RESUMO
Cells are responding to hypoxia via prolyl-4-hydroxylase domain (PHD) enzymes, which are responsible for oxygen-dependent hydroxylation of the hypoxia-inducible factor (HIF)-1α subunit. To gain further insight into PHD function, we generated knockdown cell models for the PHD2 isoform, which is the main isoform regulating HIF-1α hydroxylation and thus stability in normoxia. Induction of a PHD2 knockdown in tetracycline-inducible HeLa PHD2 knockdown cells resulted in increased F-actin formation as detected by phalloidin staining. A similar effect could be observed in the stably transfected PHD2 knockdown cell clones 1B6 and 3B7. F-actin is at least in part responsible for shaping cell morphology as well as regulating cell migration. Cell migration was impaired significantly as a consequence of PHD2 knockdown in a scratch assay. Mechanistically, PHD2 knockdown resulted in activation of the RhoA (Ras homolog gene family member A)/Rho-associated kinase pathway with subsequent phosphorylation of cofilin. Because cofilin phosphorylation impairs its actin-severing function, this may explain the F-actin phenotype, thereby providing a functional link between PHD2-dependent signaling and cell motility.
Assuntos
Fatores de Despolimerização de Actina/metabolismo , Actinas/metabolismo , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Quinases Associadas a rho/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Movimento Celular , Citoesqueleto/metabolismo , Células HeLa , Humanos , Prolina Dioxigenases do Fator Induzível por Hipóxia , Modelos Biológicos , Fosforilação , Polímeros/química , Ligação Proteica , Isoformas de Proteínas , Estrutura Terciária de ProteínaRESUMO
The hypoxia-inducible factor (HIF) is the master regulator for oxygen-dependent gene expression. The HIF signal transduction pathway can be manipulated by inhibiting the activity of the HIFalpha-regulating prolyl-4-hydroxylase domain (PHD) enzymes. The consequence of inhibiting the PHD activity for chemoresistance was studied. Inhibiting the PHD activity with the 2-oxoglutarate analog dimethyloxaloylglycine (DMOG) results in increased chemoresistance towards etoposide but not carboplatin in HeLa cells. Evidence for an etoposide-specific resistance, which develops as a consequence of inhibiting the PHD activity, was further supported in a tetracycline-inducible PHD2 knockdown HeLa cell model. The etoposide-resistance was mediated by HIF-1alpha as shown in mouse embryonic fibroblast HIF-1alpha(+/+) and HIF-1alpha(-/-) cells. Decreased cellular cytotoxicity after etoposide treatment inversely correlated with a dimethyloxaloylglycine (DMOG)-inducible, HIF-1alpha-dependent enhanced MDR-1 expression and efflux activity as determined by RT-PCR, immunoblots, and with the fluorescent dye DiOC2. Taken together, our data indicate that PHD inhibitors might increase chemoresistance of tumor cells in a HIF-1-dependent manner.
