TLR-4 signalling accelerates colon cancer cell adhesion via NF-κB mediated transcriptional up-regulation of Nox-1.

Surgery induced inflammation is a potent promoter of tumour recurrence and metastasis in colorectal cancer. The recently discovered family of Nox enzymes represent a major source of endogenous reactive oxygen species (ROS) and are now heavily implicated in tumour cell metastasis. Interestingly, Nox enzymes can be 'purposefully' activated by inflammatory cytokines and growth factors which are present in abundance in the peri-operative window. As colon cancer cells express Nox enzymes and Toll-like receptor 4 (TLR-4), we hypothesised that LPS may potentiate the ability of colon cancer cells to metastasise via Nox enzyme mediated redox signalling. In support of this hypothesis, this paper demonstrates that LPS induces a significant, transient increase of endogenous ROS in SW480, SW620 and CT-26 colon cancer cells. This increase in LPS-induced ROS activity is completely abrogated by a Nox inhibitor, diphenyleneiodonium (DPI), Nox1 siRNA and an NF-κB inhibitor, Dihydrochloride. A significant increase in Nox1 and Nox2 protein expression occurs following LPS treatment. Inhibition of NF-κB also attenuates the increase of Nox1 and Nox2 protein expression. The sub-cellular location of LPS-induced ROS generation lies mainly in the endoplasmic reticulum. LPS activates the PI3K/Akt pathway via Nox generated ROS and this signal is inhibited by DPI. This LPS activated Nox mechanism facilitates a significant increase in SW480 colon cancer cell adhesion to collagen I, which is inhibited by DPI, Nox1 siRNA and a PI3K inhibitor. Altogether, these data suggest that the LPS-Nox1 redox signalling axis plays a crucial role in facilitation of colon cancer cell adhesion, thus increasing the metastatic potential of colon cancer cells. Nox1 may represent a valuable target in which to prevent colon cancer metastasis.

H2O2 production downstream of FLT3 is mediated by p22phox in the endoplasmic reticulum and is required for STAT5 signalling.

The internal tandem duplication (ITD) of the juxtamembrane region of the FLT3 receptor has been associated with increased reactive oxygen species (ROS) generation in acute myeloid leukemia (AML). How this elevated level of ROS contributes to the leukemic phenotype, however, remains poorly understood. In this work we show that ROS in the FLT3-ITD expressing AML cell line MV4-11 is reduced by treatment with PKC412, an inhibitor of FLT3, DPI, a flavoprotein inhibitor, and VAS2870, a Nox specific inhibitor, suggesting that ROS production is both FLT3 and NADPH oxidase dependent. The majority of these ROS co-localize to the endoplasmic reticulum (ER), as determined with the H(2)O(2)-specific aryl-boronate dye Peroxyorange 1, which also corresponds to co-localization of p22phox. Moreover, knocking down p22phox dramatically reduces H(2)O(2) after 24 hours in the ER, without affecting mitochondrial ROS. Significantly, the FLT3 inhibitor PKC412 reduces H(2)O(2) in FLT3-ITD expressing cell lines (MV4-11, MOLM-13) through reduction of p22phox over 24 hours. Reduced p22phox is achieved by proteasomal degradation and is prevented upon GSK3-ß inhibition. Knockdown of p22phox resulted in reduced STAT5 signalling and reduced Pim-1 levels in the cells after 24 hours. Thus, we have shown that FLT3 driven H(2)O(2) production in AML cells is mediated by p22phox and is critical for STAT5 signalling.

Inhibition of protein-tyrosine phosphatase 1B (PTP1B) mediates ubiquitination and degradation of Bcr-Abl protein.

