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.

Coordination between the circadian clock and androgen signaling is required to sustain rhythmic expression of Elovl3 in mouse liver.

ELOVL3 is a very long-chain fatty acid elongase, and its mRNA levels display diurnal rhythmic changes exclusively in adult male mouse livers. This cyclical expression of hepatic Elovl3 is potentially controlled by the circadian clock, related hormones, and transcriptional factors. It remains unknown, however, whether the circadian clock, in conjunction with androgen signaling, functions in maintaining the rhythmic expression of Elovl3 in a sexually dimorphic manner. Under either zeitgeber or circadian time, WT mouse livers exhibited a robust circadian rhythmicity in the expression of circadian clock genes and Elovl3 In contrast, male Bmal1-/- mice displayed severely weakened expression of hepatic circadian clock genes, resulting in relatively high, but nonrhythmic, Elovl3 expression levels. ChIP assays revealed that NR1D1 binds to the Elovl3 promoter upon circadian change in WT mouse livers in vivo, and a diminished binding was observed in male Bmal1-/- mouse livers. Additionally, female mouse livers exhibited constant low levels of Elovl3 expression. Castration markedly reduced Elovl3 expression levels in male mouse livers but did not disrupt circadian variation of Elovl3 Injection of female mice with 5α-dihydrotestosterone induced Elovl3 rhythmicity in the liver. In AML12 cells, 5α-dihydrotestosterone also elevated Elovl3 expression in a time-dependent manner. In contrast, flutamide efficiently attenuated this induction effect. In conclusion, a lack of either the circadian clock or androgen signaling impairs hepatic Elovl3 expression, highlighting the observation that coordination between the circadian clock and androgen signaling is required to sustain the rhythmic expression of Elovl3 in mouse liver.

Age-related endoplasmic reticulum stress represses testosterone synthesis via attenuation of the circadian clock in Leydig cells.

Senile animals exhibit a high risk of elevated endoplasmic reticulum (ER) stress, attenuated circadian clock, and impaired steroidogenesis in testes. However, how these three processes are intertwined in mouse Leydig cells remains unclear. In this study, a mouse model of aging and hydrogen peroxide (H2O2)-induced senescent TM3 Leydig cells were used to dissect the connections among ER stress, circadian oscillators, and steroidogenesis in Leydig cells. Additionally, thapsigargin (Tg, 60 nM)/tunicamycin (Tm, 60 ng/mL)-induced ER stress were established to investigate the underlying mechanisms by which ER stress regulated testosterone synthesis via circadian clock-related signaling pathways in TM3 cells and primary Leydig cells. Elevated ER stress, attenuated circadian clock, and diminished steroidogenesis were detected in the testes of aged mice (24-month-old) and H2O2-induced (200 µM) senescent TM3 cells in comparison with their control groups. Tg/Tm-induced ER stress reduced the transcription of the circadian clock and steroidogenic genes in TM3 cells and LH-treated (100 ng/mL) primary Leydig cells. Furthermore, 4-phenylbutyric acid (4-PBA, 1 µM), an inhibitor of ER stress, alleviated the inhibitory effect of Tg-mediated ER stress on Per2:Luc oscillations in primary Leydig cells isolated from mPer2Luc knock-in mice, and attenuated the repressive effect of H2O2-induced or Tg-mediated ER stress on the transcription of circadian clock and steroidogenic genes expression and testosterone synthesis in TM3 cells. Collectively, these data indicate that age-related ER stress represses testosterone synthesis via attenuation of the circadian clock in Leydig cells.

Modulation of circadian clock by crude drug extracts used in Japanese Kampo medicine.

