Gastrodin protects against high glucose-induced cardiomyocyte toxicity via GSK-3ß-mediated nuclear translocation of Nrf2.

Diabetic cardiomyopathy (DCM) is one of the major complications of diabetes that causes mortality and morbidity in diabetic patients. Gastrodin (GSTD) is a bioactive phenolic glucoside component of an ancient Chinese herb Tianma (Gastrodia elata Bl.), which is widely used for cardiovascular and cerebrovascular diseases by ancient Chinese. Up to now, whether GSTD has a beneficial effect on DCM is unclear. Therefore, this study aimed to investigate the effect of GSTD on high glucose-induced injury in H9c2 rat cardiomyocytes and HL-1 mouse cardiomyocytes, and its underlying mechanisms. High glucose (33 mM) treatment caused cardiomyocyte toxicity, oxidative stress and apoptosis in both H9c2 and HL-1 cells. Under both normal (5.5 mM glucose) and high glucose conditions, GSTD showed protective effect against high glucose-induced cytotoxicity and promoted the nuclear translocation of Nrf2 in a concentration and time-dependent manner in H9c2 and HL-1 cells. Knockdown of Nrf2 expression using siRNA specifically targeting Nrf2 attenuated the protective effect of GSTD. Furthermore, GSTD promoted the nuclear translocation of Nrf2 via activating glycogen synthase kinse-3ß (GSK-3ß) signaling pathway. 4-benzyl, 2-methyl, 1, 2, 4-thiadiazolidine, 3, 5 dione (TDZD-8), an inhibitor of GSK-3ß, inhibited the nuclear translocation of Nrf2 induced by GSTD, and attenuated the protective effect of GSTD as Nrf2 knockdown did. In summary, GSTD could protect against high glucose-induced cardiomyocyte toxicity via GSK-3ß-mediated nuclear translocation of Nrf2.

Circadian control of the secretory pathway maintains collagen homeostasis.

Collagen is the most abundant secreted protein in vertebrates and persists throughout life without renewal. The permanency of collagen networks contrasts with both the continued synthesis of collagen throughout adulthood and the conventional transcriptional/translational homeostatic mechanisms that replace damaged proteins with new copies. Here, we show circadian clock regulation of endoplasmic reticulum-to-plasma membrane procollagen transport by the sequential rhythmic expression of SEC61, TANGO1, PDE4D and VPS33B. The result is nocturnal procollagen synthesis and daytime collagen fibril assembly in mice. Rhythmic collagen degradation by CTSK maintains collagen homeostasis. This circadian cycle of collagen synthesis and degradation affects a pool of newly synthesized collagen, while maintaining the persistent collagen network. Disabling the circadian clock causes abnormal collagen fibrils and collagen accumulation, which are reduced in vitro by the NR1D1 and CRY1/2 agonists SR9009 and KL001, respectively. In conclusion, our study has identified a circadian clock mechanism of protein homeostasis wherein a sacrificial pool of collagen maintains tissue function.

1α,25-Dihydroxyvitamin D3 Ameliorates Seawater Aspiration-Induced Lung Injury By Inhibiting The Translocation Of NF-κB and RhoA.

Our previous study have reported that 1α,25-Dihydroxyvitamin D3 (calcitriol) suppresses seawater aspiration-induced ALI in vitro and in vivo. We also have confirmed that treatment with calcitriol ameliorates seawater aspiration-induced inflammation and pulmonary edema via the inhibition of NF-κB and RhoA/Rho kinase pathway activation. In our further work, we investigated the effect of calcitriol on nuclear translocation of NF-κB and membrane translocation of RhoA in vitro. A549 cells and rat pulmonary microvascular endothelial cells (RPMVECs) were cultured with calcitriol or not for 48 h and then stimulated with 25% seawater for 40 min. After these treatments, cells were collected and performed with immunofluorescent staining to observe the translocation of NF-κB and RhoA and the cytoskeleton remodeling. In vitro, seawater stimulation activates nuclear translocation of NF-κB and membrane translocation of RhoA in A549 cells. In addition, seawater administration also induced cytoskeleton remodeling in A549 cells and RPMVECs. However, pretreatment with calcitriol significantly inhibited the activation of NF-κB and RhoA/Rho kinase pathways, as demonstrated by the reduced nuclear translocation of NF-κB and membrane translocation of RhoA in A549 cells. Meanwhile, treatment of calcitriol also regulated the cytoskeleton remodeling in both A549 cells and RPMVECs. These results demonstrated that treatment with calcitriol ameliorates seawater aspiration-induced ALI via inhibition of nuclear translocation of NF-κB and membrane translocation of RhoA and protection of alveolar epithelial and pulmonary microvascular endothelial barrier.

