Endocytosis and toxicity of clostridial binary toxins depend on a clathrin-independent pathway regulated by Rho-GDI.

Clostridial binary toxins, such as Clostridium perfringens Iota and Clostridium botulinum C2, are composed of a binding protein (Ib and C2II respectively) that recognizes distinct membrane receptors and mediates internalization of a catalytic protein (Ia and C2-I respectively) with ADP-ribosyltransferase activity that disrupts the actin cytoskeleton. We show here that the endocytic pathway followed by these toxins is independent of clathrin but requires the activity of dynamin and is regulated by Rho-GDI. This endocytic pathway is similar to a recently characterized clathrin-independent pathway followed by the interleukin-2 (IL2) receptor. We found indeed that Ib and C2II colocalized intracellularly with the IL2 receptor but not the transferrin receptor after different times of endocytosis. Accordingly, the intracellular effects of Iota and C2 on the cytoskeleton were inhibited by inactivation of dynamin or by Rho-GDI whereas inhibitors of clathrin-dependent endocytosis had no protective effect.

Use of xenofree matrices and molecularly-defined media to control human embryonic stem cell pluripotency: effect of low physiological TGF-beta concentrations.

To monitor human embryonic stem cell (hESC) self-renewal without differentiation, we used quantitative RT-PCR to study a selection of hESC genes, including markers for self-renewal, commitment/differentiation, and members of the TGF-beta superfamily and DAN gene family. Indeed, low commitment/differentiation gene expression, together with a significant self-renewal gene expres sion, provides a better pluripotency index than self-renewal genes alone. We demonstrate that matrices derived from human mesenchymal stem cells (hMSCs) can advantageously replace murine embryonic fibroblasts (MEF) or hMSC feeders. Moreover, a xenofree molecularly-defined SBX medium, containing a synthetic lipid carrier instead of albumin, can replace SR medium. The number of selected differentiation genes expressed by hESCs in these culture conditions was significantly lower than those expressed on MEF feeders in SR medium. In SBX, the positive effect of a non-physiological concentration of activin A (10-30 ng/mL) to reduce differentiation during self-renewal could also be obtained by physiological concentrations of TGF-beta(100-300 pg/mL). In contrast, these TGF-beta concentrations added to activin favored differentiation as previously observed with TGF-beta concentrations of 1 ng/mL or more. Compared to SR-containing medium, SBX medium promoted down-regulation of CER1 and LEFTIES and up-regulation of GREM1. Thus these genes better control self-renewal and pluripotency and prevent differentiation. A strategy is proposed to analyze, in more physiological, xenofree, molecularly-defined media and matrices, the numerous genes with still unknown functions controlling hESCs or human-induced pluripotent stem cells (iPS).

Interfering with interferon receptor sorting and trafficking: impact on signaling.

Interferons (IFNs) and their receptors (IFN-Rs) play fundamental roles in a multitude of biological functions. Many articles and reviews emphasize that the JAK/STAT machinery is obligatory for relay of the information transmitted by IFNs after binding to their cognate receptors at the plasma membrane. In contrast, very few studies have addressed the endocytosis and the intracellular trafficking of IFN-Rs, the immediate step following IFN binding. However, recent findings have shed light on the importance of IFN-R sorting and trafficking in the control of IFN signaling. Thus, IFN-Rs can be included in the growing family of signaling receptors for which regulation of biological activity critically involves endocytosis and trafficking.

Dynamin is involved in endolysosomal cholesterol delivery to the endoplasmic reticulum: role in cholesterol homeostasis.

Cholesterol is one of the most essential membrane components in mammalian cells and plays a critical role in several cellular functions. It is now established that intracellular cholesterol transport contributes to the regulation of cellular cholesterol homeostasis by mechanisms that are yet poorly defined. In this study, we examined the role of clathrin- and dynamin-dependent trafficking on the regulatory machinery involved in cholesterol homeostasis. Thus, expression levels of three major sterol-sensitive genes, that is sterol-regulatory element binding protein 2 (SREBP-2), hydroxymethylglutaryl-coenzyme A (HMGCoA) reductase and low-density lipoprotein (LDL) receptor, were monitored to study the cell response to the addition of LDL-derived cholesterol. We found that inhibition of clathrin-dependent endocytosis had no effect on the intracellular distribution of cholesterol and the regulation of sterol-sensitive genes. In contrast, inhibition of dynamin activity resulted in the lack of regulation of SREBP-2, HMGCoA reductase and LDL receptor genes. Immunolocalization studies along with the measure of free and esterified cholesterol indicated that dynamin inactivation led to the accumulation of free cholesterol (FC) within the late endosomal (LE)/lysosomal compartment resulting in insufficient delivery of regulatory cholesterol to the endoplasmic reticulum (ER) where the transcriptional control of sterol-sensitive genes occurs. Our data therefore indicate that dynamin plays a critical role in the delivery of cholesterol from the LE/lysosomal network to the ER and highlight the importance of LE trafficking in cholesterol homeostasis.

Stat-mediated signaling induced by type I and type II interferons (IFNs) is differentially controlled through lipid microdomain association and clathrin-dependent endocytosis of IFN receptors.

