Correction: Cigarette Smoke Affects Keratinocytes SRB1 Expression and Localization via H2O2 Production and HNE Protein Adducts Formation.

[This corrects the article DOI: 10.1371/journal.pone.0033592.].

Signaling pathways involved in isoprostane-mediated fibrogenic effects in rat hepatic stellate cells.

Despite evidence supporting a potential role for F2-isoprostanes (F2-IsoP's) in liver fibrosis, their signaling mechanisms are poorly understood. We have previously provided evidence that F2-IsoP's stimulate hepatic stellate cell (HSC) proliferation and collagen hyperproduction by activation of a modified form of isoprostane receptor homologous to the classic thromboxane receptor (TP). In this paper, we examined which signal transduction pathways are set into motion by F2-IsoP's to exert their fibrogenic effects. HSCs were isolated from rat liver, cultured to their activated myofibroblast-like phenotype, and then treated with the isoprostane 15-F2t-isoprostane (15-F2t-IsoP). Inositol trisphosphate (IP3) and adenosine 3',5'-cyclic monophosphate (cAMP) levels were determined using commercial kits. Mitogen-activated protein kinase (MAPK) and cyclin D1 expression was assessed by Western blotting. Cell proliferation and collagen synthesis were determined by measuring [(3)H]thymidine and [(3)H]proline incorporation, respectively. 15-F2t-IsoP elicited an activation of extracellular-signal-regulated kinase (ERK), p38 MAPK, and c-Jun NH2-terminal kinase (JNK), which are known to be also regulated by G-protein-coupled receptors. Preincubation with specific ERK (PD98059), p38 (SB203580), or JNK (SP600125) inhibitors prevented 15-F2t-IsoP-induced cell proliferation and collagen synthesis. 15-F2t-IsoP decreased cAMP levels within 30 min, suggesting binding to the TPß isoform and activation of Giα protein. Also, 15-F2t-IsoP increased IP3 levels within a few minutes, suggesting that the Gq protein pathway is also involved. In conclusion, the fibrogenic effects of F2-IsoP's in HSCs are mediated by downstream activation of MAPKs, through TP binding that couples via both Gqα and Giα proteins. Targeting TP receptor, or its downstream pathways, may contribute to preventing oxidative damage in liver fibrosis.

Cigarette smoke affects keratinocytes SRB1 expression and localization via H2O2 production and HNE protein adducts formation.

Scavenger Receptor B1 (SR-B1), also known as HDL receptor, is involved in cellular cholesterol uptake. Stratum corneum (SC), the outermost layer of the skin, is composed of more than 25% cholesterol. Several reports support the view that alteration of SC lipid composition may be the cause of impaired barrier function which gives rise to several skin diseases. For this reason the regulation of the genes involved in cholesterol uptake is of extreme significance for skin health. Being the first shield against external insults, the skin is exposed to several noxious substances and among these is cigarette smoke (CS), which has been recently associated with various skin pathologies. In this study we first have shown the presence of SR-B1 in murine and human skin tissue and then by using immunoblotting, immunoprecipitation, RT-PCR, and confocal microscopy we have demonstrated the translocation and the subsequent lost of SR-B1 in human keratinocytes (cell culture model) after CS exposure is driven by hydrogen peroxide (H(2)O(2)) that derives not only from the CS gas phase but mainly from the activation of cellular NADPH oxidase (NOX). This effect was reversed when the cells were pretreated with NOX inhibitors or catalase. Furthermore, CS caused the formation of SR-B1-aldheydes adducts (acrolein and 4-hydroxy-2-nonenal) and the increase of its ubiquitination, which could be one of the causes of SR-B1 loss. In conclusion, exposure to CS, through the production of H(2)O(2), induced post-translational modifications of SR-B1 with the consequence lost of the receptor and this may contribute to the skin physiology alteration as a consequence of the variation of cholesterol uptake.

Reactivity of mouse alveolar macrophages to cigarette smoke is strain dependent.

