In our previous report, PERK/NRF2/CX43/StAR/progesterone pathway activation in ovarian granulosa cells was shown to mediate cold-induced female reproductive disorders. However, how PERK is activated by low temperature remained unclear. In the present study, we found that the circadian protein E4BP4 was significantly upregulated in ovarian granulosa cells following exposure to cold or isoproterenol (ISO), a non-selective β-adrenergic receptor agonist that pharmacologically activates β-adrenergic signaling, a key component of the cold stress response. Mechanistically, E4BP4 interacted with PERK and was required for PERK activation and subsequent NRF2/CX43/StAR signaling, leading to increased progesterone secretion. Interestingly, NRF2 also acted as a transcriptional activator of E4BP4 under ISO treatment, and blocking PERK or NRF2 expression attenuated ISO-induced E4BP4 accumulation, suggesting a positive feedback loop involving PERK/NRF2/E4BP4. Collectively, these findings identify E4BP4 as a cold-responsive circadian protein that interacts with PERK and may contribute to cold-induced reproductive disorders via a bidirectional E4BP4-PERK feedback loop.

Lack of sleep is a common problem in current society, which can induce various brain dysfunctions. Neuroinflammation is a typical reaction caused by sleep deficit and is considered as a common basis for various neurological disorders and cognitive impairments, but the related mechanisms have not been fully clarified. The circadian clock protein plays a critical role in maintaining physiological homeostasis, including sleep/wake cycles. Circadian disorders induced by sleep deficit might contribute to the development of neuroinflammation. In the current study, we observed that sleep deprivation (SD) induced elevated expression of High-mobility group box 1 (HMGB1), one of the most important mediators of neuroinflammation, in the cortical microglia and cerebrospinal fluids. Moreover, acetylation-dependent nuclear export of HMGB1 was involved in up-regulation and secretion of HMGB1 after sleep deprivation. Further studies indicated that sleep deprivation induced an increase in the expression of acetyltransferase p300 and a decrease in the expression of deacetylase SIRT1, which synergistically enhanced the acetylation level of HMGB1 in the cortical microglial cells, thereby triggered the nuclear export and secretion of HMGB1. Most importantly, circadian clock protein PER2 constitutively interacted with p300 and inhibited its expression in the microglial cells, which can be interrupted by PER2 downregulation upon sleep deprivation, leading to the increased expression of p300 and acetylation and secretion of HMGB1. The truncated PER2 mutant without p300 binding ability lost its ability to regulate p300 expression, indicating that PER2 functioned as a co-suppressor of p300 in regulating acetylation and expression of HMGB1. Taken together, data in this study reveal a new mechanism by which PER2 is involved in controlling HMGB1 dependent neuroinflammation induced by sleep deprivation. Maintaining PER2 levels or blocking HMGB1 acetylation in the cortex might be prospective for preventing sleep deprivation-induced neuroinflammation and the related adverse reactions in the brain.

Sleep is critical for maintaining overall health. Insufficient sleep duration and poor sleep quality are associated with various physical and mental health risks and chronic diseases. To date, plenty of epidemiological research has shown that sleep disorders are associated with the risk of obesity, which is usually featured by the expansion of adipose tissue. However, the underlying mechanism of increased fat accumulation upon sleep disorders remains unclear. Here we demonstrated that sleep deprivation (SD) caused triglycerides (TG) accumulation in the visceral white adipose tissue (vWAT), accompanied by a remarkable decrease in the expression of adipose triglyceride lipase (ATGL) and other two rate-limiting lipolytic enzymes. Due to the key role of ATGL in initiating and controlling lipolysis, we focused on investigating the signaling pathway leading to attenuated ATGL expression in vWAT upon SD in the following study. We observed that ATGL downregulation resulted from the suppression of ATGL transcription, which was mediated by the reduction of the transcriptional factor FOXO1 and its upstream regulator SIRT1 expression in vWAT after SD. Furthermore, impairment of SIRT1/FOXO1/ATGL pathway activation and lipolysis induced by SIRT1 inhibitor EX527 in the 3 T3-L1 adipocytes were efficiently rescued by the SIRT1 activator resveratrol. Most notably, resveratrol administration in SD mice revitalized the SIRT1/FOXO1/ATGL pathway activation and lipid mobilization in vWAT. These findings suggest that targeting the SIRT1/FOXO1/ATGL pathway may offer a promising strategy to mitigate fat accumulation in vWAT and reduce obesity risk associated with sleep disorders.

