Oxidized low density lipoprotein increases RANKL level in human vascular cells. Involvement of oxidative stress.

Receptor Activator of NFκB Ligand (RANKL) and its decoy receptor osteoprotegerin (OPG) have been shown to play a role not only in bone remodeling but also in inflammation, arterial calcification and atherosclerotic plaque rupture. In human smooth muscle cells, Cu(2+)-oxidized LDL (CuLDL) 10-50 µg/ml increased reactive oxygen species (ROS) and RANKL level in a dose-dependent manner, whereas OPG level was not affected. The lipid extract of CuLDL reproduced the effects of the whole particle. Vivit, an inhibitor of the transcription factor NFAT, reduced the CuLDL-induced increase in RANKL, whereas PKA and NFκB inhibitors were ineffective. LDL oxidized by myeloperoxidase (MPO-LDL), or other pro-oxidant conditions such as ultraviolet A (UVA) irradiation, incubation with H2O2 or with buthionine sulfoximine (BSO), an inhibitor of glutathione synthesis, also induced an oxidative stress and enhanced RANKL level. The increase in RANKL in pro-oxidant conditions was also observed in fibroblasts and endothelial cells. Since RANKL is involved in myocardial inflammation, vascular calcification and plaque rupture, this study highlights a new mechanism whereby OxLDL might, by generation of an oxidative stress, exert a deleterious effect on different cell types of the arterial wall.

Oxidized low density lipoprotein enhanced RANKL expression in human osteoblast-like cells. Involvement of ERK, NFkappaB and NFAT.

BACKGROUND: Receptor activator of nuclear factor kappa-light-chain-enhancer of activated B cell ligand/osteoprotegerin ratio is of crucial importance in osteoclast differentiation and thus in bone dysregulation diseases. METHODS: Receptor activator of nuclear factor kappa-light-chain-enhancer of activated B cell ligand and osteoprotegerin were determined under oxidized low density lipoprotein treatment of human osteoblast-like cells. The involvement of oxidative stress, of the extracellular signal regulated kinase and of the transcription factors nuclear factor kappa-light-chain-enhancer of activated B cells and nuclear factor of activated T cells was demonstrated. RESULTS: Cu(2+)-oxidized low density lipoprotein increased cell-associated and extracellular receptor activator of nuclear factor kappa-light-chain-enhancer of activated B cell ligand levels whereas osteoprotegerin levels were not affected. The increase in receptor activator of nuclear factor kappa-light-chain-enhancer of activated B cell ligand was parallel to the generation of reactive oxygen species provoked by Cu(2+)-oxidized low density lipoprotein. The lipid extract of Cu(2+)-oxidized low density lipoprotein, together with other forms of oxidized low density lipoproteins such as smooth muscle cell-oxidized low density lipoprotein and myeloperoxidase-oxidized low density lipoprotein, also induced an increase in reactive oxygen species and cell-associated receptor activator of nuclear factor kappa-light-chain-enhancer of activated B cell ligand. The effect of Cu(2+)-oxidized low density lipoprotein was prevented by the antioxidant vitamin E, and mimicked by the prooxidant compounds hydrogen peroxide and buthionine sulfoximine. Inhibitors of mitogen activated protein kinase/extracellular signal regulated kinase (PD 98059), nuclear factor kappa-light-chain-enhancer of activated B cells (Ro 106-9920) and nuclear factor of activated T cells (Vivit) reduced the effect of Cu(2+)-oxidized low density lipoprotein on receptor activator of nuclear factor kappa-light-chain-enhancer of activated B cell ligand expression. Cu(2+)-oxidized low density lipoprotein signaling was also reduced by vitamin E. GENERAL SIGNIFICANCE: This work describes a new molecular mechanism and elucidates the signaling pathway whereby oxidized low density lipoprotein, by means of its lipid moiety, can modulate the crosstalk between osteoblasts/osteoclasts and bone remodeling, leading to an eventual risk of osteoporosis.

Oxidized low density lipoprotein induces cyclin A synthesis. Involvement of ERK, JNK and NFkappaB.

