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The human airway mucociliary epithelium can be recapitulated in vitro using primary cells cultured in an Air-Liquid Interface (ALI), a reliable surrogate to perform pathophysiological studies. As tremendous variations exist between media used for ALI-cultured human airway epithelial cells, our study aimed to evaluate the impact of several media (BEGMTM, PneumaCultTM, "Half&Half" and "Clancy") on cell type distribution using single-cell RNA sequencing and imaging. Our work revealed the impact of these media on cell composition, gene expression profile, cell signaling and epithelial morphology. We found higher proportions of multiciliated cells in PneumaCultTM-ALI and Half&Half, stronger EGF signaling from basal cells in BEGMTM-ALI, differential expression of the SARS-CoV-2 entry factor ACE2, and distinct secretome transcripts depending on media used. We also established that proliferation in PneumaCultTM-Ex Plus favored secretory cell fate, showing the key influence of proliferation media on late differentiation epithelial characteristics. Altogether, our data offer a comprehensive repertoire for evaluating the effects of culture conditions on airway epithelial differentiation and will help to choose the most relevant medium according to the processes to be investigated such as cilia, mucus biology or viral infection. We detail useful parameters that should be explored to document airway epithelial cell fate and morphology. This article is open access and distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives License 4.0 (http://creativecommons.org/licenses/by-nc-nd/4.0/).
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The MIR449 genomic locus encompasses several regulators of multiciliated cell (MCC) formation (multiciliogenesis). The miR-449 homologs miR-34b/c represent additional regulators of multiciliogenesis that are transcribed from another locus. Here, we characterized the expression of BTG4, LAYN, and HOATZ, located in the MIR34B/C locus using single-cell RNA-seq and super-resolution microscopy from human, mouse, or pig multiciliogenesis models. BTG4, LAYN, and HOATZ transcripts were expressed in both precursors and mature MCCs. The Layilin/LAYN protein was absent from primary cilia, but it was expressed in apical membrane regions or throughout motile cilia. LAYN silencing altered apical actin cap formation and multiciliogenesis. HOATZ protein was detected in primary cilia or throughout motile cilia. Altogether, our data suggest that the MIR34B/C locus may gather potential actors of multiciliogenesis.
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Cílios , MicroRNAs , Humanos , Camundongos , Animais , Suínos , Cílios/genética , Cílios/metabolismo , Actinas/metabolismo , Genoma , Genômica , MicroRNAs/genética , MicroRNAs/metabolismo , Lectinas Tipo C/metabolismoRESUMO
The pancreas is an abdominal gland that serves 2 vital purposes: assist food processing by secreting digestive enzymes and regulate blood glucose levels by releasing endocrine hormones. During embryonic development, this gland originates from epithelial buds located on opposite sites of the foregut endoderm. Pancreatic cell specification and maturation are coordinated by a complex interplay of extrinsic and intrinsic signaling events. In the recent years, the canonical Wnt/ß-catenin pathway has emerged as an important player of pancreas organogenesis, regulating pancreatic epithelium specification, compartmentalization and expansion. Importantly, it has been suggested to regulate proliferation, survival and function of adult pancreatic cells, including insulin-secreting ß-cells. This review summarizes recent work on the role of Wnt/ß-catenin signaling in pancreas biology from early development to adulthood, emphasizing on its relevance for the development of new therapies for pancreatic diseases.
