Toxic effects of exogenous retinoic acid on the neurodevelopment of zebrafish (Danio rerio) embryos.

Endogenous retinoic acid (RA) is essential for embryonic development and maintaining adult physiological processes. Human-caused RA residues in the environment threaten the survival of organisms in the environment. We employed zebrafish as a model to explore the developmental impacts of excess RA. We used exogenous RA to raise the amount of RA signal in the embryos and looked at the effects of excess RA on embryonic morphological development. Upregulation of the RA signal significantly reduced embryo hatching and increased embryo malformation. To further understand the neurotoxic impact of RA signaling on early neurodevelopment, we measured the expression of neurodevelopmental marker genes and cell death and proliferation markers in zebrafish embryos. Exogenous RA disrupted stem cell (SC) and neuron marker gene expression and exacerbated apoptosis in the embryos. Furthermore, we looked into the links between the transcriptional coactivator RBM14 and RA signaling to better understand the mechanism of RA neurotoxicity. There was a negative interaction between RA signaling and the transcription coactivator RBM14, and the morpholino-induced RBM14 down-regulation can partially block the effects of RAR antagonist BMS493-induced RA signaling inhibition on embryonic malformation and cell apoptosis. In conclusion, exogenous RA causes neurodevelopmental toxicity, and RBM14 may be involved in this neurotoxic process.

Oxytetracycline changes the behavior of zebrafish larvae by inhibiting NMDA receptors.

Oxytetracycline (OTC), a tetracycline antimicrobial, is one of the antimicrobial drugs frequently used in the aquaculture and livestock industries. Due to its extensive usage and emissions, OTC has been identified as a significant new emerging pollutant (EP) in a number of environments. OTC frequently causes toxic effects on the central nervous system, but it can be challenging to monitor, and it is still unclear how these toxicities are caused. We used bioinformatic analysis techniques to screen for OTC targets and discovered that NMDA receptors are potential targets of OTC neurotoxicity. To confirm this finding, we exposed zebrafish embryos to 5 mg/L OTC-containing rearing water from 2-hour post fertilization (hpf) to 8-day post fertilization (dpf), performed spontaneous movement and light-dark stimulation assays at 6 and 8 dfp, and discovered that OTC inhibited locomotor activity and attenuated anxiety-like responses in zebrafish larvae. Meanwhile, the qPCR and immunofluorescence staining results suggested that OTC inhibited the expression of multiple subtypes of NMDA receptors (grin1a, grin1b, grin2bb, grin2ca) and induced apoptosis in the brains of zebrafish embryos. Simultaneous administration of NMDA, an NMDA receptor agonist, completely antagonized the inhibitory neurobehavioral changes in zebrafish larvae, as well as the downregulation of N-methyl-D-aspartate (NMDA) receptor expression and apoptosis in the embryonic brains caused by OTC exposure. In conclusion, OTC exhibited significant inhibitory neurobehavioral toxicity in zebrafish larvae during early development, which may be dependent on its suppression of NMDA receptor activity and expression. Furthermore, OTC-induced neurodevelopmental toxicity may be associated with NMDA receptor-regulated neuronal apoptosis.

Traditional Chinese Herbal Medicine for Allergic Diseases: A Review.

The prevalence of allergic disorders has increased in recent years, lowering patients' quality of life and increasing the demand for drugs to treat these diseases. Western drugs such as glucocorticoids, antihistamines, and leukotrienes are routinely utilized in clinics. However, drawbacks like high recurrence rates and adverse effects limit their use. As one of the most promising natural medicine systems, traditional Chinese medicine offers distinct benefits in treating allergic illnesses, such as maintaining long-term treatment, preventing disease recurrence, and producing fewer adverse reactions. We analyzed and discussed recent developments in traditional Chinese medicine used in allergic diseases from three perspectives: Chinese herbal formula, Chinese patent medicine, and active ingredients of traditional Chinese medicine, and explained their main components, efficacy, and mechanisms of action. We also reviewed the modification of Chinese herbal formulas and the combined application of Chinese medicine with Western medicine or nonpharmaceutical therapies. Traditional Chinese medicines are becoming increasingly important in treating allergic disorders. Improving traditional Chinese herbal formulas and developing safe and effective Chinese patent medicines are currently the most pressing and important aspects of research on traditional Chinese medicine.