Dioxin exposure blocks lactation through a direct effect on mammary epithelial cells mediated by the aryl hydrocarbon receptor repressor.

In mammals, lactation is a rich source of nutrients and antibodies for newborn animals. However, millions of mothers each year experience an inability to breastfeed. Exposure to several environmental toxicants, including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), has been strongly implicated in impaired mammary differentiation and lactation. TCDD and related polyhalogenated aromatic hydrocarbons are widespread industrial pollutants that activate the aryl hydrocarbon receptor (AHR). Despite many epidemiological and animal studies, the molecular mechanism through which AHR signaling blocks lactation remains unclear. We employed in vitro models of mammary differentiation to recapitulate lactogenesis in the presence of toxicants. We demonstrate AHR agonists directly block milk production in isolated mammary epithelial cells. Moreover, we define a novel role for the aryl hydrocarbon receptor repressor (AHRR) in mediating this response. Our mechanistic studies suggest AHRR is sufficient to block transcription of the milk gene ß-casein. As TCDD is a prevalent environmental pollutant that affects women worldwide, our results have important public health implications for newborn nutrition.

The Alternaria mycotoxins alternariol and alternariol methyl ether induce cytochrome P450 1A1 and apoptosis in murine hepatoma cells dependent on the aryl hydrocarbon receptor.

The Alternaria mycotoxins alternariol (AOH) and alternariol methyl ether (AME) are potential carcinogens. As planar compounds, AOH and AME are preferentially metabolized by cytochrome P450 (CYP) 1A1 and 1A2. The most prominent regulator of CYP1A1 is the dimeric transcription factor complex AhR/ARNT, which is activated by planar ligands. Therefore, we studied the activation of AhR/ARNT by AOH and AME and monitored CYP1A1 induction in murine hepatoma cells (Hepa-1c1c7). Indeed, AOH and AME enhanced the levels of CYP1A1 in Hepa-1c1c7 cells but not in cells with inactivated AhR (Hepa-1c1c12) or ARNT (Hepa-1c1c4). AOH and AME did not increase the production of reactive oxygen species but reduced cell counts in Hepa-1c1c7 cells after 24 and 48 h. This effect, however, was independent of AhR/ARNT. At 48 h, AOH and AME increased apoptosis dependent on AhR and ARNT. In conclusion, AOH and AME are novel inducers of the AhR/ARNT pathway, which mediates induction of CYP1A1 and apoptosis and might thereby contribute to the toxicity of these mycotoxins.

Molecular and functional characterization of aryl hydrocarbon receptor nuclear translocator 1 (ARNT1) and ARNT2 in chicken (Gallus gallus).

