Effect of Oxidative Stress-Induced Apoptosis on Active FGF23 Levels in MLO-Y4 Cells: The Protective Role of 17-ß-Estradiol.

The discovery that osteocytes secrete phosphaturic fibroblast growth factor 23 (FGF23) has defined bone as an endocrine organ. However, the autocrine and paracrine functions of FGF23 are still unknown. The present study focuses on the cellular and molecular mechanisms involved in the complex control of FGF23 production and local bone remodeling functions. FGF23 was assayed using ELISA kit in the presence or absence of 17ß-estradiol in starved MLO-Y4 osteocytes. In these cells, a relationship between oxidative stress-induced apoptosis and up-regulation of active FGF23 levels due to MAP Kinases activation with involvement of the transcriptional factor (NF-kB) has been demonstrated. The active FGF23 increase can be due to up-regulation of its expression and post-transcriptional modifications. 17ß-estradiol prevents the increase of FGF23 by inhibiting JNK and NF-kB activation, osteocyte apoptosis and by the down-regulation of osteoclastogenic factors, such as sclerostin. No alteration in the levels of dentin matrix protein 1, a FGF23 negative regulator, has been determined. The results of this study identify biological targets on which drugs and estrogen may act to control active FGF23 levels in oxidative stress-related bone and non-bone inflammatory diseases.

Blueberry Juice Antioxidants Protect Osteogenic Activity against Oxidative Stress and Improve Long-Term Activation of the Mineralization Process in Human Osteoblast-Like SaOS-2 Cells: Involvement of SIRT1.

Diets rich in fruits and vegetables with many antioxidants can be very important in the prevention and treatment of osteoporosis. Studies show that oxidative stress, often due to lack of antioxidants, is involved in alteration of bone remodeling and reduction in bone density. This study demonstrates in human osteoblast-like SaOS-2 cells that blueberry juice (BJ), containing 7.5 or 15 µg∙mL-1 total soluble polyphenols (TSP), is able to prevent the inhibition of osteogenic differentiation and the mineralization process due to oxidative stress induced by glutathione depletion. This situation mimics a metabolic condition of oxidative stress that may occur during estrogen deficiency. The effect of BJ phytochemicals occurs through redox- and non-redox-regulated mechanisms. BJ protects from oxidative damage factors related to bone remodeling and bone formation, such as alkaline phosphatase and Runt-related transcription factor 2. It upregulates these factors by activation of sirtuin type 1 deacetylase expression, a possible molecular target for anti-osteoporotic drugs. Quantitative analysis of TSP in BJ shows high levels of anthocyanins with high antioxidant capacity and bioavailability. These novel data may be important to elucidate the molecular and cellular beneficial effects of blueberry polyphenols on bone regeneration, and they suggest their use as a dietary supplement for osteoporosis prevention and therapies.

Resveratrol decreases TNFα-induced ICAM-1 expression and release by Sirt-1-independent mechanism in intestinal myofibroblasts.

Up-regulation of intercellular adhesion molecule-1 (ICAM-1) and its soluble form are involved in the chronic inflammation. For the first time, we demonstrated that resveratrol (RE), a natural polyphenol with antioxidant and anti-inflammatory properties, reduces the increase of expression and release of ICAM-1, due to TNFα-induced oxidative stress, in a myofibroblast cell line derived from human colonic (18Co cells). RE is scavenger of radical oxygen species (ROS) and modulates signaling pathways in which Sirt-1 and NF-κB are involved. Effectively, in TNFα-stimulated 18Co cells RE decreases ROS production and increases Sirt-1 expression and activity, but it reduces TNFα-induced ICAM-1 up-regulation by a Sirt-1-independent mechanism, as demonstrated by EX527 and Sirt-1 siRNA treatments. RE inhibits TNFα-induced activation of NF-κB by reducing both ROS and the degradation of IκB-α, an endogenous inhibitor of NF-κB, with consequent decrease of NF-κB nuclear translocation. This study also shows that NF-κB is not the only factor involved in the TNFα-induced ICAM-1 up-regulation and confirms our previous evidence according to which TNFα increases ICAM-1 levels by redox- and non-redox-regulated mechanisms. RE can represent good and useful support in therapies for intestinal inflammatory diseases in which TNFα plays a crucial role in the increase of adhesion molecule expression.

Blueberry juice protects osteocytes and bone precursor cells against oxidative stress partly through SIRT1.