Chronic myelogenous leukemia (CML) is a myeloproliferative disorder characterized at the molecular level by the expression of Bcr-Abl, a chimeric protein with deregulated tyrosine kinase activity. The protein-tyrosine phosphatase 1B (PTP1B) is up-regulated in Bcr-Abl-expressing cells, suggesting a regulatory link between the two proteins. To investigate the interplay between these two proteins, we inhibited the activity of PTP1B in Bcr-Abl-expressing TonB.210 cells by either pharmacological or siRNA means and examined the effects of such inhibition on Bcr-Abl expression and function. Herein we describe a novel mechanism by which the phosphatase activity of PTP1B is required for Bcr-Abl protein stability. Inhibition of PTP1B elicits tyrosine phosphorylation of Bcr-Abl that triggers the degradation of Bcr-Abl through ubiquitination via the lysosomal pathway. The degradation of Bcr-Abl consequently inhibits tyrosine phosphorylation of Bcr-Abl substrates and the downstream production of intracellular reactive oxygen species. Furthermore, PTP1B inhibition reduces cell viability and the IC(50) of the Bcr-Abl inhibitor imatinib mesylate. Degradation of Bcr-Abl via PTP1B inhibition is also observed in human CML cell lines K562 and LAMA-84. These results suggest that inhibition of PTP1B may be a useful strategy to explore in the development of novel therapeutic agents for the treatment of CML, particularly because host drugs currently used in CML such as imatinib focus on inhibiting the kinase activity of Bcr-Abl.

bFGF-mediated redox activation of the PI3K/Akt pathway in retinal photoreceptor cells.

In many retinal diseases, it is the death of photoreceptors that leads to blindness. In previous in vitro and in vivo studies, basic fibroblast growth factor (bFGF) has been shown to increase retinal cell survival. More recently, reactive oxygen species (ROS) have also been shown to promote cell survival, contrary to the traditional view that they are solely destructive molecules. Due to this possible link, we hypothesised that bFGF could stimulate the production of ROS, which in turn stimulates the protein kinase B (Akt) survival pathway. Flow cytometry was used to measure the fluorescence of oxidised dihydrorhodamine, a ROS indicator, in the murine 661W photoreceptor cell line under several different conditions. Expression of cyclooxygenase (Cox) enzymes was evaluated by immunohistochemistry, and the response of photoreceptor cells to exogenous bFGF in the explanted mouse retina was studied by confocal microscopy. Exogenous addition of bFGF to 661W cells resulted in an increase in ROS production that lasted for 24 h. When this ROS production was inhibited, bFGF-induced phosphorylation of Akt was prevented. Through the use of inhibitors and small interfering RNA in the cell line, the source of this production was shown to be Cox and to involve the activation of phospholipases A(2) + C. This pathway may also occur in the mouse retina, as we showed that the retina expressed Cox1&2, and that photoreceptors in explanted retina respond to bFGF by increasing their ROS levels. These results demonstrate that exogenous bFGF can stimulate ROS production through the activation of Cox, and activate the Akt pathway.

N-glycosylation is important for the correct intracellular localization of HFE and its ability to decrease cell surface transferrin binding.

HFE is a type 1 transmembrane protein that becomes N-glycosylated during transport to the cell membrane. It influences cellular iron concentrations through multiple mechanisms, including regulation of transferrin binding to transferrin receptors. The importance of glycosylation in HFE localization and function has not yet been studied. Here we employed bioinformatics to identify putative N-glycosylation sites at residues N110, N130 and N234 of the human HFE protein, and used site-directed mutagenesis to create combinations of single, double or triple mutants. Compared with the wild-type protein, which co-localizes with the type 1 transferrin receptor in the endosomal recycling compartment and on distributed punctae, the triple mutant co-localized with BiP in the endoplasmic reticulum. This was similar to the localization pattern described previously for the misfolding HFE-C282Y mutant that causes type 1 hereditary haemachromatosis. We also observed that the triple mutant was functionally deficient in beta2-microglobulin interactions and incapable of regulating transferrin binding, once again, reminiscent of the HFE-C282Y variant. Single and double mutants that undergo limited glycosylation appeared to have a mixed phenotype, with characteristics primarily of the wild-type, but also some from the glycosylation-deficient protein. Therefore, although they displayed an endosomal recycling compartment/punctate localization like the wild-type protein, many cells simultaneously displayed additional reticular localization. Furthermore, although the majority of cells expressing these single and double mutants showed decreased surface binding of transferrin, a number appeared to have lost this ability. We conclude that glycosylation is important for the normal intracellular trafficking and functional activity of HFE.

Rod and cone photoreceptor cells produce ROS in response to stress in a live retinal explant system.