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Via de Sinalização Wnt , beta Catenina , Gravidez , Feminino , Humanos , beta Catenina/metabolismo , Pâncreas/metabolismo , Organogênese , Desenvolvimento EmbrionárioRESUMO
Background: Although several approaches have revealed much about individual factors that regulate pancreatic development, we have yet to fully understand their complicated interplay during pancreas morphogenesis. Gfi1 is transcription factor specifically expressed in pancreatic acinar cells, whose role in pancreas cells fate identity and specification is still elusive. Methods: In order to gain further insight into the function of this factor in the pancreas, we generated animals deficient for Gfi1 specifically in the pancreas. Gfi1 conditional knockout animals were phenotypically characterized by immunohistochemistry, RT-qPCR, and RNA scope. To assess the role of Gfi1 in the pathogenesis of diabetes, we challenged Gfi1-deficient mice with two models of induced hyperglycemia: long-term high-fat/high-sugar feeding and streptozotocin injections. Results: Interestingly, mutant mice did not show any obvious deleterious phenotype. However, in depth analyses demonstrated a significant decrease in pancreatic amylase expression, leading to a diminution in intestinal carbohydrates processing and thus glucose absorption. In fact, Gfi1-deficient mice were found resistant to diet-induced hyperglycemia, appearing normoglycemic even after long-term high-fat/high-sugar diet. Another feature observed in mutant acinar cells was the misexpression of ghrelin, a hormone previously suggested to exhibit anti-apoptotic effects on ß-cells in vitro. Impressively, Gfi1 mutant mice were found to be resistant to the cytotoxic and diabetogenic effects of high-dose streptozotocin administrations, displaying a negligible loss of ß-cells and an imperturbable normoglycemia. Conclusions: Together, these results demonstrate that Gfi1 could turn to be extremely valuable for the development of new therapies and could thus open new research avenues in the context of diabetes research.
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Proteínas de Ligação a DNA/deficiência , Diabetes Mellitus/metabolismo , Diabetes Mellitus/prevenção & controle , Fatores de Transcrição/deficiência , Células Acinares/citologia , Células Acinares/metabolismo , Amilases/metabolismo , Animais , Diferenciação Celular/genética , Proliferação de Células/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Diabetes Mellitus/genética , Modelos Animais de Doenças , Regulação da Expressão Gênica , Grelina/metabolismo , Proteínas de Homeodomínio/metabolismo , Hiperglicemia/complicações , Hiperglicemia/genética , Integrases/metabolismo , Camundongos Transgênicos , Mutação/genética , Pâncreas/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Background: In line with our recent discovery of an efficient anticancer thiazolebenzenesulfonamide framework HA15 (1) based on a remarkable endoplasmic reticulum stress inducement mode of action, we report herein a series of innovative constrained HA15 analogs, featuring four types of bicylic derivatives. Results: The structure-activity relationship analysis, using a cell line assay, led us to identify a novel version of HA15: a new benzothiazole derivative (10b) exhibiting important anti-melanoma effect against sensitive and resistant melanoma cells. Meanwhile, compound 10b induced a significant tumor growth inhibition in vivo with no apparent signs of toxicity. Conclusion: These results consistently open new directions to improve and develop more powerful anticancer therapeutics harboring this type of fused framework.
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Antineoplásicos/uso terapêutico , Benzotiazóis/química , Melanoma/tratamento farmacológico , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Benzotiazóis/farmacologia , Benzotiazóis/uso terapêutico , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Humanos , Melanoma/patologia , Camundongos , Camundongos Nus , Relação Estrutura-Atividade , Transplante HeterólogoRESUMO
Rationale: The respiratory tract constitutes an elaborate line of defense that is based on a unique cellular ecosystem.Objectives: We aimed to investigate cell population distributions and transcriptional changes along the airways by using single-cell RNA profiling.Methods: We have explored the cellular heterogeneity of the human airway epithelium in 10 healthy living volunteers by single-cell RNA profiling. A total of 77,969 cells were collected at 35 distinct locations, from the nose to the 12th division of the airway tree.Measurements and Main Results: The resulting atlas is composed of a high percentage of epithelial cells (89.1%) but also immune (6.2%) and stromal (4.7%) cells with distinct cellular proportions in different regions of the airways. It reveals differential gene expression between identical cell types (suprabasal, secretory, and multiciliated cells) from the nose (MUC4, PI3, SIX3) and tracheobronchial (SCGB1A1, TFF3) airways. By contrast, cell-type-specific gene expression is stable across all tracheobronchial samples. Our atlas improves the description of ionocytes, pulmonary neuroendocrine cells, and brush cells and identifies a related population of NREP-positive cells. We also report the association of KRT13 with dividing cells that are reminiscent of previously described mouse "hillock" cells and with squamous cells expressing SCEL and SPRR1A/B.Conclusions: Robust characterization of a single-cell cohort in healthy airways establishes a valuable resource for future investigations. The precise description of the continuum existing from the nasal epithelium to successive divisions of the airways and the stable gene expression profile of these regions better defines conditions under which relevant tracheobronchial proxies of human respiratory diseases can be developed.
