Gaetano Calì

An Oscillatory Switch in mTOR Kinase Activity Sets Regulatory T Cell Responsiveness

There is a discrepancy between the inxa0vitro anergic state of CD4(+)CD25(hi)FoxP3(+) regulatory T (Treg) cells and their inxa0vivo proliferative capability. The underlying mechanism of this paradox is unknown. Here we show that the anergic state of Treg cells depends on the elevated activity of the mammalian target of rapamycin (mTOR) pathway induced by leptin: a transient inhibition of mTOR with rapamycin, before Txa0cell receptor (TCR) stimulation, made Treg cells highly proliferative in the absence of exogenous interleukin-2 (IL-2). This was a dynamic and oscillatory phenomenon characterized by an early downregulation of the leptin-mTOR pathway followed by an increase in mTOR activation necessary for Treg cell expansion to occur. These data suggest that energy metabolism, through the leptin-mTOR-axis, sets responsiveness of Treg cells that use this information to control immune tolerance and autoimmunity.

Leptin-Induced mTOR Activation Defines a Specific Molecular and Transcriptional Signature Controlling CD4+ Effector T Cell Responses

The sensing by T cells of metabolic and energetic changes in the microenvironment can determine the differentiation, maturation, and activation of these cells. Although it is known that mammalian target of rapamycin (mTOR) gauges nutritonal and energetic signals in the extracellular milieu, it is not known how mTOR and metabolism influence CD4+CD25−FOXP3− effector T cell (Teff) responses. In this article, we show that leptin-induced activation of mTOR, which, in turn, controls leptin production and signaling, causes a defined cellular, biochemical, and transcriptional signature that determine the outcome of Teff responses, both in vitro and in vivo. The blockade of leptin/leptin receptor signaling, induced by genetic means or by starvation, leads to impaired mTOR activity that inhibits the proliferation of Teffs in vivo. Notably, the transcriptional signature of Teffs in the presence of leptin blockade appears similar to that observed in rapamycin-treated Teffs. These results identify a novel link between nutritional status and Teff responses through the leptin–mTOR axis and define a potential target for Teff modulation in normal and pathologic conditions.

Increased mucosal nitric oxide production in ulcerative colitis is mediated in part by the enteroglial‐derived S100B protein

Abstractu2002 In the central nervous system glial‐derived S100B protein has been associated with inflammation via nitric oxide (NO) production. As the role of enteroglial cells in inflammatory bowel disease has been poorly investigated in humans, we evaluated the association of S100B and NO production in ulcerative colitis (UC). S100B mRNA and protein expression, inducible NO synthase (iNOS) expression, and NO production were evaluated in rectal biopsies from 30 controls and 35 UC patients. To verify the correlation between S100B and NO production, biopsies were exposed to S100B, in the presence or absence of specific receptor for advanced glycation end‐products (RAGE) blocking antibody, to measure iNOS expression and nitrite production. S100B and iNOS expression were evaluated after incubation of biopsies with lipopolysaccharides (LPS)u2003+u2003interferon‐gamma (IFN‐γ) in the presence of anti‐RAGE or anti‐S100B antibodies or budesonide. S100B mRNA and protein expression, iNOS expression and NO production were significantly higher in the rectal mucosa of patients compared to that of controls. Exogenous S100B induced a significant increase in both iNOS expression and NO production in controls and UC patients; this increase was inhibited by specific anti‐RAGE blocking antibody. Incubation with LPSu2003+u2003IFN‐γ induced a significant increase in S100B mRNA and protein expression, together with increased iNOS expression and NO production. LPSu2003+u2003IFN‐γ‐induced S100B up‐regulation was not affected by budesonide, while iNOS expression and NO production were significantly inhibited by both specific anti‐RAGE and anti‐S100B blocking antibodies. Enteroglial‐derived S100B up‐regulation in UC participates in NO production, involving RAGE in a steroid insensitive pathway.