Oxidative stress and abnormal osteocyte apoptosis are often related to dysregulation of bone turnover and chronic bone loss, and so fruit and vegetables with high antioxidant potential may play an important role in the prevention and/or management of osteoporosis. Osteocytes are the main regulators of bone remodelling. For the first time, we demonstrate here that blueberry juice (BJ), obtained from Vaccinium myrtillus, rich in polyphenols, shows antioxidant and antiosteoclastogenic properties in MLO-Y4 osteocytes. We report that BJ prevents oxidative stress-induced apoptosis and reverses the increase in receptor activator of nuclear factor κB ligand and sclerostin expression, crucial factors for osteoclast activation and bone resorption. BJ is also able to prevent oxidative stress-induced cell cytotoxicity in bone marrow mesenchymal stromal cells (MSCs), which are considered to be an important tool for cell therapy in bone disorders. No significant difference in preventing these events was observed between BJ and blueberry dry extract containing equal amounts of total soluble polyphenols. We have also shown that blueberry acts as both an antioxidant and an activator of sirtuin type 1, a class III histone deacetylase involved in cell death regulation and considered a molecular target for blocking bone resorption without affecting osteoclast survival. Overall, these novel data obtained in osteocytes and MSCs may help us clarify the mechanisms by which blueberry counteracts oxidative stress-induced damage in bone remodelling and osteogenesis at the cellular and molecular level. Our findings are consistent with the reported beneficial effects of blueberry on bone tissue reported in animal studies, which suggest that blueberry may be a useful supplement for the prevention and/or management of osteoporosis and osteogenic process.

Oxidative stress in bone remodeling: role of antioxidants.

ROS are highly reactive molecules which consist of a number of diverse chemical species, including radical and non-radical oxygen species. Oxidative stress occurs as a result of an overproduction of ROS not balanced by an adequate level of antioxidants. The natural antioxidants are: thiol compounds among which GSH is the most representative, and non-thiol compounds such as polyphenols, vitamins and also various enzymes. Many diseases have been linked to oxidative stress including bone diseases among which one of the most important is the osteoporosis. The redox state changes are also related to the bone remodeling process which allows the continuous bone regeneration through the coordinated action of bone cells: osteoclasts, osteoblasts and osteocytes. Changes in ROS and/or antioxidant systems seem to be involved in the pathogenesis of bone loss. ROS induce the apoptosis of osteoblasts and osteocytes, and this favours osteoclastogenesis and inhibits the mineralization and osteogenesis. Excessive osteocyte apoptosis correlates with oxidative stress causing an imbalance in favor of osteoclastogenesis which leads to increased turnover of bone remodeling and bone loss. Antioxidants either directly or by counteracting the action of oxidants contribute to activate the differentiation of osteoblasts, mineralization process and the reduction of osteoclast activity. In fact, a marked decrease in plasma antioxidants was found in aged or osteoporotic women. Some evidence shows a link among nutrients, antioxidant intake and bone health. Recent data demonstrate the antioxidant properties of various nutrients and their influence on bone metabolism. Polyphenols and anthocyanins are the most abundant antioxidants in the diet, and nutritional approaches to antioxidant strategies, in animals or selected groups of patients with osteoporosis or inflammatory bone diseases, suggest the antioxidant use in anti-resorptive therapies for the treatment and prevention of bone loss.

Protective role of benzoselenophene derivatives of resveratrol on the induced oxidative stress in intestinal myofibroblasts and osteocytes.

Resveratrol (RE), a polyphenolic compound present in some food and plants, is characterized by anti-inflammatory and antioxidant properties. However, it is quickly metabolized with consequent loss of its efficacy. In this study, the antioxidant effect of 2-phenyl-benzoselenophene derivatives (VD0, VD1 and VD2) was detected in intestinal myofibroblast and osteocyte cell lines in which the oxidative stress was induced by GSH depletion or starvation, respectively. In fact, the oxidative stress is involved in pathogenesis of inflammatory bowel diseases (IBD) and in increased osteoclastogenesis in osteoporosis. Our results show that these derivatives have major antioxidant power in reducing and/or restoring radical oxygen species to control values than RE itself in both cell types. Moreover, derivatives have different antioxidant capacity in myofibroblasts and in osteocytes and this can be due to different degree of oxidative stress and structural characteristics of these compounds. Some of the synthesized RE analogs have shown anti-bacterial role in IBD and anti-resorptive activity in bone pathologies related to inflammatory and osteoporotic processes. Thus, we suggest benzoselenophene derivatives as good candidates for alternative therapy and/or therapeutic support in these pathologies.

