Inhibition of Hedgehog Signaling Decreases Proliferation and Clonogenicity of Human Mesenchymal Stem Cells

Human mesenchymal stem cells (hMSC) have the ability to differentiate into osteoblasts, adipocytes and chondrocytes. We have previously shown that hMSC were endowed with a basal level of Hedgehog signaling that decreased after differentiation of these cells. Since hMSC differentiation is associated with growth-arrest we investigated the function of Hh signaling on cell proliferation. Here, we show that inhibition of Hh signaling, using the classical inhibitor cyclopamine, or a siRNA directed against Gli-2, leads to a decrease in hMSC proliferation. This phenomenon is not linked to apoptosis but to a block of the cells in the G0/G1 phases of the cell cycle. At the molecular level, it is associated with an increase in the active form of pRB, and a decrease in cyclin A expression and MAP kinase phosphorylation. Inhibition of Hh signaling is also associated with a decrease in the ability of the cells to form clones. By contrast, inhibition of Hh signaling during hMSC proliferation does not affect their ability to differentiate. This study demonstrates that hMSC are endowed with a basal Hedgehog signaling activity that is necessary for efficient proliferation and clonogenicity of hMSC. This observation unravels an unexpected new function for Hedgehog signaling in the regulation of human mesenchymal stem cells and highlights the critical function of this morphogen in hMSC biology

A Serum Factor Induces Insulin-Independent Translocation of GLUT4 to the Cell Surface which Is Maintained in Insulin Resistance

In response to insulin, glucose transporter GLUT4 translocates from intracellular compartments towards the plasma membrane where it enhances cellular glucose uptake. Here, we show that sera from various species contain a factor that dose-dependently induces GLUT4 translocation and glucose uptake in 3T3-L1 adipocytes, human adipocytes, myoblasts and myotubes. Notably, the effect of this factor on GLUT4 is fully maintained in insulin-resistant cells. Our studies demonstrate that the serum-induced increase in cell surface GLUT4 levels is not due to inhibition of its internalization and is not mediated by insulin, PDGF, IGF-1, or HGF. Similarly to insulin, serum also augments cell surface levels of GLUT1 and TfR. Remarkably, the acute effect of serum on GLUT4 is largely additive to that of insulin, while it also sensitizes the cells to insulin. In accordance with these findings, serum does not appear to activate the same repertoire of downstream signaling molecules that are implicated in insulin-induced GLUT4 translocation. We conclude that in addition to insulin, at least one other biological proteinaceous factor exists that contributes to GLUT4 regulation and still functions in insulin resistance. The challenge now is to identify this factor

The Primary Cilium of Adipose Progenitors Is Necessary for Their Differentiation into Cancer-Associated Fibroblasts that Promote Migration of Breast Cancer Cells In Vitro

Cancer associated fibroblasts (CAFs) are central elements of the microenvironment that control tumor development. In breast cancer, CAFs can originate from adipose progenitors (APs). We, and others, have shown that the primary cilium, an antenna-shaped organelle, controls several aspects of APs’ biology. We studied the conversion of human APs into CAFs by breast cancer cell lines (BCCs). Deletion of the cilium of APs by a pharmacological inhibitor, or by siRNA, allow us to demonstrate that the cilium is necessary for the differentiation of APs into CAFs. BCCs increase production of TGF-β1 by APs, which is a known inducer of CAFs. Pharmacological inhibition of TGF-β1 signaling in APs prevents their conversion into CAFs. Since we previously showed that deletion of the APs’ cilium inhibits TGF-β1 signaling, we propose that BCCs induce TGF-β1 production in Aps, which binds to the primary cilium of Aps and leads to their differentiation into CAFs. Inhibition of APs conversion into CAFs induces a loss in some of the biological effects of CAFs since deletion of the cilium of APs decreases their effect on the migration of BCCs. This is the first observation of a function of the cilium of APs in their conversion into CAFs, and its consequences on BCCs

Régulation de l'activité du facteur de transcription HIF-1 et de l'expression du VEGF