Tissue Factor Binding of Activated Factor VII Triggers Smooth Muscle Cell Proliferation via Extracellular Signal–Regulated Kinase Activation

Background—Tissue factor (TF) is the main initiator of coagulation in vivo. Recently, however, a role for TF as a cell receptor involved in signal transduction has been suggested. The aim of the present study was to assess whether activated factor VII (FVIIa) binding to TF could induce smooth muscle cell (SMC) proliferation and to clarify the possible intracellular mechanism(s) responsible for this proliferation. Methods and Results—Cell proliferation was induced by FVIIa in a dose-dependent manner, as assessed by [3H]thymidine incorporation and direct cell counting, whereas no response was observed with active site–inhibited FVIIa (FVIIai), which is identical to FVIIa but is devoid of enzymatic activity. Similarly, no proliferation was observed when binding of FVIIa to TF was prevented by the monoclonal anti-TF antibody AP-1. Activation of the p44/42 mitogen-activated protein (MAP) kinase (extracellular signal–regulated kinases 1 and 2 [ERK 1/2]) pathway on binding of FVIIa to TF was demonstrated by transient ERK phosphorylation in Western blots and by suppression of proliferation with the specific MEK (MAP kinase/ERK kinase) inhibitor UO126. ERK phosphorylation was not observed with FVIIai or when cells were pretreated with AP-1. Conclusions—These data indicate a specific effect by which binding of FVIIa to TF on the surface of SMCs induces proliferation via a coagulation-independent mechanism and possibly indicate a new link between coagulation, inflammation, and atherosclerosis.

FK506 can activate transforming growth factor-β signalling in vascular smooth muscle cells and promote proliferation

AIMSnFK506-binding protein (FKBP) 12 is an inhibitor of transforming growth factor (TGF)-beta type I receptors. Several lines of evidence support the view that TGF-beta stimulates vascular smooth muscle cell (VSMC) proliferation and matrix accumulation. We investigated the effect of FK506, also known as tacrolimus, on cellular proliferation and on matrix protein production in human VSMCs.nnnMETHODS AND RESULTSnWe measured cell proliferation with flow cytometry using BrdU incorporation and fluorimetrically by measuring DNA concentration with Hoechst 33258. Western blot assay of whole-cell lysates was used to measure the levels of signalling proteins involved in proliferative pathways, in particular beta-catenin, pErk, pAkt, pmTOR, and cyclin D1. Collagen synthesis was also investigated by Western blotting. The TGF-beta signal was studied by both Western blotting and confocal microscopy. We used the SiRNA technique for FKBP12 gene silencing. Our results show that FK506 stimulates VSMC proliferation and collagen type I production. FK506 enhanced beta-catenin levels and activated the extracellular signal-regulated kinase, Akt, and mammalian target of rapamycin kinase, which are important effectors of proliferation. Accordingly, cyclin D1 expression was increased. We also demonstrate that FK506 activates the TGF-beta signal in VSMCs and that, through this mechanism, it stimulates cell proliferation.nnnCONCLUSIONnFK506 can act as a growth factor for VSMCs.

Nuclear factor-κB activation by reactive oxygen species mediates voltage-gated K+ current enhancement by neurotoxic β-amyloid peptides in nerve growth factor-differentiated PC-12 cells and hippocampal neurones

Increased activity of plasma membrane K+ channels, leading to decreased cytoplasmic K+ concentrations, occurs during neuronal cell death. In the present study, we showed that the neurotoxic β‐amyloid peptide Aβ25−35 caused a dose‐dependent (0.1–10u2003µm) and time‐dependent (>u200312u2003h) enhancement of both inactivating and non‐inactivating components of voltage‐dependent K+ (VGK) currents in nerve growth factor‐differentiated rat phaeochromocytoma (PC‐12) cells and primary rat hippocampal neurones. Similar effects were exerted by Aβ1−42, but not by the non‐neurotoxic Aβ35−25 peptide. Aβ25−35 and Aβ1−42 caused an early (15–20u2003min) increase in intracellular Ca2+ concentration. This led to an increased production of reactive oxygen species (ROS), which peaked at 3u2003h and lasted for 24u2003h; ROS production seemed to trigger the VGK current increase as vitamin E (50u2003µm) blocked both the Aβ25−35‐ and Aβ1−42‐induced ROS increase and VGK current enhancement. Inhibition of protein synthesis (cycloheximide, 1u2003µg/mL) and transcription (actinomycin D, 50u2003ng/mL) blocked Aβ25−35‐induced VGK current enhancement, suggesting that this potentiation is mediated by transcriptional activation induced by ROS. Interestingly, the specific nuclear factor‐κB inhibitor SN‐50 (5u2003µm), but not its inactive analogue SN‐50M (5u2003µm), fully counteracted Aβ1−42‐ or Aβ25−35‐induced enhancement of VGK currents, providing evidence for a role of this family of transcription factors in regulating neuronal K+ channel function during exposure to Aβ.