Our previous studies have provided evidence that birds have two isoforms of aryl hydrocarbon receptors (AHR1 and AHR2) and AHR nuclear translocators (ARNT1 and ARNT2) that potentially mediate toxic responses to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds. We have also shown that while both in vitro-expressed chicken AHR1 (ckAHR1) and AHR2 (ckAHR2) exhibit binding affinities to TCDD, only ckAHR1 but not ckAHR2 showed a TCDD-dose-dependent transactivation potency of chicken cytochrome P450 1A5 (ckCYP1A5) in in vitro reporter gene assays. To explore the molecular mechanism of functional difference in the two ckAHRs, the present study investigated the molecular characteristics and function of chicken ARNT (ckARNT) that is a potential dimerization partner for the activation of ckAHR. The full-length ckARNT1 and ckARNT2 cDNAs were isolated and their alternative splice variants were also identified. The ckARNT1 transcript was ubiquitously expressed in various tissues, but ckARNT2 showed restricted expressions in brain, kidney and eye, indicating a similar expression pattern to mammalian ARNTs. The expressions of tagged-ckARNT1 and -ckARNT2 were confirmed in a chicken hepatoma LMH cells by western blot analyses, and their interactions with each ckAHR and a specific recognition DNA element, xenobiotic response element (XRE), were examined by gel shift assays. The result showed that ckARNT1 and ckARNT2 dimerize with each ckAHR isoform and bind with the XRE in a TCDD-dependent manner. Hence, we conclude that functional loss on the dimerization with ckARNTs or the XRE binding is not the major cause of the deficient TCDD-dependency of ckAHR2 for the transactivation. Furthermore, in vitro reporter gene assays showed that transfected ckARNT1 failed to modulate the transcriptional induction of ckAHR-mediated ckCYP1A5 gene by TCDD in COS-7 and LMH cells, whereas ckARNT2 could potentiate the TCDD-dependent response in COS-7 but not in LMH cells. This suggests that ckARNT2 has a distinct role from ckARNT1 in AHR signaling pathway and in a cell-specific mode of action.

Disruption of period gene expression alters the inductive effects of dioxin on the AhR signaling pathway in the mouse liver.

The aryl hydrocarbon receptor (AhR) and AhR nuclear translocator (ARNT) are transcription factors that express Per-Arnt-Sim (PAS) DNA-binding motifs and mediate the metabolism of drugs and environmental toxins in the liver. Because these transcription factors interact with other PAS genes in molecular feedback loops forming the mammalian circadian clockworks, we determined whether targeted disruption or siRNA inhibition of Per1 and Per2 expression alters toxin-mediated regulation of the AhR signaling pathway in the mouse liver and Hepa1c1c7 hepatoma cells in vitro. Treatment with the prototypical Ahr ligand, 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), had inductive effects on the primary targets of AhR signaling, Cyp1A1 and Cyp1B1, in the liver of all animals, but genotype-based differences were evident such that the toxin-mediated induction of Cyp1A1 expression was significantly greater (2-fold) in mice with targeted disruption of Per1 (Per1(ldc) and Per1(ldc)/Per2(ldc)). In vitro experiments yielded similar results demonstrating that siRNA inhibition of Per1 significantly increases the TCDD-induced expression of Cyp1A1 and Cyp1B1 in Hepa1c1c7 cells. Per2 inhibition in siRNA-infected Hepa1c1c7 cells had the opposite effect and significantly decreased both the induction of these p450 genes as well as AhR and Arnt expression in response to TCDD treatment. These findings suggest that Per1 may play a distinctive role in modulating AhR-regulated responses to TCDD in the liver.

Regulation of CYP1A1 gene expression by the antioxidant tert-butylhydroquinone.

CYP1A1, a major phase I enzyme, plays an important role in the metabolism of polycyclic aromatic hydrocarbons and in the chemical activation of xenobiotics to carcinogenic derivatives. The phenolic antioxidant tert-butylhydroquinone (tBHQ), often used as a food preservative, is generally considered to act only as a mono-functional inducer of phase II enzymes, thereby exerting chemo-protection. However, we recently observed that tBHQ elevated the activity of an aryl hydrocarbon receptor (AhR) response element (DRE)-driven luciferase reporter in human colon carcinoma cells (Caco-2). Therefore, we studied the effects of tBHQ on the activity of a DRE-driven reporter, CYP1A1 mRNA expression, and CYP1A enzyme activity in Caco-2 cells and human HepG2 hepatoma cells. We found tBHQ caused induction of reporter activity and CYP1A1 expression and activity in Caco-2 and HepG2 cells. Moreover, tBHQ combined with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) increased reporter activity and mRNA expression in Caco-2 cells in an additive manner. By contrast, tBHQ decreased TCDD-mediated induction of reporter activity and CYP1A1 mRNA expression in HepG2 cells. Resveratrol, an AhR antagonist, repressed the induction of CYP1A1 by tBHQ. Cotransfection of HepG2 cells with a dominant negative AhR nuclear translocator mutant abolished the tBHQ-induced CYP1A1 reporter activity. These findings indicate that CYP1A1 may be induced by the antioxidant tBHQ via an AhR-dependent mechanism.