Estrogen inhibits starvation-induced apoptosis in osteocytes by a redox-independent process involving association of JNK and glutathione S-transferase P1-1.

Estrogen deficiency causes bone loss as a result of microdamage, oxidative stress, and osteocyte apoptosis. A relationship between oxidative stress-induced apoptosis, c-Jun N-terminal kinase (JNK) activation, and expression of factors involved in bone remodeling has been demonstrated in osteocytes. However, the molecular regulation of these events in osteocytes treated with 17ß-estradiol (17ß-E2) remains unexplored. The MLO-Y4 murine osteocyte-like cell line was used as a model to study starvation-induced apoptosis and ROS production during 17ß-E2 treatment. Expression of glutathione S-transferase P1-1 (GSTP1-1), receptor activator kB ligand (RANKL), osteoprotegerin (OPG), sclerostin, and kinases activation were measured by western blot. In addition, the GSTP1-1/JNK association was assessed by immunoprecipitation, and GSTP1-1 involvement in the osteocyte response to 17ß-E2 was detected by specific siRNA transfection. 17ß-E2 prevents starvation-induced apoptosis (DNA fragmentation and caspase activation), the increase in sclerostin expression and the RANKL/OPG ratio, which are all related to JNK activation due to oxidative stress in osteocytes. This occurs through GSTP1-1 overexpression, which can inhibit JNK activation by formation of a GSTP1-1/JNK complex. No early antioxidant action of 17ß-E2 has been found but the estrogen effect is similar to N-acetylcysteine which, by increasing the intracellular redox state, maintains JNK bound to GSTP1-1. Thus, the antiapoptotic and osteogenic effect of 17ß-E2 in MLO-Y4 occurs by a redox-independent process involving GSTP1-1/JNK association. This study clarifies at molecular level the effect of 17ß-E2 on osteocyte activity and identifies a possible role of GSTP1-1 and JNK activity in bone remodeling and repair mechanisms.

Tumor Necrosis Factor-Alpha Up-Regulates ICAM-1 Expression and Release in Intestinal Myofibroblasts by Redox-Dependent and -Independent Mechanisms.

Intercellular adhesion molecule-1 (ICAM-1) is distributed and expressed on cell surface and is present in circulation as soluble form (sICAM-1). Tumor necrosis factor-alpha (TNFα) and radical oxygen species (ROS) up-regulate the expression of ICAM-1. This study demonstrates for the first time in 18 Co cells, a myofibroblast cell line derived from human colonic mucosa, an up-regulation of ICAM-1 expression and sICAM-1 release induced by oxidative stress and TNFα stimulation. The intracellular redox state was modulated by L-buthionine-S,R-sulfoximine (BSO) or N-acetylcysteine (NAC), inhibitor and precursor respectively of GSH synthesis. ROS production increases in cells treated with BSO or TNFα, and this has been related to an up-regulation of ICAM-1 expression and sICAM-1 release. The involvement of metalloproteinases in ICAM-1 release has been demonstrated. Moreover, also expression and activation of A disintegrin and metalloproteinase 17, a membrane-bound enzyme known as TNFα-converting enzyme (TACE), have been related to ROS levels. This suggests the possible involvement of TACE in the cleavage of ICAM-1 on cell surface in condition of oxidative stress. NAC down-regulates the expression and release of ICAM-1 as well as the expression and activation of TACE. However, in TNFα stimulated cells NAC treatment reduces only in part ICAM-1 expression and sICAM-1 release. Given this TNFα may also act on these events by a redox-independent mechanism.

Glutathione, N-acetylcysteine and lipoic acid down-regulate starvation-induced apoptosis, RANKL/OPG ratio and sclerostin in osteocytes: involvement of JNK and ERK1/2 signalling.

Osteocyte apoptosis due to microdamage and/or oxidative stress is related to increased local bone turnover and resorption observed in various bone diseases. Previous data on osteoblasts and osteoclasts have linked reactive oxygen species and antioxidants to bone remodelling. This study performs a comprehensive analysis on the effect of antioxidants such as glutathione (GSH), N-acetylcysteine and lipoic acid (LA) on starvation-induced osteocyte apoptosis and on cytokines involved in bone remodelling such as the receptor activator kB ligand (RANKL), osteoprotegerin (OPG) and sclerostin. For this study, apoptosis was induced by serum starvation in a murine osteocyte-like cell line MLO-Y4; this condition mimics in part osteocyte apoptosis due to microdamage. The results show that starvation-induced apoptosis and expression of RANKL, OPG and sclerostin are redox regulated processes. All antioxidants are able to inhibit the apoptosis due to starvation. They down-regulate the expression and the release of RANKL, the expression of sclerostin and RANKL/OPG ratio, whereas they only in part up-regulate OPG expression. Antioxidants mediate their effect on starvation-induced apoptosis by JNK signalling and on cytokine expression by both JNK and ERK1/2 activities. This study shows the possible involvement of biological antioxidants such as GSH and LA on redox regulated mechanisms related to apoptosis and expression of cytokines involved in bone remodelling. Moreover, it suggests that both JNK and ERK1/2 may be useful biological targets for drugs affecting bone diseases associated with increased oxidative stress.

