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1.
J Invest Dermatol ; 139(7): 1497-1505.e5, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30684555

RESUMO

Mutations in the gene encoding collagen VII cause the devastating blistering disease recessive dystrophic epidermolysis bullosa (RDEB). RDEB is characterized by severe skin fragility and nonhealing wounds aggravated by scarring and fibrosis. We previously showed that TSP1 is increased in RDEB fibroblasts. Because transforming growth factor-ß (TGF-ß) signaling is also increased in RDEB, and TSP1 is known to activate TGF-ß, we investigated the role of TSP1 in TGF-ß signaling in RDEB patient cells. Knockdown of TSP1 reduced phosphorylation of smad3 (a downstream target of TGF-ß signaling) in RDEB primary fibroblasts, whereas overexpression of collagen VII reduced phosphorylation of smad3. Furthermore, inhibition of TSP1 binding to the LAP/TGF-ß complex decreased fibrosis in engineered extracellular matrix formed by RDEB fibroblasts, as evaluated by picrosirius red staining and analyses of birefringent collagen fibrillar deposits. We show that collagen VII binds TSP1, which could potentially limit TSP1-LAP association and subsequent TGF-ß activation. Our study suggests a previously unreported mechanism for increased TGF-ß signaling in the absence of collagen VII in RDEB patient skin. Moreover, these data identify TSP1 as a possible target for reducing fibrosis in the tumor-promoting dermal microenvironment of RDEB patients.


Assuntos
Epidermólise Bolhosa Distrófica/metabolismo , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Pele/patologia , Trombospondina 1/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Células Cultivadas , Criança , Pré-Escolar , Colágeno Tipo VII/genética , Epidermólise Bolhosa Distrófica/genética , Feminino , Fibroblastos/patologia , Fibrose , Técnicas de Silenciamento de Genes , Genes Recessivos , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Mutação/genética , Fosforilação , Ligação Proteica , Transdução de Sinais , Proteína Smad3/metabolismo , Trombospondina 1/genética , Microambiente Tumoral , Adulto Jovem
2.
Int J Biochem Cell Biol ; 53: 450-8, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24955488

RESUMO

Squamous cell carcinoma (SCC) represents one of the most frequently diagnosed tumours and contributes significant mortality worldwide. Recent deep sequencing of cancer genomes has identified common mutations in SCC arising across different tissues highlighting perturbation of squamous differentiation as a key event. At the same time significant data have been accumulating to show that common tumour-stroma interactions capable of driving disease progression are also evident when comparing SCC arising in different tissues. We and others have shown altered matrix composition surrounding SCC can promote tumour development. This review focuses on some of the emerging data with particular emphasis on SCC of head and neck and skin with discussion on the potential tumour suppressive properties of a normal microenvironment. Such data indicate that regardless of the extent and type of somatic mutation it is in fact the tumour context that defines metastatic progression.


Assuntos
Carcinogênese/genética , Carcinoma de Células Escamosas/genética , Sequenciamento de Nucleotídeos em Larga Escala , Microambiente Tumoral/genética , Carcinoma de Células Escamosas/patologia , Matriz Extracelular/genética , Fibroblastos/metabolismo , Fibroblastos/patologia , Genoma Humano , Humanos , Mutação
3.
J Cell Sci ; 127(Pt 4): 740-51, 2014 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-24357722

RESUMO

Type VII collagen is the main component of anchoring fibrils, structures that are integral to basement membrane homeostasis in skin. Mutations in the gene encoding type VII collagen COL7A1 cause recessive dystrophic epidermolysis bullosa (RDEB) an inherited skin blistering condition complicated by frequent aggressive cutaneous squamous cell carcinoma (cSCC). OATP1B3, which is encoded by the gene SLCO1B3, is a member of the OATP (organic anion transporting polypeptide) superfamily responsible for transporting a wide range of endogenous and xenobiotic compounds. OATP1B3 expression is limited to the liver in healthy tissues, but is frequently detected in multiple cancer types and is reported to be associated with differing clinical outcome. The mechanism and functional significance of tumour-specific expression of OATP1B3 has yet to be determined. Here, we identify SLCO1B3 expression in tumour keratinocytes isolated from RDEB and UV-induced cSCC and demonstrate that SLCO1B3 expression and promoter activity are modulated by type VII collagen. We show that reduction of SLCO1B3 expression upon expression of full-length type VII collagen in RDEB cSCC coincides with acquisition of front-to-rear polarity and increased organisation of 3D spheroid cultures. In addition, we show that type VII collagen positively regulates the abundance of markers implicated in cellular polarity, namely ELMO2, PAR3, E-cadherin, B-catenin, ITGA6 and Ln332.