Chronic Neuroinflammation Induced by Lipopolysaccharide Injection into the Third Ventricle Induces Behavioral Changes.

The existence of Gram-negative bacteria in the brain, regardless of underlying immune status has been demonstrated by recent studies. The colocalization of lipopolysaccharide (LPS) with Aß1-40/42 in amyloid plaques supports the hypothesis that brain microbes may be the cause, triggering chronic neuroinflammation, leading to Alzheimer's disease (AD). To investigate the behavioral changes induced by infectious neuroinflammation, we chose the third ventricle as the site of a single LPS injection (20 µg or 80 µg) in male Wistar rats to avoid mechanical injury to forebrain structures while inducing widespread inflammation throughout the brain. Chronic neuroinflammation induced by LPS resulted in depressive-like behaviors and the impairment of spatial learning; however, there was no evidence of the development of pathological hallmarks (e.g., the phosphorylation of tau) for 10 months following LPS injection. The acceleration of cholesterol metabolism via CYP46A1 and the retardation of cholesterol synthesis via HMGCR were observed in the hippocampus of rats treated with either low-dose or high-dose LPS. The rate-limiting enzymes of cholesterol metabolism (CYP46A1) in SH-SY5Y cells and synthesis (HMGCR) in U251 cells were altered by inflammation stimulators, including LPS, IL-1ß, and TNF-α, through the TLR4/MyD88/NF-κB signaling pathway. The data suggest that chronic neuroinflammation provoked by the administration of LPS into the third ventricle may induce depressive-like symptoms and that the loss of cholesterol might be a biomarker of chronic neuroinflammation. The lack of pathological hallmarks of AD in our model indicates that Gram-negative bacteria infection might not be a single cause of AD.

The Inhibition of miR-873 Provides Therapeutic Benefit in a Lipopolysaccharide-Induced Neuroinflammatory Model of Parkinson's Disease.

Background and Purpose. Alterations in cholesterol homeostasis have been reported in cell and animal models of Parkinson's disease (PD), although there are inconsistent data about the association between serum cholesterol levels and risk of PD. Here, we investigated the effects of miR-873 on lysosomal cholesterol homeostasis and progressive dopaminergic neuron damage in a lipopolysaccharide-(LPS) induced model of PD. Experimental Approach. To evaluate the therapeutic benefit of the miR-873 sponge, rats were injected with a LV-miR-873 sponge or the control vector 3 days before the right-unilateral injection of LPS into the substantia nigra (SN) pars compacta, or 8 and 16 days after LPS injection. Normal SH-SY5Y cells or SH-SY5Y cells overexpressing α-synuclein were used to evaluate the distribution of α-synuclein and cholesterol in lysosomes and to assess the autophagic flux after miR-873 transfection or ABCA1 silencing. The inhibition of miR-873 significantly ameliorated the LPS-induced accumulation of α-synuclein and loss of dopaminergic neurons in the SN at the early stage. miR-873 mediated the inhibition of ABCA1 by LPS. miR-873 transfection or ABCA1 silencing increased the lysosomal cholesterol and α-synuclein levels, and decreased the autophagic flux. The knockdown of ABCA1 or A20, which are the downstream target genes of miR-873, exacerbated the damage to LPS-induced dopaminergic neurons. Conclusion and Implications. The results suggest that the inhibition of miR-873 may play a dual protective role by improving intracellular cholesterol homeostasis and neuroinflammation in PD. The therapeutic effects of the miR-873 sponge in PD may be due to the upregulation of ABCA1 and A20.

Glutamate affects cholesterol homeostasis within the brain via the up-regulation of CYP46A1 and ApoE.