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Brônquios/citologia , Brônquios/crescimento & desenvolvimento , Diferenciação Celular/genética , Proliferação de Células/genética , Células Epiteliais/citologia , Mucosa Nasal/citologia , Mucosa Nasal/crescimento & desenvolvimento , Células Estromais/citologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Regulação da Expressão Gênica , Voluntários Saudáveis , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
There is pressing urgency to understand the pathogenesis of the severe acute respiratory syndrome coronavirus clade 2 (SARS-CoV-2), which causes the disease COVID-19. SARS-CoV-2 spike (S) protein binds angiotensin-converting enzyme 2 (ACE2), and in concert with host proteases, principally transmembrane serine protease 2 (TMPRSS2), promotes cellular entry. The cell subsets targeted by SARS-CoV-2 in host tissues and the factors that regulate ACE2 expression remain unknown. Here, we leverage human, non-human primate, and mouse single-cell RNA-sequencing (scRNA-seq) datasets across health and disease to uncover putative targets of SARS-CoV-2 among tissue-resident cell subsets. We identify ACE2 and TMPRSS2 co-expressing cells within lung type II pneumocytes, ileal absorptive enterocytes, and nasal goblet secretory cells. Strikingly, we discovered that ACE2 is a human interferon-stimulated gene (ISG) in vitro using airway epithelial cells and extend our findings to in vivo viral infections. Our data suggest that SARS-CoV-2 could exploit species-specific interferon-driven upregulation of ACE2, a tissue-protective mediator during lung injury, to enhance infection.
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Células Epiteliais Alveolares/metabolismo , Enterócitos/metabolismo , Células Caliciformes/metabolismo , Interferon Tipo I/metabolismo , Mucosa Nasal/citologia , Peptidil Dipeptidase A/genética , Adolescente , Células Epiteliais Alveolares/imunologia , Enzima de Conversão de Angiotensina 2 , Animais , Betacoronavirus/fisiologia , COVID-19 , Linhagem Celular , Células Cultivadas , Criança , Infecções por Coronavirus/virologia , Enterócitos/imunologia , Células Caliciformes/imunologia , Infecções por HIV/imunologia , Humanos , Influenza Humana/imunologia , Interferon Tipo I/imunologia , Pulmão/citologia , Pulmão/patologia , Macaca mulatta , Camundongos , Mycobacterium tuberculosis , Mucosa Nasal/imunologia , Pandemias , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/virologia , Receptores Virais/genética , SARS-CoV-2 , Serina Endopeptidases/metabolismo , Análise de Célula Única , Tuberculose/imunologia , Regulação para CimaRESUMO
Mutations in the polycystins cause autosomal dominant polycystic kidney disease (ADPKD). Here we show that transmembrane protein 33 (TMEM33) interacts with the ion channel polycystin-2 (PC2) at the endoplasmic reticulum (ER) membrane, enhancing its opening over the whole physiological calcium range in ER liposomes fused to planar bilayers. Consequently, TMEM33 reduces intracellular calcium content in a PC2-dependent manner, impairs lysosomal calcium refilling, causes cathepsins translocation, inhibition of autophagic flux upon ER stress, as well as sensitization to apoptosis. Invalidation of TMEM33 in the mouse exerts a potent protection against renal ER stress. By contrast, TMEM33 does not influence pkd2-dependent renal cystogenesis in the zebrafish. Together, our results identify a key role for TMEM33 in the regulation of intracellular calcium homeostasis of renal proximal convoluted tubule cells and establish a causal link between TMEM33 and acute kidney injury.
