Arterial Tortuosity Syndrome: a vitamin C compartmentation disease?

Arterial tortuosity syndrome (ATS, MIM #208050) is a rare autosomal recessive connective tissue disorder characterized by tortuosity and elongation of the large and medium-sized arteries and a propensity towards aneurysm formation and vascular dissection. ATS is caused by mutations in SLC2A10 encoding the facilitative glucose transporter 10 (GLUT10), whose role in the ATS pathogenesis remains still controversial. We recently showed that GLUT10 deficiency causes the dysregulation of several genes/proteins involved in TGFβ signaling, extracellular matrix architecture and pathways that control oxidative stress response. GLUT10 should be located intracellularly; however, neither the exact localization, i.e., nuclear membrane, mitochondria, or endoplasmic reticulum (ER), nor the transported substances, i.e., glucose or dehydroascorbic acid (DAA), have been demonstrated. Here, we demonstrate that GLUT10 facilitates DAA uptake into the endomembranes and, in particular, into ER. GLUT10 produced by in vitro translation and incorporated into proteoliposomes efficiently transports DAA. Silencing of GLUT10 in hTERT immortalized human fibroblasts compromised DAA transport activity through the endomembranes. Similarly, in plasma membrane-permeabilized ATS fibroblasts a huge decrease in DAA transport was observed and the stable re-expression of GLUT10 restored the impaired DAA transport activity. Immunocytochemistry of human control fibroblasts showed a perinuclear abundance of GLUT10. Immunoblotting of subcellular fractions from human control fibroblasts revealed that GLUT10 was principally present in the microsomal fraction, containing ER-derived vesicles, as showed by the presence of the specific ER marker proteins GRP78 and GRP94, and by the almost complete absence of mitochondrial and cytoplasmic markers, VDAC1, cyclophilin D, and GAPDH, respectively. Transient expression of V5-tagged GLUT10 in ATS patients’ fibroblasts and co-localization experiments with the specific ER marker PDI definitely confirmed the ER localization of GLUT10. Overall, the present findings demonstrate that GLUT10 facilitates DAA uptake into the ER lumen and likely to the nucleoplasm through the nuclear envelope, which is a subdomain of the ER. Our findings support both “antioxidant-” and “enzyme cofactor-” models of a vitamin C-related pathology. Indeed, AA acts as an antioxidant/electron acceptor protecting against oxidative stress-induced cellular damage by scavenging free radicals also during the process of oxidative protein folding. Furthermore, AA is an essential cofactor for α-ketoglutarate-dependent dioxygenases, such as prolyl and lysyl hydroxylases inside the ER and for ten-eleven translocation demethylases and the Jumonji protein family present in the nucleus. Thus, shortage of AA in the lumenal compartments of the secretory pathway and in the nucleoplasm can depress the production of extracellular matrix proteins at both post-translational and epigenetic levels

Searching novel therapeutic targets for scleroderma: P2X7-receptor is UP-regulated and promotes a fibrogenic phenotype in systemic sclerosis fibroblasts

Objectives: Systemic sclerosis (SSc) is a connective tissue disorder presenting fibrosis of the skin and internal organs, for which no effective treatments are currently available. Increasing evidence indicates that the P2X7 receptor (P2X7R), a nucleotide-gated ionotropic channel primarily involved in the inflammatory response, may also have a key role in the development of tissue fibrosis in different body districts. This study was aimed at investigating P2X7R expression and function in promoting a fibrogenic phenotype in dermal fibroblasts from SSc patients, also analyzing putative underlying mechanistic pathways. Methods: Fibroblasts were isolated by skin biopsy from 9 SSc patients and 8 healthy controls. P2X7R expression, and function (cytosolic free Ca2+ fluxes, α-smooth muscle actin [α-SMA] expression, cell migration, and collagen release) were studied. Moreover, the role of cytokine (interleukin-1β, interleukin-6) and connective tissue growth factor (CTGF) production, and extracellular signal-regulated kinases (ERK) activation in mediating P2X7R-dependent pro-fibrotic effects in SSc fibroblasts was evaluated. Results: P2X7R expression and Ca2+ permeability induced by the selective P2X7R agonist 2'-3'-O-(4-benzoylbenzoyl)ATP (BzATP) weremarkedly higher in SSc than control fibroblasts. Moreover, increased aSMA expression, cell migration, CTGF, and collagen release were observed in lipopolysaccharides-primed SSc fibroblasts after BzATP stimulation. While P2X7-induced cytokine changes did not affect collagen production, it was completely abrogated by inhibition of the ERK pathway. Conclusion: In SSc fibroblasts, P2X7R is overexpressed and its stimulation induces Ca2+-signaling activation and a fibrogenic phenotype characterized by increased migration and collagen production. These data point to the P2X7R as a potential, novel therapeutic target for controlling exaggerated collagen deposition and tissue fibrosis in patients with SSc