Type I (alpha/beta) and type II (gamma) interferons (IFNs) bind to distinct receptors, although they activate the same signal transducer and activator of transcription, Stat1, raising the question of how signal specificity is maintained. Here, we have characterized the sorting of IFN receptors (IFN-Rs) at the plasma membrane and the role it plays in IFN-dependent signaling and biological activities. We show that both IFN-alpha and IFN-gamma receptors are internalized by a classical clathrin- and dynamin-dependent endocytic pathway. Although inhibition of clathrin-dependent endocytosis blocked the uptake of IFN-alpha and IFN-gamma receptors, this inhibition only affected IFN-alpha-induced Stat1 and Stat2 signaling. Furthermore, the antiviral and antiproliferative activities induced by IFN-alpha but not IFN-gamma were also affected. Finally, we show that, unlike IFN-alpha receptors, activated IFN-gamma receptors rapidly become enriched in plasma membrane lipid microdomains. We conclude that IFN-R compartmentalization at the plasma membrane, through clathrin-dependent endocytosis and lipid-based microdomains, plays a critical role in the signaling and biological responses induced by IFNs and contributes to establishing specificity within the Jak/Stat signaling pathway.

Long-term expansion of human functional epidermal precursor cells: promotion of extensive amplification by low TGF-beta1 concentrations.

We have previously introduced the concept of high proliferative potential-quiescent (HPP-Q) cells to refer to primitive human hematopoietic progenitors, on which transforming growth factor-beta1 (TGF-beta1) exerts a pleiotropic effect. TGF-beta1 confers to these slow-dividing cells a mitogenic receptor(low) phenotype and maintains immature properties by preventing differentiation and apoptosis. However, the effect of TGF-beta1 on long-term expansion has not yet been clearly demonstrated. Here, we describe the characterization of a human skin keratinocyte subpopulation, highly enriched for primitive epidermal precursors, on the basis of high adhesion capacity (Adh+++) and low expression of the epidermal growth factor receptor (Adh+++EGF-Rlow). In our standard culture condition without feeder cells, the mean estimated output for cells from an unfractionated population of primary foreskin keratinocytes was 10(7)-10(8), increasing to 10(12)-10(13) in cultures initiated with selected Adh+++EGF-Rlow precursors. Characterization of these cells revealed a hitherto unknown property of TGF-beta1: its addition at a very low concentration (10 pg/ml) in long-term cultures induces a very significant additional increase of expansion. In this optimized system, outputs obtained in cultures initiated with Adh+++EGF-Rlow cells repeatedly reached 10(16)-10(17) ( approximately 60 population doublings, approximately 4 x 10(18) keratinocytes produced per clonogenic cell present in the initial population). At the molecular level, this effect is associated with an increase in Smad1, Smad2 and Smad3 phosphorylation and an increase in alpha6 and beta1 integrin expression. No such effect could be observed on mature keratinocytes with low adhesion capacity (Adh-/+). We finally demonstrated that the progeny of Adh+++EGF-Rlow precursors after long-term expansion is still capable of generating a pluristratified epidermis in a model for skin reconstruction. In conclusion, after further characterizing the phenotype of primitive epidermal precursors, we demonstrated a new function of TGF-beta1, which is to promote undifferentiated keratinocyte amplification.

Control of hematopoietic stem/progenitor cell fate by transforming growth factor-beta.

A major obstacle to the use of adult somatic stem cells for cell therapy is our current inability to fully exploit stem cell self-renewal properties. The challenge is to obtain defined culture systems where cycling of primitive stem/progenitor cells is stimulated, while their differentiation and senescence are prevented. The cytokine transforming growth factor-beta1 (TGF-beta1) appears as a potential regulator of hematopoietic stem/ progenitor cell self-renewal, as it participates in the control of cell proliferation, survival/apoptosis, and cell immaturity/differentiation. TGF-beta1 acts via a complex regulatory network involving intracellular signaling molecules and cell surface receptors. According to the High Proliferative Potential-Quiescent (HPP-Q) cell working model that we introduced previously, TGF-beta1 maintains primitive hematopoietic stem/progenitor cells in a quiescent or slow cycling state, in part by downmodulating the cell surface expression of mitogenic cytokine receptors, thus preventing cells from responding rapidly to a mitogenic signal. We have established that this modulation concerns the tyrosine kinase receptors KIT and FLT3, and the IL-6 receptor (IL-6R), three important cytokine receptors controlling early human hematopoietic stem/progenitor cell development. In this article. we show a similar modulation by TGF-beta1 of a fourth receptor: the TPO receptor (MPL). As a consequence, TGF-beta1 decreased the cell cycle entry of stem/progenitor cells stimulated by the respective ligands of these receptors, the cytokines SF, FL, IL-6, and TPO, whereas neutralization of TGF-beta1 increased the cell responsiveness to these mitogenic cytokines. Other aspects of the function of TGF-beta1 in the regulation of early hematopoiesis (i.e., the control of stem/progenitor cell survival and immaturity) are reviewed in the discussion.