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Injúria Renal Aguda/patologia , Cálcio/metabolismo , Túbulos Renais Proximais/metabolismo , Proteínas de Membrana/metabolismo , Canais de Cátion TRPP/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Injúria Renal Aguda/genética , Animais , Membrana Celular/metabolismo , Modelos Animais de Doenças , Embrião não Mamífero , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , Túbulos Renais Proximais/citologia , Lisossomos/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Camundongos , Camundongos Knockout , Mutação , Rim Policístico Autossômico Dominante/genética , Rim Policístico Autossômico Dominante/patologia , RNA Interferente Pequeno/metabolismo , Canais de Cátion TRPP/genética , Canais de Cátion TRPP/fisiologia , Peixe-Zebra , Proteínas de Peixe-Zebra/fisiologiaRESUMO
We recently described a new family of bioactive molecules with interesting anti-cancer activities: the N-(4-(3-aminophenyl)thiazol-2-yl)acetamides. The lead compound of the series (1) displays significant anti-proliferative and cytotoxic activities against a panel of cancer cell lines, either sensitive or resistant to standard treatments. This molecule also shows a good pharmacological profile and high in vivo potency towards mice xenografts, without signs of toxicity on the animals. In the present article, we disclose the structure-activity relationships of this lead compound, which have provided clear information about the replacement of the acetamide function and the substitution pattern of the benzenesulfonamide ring. An improved high-yielding synthetic procedure towards these compounds has also been developed. Our drug design resulted in potency enhancement of 1, our new optimized lead compound being 19. These findings are of great interest to further improve this scaffold for the development of future clinical candidates.
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Antineoplásicos/química , Sulfonamidas/química , Antineoplásicos/síntese química , Antineoplásicos/toxicidade , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Desenho de Fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Humanos , Relação Estrutura-Atividade , Sulfonamidas/síntese química , Sulfonamidas/toxicidade , BenzenossulfonamidasRESUMO
Cancer is the second cause of deaths worldwide and is forecasted to affect more that 22 million people in 2020. Despite dramatic improvement in its care over the last two decades, the treatment of resistant forms of cancer is still an unmet challenge. Thus, innovative and efficient treatments are still needed. In this context, we report herein the synthesis and the evaluation of a new class of bioactive molecules belonging to the N-(4-(3-aminophenyl(thiazol-2-yl)acetamide family. Structure-activity relationships could be driven and resulted in the discovery of lead compound 6b. The latter display high in vitro potency against both sensitive and resistant cancer cell lines on three models: melanoma, pancreatic cancer, and chronic myeloid leukemia (CML). 6b leads to cell death by concomitant induction of apoptosis and autophagy, shows good pharmacokinetic properties, and demonstrates a significant reduction of tumor growth in vivo on A375 xenograft model in mice.
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Acetamidas/química , Acetamidas/farmacologia , Antineoplásicos/química , Antineoplásicos/farmacologia , Acetamidas/farmacocinética , Acetamidas/uso terapêutico , Animais , Antineoplásicos/farmacocinética , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Melanoma/tratamento farmacológico , Camundongos , Neoplasias Pancreáticas/tratamento farmacológico , Relação Estrutura-Atividade , Tiazóis/química , Tiazóis/farmacocinética , Tiazóis/farmacologia , Tiazóis/uso terapêuticoRESUMO
We have discovered and developed a series of molecules (thiazole benzenesulfonamides). HA15, the lead compound of this series, displayed anti-cancerous activity on all melanoma cells tested, including cells isolated from patients and cells that developed resistance to BRAF inhibitors. Our molecule displayed activity against other liquid and solid tumors. HA15 also exhibited strong efficacy in xenograft mouse models with melanoma cells either sensitive or resistant to BRAF inhibitors. Transcriptomic, proteomic, and biochemical studies identified the chaperone BiP/GRP78/HSPA5 as the specific target of HA15 and demonstrated that the interaction increases ER stress, leading to melanoma cell death by concomitant induction of autophagic and apoptotic mechanisms.
