Glucocorticoid regulation of beta-adrenergic receptors in 3T3-L1 preadipocytes

Treatment of 3T3-L1 preadipocytes (fibroblasts) with 250 nM dexamethasone for 48 hr caused a doubling of total beta-adrenergic receptors and an increase in beta 2-adrenergic receptor subtype proportion from approximately 50% in controls to 85% in treated cells. The responses to epinephrine and norepinephrine in a whole cell cAMP accumulation assay reflected these changes. The effects of dexamethasone on beta-adrenergic receptors were mediated through the glucocorticoid receptor and were time and dose dependent with an EC50 of 2.77 +/- 0.73 nM for an increase in the proportion of beta 2-adrenergic receptors. The rank order of potency of steroids to effect these changes (betamethasone = dexamethasone greater than fludrocortisone greater than hydrocortisone = triamcinolone greater than aldosterone) correlated with their glucocorticoid potency. [3H]Dexamethasone binding to intact cells yielded a KD value of 3.47 +/- 0.38 nM for binding to the glucocorticoid receptor which correlated well with the EC50 for dexamethasone to alter beta-adrenergic receptors. Inhibition of [3H]dexamethasone binding by other steroids confirmed that the ability of steroids to regulate beta-adrenergic receptors correlated with the affinity of each compound for the 3T3-L1 glucocorticoid receptor. Progesterone, which can bind to the glucocorticoid receptor but has only weak agonist activity, competitively inhibited the ability of dexamethasone to alter beta-adrenergic receptors. Protein synthesis, RNA synthesis, and N-linked glycosylation appeared to be necessary for the change in receptor subtype expression and the increase in beta-adrenergic receptor number induced by dexamethasone. The present study suggests that regulation of beta-adrenergic receptor expression in 3T3-L1 preadipocytes by dexamethasone is a glucocorticoid-specific effect which may require gene activation.     

基于报告基因的雌激素受体β亚型激动剂细胞筛选模型的建立

目的建立基于报告基因和雌激素受体β(estrogen receptor β，ERβ)亚型信号通路的药物筛选模型，以期发现ERβ亚型激动剂。方法构建带有雌激素应答序列(estrogen responsive element，ERE)靶序列和报告基因的诱导性表达载体pTAL-ERE-SEAP，并将其转入人胚肾(HEK293)细胞中，筛选报告基因分泌型碱性磷酸酶(secreted alkaline phosphatase，SEAP)表达受雌二醇(17β-estradiol，E₂)诱导的阳性克隆。选取相关核受体配体进行模型的特异性考察，同时考察模型的稳定性及时效量效关系，以及免疫细胞化学染色鉴定转染后ERβ的表达情况。利用此模型对本实验室样品库中的2 622个化合物进行筛选。结果转染pTAL-ERE-SEAP的HEK293细胞中，SEAP报告基因的表达受E₂的诱导并呈剂量与时间依赖关系，E₂对阳性克隆细胞无增殖作用，本模型的Z′因子值为0.7，免疫细胞化学染色结果表明，转染后ERβ表达、地塞米松以及其他配体的诱导表达率低。结论利用此模型通过测定SEAP报告基因的诱导表达水平可筛选ERβ亚型激动剂。     

Increased glucocorticoid receptor gene promoter activity after antidepressant treatment.

We have tested the hypothesis that antidepressants affect the expression of the glucocorticoid receptor gene, by looking at glucocorticoid receptor gene promoter activity, glucocorticoid receptor mRNA levels, and glucocorticoid-binding activity after treatment of different cell lines with desipramine. Treatment of LTK- cells or Neuro 2A cells with desipramine produced a 50-200% increase in chloramphenicol acetyltransferase activity transcribed from a 2.7-kilobase glucocorticoid receptor gene promoter region. In cell lines derived from both neuronal and non-neuronal sources, glucocorticoid receptor mRNA concentration doubled after desipramine treatment, and this was associated with a 2-fold higher functional glucocorticoid binding capacity and increased glucocorticoid sensitivity, as measured with the reporter plasmid pMMTVCAT. Antidepressant-induced increases in glucocorticoid receptor gene promoter activity, glucocorticoid receptor mRNA levels, and functional glucocorticoid binding activity suggest a novel mechanism of action for these drugs on the hypothalamic-pituitary-adrenal axis.     

Dexamethasone suppresses the expression of multiple rat carboxylesterases through transcriptional repression: evidence for an involvement of the glucocorticoid receptor

Carboxylesterases play important roles in the metabolism of xenobiotics and detoxication of insecticides. Without exception, all mammalian species studied express multiple forms of carboxylesterases. Several rat carboxylesterases are well-characterized including hydrolase A, B and S, and the expression of these enzymes is significantly suppressed by glucocorticoid dexamethasone. In this study, we used multiple experimental systems and presented a molecular mechanism for the suppression. Rats receiving one or more daily injections of dexamethasone consistently expressed lower HA, HB and HS. The suppression occurred at the levels of mRNA, protein and hydrolytic activity. In hepatoma cell line H4-II-E-C3, nanomolar dexamethasone caused significant decreases in HA, HB and HS mRNA, and the decreases were abolished by antiglucocorticoid RU486. Additionally, dexamethasone at nanomolar concentrations repressed the promoters of carboxylesterases, and the repression was reduced by glucocorticoid receptor-beta, a dominant negative regulator of the glucocorticoid receptor (GR). In contrast, co-transfection of the pregnane X receptor (PXR) increased the reporter activities, but the increase occurred only at micromolar concentrations of dexamethasone. These findings establish that both GR and PXR are involved in the regulated expression of rat carboxylesterases by dexamethasone but their involvement depends on the concentrations.