PURPOSE: The production of reactive oxygen species (ROS) can lead to oxidative stress, which is a strong contributory factor to many ocular diseases. In this study, the removal of trophic factors is used as a model system to investigate the effects of stress in the retina. The aims were to determine if both rod and cone photoreceptor cells produce ROS when they are deprived of trophic factor support and to demonstrate if the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox) enzymes are responsible for this ROS production. METHODS: Retinas were explanted from mice aged between postnatal days 8-10 and cultured overnight. The following morning, confocal microscopy combined with various fluorescent probes was used to detect the production of ROS. Each time peanut agglutinin (PNA), a cone photoreceptor marker, was used to facilitate orientation of the retina. Dihydroethidium and dihydrorhodamine 123 (DHR123) were used to determine which cells produce ROS. Subsequently, western blots of retinal serial sections were used to detect the presence of Noxs in the different retinal layers. The Nox inhibitor apocynin was then tested to determine if it altered the production of ROS within these cells. RESULTS: Live retinal explants, viewed at high magnifications using confocal microscopy, displayed an increase in the fluorescent products of dihydroethidium and DHR123 upon serum removal when compared to controls. DHR123 fluorescence, once oxidized, localized to mitochondria and was found in the same focal plane as the PNA staining. This showed that cones and rods produced ROS when stressed. Retinal serial sectioning established that the photoreceptor layer expressed Nox4, dual oxidase (Duox) 1, and Duox2 at varying levels. Finally, the Nox inhibitor apocynin decreased the burst stimulated by the stress of serum removal. CONCLUSIONS: Confocal microscopy and PNA staining allowed differentiation of cell types within the outermost layers of the retina, demonstrating that both rods and cones generated ROS in response to the stress of serum deprivation. Nox4 was the most abundantly expressed Nox in the photoreceptor layer, but Duox1 and Duox2 were also present at detectable levels, and as apocynin reduced the levels of ROS produced, this implied that these proteins may play some role in this production.

Stress-induced activation of Nox contributes to cell survival signalling via production of hydrogen peroxide.

Reactive oxygen species (ROS) have traditionally been viewed as a toxic group of molecules; however, recent publications have shown that these molecules, including H(2)O(2), can also strongly promote cell survival. Even though the retina has a large capacity to produce ROS, little is known about its non-mitochondrial sources of these molecules, in particular the expression and function of NADPH oxidase (Nox) proteins which are involved in the direct generation of superoxide and indirectly H(2)O(2). This study demonstrated that 661W cells, a retina-derived cell line, and mouse retinal explants express Nox2, Nox4 and certain of their well-established regulators. The roles of Nox2 and Nox4 in producing pro-survival H(2)O(2) were determined using 661W cells and some of the controlling factors were identified. To ascertain if this phenomenon could have physiological relevance, the novel technique of time-lapse imaging of dichlorofluorescein fluorescence (generated upon H(2)O(2) production) in retinal explants was established and it showed that explants also produce a burst of H(2)O(2). The increase in H(2)O(2) production was partly blocked by an inhibitor of Nox proteins. Overall, this study demonstrates a pro-survival role of Nox2 and Nox4 in retina-derived cells, elucidates some of the regulatory mechanisms and reveals that a similar phenomenon exists in retinal tissue as a whole.

Knockdown of beta2-microglobulin perturbs the subcellular distribution of HFE and hepcidin.

Hereditary Haemochromatosis is an iron overload disorder associated with mutations in the HFE gene, and to a lesser degree, the gene encoding its chaperone protein beta-2 microglobulin (beta2M). Here, we report that knockdown of beta2M by RNAi restricts HFE distribution to the endoplasmic reticulum (ER). Additionally, we demonstrate that hepcidin, an iron homeostasis-associated protein, localises predominantly to LBPA-positive late endosomes. Interestingly, we show that knockdown of beta2M by RNAi perturbs hepcidin localisation to late endosomes. In summary, our data suggest that beta2M is essential for the correct subcellular distribution of both HFE and hepcidin, two proteins, which are critical for iron homeostasis.

The hereditary hemochromatosis protein HFE and its chaperone beta2-microglobulin localise predominantly to the endosomal-recycling compartment.

Hereditary Hemochromatosis is an iron overload disease most frequently associated with mutations in the HFE gene. While clinical studies of the disease have received extensive attention by various groups, the localisation, trafficking and function of the HFE protein, and its chaperone beta2-microglobulin (beta2M), require further investigation. In this study, we present data on the cellular localisation of HFE and its clinically relevant mutants in HuTu 80 cells. We find by confocal microscopy that HFE localises to the endosomal-recycling compartment (ERC), with minimal localisation to sorting or late endosomes. Interestingly, we also demonstrate that beta2M localises to the ERC where it co-localises with HFE. We find that exogenous expression of HFE results in enhanced beta2M cellular levels and that beta2M is necessary for cell surface expression of HFE. Finally, we have analysed the functional effects of exogenous expression of HFE and beta2M on transferrin binding to the cell surface. In summary, our study sheds light on the localisation and functional effects of the HFE and its chaperone protein beta2M.