Ambient air temperature is a key factor affecting human health. Female reproductive disorders are representative health risk events under low temperature. However, the mechanism involving in cold-induced female reproductive disorders remains largely unknown. Female mice were intermittently exposed to cold conditions (4 °C) to address the health risk of low temperature on female reproductive system. Primary granulosa cells (GCs) were prepared and cultured under low temperature (35 °C) or exposed to β3-adrenoreceptor agonist, isoproterenol, to mimic the condition of cold exposure. Western-blot, RT-PCR, co-IP, ELISA, pharmacological inhibition or siRNA-mediated knockdown of target gene were performed to investigate the possible role of hormones, gap conjunction proteins, and ER stress sensor protein in regulating female reproductive disorders under cold exposure. Cold exposure induced estrous cycle disorder and follicular dysplasia in female mice, accompanying with abnormal upregulation of progesterone and its synthetic rate-limiting enzyme, StAR, in the ovarian granulosa cells. Under the same conditions, an increase in connexin 43 (CX43) expressions in the GCs was also observed, which contributed to elevated progesterone levels in the ovary. Moreover, ER stress sensor protein, PERK, was activated in the ovarian GCs after cold exposure, leading to the upregulation of downstream NRF2-dependent CX43 transcription and aberrant increase in progesterone synthesis. Most importantly, blocking PERK expression in vivo significantly inhibited NRF2/CX43/StAR/progesterone pathway activation in the ovary and efficiently rescued the prolongation of estrous cycle and the increase in follicular atresia of the female mice induced by cold stress. We have elucidated the mechanism of ovarian PERK/NRF2/CX43/StAR/progesterone pathway activation in mediating female reproductive disorder under cold exposure. Targeting PERK might be helpful for maintaining female reproductive health under cold conditions.

Chronic stress enhances the risk for psychiatric disorders and induces depression and cognitive impairment. Gamma oscillations are essential for neurocircuit function, emotion, and cognition. However, the influence of gamma entrainment by sensory stimuli on specific aspects of chronic stress-induced responses remains unclear. Mice were subjected to corticosterone (CORT) administration and chronic restraint stress (CRS) for weeks, followed by rhythmic gamma frequency light flickering exposure. Local field potentials (LFPs) were recorded from the V1, CA1, and PFC regions to verify the light flicker on gamma oscillations. Behavioral tests were used to examine stress-related and memory-related behaviors. Golgi staining was performed to observe changes in spine morphology. Synaptosomes were isolated to determine the expression of synapse-related proteins through immunoblotting. RNA sequencing (RNA-seq) was applied to explore specific changes in the transcriptome. Immunofluorescence staining, real-time quantitative polymerase chain reaction (qPCR), and ELISA were used to evaluate microglial activation and cytokine levels. In this study, we demonstrated that rhythmic 40 Hz LF attenuated stress-related behavior and cognitive impairments by ameliorating the microstructural alterations in spine morphology and increasing the expression of GluN2A and GluA1 in chronically stressed mice. Transcriptome analysis revealed that significantly downregulated genes in LF-exposed CRS mice were enriched in neuroimmune-related signaling pathways. Rhythmic 40 Hz LF exposure significantly decreased the number of Iba1-positive microglia in the PFC and hippocampus, and the expression levels of the M1 markers of microglia iNOS and CD68 were reduced significantly in CRS mice. In addition, 40 Hz LF exposure suppressed the secretion of cytokines IL-12, which could regulate the production of IFN-gamma gamma and IL-10 in stressed mice. Our results demonstrate that exposure to rhythmic 40 Hz LF induces the neuroimmune response and downregulation of neuroinflammation with attenuated stress-related behaviors and cognitive function in CRS-induced mice. Our findings highlight the importance of sensory-evoked gamma entrainment as a potential therapeutic strategy for stress-related disorders treatment. Abbreviations: CORT, Chronic corticosterone treatment; CRS, Chronic restraint stress; IACUC, Institutional Animal Care and Use Committee; LF, light flickers; FST, Forced swim test; NSFT, Novelty-suppressed feeding test; SPT, Sucrose preference test; NSFT, Novelty-suppressed feeding; qPCR, Quantitative real-time polymerase chain reaction; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; PVDF, polyvinylidene fluoride; PBS, phosphate-buffered saline; PBS-T, phosphate-buffered saline plus 0.1% Tween 20; PVDF, polyvinylidene fluoride; GFAP, Glial fibrillary acidic protein; DAPI, 4 ',6-Diamid- ' ,6-Diamid- ino-2-phenylindole; Iba1, Ionized calcium- binding adaptor molecule 1; iNOS, Inducible nitric oxide synthase; IL-10, Interleukin-10; IL6, Interleukin 6; IL-1 (3, Interleukin 1(3; (3 ; IL-12, Interleukin 12; TNF-alpha, Tumor necrosis factor alpha; IFN-gamma, gamma, Interferon-gamma; TLR6 and 9, Toll-like Receptor 6 and 9.

