Recent progress in understanding the influence of hypoxia on cell function has revealed new information about the interrelationship between the actin cytoskeleton and hypoxia; nevertheless, details remain cloudy. The dynamic regulation of the actin cytoskeleton during hypoxia is complex, varies in different cells and tissues, and also depends on the mode of hypoxia. Several molecular players and pathways are emerging that contribute to the modulation of the actin cytoskeleton and that affect the large repertoire of actin-binding proteins in hypoxia. This review describes and discusses the accumulated knowledge about actin cytoskeleton dynamics in hypoxia, placing special emphasis on the Rho family of small guanosine triphosphatases (Rho GTPases). Given that RhoA, Rac and Cdc42 are very well characterized, the review is focused on these family members of Rho GTPases. Notably, in several cell types and tissues, hypoxia, presumably via Rho GTPase signaling, induces actin rearrangement and actin stress fiber assembly, which is a prevalent modulation of the actin cytoskeleton in hypoxia.

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.

Fibroblasts show a high range of phenotypic plasticity including the transdifferentiating into myofibroblasts. Myofibroblasts are responsible for the generation of the contraction forces that are important for wound healing and scar formation. Overactive myofibroblasts on the other hand are involved in abnormal scarring. Cell stretching and extracellular signals such as transforming growth factor β can induce the myofibroblastic program whereas microenvironmental conditions such as reduced tissue oxygenation have an inhibitory effect. We investigated the effects of hypoxia on myofibroblastic properties and linked this to RhoA activity. Hypoxia reversed the myofibroblastic phenotype of primary fibroblasts. This was accompanied by decreased αSMA expression, alterations in cell contractility, actin reorganization, and RhoA activity. We identified a hypoxia-inducible induction of ArhGAP29, which is critically involved in MRTF-A (myocardin-related transcription factor-A) signaling, the differentiation state of myofibroblasts and modulates RhoA activity. This novel link between hypoxia and MRTF-A signaling is likely to be important for ischemia-induced tissue remodeling and the fibrotic response.

Aims: The prolyl-4-hydroxylase domain (PHD) enzymes are representing novel therapeutic targets for ischemic tissue protection. Whereas the consequences of a knock out of the PHDs have been analyzed in the context of cardioprotection, the implications of PHD overexpression is unknown so far. Methods and Results: We generated cardiomyocyte-specific PHD3transgenic mice (cPhd3tg). Resting cPhd3tg mice did not show constitutive accumulation of HIF-1α or HIF-2α or changes in HIF target gene expression in the heart. Cardiac function was followed up for 14 months in these mice and found to be unchanged. After challenging the cPhd3tg mice with ligation of the left anterior descending artery, HIF-1α/-2α accumulation in the left ventricles was blunted. This was associated with a significantly increased infarct size of the cPhd3tg compared to wild type mice. Conclusion: Whereas overexpression of PHD3 in the resting state does not significantly influence cardiac function, it is crucial for the cardiac response to ischemia by affecting HIFα accumulation in the ischemic tissue.

The purpose of this study was to investigate whether aquaporin-3 (AQP3) expression is altered in hypoxia and whether hypoxia-inducible transcription factor (HIF)-1 regulates the hypoxic expression. AQP3 mRNA expression was studied in L929 fibrosarcoma cells and in several tissues derived from mice that were subjected to hypoxia. Computational analysis of the AQP3 promoter revealed conserved HIF binding sites within close proximity to the translational start site, and chromatin immunoprecipitation assays confirmed binding of HIF-1 to the endogenous hypoxia response elements. Furthermore, hypoxia resulted in increased expression of AQP3 mRNA in L929 fibrosarcoma cells. Consistently, shRNA-mediated knockdown of HIF-1 greatly reduced the hypoxic induction of AQP3. In addition, mRNA analysis of organs from mice exposed to inspiratory hypoxia demonstrated pronounced hypoxia-inducible expression of AQP3 in the kidney. Overall, our findings suggest that AQP3 expression can be regulated at the transcriptional level and that AQP3 represents a novel HIF-1 target gene.