The cytotoxicity of oxidized LDL (OxLDL) towards different cell types of the arterial wall results in atherosclerotic plaque fissuring or rupture. The effects of OxLDL on cyclins A, E and D1 levels were investigated in human fibroblasts. A 24h incubation with Cu(2+)-oxidized (CuLDL) or monocyte-oxidized LDL (M-LDL), within the range of 10-50µg ApoB/ml, increased the intracellular level of cyclin A, both in the nuclear and in the cytoplasmic fraction. This increase is due to a stimulation of cyclin A mRNA synthesis, and cycloheximide had a preventive effect. The CuLDL-induced rise in cyclin A was accompanied by an augmentation of reactive oxygen species ROS and of lipid peroxidation end products. Furthermore, this effect was reproduced by the lipid extract of CuLDL and prevented by the antioxidant vitamin E, demonstrating the role of oxidized lipids and the involvement of oxidative stress. In addition, the use of specific inhibitors indicated that the increase in cyclin A involved ERK/JNK kinases and NFkappaB transcription factor. The augmentation of cyclin A was observed not only in fibroblasts but also in other cell types such as macrophages, T lymphocytes, endothelial and smooth muscle cells. Since cyclin A has been shown to be involved in cell cycle arrest, the described phenomenon might be related to the harmful effect of OxLDL, leading to plaque rupture.

Oxidized low density lipoprotein inhibits phosphate signaling and phosphate-induced mineralization in osteoblasts. Involvement of oxidative stress.

BACKGROUND: It is well admitted that oxidized LDL (OxLDL) plays a major role in the generation and progression of atherosclerosis. Since atherosclerosis is often accompanied by osteoporosis, the effects of OxLDL on phosphate-induced osteoblast mineralization were investigated. METHODS: Calcium deposition, expression of osteoblast markers and inorganic phosphate (Pi) signaling were determined under OxLDL treatment. RESULTS: OxLDL, within the range of 10-50 µg protein/ml, inhibited Pi-induced UMR106 rat osteoblast mineralization. In parallel, the expression of Cbfa1/Runx2 transcription factor was decreased, and the intracellular level of the osteoblast marker osteopontin (OPN) was reduced. The extracellular level of another marker, receptor activator of nuclear factor kappa B ligand (RANKL), was also diminished. OxLDL inhibited Pi signaling via ERK/JNK kinases and AP1/CREB transcription factors. OxLDL triggered the generation of reactive oxygen species (ROS), either in the absence or presence of Pi. Furthermore, the effects of OxLDL on Pi-induced mineralization, generation of ROS and extracellular level OPN were reproduced by the lipid extract of the particle, whereas the antioxidant vitamin E prevented them. CONCLUSIONS: This work demonstrates that OxLDL, by generation of an oxidative stress, inhibits of Pi signaling and impairs Pi-induced osteoblast differentiation. GENERAL SIGNIFICANCE: This highlights the role of OxLDL in bone remodeling and in degenerative disorders other than atherosclerosis, especially in osteoporosis.

Oxidized low-density lipoprotein increases osteopontin expression by generation of oxidative stress.

Osteopontin (OPN) is an important mediator of inflammation and is involved in the generation of atherosclerotic lesions. Oxidized LDL (OxLDL) increased the intracellular and secreted levels of OPN in rat smooth muscle cells in a dose- and time-dependent manner. Experiments with kinase inhibitors demonstrated that this effect was mediated by ERK and JNK, but not p38. OxLDL induced oxidative stress, measured by the intracellular levels of reactive oxygen species (ROS) and lipid peroxidation products. The increase in OPN levels was reproduced by the lipid extract of the particle and prevented by the antioxidant vitamin E. Furthermore, ROS generated by UVA irradiation or treatment with pro-oxidant compounds such as buthionine sulfoximine or H(2)O(2) also enhanced intracellular and secreted OPN. Finally, OxLDL also augmented OPN levels in other cell types such as fibroblasts, keratinocytes, and endothelial cells. This work demonstrates the role of OxLDL in the expression of the OPN gene and further highlights the role of oxidative stress in the regulation of this cytokine. This might be related to the proinflammatory effects of OxLDL in the initiation and progression of atherosclerotic plaque.

Oxidized low density lipoprotein decreases Rankl-induced differentiation of osteoclasts by inhibition of Rankl signaling.

