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

Fructose, glucocorticoids and adipose tissue: Implications for the metabolic syndrome

The modern Western society lifestyle is characterized by a hyperenergetic, high sugar containing food intake. Sugar intake increased dramatically during the last few decades, due to the excessive consumption of high-sugar drinks and high-fructose corn syrup. Current evidence suggests that high fructose intake when combined with overeating and adiposity promotes adverse metabolic health effects including dyslipidemia, insulin resistance, type II diabetes, and inflammation. Similarly, elevated glucocorticoid levels, especially the enhanced generation of active glucocorticoids in the adipose tissue due to increased 11β-hydroxysteroid dehydrogenase 1 (11β -HSD1) activity, have been associated with metabolic diseases. Moreover, recent evidence suggests that fructose stimulates the 11β -HSD1-mediated glucocorticoid activation by enhancing the availability of its cofactor NADPH. In adipocytes, fructose was found to stimulate 11β -HSD1 expression and activity, thereby promoting the adipogenic effects of glucocorticoids. This article aims to highlight the interconnections between overwhelmed fructose metabolism, intracellular glucocorticoid activation in adipose tissue, and their metabolic effects on the progression of the metabolic syndrome. © 2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license

Proton pump inhibitors and serum magnesium levels in patients with Torsades de Pointes

Background: Torsades de pointes (TdP) is a life-threatening ventricular tachycardia occurring in long QT-syndrome patients. It usually develops when multiple QT-prolonging factors are concomitantly present, more frequently drugs and electrolyte imbalances. Since proton-pump inhibitors (PPIs)-associated hypomagnesemia is an increasingly recognized adverse event, PPIs were recently included in the list of drugs with conditional risk of TdP, despite only few cases of TdP in PPI users have been reported so far. Objectives: Aim of the present study is to evaluate whether PPI-induced hypomagnesemia actually has a significant clinical impact on the risk of TdP in the general population. Methods: Forty-eight unselected patients who experienced TdP were consecutively enrolled (2008-2017). Shortly after the first TdP episode, in those patients who did not receive magnesium sulfate and/or potassium or calcium replacement therapy, serum electrolytes were measured and their relationship with PPI usage analyzed. Results: Many patients (28/48, 58%) were under current PPI treatment when TdP occurred. Among TdP patients in whom serum electrolyte determinations were obtained before replacement therapy (27/48), those taking PPIs had significantly lower serum magnesium levels than those who did not. Hypomagnesemia occurred in ~40% of patients receiving PPIs (6/14), in all cases after an extended treatment (> 2 weeks). In patients taking PPIs the mean QT-prolonging risk factor number was significantly higher than in those who did not, a difference which was mainly driven by lower magnesium levels. Conclusions: In unselected TdP patients, PPI-induced hypomagnesemia was common and significantly contributed to their cumulative arrhythmic risk. By providing clinical support to current recommendations, our data confirm that more awareness is needed when a PPI is prescribed, specifically as regards the risk of life-threatening arrhythmias

GLUT10-Lacking in Arterial Tortuosity Syndrome-Is Localized to the Endoplasmic Reticulum of Human Fibroblasts.

GLUT10 belongs to a family of transporters that catalyze the uptake of sugars/polyols by facilitated diffusion. Loss-of-function mutations in the SLC2A10 gene encoding GLUT10 are responsible for arterial tortuosity syndrome (ATS). Since subcellular distribution of the transporter is dubious, we aimed to clarify the localization of GLUT10. In silico GLUT10 localization prediction suggested its presence in the endoplasmic reticulum (ER). Immunoblotting showed the presence of GLUT10 protein in the microsomal, but not in mitochondrial fractions of human fibroblasts and liver tissue. An even cytosolic distribution with an intense perinuclear decoration of GLUT10 was demonstrated by immunofluorescence in human fibroblasts, whilst mitochondrial markers revealed a fully different decoration pattern. GLUT10 decoration was fully absent in fibroblasts from three ATS patients. Expression of exogenous, tagged GLUT10 in fibroblasts from an ATS patient revealed a strict co-localization with the ER marker protein disulfide isomerase (PDI). The results demonstrate that GLUT10 is present in the ER

Glucose transporter type 10—lacking in arterial tortuosity syndrome—facilitates dehydroascorbic acid transport

Loss-of-function mutations in the gene encoding GLUT10 are responsible for arterial tortuosity syndrome (ATS), a rare connective tissue disorder. In this study GLUT10-mediated dehydroascorbic acid (DAA) transport was investigated, supposing its involvement in the pathomechanism. GLUT10 protein produced by in vitro translation and incorporated into liposomes efficiently transported DAA. Silencing of GLUT10 decreased DAA transport in immortalized human fibroblasts whose plasma membrane was selectively permeabilized. Similarly, the transport of DAA through endomembranes was markedly reduced in fibroblasts from ATS patients. Re-expression of GLUT10 in patients’ fibroblasts restored DAA transport activity. The present results demonstrate that GLUT10 is a DAA transporter and DAA transport is diminished in the endomembranes of fibroblasts from ATS patients