Merging mouse transcriptome analyses with Parkinson's disease linkage studies.

The hallmark of Parkinson's disease (PD OMIM #168600) is the degeneration of the nigral dopaminergic system affecting approximately 1% of the human population older than 65. In pursuit of genetic factors contributing to PD, linkage and association studies identified several susceptibility genes. The majority of these genes are expressed by the dopamine-producing neurons in the substantia nigra. We, therefore, propose expression by these neurons as a selection criterion, to narrow down, in a rational manner, the number of candidate genes in orphan PD loci, where no mutation has been associated thus far. We determined the corresponding human chromosome locations of 1435 murine cDNA fragments obtained from murine expression analyses of nigral dopaminergic neurons and combined these data with human linkage studies. These fragments represent 19 genes within orphan OMIM PD loci. We used the same approach for independent association studies and determined the genes in neighborhood to the peaks with the highest LOD score value. Our approach did not make any assumptions about disease mechanisms, but it, nevertheless, revealed alpha-synuclein, NR4A2 (Nurr1), and the tau genes, which had previously been associated to PD. Furthermore, our transcriptome analysis identified several classes of candidate genes for PD mutations and may also provide insight into the molecular pathways active in nigral dopaminergic neurons.

Identification and developmental analysis of genes expressed by dopaminergic neurons of the substantia nigra pars compacta.

The hallmark of Parkinson's Disease is the degenerative loss of mesencephalic dopaminergic (mDA) neurons. Previous studies have shown that the homeobox transcription factors, engrailed-1 and -2, are essential for the survival of these cells. To identify genes downstream of engrailed-1 and -2, we performed a PCR-based differential display, comparing RNA from engrailed-1/2 double mutant and wild type ventral midbrain of different embryonic ages to adult olfactory bulb, a source of unrelated DA neurons. Here, we report the result of this experiment and describe the developmental expression pattern in the ventral midbrain of three of the isolated genes, HNF3alpha, synaptotagmin I, and Ebf3. Though not regulated by engrailed-1 and -2, the expression of all three genes is limited to mDA neurons and a few other brain areas. HNF3alpha appears in the precursors of mDA neurons at E9 and is expressed in the adult brain almost exclusively by this neuronal population. Synaptotagmin I is expressed from E14 into adulthood. Ebf3, in contrast, is transiently expressed during early postmitotic differentiation.

Midbrain dopaminergic neurons: determination of their developmental fate by transcription factors.

Midbrain dopaminergic neurons are the main source of dopamine in the mammalian central nervous system and are associated with one of the most prominent human neurological disorders, Parkinson's disease. During development, they are induced in the ventral midbrain by an interaction between two diffusible factors, SHH and FGF8. The local identity of this part of the midbrain is probably determined by the combinatorial expression of three transcription factors, Otx2, Pax2, and Pax5. After the last cell division, the neurons start to express transcription factors that control further differentiation and the manifestation of cellular properties characteristic for adult dopaminergic neurons of the substantia nigra compacta and the ventral tegmentum. The first to appear is the LIM-homeodomain transcription factor, Lmx1b. It is essential for the survival of these neurons, and it regulates the expression of another transcription factor, Pitx3, an activator of tyrosine hydroxylase. Lmx1b is followed by the orphan steroid receptor Nurr1. It is essential for the expression of the dopaminergic phenotype. Several genes involved in dopamine synthesis, transport, release, and reuptake are regulated by Nurr1. This requirement is specific to the midbrain dopaminergic neurons, since other populations of the same neurotransmitter phenotype develop normally in absence of the gene. A day after Nurr1, two homeodomain transcription factors, engrailed-1 and -2, are expressed. In animals deficient in the two genes, the midbrain dopaminergic neurons are generated, but then fail to differentiate and disappear very rapidly. Interestingly, alpha-synuclein, a gene recently linked to familial forms of Parkinson's disease, is regulated by engrailed-1 and -2.