Nous avons étudié les mécanismes moléculaires impliqués dans la régulation de l'expression du VEGF (Vascular Endothelial Growth Factor) en réponse aux produits de glycation, à l'insuline et à l'IGF-1 (Insulin-like Growth Factor-1), dans des cellules épithéliales rétiniennes humaines. Nous nous sommes particulièrement intéressés au rôle du facteur de transcription HIF-1 (Hypoxia Inducible Factor-1). L'albumine glyquée induit l'expression génique et protéique du VEGF. L'utilisation d'inhibiteurs spécifiques nous a permis de montrer que les produits de glycation activent le facteur de transcription HIF-1 et l'expression du VEGF par une voie dépendante des MAP-kinases, ERK1/2. HIF-1, acteur majeur de la régulation hypoxique, est formé de deux sous-unités, HIF-1a qui est dégradée sous normoxie, et HIF-1b qui est constitutivement exprimée. Nous avons montré que l'insuline et l'IGF-1 stimulent la traduction de HIF-1a permettant ainsi son accumulation, activent le facteur de transcription HIF-1 et stimulent l'expression du VEGF, mais les mécanismes de régulation semblent être différents. En effet, l'action de l'insuline est dépendante de la voie PI-3-kinase/PKB/mTOR, alors que l'action de l'IGF-1 est dépendante à la fois de la voie PI-3-kinase/PKB/mTOR et de celle des MAP-kinases. L'ensemble de nos résultats suggère que les produits de glycation, l'insuline et l'IGF-1 activent le facteur de transcription HIF-1 par des cascades de signalisation et des mécanismes distincts qui semblent être pour l'essentiel différents de ceux utilisés par l'hypoxie. L'action conjuguée de ces différents facteurs doit avoir des effets dévastateurs sur le développement de la rétinopathie diabétique.The diabetic retinopathy is the major cause of blindness in adult. It is characterized by an increased retinal neovascularization due to the action of the angiogenic factor, Vascular Endothelial Growth Factor (VEGF). We have studied the implication of advanced glycation end products (AGE), insulin and Insulin-like Growth Factor-1 (IGF-1) in the regulation of VEGF expression and in the activation of the transcription factor Hypoxia Inducible Factor-1 (HIF-1), in human retinal epithelial cells (ARPE-19). HIF-1 is constituted of two subunits, HIF-1a, which is degraded during normoxia, and HIF-1ß, which is constitutively expressed. We have shown that, in response to glycated albumin, HIF-1 activation is mediated by an increase in the accumulation of the HIF-1a protein through an ERK-dependent pathway leading to VEGF mRNA and protein expression. Insulin and IGF-1 activate HIF-1a protein expression through a translation-dependent pathway, leading to HIF-1 activation and VEGF mRNA expression. Insulin regulates HIF-1 activation through a PI-3-kinase/mTOR-dependent pathway, resulting in increased VEGF expression. Moreover, IGF-1 regulates HIF-1 activity and VEGF mRNA expression through a PI-3-Kinase/mTOR and an ERK-dependent pathway. The study of the regulation of HIF-1 activity, led us to study the regulation of HIF-hydroxylases which are involved in the regulation of HIF-1 stability and activity. Taken together, our data reveal that AGE, insulin, IGF-1 and hypoxia lead to HIF-1 activation and ensuing VEGF expression by different mechanisms. Their joined actions are likely to have devastating effects on the development of diabetic retinopathy.NICE-BU Sciences (060882101) / SudocSudocFranceF

Regulated in Development and DNA Damage Responses -1 (REDD1) Protein Contributes to Insulin Signaling Pathway in Adipocytes.

International audienceREDD1 (Regulated in development and DNA damage response 1) is a hypoxia and stress response gene and is a negative regulator of mTORC1. Since mTORC1 is involved in the negative feedback loop of insulin signaling, we have studied the role of REDD1 on insulin signaling pathway and its regulation by insulin. In human and murine adipocytes, insulin transiently stimulates REDD1 expression through a MEK dependent pathway. In HEK-293 cells, expression of a constitutive active form of MEK stabilizes REDD1 and protects REDD1 from proteasomal degradation mediated by CUL4A-DDB1 ubiquitin ligase complex. In 3T3-L1 adipocytes, silencing of REDD1 with siRNA induces an increase of mTORC1 activity as well as an inhibition of insulin signaling pathway and lipogenesis. Rapamycin, a mTORC1 inhibitor, restores the insulin signaling after downregulation of REDD1 expression. This observation suggests that REDD1 positively regulates insulin signaling through the inhibition of mTORC1 activity. In conclusion, our results demonstrate that insulin increases REDD1 expression, and that REDD1 participates in the biological response to insulin

Is REDD1 a metabolic double agent? Lessons from physiology and pathology

International audienceThe Akt/mTOR signaling pathway governs macromolecules synthesis, cell growth and metabolism in response to nutrients and growth factors. REDD1 is a conserved and ubiquitous protein, which is transiently induced in response to multiple stimuli. Acting like an endogenous inhibitor of the Akt/mTOR signaling pathway, REDD1 protein has been shown to regulate cell growth, mitochondrial function, oxidative stress and apoptosis. Recent studies also indicate that timely REDD1 expression limits Akt/mTOR-dependent synthesis processes to spare energy during metabolic stresses, avoiding energy collapse and detrimental consequences. In contrast to this beneficial role for metabolic adaptation, REDD1 chronic expression appears involved in the pathogenesis of several diseases. Indeed, REDD1 expression is found as an early biomarker in many pathologies including inflammatory diseases, cancer, neurodegenerative disorders, depression, diabetes and obesity. Moreover, prolonged REDD1 expression is associated with cell apoptosis, excessive ROS production and inflammation activation leading to tissue damages. In this review, we decipher several mechanisms that make REDD1 a likely metabolic double agent depending on its duration of expression in different physiological and pathological contexts. We also discuss the role played by REDD1 in the crosstalk between the Akt/mTOR signaling pathway and the energetic metabolism

Implication of REDD1 in the activation of inflammatory pathways

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Implication des MAP kinases dans l’inflammation et l’insulino-résistance associées à l’obésité

La résistance à l’action de l’insuline ou insulino-résistance est souvent associée à l’obésité et c’est un facteur de risque important pour le développement du diabète de type 2, de maladies cardiovasculaires et hépatiques; elle pourrait également favoriser l’apparition et/ou l’agressivité de certains cancers. L’insulino-résistance est due à des altérations de la signalisation de l’insuline dans ses tissus cibles. Au cours de ces dernières années, l’inflammation chronique de bas grade associée à l’obésité est apparue comme acteur important dans le développement de l’insulino-résistance. En effet, les cytokines inflammatoires activent différentes voies de signalisation qui interférent avec la signalisation insulinique. Parmi les différentes voies de signalisation activées par les cytokines inflammatoires, cette revue détaillera plus particulièrement l’implication des voies de signalisation des MAP (Mitogen Activated Protein kinases) JNK (c-Jun N-terminal kinases) et ERK1/2 (Extracellular signal-regulated kinases) dans le développement des altérations de la signalisation insulinique et discutera la possibilité de cibler ces protéines pour réduire l’insulino-résistance