RESUMO
Uncontrolled activation of the Hedgehog (Hh) signaling pathway, operating through GLI transcription factors, plays a central role in the pathogenesis of cutaneous basal cell carcinoma and contributes to the development of several malignancies arising in extracutaneous sites. We now report that K5-tTA;tetO-Gli2 bitransgenic mice develop distinctive epithelial tumors within their jaws. These tumors consist of large masses of highly proliferative, monomorphous, basaloid cells with scattered foci of keratinization and central necrosis, mimicking human basaloid squamous cell carcinoma (BSCC), an aggressive upper aerodigestive tract tumor. Like human BSCC, these tumors express epidermal basal keratins and differentiation-specific keratins within squamous foci. Mouse BSCCs express high levels of Gli2 and Hh target genes, including Gli1 and Ptch1, which we show are also upregulated in a subset of human BSCCs. Mouse BSCCs appear to arise from distinct epithelial sites, including the gingival junctional epithelium and epithelial rests of Malassez, a proposed stem cell compartment. Although Gli2 transgene expression is restricted to epithelial cells, we also detect striking alterations in bone adjacent to BSCCs, with activated osteoblasts, osteoclasts and osteal macrophages, indicative of active bone remodeling. Gli2 transgene inactivation resulted in rapid BSCC regression and reversal of the bone remodeling phenotype. This first-reported mouse model of BSCC supports the concept that uncontrolled Hh signaling plays a central role in the pathogenesis of a subset of human BSCCs, points to Hh/GLI2 signaling as a potential therapeutic target and provides a powerful new tool for probing the mechanistic underpinnings of tumor-associated bone remodeling.
Assuntos
Remodelação Óssea , Carcinoma de Células Escamosas/patologia , Proteínas Hedgehog/metabolismo , Proteínas Nucleares/metabolismo , Neoplasias Cutâneas/patologia , Proteína Gli2 com Dedos de Zinco/metabolismo , Animais , Carcinoma de Células Escamosas/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , Neoplasias Cutâneas/metabolismoRESUMO
Notch signaling is essential for lymphocyte development and is also implicated in myelopoiesis. Notch receptors are modified by O-fucosylation catalyzed by protein O-fucosyltransferase 1 (Pofut1). Fringe enzymes add N-acetylglucosamine to O-fucose and modify Notch signaling by altering the sensitivity of Notch receptors to Notch ligands. To address physiologic functions in hematopoiesis of Notch modified by O-fucose glycans, we examined mice with inducible inactivation of Pofut1 using Mx-Cre. These mice exhibited a reduction in T lymphopoiesis and in the production of marginal-zone B cells, in addition to myeloid hyperplasia. Restoration of Notch1 signaling rescued T lymphopoiesis and the marrow myeloid hyperplasia. After marrow transfer, both cell-autonomous and environmental cues were found to contribute to lymphoid developmental defects and myeloid hyperplasia in Pofut1-deleted mice. Although Pofut1 deficiency slightly decreased cell surface expression of Notch1 and Notch2, it completely abrogated the binding of Notch receptors with Delta-like Notch ligands and suppressed downstream Notch target activation, indicating that O-fucose glycans are critical for efficient Notch-ligand binding that transduce Notch signals. The combined data support a key role for the O-fucose glycans generated by Pofut1 in Notch regulation of hematopoietic homeostasis through modulation of Notch-ligand interactions.
Assuntos
Fucosiltransferases/fisiologia , Homeostase/fisiologia , Linfopoese/fisiologia , Mielopoese/fisiologia , Receptores Notch/metabolismo , Animais , Transplante de Medula Óssea , Diferenciação Celular , Células Cultivadas , Citometria de Fluxo , Fucose/metabolismo , Humanos , Hidroliases/metabolismo , Hiperplasia/enzimologia , Hiperplasia/patologia , Integrases/metabolismo , Ligantes , Camundongos , Camundongos Knockout , Camundongos Transgênicos , RNA Mensageiro/genética , Receptores Notch/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Linfócitos T/enzimologia , Linfócitos T/patologiaRESUMO
Notch receptors are cell surface molecules essential for cell fate determination. Notch signaling is subject to tight regulation at multiple levels, including the posttranslational modification of Notch receptors by O-linked fucosylation, a reaction that is catalyzed by protein O-fucosyltransferase-1 (Pofut1). Our previous studies identified a myeloproliferative phenotype in mice conditionally deficient in cellular fucosylation that is attributable to a loss of Notch-dependent suppression of myelopoiesis. Here, we report that hematopoietic stem cells deficient in cellular fucosylation display decreased frequency and defective repopulating ability as well as decreased lymphoid but increased myeloid developmental potential. This phenotype may be attributed to suppressed Notch ligand binding and reduced downstream signaling of Notch activity in hematopoietic stem cells. Consistent with this finding, we further demonstrate that mouse embryonic stem cells deficient in Notch1 (Notch1(-/-)) or Pofut1 (Pofut1(-/-)) fail to generate T lymphocytes but differentiate into myeloid cells while coculturing with Notch ligand-expressing bone marrow stromal cells in vitro. Moreover, in vivo hematopoietic reconstitution of CD34(+) progenitor cells derived from either Notch1(-/-) or Pofut1(-/-) embryonic stem cells show enhanced granulopoiesis with depressed lymphoid lineage development. Together, these results indicate that Notch signaling maintains hematopoietic lineage homeostasis by promoting lymphoid development and suppressing overt myelopoiesis, in part through processes controlled by O-linked fucosylation of Notch receptors.