Chronic glutamate excitotoxicity has been thought to be involved in numerous neurodegenerative disorders. A small but significant loss of membrane cholesterol has been reported following a short stimulation of ionotropic glutamate receptors (iGluRs). We investigated the alteration of brain cholesterol following chronic glutamate treatment. The alteration of cholesterol levels was evaluated in the hippocampus from the adult rats that received the subcutaneous injection with monosodium l-glutamate at 1, 3, 5, and 7 days of age. The regulation of CYP46A1, LXRα, and ApoE levels were assayed following subtoxic glutamate treatment in SH-SY5Y cells as well as HT-22 cells lacking iGluRs. The ratio of 24S-hydroxycholesterol to cholesterol was elevated in the adult rats exposed to monosodium l-glutamate before the weaning age, compared to the control. The blockers of NMDA receptor (MK801) and mGluR5 (MPEP) attenuated the glutamate-induced loss of cholesterol and elevation of 24S-hydroxycholesterol level in SH-SY5Y cells. The induction of the mRNA levels of CYP46A1, LXRα, and ApoE by glutamate was observed in both SH-SY5Y cells and HT-22 cells; additionally, MK801 and MPEP attenuated the increases in these genes in SH-SY5Y cells. The increase in the binding of LXRα proteins with ApoE promoter following glutamate treatment was attenuated by MK801. The luciferase assay indicated the binding of CREB protein with CYP46A1 promoter, and the glutamate-induced CREB expression was inhibited by MK801. The results suggest that glutamate, the major excitatory neurotransmitter, may affect the metabolism and redistribution of cholesterol in the neuronal cells via its specific receptors during chronic exposure.

Glutamate affects the CYP1B1- and CYP2U1-mediated hydroxylation of arachidonic acid metabolism via astrocytic mGlu5 receptor.

The extrahepatic CYP enzymes, CYP1B1 and CYP2U1, have been predominantly found in both astrocytes and brain microvessels. We investigated the alteration in the production of hydroxyeicosatetraenoic acids (HETEs) from arachidonic acid (AA) mainly via CYP1B1 and CYP2U1 by glutamate. CYP1B1 and CYP2U1 mRNA levels were dose-dependently induced by glutamate in human U251 glioma cells and hCMEC/D3 blood-brain barrier cells. The increases in the CYP1B1 and CYP2U1 mRNA levels and the binding of CREB to CYP1B1 and CYP2U1 promoters following glutamate treatment were attenuated by mGlu5 receptor antagonist. The mRNA levels of CYP1B1 and CYP2U1 were increased in the cortex, hippocampus, and cerebellum from adult rats that received a subcutaneous injection of monosodium l-glutamate at 1, 3, 5, and 7 days of age; meanwhile, the protein levels of CYP1B1 and CYP2U1 in the astrocytes were induced by glutamate. Glutamate treatment significantly increased the production of 5-HETE, 8-HETE, 11-HETE, and 20-HETE in the cortex and cerebellum. These data suggested that the neuron-astrocyte reciprocal signaling can change the CYP-mediated AA metabolism (e.g. EETs and HETEs) in astrocytes via its specific receptor.

Effects of norfloxacin exposure on neurodevelopment of zebrafish (Danio rerio) embryos.

In view of the wide application of fluoroquinolones (FQs), a group of broad-spectrum synthetic antibacterial agents, and their large ingress into the environment, the toxic effects on non-target organisms caused by FQs have received great attention. In this study, we used zebrafish embryo as a model, measured the general toxic effects of norfloxacin, a commonly used FQs, and investigated the effects of norfloxacin on the neurodevelopment of zebrafish embryos. Our data showed that norfloxacin significantly inhibited the hatching rate of zebrafish embryos, and increased the mortality and malformation rate of the embryos. To discuss the developmental neurotoxicity of norfloxacin, we measured the expression of several stem cell and neuron lineage markers in the zebrafish embryos. We found that norfloxacin exposure inhibited the expression of GFAP (glial cell marker), and enhanced the expression of Sox 2 (stem cell marker) and Eno2 (mature neuron marker). By measuring the level of active Caspase 3 and the expression ratio of Bax to Bcl2, we discovered that norfloxacin induced obvious cell apoptosis in the brain of zebrafish embryos. To explore the mechanism of the developmental neurotoxic effects of norfloxacin, we applied MK-801, a non-competitive NMDA receptors antagonist, to block the actions of NMDA receptors. The results indicated that MK-801 could rescue the upregulated cell apoptosis and disrupted balance of neuro-glial differentiation induced by norfloxacin in the brain of zebrafish embryos. Our results suggest that the activation of NMDA receptors mediates the developmental neurotoxicity of norfloxacin.