Assuntos
Carcinoma de Células Escamosas/metabolismo , Polaridade Celular , Colágeno Tipo VII/fisiologia , Epidermólise Bolhosa Distrófica/metabolismo , Transportadores de Ânions Orgânicos Sódio-Independentes/metabolismo , Neoplasias Cutâneas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Antígenos CD , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Caderinas/genética , Caderinas/metabolismo , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Técnicas de Cocultura , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Epidermólise Bolhosa Distrófica/genética , Epidermólise Bolhosa Distrófica/patologia , Regulação Neoplásica da Expressão Gênica , Humanos , Integrina alfa6/genética , Integrina alfa6/metabolismo , Queratinócitos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Transplante de Neoplasias , Transportadores de Ânions Orgânicos Sódio-Independentes/genética , Regiões Promotoras Genéticas , Transporte Proteico , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Membro 1B3 da Família de Transportadores de Ânion Orgânico Carreador de Soluto , Transcrição Gênica , Células Tumorais Cultivadas , beta Catenina/genética , beta Catenina/metabolismo , Calinina
4.
Biochimie ; 89(1): 21-9, 2007 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17097793

RESUMO

In a previous paper, we reported more efficient enterokinase cleavage at a C-terminal non-target LKGDR(201) site compared with an internally sited canonical recognition site, DDDDK(156). When this non-target site was placed internally to replace DDDDK(156) between the thioredoxin moiety and mouse NT-proCNP(1-50), this site was poorly processed leading us to conclude that efficient processing at LKGDR(201) in the first instance was due to its accessibility at the C-terminus of the fusion protein. Subsequently, we reasoned that treatment of thioredoxin-fused NT-proCNP(1-81) would allow us to retrieve full-length NT-proCNP(1-81) without undue processing at the LKGDR(201) site since this non-target site would now be located internally about 36 residues away from the C-terminus and hence not be hydrolyzed efficiently. Surprisingly, ESI-MS data showed that the LKGDR site in thioredoxin-fused human NT-proCNP(1-81) was still very efficiently cleaved and revealed a new but slow hydrolysis site with the sequence RVDTK/SRAAW to yield a peptide consistent with NT-proCNP(58-81). The evidence obtained from these experiments led us to postulate that efficient cleavage at the non-target LKGDR(201) site was not merely influenced by steric constraints but also by the sequence context downstream of the scissile bond. Hence, we constructed variants of thioredoxin-mouse NT-proCNP(1-50) where SRLLR residues (i.e. those immediately downstream from the LKGDR(201) site in NT-proCNP(1-50)) were systematically added one at a time downstream of the internal DDDDK(156) site. To evaluate the relative effects of site accessibility and downstream sequence context on the efficiency of enterokinase cleavage, we have also replaced the native LKGDR(201) sequence with DDDDK(201). Our results showed that incremental addition of SRLLR residues led to a steady increase in the rate of hydrolysis at DDDDK(156). Further variants comprising DDDDK(156)SS, DDDDK(156)SD and DDDDK(156)RR showed that the minimal critical determinants for enhanced enterokinase cleavage are serine in the P1' position followed by a serine or a basic residue, lysine or arginine, in the P2' position. Our data provided conclusive evidence that the influence of downstream sequences on recombinant light chain enterokinase activity was greater than accessibility of the target site at the terminus region of the protein. We further showed that the catalytic efficiency of the native holoenzyme was influenced primarily by residues on the N-terminal side of the scissile bond while being neutral to residues on the C-terminal side. Finally, we found that cleavage of all nine fusion proteins reflects accurate hydrolysis at the DDDDK(156) and DDDDK(201) sites when recombinant light chain enterokinase was used while non-specific processing at secondary sites were observed when these fusion proteins were treated with the native holoenzyme.


Assuntos
Enteropeptidase/genética , Enteropeptidase/metabolismo , Peptídeo Natriurético Tipo C/metabolismo , Tiorredoxinas/metabolismo , Motivos de Aminoácidos , Animais , Cromatografia Líquida de Alta Pressão , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Enteropeptidase/química , Humanos , Camundongos , Dados de Sequência Molecular , Peptídeo Natriurético Tipo C/isolamento & purificação , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização por Electrospray
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