RESUMO
The contribution of nutrient availability to control epidermal cell proliferation, inflammation, and hyperproliferative diseases remains unknown. Here, we studied extracellular serine and serine/glycine metabolism using human keratinocytes, human skin biopsies, and a mouse model of psoriasis-like disease. We focused on a metabolic enzyme, serine hydroxymethyltransferase (SHMT), that converts serine into glycine and tetrahydrofolate-bound onecarbon units to support cell growth. We found that keratinocytes are both serine and glycine auxotrophs. Metabolomic profiling and hypoxanthine supplementation indicated that SHMT silencing/inhibition reduced cell growth through purine depletion, leading to nucleotide loss. In addition, topical application of an SHMT inhibitor suppressed both keratinocyte proliferation and inflammation in the imiquimod model and resulted in a decrease in psoriasis-associated gene expression. In conclusion, our study highlights SHMT2 activity and serine/glycine availability as an important metabolic hub controlling both keratinocyte proliferation and inflammatory cell expansion in psoriasis and holds promise for additional approaches to treat skin diseases.
Assuntos
Psoríase , Dermatopatias , Camundongos , Animais , Humanos , Serina/metabolismo , Glicina Hidroximetiltransferase/genética , Glicina Hidroximetiltransferase/metabolismo , Psoríase/patologia , Glicina/farmacologia , Glicina/metabolismo , Inflamação/patologia , Proliferação de CélulasRESUMO
Preadipocytes can give rise to either white adipocytes or beige adipocytes. Owing to their distinct abilities in nutrient storage and energy expenditure, strategies that specifically promote "beiging" of adipocytes hold great promise for counterbalancing obesity and metabolic diseases. Yet, factors dictating the differentiation fate of adipocyte progenitors remain to be elucidated. We found that stearoyl-coenzyme A desaturase 1 (Scd1)-deficient mice, which resist metabolic stress, possess augmentation in beige adipocytes under basal conditions. Deletion of Scd1 in mature adipocytes expressing Fabp4 or Ucp1 did not affect thermogenesis in mice. Rather, Scd1 deficiency shifted the differentiation fate of preadipocytes from white adipogenesis to beige adipogenesis. Such effects are dependent on succinate accumulation in adipocyte progenitors, which fuels mitochondrial complex II activity. Suppression of mitochondrial complex II by Atpenin A5 or oxaloacetic acid reverted the differentiation potential of Scd1-deficient preadipocytes to white adipocytes. Furthermore, supplementation of succinate was found to increase beige adipocyte differentiation both in vitro and in vivo. Our data reveal an unappreciated role of Scd1 in determining the cell fate of adipocyte progenitors through succinate-dependent regulation of mitochondrial complex II.
Assuntos
Complexo II de Transporte de Elétrons/metabolismo , Gorduras/metabolismo , Obesidade/enzimologia , Estearoil-CoA Dessaturase/genética , Ácido Succínico/metabolismo , Adipócitos Bege/citologia , Adipócitos Bege/metabolismo , Adipogenia , Animais , Metabolismo Energético , Proteínas de Ligação a Ácido Graxo/genética , Proteínas de Ligação a Ácido Graxo/metabolismo , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Obesidade/genética , Obesidade/metabolismo , Obesidade/fisiopatologia , Estearoil-CoA Dessaturase/metabolismo , TermogêneseRESUMO
Medulloblastoma is the most common solid primary brain tumor in children. Remarkable advancements in the understanding of the genetic and epigenetic basis of these tumors have informed their recent molecular classification. However, the genotype/phenotype correlation of the subgroups remains largely uncharacterized. In particular, the metabolic phenotype is of great interest because of its druggability, which could lead to the development of novel and more tailored therapies for a subset of medulloblastoma. p73 plays a critical role in a range of cellular metabolic processes. We show overexpression of p73 in a proportion of non-WNT medulloblastoma. In these tumors, p73 sustains cell growth and proliferation via regulation of glutamine metabolism. We validated our results in a xenograft model in which we observed an increase in survival time in mice on a glutamine restriction diet. Notably, glutamine starvation has a synergistic effect with cisplatin, a component of the current medulloblastoma chemotherapy. These findings raise the possibility that glutamine depletion can be used as an adjuvant treatment for p73-expressing medulloblastoma.
