Prolyl Hydroxylase Domain (PHD) 2 Affects Cell Migration and F-actin Formation via RhoA/Rho-associated Kinase-dependent Cofilin Phosphorylation

Abstract

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)-1␣ subunit. To gain further insight into PHD function, we generated knockdown cell models for the PHD2 isoform, which is the main isoform regulating HIF-1␣ 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.

Hypoxia Modulates Fibroblastic Architecture, Adhesion and Migration: A Role for HIF-1α in Cofilin Regulation and Cytoplasmic Actin Distribution

“…This results in severing of actin filaments and sequestering of actin monomers from the pointed end of the filament [28]. Elevated cofilin phosphorylation was observed in hypoxic cells [29], [30] and tissues [31]. This led us to investigate the expression and phosphorylation status of cofilin in L929 cells by Western blot.…”

A Novel Small Molecular STAT3 Inhibitor, LY5, Inhibits Cell Viability, Cell Migration, and Angiogenesis in Medulloblastoma Cells

“…5B). In addition, considering F-actin and microtubule cytoskeletal elements that are regulated by STAT3 both involve the function of cell migration (50,51), the ability of LY5 to inhibit F-actin fiber and microtubule formation was evaluated by confocal microscopy as described under "Experimental Procedures." In the presence of LY5 treatment, Fig.…”

Prolyl Hydroxylase Domain 2 Protein Is a Strong Prognostic Marker in Human Gastric Cancer

“…Antigen retrieval was achieved by pressure cooking in 0.01 M citrate buffer for 5 min. Slides were incubated with an established polyclonal PHD2 anti-rabbit antibody (NB100-137; Novus Biologicals, Littleton, Colo., USA) [19,20] diluted 1: 4,000 in background reducing dilution buffer (Zymed, San Francisco, Calif., USA) at room temperature for 1 h. Detection took place according to the manufacturer's instructions using a streptavidin-biotin system (BioGenex, San Ramon, Calif., USA) with alkaline phosphatase as the reporting enzyme. Either diaminobenzidine chromogen (Dako, Germany) or Fast-red (Sigma-Aldrich, Munich, Germany) served as chromogen.…”