RESUMO
Collagen IV is a major component of the dermo-epidermal junction (DEJ). To study expression of collagen IV upon aging in the DEJ and dermal fibroblasts isolated from the same patients. A model of senescent fibroblasts was developed in order to identify biological compounds that might restore the level of collagen IV. Skin fragments of women (30 to 70 years old) were collected. Localisation of collagen IV expression in the DEJ was studied by immunofluorescence. Fibroblast collagen IV expression was studied by real-time PCR, ELISA, and western blotting. Premature senescence was simulated by exposing fibroblasts to subcytotoxic H2O2 concentrations. Collagen IV decreased in the DEJ and fibroblasts relative to age. TGF-ß1 treatment significantly increased collagen IV gene and protein expression in fibroblasts and restored expression in the model of senescence. Addition of TGF-ß1-neutralizing antibody to fibroblast cultures decreased collagen IV expression. Taken together, the results suggest that the decrease in collagen IV in the DEJ, relative to age, could be due to a decrease in collagen IV expression by senescent dermal fibroblasts and may involve TGF-ß1 signalling.
Assuntos
Envelhecimento/fisiologia , Colágeno Tipo IV/metabolismo , Derme/metabolismo , Epiderme/metabolismo , Fibroblastos/metabolismo , Fator de Crescimento Transformador beta1/farmacologia , Adulto , Idoso , Envelhecimento/genética , Membrana Basal/metabolismo , Células Cultivadas , Senescência Celular , Colágeno Tipo IV/genética , Derme/citologia , Feminino , Expressão Gênica/efeitos dos fármacos , Humanos , Peróxido de Hidrogênio/farmacologia , Pessoa de Meia-Idade , Proteínas Serina-Treonina Quinases/genética , RNA Mensageiro/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo II , Receptores de Fatores de Crescimento Transformadores beta/genética , Transdução de Sinais , Fator de Crescimento Transformador beta1/metabolismoRESUMO
During chronological skin aging, alterations in dermal structural proteins cause morphological modifications. Modifications are probably due to collagen fiber (type I collagen) rearrangement and reorientation with aging that have not been researched until now. FTIR microspectroscopy appears as an interesting method to study protein structure under normal and pathological conditions. Associated with a polarizer, this vibrational technique permits us to probe collagen orientation within skin tissue sections, by computing the ratio of integrated intensities of amide I and amide II bands. In this study, we used the polarized-FTIR imaging to evaluate molecular modifications of dermal collagen during chronological aging. The data processing of polarized infrared data revealed that type I collagen fibers become parallel to the skin surface in aged skin dermis. Our approach could find innovative applications in dermatology as well as in cosmetics.
Assuntos
Envelhecimento/metabolismo , Colágeno/metabolismo , Pele/metabolismo , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Análise por Conglomerados , Humanos , Pessoa de Meia-Idade , RatosRESUMO
BACKGROUND: NC1 domains from α1, α2, α3 and α6(IV) collagen chains were shown to exert anti-tumor or anti-angiogenic activities, whereas the NC1 domain of the α4(IV) chain did not show such activities so far. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrate in the present paper that the NC1 α4(IV) domain exerts a potent anti-tumor activity both in vitro and in an experimental human melanoma model in vivo. The overexpression of NC1 α4(IV) in human UACC-903 melanoma cells strongly inhibited their in vitro proliferative (-38%) and invasive (-52%) properties. MT1-MMP activation was largely decreased and its cellular distribution was modified, resulting in a loss of expression at the migration front associated with a loss of migratory phenotype. In an in vivo xenograft model in athymic nude mice, the subcutaneous injection of NC1 α4(IV)-overexpressing melanoma cells induced significantly smaller tumors (-80% tumor volume) than the Mock cells, due to a strong inhibition of tumor growth. Exogenously added recombinant human NC1 α4(IV) reproduced the inhibitory effects of NC1 α4(IV) overexpression in UACC-903 cells but not in dermal fibroblasts. An anti-αvß3 integrin blocking antibody inhibited cell adhesion on recombinant human NC1 α4(IV) substratum. The involvement of αvß3 integrin in mediating NC1 α4(IV) effect was confirmed by surface plasmon resonance (SPR) binding assays showing that recombinant human NC1 α4(IV) binds to αvß3 integrin (K(D) = 148 ± 9.54 nM). CONCLUSION/SIGNIFICANCE: Collectively, our results demonstrate that the NC1 α4(IV) domain, named tetrastatin, is a new endogenous anti-tumor matrikine.