Thomas Hellwig-Bürgel

Intracellular localisation of human HIF-1 alpha hydroxylases: implications for oxygen sensing

Hypoxia-inducible factor1 (HIF-1) is an essential transcription factor for cellular adaptation to decreased oxygen availability. In normoxia the oxygen-sensitive α-subunit of HIF-1 is hydroxylated on Pro564 and Pro402 and thus targeted for proteasomal degradation. Three human oxygen-dependent HIF-1α prolyl hydroxylases (PHD1, PHD2, and PHD3) function as oxygen sensors in vivo. Furthermore, the asparagine hydroxylase FIH-1 (factor inhibiting HIF) has been found to hydroxylate Asp803 of the HIF-1 C-terminal transactivation domain, which results in the decreased ability of HIF-1 to bind to the transcriptional coactivator p300/CBP. We have fused these enzymes to the N-terminus of fluorescent proteins and transiently transfected the fusion proteins into human osteosarcoma cells (U2OS). Three-dimensional 2-photon confocal fluorescence microscopy showed that PHD1 was exclusively present in the nucleus, PHD2 and FIH-1 were mainly located in the cytoplasm and PHD3 was homogeneously distributed in cytoplasm and nucleus. Hypoxia did not influence the localisation of any enzyme under investigation. In contrast to FIH-1, each PHD inhibited nuclear HIF-1α accumulation in hypoxia. All hydroxylases suppressed activation of a cotransfected hypoxia-responsive luciferase reporter gene. Endogenous PHD2mRNA and PHD3mRNA were hypoxia-inducible, whereas expression of PHD1mRNA and FIH-1mRNA was oxygen independent. We propose that PHDs and FIH-1 form an oxygen sensor cascade of distinct subcellular localisation.

Normoxic induction of the hypoxia-inducible factor 1α by insulin and interleukin-1β involves the phosphatidylinositol 3-kinase pathway

Hypoxia‐inducible factor 1 (HIF‐1) is a heterodimeric DNA‐binding complex of the subunits α and β with relevance in O2 and energy homeostasis. The labile component, HIF‐1α, is not only activated by hypoxia but also by peptides such as insulin and interleukin‐1 (IL‐1) in normoxia. We investigated whether inhibitors of mitogen‐activated protein kinase kinases (MAPKKs: PD 98059, U0126) and phosphatidylinositol 3‐kinase (PI3K: LY 294002) do not only lower the hypoxia‐induced, but also the insulin‐ and IL‐1‐induced HIF‐1α accumulation and HIF‐1 DNA‐binding in human hepatoma cell cultures (line HepG2). The results show that LY 294002 suppressed HIF‐1 activation in a dose‐dependent manner irrespective of the stimulus. With respect to target proteins controlled by HIF‐1, the production of erythropoietin was fully blocked and that of vascular endothelial growth factor reduced following inhibition of the PI3K pathway. The role of MAPKKs in this process remained in question, because PD 98059 and U0126 did not significantly reduce HIF‐1α levels at non‐toxic doses. We propose that PI3K signaling is not only important in the hypoxic induction of HIF‐1 but it is also crucially involved in the response to insulin and IL‐1.

Biology of erythropoietin

The glycoprotein hormone erythropoietin is an essential survival and growth factor for the erythrocytic progenitors in the bone marrow. Its peptide sequence of 165 amino acids is stabilized by 4 carbohydrate side chains (total molecular mass 30 kDa, 60% protein). Erythropoietin is synthesized mainly in the kidney. Its formation increases greatly in response to hypoxia. The mechanism by which lack of oxygen induces erythropoietin gene expression is only partly understood. Erythropoietin deficiency is the primary cause of the anemia in chronic renal failure. Erythropoietin production is lowered in severe acidosis and in inflammatory states. Genetical engineering has enabled the isolation of the human erythropoietin gene and its in vitro expression in mammalian cell cultures. Recombinant human erythropoietin has been approved to be an efficient and safe drug for treatment of renal anemia. This paper reviews some of the recent advances in the understanding of the biology of erythropoietin with respect to its possible application as an alternative to blood transfusion in surgical patients.

Chlamydia pneumoniae directly interferes with HIF‐1α stabilization in human host cells

Chlamydiaceae are obligate intracellular bacteria that cause endemic trachoma, sexually transmitted diseases and respiratory infections. The course of the diseases is determined by local inflammatory immune responses and the propensity of the pathogen to replicate within infected host cells. Both features require energy which is inseparably coupled to oxygen availability in the microenvironment. Hypoxia‐inducible factor‐1 (HIF‐1) regulates crucial genes involved in the adaptation to low oxygen concentrations, cell metabolism and the innate immune response. Here we report that Chlamydia pneumoniae directly interferes with host cell HIF‐1α regulation in a biphasic manner. In hypoxia, C. pneumoniae infection had an additive effect on HIF‐1α stabilization resulting in enhanced glucose uptake during the early phase of infection. During the late phase of intracellular chlamydial replication, host cell adaptation to hypoxia was actively silenced by pathogen‐induced HIF‐1α degradation. HIF‐1α was targeted by the chlamydial protease‐like activity factor, which was secreted into the cytoplasm of infected cells. Direct interference with HIF‐1α stabilization was essential for efficient C. pneumoniae replication in hypoxia and highlights a novel strategy of adaptive pathogen–host interaction in chlamydial diseases.