The Protective Role of Brain CYP2J in Parkinson's Disease Models.

CYP2J proteins are present in the neural cells of human and rodent brain regions. The aim of this study was to investigate the role of brain CYP2J in Parkinson's disease. Rats received right unilateral injection with lipopolysaccharide (LPS) or 6-hydroxydopamine (6-OHDA) in the substantia nigra following transfection with or without the CYP2J3 expression vector. Compared with LPS-treated rats, CYP2J3 transfection significantly decreased apomorphine-induced rotation by 57.3% at day 12 and 47.0% at day 21 after LPS treatment; moreover, CYP2J3 transfection attenuated the accumulation of α-synuclein. Compared with the 6-OHDA group, the number of rotations by rats transfected with CYP2J3 decreased by 59.6% at day 12 and 43.5% at day 21 after 6-OHDA treatment. The loss of dopaminergic neurons and the inhibition of the antioxidative system induced by LPS or 6-OHDA were attenuated following CYP2J3 transfection. The TLR4-MyD88 signaling pathway was involved in the downregulation of brain CYP2J induced by LPS, and CYP2J transfection upregulated the expression of Nrf2 via the inhibition of miR-340 in U251 cells. The data suggest that increased levels of CYP2J in the brain can delay the pathological progression of PD initiated by inflammation or neurotoxins. The alteration of the metabolism of the endogenous substrates (e.g., AA) could affect the risk of neurodegenerative disease.

The Involvement of PPARs in the Selective Regulation of Brain CYP2D by Growth Hormone.

Brain CYP2D is responsible for the synthesis of endogenous neurotransmitters such as dopamine and serotonin. This study is to investigate the effects of cerebral CYP2D on mouse behavior and the mechanism whereby growth hormone regulates brain CYP2D. The inhibition of cerebellar CYP2D significantly affected the spatial learning and exploratory behavior of mice. CYP2D expression was lower in the brain in GHR-/- mice than that in WT mice; however, hepatic CYP2D levels were similar. Brain PPARα expression in male GHR-/- mice were markedly higher than those in WT mice, while brain PPARγ levels were decreased or unchanged in different regions. However, both hepatic PPARα and PPARγ in male GHR-/- mice were markedly higher than those in WT mice. Pulsatile GH decreased the PPARα mRNA level and increased the mRNA levels of CYP2D6 and PPARγ in SH-SY5Y cells. A luciferase assay showed that PPARγ activated the CYP2D6 gene promoter while PPARα inhibited its function. Pulsatile GH decreased the binding of PPARα to the CYP2D6 promoter by 40% and promoted the binding of PPARγ to the CYP2D6 promoter by approximately 60%. The male GH secretory pattern altered PPAR expression and the binding of PPARs to the CYP2D promoter, leading to the elevation of brain CYP2D in a tissue-specific manner. Growth hormone may alter the learning and memory functions in patients receiving GH replacement therapy via brain CYP2D.

The induction of cytochrome P450 2E1 by ethanol leads to the loss of synaptic proteins via PPARα down-regulation.