The role of OxLDL in the generation and progression of atherosclerosis is well admitted. In addition, it is well known that atherosclerosis is often accompanied by perturbations in bone remodeling, resulting in osteoporosis. In the current studies, the effect of Cu(2+)-oxidized LDL (OxLDL) on RANKL-induced RAW264.7 mouse monocytes-macrophages differentiation to osteoclasts and on RANKL signaling pathway was investigated. OxLDL, within the range of 10-50 microg protein/ml, prevented RANKL-induced generation of multinucleated osteoclast-like cells and RANKL-induced tartrate resistant acid phosphatase (TRAP) activity. OxLDL also prevented the RANKL-induced phosphorylation of ERK, p38 and JNK kinases, together with the RANKL-induced DNA binding activities of NFkappaB and NFAT transcription factors. Concomitantly, OxLDL enhanced RANKL-induced generation of reactive oxygen species in a dose-dependent manner. The antioxidant glutathione (GSH) prevented whereas the prooxidant compound buthionine-sulfoximine (BSO) enhanced the effect of OxLDL on RANKL-induced oxidative stress and RANKL-induced differentiation. Finally, OxLDL also prevented RANKL-induced TRAP activity and RANKL-induced bone resorbing activity of human peripheral blood mononuclear cells. These results demonstrate that OxLDL, by generation of an intracellular oxidative stress, prevents the differentiation of osteoclasts by inhibition of RANKL signaling pathway. This might be related to the fact that atherosclerosis is accompanied by perturbations in bone and vascular remodeling, leading to osteoporosis and vascular calcification.

Activation of transcription factors and gene expression by oxidized low-density lipoprotein.

It is well recognized that oxidized LDL (OxLDL) plays a crucial role in the initiation and progression of atherosclerosis. Many biological effects of OxLDL are mediated through signaling pathways, especially via the activation of transcription factors, which in turn stimulate the expression of genes involved in the inflammatory and oxidative stress response or in cell cycle regulation. In this review, we will discuss the various transcription factors activated by OxLDL, the studied cell types, the active compounds of the OxLDL particle, and the downstream genes when identified. Identification of the transcription factors and some of the downstream genes regulated by OxLDL has helped us understand the molecular mechanism involved in generation of the atherosclerotic plaque.

High extracellular inorganic phosphate concentration inhibits RANK-RANKL signaling in osteoclast-like cells.

In this work, we investigated the effect of inorganic phosphate (Pi) on the differentiation of monocyte/macrophage precursors into an "osteoclastic" phenotype, and we delineated the molecular mechanisms which could be involved in this phenomenon. This was achieved by stimulating human peripheral blood monocytic cells and RAW 264.7 monocyte-macrophage precursor cells to differentiate into osteoclast-like cells in the presence of receptor activator of NF-kappaB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). RANKL has been previously reported to stimulate the signaling kinases ERK 1/2, p38, Akt, JNK, and the DNA-binding activity of the transcription factors AP-1 and NF-kappaB. Increase in extracellular Pi concentration (1.5-4.5 mM) dose-dependently inhibits both osteoclastic differentiation and bone resorption activity induced by RANKL and M-CSF. Pi was found to specifically inhibit the RANKL-induced JNK and Akt activation, while RANKL-induced p38 and ERK 1/2 phosphorylation were not significantly affected. Moreover, we found that Pi significantly reduced the RANKL-stimulated DNA-binding activity of NF-kappaB. The effects of Pi on osteoclast differentiation and DNA-binding activity of NF-kappaB were prevented by Foscarnet, a sodium-phosphate cotransport inhibitor, suggesting that the effects of Pi occur subsequently to its intake. These results demonstrate that Pi downregulates the differentiation of osteoclasts via a negative feedback exerted on RANK-RANKL signaling.

Enhancement of the photodynamic activity of tri-cationic porphyrins towards proliferating keratinocytes by conjugation to poly-S-lysine.

A fast uptake of the tri-cationic 5-(4-carboxyphenyl)-10,15,20-tris(4-methylpyridinium-4-yl)porphyrin tri-iodide (P-H), independent of the presence or absence of proteins in the culture medium, occurs during incubation of NCTC 2544 human keratinocytes with this porphyrin. By contrast, the uptake of the poly-S-lysine conjugate (P-(Lys)(n)) is faster in serum-free medium than in the supplemented medium suggesting that P-(Lys)(n) interacts with serum proteins. The P-(Lys)(n) uptake is almost an order of magnitude greater than that of P-H in serum-free or supplemented culture medium. With histidine as a specific probe of type II photodynamic reactions, the relative photosensitizing effectiveness of the conjugate is only one fourth that of P-H. Nevertheless, the photocytotoxicity of the conjugate is strongly enhanced as compared to that of P-H as a result of its larger uptake. Thus, the doses achieving 50% of photocytotoxicity after incubation with 5 microM of the conjugate and its parent cationic porphyrin are about 20 min and 1 h, respectively. Similarly, the initial rate of the cell lipid peroxidation induced by photosensitization with P-(Lys)(n) is about 8 times higher than that obtained with P-H. Fluorescence microscopy reveals that P-H is more diffusely located in the cytoplasm than P-(Lys)(n) which seems to accumulate in lysosome-like structures. Little if any staining of the nucleus is observed with both photosensitizers.