Assuntos
Diferenciação Celular/fisiologia , Linhagem da Célula , Fucose/metabolismo , Células-Tronco Hematopoéticas/fisiologia , Receptor Notch1/metabolismo , Animais , Transplante de Medula Óssea , Linhagem Celular , Fucose/química , Fucosiltransferases/genética , Fucosiltransferases/metabolismo , Células-Tronco Hematopoéticas/citologia , Camundongos , Camundongos Knockout , Transdução de Sinais/fisiologiaRESUMO
BACKGROUND: Modification of Notch receptors by O-linked fucose and its further elongation by the Fringe family of glycosyltransferase has been shown to be important for Notch signaling activation. Our recent studies disclose a myeloproliferative phenotype, hematopoietic stem cell (HSC) dysfunction, and abnormal Notch signaling in mice deficient in FX, which is required for fucosylation of a number of proteins including Notch. The purpose of this study was to assess the self-renewal and stem cell niche features of fucose-deficient HSCs. STUDY DESIGN AND METHODS: Homeostasis and maintenance of HSCs derived from FX(-/-) mice were studied by serial bone marrow transplantation, homing assay, and cell cycle analysis. Two-photon intravital microscopy was performed to visualize and compare the in vivo marrow niche occupancy by fucose-deficient and wild-type (WT) HSCs. RESULTS: Marrow progenitors from FX(-/-) mice had mild homing defects that could be partially prevented by exogenous fucose supplementation. Fucose-deficient HSCs from FX(-/-) mice displayed decreased self-renewal capability compared with the WT controls. This is accompanied with their increased cell cycling activity and suppressed Notch ligand binding. When tracked in vivo by two-photon intravital imaging, the fucose-deficient HSCs were found localized farther from the endosteum of the calvarium marrow than the WT HSCs. CONCLUSIONS: The current reported aberrant niche occupancy by HSCs from FX(-/-) mice, in the context of a faulty blood lineage homeostasis and HSC dysfunction in mice expressing Notch receptors deficient in O-fucosylation, suggests that fucosylation-modified Notch receptor may represent a novel extrinsic regulator for HSC engraftment and HSC niche maintenance.
Assuntos
Medula Óssea/fisiologia , Movimento Celular/fisiologia , Proliferação de Células , Fucose/deficiência , Células-Tronco Hematopoéticas/fisiologia , Nicho de Células-Tronco/citologia , Animais , Medula Óssea/metabolismo , Células Cultivadas , Simulação por Computador , Feminino , Fucose/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Humanos , Hidroliases/genética , Hidroliases/metabolismo , Camundongos , Camundongos Knockout , Processamento de Proteína Pós-Traducional/fisiologia , Receptores Notch/metabolismo , Nicho de Células-Tronco/metabolismoRESUMO
Odontogenic keratocysts in humans are aggressive, noninflammatory jaw cysts that may harbor PTCH1 mutations, leading to constitutive activity of the embryonic Hedgehog (Hh) signaling pathway. We show here that epithelial expression of the Hh transcriptional effector Gli2 is sufficient for highly penetrant keratocyst development in transgenic mice. Mouse and human keratocysts expressed similar markers, leading to tooth misalignment, bone remodeling, and craniofacial abnormalities. We detected Hh target gene expression in epithelial cells lining keratocysts from both species, implicating deregulated Hh signaling in their development. Most mouse keratocysts arose from rests of Malassez--quiescent, residual embryonic epithelial cells that remain embedded in the periodontal ligament surrounding mature teeth. In Gli2-expressing mice, these rests were stimulated to proliferate, stratify, and form a differentiated squamous epithelium. The frequent development of keratocysts in Gli2-expressing mice supports the idea that GLI transcription factor activity mediates pathological responses to deregulated Hh signaling in humans. Moreover, Gli2-mediated reactivation of quiescent epithelial rests to form keratocysts indicates that these cells retain the capacity to function as progenitor cells on activation by an appropriate developmental signal.
