RESUMO
Hyaluronan is a component of the extracellular matrix, which affects tissue homeostasis. In this study, we investigated the regulatory mechanisms of one of the hyaluronan-synthesizing enzymes, HAS2. Ectopic expression of Flag- and 6myc-HAS2 in COS-1 cells followed by immunoprecipitation and immunoblotting revealed homodimers; after co-transfection with Flag-HAS3, also heterodimers were seen. Furthermore, the expressed HAS2 was ubiquitinated. We identified one acceptor site for ubiquitin on lysine residue 190. Mutation of this residue led to inactivation of the enzymatic activity of HAS2. Interestingly, K190R-mutated HAS2 formed dimers with wt HAS2 and quenched the activity of wt HAS2, thus demonstrating a functional role of the dimeric configuration.
Assuntos
Regulação Enzimológica da Expressão Gênica , Glucuronosiltransferase/química , Glucuronosiltransferase/metabolismo , Ubiquitina/química , Animais , Sítios de Ligação , Células COS , Catálise , Linhagem Celular Transformada , Chlorocebus aethiops , Dimerização , Hialuronan Sintases , Camundongos , Modelos Biológicos , Mutagênese Sítio-Dirigida , Mutação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
PURPOSE: To establish human corneal stroma- and sclera-derived cells as models for studying diseases of the anterior segment of the eye. METHODS: Using a recombinant retrovirus system, we transfected human papilloma virus 16 E6 and E7 (HPV16 E6/E7) into human corneal stroma- and sclera-derived cells. The primary cells and established cell strains were characterized by assessing the mRNA expression of collagen, matrix metalloproteinase, and tissue inhibitor of metalloproteinase by reverse transcription-polymerase chain reaction. We also examined the effects of inflammatory cytokines on hyaluronan synthase expression and hyaluronan products. RESULTS: Both a corneal stroma-derived cell strain, Cs3, and a sclera-derived cell strain, Sc1, were obtained, and both cell strains could be passaged up to 25 times. The mRNA expression pattern observed in the primary cells was identical to that observed in the cell strains. Hyaluronan synthase 1 and 2 mRNAs were increased by transforming growth factor beta and platelet-derived growth factor BB. Significant differences were observed between the hyaluronan products with and without cytokine treatment. CONCLUSION: Cell strains derived from corneal stroma and sclera fibroblast cells can be established using HPV16 E6/E7 immortalized genes of the same origin. The phenotypic cell characteristics did not change after transfection, immortalization, or successive passages in culture.
Assuntos
Substância Própria/citologia , Fibroblastos/citologia , Esclera/citologia , Idoso , Células Cultivadas , Clonagem Molecular , Colágeno/genética , Substância Própria/metabolismo , Feminino , Fibroblastos/metabolismo , Regulação Viral da Expressão Gênica/fisiologia , Glucuronosiltransferase/genética , Humanos , Hialuronan Sintases , Ácido Hialurônico/metabolismo , Metaloproteinases da Matriz/genética , Proteínas Oncogênicas Virais/genética , Proteínas E7 de Papillomavirus/genética , RNA Mensageiro/metabolismo , Proteínas Repressoras/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Esclera/metabolismo , Inibidores Teciduais de Metaloproteinases/genética , TransfecçãoRESUMO
Hyaluronan is a glycosaminoglycan of the extracellular matrix. In tumors and during chronic inflammatory diseases, hyaluronan is degraded to smaller fragments, which are known to stimulate endothelial cell differentiation. In this study, we have compared the molecular mechanisms through which hyaluronan dodecasaccharides (HA12), and the known angiogenic factor, fibroblast growth factor 2 (FGF-2), induce capillary endothelial cell sprouting in a three-dimensional collagen gel. The gene expression profiles of unstimulated and HA12- or FGF-2-stimulated endothelial cells were compared using a microarray analysis approach. The data revealed that both FGF-2 and HA12 promoted endothelial cell morphogenesis in a process depending on the expression of ornithine decarboxylase (Odc) and ornithine decarboxylase antizyme inhibitor (Oazi) genes. Among the genes selectively up-regulated in response to HA12 was the chemokine CXCL1/GRO1 gene. The notion that the induction of CXCL1/GRO1 is of importance for HA12-induced endothelial cell sprouting was supported by the fact that morphogenesis was inhibited by antibodies specifically neutralizing the CXCL1/GRO1 protein product. HA12-stimulated endothelial cell differentiation was exerted via binding to CD44 since it was inhibited by antibodies blocking CD44 function. Our data show that hyaluronan fragments and FGF-2 affect endothelial cell morphogenesis by the induction of overlapping but also by distinct sets of genes.