Low UVA doses activate the transcription factor NFAT in human fibroblasts by a calcium-calcineurin pathway.

UVA radiation induces an inflammatory response as observed in erythema, and the cytokine genes involved in this response are under the control of the transcription factor NFAT (nuclear factor of activated T lymphocytes). The effects of UVA on NFAT DNA binding activity were investigated in cultured human fibroblasts. A dose-dependent increase was observed within the range of 0.6-4.5 J/cm2 UVA. Beyond this value, the activity decreased and a value of 60% of control was found at 13.5 J/cm2. The enhancement of NFAT activity was transient and peaked 45 min after irradiation. Furthermore, immunoblot analysis demonstrated a nuclear translocation of NFAT under low UVA doses. Concomitantly, as assessed by the fluorescent probe Fluo3, UVA induced an increase in intracellular free calcium, with a maximum increase found at 9 J/cm2. The UVA-induced activation of NFAT was prevented by the intracellular calcium trapping drug BAPTA, whereas the extracellular calcium chelator EGTA had no significant effect. In addition, the calcineurin inhibitors cyclosporin A and FK506 both prevented the UVA-induced NFAT activation. Furthermore, the antioxidant vitamin E prevented the UVA-induced increase in both intracellular free calcium and NFAT binding activity. Finally, the cytotoxicity of UVA was enhanced in the presence of the inhibitors cyclosporin and FK506, suggesting that the activation of NFAT might play a protective role after the UVA-induced oxidative stress. These results demonstrate that UVA activates the calcium-calcineurin signaling pathway of NFAT activation, that the calcium ions are mainly released from intracellular stores, and that the increase in calcium is, at least partially, due to the oxidative stress generated under UVA. Because NFAT regulates several genes implicated in the inflammatory response, the enhancement of NFAT activity by low UVA doses might be interpreted in view of the proinflammatory action of solar radiation.

Oxidized low-density lipoprotein elicits an intracellular calcium rise and increases the binding activity of the transcription factor NFAT.

Oxidized low-density lipoprotein (OxLDL) plays a key role in the generation and progression of atherosclerosis, which might be considered as an inflammatory disease. The transcription factor NFAT(Nuclear Factor of Activated T cells) plays an important role in the control of cytokine genes involved in the inflammatory response. The effect of copper-oxidized LDL (CuLDL) and monocyte-oxidized LDL (M-LDL) on the DNA-binding activity of NFAT was investigated in the T lymphocyte cell line Jurkat. Both OxLDL increased NFAT-binding activity in a dose-dependent manner within the range of 25-75 microg LDL protein/ml. This effect reached a maximum 1 h after the introduction of OxLDL in the medium. CuLDL and M-LDL both induce an intracellular calcium rise in a dose-dependent manner, with a maximum increase 15 min after the addition of OxLDL. The CuLDL-induced NFAT-binding activity was abolished in the presence of the calcium chelator EGTA or of the intracellular calcium trapping drug BAPTA, further indicating the involvement of calcium ions in the effect of OxLDL. In addition, cyclosporin A and FK 506, two inhibitors of calcineurin, a calcium-dependent phosphatase upstream of NFAT, also prevented the CuLDL-induced NFAT-binding activity, thus demonstrating the role of calcineurin. CuLDL and M-LDL also induced an increase in the intracellular level of reactive oxygen species (ROS), which reached a maximum 30 min after the addition of OxLDL. Finally, a pretreatment of cells with the antioxidant vitamin E blocked the CuLDL-induced increase in reactive oxygen species, in intracellular calcium rise and the CuLDL-induced NFAT-binding activity. The lipid extract of CuLDL, which includes the lipid peroxidation products, reproduced the effect of the CuLDL itself. These results suggest that the effect of OxLDL on NFAT is initiated by an oxidative stress, which then in turn activates the calcium-calcineurin signaling pathway of the transcription factor NFAT. This effect of OxLDL might be involved in the inflammatory process observed in atherosclerotic lesions.

