RESUMO
A first step in hematopoiesis is the specification of the lymphoid and myeloid lineages from multipotent progenitor cells in the bone marrow. Using a conditional ablation strategy in adult mice, we show that this differentiation step requires Patched (Ptch), the cell surface-bound receptor for Hedgehog (Hh). In the absence of Ptch, the development of T- and B-lymphoid lineages is blocked at the level of the common lymphoid progenitor in the bone marrow. Consequently, the generation of peripheral T and B cells is abrogated. Cells of the myeloid lineage develop normally in Ptch mutant mice. Finally, adoptive transfer experiments identified the stromal cell compartment as a critical Ptch-dependent inducer of lymphoid versus myeloid lineage commitment. Our data show that Ptch acts as a master switch for proper diversification of hematopoietic stem cells in the adult organism.
Assuntos
Linfócitos B/metabolismo , Linhagem da Célula , Células-Tronco Multipotentes/metabolismo , Receptores de Superfície Celular/fisiologia , Linfócitos T/metabolismo , Transferência Adotiva , Animais , Linfócitos B/patologia , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Diferenciação Celular , Células Cultivadas , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Feminino , Citometria de Fluxo , Granulócitos/citologia , Granulócitos/metabolismo , Hematopoese , Imunofenotipagem , Integrases/metabolismo , Macrófagos/citologia , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células-Tronco Multipotentes/citologia , Células Mieloides/citologia , Células Mieloides/metabolismo , Receptores Patched , Receptor Patched-1 , Receptores de Superfície Celular/genética , Células-Tronco/citologia , Células Estromais/citologia , Células Estromais/metabolismo , Linfócitos T/patologia , Timo/patologia , Fatores de TempoRESUMO
Mutations in the human homologue of Drosophila Patched1 (PTCH1) have been found in several common tumours including basal cell carcinoma, medulloblastoma, and rhabdomyosarcoma (RMS). Medulloblastoma and RMS are also present in the murine model for Ptch1 deficiency. Tumours in heterozygous Ptch1(neo67/+) mice consistently exhibit elevated transcript levels of the proto-oncogene Gli1, of Ptch1 itself, and of the insulin-like growth factor 2 (Igf2). The present study has investigated additional molecular changes in RMSs of Ptch1 mutant mice by means of microarray analysis and protein expression analysis. The data show activation of the cell survival-promoting Akt/protein kinase B (Pkb). Furthermore, RMSs express increased levels of the anti-apoptotic protein Bcl-2 and of genes and proteins known to inhibit cell proliferation, including Gadd45a and p27kip1. Taken together, the data suggest that the formation of RMSs in Ptch1 mutants is associated with the ability of tumour cells to resist apoptosis.
Assuntos
Rabdomiossarcoma/genética , Animais , Apoptose/fisiologia , Northern Blotting , Western Blotting/métodos , DNA de Neoplasias/análise , Modelos Animais de Doenças , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Imuno-Histoquímica/métodos , Hibridização In Situ/métodos , Camundongos , Microscopia Eletrônica/métodos , Músculo Esquelético/metabolismo , Mutação/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Proto-Oncogene Mas , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Rabdomiossarcoma/fisiopatologia , Transdução de Sinais , Células Tumorais CultivadasRESUMO
BACKGROUND & AIMS: In normal rat liver, anaphylatoxin C5a induces glucose output from hepatocytes indirectly via prostanoids released from Kupffer cells. Correspondingly, it was found that hepatocytes, in contrast to Kupffer cells, did not express C5a receptors. Lipopolysaccharide (LPS) has been reported to enhance C5a receptor expression in murine livers. This might be the result of de novo expression in hepatocytes. METHODS: C5a receptor expression was investigated in hepatocytes after in vivo treatment of rats with LPS and in vitro stimulation of isolated cells with LPS and proinflammatory cytokines on messenger RNA (mRNA) and protein level, and functionally in isolated hepatocytes and perfused liver. RESULTS: In vivo treatment of rats with LPS induced C5a receptor mRNA and protein in hepatocytes with a maximum after 8-10 hours. At this time-point, C5a directly activated glycogen phosphorylase in isolated hepatocytes and enhanced glucose output in perfused livers without the involvement of prostanoids. LPS failed to induce C5a receptors in cultured hepatocytes in vitro, whereas interleukin (IL) 6 and IL-1beta, which are known to be released from Kupffer cells on stimulation with LPS, did so. In cocultures of hepatocytes with Kupffer cells, LPS induced C5a receptors in hepatocytes in an IL-6-dependent manner. CONCLUSIONS: Thus, IL-6 from Kupffer cells appears to be the main mediator of LPS-induced de novo expression of C5a receptors in hepatocytes.