Redox regulation of MMP-3/TIMP-1 ratio in intestinal myofibroblasts: effect of N-acetylcysteine and curcumin.

Matrix metalloproteinases (MMPs) play a critical role in inflammation and ulcerations in gut of Crohn׳s disease (CD) patients. Intestinal subepithelial myofibroblasts (ISEMFs) secrete MMPs in response to inflammatory stimuli. Previous data showed in CD-ISEMFs increased oxidative status. The aim of this study was to investigate the role of ISEMFs in modulating the production of MMP-3 and TIMP-1, an inhibitor of MMPs activity. A relationship among oxidative stress, activity of antioxidants and MMP-3/TIMP-1 was also studied. ISEMFs isolated from CD patient colon and human colonic cell line of myofibroblasts (18Co) were used. Oxidative state was modulated by buthionine sulfoximine, an inhibitor of glutathione (GSH) synthesis, and N-acetylcysteine (NAC), GSH precursor. An up-regulation of MMP-3 due to increased oxidative state was found in CD-ISEMFs. Stimulation by tumor necrosis factor (TNF)α increased further MMP-3 levels. On the contrary, no change in TIMP-1 production was determined. NAC treatment decreased MMP-3 production in CD-ISMEFs and removed the enhancement due to TNFα. Similar effects were observed in 18Co cells treated with curcumin, antioxidant with anti-inflammatory properties. The involvement of MAPKs on MMP-3 redox regulation was also shown. This study demonstrates the involvement of ISEMFs and high oxidative state in the increased MMP-3 production found in intestinal mucosa of CD patients. NAC and curcumin normalize MMP-3 levels mainly in TNFα stimulated cells. A modulation of MMP-3 production by NAC and curcumin due to their direct action on transcriptional factors has been also suggested. Therefore, they could have a therapeutic use for the prevention and treatment of fistulaes in CD.

Role of N-acetylcysteine and GSH redox system on total and active MMP-2 in intestinal myofibroblasts of Crohn's disease patients.

PURPOSE: Intestinal subepithelial myofibroblasts (ISEMFs)(1) are the predominant source of matrix metalloproteinase-2 (MMP-2) in gut, and a decrease in glutathione/oxidized glutathione (GSH/GSSG) ratio, intracellular redox state index, occurs in the ISEMFs of patients with Crohn's disease (CD). The aim of this study is to demonstrate a relationship between MMP-2 secretion and activation and changes of GSH/GSSG ratio in ISEMFs stimulated or not with tumor necrosis factor alpha (TNFα). METHODS: ISEMFs were isolated from ill and healthy colon mucosa of patients with active CD. Buthionine sulfoximine, GSH synthesis inhibitor, and N-acetylcysteine (NAC), precursor of GSH synthesis, were used to modulate GSH/GSSG ratio. GSH and GSSG were measured by HPLC and MMP-2 by ELISA Kit. RESULTS: In cells, stimulated or not with TNFα, a significant increase in MMP-2 secretion and activation, related to increased oxidative stress, due to low GSH/GSSG ratio, was detected. NAC treatment, increasing this ratio, reduced MMP-2 secretion and exhibited a direct effect on the secreted MMP-2 activity. In NAC-treated and TNFα-stimulated ISEMFs of CD patients' MMP-2 activity were restored to physiological value. The involvement of c-Jun N-terminal kinase pathway on redox regulation of MMP-2 secretion has been demonstrated. CONCLUSION: For the first time, in CD patient ISEMFs, a redox regulation of MMP-2 secretion and activation related to GSH/GSSG ratio and inflammatory state have been demonstrated. This study suggests that compounds able to maintain GSH/GSSG ratio to physiological values can be useful to restore normal MMP-2 levels reducing in CD patient intestine the dysfunction of epithelial barrier.

Oxidative state and IL-6 production in intestinal myofibroblasts of Crohn's disease patients.