Assuntos
Neoplasias Cerebelares/dietoterapia , Neoplasias Cerebelares/fisiopatologia , Glutamina/metabolismo , Meduloblastoma/dietoterapia , Meduloblastoma/fisiopatologia , Proteína Tumoral p73/genética , Proteína Tumoral p73/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células/genética , Modelos Animais de Doenças , Regulação Neoplásica da Expressão Gênica/genética , Glutaminase/genética , Glutaminase/metabolismo , Xenoenxertos , Humanos , Camundongos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Análise de Sobrevida , Serina-Treonina Quinases TOR/metabolismo , Resultado do Tratamento , Células Tumorais CultivadasRESUMO
p63, a member of the p53 family, is essential for skin morphogenesis and epithelial stem cell maintenance. Here, we report an unexpected role of TAp63 in cardiogenesis. p63 null mice exhibit severe defects in embryonic cardiac development, including dilation of both ventricles, a defect in trabeculation and abnormal septation. This was accompanied by myofibrillar disarray, mitochondrial disorganization, and reduction in spontaneous calcium spikes. By the use of embryonic stem cells (ESCs), we show that TAp63 deficiency prevents expression of pivotal cardiac genes and production of cardiomyocytes. TAp63 is expressed by endodermal cells. Coculture of p63-knockdown ESCs with wild-type ESCs, supplementation with Activin A, or overexpression of GATA-6 rescue cardiogenesis. Therefore, TAp63 acts in a non-cell-autonomous manner by modulating expression of endodermal factors. Our findings uncover a critical role for p63 in cardiogenesis that could be related to human heart disease.
Assuntos
Diferenciação Celular/fisiologia , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Coração/embriologia , Fosfoproteínas/metabolismo , Transativadores/metabolismo , Animais , Diferenciação Celular/genética , Linhagem Celular , Células-Tronco Embrionárias/ultraestrutura , Citometria de Fluxo , Imunofluorescência , Fator de Transcrição GATA6/genética , Fator de Transcrição GATA6/metabolismo , Proteínas HMGB/genética , Proteínas HMGB/metabolismo , Coração/crescimento & desenvolvimento , Immunoblotting , Camundongos , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Fosfoproteínas/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição SOXF/genética , Fatores de Transcrição SOXF/metabolismo , Transativadores/genéticaRESUMO
Transglutaminase 2 (TG2) is a protein crosslinking enzyme with several additional biochemical functions. Loss of TG2 in vivo results in impaired phagocytosis of apoptotic cells and altered proinflammatory cytokine production by macrophages engulfing apoptotic cells leading to autoimmunity. It has been proposed that TG2 acts as an integrin ß(3) coreceptor in the engulfment process, while altered proinflammatory cytokine production is related to the lack of latent TGFß activation by TG2 null macrophages. Here we report that TG2 null macrophages respond to lipopolysaccharide treatment by elevated IL-6 and TNFα production. Though TGFß has been proposed to act as a feed back regulator of proinflammatory cytokine production in LPS-stimulated macrophages, this phenomenon is not related to the lack of active TGFß production. Instead, in the absence of TG2 integrin ß(3) maintains an elevated basal Src family kinase activity in macrophages, which leads to enhanced phosphorylation and degradation of the IκBα. Low basal levels of IκBα explain the enhanced sensitivity of TG2 null macrophages to signals that regulate NF-κB. Our data suggest that TG2 null macrophages bear a proinflammatory phenotype, which might contribute to the enhanced susceptibility of these mice to develop autoimmunity and atherosclerosis.
