RESUMO
In vivo function of CDK5 and Abl enzyme substrate 2 (Cables2), belonging to the Cables protein family, is unknown. Here, we found that targeted disruption of the entire Cables2 locus (Cables2d) caused growth retardation and enhanced apoptosis at the gastrulation stage and then induced embryonic lethality in mice. Comparative transcriptome analysis revealed disruption of Cables2, 50% down-regulation of Rps21 abutting on the Cables2 locus, and up-regulation of p53-target genes in Cables2d gastrulas. We further revealed the lethality phenotype in Rps21-deleted mice and unexpectedly, the exon 1-deleted Cables2 mice survived. Interestingly, chimeric mice derived from Cables2d ESCs carrying exogenous Cables2 and tetraploid wild-type embryo overcame gastrulation. These results suggest that the diminished expression of Rps21 and the completed lack of Cables2 expression are intricately involved in the embryonic lethality via the p53 pathway. This study sheds light on the importance of Cables2 locus in mouse embryonic development.
Assuntos
Proteínas de Ciclo Celular/genética , Gastrulação/genética , Expressão Gênica , Proteínas Ribossômicas/genética , Transdução de Sinais , Proteína Supressora de Tumor p53/metabolismo , Animais , Feminino , Masculino , Camundongos , Camundongos Endogâmicos ICR , Fenótipo , Ativação Transcricional , Proteína Supressora de Tumor p53/genética , Regulação para CimaRESUMO
The cytoskeletal protein Flightless (Flii) is a negative regulator of wound healing. Upregulation of Flii is associated with impaired migration, proliferation and adhesion of both fibroblasts and keratinocytes. Importantly, Flii translocates from the cytoplasm to the nucleus in response to wounding in fibroblasts but not keratinocytes. This cell-specific nuclear translocation of Flii suggests that Flii may directly regulate gene expression in fibroblasts, providing one potential mechanism of action for Flii in the wound healing response. To determine whether the tissue-specific upregulation of Flii in fibroblasts was important for the observed inhibitory effects of Flii on wound healing, an inducible fibroblast-specific Flii overexpressing mouse model was generated. The inducible ROSA26 system allowed the overexpression of Flii in a temporal and tissue-specific manner in response to tamoxifen treatment. Wound healing in the inducible mice was impaired, with wounds at day 7 postwounding significantly larger than those from non-inducible controls. There was also reduced collagen maturation, increased myofibroblast infiltration and elevated inflammation. The impaired healing response was similar in magnitude to that observed in mice with non-tissue-specific upregulation of Flii suggesting that fibroblast-derived Flii may have an important role in the wound healing response.
Assuntos
Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Fibroblastos/metabolismo , Pele/metabolismo , Cicatrização/genética , Animais , Antineoplásicos Hormonais/farmacologia , Proteínas de Transporte , Movimento Celular , Proliferação de Células , Células Cultivadas , Colágeno/ultraestrutura , Fibroblastos/efeitos dos fármacos , Fibroblastos/ultraestrutura , Expressão Gênica/efeitos dos fármacos , Camundongos , Proteínas dos Microfilamentos , Modelos Animais , Recombinação Genética/efeitos dos fármacos , Pele/efeitos dos fármacos , Pele/lesões , Tamoxifeno/farmacologia , Transativadores , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genética , Cicatrização/efeitos dos fármacosRESUMO
Development of an intact epidermis is critical for maintaining the integrity of the skin. Patients with epidermolysis bullosa (EB) experience multiple erosions, which breach the epidermal barrier and lead to increased microbial colocalization of wounds, infections and sepsis. The cytoskeletal protein Flightless I (Flii) is a known regulator of both development and wound healing. Using Flii(+/-), WT and Flii(Tg/Tg) mice, we investigated the effect of altering Flii levels in embryos and adult mice on the development of the epidermal barrier and, consequently, how this affects the integrity of the skin in EB. Flii over-expression resulted in delayed formation of the epidermal barrier in embryos and decreased expression of tight junction (TJ) proteins Claudin-1 and ZO-2. Increased intercellular space and transepidermal water loss was observed in Flii(Tg)(/Tg) adult mouse skin, while Flii(Tg/Tg) keratinocytes showed altered TJ protein localization and reduced transepithelial resistance. Flii is increased in the blistered skin of patients with EB, and over-expression of Flii in experimental EBA showed impaired Claudin-1 and -4 TJ protein expression and delayed recovery of functional barrier post-blistering. Immunoprecipitation confirmed Flii associated with TJ proteins and in vivo actin assays showed that the effect of Flii on actin polymerization underpinned the impaired barrier function observed in Flii(Tg/Tg) mice. These results therefore demonstrate an important role for Flii in the development and regulation of the epidermal barrier, which may contribute to the impaired healing and skin fragility of EB patients.
