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

Abstract

Cells can adapt to hypoxia by various mechanisms. Yet, hypoxia-induced effects on the cytoskeleton-based cell architecture and functions are largely unknown. Here we present a comprehensive analysis of the architecture and function of L929 fibroblasts under hypoxic conditions (1% O2). Cells cultivated in hypoxia showed striking morphological differences as compared to cells cultivated under normoxic conditions (20% O2). These changes include an enlargement of cell area and volume, increased numbers of focal contacts and loss of cell polarization. Furthermore the β- and γ-actin distribution is greatly altered. These hypoxic adjustments are associated with enhanced cell spreading and a decline of cell motility in wound closure and single cell motility assays. As the hypoxia-inducible factor-1α (HIF-1α) is stabilised in hypoxia and plays a pivotal role in the transcriptional response to changes in oxygen availability we used an shRNA-approach to examine the role of HIF-1α in cytoskeleton-related architecture and functions. We show that the observed increase in cell area, actin filament rearrangement, decrease of single cell migration in hypoxia and the maintenance of p-cofilin levels is dependent on HIF-1α stabilisation.

Oxygen-dependent regulation of aquaporin-3 expression

“…Generation and characterization of these stable shRNA-mediated knockdown clones have been described by Vogler et al21 Similar to the HIF-1 target genes Glut1 and PHD3, knockdown of HIF-1α in L929 cells prevented the hypoxia-stimulated induction of AQP3 in both HIF-1α-deficient clones. However, as seen before, in the wild-type cells, AQP3, Glut1, and PHD3 mRNA expression levels are significantly induced after hypoxic incubation ( P <0.001) (Figure 2).…”

“…Western blot experiments were performed as described by Vogler et al21 Briefly, cells were lysed in 400 mM NaCl, 1 mM EDTA, 10 mM Tris/HCl, pH 8.0, and 0.1% Triton X-100 including the protease inhibitor cocktail complete mini (Roche Applied Science, Mannheim, Germany). Protein concentrations were quantified with the Bradford method using bovine serum albumin as a standard.…”

Effects of an Acetic Acid and Acetone Mixture on the Characteristics and Scaffold–Cell Interaction of Electrospun Polycaprolactone Membranes

“…Although the larger fiber diameters in the PCL-AC membrane also increased the surface area for cell adhesion, this effect might have been restricted by the hydrophobicity of the fibers [23] and outweighed by the large mean pore size [24]. Moreover, compared to the size of L929 cell pellets shortly post-seeding (8-10 µm) [25], the PCL-AC pores were larger, while the PCL-7AA/3AC membrane acted as a barrier, retaining the cells on the surface. This indicates that the seeded cell pellets might infiltrate directly through the large pores of the PCL-AC sample [26] and helps explain the equal proliferative rates of the two membranes, which needs to be examined further in subsequent studies.…”

Autologous blood transfusion stimulates wound healing in diabetic mice through activation of the HIF‐1α pathway by improving the blood preservation solution

“…Skin fibroblasts play a crucial role in wound contraction, tissue remodeling, and extracellular matrix deposition of wound healing . A recent study has linked HIF‐1a stabilization to the increased cell area, actin filament rearrangement, decreased single cell migration in hypoxia and the maintenance of p‐cofilin levels . A previous report provided verification that both baric oxygen‐dependent HIF‐1 and hypoxia‐dependent HIF‐1 increase fibroblast proliferation which represents a pivotal cellular involved in the stimulation of wound healing …”