Inhibition of insulin signaling by oxidized low density lipoprotein. Protective effect of the antioxidant Vitamin E.

Oxidative stress is involved in several pathological conditions, including diabetes. Reactive oxygen species (ROS) have been demonstrated to act as second messengers for several hormones and cytokines, including insulin (INS). The effect of Cu(2+)-oxidized LDL (CuLDL) on INS-induced generation of ROS and on INS signaling was investigated on cultured human fibroblasts. Intracellular ROS generation was observed either in CuLDL- or in INS-treated cells. Moreover, CuLDL and INS had an additive effect on ROS formation in human fibroblasts. CuLDL by itself increased the phosphorylation of ERK without affecting the PKB/Akt phosphorylation. CuLDL also stimulated the DNA binding activities of the transcription factors AP1 and NFkappaB. However, CuLDL dose-dependently prevented the INS-signaling pathway, by inhibiting the INS-induced phosphorylation of the signaling kinases ERK and PKB/Akt and the INS-induced activation of the transcription factors AP1 and NFkappaB. Finally, the lipophilic antioxidant Vitamin E (Vit E) partially restored all the studied signaling events initiated by INS and impaired after pretreatment with CuLDL. These studies demonstrate that the oxidative stress generated by CuLDL has a negative effect on the INS-signaling pathway, independently of the INS-induced generation of ROS. Thus, oxidized LDL might be involved not only in atherosclerosis, as it is commonly admitted, but also in the INS-resistance observed in type 2 diabetes mellitus.

Anti- and pro-oxidant effects of quercetin in copper-induced low density lipoprotein oxidation. Quercetin as an effective antioxidant against pro-oxidant effects of urate.

We recently reported that, depending on its concentration, urate is either a pro- or an antioxidant in Cu(2+)-induced low-density lipoprotein (LDL) oxidation. We also previously demonstrated an antioxidant synergy between urate and some flavonoids in the Cu(2+)-induced oxidation of diluted serum. As a result, the effect of the flavonoid quercetin on the Cu(2+)-induced oxidation of isolated LDL has been studied either in the presence or absence of urate. We demonstrate that, like urate, quercetin alone, at low concentration, exhibits a pro-oxidant activity. The pro-oxidant behavior depends on the Cu(2+) concentration but it is not observed at high Cu(2+) concentration. When compared with urate, the switch between the pro- and the antioxidant activities occurs at much lower quercetin concentrations. As for urate, the pro-oxidant character of quercetin is related to its ability to reduce Cu(2+) with the formation of semioxidized quercetin and Cu(+) with an expected yield larger than that obtained with urate owing to a more favorable redox potential. It is also shown that the pro-oxidant activity of urate can be inhibited by quercetin. An electron transfer between quercetin and semioxidized urate leading to the repair of urate could account for this observation as suggested by recently published pulse radiolysis data. It is anticipated that the interactions between quercetin-Cu(2+)-LDL and urate, which are tightly controlled by their respective concentration, determine the balance between the pro- and antioxidant behaviors. Moreover, as already observed with other antioxidants, it is demonstrated that quercetin alone behaves as a pro-oxidant towards preoxidized LDL.

The inhibition of cell spreading on a cellulose substrate (cuprophan) induces an apoptotic process via a mitochondria-dependent pathway.

Cell shape was found to be a strong indicator of whether individual cells grow or die, and may play an important role in controlling apoptosis as well as cell growth. We compared here the behaviour of rounded Swiss 3T3 cells aggregated on a cellulose cuprophan membrane to those cultured on dish polystyrene. We demonstrated that cells aggregated on cellulose substrates for up to 48 h underwent programmed cell death that was associated with phosphatidylserine flipping and caspase 9 and caspase 3 activation, suggesting a mitochondria-dependent apoptotic process. In addition, we found that this phenomenon cannot be entirely explained by disengagement of alpha 5 beta 1 integrin ligation.

Interleukin-7 induces apoptosis of 697 pre-B cells expressing dominant-negative forms of STAT5: evidence for caspase-dependent and -independent mechanisms.