Ethanol, one of the most commonly abused substances throughout history, is a substrate and potent inducer of cytochrome P450 2E1 (CYP2E1). Our previous study showed that brain CYP2E1 was induced by chronic ethanol treatment and was associated with ethanol-induced neurotoxicity in rats. We therefore investigated the possible mechanism of brain CYP2E1 involvement in ethanol-induced neurodegeneration. Compared with the controls, chronic ethanol treatment (3.0g/kg, i.g., 160days) significantly increased CYP2E1 mRNA levels in the rat cortex, but the mRNA levels of peroxisome proliferator-activated receptor α (PPARα) and the pre- and post-synaptic proteins (synaptophysin, SYP, and drebrin1, DBN1) were decreased. Ethanol treatment dose-dependently induced CYP2E1 mRNA expression, and CYP2E1 overexpression exacerbated the ethanol-induced neurotoxicity. Pretreatment with p38 inhibitor (SB202190) and ERK1/2 inhibitor (U0126) attenuated the induction of CYP2E1 mRNA and protein levels by ethanol; however, no change was observed with JNK inhibitor pretreatment. Ethanol exposure or CYP2E1 overexpression significantly decreased PPARα, SYP, and DBN1 expression as indicated by the data from real-time RT-PCR, Western blotting and immunocytochemistry. The activation of PPARα by WY14643 increased the activity of the SYP and DBN1 promoters and attenuated the inhibition of these genes by ethanol. The specific siRNA for CYP2E1 significantly attenuated the ethanol-induced inhibition of PPARα, SYP and DBN1 mRNA levels. These results suggest that the induction of CYP2E1 by ethanol may be mediated via the p38 and ERK1/2 signaling pathways in neurons but not via the JNK pathway. The CYP2E1-PPARα axis may play a role in ethanol-induced neurotoxicity via the alteration of the genes related with synaptic function.

Glutamate affects the production of epoxyeicosanoids within the brain: The up-regulation of brain CYP2J through the MAPK-CREB signaling pathway.

Glutamate is the major excitatory neurotransmitter in the brain, and chronic glutamate excitotoxicity has been thought to be involved in numerous neurodegenerative diseases. We investigated the effects of glutamate at concentrations lower than the usual extrasynaptic concentrations on the production of epoxyeicosanoids mediated by brain CYP2J. Glutamate increased CYP2J2 mRNA levels in astrocytes in a dose-dependent manner, while an antagonist of the metabotropic glutamate receptor subtype 5 (mGlu5 receptor) attenuated the glutamate-induced increases in CYP2J2 levels by glutamate. Glutamate increased the binding of cAMP response element-binding protein (CREB) with the CYP2J2 promoter, and the inhibition of the MAPK signaling pathway (ERK1/2, p38, and JNK) decreased the binding of CREB with the CYP2J2 promoter following the glutamate treatment. CREB activated the CYP2J2 promoter located at -1522 to -1317bp, and CREB overexpression significantly increased CYP2J2 mRNA levels. The CYP2J2 and mGlu5 mRNA levels were higher in the frontal cortex, hippocampus, cerebellum, and brainstem in adult rats that received a subcutaneous injection of monosodium l-glutamate at 1, 3, 5, and 7days of age. The data from the partial least-squares-discriminant analysis showed the epoxyeicosanoid profile of the hippocampus from the cerebellum, brain stem, and frontal cortex. The sum of the epoxyeicosatrienoic acids (EETs) and dihydroxyeicosatrienoic acids (DHETs) was increased by 1.16-fold, 1.18-fold, and 1.19-fold in the frontal cortex, cerebellum, and brain stem, respectively, in rats treated with monosodium l-glutamate compared with the control group. The results suggest that brain CYP2J levels and CYP2J-mediated epoxyeicosanoid production can be regulated by extrasynaptic glutamate. The glutamate receptors expressed in astrocytes may mediate the regulation of drug-metabolizing enzymes and the metabolome of endogenous substances by glutamate.

Negative effects of retinoic acid on stem cell niche of mouse incisor.