Cigarette smoke (CS) is a main risk factor in chronic obstructive pulmonary disease (COPD), but only 20% of smokers develop COPD, suggesting genetic predisposition. Animal studies have shown that C57BL/6J mice are sensitive to CS and develop emphysema, whereas Institute of Cancer Research (ICR) mice are not. To investigate the potential factors responsible for the different susceptibility of ICR and C57BL/6J mice to CS, we evaluated in alveolar macrophages (AMs) isolated from these strains of mice the possible mechanisms involved in the inflammatory and oxidative responses induced by CS. Lactate dehydrogenase (LDH) release revealed that C57BL/6J AMs were more susceptible to CS extract (CSE) toxicity than ICR. Differences were observed in inflammatory and oxidative response after CSE exposure. Proinflammatory cytokines and matrix metalloproteinases (MMPs) were increased in C57BL/6J but not ICR AMs. Control C57BL/6J AMs showed a higher baseline production of reactive oxygen species (ROS) and H(2)O(2) with lower baseline levels of GSH, nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), and glutathione peroxidase (GPX2). This was associated with reduced histone deacetylase-2 (HDAC2) expression, activation of NF-κB, and higher basal levels of TNF-α and IL-6. CSE induced a decrease in HDAC2 protein levels in both C57BL/6J and ICR AMs; however, the level of HDAC2 was significantly lower in C57BL/6 than in ICR AMs. Furthermore, CSE enhanced NF-κB-dependent cytokine release only in C57BL/6J AMs. We suggest that an imbalance in oxidative stress decreases HDAC2 levels and facilitates NF-κB binding, resulting in a proinflammatory response in C57BL/6J but not in ICR AMs. These results could contribute in understanding the different susceptibility to CS of these strains of mice.

Synthesis and characterization of glucosyl-curcuminoids as Fe3+ suppliers in the treatment of iron deficiency.

The Fe(3+) chelating ability of some curcumin glucosyl derivatives (Glc-H; Glc-OH; Glc-OCH(3)) is tested by means of UV and NMR study. The pK(a) values of the ligands and the overall stability constants of Fe(3+) and Ga(3+) complexes are evaluated from UV spectra. The only metal binding site of the ligand is the beta-diketo moiety in the keto-enolic form; the glucosyl moiety does not interact with metal ion but it contributes to the stability of metal/ligand 1:2 complexes by means of hydrophilic interactions. These glucosyl derivatives are able to bind Fe(3+) in a wide pH rage, forming complex species thermodynamically more stable than those of other ligands commonly used in the treatment of iron deficiency. In addition they demonstrate to have a poor affinity for competitive biological metal ions such as Ca(2+). All ligands and their iron complexes have a good lypophilicity (log P > -0.7) suggesting an efficient gastrointestinal absorption in view of their possible use as iron supplements in oral therapy. The ligand molecules are also tested for their antioxidant properties in "ex vivo" biological system.

Synthesis, chemical and biological studies on new Fe(3+)-glycosilated beta-diketo complexes for the treatment of iron deficiency.

A simple synthetic pathway to obtain glycosilated beta-diketo derivatives is proposed. These compounds show a good iron(III) affinity therefore we may suggest the use of their Fe(3+)-complexes as oral iron supplements in the treatment of anaemia. The glycosilated compounds (6-GlcH, 6-GlcOH and 6-GlcOCH(3)) are characterized by means of spectroscopic (UV, (1)H and (13)C NMR) and potentiometric techniques; they have a good water solubility, are kinetically stable in physiological condition (t(1/2)>100h) and show a low cytotoxicity also in high concentrations (IC(50)>400 microM). They are able to bind Fe(3+) ion in acid condition (pH approximately 2) forming complex species thermodynamically more stable than those of other ligands commonly used in the treatment of iron deficiency. The iron complexes show also a good kinetic stability both in acidic and physiological pH and have a good lypophilicity (logP>-0.7) that suggests an efficient gastrointestinal absorption in view of their possible use in oral therapy. In addition they demonstrate a poor affinity for competitive biological metal ion such as Ca(2+), and in particular 6-GlcOCH(3) is able to inhibit lipid peroxidation.

Iron overload enhances the development of experimental liver cirrhosis in mice.