The transcription factors STAT5A and STAT5B (STAT: signal transducer and activator of transcription) play a major role in the signaling events elicited by a number of growth factor and cytokine receptors. In this work, we aimed to investigate the role of STAT5 in human precursor B cell survival by introducing dominant-negative (DN) forms of STAT5A or STAT5B in the 697 pre-B cell line. All clones expressing DN forms of either transcription factor exhibited a higher spontaneous apoptotic rate that was massively enhanced upon interleukin-7 (IL-7) stimulation. This was associated with caspase 8 cleavage, mitochondrial transmembrane potential disruption and caspase 3 activation. However, the DN forms of STAT5 did not alter the expression of Bcl-2, Bax, Bcl-x, Bim, A1 and Mcl1 proteins in IL-7-stimulated cells. The pancaspase inhibitor Z-Val-Ala-Asp-fluoromylmethyl ketone partially suppressed IL-7-mediated mitochondrial transmembrane potential disruption and cell death, suggesting that IL-7 induced the death of DN STAT5 expressing 697 cells through caspase-dependent and -independent mechanisms that both require mitochondrial activation.

Stimulatory effect of naturally occurring flavonols quercetin and kaempferol on alkaline phosphatase activity in MG-63 human osteoblasts through ERK and estrogen receptor pathway.

Many plant-derived substances have estrogenic activities. Due to their ability to bind the estrogen receptor (ER), these compounds have the potential to counteract the deleterious effects of estrogen deficiency on bone. In this study, we investigated the in vitro effect of two widespread flavonols, quercetin and kaempferol, on alkaline phosphatase (ALP) activity in MG-63 cultured human osteoblasts. We found that both flavonols significantly increased ALP activity. This effect was markedly reduced by PD 98059, an inhibitor of the extracellular regulated kinase (ERK) pathway, and by ICI 182780, an antagonist of ERs. Western blot studies confirmed that ERK is rapidly activated in cells treated by both flavonols. Finally, ICI 182780 markedly inhibits the flavonol-induced ERK activation. The data presented in this study support the conclusion that, in MG-63 osteoblasts (i) the increase in ALP activity by flavonols involves a rapid stimulation of ERK activation but also involves the ER, and that (ii) the activation of ERK by flavonols occurs most likely downstream of the ERs activation. Taken together, these results suggest that flavonols derivatives as quercetin and kaempferol can stimulate osteoblastic activity. Such compounds may represent new pharmacological tools for the treatment of osteoporosis.

TGF-beta1 modulates Fas (APO-1/CD95)-mediated apoptosis of human pre-B cell lines.

We have previously shown that Fas-induced apoptosis is markedly enhanced by IL-7 in human pre-B but not pro-B cell lines. In addition, pre-B cell receptor (pre-BCR) ligation significantly potentiates the IL-7 effects on Fas-triggered pre-B cell death. We show herein that transforming growth factor (TGF)-beta 1 sharply reduces Fas-induced death rate of pre-B but not pro-B cells. TGF-beta 1 causes inhibition of Fas-mediated disruption of mitochondrial transmembrane potential and cleavage of caspase 8, Bid and caspase 3. Bcl2 expression is markedly increased in TGF-beta 1-treated pre-B cells, whereas cellular FLICE-like inhibitory protein long (c-FLIPL), Bcl-XL, Bax, and Bad expression remains unchanged. TGF-beta 1 causes a selective growth arrest of pre-B cells in G0/G1 phase of the cell cycle and induces a partial down-modulation of both Fas and pre-BCR expression. All TGF-beta 1-mediated effects, but Bcl2 up-regulation, can be reproduced by the LY294002 phosphatidylinositol 3-kinase (PI3K)/Akt inhibitor but not by inhibitors of the MAPK/ERK (MEK) and Janus kinase (Jak)/STAT pathways, which promote cell death. Akt phosphorylation is strongly inhibited by TGF-beta1 in pre-B but not pro-B cells and is not modified by Fas engagement. Altogether, our findings suggest that TGF-beta1 prevents Fas-induced apoptosis of pre-B lines by inhibiting PI3K pathway and by enhancing expression of Bcl2. They also suggest that the PI3K/Akt pathway is involved in the control of Fas and pre-BCR expression, a checkpoint in B cell development.

Impairment of the EGF signaling pathway by the oxidative stress generated with UVA.