The continuous growth of mouse incisors depends on epithelial stem cells (SCs) residing in the SC niche, called labial cervical loop (LaCL). The homeostasis of the SCs is subtly regulated by complex signaling networks. In this study, we focus on retinoic acid (RA), a derivative of Vitamin A and a known pivotal signaling molecule in controlling the functions of stem cells (SCs). We analyzed the expression profiles of several key molecules of the RA signaling pathway in cultured incisor explants upon exogenous RA treatment. The expression patterns of these molecules suggested a negative feedback regulation of RA signaling in the developing incisor. We demonstrated that exogenous RA had negative effects on incisor SCs and that this was accompanied by downregulation of Fgf10, a mesenchymally expressed SC survival factor in the mouse incisor. Supplement of Fgf10 in incisor cultures completely blocked RA effects by antagonizing apoptosis and increasing proliferation in LaCL epithelial SCs. In addition, Fgf10 obviously antagonized RA-induced downregulation of the SC marker Sox2 in incisor epithelial SCs. Our findings suggest that the negative effects of RA on incisor SCs result from inhibition of mesenchymal Fgf10.

GT198 Expression Defines Mutant Tumor Stroma in Human Breast Cancer.

Human breast cancer precursor cells remain to be elucidated. Using breast cancer gene product GT198 (PSMC3IP; alias TBPIP or Hop2) as a unique marker, we revealed the cellular identities of GT198 mutant cells in human breast tumor stroma. GT198 is a steroid hormone receptor coactivator and a crucial factor in DNA repair. Germline mutations in GT198 are present in breast and ovarian cancer families. Somatic mutations in GT198 are present in ovarian tumor stromal cells. Herein, we show that human breast tumor stromal cells carry GT198 somatic mutations and express cytoplasmic GT198 protein. GT198(+) stromal cells share vascular smooth muscle cell origin, including myoepithelial cells, adipocytes, capillary pericytes, and stromal fibroblasts. Frequent GT198 mutations are associated with GT198(+) tumor stroma but not with GT198(-) tumor cells. GT198(+) progenitor cells are mostly capillary pericytes. When tested in cultured cells, mutant GT198 induces vascular endothelial growth factor promoter, and potentially promotes angiogenesis and adipogenesis. Our results suggest that multiple lineages of breast tumor stromal cells are mutated in GT198. These findings imply the presence of mutant progenitors, whereas their descendants, carrying the same GT198 mutations, are collectively responsible for forming breast tumor microenvironment. GT198 expression is, therefore, a specific marker of mutant breast tumor stroma and has the potential to facilitate diagnosis and targeted treatment of human breast cancer.

Sex hormones regulate cerebral drug metabolism via brain miRNAs: down-regulation of brain CYP2D by androgens reduces the analgesic effects of tramadol.

BACKGROUND AND PURPOSE: Brain cytochrome P450 2D (CYP2D) metabolises exogenous neurotoxins, endogenous substances and neurotransmitters. Brain CYP2D can be regulated in an organ-specific manner, but the possible regulatory mechanisms are poorly understood. We investigated the involvement of miRNAs in the selective regulation of brain CYP2D by testosterone and the corresponding alteration of the pharmacological profiles of tramadol by testosterone. EXPERIMENTAL APPROACH: The regulation of CYP2D and brain-enriched miRNAs by testosterone was investigated using SH-SY5Y cells, U251 cells, and HepG2 cells as well as orchiectomized growth hormone receptor knockout (GHR-KO) mice and rats. Concentration-time curves of tramadol in rat brain were determined using a microdialysis technique. The analgesic action of tramadol was assessed by the tail-flick test in rats. KEY RESULTS: miR-101 and miR-128-2 bound the 3'-untranslated region of the CYP2D6 mRNA and decreased its level. Testosterone decreased CYP2D6 catalytic function via the up-regulation of miR-101 and miR-128-2 in SH-SY5Y and U251 cells, but not in HepG2 cells. Orchiectomy decreased the levels of miR-101 and miR-128-2 in the hippocampus of male GHR-KO mice, indicating that androgens regulate miRNAs directly, not via the alteration of growth hormone secretion patterns. Changes in the pharmacokinetic and pharmacodynamic profiles of tramadol by orchiectomy was attenuated by either testosterone supplementation or a specific brain CYP2D inhibitor. CONCLUSIONS AND IMPLICATIONS: The selective regulation of brain CYP2D via brain-enriched miRNAs, following changes in androgen levels, such as in testosterone therapy, androgen deprivation therapy and/or ageing may alter the response to centrally active substances.