The microRNA-Processing Enzyme Dicer Is Essential for Thyroid Function

Dicer is a type III ribonuclease required for the biogenesis of microRNAs (miRNAs), a class of small non-coding RNAs regulating gene expression at the post-transcriptional level. To explore the functional role of miRNAs in thyroid gland function, we generated a thyrocyte-specific Dicer conditional knockout mouse. Here we show that development and early differentiation of the thyroid gland are not affected by the absence of Dicer, while severe hypothyroidism gradually develops after birth, leading to reduced body weight and shortened life span. Histological and molecular characterization of knockout mice reveals a dramatic loss of the thyroid gland follicular architecture associated with functional aberrations and down-regulation of several differentiation markers. The data presented in this study show for the first time that an intact miRNAs processing machinery is essential for thyroid physiology, suggesting that deregulation of specific miRNAs could be also involved in human thyroid dysfunctions.

Intracranial gene delivery of LV-NAGLU vector corrects neuropathology in murine MPS IIIB†‡

Mucopolysacccharidosis (MPS) IIIB is an inherited lysosomal storage disorder caused by the deficiency of alpha‐N‐acetylglucosaminidase (NAGLU). The disease is characterized by mild somatic features and severe neurological involvement with high mortality. Although several therapeutic approaches have been applied to the murine model of the disease, no effective therapy is available for patients. In this study, we used the lentiviral‐NAGLU vector to deliver the functional human NAGLU gene into the brain of young adult MPS IIIB mice. We report the restoration of active enzyme with a sustained expression throughout a large portion of the brain, and a significantly improved behavioral performance of treated animals. Moreover, we analyzed the effect of therapy on the expression profile of some genes related to neurotrophic signaling molecules and inflammatory cytokines previously found altered in MPS IIIB mice. At 1 month from treatment, the level of cerebellin 1 (Cbln1) was decreased while the brain‐derived neurotrophic factor (Bdnf) expression was increased, both reaching normal values. At 6 months from treatment a significant reduction in the expression of all the inflammation‐ and oxidative stress‐related genes was observed, as well as the maintenance of the correction of the Bdnf gene expression. These results indicate that NAGLU delivery from intracerebral sources has the capacity to alleviate most disease manifestations in MPS IIIB mice; furthermore, Bdnf might be a response‐to‐therapy biomarker for MPS IIIB.

Intracellular route and mechanism of action of ERB-hRNase, a human anti-ErbB2 anticancer immunoagent.

Human anti‐ErbB2 immunoRNase with Erbicin fused to HP‐RNase (ERB‐hRNase) is a fully human immunoRNase made up of human pancreatic RNase fused to a human anti‐ErbB2 scFv. It binds selectively with high affinity to ErbB2‐positive cells, and specifically inhibits their proliferation, in vitro and in vivo. An investigation of its mechanism of action and its intracellular destination has revealed that ERB‐hRNase depends on its RNase activity for cytotoxic action; it reaches the cytosol directly from the endosomal compartment; it is inhibited by the cytosolic RNase inhibitor (cRI), but the levels that ERB‐hRNase reaches in the cytosol neutralize cRI, thus inducing cell death by apoptosis.

Pro-atherothrombotic effects of leptin in human coronary endothelial cells

Adipocytes are nowadays recognised as cells able to produce and secrete a large variety of active substances termed adipokines, which exert direct effects on vascular cells. Among these adipokines, leptin has been proposed to play a role in the pathophysiology of acute coronary syndromes, as well as in increasing cardiovascular risk. At the moment, however, the mechanisms linking leptin to cardiovascular disease are not completely understood. This study investigates the effects of leptin, in a concentration range usually observed in the plasma of patients with increased cardiovascular risk or measurable in patients with acute coronary syndromes, on tissue factor (TF) and cellular adhesion molecules (CAMs) expression in human coronary endothelial cells (HCAECs). We demonstrate that leptin induces transcription of mRNA for TF and CAMs by real-time PCR. In addition, we show that this adipokine promotes surface expression of TF and CAMs that are functionally active since we observed increased procoagulant activity and leukocyte adhesion on cell surface. Leptin effects appear modulated by eNOS-production of oxygen free radicals through the activation of the transcription factor, nuclear factor(NF)-kappaB, since L-NAME, Superoxide Dismutase and NF-kappaB inhibitors suppressed CAMs and TF expression. Data of the present study, although in vitro , indicate that leptin may exert direct effects on human coronary endothelial cells by promoting CAMs and TF expression and support the hypothesis that this adipokines, besides being involved in the pathophysiology of obesity, might play a relevant role as an active mediator linking obesity to cardiovascular disease.