Ultraviolet A (UVA) is a component of sunlight reaching the surface of the earth and involved in photodegenerescence and photocarcinogenesis. The effect of UVA was investigated on the EGF-induced activation of the signaling kinase ERK and the transcription factors AP1, NFkappaB, and STAT1. UVA prevented the Epidermal Growth Factor (EGF)-induced stimulation of ERK in a dose-dependent manner within the range of 1.5-9 J/cm(2). Concomitantly, the DNA binding activity of AP1, NFkappaB, and STAT1 under EGF were markedly inhibited by UVA within the same dose range. UVA by itself induced an activation of ERK activity, and a stimulation of AP1, NFkappaB, and STAT1 binding activity. UVA decreased EGF binding in a dose-dependent manner. Furthermore, the highest dose of UVA (9 J/cm(2)) prevented the EGF-induced Tyr-phosphorylation of the EGF-receptor (EGF-R). The generation of reactive oxygen species (ROS), as assessed by the fluorescent probe dichloro-fluorescein, showed an additive effect of EGF and UVA, within the studied range of UVA doses. Finally, the antioxidant Vitamin E prevented the inhibitory effect of UVA on ERK, AP1, NFkappaB, and STAT1. These results demonstrate that an overproduction of ROS, initiated by two different and successive triggering agents such as UVA and EGF, leads to inactivation of the EGF signaling pathway. This inhibition of gene expression control by EGF might play a role in the photodegenerative processes observed after exposition of skin cells to solar radiation.

Anti- and pro-oxidant effects of urate in copper-induced low-density lipoprotein oxidation.

We reported earlier that urate may behave as a pro-oxidant in Cu2+-induced oxidation of diluted plasma. Thus, its effect on Cu2+-induced oxidation of isolated low-density lipoprotein (LDL) was investigated by monitoring the formation of malondialdehyde and conjugated dienes and the consumption of urate and carotenoids. We show that urate is antioxidant at high concentration but pro-oxidant at low concentration. Depending on Cu2+ concentration, the switch between the pro- and antioxidant behavior of urate occurs at different urate concentrations. At high Cu2+ concentration, in the presence of urate, superoxide dismutase and ferricytochrome c protect LDL from oxidation but no protection is observed at low Cu2+ concentration. The use of Cu2+ or Cu+ chelators demonstrates that both copper redox states are required. We suggest that two mechanisms occur depending on the Cu2+ concentration. Urate may reduce Cu2+ to Cu+, which in turn contributes to formation. The Cu2+ reduction is likely to produce the urate radical (UH.-). It is proposed that at high Cu2+ concentration, the reaction of UH.- radical with generates products or intermediates, which trigger LDL oxidation. At low Cu2+ concentration, we suggest that the Cu+ ions formed reduce lipid hydroperoxides to alkoxyl radicals, thereby facilitating the peroxidizing chain reaction. It is anticipated that these two mechanisms are the consequence of complex LDL-urate-Cu2+ interactions. It is also shown that urate is pro-oxidant towards slightly preoxidized LDL, whatever its concentration. We reiterate the conclusion that the use of antioxidants may be a two-edged sword.

Repair of amino acid radicals of apolipoprotein B100 of low-density lipoproteins by flavonoids. A pulse radiolysis study with quercetin and rutin.

Selective oxidative damage to apolipoprotein B in LDL can be effected radiolytically by (*)Br(2)(-) radicals. Twenty-seven Trp residues constitute major primary sites of oxidation, but two-thirds of oxidized Trps ((*)Trp) that are formed are repaired by intramolecular electron transfer from Tyr residues with formation of phenoxyl radicals (TyrO(*)). Analysis of (*)Trp and TyrO(*) transient absorbance changes suggests that other apolipoprotein B residues, probably Cys, are oxidized. LDL-bound quercetin can efficiently repair this damage. Absorption studies show that a LDL particle has the capacity to bind approximately 10 quercetin molecules through interaction with apolipoprotein B. The repair occurs by intramolecular electron transfer characterized by a rate constant of 2 x 10(3) s(-)(1). In contrast, rutin, a related flavonoid which does not bind to LDL, cannot repair oxidized apolipoprotein B. Urate is a hydrophilic plasma antioxidant which displays synergistic antioxidant properties with flavonoids. Urate radicals produced by (*)Br(2)(-) can also be repaired by LDL-bound quercetin. This repair occurs with a reaction rate constant of 6.8 x 10(7) M(-)(1) s(-)(1). Comparison with previous studies conducted with human serum albumin-bound quercetin suggests that quercetin analogues tailored to be carried preferentially by lipoproteins might be more powerful plasma antioxidants than natural quercetin carried by serum albumin.