Effects of GSK3 inhibitors on in vitro expansion and differentiation of human adipose-derived stem cells into adipocytes

<p>Abstract</p> <p>Background</p> <p>Multipotent stem cells exist within adipose tissue throughout life. An abnormal recruitment of these adipose precursor cells could participate to hyperplasia of adipose tissue observed in severe obesity or to hypoplasia of adipose tissue observed in lipodystrophy. Therefore, pharmacological molecules that control the pool of stem cells in adipose tissue are of great interest. Glycogen Synthase Kinase (GSK) 3 has been previously described as involved in differentiation of preadipose cells and might be a potential therapeutic target to modulate proliferation and differentiation of adipocyte precursors. However, the impact of GSK3 inhibition on human adipose-derived stem cells remained to be investigated. The aim of this study was to investigate GSK3 as a possible target for pharmacological inhibition of stem cell adipogenesis. To reach this goal, we studied the effects of pharmacological inhibitors of GSK3, i.e. lithium chloride (LiCl) and BIO on proliferation and adipocyte differentiation of multipotent stem cells derived from human adipose tissue.</p> <p>Results</p> <p>Our results showed that GSK3 inhibitors inhibited proliferation and clonogenicity of human stem cells, strongly suggesting that GSK3 inhibitors could be potent regulators of the pool of adipocyte precursors in adipose tissue. The impact of GSK3 inhibition on differentiation of hMADS cells was also investigated. Adipogenic and osteogenic differentiations were inhibited upon hMADS treatment with BIO. Whereas a chronic treatment was required to inhibit osteogenesis, a treatment that was strictly restricted to the early step of differentiation was sufficient to inhibit adipogenesis.</p> <p>Conclusion</p> <p>These results demonstrated the feasibility of a pharmacological approach to regulate adipose-derived stem cell function and that GSK3 could represent a potential target for controlling adipocyte precursor pool under conditions where fat tissue formation is impaired.</p

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

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&rsquo; 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-&beta;1 by APs, which is a known inducer of CAFs. Pharmacological inhibition of TGF-&beta;1 signaling in APs prevents their conversion into CAFs. Since we previously showed that deletion of the APs&rsquo; cilium inhibits TGF-&beta;1 signaling, we propose that BCCs induce TGF-&beta;1 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

Inhibition of the anti-adipogenic Hedgehog signaling pathway by cyclopamine does not trigger adipocyte differentiation.

International audienceDysregulation of Hedgehog signaling can lead to several pathologies such as congenital defects and cancer. Here, we show that Hedgehog signaling is active in undifferentiated 3T3-L1 cells and decreases during adipocyte differentiation. Interestingly, this is paralleled by a decrease in Indian Hedgehog expression. We then tested if this down-regulation was sufficient to induce adipocyte differentiation. To this end, we demonstrate that the well-characterized Hedgehog inhibitor cyclopamine induced a decrease in Hedgehog signaling, similar to the one observed during adipocyte differentiation. However, cyclopamine did not induce nor potentiate adipocyte differentiation, as monitored by triglyceride staining and by the expression of several adipocyte markers: aP2, adipsin, C/EBPalpha, and Pref-1. Moreover, cyclopamine cannot substitute for other components of the differentiation medium: insulin, dexamethasone or IBMX. These results indicate that although Hedgehog signaling decreases during adipocyte differentiation, this down-regulation is not sufficient to trigger adipocyte differentiation. This suggests that Hedgehog signaling is an inadequate pharmacological target for patient suffering from syndromes associated with a decrease in fat mass, such as the ones observed in lipodystrophies

Identification de socs-3 comme un nouvel élément de la voie de signalisation de l'insuline (implication dans les mécanismes de résistance à cette hormone)

Au cours de ma thèse, nous avons identifié la protéine SOCS-3 comme un nouvel élément de la voie de signalisation de l'insuline. La famille des SOCS a été originellement découverte pour son action inhibitrice sur la voie de signalisation des cytokines. Nous avons montré que SOCS-3 est un nouveau gène-cible de l'insuline, dont la transcription est dépendante du facteur de transcription STAT5b. SOCS-3 interagit avec le récepteur de l'insuline, et diminue l'activité de liaison à l'ADN de STAT5b et la phosphorylation sur tyrosine d'IRS1, stimulées par l'insuline. SOCS-3 exerce donc un effet inhibiteur sur la voie de signalisation de l'insuline. De plus, son expression est augmentée dans le tissu adipeux de souris obèses et résistantes à l'insuline. Le TNF-a, dont le rôle dans la résistance à l'insuline liée à l'obésité est reconnu, est responsable de cette surexpression. L'ensemble de nos résultats suggère que SOCS-3 est un nouvel acteur de la résistance à l'insuline associée à l'obésité.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

Distinct Shades of Adipocytes Control the Metabolic Roles of Adipose Tissues: From Their Origins to Their Relevance for Medical Applications

Adipose tissue resides in specific depots scattered in peripheral or deeper locations all over the body and it enwraps most of the organs. This tissue is always in a dynamic evolution as it must adapt to the metabolic demand and constraints. It exhibits also endocrine functions important to regulate energy homeostasis. This complex organ is composed of depots able to produce opposite functions to monitor energy: the so called white adipose tissue acts to store energy as triglycerides preventing ectopic fat deposition while the brown adipose depots dissipate it. It is composed of many cell types. Different types of adipocytes constitute the mature cells specialized to store or burn energy. Immature adipose progenitors (AP) presenting stem cells properties contribute not only to the maintenance but also to the expansion of this tissue as observed in overweight or obese individuals. They display a high regeneration potential offering a great interest for cell therapy. In this review, we will depict the attributes of the distinct types of adipocytes and their contribution to the function and metabolic features of adipose tissue. We will examine the specific role and properties of distinct depots according to their location. We will consider their cellular heterogeneity to present an updated picture of this sophisticated tissue. We will also introduce new trends pointing out a rational targeting of adipose tissue for medical applications

Identification of Human Breast Adipose Tissue Progenitors Displaying Distinct Differentiation Potentials and Interactions with Cancer Cells

Breast adipose tissue (AT) participates in the physiological evolution and remodeling of the mammary gland due to its high plasticity. It is also a favorable microenvironment for breast cancer progression. However, information on the properties of human breast adipose progenitor cells (APCs) involved in breast physiology or pathology is scant. We performed differential enzymatic dissociation of human breast AT lobules. We isolated and characterized two populations of APCs. Here we report that these distinct breast APC populations selectively expressed markers suitable for characterization. The population preferentially expressing ALPL (MSCA1) showed higher adipogenic potential. The population expressing higher levels of INHBA and CD142 acquired myofibroblast characteristics upon TGF-&beta; treatment and a myo-cancer-associated fibroblast profile in the presence of breast cancer cells. This population expressed the immune checkpoint CD274 (PD-L1) and facilitated the expansion of breast cancer mammospheres compared with the adipogenic population. Indeed, the breast, as with other fat depots, contains distinct types of APCs with differences in their ability to specialize. This indicates that they were differentially involved in breast remodeling. Their interactions with breast cancer cells revealed differences in the potential for tumor dissemination and estrogen receptor expression, and these differences might be relevant to improve therapies targeting the tumor microenvironment