Assuntos
Células Epiteliais/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Proteínas Nucleares/metabolismo , Cistos Odontogênicos/metabolismo , Ligamento Periodontal/metabolismo , Transdução de Sinais , Células-Tronco/metabolismo , Animais , Remodelação Óssea/genética , Diferenciação Celular/genética , Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/patologia , Células Epiteliais/patologia , Proteínas Hedgehog , Humanos , Fatores de Transcrição Kruppel-Like/genética , Camundongos , Camundongos Transgênicos , Proteínas Nucleares/genética , Odontogênese/genética , Cistos Odontogênicos/genética , Cistos Odontogênicos/patologia , Receptores Patched , Receptor Patched-1 , Ligamento Periodontal/patologia , Receptores de Superfície Celular/metabolismo , Transdução de Sinais/genética , Células-Tronco/patologia , Dente/embriologia , Dente/metabolismo , Dente/patologia , Transativadores/metabolismo , Proteína Gli2 com Dedos de ZincoRESUMO
Temporally and spatially constrained Hedgehog (Hh) signaling regulates cyclic growth of hair follicle epithelium while constitutive Hh signaling drives the development of basal cell carcinomas (BCCs), the most common cancers in humans. Using mice engineered to conditionally express the Hh effector Gli2, we show that continued Hh signaling is required for growth of established BCCs. Transgene inactivation led to BCC regression accompanied by reduced tumor cell proliferation and increased apoptosis, leaving behind a small subset of nonproliferative cells that could form tumors upon transgene reactivation. Nearly all BCCs arose from hair follicles, which harbor cutaneous epithelial stem cells, and reconstitution of regressing tumor cells with an inductive mesenchyme led to multilineage differentiation and hair follicle formation. Our data reveal that continued Hh signaling is required for proliferation and survival of established BCCs, provide compelling support for the concept that these tumors represent an aberrant form of follicle organogenesis, and uncover potential limitations to treating BCCs using Hh pathway inhibitors.
Assuntos
Carcinoma Basocelular/genética , Proliferação de Células , Regulação Neoplásica da Expressão Gênica/genética , Neoplasias Cutâneas/genética , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Animais , Apoptose/genética , Carcinoma Basocelular/metabolismo , Carcinoma Basocelular/patologia , Diferenciação Celular/genética , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Folículo Piloso/citologia , Folículo Piloso/metabolismo , Folículo Piloso/patologia , Proteínas Hedgehog , Fatores de Transcrição Kruppel-Like , Camundongos , Camundongos Transgênicos , Transdução de Sinais/genética , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Células-Tronco/metabolismo , Células-Tronco/patologia , Transativadores/genética , Fatores de Transcrição/genética , Proteína Gli2 com Dedos de ZincoRESUMO
Gain-of-function mutations in SMO have been implicated in constitutive activation of the hedgehog signaling pathway in human basal cell carcinomas (BCCs). We used a truncated keratin 5 (DeltaK5) promoter to assess the potential role of the human M2SMO mutant in BCC development in adult transgenic mice. DeltaK5-M2SMO mouse epidermis is hyperproliferative, ex presses BCC protein markers and gives rise to numerous epithelial downgrowths invading the underlying dermis. Lesions strikingly similar to human basaloid follicular hamartomas develop, but BCCs do not arise even in elderly mice. Hedgehog target gene transcripts were only modestly upregulated in mouse and human follicular hamartomas, in contrast to the high levels detected in BCCs. Cyclins D1 and D2 were selectively upregulated in mouse BCCs. Our data suggest that the levels of hedgehog pathway activation and G(1) cyclins are major determinants of tumor phenotype in skin, and strongly implicate deregulated hedgehog signaling in the genesis of human basaloid follicular hamartomas. Expression of an activated SMO mutant in keratinocytes appears to be insufficient for the development and/or maintenance of full-blown BCCs.
Assuntos
Receptores de Superfície Celular/fisiologia , Receptores Acoplados a Proteínas G , Neoplasias Cutâneas/fisiopatologia , Transativadores/fisiologia , Alopecia/etiologia , Alopecia/genética , Alopecia/patologia , Animais , Carcinoma Basocelular/genética , Carcinoma Basocelular/patologia , Carcinoma Basocelular/fisiopatologia , Diferenciação Celular , Hamartoma/genética , Hamartoma/patologia , Hamartoma/fisiopatologia , Proteínas Hedgehog , Humanos , Hiperplasia , Queratina-15 , Queratina-5 , Queratinócitos/metabolismo , Queratinas/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação , Fenótipo , Regiões Promotoras Genéticas , Receptores de Superfície Celular/genética , Transdução de Sinais , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Receptor Smoothened , Transativadores/genéticaRESUMO
Epithelial progenitor cells in skin give rise to multiple lineages, comprising the hair follicle, an associated sebaceous gland, and overlying epidermis; however, the signals that regulate sebocyte development are poorly understood. We tested the potential involvement of the Hedgehog pathway in sebaceous gland development using transgenes designed to either block or stimulate Hedgehog signaling in cutaneous keratinocytes in vivo. Whereas inhibition of the Hedgehog pathway selectively suppressed sebocyte development, Hedgehog pathway activation led to a striking increase both in size and number of sebaceous glands. Remarkably, ectopic Hedgehog signaling also triggered the formation of sebaceous glands from footpad epidermis, in regions normally devoid of hair follicles and associated structures. These ectopic sebaceous glands expressed molecular markers of sebocyte differentiation and were functional, secreting their contents directly onto the skin's surface instead of into a hair canal. The Hedgehog pathway thus plays a key role in sebocyte cell fate decisions and is a potential target for treatment of skin disorders linked to abnormal sebaceous gland function, such as acne.