Hypoxia abrogates antichlamydial properties of IFN-γ in human fallopian tube cells in vitro and ex vivo

IFN-γ has an important role in the adaptive immune response against intracellular pathogens. In urogenital tract (UGT) infections with the obligate intracellular pathogen Chlamydia trachomatis, IFN-γ–mediated control of chlamydial growth implies the JAK-STAT signaling cascades and subsequent induction of the indoleamine 2,3-dioxygenase (IDO). As oxygen concentrations in the UGT are low under physiological conditions (O2 < 5%) and further decrease during an inflammatory process, we wondered whether antibacterial properties of IFN-γ are maintained under hypoxic conditions. Using primary cells that were isolated from human fallopian tubes and an ex vivo human fallopian tube model (HFTM), we found that even high IFN-γ concentrations (200 units/mL) were not sufficient to limit growth of C. trachomatis under hypoxia. Reduced antibacterial activity of IFN-γ under hypoxia was restricted to the urogenital serovars D and L2, but was not observed with the ocular serovar A. Impaired effectiveness of IFN-γ on chlamydial growth under hypoxia was accompanied by reduced phosphorylation of Stat-1 on Tyr701 and diminished IDO activity. This study shows that IFN-γ effector functions on intracellular C. trachomatis depend on the environmental oxygen supply, which could explain inadequate bacterial clearance and subsequent chronic infections eventually occurring in the UGT of women.

Dexamethasone impairs hypoxia-inducible factor-1 function

Hypoxia-inducible factor-1 (HIF-1) is a heterodimeric transcription-factor composed of alpha- and beta-subunits. HIF-1 is not only necessary for the cellular adaptation to hypoxia, but it is also involved in inflammatory processes and wound healing. Glucocorticoids (GC) are therapeutically used to suppress inflammatory responses. Herein, we investigated whether GC modulate HIF-1 function using GC receptor (GR) possessing (HepG2) and GR deficient (Hep3B) human hepatoma cell cultures as model systems. Dexamethasone (DEX) treatment increased HIF-1alpha levels in the cytosol of HepG2 cells, while nuclear HIF-1alpha levels and HIF-1 DNA-binding was reduced. In addition, DEX dose-dependently lowered the hypoxia-induced luciferase activity in a reporter gene system. DEX suppressed the hypoxic stimulation of the expression of the HIF-1 target gene VEGF (vascular endothelial growth factor) in HepG2 cultures. DEX did not reduce hypoxically induced luciferase activity in HRB5 cells, a Hep3B derivative lacking GR. Transient expression of the GR in HRB5 cells restored the susceptibility to DEX. Our study discloses the inhibitory action of GC on HIF-1 dependent gene expression, which may be important with respect to the impaired wound healing in DEX-treated patients.

Thyrotropin-Releasing Hormone Controls Mitochondrial Biology in Human Epidermis

CONTEXT Mitochondrial capacity and metabolic potential are under the control of hormones, such as thyroid hormones. The most proximal regulator of the hypothalamic-pituitary-thyroid (HPT) axis, TRH, is the key hypothalamic integrator of energy metabolism via its impact on thyroid hormone secretion. OBJECTIVE Here, we asked whether TRH directly modulates mitochondrial functions in normal, TRH-receptor-positive human epidermis. METHODS Organ-cultured human skin was treated with TRH (5-100 ng/ml) for 12-48 h. RESULTS TRH significantly increased epidermal immunoreactivity for the mitochondria-selective subunit I of respiratory chain complex IV (MTCO1). This resulted from an increased MTCO1 transcription and protein synthesis and a stimulation of mitochondrial biogenesis as demonstrated by transmission electron microscopy and TRH-enhanced mitochondrial DNA synthesis. TRH also significantly stimulated the transcription of several other mitochondrial key genes (TFAM, HSP60, and BMAL1), including the master regulator of mitochondrial biogenesis (PGC-1α). TRH significantly enhanced mitochondrial complex I and IV enzyme activity and enhanced the oxygen consumption of human skin samples, which shows that the stimulated mitochondria are fully vital because the main source for cellular oxygen consumption is mitochondrial endoxidation. CONCLUSIONS These findings identify TRH as a potent, novel neuroendocrine stimulator of mitochondrial activity and biogenesis in human epidermal keratinocytes in situ. Thus, human epidermis offers an excellent model for dissecting neuroendocrine controls of human mitochondrial biology under physiologically relevant conditions and for exploring corresponding clinical applications.