Ion channel clustering enhances weak electric field detection by neutrophils: apparent roles of SKF96365-sensitive cation channels and myeloperoxidase trafficking in cellular responses

We have tested Galvanovskis and Sandblom’s prediction that ion channel clustering enhances weak electric field detection by cells as well as how the elicited signals couple to metabolic alterations. Electric field application was timed to coincide with certain known intracellular chemical oscillators (phase-matched conditions). Polarized, but not spherical, neutrophils labeled with anti-K v 1.3, FL-DHP, and anti-TRP1, but not anti-T-type Ca 2+ channels, displayed clusters at the lamellipodium. Resonance energy transfer experiments showed that these channel pairs were in close proximity. Dose-field sensitivity studies of channel blockers suggested that K + and Ca 2+ channels participate in field detection, as judged by enhanced oscillatory NAD(P)H amplitudes. Further studies suggested that K + channel blockers act by reducing the neutrophil’s membrane potential. Mibefradil and SKF93635, which block T-type Ca 2+ channels and SOCs, respectively, affected field detection at appropriate doses. Microfluorometry and high-speed imaging of indo-1-labeled neutrophils was used to examine Ca 2+ signaling. Electric fields enhanced Ca 2+ spike amplitude and triggered formation of a second traveling Ca 2+ wave. Mibefradil blocked Ca 2+ spikes and waves. Although 10 μM SKF96365 mimicked mibefradil, 7 μM SKF96365 specifically inhibited electric field-induced Ca 2+ signals, suggesting that one SKF96365-senstive site is influenced by electric fields. Although cells remained morphologically polarized, ion channel clusters at the lamellipodium and electric field sensitivity were inhibited by methyl-β-cyclodextrin. As a result of phase-matched electric field application in the presence of ion channel clusters, myeloperoxidase (MPO) was found to traffic to the cell surface. As MPO participates in high amplitude metabolic oscillations, this suggests a link between the signaling apparatus and metabolic changes. Furthermore, electric field effects could be blocked by MPO inhibition or removal while certain electric field effects were mimicked by the addition of MPO to untreated cells. Therefore, channel clustering plays an important role in electric field detection and downstream responses of morphologically polarized neutrophils. In addition to providing new mechanistic insights concerning electric field interactions with cells, our work suggests novel methods to remotely manipulate physiological pathways.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/46726/1/249_2005_Article_1.pd

Inhibition of store-dependent capacitative Ca2+ influx by unsaturated fatty acids

The effects of the unsaturated fatty acids, arachidonic and oleic acid, on the influx of Ca2+ activated by depletion of intracellular stores with thapsigargin were investigated in various cell types. By using a Ca2+ free/Ca2+ reintroduction protocol, we observed that arachidonic acid (2 to 5 microM) inhibited thapsigargin-induced rises in cytosolic free Ca2+ ([Ca2+]i) in Ehrlich tumor cells, Jurkat T lymphocytes, rat thymocytes, and Friend erythroleukemia and PC12 rat pheochromocytoma cells. This effect was attributed to the inhibition of Ca2+ entry, since arachidonate also inhibited thapsigargin-stimulated unidirectional entry of the Ca2+ surrogates Ba2+ and Mn2+. In Ehrlich cells, the IC50 for arachidonic and oleic acid was 1.2 and 1.8 microM, respectively. The inhibition appeared to depend on the ratio [fatty acid]/[cells] rather than on the absolute fatty acid concentration. Experiments with [3H]-oleic acid revealed that the inhibitory activity was not correlated with cell internalisation and metabolism of the fatty acid. The inhibition was reverted by removal of the fatty acid bound to cell membrane by fatty acid-free albumin treatment. The unsaturated fatty acids had no effect on ATP/ADP cell levels and plasma membrane potential. Pharmacological evidence indicated that cell phosphorylation/dephosphorylation events, and pertussis toxin-sensitive G proteins were not involved. Other amphipathic lipophilic compounds, i.e. 2-bromopalmitic acid, retinoic acid, sphingosine, and dihydrosphingosine, mimicked arachidonic/oleic acid as they inhibited thapsigargin-stimulated Ca2+ influx in an albumin-reversible fashion. These results suggest that physiologically relevant (unsaturated) fatty acids can inhibit capacitative Ca2+ influx possibly because they intercalate into the plasma membrane and directly affect the activity of the channels involved