Mesenchymal Wnt/ß-Catenin Signaling Controls Epithelial Stem Cell Homeostasis in Teeth by Inhibiting the Antiapoptotic Effect of Fgf10.

Continuous growth of rodent incisors relies on epithelial stem cells (SCs) located in the SC niche called labial cervical loop (LaCL). Here, we found a population of apoptotic cells residing in a specific location of the LaCL in mouse incisor. Activated Caspase 3 and Caspase 9, expressed in this location colocalized in part with Lgr5 in putative SCs. The addition of Caspase inhibitors to incisors ex vivo resulted in concentration dependent thickening of LaCL. To examine the role of Wnt signaling in regulation of apoptosis, we exposed the LaCL of postnatal day 2 (P2) mouse incisor ex vivo to BIO, a known activator of Wnt/ß-catenin signaling. This resulted in marked thinning of LaCL as well as enhanced apoptosis. We found that Wnt/ß-catenin signaling was intensely induced by BIO in the mesenchyme surrounding the LaCL, but, unexpectedly, no ß-catenin activity was detected in the LaCL epithelium either before or after BIO treatment. We discovered that the expression of Fgf10, an essential growth factor for incisor epithelial SCs, was dramatically downregulated in the mesenchyme around BIO-treated LaCL, and that exogenous Fgf10 could rescue the thinning of the LaCL caused by BIO. We conclude that the homeostasis of the epithelial SC population in the mouse incisor depends on a proper rate of apoptosis and that this apoptosis is controlled by signals from the mesenchyme surrounding the LaCL. Fgf10 is a key mesenchymal signal limiting apoptosis of incisor epithelial SCs and its expression is negatively regulated by Wnt/ß-catenin. Stem Cells 2015;33:1670-1681.

Sex-dependent regulation of hepatic CYP3A by growth hormone: Roles of HNF6, C/EBPα, and RXRα.

Sex-based differences in the pharmacological profiles of many drugs are due in part to the female-predominant expression of CYP3A4, which is the most important CYP isoform responsible for drug metabolism. Transcription factors trigger the sexually dimorphic expression of drug-metabolizing enzymes in response to sex-dependent growth hormone (GH) secretion. We investigated the roles of HNF6, C/EBPα, and RXRα in the regulation of human female-predominant CYP3A4, mouse female-specific CYP3A41, and rat male-specific CYP3A2 expression by GH secretion patterns using HepG2 cells, growth hormone receptor (GHR) knockout mice as well as rat models of orchiectomy and hypophysectomy. The constitutive expression of HNF6 and RXRα was GH-dependent, and GHR deficiency decreased HNF6/C/EBPα complex levels and increased HNF6/RXRα complex levels. Feminine GH secretion induced the binding of HNF6 and C/EBPα to the CYP3A4 and Cyp3a41 promoters and HNF6/C/EBPα complex levels was more efficiently compared with masculine pattern. Additionally, a greater inhibition of the binding of RXRα to the CYP3A4 and Cyp3a41 promoters and HNF6/RXRα complex levels was observed by feminine GH secretion, but less inhibition was observed by masculine pattern. The binding of HNF6, C/EBPα, and RXRα to the CYP3A2 promoter was not directly regulated by androgens. RXRα completely abolished the synergistic activation of the CYP3A4, Cyp3a41, and CYP3A2 promoters by HNF6 and C/EBPα. The results demonstrate that sex-dependent GH secretion patterns affect the expressions and interactions of HNF6, C/EBPα, and RXRα as well as their binding to CYP3A genes. RXRα mediates the sex-dependent influence of GH on CYP3A expression as an important signalling molecule.

Reduction of thyroid hormones triggers down-regulation of hepatic CYP2B through nuclear receptors CAR and TR in a rat model of acute stroke.