BACKGROUND: Intestinal subepithelial myofibroblasts (ISEMFs) produce inflammatory cytokines in response to certain stimuli. In the intestine of patients with Crohn's disease (CD), cytokine synthesis is modified and an increased number of myofibroblasts has been observed. The intracellular redox state influences cytokine production and oxidative stress is present in the intestinal mucosa of CD patients. METHODS: This study was performed in ISEMFs isolated from the colon of patients with active CD and in a myofibroblast cell line derived from human colonic mucosa: 18Co cells. Cellular glutathione (GSH) levels were modulated by treatment with buthionine sulfoximine, an inhibitor of GSH synthesis, or N-acetylcysteine, a GSH precursor. GSH and oxidized glutathione (GSSG) levels were measured by high-performance liquid chromatography (HPLC) methods. Interleukin (IL)-6 production was detected by enzyme-linked immunosorbent assay (ELISA). RESULTS: ISEMFs of CD patients exhibited an increased oxidative state due to a decrease in the GSH/GSSG ratio, which is related to an increase in basal IL-6 production or is stimulated by tumor necrosis factor alpha (TNFα) or bacterial products. This relationship was also confirmed in 18Co cells. Phosphorylation and activation of ERK1/2 and p38 MAPK, which are signaling factors involved in the IL-6 synthesis, were also increased when there is oxidative stress in ISEMFs. CONCLUSIONS: This study shows for the first time in ISEMFs of CD patients an increased production of IL-6 synthesis related to the decrease in the GSH/GSSH ratio, suggesting redox regulation with the involvement of specific kinase activation. The present data shed light on the pathogenesis of inflammatory chronic processes and relapses that occur in this pathology.

Apoptosis and Bax, Bcl-2, Mcl-1 expression in neutrophils of Crohn's disease patients.

BACKGROUND: The etiology of Crohn's disease (CD) remains unknown, and the defective function of neutrophils appears to be associated with this pathology. Neutrophils undergo spontaneous apoptosis which, if not tightly regulated, can induce the development of chronic inflammatory disease. The Bcl-2 protein family is also involved in the regulation of neutrophil apoptosis. METHODS: This study investigated the apoptosis and expression of some regulatory factors in CD patient and control polymorphonuclear neutrophils (PMN) in suspension and in adhesion on fibronectin, an extracellular matrix protein. These 2 conditions mimic circulating neutrophils before they are recruited at the intestinal levels, and their adhesion to tissue. RESULTS: Apoptosis in CD patient PMN was delayed in suspension and accelerated in adhesion, which is the opposite of what happens in controls. Higher levels of Bax, Bcl-2, and Mcl-1 proteins were registered in freshly isolated CD patient PMN, in contrast to controls, in which Bcl-2 protein was undetectable. Among the studied pro- and antiapoptotic factors, Bax levels seem to be mainly related to the difference in apoptosis between PMN of CD patients and controls. CONCLUSIONS: For the first time it has been demonstrated by direct experimental evidence that apoptosis in CD patient PMN is regulated differently from that of control PMN. Abnormal expression of regulating apoptosis proteins is shown in CD patient PMN. These data suggest that the defective functionality of neutrophils can be the early event responsible for the altered mucosal immune response in CD, and that neutrophil apoptosis may offer a new target for specific drugs and therapy tools.

Redox regulation of platelet-derived-growth-factor-receptor: role of NADPH-oxidase and c-Src tyrosine kinase.

This study identifies some early events contributing to the redox regulation of platelet-derived growth factor receptor (PDGFr) activation and its signalling in NIH3T3 fibroblasts. We demonstrate for the first time that the redox regulation of PDGFr tyrosine autophosphorylation and its signalling are related to NADPH oxidase activity through protein kinase C (PKC) and phosphoinositide-3-kinase (PI3K) activation and H2O2 production. This event is also essential for complete PDGF-induced activation of c-Src kinase by Tyr416 phosphorylation, and the involvement of c-Src kinase on H2O2-induced PDGFr tyrosine phosphorylation is demonstrated, suggesting a role of this kinase on the redox regulation of PDGFr activation. Finally, it has been determined that not only PI3K activity, but also PKC activity, are related to NADPH oxidase activation due to PDGF stimulation in NIH3T3 cells, as it occurs in non-phagocyte cells. Therefore, we suggest a redox circuit whereby, upon PDGF stimulation, PKC, PI3K and NADPH oxidase activity contribute to complete c-Src kinase activation, thus promoting maximal phosphorylation and activation of PDGFr tyrosine phosphorylation.