RESUMO
Medulloblastoma is the most common malignant brain tumor in children. A subset of medulloblastoma originates from granule cell precursors (GCPs) of the developing cerebellum and demonstrates aberrant hedgehog signaling, typically due to inactivating mutations in the receptor PTCH1, a pathomechanism recapitulated in Ptch1(+/-) mice. As nitric oxide may regulate GCP proliferation and differentiation, we crossed Ptch1(+/-) mice with mice lacking inducible nitric oxide synthase (Nos2) to investigate a possible influence on tumorigenesis. We observed a two-fold higher medulloblastoma rate in Ptch1(+/-) Nos2(-/-) mice compared to Ptch1(+/-) Nos2(+/+) mice. To identify the molecular mechanisms underlying this finding, we performed gene expression profiling of medulloblastomas from both genotypes, as well as normal cerebellar tissue samples of different developmental stages and genotypes. Downregulation of hedgehog target genes was observed in postnatal cerebellum from Ptch1(+/+) Nos2(-/-) mice but not from Ptch1(+/-) Nos2(-/-) mice. The most consistent effect of Nos2 deficiency was downregulation of growth-associated protein 43 (Gap43). Functional studies in neuronal progenitor cells demonstrated nitric oxide dependence of Gap43 expression and impaired migration upon Gap43 knock-down. Both effects were confirmed in situ by immunofluorescence analyses on tissue sections of the developing cerebellum. Finally, the number of proliferating GCPs at the cerebellar periphery was decreased in Ptch1(+/+) Nos2(-/-) mice but increased in Ptch1(+/-) Nos2(-/) (-) mice relative to Ptch1(+/-) Nos2(+/+) mice. Taken together, these results indicate that Nos2 deficiency promotes medulloblastoma development in Ptch1(+/-) mice through retention of proliferating GCPs in the external granular layer due to reduced Gap43 expression. This study illustrates a new role of nitric oxide signaling in cerebellar development and demonstrates that the localization of pre-neoplastic cells during morphogenesis is crucial for their malignant progression.
Assuntos
Cerebelo , Proteína GAP-43 , Meduloblastoma , Óxido Nítrico Sintase Tipo II/genética , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Animais , Movimento Celular , Proliferação de Células , Transformação Celular Neoplásica , Cerebelo/citologia , Cerebelo/crescimento & desenvolvimento , Cerebelo/metabolismo , Proteína GAP-43/genética , Proteína GAP-43/metabolismo , Regulação Neoplásica da Expressão Gênica , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Meduloblastoma/genética , Meduloblastoma/metabolismo , Camundongos , Camundongos Mutantes , Neurônios/citologia , Neurônios/metabolismo , Óxido Nítrico , Óxido Nítrico Sintase Tipo II/deficiência , Óxido Nítrico Sintase Tipo II/metabolismo , Receptores Patched , Receptor Patched-1 , Transdução de Sinais , Células-Tronco/citologia , Células-Tronco/metabolismoRESUMO
Hyaluronan (HA) and versican are key components of the dermis and are responsive to ultraviolet (UV)B-induced remodeling. The aim of this study was to explore the molecular mechanisms mediating the effects of estrogen (E(2)) on HA-rich extracellular matrix during photoaging. Hairless skh-1 mice were irradiated with UVB (three times, 1 minimal erythema dose (80 mJ/cm(2)), weekly) for 10 weeks, and endogenous sex hormone production was abrogated by ovariectomy. Subcutaneous substitution of E(2) by means of controlled-release pellets caused a strong increase in the dermal HA content in both irradiated and nonirradiated skin. The increase in dermal HA correlated with induction of HA synthase HAS3 by E(2). Expression of splice variant 2 of the HA-binding proteoglycan versican was also increased by E(2). In search of candidate mediators of these effects, it was found that E(2) strongly induced the expression of epidermal growth factor (EGF) in UVB-irradiated epidermis in vivo and in keratinocytes in vitro. EGF in turn up-regulated the expression of HAS3 and versican V2 in dermal fibroblasts. HAS3 knockdown by shRNA caused inhibition of fibroblast proliferation. Furthermore, HAS3 and versican V2 induction by E(2) correlated positively with proliferation in vivo. In addition, the accumulation of inflammatory macrophages, expression of inducible cyclooxygenase 2, as well as proinflammatory monocyte chemotactic protein 1 were decreased in response to E(2) in the dermis. Collectively, these data suggest that E(2) treatment increases the amount of dermal HA and versican V2 via paracrine release of EGF, which may be implicated in the pro-proliferative and anti-inflammatory effects of E(2) during photoaging.