Stroke is a neurological condition and may cause changes in hepatic drug-metabolizing enzymes. Hepatic CYP2B is involved in the metabolism of a variety of centrally active substances. The purpose of this study was to investigate the possible down-regulation mechanism of hepatic CYP2B after acute stroke. Using a rat model of acute stroke induced by middle cerebral artery occlusion, we studied the influence of brain ischemia/reperfusion (I/R) injury on CYP2B expression. Effects of 3,5,3'-triiodo-L-thyronine (T3) treatment on constitutive androstane receptor (CAR) and thyroid hormone receptors (TRs, including TRα and TRß) proteins were detected in Huh7 cells. We found dramatic decreases in the levels of plasma free triiodthyronine, free thyroxine and hepatic CYP2B expression. Both CAR and retinoid X receptor alpha (RXRα) were significantly dissociated from the phenobarbital-responsive enhancer module (PBREM) of the CYP2B1 promoter in the early stages of the acute stroke. The levels of the polymer of TRs, CAR, and RXRα were time-dependently decreased after brain I/R injury. T3 regulated the CAR expression at the transcriptional level, and facilitated the translocation of TRα/ß proteins as well as the binding of TRs, RXRα, and CAR to PBREM region. The reduction of thyroid hormone levels after a brain I/R injury may be the initial trigger for the down-regulation of hepatic CYP2B1 via induction of the dissociation of CAR from the TRs and from the PBREM region. Our data suggest that patients with acute ischemic stroke may have a decreased CYP2B-mediated metabolism of exogenous and endogenous compounds because of the low level of thyroid hormones.

GT198 Splice Variants Display Dominant-Negative Activities and Are Induced by Inactivating Mutations.

Alternative pre-mRNA splicing yields functionally distinct splice variants in regulating normal cell differentiation as well as cancer development. The putative tumor suppressor gene GT198 (PSMC3IP), encoding a protein also known as TBPIP and Hop2, has been shown to regulate steroid hormone receptor-mediated transcription and to stimulate homologous recombination in DNA repair. Here, we have identified 6 distinct GT198 splice variant transcripts generated by alternative promoter usage or alternative splicing. Various splice variant transcripts preserve a common open reading frame, which encodes the DNA binding domain of GT198. The splice variants act as dominant negatives to counteract wild-type GT198 activity in transcription and to abolish Rad51 foci formation during radiation-induced DNA damage. In fallopian tube cancer, we have identified 44 point mutations in GT198 clustered in 2 mutation hotspot sequences. The mutation hotspots coincide with the regulatory sequences responsible for alternative splicing, strongly supporting that imbalanced alternative splicing is a selected consequence in cancer. In addition, splice variant-associated cytoplasmic expression is found in tumors carrying germline or somatic GT198 mutations. An altered alternative splicing pattern with increased variants is also present in lymphoblastoid cells derived from familial breast cancer patients carrying GT198 germline mutations. Furthermore, GT198 and its variant are reciprocally expressed during mouse stem cell differentiation. The constitutive expression of the GT198 variant but not the wild type induces tumor growth in nude mice. Our results collectively suggest that mutations in the GT198 gene deregulate alternative splicing. Defective alternative splicing promotes antagonizing variants and in turn induces a loss of the wild type in tumorigenesis. The study highlights the role of alternative splicing in tumor suppressor gene inactivation.

Suppressive effect of triadimefon, a triazole fungicide, on spatial learning and reference memory in rats.

Although some central effects of triadimefon, a triazole fungicide, have been reported, its effects on spatial memory have not been examined. In this study, we used the Morris water maze to study the effect of triadimefon on spatial learning and memory in rats. To elucidate the mechanism of this effect, we also measured the retinoic acid concentration in the hippocampus by high-performance liquid chromatography. Our data showed that triadimefon inhibited spatial learning and impaired spatial reference memory, and decreased hippocampal retinoic acid concentration. There is evidence that triadimefon can regulate the metabolism of retinoic acid, which serves a critical function in the development and maintenance of spatial memory. Therefore, we speculate that the reduction in hippocampal retinoic acid concentration induced by triadimefon might be responsible for its suppressive effect on spatial learning and reference memory.