[Show abstract][Hide abstract] ABSTRACT: Erythropoietin (Epo) is required for erythroid progenitor differentiation. Although Epo crosslinking experiments have revealed the presence of Epo receptor (EpoR)-associated proteins that could never be identified, EpoR is considered to be a paradigm for homodimeric cytokine receptors. We purified EpoR-binding partners and identified the type 2 transferrin receptor (TfR2) as a component of the EpoR complex corresponding to proteins previously detected in cross-linking experiments. TfR2 is involved in iron metabolism by regulating hepcidin production in liver cells. We show that TfR2 and EpoR are synchronously coexpressed during the differentiation of erythroid progenitors. TfR2 associates with EpoR in the endoplasmic reticulum and is required for the efficient transport of this receptor to the cell surface. Erythroid progenitors from TfR2(-/-)mice show a decreased sensitivity to Epo and increased circulating Epo levels. In human erythroid progenitors, TfR2 knockdown delays the terminal differentiation. Erythroid cells produce growth differentiation factor-15, a cytokine that suppresses hepatic hepcidin production in certain erythroid diseases such as thalassemia. We show that the production of growth differentiation factor-15 by erythroid cells is dependent on both Epo and TfR2. Taken together, our results show that TfR2 exhibits a non hepatic function as a component of the EpoR complex and is required for efficient erythropoiesis.
[Show abstract][Hide abstract] ABSTRACT: Five geranylflavonoids, one prenylated flavonoid, and a simple flavanone were isolated from an ethanolic extract of Paulownia tomentosa fruit. Tomentodiplacol (1), 3'-O-methyl-5'-methoxydiplacol (2), 6-isopentenyl-3'-O-methyltaxifolin (3), and dihydrotricin (4) are reported from a natural source for the first time and 3'-O-methyldiplacone (6) for the first time from the genus Paulownia. The structures of the compounds were determined by mass spectrometry, including HRMS, and by 1D and 2D NMR spectroscopy. The cytotoxicity and DPPH (2,2-diphenyl-1-picrylhydrazyl)-quenching activity of some of these compounds were tested, with diplacone proving to be the best antioxidant, although the most cytotoxic compound.
[Show abstract][Hide abstract] ABSTRACT: Control of intensity and duration of erythropoietin (Epo) signaling is necessary to tightly regulate red blood cell production. We have recently shown that the ubiquitin/proteasome system plays a major role in the control of Epo-R signaling. Indeed, after Epo stimulation, Epo-R is ubiquitinated and its intracellular part is degraded by the proteasome, preventing further signal transduction. The remaining part of the receptor and associated Epo are internalized and degraded by the lysosomes. We show that beta-Trcp is responsible for Epo-R ubiquitination and degradation. After Epo stimulation, beta-Trcp binds to the Epo-R. This binding, like Epo-R ubiquitination, requires Jak2 activation. The Epo-R contains a typical DSG binding sequence for beta-Trcp that is highly conserved among species. Interestingly, this sequence is located in a region of the Epo-R that is deleted in patients with familial polycythemia. Mutation of the serine residue of this motif to alanine (Epo-RS462A) abolished beta-Trcp binding, Epo-R ubiquitination, and degradation. Epo-RS462A activation was prolonged and BaF3 cells expressing this receptor are hypersensitive to Epo, suggesting that part of the hypersensitivity to Epo in familial polycythemia could be the result of the lack of beta-Trcp recruitment to the Epo-R.
[Show abstract][Hide abstract] ABSTRACT: Cytochrome P4501A activity, oxidative stress and inhibition of gap junctional intercellular communication (GJIC) are involved in metabolic activation of promutagens and tumor-promoting activity of various xenobiotics, and their prevention is considered to be an important characteristic of chemoprotective compounds. In this study, a series of 31 chalcones and their corresponding dihydroderivatives, substituted in 2,2'-, 3,3'-, 4- or 4'-position by hydroxyl or methoxy group, were tested for their ability to inhibit Fe(II)/NADPH-enhanced lipid peroxidation and cytochrome P4501A-dependent 7-cethoxyresorufin-O-deethylase (EROD) activity in rat hepatic microsomes. Effects of the compounds on GJIC were determined in rat liver epithelial WB-F344 cells. Most of the chalcones and dihydrochalcones inhibited EROD activity in a dose-dependent manner at the range 0.25-25 microM, which was comparable to model flavonoid inhibitors alpha-naphthoflavone and quercetin. The chalcones exhibited higher inhibition activity than the corresponding dihydroderivatives. Mono and dihydroxylated chalcones, and dihydrochalcones showed none or only a weak antioxidant activity; trihydroxyderivatives inhibited in vitro lipid peroxidation significantly only at 50 microM concentration. Potential adverse effects, namely inhibition of GJIC and/or cytotoxicity were detected after treatment of WB-F344 cells with a number of chalcone and dihydrochalcone derivatives, suggesting that they should be excluded from additional screening as chemoprotective compounds. Chalcones and dihydrochalcones substituted at 4- and/or 4'-position, which elicited no inhibition of GJIC, were further tested for the potential enhancing effects on GJIC. The present data seem to suggest that 4-hydroxy, 2',4'-dihydroxy-3-methoxy, 2,4,4'-trihydroxy, and 2',4,4'-trihydroxychalcone, 2',4-dihydroxy and 2'-hydroxy-3,4-dimethoxydihydrochalcone might be promising chemoprotective compounds against CYP1A activity, and partly also against oxidative damage without inducing adverse effects, such as GJIC inhibition. In general, determination of potencies of tested compounds to inhibit GJIC should be involved in any set of methods for the in vitro screening of chemoprotective characteristics of potential drugs, in order to reveal their potential adverse effects associated with tumor promotion.
[Show abstract][Hide abstract] ABSTRACT: Both epigenetic and mutagenic events influence the process of carcinogenesis. Any mutagenic event irreversibly alters the genomic information of the cell. Epigenetic activities of compounds do not damage genes directly, but they can alter the gene expression. The most important consequences include alteration of cell proliferation, differentiation, or apoptosis. One of them, which apparently link to the promotion of carcinogenesis is the inhibition of gap-junctional intercellular communication (GJIC) in vitro. These gap junctions allow ions and low-molecular-weight molecules to move between coupled cells, thereby facilitating synchronization of electrotonic or metabolic cooperation. Transient down-regulation by endogenous or exogenous chemicals can bring about adaptive or maladaptive consequences depending on circumstances. Tumor promotors (e.g. DDT) and growth factors reduce GJIC, whereas anticarcinogens and growth inhibitors (e.g., flavonoids, resveratrole, carotenoids) enhance GJIC. Prevention of inhibition of GJIC could be an important chemopreventive strategy of cancer therapy.