Assuntos
Vesícula/metabolismo , Epiderme/metabolismo , Epidermólise Bolhosa/metabolismo , Proteína Proto-Oncogênica c-fli-1/deficiência , Proteína Proto-Oncogênica c-fli-1/metabolismo , Cicatrização , Actinas/metabolismo , Animais , Vesícula/genética , Vesícula/patologia , Células Cultivadas , Modelos Animais de Doenças , Impedância Elétrica , Epiderme/patologia , Epidermólise Bolhosa/genética , Epidermólise Bolhosa/patologia , Genótipo , Humanos , Queratinócitos/metabolismo , Queratinócitos/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Camundongos Transgênicos , Permeabilidade , Fenótipo , Polimerização , Proteína Proto-Oncogênica c-fli-1/genética , Proteínas de Junções Íntimas/metabolismo , Junções Íntimas/metabolismo , Junções Íntimas/patologia , Fatores de Tempo , Regulação para Cima , Perda Insensível de ÁguaRESUMO
AIMS/HYPOTHESIS: Skin lesions and ulcerations are severe complications of diabetes that often result in leg amputations. In this study we investigated the function of the cytoskeletal protein flightless I (FLII) in diabetic wound healing. We hypothesised that overexpression of FLII would have a negative effect on diabetic wound closure and modulation of this protein using specific FLII-neutralising antibodies (FnAb) would enhance cellular proliferation, migration and angiogenesis within the diabetic wound. METHODS: Using a streptozotocin-induced model of diabetes we investigated the effect of altered FLII levels through Flii genetic knockdown, overexpression or treatment with FnAb on wound healing. Diabetic wounds were assessed using histology, immunohistochemistry and biochemical analysis. In vitro and in vivo assays of angiogenesis were used to assess the angiogenic response. RESULTS: FLII levels were elevated in the wounds of both diabetic mice and humans. Reduction in the level of FLII improved healing of murine diabetic wounds and promoted a robust pro-angiogenic response with significantly elevated von Willebrand factor (vWF) and vascular endothelial growth factor (VEGF)-positive endothelial cell infiltration. Diabetic mouse wounds treated intradermally with FnAb showed improved healing and a significantly increased rate of re-epithelialisation. FnAb improved the angiogenic response through enhanced formation of capillary tubes and functional neovasculature. Reducing the level of FLII led to increased numbers of mature blood vessels, increased recruitment of smooth muscle actin-α-positive cells and improved tight junction formation. CONCLUSIONS/INTERPRETATION: Reducing the level of FLII in a wound may be a potential therapeutic approach for the treatment of diabetic foot ulcers.
Assuntos
Proteínas do Citoesqueleto/farmacologia , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Tipo 1/patologia , Angiopatias Diabéticas/patologia , Pele/patologia , Cicatrização/imunologia , Indutores da Angiogênese , Animais , Anticorpos Neutralizantes/metabolismo , Proteínas de Transporte , Proliferação de Células , Diabetes Mellitus Experimental/imunologia , Diabetes Mellitus Tipo 1/imunologia , Angiopatias Diabéticas/imunologia , Feminino , Humanos , Imuno-Histoquímica , Inflamação , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Proteínas dos Microfilamentos , Pele/lesões , Transativadores , Úlcera/patologiaRESUMO
We identified two novel mouse mutants with abnormal head-shaking behavior and neural tube defects during the course of independent ENU mutagenesis experiments. The heterozygous and homozygous mutants exhibit defects in the orientation of sensory hair cells in the organ of Corti, indicating a defect in planar cell polarity. The homozygous mutants exhibit severe neural tube defects as a result of failure to initiate neural tube closure. We show that these mutants, spin cycle and crash, carry independent missense mutations within the coding region of Celsr1, encoding a large protocadherin molecule [1]. Celsr1 is one of three mammalian homologs of Drosophila flamingo/starry night, which is essential for the planar cell polarity pathway in Drosophila together with frizzled, dishevelled, prickle, strabismus/van gogh, and rhoA. The identification of mouse mutants of Celsr1 provides the first evidence for the function of the Celsr family in planar cell polarity in mammals and further supports the involvement of a planar cell polarity pathway in vertebrate neurulation.