Circadian rhythm is an approximately 24 h endogenous biological rhythm. Chronic disruption of the circadian clock leads to an increased risk of diabetes, cardiovascular disease, and cancer. Hence, it is important to develop circadian clock modulators. Natural organisms are a good source of several medicines currently in use. Crude drugs used in Japanese traditional Kampo medicine or folk medicines are an excellent source for drug discovery. Furthermore, identifying new functions for existing drugs, known as the drug repositioning approach, is a popular and powerful tool. In this study, we screened 137 crude drug extracts to act as circadian clock modulators in human U2OS cells stably expressing the clock reporter Bmal1-dLuc, and approximately 12% of these modulated the circadian rhythm. We further examined the effects of several crude drugs in Rat-1 fibroblasts stably expressing Per2-luc, explant culture of lung from Per2::Luciferase knockin mice, and zebrafish larvae in vivo. Notably, more than half of the major ingredients of these crude drugs were reported to target AKT and its relevant signaling pathways. As expected, analysis of the major ingredients targeting AKT signaling confirmed the circadian clock-modulating effects. Furthermore, activator and inhibitor of AKT, and triple knockdown of AKT isoforms by siRNA also modulated the circadian rhythm. This study, by employing the drug repositioning approach, shows that Kampo medicines are a useful source for the identification of underlying mechanisms of circadian clock modulators and could potentially be used in the treatment of circadian clock disruption.

ER stress activation impairs the expression of circadian clock and clock-controlled genes in NIH3T3 cells via an ATF4-dependent mechanism.

Endoplasmic reticulum (ER) stress and circadian clockwork signaling pathways mutually regulate various cellular functions, but the details regarding the cross-talk between these pathways in mammalian cells are unclear. In this study, whether perturbation of ER stress signaling affects the cellular circadian clockwork and transcription of clock-controlled genes was investigated in NIH3T3 mouse fibroblasts. An NIH3T3 cell model stably expressing luciferase (Luc) under the control of the Bmal1 clock gene promoter was established using a lentiviral system. Then, Luc activity was monitored in real-time to detect Bmal1-Luc oscillations. The ER stress activators thapsigargin (Tg) and tunicamycin (Tm) markedly reduced Bmal1-Luc oscillation amplitudes and induced phase delay shifts in NIH3T3 cells. Treatment with Tg/Tm activated ER stress signaling by upregulating GRP78, CHOP, ATF6, and ATF4 and simultaneously significantly decreased BMAL1 protein levels and inhibited the transcription of circadian clock (Bmal1, Per2, Nr1d1, and Dbp) and clock-controlled (Scad1, Fgf7, and Arnt) genes. 4-Phenylbutyric acid, an ER stress inhibitor, alleviated the transcriptional repression of the circadian clock genes and partially restored Bmal1-Luc oscillation amplitudes in Tg- or Tm-treated NIH3T3 cells. More importantly, knock-down of ATF4, but not ATF6, in Tg-treated NIH3T3 cells partially rescued Bmal1-Luc oscillation amplitudes and mRNA expression of the four circadian clock genes. Taken together, our study demonstrates that ER stress activation inhibits the transcription of circadian clock and clock-controlled genes via an ATF4-dependent mechanism.

Adipose tissue-derived stem cells ameliorates dermal fibrosis in a mouse model of scleroderma.

OBJECTIVE: To investigate the therapeutic potential of adipose-derived stem cells (ADSCs) for limited cutaneous scleroderma (LS) in mouse models. METHODS: ADSCs were isolated from pathogen-free female C57BL/6 mice and LS was induced in wild type (WT) C57BL/6 mice via daily injection of bleomycin (0.1 mL × 300 µg/mL) for 4 weeks; then the ADSCs were subcutaneously injected into the dorsal area in the model treatment group, and 100 µL of phosphate-buffered saline (PBS) solution was injected into the same site in the model control group. Green fluorescent protein (GFP) was used to track the cells using an in vivo imaging system on days 7, 14, 21, and 28 after transplantation. All mice were sacrificed and histologic analyses were performed after 4 weeks, and the skin thickness, collagen deposition and the total content of hydroxyproline were evaluated. Additionally, immunohistochemistry were performed to compare the tissue expression and distribution of TGF-ß1 and VEGF between the ADSCs treatment group and the treatment control group. RESULTS: WT C57BL/6 LS mouse model were successfully established and GFP in vivo fluorescence imaging showed that the translated ADSCs survived at the local for at least 4 weeks. Compared with the control group, the ADSCs treatment group significantly attenuated bleomycin-induced dermal fibrosis, reduced the skin thickness and the total content of hydroxyproline (P < 0.05). The ADSCs treatment group displayed significantly lower levels of TGF-ß1 and higher levels of VEGF than the control group (P < 0.05). CONCLUSIONS: ADSCs may provide a feasible and practical treatment for autoimmune diseases such as LS and ameliorate dermal fibrosis.