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Antineoplásicos/administração & dosagem , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Melanoma/tratamento farmacológico , Sulfonamidas/administração & dosagem , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Chaperona BiP do Retículo Endoplasmático , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Melanoma/metabolismo , Camundongos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Sulfonamidas/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Adipose tissue is an alternative source of mesenchymal stem cells and human adipose-derived stem cells (ASCs) display an attractive and substantial therapeutic potential when transplanted in animal models. To this end, an understanding of ASC biology is necessary and the knowledge of mechanisms that maintain ASCs in an undifferentiated state with no loss of differentiation potential during ex vivo expansion represents a crucial step. However, these mechanisms remain to be identified because appropriate human cellular models are scant. In this review we will describe a cellular model isolated from human adipose tissue displaying all the features of stem cells. Then, we will focus on the identification of intrinsic and extrinsic factors regulating the balance between human ASC proliferation and differentiation. We will point out the role of factors secreted by undifferentiated ASCs, such a FGF2, activin A, BMP4, Hedgehog molecules and secreted by adipose tissue macrophages. Finally, we will outline the role of miRNAs in these processes.
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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.
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Proliferação de Células , Proteínas Hedgehog/antagonistas & inibidores , Células-Tronco Mesenquimais/fisiologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Sobrevivência Celular/fisiologia , Células Cultivadas , Células Clonais , Ensaio de Unidades Formadoras de Colônias , Relação Dose-Resposta a Droga , Regulação para Baixo/efeitos dos fármacos , Proteínas Hedgehog/metabolismo , Humanos , Fatores de Transcrição Kruppel-Like/antagonistas & inibidores , Fatores de Transcrição Kruppel-Like/genética , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , RNA Interferente Pequeno/farmacologia , Transdução de Sinais/efeitos dos fármacos , Teratogênicos/farmacologia , Alcaloides de Veratrum/farmacologia , Proteína Gli2 com Dedos de ZincoRESUMO
Skeletal muscle cells constitute a heterogeneous population that maintains muscle integrity through a high myogenic regenerative capacity. More unexpectedly, this population is also endowed with an adipogenic potential, even in humans, and intramuscular adipocytes have been found to be present in several disorders. We tested the distribution of myogenic and adipogenic commitments in human muscle-derived cells to decipher the cellular basis of the myoadipogenic balance. Clonal analysis showed that adipogenic progenitors can be separated from myogenic progenitors and, interestingly, from myoadipogenic bipotent progenitors. These progenitors were isolated in the CD34(+) population on the basis of the expression of CD56 and CD15 cell surface markers. In vivo, these different cell types have been found in the interstitial compartment of human muscle. In vitro, we show that the proliferation of bipotent myoadipogenic CD56(+)CD15(+) progenitors gives rise to myogenic CD56(+)CD15(-) progenitors and adipogenic CD56(-)CD15(+) progenitors. A cellular hierarchy of muscle and fat progenitors thus occurs within human muscle. These results provide cellular bases for adipogenic differentiation in human skeletal muscle, which may explain the fat development encountered in different muscle pathological situations.