Progesterone inhibits capacitative Ca2+ entry in Jurkat T lymphocytes by a membrane delimited mechanism, independently of plasma membrane depolarization

The non-genomic inhibitory effect of progesterone on capacitative calcium entry was studied in Jurkat T lymphocytes. Capacitative calcium entry was induced by depleting intracellular calcium stores with thapsigargin and evaluated by a calcium free/calcium readmission protocol, in Fura-2 loaded cells. Progesterone (10-40 microg/ml) inhibited calcium entry and concomitantly depolarized cells, as revealed by measuring the plasma membrane potential with the fluorescent probe bis-oxonol. However, experiments run under depolarizing conditions (i.e. by substituting for Na+ with K+ ions in the medium) revealed that progesterone (10-40 microg/ml) could inhibit capacitative calcium entry independently of plasma membrane depolarization. The direct inhibition of calcium entry by progesterone was: (i) reverted by a treatment suitable to remove progesterone bound to cell surface, (ii) apparently related to the extent of membrane bound progesterone (measured radioisotopically), and (iii) specific, in that other related steroid compounds did not inhibit calcium entry

The autophagy inducer trehalose stimulates macropinocytosis in NF1-deficient glioblastoma cells

Abstract Background Glioblastoma is a highly aggressive brain tumor. A big effort is required to find novel molecules which can cross the blood–brain barrier and efficiently kill these tumor cells. In this perspective, trehalose (α-glucopyranosyl‐[1→1]‐α‐d‐glucopyranoside), found in various dietary sources and used as a safe nutrient supplement, attracted our attention for its pleiotropic effects against tumor cells. Methods Human glioblastoma cell lines U373-MG and T98G were exposed to trehalose and analyzed at different time points. Cell proliferation was evaluated at medium term, and clonogenic capacity and cell morphology were evaluated at long term. Western blot was used to evaluate biochemical markers of autophagy (also measured in cells co-treated with EIPA or chloroquine), and mTOR, AMPK and ERK 1/2 signalling. Macropinocytosis was evaluated morphologically by bright-field microscopy; in cells loaded with the fluorescein-conjugated fluid-phase tracer dextran, macropinocytic vacuoles were also visualized by fluorescence microscopy, and the extent of macropinocytosis was quantified by flow cytometry. Results The long-term effect of trehalose on U373-MG and T98G cell lines was impressive, as indicated by a dramatic reduction in clonogenic efficiency. Mechanistically, trehalose proved to be an efficient autophagy inducer in macropinocytosis-deficient T98G cells and an efficient inducer of macropinocytosis and eventual cell death by methuosis in U373-MG glioblastoma cells, proved to be poorly responsive to induction of autophagy. These two processes appeared to act in a mutually exclusive manner; indeed, co-treatment of U373-MG cells with the macropinocytosis inhibitor, EIPA, significantly increased the autophagic response. mTOR activation and AMPK inhibition occurred in a similar way in the two trehalose-treated cell lines. Interestingly, ERK 1/2 was activated only in macropinocytosis-proficient U373-MG cells harbouring loss-of-function mutations in the negative RAS regulator, NF1, suggesting a key role of RAS signalling. Conclusions Our results indicate that trehalose is worthy of further study as a candidate molecule for glioblastoma therapy, due to its capacity to induce a sustained autophagic response, ultimately leading to loss of clonogenic potential, and more interestingly, to force macropinocytosis, eventually leading to cell death by methuosis, particularly in tumor cells with RAS hyperactivity. As a further anticancer strategy, stimulation of macropinocytosis may be exploited to increase intracellular delivery of anticancer drugs

Biochemical and functional heterogeneity of rat cerebrum microsomal membranes in relation to SERCA Ca2+-ATPases and Ca2+ release channels

Rat cerebrum microsomes were subfractionated on isopycnic linear sucrose (20-42%) density gradients. The Ca2+ loading/release properties and the distribution of intracellular Ca2+ store channels, inositol 1,4,5-trisphosphate (IP3) receptor and ryanodine (Ry) receptor, and SERCA pumps, were monitored in each subfraction by ligand binding and 45Ca2+ loading/release assays. Three different classes of vesicles were identified: (i) heavy density vesicles with high content of Ry receptors and Ca2+ pumps and high thapsigargin (TG)-sensitivity of Ca2+ loading; (ii) intermediate sucrose density vesicles with high content of IP3 receptor, high IP(S)3-sensitivity of Ca2+ loading and low content of Ry receptors; and (iii) light sucrose density vesicles with high content of Ry receptors, low content of IP3 receptors and low content of SERCA pumps highly sensitive to TG. Isolation of molecularly heterogeneous rat cerebrum microsomes and identification of specific Ca2+ loading/release properties support the presence of multiple, potentially active, heterogeneous rapidly exchanging Ca2+ stores in rat cerebru