The role of iron in initiating liver fibrosis in iron overload diseases is not clearly established. Partly, this is due to the lack of suitable animal models that can produce the full liver pathology seen in genetic hemochromatosis. Recent advances in this field have demonstrated that iron may be interacting with other potential liver-damaging agents. The aim of this study was to investigate if feeding with carbonyl iron (CI) facilitates the development of carbon tetrachloride (CCl4)-induced liver fibrosis in the mouse. Mice were given a diet containing 3% CI and treated with CCl4 intraperitoneally twice weekly and 5% alcohol added to the drinking water for 12 weeks. Hepatic iron content increased 15- and 22-fold in animals receiving CI and CI + CCl4. At histological examination, iron-laden hepatocytes were found in CI treated animals, whereas these were absent in animals not exposed to CI. Mice receiving iron-enriched diet alone showed a mild fibrosis. Conversely, a marked collagen deposition was observed in CCl4 and CI + CCl4 groups. In particular, in this latter group, there was evidence of liver cirrhosis. Biochemical evaluation of collagen content substantiated histologic analysis. These results demonstrate that the addition of iron facilitates the development of cirrhosis in animals exposed to subtoxic doses of CCl4. This model may be useful in exploring the pathogenesis of liver cirrhosis. Moreover, its use in genetically altered mouse strains might provide new insight on the role of iron in fibrosis.

Effect of free iron on collagen synthesis, cell proliferation and MMP-2 expression in rat hepatic stellate cells.

Various studies on hepatic fibrosis occurring in iron overload suggest that excess of tissue iron may be involved in the stimulation of collagen synthesis. Anyway, up to date, direct evidence on the role of iron in hepatic fibrosis is lacking. Moreover, it is not clear whether iron acts as direct initiator of fibrogenesis or as mediator of hepatocellular necrosis. In the present study, we investigated the effect of nontoxic doses of iron on collagen metabolism and proliferation, key features of liver fibrosis, by means of cultures of hepatic stellate cells, the liver cells responsible for collagen production. Iron treatment increased collagen synthesis without affecting noncollagen proteins. The maximum effect was observed at 5 microM iron (+132%). At this dose, no cell damage or proliferation was detected. Conversely, higher doses of iron (10 and 25 microM) induced cell proliferation and a lower increase in collagen synthesis, suggesting the prevalence of proliferative effect on the synthetic one. These effects occurred without the intervention of serum factors and were not mediated by lipid peroxidation. Our results strongly support the hypothesis that iron "per sé" may act as a profibrogenic agent. Finally, we provide evidence that iron plays a role also in matrix degradation, by stimulating some metalloprotease activities. Iron treatment increased metalloprotease-2 activity in hepatic stellate cells, while no changes were observed for interstitial collagenase activity suggesting that, in these conditions, a pathological accumulation of hepatic extracellular matrix may occur.

Effect of parathyroid hormone-related protein on fibroblast proliferation and collagen metabolism in human skin.

The parathyroid hormone-related protein (PTHrp), structurally similar to the parathyroid hormone (PTH) in its NH(2)-terminal part, was first identified as a tumour-derived peptide responsible for a paraneoplastic syndrome known as humoral hypercalcemia of malignancy. The PTHrp gene is expressed not only in cancer but also in normal tissues during adult and/or fetal life, where it plays predominantly paracrine and/or autocrine roles. In the skin PTHrp produced by keratinocytes acts on fibroblasts by complex cooperative circuits involving cytokines and growth factors. In this report, we studied the direct effects of synthetic PTHrp 1-40 on proliferation and collagen synthesis and matrix metalloproteinase-2 (MMP-2) activity in cultures of fibroblasts isolated from normal human skin. Fibroblasts exposure to varying doses of PTHrp for 48 h, significantly and dose-dependently inhibited proliferation evaluated by [(3)H]-thymidine incorporation into DNA. A dose-dependent stimulation of cAMP released into the medium was concomitantly observed. In contrast, PTHrp had no effect on collagen synthesis evaluated either by [(3)H]-proline incorporation or by radioimmunoassay (RIA) of the carboxyterminal fragment of type I procollagen (PICP). MMP-2 activity, evaluated by quantitative zymographic analysis, was significantly increased by PTHrp treatment at doses of 160 and 320 nM. These findings indicate that PTHrp may play a role in normal dermal physiology by controlling both fibroblast proliferation and extracellular matrix degradation.