Changes induced with transgenic cardiac HIF-1α possibly mediate beneficial effects in the short term; however, with increased mechanical load and ageing they become detrimental for cardiac function. Together with the finding of increased HIF-1α protein levels in samples from human patients with cardiomyopathy, these data indicate that chronic HIF-1α stabilization drives autonomous pathways that add to disease progression.
Prolylhydroxylase domain proteins (PHD) are cellular oxygen-sensing molecules that regulate the stability of the ␣-subunit of the transcription factor hypoxia inducible factor (HIF)-1. HIF-1 affects cardiac development as well as adaptation of the heart toward increased pressure overload or myocardial infarction. We have disrupted PHD2 in cardiomyocytes (cPhd ؊/؊ ) When oxygen availability is impaired, the resulting hypoxia activates homeostatic mechanisms at the systemic and cellular level (1). Hypoxia-inducible factors (HIFs) 2 are essential players in these responses because they regulate the transcription of a large number of genes that affect a myriad of cellular processes, including angiogenesis, metabolism, cell survival, and oxygen delivery (2). HIF is a heterodimeric protein comprising the oxygen-sensitive ␣-subunit HIF-1␣ or the more cell type-specifically expressed HIF-2␣ or HIF-3␣ and the oxygen-insensitive -subunit (3). In the presence of oxygen, HIF␣ becomes hydroxylated at two critical proline residues by prolylhydroxylase domain (PHD) enzymes (4, 5). The PHD protein family responsible for HIF␣ regulation consists of three members called prolylhydroxylase domain (PHD)1, PHD2, and PHD3 (6, 7). Following prolyl-4-hydroxylation of the critical prolyl residues under normoxic conditions, the ubiquitin ligase von Hippel-Lindau tumor suppressor protein recognizes ⌯⌱F-1␣ subunits and targets them for rapid ubiquitination and proteasomal degradation (8 -10).Based on the ubiquitous expression pattern and its dominant effect in normoxia, it had to be assumed that PHD2 is the most critical HIF-1␣-regulating PHD isoform in most tissues (11)(12)(13). This notion, learned from in vitro studies, was confirmed by the up to now available genetically modified Phd2 mouse models (14). Phd2 knock-out embryos die between embryonic day (E) 12.5 and E14.5 (15). This time point coincides with the increased levels of PHD2 in wild-type (wt) mice starting from E9.0. A major role of PHD2 in regulating the HIF system is further underscored by mouse models with a somatic Phd2 Ϫ/Ϫ knock out, which enable to analyze the in vivo function of PHD2 in the adult mice. Two independent inducible Phd2 Ϫ/Ϫ mouse models were developed by Takeda et al. (16) and Minamishima et al. (17). The phenotype of these mice most obviously resembles the consequences of HIF␣ overexpression with increased angiogenesis, erythropoiesis, and extramedullar hematopoiesis (17,18). Most interestingly, these mice also develop a cardiac phenotype with symptoms of dilated cardiomyopathy. In the heart, HIF-1␣ and thereby also the PHDs are known to influence key components of heart development, morphogenesis, and function (19,20). Long term activation of HIF-1␣ in the heart seems to activate detrimental pathways resulting in the development of heart failure (21). Thus, it is tempting to speculate that loss of PHD2 in the heart is responsible for the dilated cardiomyopathy as observed in the inducible Phd2 Ϫ/Ϫ mice. However, because these mice also develop an inc...
Lysosomal acid phosphatase (LAP) is rapidly internalized from the cell surface due to a tyrosine‐containing internalization signal in its 19 amino acid cytoplasmic tail. Measuring the internalization of a series of LAP cytoplasmic tail truncation and substitution mutants revealed that the N‐terminal 12 amino acids of the cytoplasmic tail are sufficient for rapid endocytosis and that the hexapeptide 411‐PGYRHV‐416 is the tyrosine‐containing internalization signal. Truncation and substitution mutants of amino acid residues following Val416 can prevent internalization even though these residues do not belong to the internalization signal. It was shown recently that part of the LAP cytoplasmic tail peptide corresponding to 410‐PPGY‐413 forms a well‐ordered beta turn structure in solution. Two‐dimensional NMR spectroscopy of two modified LAP tail peptides, in which the single tyrosine was substituted either by phenylalanine or by alanine, revealed that the tendency to form a beta turn is reduced by 25% in the phenylalanine‐containing peptide and by approximately 50% in the alanine‐containing mutant peptide. Our results suggest, that in the short cytoplasmic tail of LAP tyrosine is required for stabilization of the right turn and that the aromatic ring system of the tyrosine residue is a contact point to the putative cytoplasmic receptor.
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 transdifferentiation into myofibroblasts. Myofibroblasts are responsible for generation of the contraction forces that are important for wound healing and scar formation. Overactive myofibroblasts, by contrast, 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 (ACTA2) expression, alterations in cell contractility, actin reorganization and RhoA activity. We identified a hypoxia-inducible induction of ARHGAP29, which is critically involved in myocardin-related transcription factor-A (MRTF-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 ischemiainduced tissue remodeling and the fibrotic response. This article has an associated First Person interview with the first author of the paper.
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