Fine particulate matter (PM) 2.5 is the main component of air pollution causing pathological responses primarily in the respiratory and cardiovascular systems. Therefore, it is urgent to explore valid strategies to inhibit the adverse reactions induced by PM2.5. In our previous studies, we have revealed that intratracheal instillation of PM2.5 evoked airway remodeling, pulmonary inflammatory, and oxidative stress responses in rat lungs by upregulating VEGFA levels in bronchial epithelial cells and by activating ANGII/AT1R axis activation in vascular endothelial cells. The same results were obtained when human bronchial epithelial cells (Beas−2B) and human umbilical vein endothelial cells (HUVECs) cells were exposed to PM2.5 in vitro. Curcumin is a dietary polyphenol with protective properties, including anti−inflammatory and antioxidant effects. This study aims to determine the potential role of curcumin in protecting against PM2.5−induced adverse responses in the bronchial epithelium and vascular endothelium and the mechanism involved. To this end, we pretreated cells with curcumin (diluted 1000 times in sterile saline) for 2 h and then exposed them to PM2.5. Our results from RT−PCR, a luciferase reporter assay, and ELISA indicated that curcumin pretreatment effectively inhibited PM2.5−induced VEGFA elevation in Beas−2B cells by over 60% via blocking HIF1α accumulation and HIF1 transactivity, Moreover, curcumin also exerted a protective role in suppressing PM2.5−induced ANGII/AT1R axis components expression in HUVEC by over 90% via targeting the transcriptional factors, AP−1 and HIF1. Under the same conditions, curcumin pretreatment also blocked the downstream signaling events following ANGII/AT1R pathway activation, the increase in chemokines and cell adhesion molecules (sICAM−1, VCAM−1, E−Selectin, P−Selectin, IL−8, MCP−1) that drive monocyte−endothelial cell adhesion, as well as the elevated production of oxidative stress mediators (ROS and MDA) in HUVECs according to the data from immunofluorescence and flow cytometric assays. Most importantly, administration of curcumin resulted in an 80% reduction of the HIF1− and AP−1−dependent upregulation of VEGFA and AGT/AT1R axis components and impeding the resultant pro−inflammatory and oxidative responses in the lung of the rats exposed to PM2.5. Taking these data together, we disclosed the important role and mechanism of curcumin in protecting against PM2.5−induced adverse reactions in the bronchial epithelium and vascular endothelium. Curcumin might be used as a feasible and safe dietary agent to reduce the health risk of PM2.5.

Dysregulated IL-10 producing regulatory B cells (Bregs) are associated with the progression of systemic lupus erythematosus. An immunomodulatory role of heat shock proteins (HSPs) is implicated in autoimmune diseases. However, the molecular basis underlying the role of Hspa13 in regulating Bregs function and lupus pathogenesis remains unclear. In this study, Bregs display higher Hspa13 expression than IL-10- B cells. Induction of IL-10 production is weakened in B cells with Hspa13 knockdown or knockout. Hspa13 binds to the IL-10 promoter via the TATA or CAAT box and activates IL-10 transcription in the nucleus. Furthermore, Hspa13 positive cells are enriched in marginal zone (MZ) B cells to regulate IL-10 production. Stimulated B220+ B or MZ B cells from CD19creHspa13fl/fl mice for Breg induction show an impaired capacity to promote CD4+Foxp3+ regulatory T cells (Treg) differentiation. In lupus MRL/lpr mice, a decline in Treg differentiation is accompanied by decreased Hspa13 expression in both Bregs and MZ B cells. Moreover, adoptive transfusion of Bregs and MZ B cells from CD19creHspa13fl/fl mice fails to increase the frequency of Tregs, attenuate renal pathology, or decrease anti-dsDNA antibody levels. These results explain the unique role of Hspa13 in determining MZ regulatory function and affecting lupus pathogenesis.

