circadian clock system were found to be broadly conserved in goats and not as robust as those found in mice, at least in the current experimental models. Further studies are warranted to elucidate the specific molecular mechanisms involved.

Circadian clock gene BMAL1 controls testosterone production by regulating steroidogenesis-related gene transcription in goat Leydig cells.

Testosterone is produced by Leydig cells (LCs) and undergoes diurnal changes in serum levels in rats, mice, and humans, but little is known in goats. The present study revealed that goat serum testosterone levels displayed diurnal rhythmic changes (peak time at ZT11.2). Immunohistochemical staining showed that BMAL1, a circadian clock protein, is highly expressed in goat LCs. ELISA revealed that both hCG (0-5 IU/ml) and 22R-OH-cholesterol (0-30 µM) addition stimulated testosterone synthesis in primary goat LCs in a dose-dependent manner. Treating goat LCs with hCG (5 IU/ml) significantly increased intracellular cAMP levels. Additionally, real-time quantitative polymerase chain reaction (PCR) analysis revealed that the circadian clock (BMAL1, PER1, PER2, DBP, and NR1D1) and steroidogenesis-related genes (SF1, NUR77, StAR, HSD3B2, CYP17A1, CYP11A1, and HSD17B3) showed rhythmic expression patterns in goat LCs following dexamethasone synchronization. Several Bmal1-Luc circadian oscillations were clearly observed in dexamethasone-treated goat LCs transfected with the pLV6-Bmal1-Luc plasmid. BMAL1 knockdown significantly downregulated mRNA levels of PER2, NR1D1, DBP, StAR, HSD3B2, SF1, NUR77, and GATA4, and dramatically decreased StAR and HSD3B2 protein levels and testosterone production. In contrast, BMAL1 overexpression significantly increased the mRNA and protein expression levels of StAR and HSD17B3 and enhanced testosterone production. Reporter assays revealed that goat BMAL1, or in combination with mouse CLOCK, activated goat HSD17B3 transcription in vitro. These data indicate that BMAL1 contributes to testosterone production by regulating transcription of steroidogenesis-related genes in goat LCs, providing a basis for further exploring the underlying mechanism by which the circadian clock regulates ruminant reproductive capability.

Zearalenone perturbs the circadian clock and inhibits testosterone synthesis in mouse Leydig cells.

Zearalenone (ZEA), a mycotoxin, is known to impair reproductive capability by disrupting the synthesis and secretion of testosterone by Leydig cells (LCs), although the mechanism is unknown. Robust rhythmicity of circadian clock and steroidogenic genes were identified in LCs. The aim of this study was to examine whether ZEA significantly attenuated the transcription of core clock genes (Bmal1, Dbp, Per2, and Nr1d1) as well as steroidogenic genes (StAR, Hsd3b2, and Cyp11a1) in mouse testis Leydig cell line (TM3). Western blotting confirmed declines in BMAL1, NR1D1, and StAR protein levels. ZEA also suppressed secreted testosterone levels. In primary LCs, isolated from PER2::LUCIFERASE reporter gene knock in mice, ZEA diminished the amplitude of PER2::LUC expression, and induced a phase shift and period extension. In primary LCs, ZEA also suppressed the expression levels of core clock and steroidogenic genes, reduced protein levels of BMAL1, and decreased testosterone secretion. In vivo expression of core clock and steroidogenic genes were reduced in testes of mice exposed to ZEA for 1 week leading to decreased serum testosterone levels. In summary, data suggest that ZEA may impair testosterone synthesis through attenuation of the circadian clock in LCs culminating in reproductive dysfunction in male mammals .

Bisphenol A attenuates testosterone production in Leydig cells via the inhibition of NR1D1 signaling.

Bisphenol A (BPA) is an endocrine-disrupting compound that impairs testosterone synthesis in male mammals. A circadian clock gene deficiency leads to diminished fertility and even infertility in male mice. However, whether circadian clock signaling pathways mediate the suppressive effect of BPA on testosterone synthesis in Leydig cells (LCs) remains unknown. The present study aims to detect the effect of BPA on cellular circadian clock and testosterone synthesis in mouse LCs, and examine the mechanisms underlying NR1D1 signaling. BPA treatment significantly attenuated the transcription levels of Nr1d1 and steroidogenic genes (Hsd3b2 and Hsd17b3) in TM3 cells, but increased other circadian clock gene levels (Per2 and Dbp). BPA treatment also significantly downregulated NR1D1 and StAR protein expression, but upregulated BMAL1 protein expression in TM3 cells. Furthermore, there was a marked decline in testosterone production in BPA-treated TM3 cells. Intraperitoneal injection of BPA profoundly reduced NR1D1 and StAR protein levels and steroidogenic gene transcription levels (Cyp11a1, Hsd3b2, and Hsd17b3), while enhancing BMAL1 protein and other circadian clock gene (Per2 and Dbp) levels in mouse testes. Notably, serum testosterone levels were also drastically reduced in BPA-treated mice. Moreover, SR9009, an NR1D1 agonist, augmented testosterone production in TM3 cells via elevated expression of steroidogenic genes (StAR, Cyp11a1 and Hsd17b3). Conversely, Nr1d1 knockdown inhibited testosterone accumulation and attenuated steroidogenic gene expression. Moreover, treatment with SR9009 partially reversed the BPA effect on the circadian clock and testosterone production. Taken together, our study demonstrates that BPA perturbs testosterone production, at least partially, via inhibiting NR1D1 signaling in LCs.