Vascular endothelial growth factor gene expression in the human breast cancer cell line MX-1 is controlled by O2 availability in vitro and in vivo

The vascular endothelial growth factor (VEGF) plays an important role in angiogenesis. Mediated by the hypoxia-inducible transcription factor HIF-1alpha/beta, a reduction in O2 tension (pO2) leads to increased VEGF gene expression in nonmalignant tissues. In tumor cells VEGF mRNA levels are often constitutively elevated. We examined pO2-dependent VEGF mRNA expression and VEGF protein formation in the human breast cancer cell line MX-1 in vitro and in vivo. For in vitro study MX-1 cultures were grown on dishes with a gas-permeable bottom to expose the cells to defined O2 concentrations (from 95% to 0%) for 4 h. Northern blot analysis showed significant VEGF mRNA in MX-1 cultures under normoxic conditions which was further increased by hypoxia. The amount of secreted VEGF was also elevated in hypoxic cultures. Western blot analysis revealed a correlation between the severity of hypoxia and HIF-1alpha protein amounts in the nucleus. Furthermore, DNA-binding activity of HIF-1 could be demonstrated by gel-shift assays. For in vivo study immunodeficient nude mice bearing MX-1 tumor transplants were exposed to inspiratory hypoxia (10% O2). Northern blot and immunohistochemical analyses of MX-1 tumor transplants showed that VEGF mRNA and VEGF protein levels were increased in mice 17 h after the induction of inspiratory hypoxia. Thus, pO2-dependence of VEGF gene expression can be maintained in cancer cells, even in vivo, which may be relevant in regard to therapeutic attempts to inhibit tumor angiogenesis by increasing tumor oxygenation.

Timing and Targeting of Cell-Based VEGF165 Gene Expression in Ischemic Tissue

BACKGROUND Therapeutic angiogenesis has become a key technology in experimental and clinical medicine. Only few data are available on the effects of timing and targeting of therapeutic proteins after cell-based gene transfer. This work investigates such effects after temporary expression of vascular endothelial growth factor 165 (VEGF(165)), the most commonly used angiogenic protein for therapeutic purposes. METHODS We established a cell-based gene-transfer model using fibroblasts to temporarily produce VEGF(165). Cells were implanted into 40 rats. Protein expression and angiogenic effects were measured by PCR, immunohistology, and microangiography. To determine an improvement for survival of ischemically challenged tissue, cells were implanted in an ischemic flap model at different locations and time points. RESULTS After implantation of modified cells, a temporary increase was found in the target tissue for VEGF(165), endothelial cell counts, and capillary network formations. Four wk later, histological alterations in the target tissue area were not different from controls. Implantation of modified cells into flap plus wound margin 1 wk before surgery showed significant improvement of tissue survival demonstrated by planimetric measurements and blood vessels counting in the target tissue. CONCLUSION In our model, temporary expression of VEGF(165) induces therapeutically relevant angiogenesis and improves blood supply only if applied 1 wk before ischemia. It is essential to include the surrounding area for induction of angiogenesis in this model. In contrast, the angiogenic effects are not effective in the target area and its surrounding tissue, if therapeutic gene expression is started during onset of ischemia or 2 wk before ischemia in this model.

VEGF Production by Primary Human Renal Proximal Tubular Cells: Requirement of HIF-1, PI3-Kinase and MAPKK-1 Signaling

Renal proximal tubular epithelial cells (PTEC) respond to hypoxia exposure or interleukin-1β (IL-1β) treatment with increased vascular endothelial growth factor (VEGF) production. With respect to O2 deprivation, the hypoxia-inducible factor 1α/ β (HIF-1) is the most important transcription factor driving VEGF mRNA expression. HIF-1 is also activated by IL-1β and may thus be involved in the stimulation of VEGF production by this cytokine. However, the molecular mechanisms of HIF-1 dependent VEGF synthesis are poorly understood. Herein, human PTEC in primary culture were challenged by hypoxic incubation and/or IL-1β treatment in absence or presence of specific phosphatidylinositol 3-kinase (PI3K) or mitogen activated protein kinase kinase-1 (MAPKK-1) inhibitors for assay of VEGF protein, VEGF mRNA and detection of HIF-1α by Western Blotting, EMSA and fluorescence microscopy. In addition, the activities of PI3K and MAPKK-1 were studied following hypoxia and IL-1β treatment of the cultures. The study shows that PI3K but not MAPKK-1 inhibition resulted in the loss of hypoxic and IL-1β induced HIF-1α accumulation, whereas VEGF synthesis was reduced by either intervention. Thus, PI3K signaling is required for HIF-1α accumulation and VEGF synthesis, whereas MAPKK-1 signaling is required for VEGF synthesis only. Furthermore, hypoxia alone was sufficient to activate PI3K in PTEC in contrast to MAPKK-1, whose activity was lowered in hypoxia.