Our previous studies have revealed that GADD45α is a liable proapoptotic protein, which undergoes MDM2-dependent constitutive ubiquitylation and degradation in resting cancer cells. Under chemotherapeutic agent (such as arsenite, 5-Fu and VP-16) exposure, DAPK1 functions as a novel p53 (also known as TP53) kinase, which induces phosphorylation of p53 at Ser15 and transactivates the p53 target Ets-1, to synergistically repress IKKβ-dependent MDM2 stability, and ultimately removes the inhibitory effect of MDM2 on GADD45α, resulting in GADD45α accumulation and cell apoptosis. In the current study, we show that there is a strong induction of ISG20L1 (also known as AEN) expression in several cancer cell lines under exposure of arsenite and other chemotherapeutic agents. Surprisingly, although originally identified as a transcriptional target of p53, ISG20L1 induction was not controlled by p53. Instead, ISG20L1 functioned as upstream activator of p53 by interacting with DAPK1, and plays an essential role in promoting DAPK1-p53 complex formation and the subsequent activation of Ets-1/IKKβ/MDM2/GADD45α cascade. Therefore, our findings have revealed novel function of ISG20L1 in mediating cancer cell apoptosis induced by chemotherapeutic agents via modulating activation of the DAPK1- and p53-dependent cell death pathway.

PM2.5 has been accepted as a strong risk factor for cardiovascular diseases. Activation of the renin-angiotensin system (RAS) has been proved to be a key factor in triggering vascular endothelial dysfunction upon PM2.5 exposure in our previous reports. In the current study, we observed the concurrent induction of hemoxygenase (HO)-1 and RAS components (ANGII and AT1R) expression both in the vascular endothelial cell lines and in rat lung tissue after PM2.5 exposure. Furthermore, HO-1 inhibited RAS activation by suppressing the expression and activity of HIF1 alpha, the upstream transcriptional activator of ANGII and AT1R. In addition, HO-1 blocked signif-icantly increased the release of cell adhesion molecules and chemokines (VCAM-1, E-Selectin, P-Selectin, IL-8, MCP-1) that drive monocyte-endothelium adhesion, along with the enhanced the generation of oxidative stress response mediators in the vascular endothelium. These data together indicate that PM2.5 induced HO-1 upregulation functions as a self-defense response to antagonize endothelial dysfunction by inhibiting HIF1 alpha-mediated RAS activation. Targeting endogenous protective pathway might be helpful to protect from PM2.5-induced cardiovascular injury.

Particulate matter (PM) 2.5 has long been regarded as a major risk factor of the respiratory system, which constitutes a threat to human health. Although the positive relationship between PM2.5 exposure and the development of respiratory diseases has been well established, limited studies investigate the intrinsic self‐protection mechanisms against PM2.5‐induced respiratory injuries. Excessive pulmonary inflammation served as a key pathogenic mechanism in PM2.5‐induced airway dysfunction, and we have previously shown that PM2.5 induced the production of vascular endothelial growth factor A (VEGFA) in the bronchial epithelial cells, which subsequently led to pulmonary inflammatory responses. In the current study, we found that PM2.5 also concurrently induced the expression of the stress‐responsive protein heme oxygenase‐1 (HO‐1) along with VEGFA in the bronchial epithelial cells both in vivo and in vitro. Importantly, knocking down of HO‐1 expression significantly increased the synthesis and secretion of VEGFA; while overexpression of HO‐1 showed the opposite effects, indicating that HO‐1 induction can antagonize VEGFA production in the bronchial epithelial cells upon PM2.5 exposure. Mechanistically, HO‐1 inhibited PM2.5‐evoked VEGFA induction through modulating hypoxia‐inducible factor 1 alpha (HIF‐1α), which was the upstream transcriptional factor of VEGFA. More specifically, HO‐1 could not only inhibit HIF‐1α expression, but also suppress its transactivity. Taken together, our results suggested that HO‐1 was an intrinsic protective factor against PM2.5‐induced pulmonary VEGFA production with a mechanism relating to HIF‐1α, thus providing a potential treatment strategy against PM2.5 triggered airway injuries.