Assuntos
Citocinas/metabolismo , Proteínas de Ligação ao GTP/deficiência , Integrina alfaVbeta3/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos , Transdução de Sinais , Transglutaminases/deficiência , Quinases da Família src/metabolismo , Adjuvantes Imunológicos/farmacologia , Animais , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Proteínas I-kappa B/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/enzimologia , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NF-kappa B/metabolismo , Proteína 2 Glutamina gama-Glutamiltransferase , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta/metabolismo , Transglutaminases/genética , Transglutaminases/metabolismoRESUMO
We have found previously that ascorbic acid (vitamin C), as well as acting as a radical scavenger, may modulate the expression of several genes [i.e. fra-1, glutathione S-transferase Pi (GSTpi) and Mut L homologue-1 (MLH1)] in human keratinocytes. In the present paper, we demonstrate that MLH1, as well as its downstream target p73, can be positively modulated by this antioxidant vitamin, indeed, up-regulation of the two mRNAs was observed after just 2 h, and increased further up to 16 h of treatment. Modulation of MLH1 and p73 gene expression improves cellular susceptibility to apoptosis triggered by the DNA-damaging agent cisplatin. Indeed, in ascorbate-supplemented cells, increased cisplatin-induced apoptosis was seen, involving activation of the MLH1/c-Abl/p73 signalling cascade. Our results were further confirmed by studies performed on genetically defined mutants, i.e. mouse embryo fibroblasts derived from knock-out animals for c-Abl or p53, as well as human colon carcinoma cell lines deficient in MLH1. The increased sensitivity to cisplatin observed in ascorbate-loaded cells appeared to be dependent exclusively on MLH1 and c-Abl expression, and independent of p53. These data suggest a potential mechanism accounting for the anti-carcinogenic and anti-cancer activities of vitamin C.
Assuntos
Adenosina Trifosfatases , Ácido Ascórbico/farmacologia , Proteínas de Bactérias/fisiologia , Dano ao DNA , Proteínas de Ligação a DNA/fisiologia , Proteínas de Escherichia coli , Proteínas Nucleares/fisiologia , Regulação para Cima/efeitos dos fármacos , Animais , Proteínas de Bactérias/genética , Sequência de Bases , Morte Celular/efeitos dos fármacos , Linhagem Celular , Cisplatino/farmacologia , Primers do DNA , Proteínas de Ligação a DNA/genética , Genes Supressores de Tumor , Humanos , Camundongos , Camundongos Knockout , Proteínas MutL , Proteínas Nucleares/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína Tumoral p73 , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de TumorRESUMO
Epidermal keratinocytes undergo differentiation in response to several stimuli to form the cornified envelope, a structure that contributes to the barrier function of skin. Although differentiation has been extensively analyzed, the precise role of vitamin C during this process is still not defined. Ascorbic acid, besides acting as a radical scavenger, has been shown to promote mesenchymal differentiation. In this study, we found that keratinocytes grown in ascorbate-supplemented medium developed a differentiated phenotype, as demonstrated by enhanced expression of marker genes and increase in cornified envelope content. The pro-differentiating effects of ascorbate were mediated by the protein-kinase-C-dependent induction of activating protein 1 DNA binding activity; indeed, down-modulation of protein kinase C activity abolished differentiation triggered by ascorbic acid. Although vitamin C appeared to regulate the same signaling pathway modulated by calcium, a classical in vitro inducer of epidermal differentiation, nonetheless terminally differentiated keratinocytes exhibited different ascorbate homeostasis and cellular antioxidant status. Indeed, we found that, unlike calcium, differentiation promoted by ascorbate was accompanied by (i) an enhanced ascorbate transport, due to overexpression of specific transporters, (ii) a great efficiency of dehydroascorbate uptake, and (iii) an increase in glutathione content with respect to proliferating cells. Ascorbic acid may be useful to promote epidermal differentiation, avoiding depletion of hydrophilic antioxidant stores.