Bisphenol A [2,2-bis-(4-hydroxyphenyl) propane; BPA] is a versatile industrial material for plastic products, but is increasingly being recognized as a pervasive industrial pollutant as well. Accumulating evidence indicates that the environmental contaminant BPA is one of the endocrine-disrupting chemicals that potentially can adversely affect humans as well as wildlife. To define the molecular aspects of BPA action, we first investigated the molecules with which it physically interacts. High BPA-binding activity was detected in the P2 membrane fraction prepared from rat brains. As determined by SDS-PAGE analysis, the molecular mass of a BPA-binding protein purified from the rat brain P2 fraction was 53 kDa. The N-terminal amino acid sequence of the purified BPA-binding protein was identical with that of the rat protein disulfide isomerase (PDI), which is a multifunctional protein that is critically involved in the folding, assembly, and shedding of many cellular proteins via its isomerase activity in addition to being considered to function as an intracellular hormone reservoir. The Kd value of BPA binding to recombinant rat PDI was 22.6 +/- 6.6 microm. Importantly, the binding activity of L-T3 and 17beta-estradiol hormones to PDI was competitively inhibited by BPA in addition to abolishing its isomerase activities. In this paper we report that the ubiquitous and multifunctional protein PDI is a target of BPA and propose that binding to PDI and subsequent inhibition of PDI activity might be mechanistically responsible for various actions of BPA.
Apigenin, a plant-derived flavone, is a potent inhibitor of cell proliferation and angiogenesis, but the mechanisms leading to the pathological anti-angiogenic effects of apigenin are still unclear. In this study, we found that apigenin inhibited the hypoxia-induced expression of vascular endothelial growth factor (VEGF) mRNA in human umbilical artery endothelial cells. Apigenin also suppressed the expression of erythropoietin mRNA, which is a typical hypoxia-inducible gene, via the degradation of hypoxia-inducible factor 1 (HIF-1) a. We investigated the effect of apigenin on the interaction of HIF-1a with heat shock protein 90 (Hsp90), which is reported to be important for the stabilization of HIF-1a, and found that VEGF expression was inhibited via degradation of HIF-1a through interference with the function of Hsp90.
mRNA of cytochrome P450 21-hydroxylase (P450c21) is expressed in the brain, but little is known about the enzymatic properties of P450c21 in the brain. In the present study, we showed, by using various recombinant cytochrome P450 (CYP)2D enzymes and anti-CYP2D4- or P450c21-specific antibodies, that rat brain microsomal steroid 21-hydroxylation is catalyzed not by P450c21, but by CYP2D isoforms. Rat CYP2D4 and human CYP2D6, which are the predominant CYP2D isoforms in the brain, possess 21-hydroxylation activity for both progesterone and 17alpha-hydroxyprogesterone. In rat brain microsomes, these activities were not inhibited by anti-P450c21 antibodies, but they were effectively inhibited by the CYP2D-specific chemical inhibitor quinidine and by anti-CYP2D4 antibodies. mRNA and protein of CYP2D4 were expressed throughout the brain, especially in cerebellum, striatum, pons, and medulla oblongata, whereas the mRNA and protein levels of P450c21 were extremely low or undetectable. These results support the idea that CYP2D4, not P450c21, works as steroid 21-hydroxylase in the brain. Allopregnanolone, a representative gamma-aminobutyric acid receptor modulator, was also hydroxylated at the C-21 position by recombinant CYP2D4 and CYP2D6. Rat brain microsomal allopregnanolone 21-hydroxylation was inhibited by fluoxetine with an IC(50) value of 2 microm, suggesting the possibility that the brain CYP2D isoforms regulate levels of neurosteroids such as allopregnanolone, and that this regulation is modified by central nervous system-active drugs such as fluoxetine.
Hypoxia induces a group of physiologically important genes that include erythropoietin (EPO) and vascular endothelial growth factor (VEGF). Hypoxia-inducible factor 1 (HIF-1) was identified as a hypoxia-activated transcription factor; however, the molecular mechanisms that underlie hypoxia signal transduction in mammalian cells remain undefined. In this study, we found that a flavoprotein, NADPH-P450 reductase (NPR), could regulate the induction of EPO mRNA under hypoxic conditions. Hypoxic EPO mRNA induction in Hep3B cells was inhibited by diphenyleneiodonium chloride, which is an inhibitor of NADPH-dependent enzymes. NPR antisense cDNA was transfected into Hep3B cells, and NPR-deficient hepatocyte cells (NPR These results suggested that NPR located at the plasma membrane regulates EPO expression in hypoxia, including HIF-1 activation and translocation. We further studied the expression of NPR and VEGF mRNAs in human tumor tissues and found that the NPR mRNA levels were correlated with the VEGF mRNA levels, suggesting that NPR might be an important factor in the hypoxic induction of genes such as VEGF in vivo.
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