Assuntos
Senescência Celular/efeitos dos fármacos , Senescência Celular/efeitos da radiação , Fator de Crescimento Epidérmico/metabolismo , Estradiol/farmacologia , Estrogênios/farmacologia , Ácido Hialurônico/metabolismo , Queratinócitos/metabolismo , Raios Ultravioleta/efeitos adversos , Versicanas/metabolismo , Animais , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Derme/metabolismo , Derme/patologia , Fator de Crescimento Epidérmico/genética , Epiderme/metabolismo , Epiderme/patologia , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Técnicas de Silenciamento de Genes , Glucuronosiltransferase/genética , Glucuronosiltransferase/metabolismo , Humanos , Hialuronan Sintases , Ácido Hialurônico/genética , Queratinócitos/patologia , Macrófagos/metabolismo , Macrófagos/patologia , Camundongos , Camundongos Mutantes , Comunicação Parácrina/efeitos dos fármacos , Comunicação Parácrina/efeitos da radiação , Versicanas/genéticaRESUMO
Chronic UVB-exposure and declined estradiol production after menopause represent important factors leading to extrinsic and intrinsic aging, respectively. Remodeling of the extracellular matrix (ECM) plays a crucial role in both responses. Whether the dermal ECM is able to recover after cessation of UVB-irradiation in dependence of estradiol is not known, however of relevance when regarding possible treatment options. Therefore, the endogenous sex hormone production was depleted by ovariectomy in female mice. Half of the mice received estradiol substitution. Mice were UVB-irradiated for 20 weeks and afterwards kept for 10 weeks without irradiation. The collagen-, hyaluronan- and proteoglycan- (versican, biglycan, lumican) matrix, collagen cleavage products and functional skin parameters were analyzed. The intrinsic aging process was characterized by increased collagen fragmentation and accumulation of biglycan. Chronic UVB-irradiation additionally augmented the lumican, versican and hyaluronan content of the dermis. In the absence of further UVB-irradiation the degradation of collagen and accumulation of biglycan in the extrinsically aged group was perpetuated in an excessive matter. Whereas estradiol increased the proteoglycan content, it reversed the effects of the perpetuated extrinsic response on collagen degradation. Suspension of the intrinsic pathway might therefore be sufficient to antagonize UVB-evoked long-term damage to the dermal ECM.
Assuntos
Derme/metabolismo , Derme/efeitos da radiação , Estrogênios/farmacologia , Substâncias Protetoras/farmacologia , Raios Ultravioleta , Animais , Biópsia , Proliferação de Células/efeitos dos fármacos , Colágeno/metabolismo , Derme/efeitos dos fármacos , Derme/patologia , Feminino , Ácido Hialurônico/metabolismo , Inflamação/patologia , Camundongos Pelados , Ovariectomia , Proteoglicanas/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
EGFRvIII, a frequently occurring mutation in primary glioblastoma, results in a protein product that cannot bind ligand, but signals constitutively. Deducing how EGFRvIII causes transformation has been difficult because of autocrine and paracrine loops triggered by EGFRvIII alone or in heterodimers with wild-type EGFR. Here, we document coexpression of EGFR and EGFRvIII in primary human glioblastoma that drives transformation and tumorigenesis in a cell-intrinsic manner. We demonstrate enhancement of downstream STAT signaling triggered by EGFR-catalyzed phosphorylation of EGFRvIII, implicating EGFRvIII as a substrate for EGFR. Subsequent phosphorylation of STAT3 requires nuclear entry of EGFRvIII and formation of an EGFRvIII-STAT3 nuclear complex. Our findings clarify specific oncogenic signaling relationships between EGFR and EGFRvIII in glioblastoma.
Assuntos
Neoplasias Encefálicas/metabolismo , Receptores ErbB/metabolismo , Glioblastoma/metabolismo , Fator de Transcrição STAT3/metabolismo , Fator de Transcrição STAT5/metabolismo , Alelos , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Humanos , Mutação , Transplante de Neoplasias , Fosforilação , Transdução de SinaisRESUMO
Remodeling of the dermal extracellular matrix occurs during photoaging. Here, the effect of repetitive UVB irradiation on dermal hyaluronic acid (HA) was examined. C57/BL6 mice were chronically (182 days) irradiated with UVB, and consecutive skin biopsies were collected during the irradiation period and afterward (300 and 400 days of age). UVB caused marked loss of HA from the papillary dermis and down-regulation of HA synthase 1 (HAS1), HAS2, and HAS3 mRNA expression. In contrast, hyaluronidases (HYAL) 1, HYAL2, and HA receptor CD44 were unchanged. Furthermore, transforming growth factor beta-1 (TGF-beta1) and TGF-beta1-receptor II expression were decreased in UVB-irradiated biopsies, and TGF-beta1 strongly induced HAS1 and HAS2 expression in cultured dermal fibroblasts. Therefore, TGF-beta1 might be one factor involved in UVB-induced down-regulation of HAS enzymes. In addition, total cell number and the percentage of proliferating fibroblasts in the papillary dermis of UVB-irradiated mice were decreased. Down-regulation of HAS2 by lentiviral overexpression of short hairpin RNA in vitro caused inhibition of HA synthesis, DNA synthesis, and migration of dermal fibroblasts. In conclusion, chronic UVB irradiation induces loss of HA from the dermis, thereby contributing to the quiescent phenotype of dermal fibroblasts.