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Adipócitos/citologia , Diferenciação Celular , Linhagem da Célula , Células Musculares/citologia , Músculo Esquelético/citologia , Células-Tronco/citologia , Adipócitos/metabolismo , Adolescente , Adulto , Idoso , Antígenos CD/metabolismo , Biópsia , Antígeno CD56/metabolismo , Criança , Pré-Escolar , Células Clonais , Humanos , Lactente , Pessoa de Meia-Idade , Modelos Biológicos , Células Musculares/metabolismo , Músculo Esquelético/patologia , Células-Tronco/metabolismo , Adulto JovemRESUMO
Mesenchymal stem cells within the bone are responsible for the generation of osteoblasts, chondrocytes, and adipocytes. In rodents, Indian hedgehog has been shown to play a role in osteoblast differentiation. However, evidence for a direct function of hedgehog (Hh) in human osteoblastic differentiation is missing. Using different models of human mesenchymal stem cells we show that Hh signaling decreases during osteoblast differentiation. This is associated with a decrease in Smoothened expression, a key partner that triggers Hh signaling, and in the number of cells displaying a primary cilium, an organelle necessary for Hh signaling. Remarkably, treatment of human mesenchymal stem cells with sonic hedgehog or two molecules able to activate Hh signaling inhibits osteoblast differentiation. This inhibition is visualized through a decrease in mineralization and in the expression of osteoblastic genes. In particular, activation of Hh signaling induces a decrease in Runx2 expression, a key transcriptional factor controlling the early stage of osteoblast differentiation. Consistently, the activation of Hh signaling during the first days of differentiation is sufficient to inhibit osteoblast differentiation, whereas differentiated osteoblasts are not affected by Hh signaling. In summary, we show here, using various inducers of Hh signaling and mesenchymal stem cells of two different origins, that Hh signaling inhibits human osteoblast differentiation, in sharp contrast to what has been described in rodent cells. This species difference should be taken into account for screening for pro-osteogenic molecules.
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Diferenciação Celular/fisiologia , Proteínas Hedgehog/metabolismo , Células-Tronco Mesenquimais/citologia , Transdução de Sinais/fisiologia , Fosfatase Alcalina/metabolismo , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Linhagem Celular , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Meios de Cultivo Condicionados/farmacologia , Regulação da Expressão Gênica , Humanos , Hidroxicolesteróis/farmacologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Morfolinas/farmacologia , Células T Matadoras Naturais , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Purinas/farmacologia , Transdução de Sinais/efeitos dos fármacosRESUMO
Human stem cells are powerful tools by which to investigate molecular mechanisms of cell growth and differentiation under normal and pathological conditions. Hedgehog signaling, the dysregulation of which causes several pathologies, such as congenital defects and cancer, is involved in several cell differentiation processes and interferes with adipocyte differentiation of rodent cells. The present study was aimed at investigating the effect of Hedgehog pathway modulation on adipocyte phenotype using different sources of human mesenchymal cells, such as bone marrow stromal cells and human multipotent adipose-derived stem cells. We bring evidence that Hedgehog signaling decreases during human adipocyte differentiation. Inhibition of this pathway is not sufficient to trigger adipogenesis, but activation of Hedgehog pathway alters adipocyte morphology as well as insulin sensitivity. Analysis of glycerol-3-phosphate dehydrogenase activity and expression of adipocyte marker genes indicate that activation of Hedgehog signaling by purmorphamine impairs adipogenesis. In sharp contrast to reports in rodent cells, the maturation process, but not the early steps of human mesenchymal stem cell differentiation, is affected by Hedgehog activation. Hedgehog interferes with adipocyte differentiation by targeting CCAAT enhancer-binding protein alpha and peroxisome proliferator-activated receptor (PPAR) gamma2 expression, whereas PPARgamma1 level remains unaffected. Although Hedgehog pathway stimulation does not modify the total number of adipocytes, adipogenesis appears dramatically impaired, with reduced lipid accumulation, a decrease in adipocyte-specific markers, and acquisition of an insulin-resistant phenotype. This study indicates that a decrease in Hedgehog signaling is necessary but not sufficient to trigger adipocyte differentiation and unveils a striking difference in the adipocyte differentiation process between rodent and human mesenchymal stem cells.