Biomedical subjects

Lorenz Poellinger

Publications and source records attributed to Lorenz Poellinger.

35 records · Page 2Linked to original sources

PubMed2004

Hyperglycemia regulates hypoxia-inducible factor-1alpha protein stability and function.

Hyperglycemia and hypoxia are suggested to play essential pathophysiological roles in the complications of diabetes, which may result from a defective response of the tissues to low oxygen tension. In this study, we show that in primary dermal fibroblasts and endothelial cells, hyperglycemia interferes with the function of hypoxia-inducible factor-1 (HIF-1), a transcription factor that is essential for adaptive responses of the cell to hypoxia. Experiments using proteasomal and prolyl hydroxylases inhibitors indicate that hyperglycemia inhibits hypoxia-induced stabilization of HIF-1alpha protein levels against degradation and suggest that mechanisms in addition to proline hydroxylation may be involved. This effect of hyperglycemia was dose dependent and correlates with a lower transcription activation potency of HIF-1alpha, as assessed by transient hypoxia-inducible reporter gene assay. Regulation of HIF-1alpha function by hyperglycemia could be mimicked by mannitol, suggesting hyperosmolarity as one critical parameter. The interference of hyperglycemia with hypoxia-dependent stabilization of HIF-1alpha protein levels was confirmed in vivo, where only very low levels of HIF-1alpha protein could be detected in diabetic wounds, as compared with chronic venous ulcers. In conclusion, our data demonstrate that hyperglycemia impairs hypoxia-dependent protection of HIF-1alpha against proteasomal degradation and suggest a mechanism by which diabetes interferes with cellular responses to hypoxia.

Cell Hypoxia↗

PubMed2003

Hypoxia-inducible factor regulates survival of antigen receptor-driven T cells.

Peripheral T lymphocytes undergo activation by antigenic stimulation and function in hypoxic areas of inflammation. We demonstrated in CD3-positive human T cells accumulating in inflammatory tissue expression of the hypoxia-inducible factor-1alpha (HIF-1alpha), indicating a role of hypoxia-mediated signals in regulation of T cell function. Surprisingly, accumulation of HIF-1alpha in human T cells required not only hypoxia but also TCR/CD3-mediated activation. Moreover, hypoxia repressed activation-induced cell death (AICD) by TCR/CD3 stimulation, resulting in an increased survival of the cells. Microarray analysis suggested the involvement of HIF-1 target gene product adrenomedullin (AM) in this process. Indeed, AM receptor antagonist abrogated hypoxia-mediated repression of AICD. Moreover, synthetic AM peptides repressed AICD even in normoxia. Taken together, we propose that hypoxia is a critical determinant of survival of the activated T cells via the HIF-1alpha-AM cascade, defining a previously unknown mode of regulation of peripheral immunity.

Adrenomedullin↗

PubMed2003

Hypoxia-inducible factor-1alpha polymorphisms associated with enhanced transactivation capacity, implying clinical significance.

Hypoxia-inducible factor-1 (HIF-1) is a pivotal factor that regulates cellular responses to hypoxia and is presumably linked to regulation of angiogenesis and tumor growth. We assessed the difference in transcription activity of two HIF-1alpha polymorphic variants (P582S and A588T), along with molecular epidemiological study among head and neck squamous cell carcinoma (HNSCC) patients. Both HIF-1alpha variants revealed significantly higher transcription activity than wild-type (WT) did, under normoxic and hypoxic conditions (P < 0.02). Furthermore, tumors from HNSCC patients with heterozygous alleles having P582S or A588T had significantly increased numbers of microvessels compared with those with homozygous WT (P = 0.02). In addition, all patients with tumors of T1 (below 2 cm diameter) were WT, while 14 of 47 patients with tumors of > or =T2 were heterozygous. The elevated transactivation capacity of variant forms of HIF-1alpha implies a role of HIF-1alpha polymorphisms in generating individually different tumor progression.

Amino Acid Sequence↗

PubMed2003

Hypoxia-induced dedifferentiation in neuroblastoma cells.

Hypoxia in solid tumors is associated with aggressive behavior and poor outcome. We recently discovered that hypoxia alters the expression of differentiation marker genes in neuroblastoma cells, in that the tumor cells adjust to the hypoxic environment by down-regulating genes associated with a neuronal and upregulating genes associated with a neural crest-like phenotype. As there is a correlation in neuroblastoma between low stage of differentiation and high (aggressive) clinical stage, we propose that dedifferentiation of neuroblastoma cells in hypoxic tumor regions contribute to the malignancy of the tumor.

Cell Differentiation↗

PubMed2003

Up-regulation of hypoxia-inducible factor 2alpha in renal cell carcinoma associated with loss of Tsc-2 tumor suppressor gene.

In the Eker rat model, inactivation of the Tuberous Sclerosis-2 (Tsc-2) tumor suppressor gene leads to high frequency of spontaneous renal cell carcinoma (RCC). By analogy to human RCC in which mutations in the von Hippel-Lindau (VHL) tumor suppressor gene result in accumulation of hypoxia-inducible factor alpha (HIFalpha) and up-regulation of vascular endothelial growth factor (VEGF), we investigated the regulation of HIF and its target gene VEGF in rat RCC resulting from Tsc-2 defects. To examine HIFalpha activity, a panel of rat renal epithelial cells were analyzed for expression of HIF1alpha and the homologous protein, HIF2alpha, under normoxic and hypoxic conditions. RCC-derived cell lines exhibited high basal levels of HIF activity as determined using hypoxia response element-luciferase reporter constructs. HIF2alpha was stabilized in RCC-derived cell lines and in five of six primary tumors compared with normal kidney, which was consistent with the high levels of hypoxia response element-reporter activity observed in the cell lines. Primary RCCs that developed in Eker rats were highly vascularized, which was similar to their human counterparts. Furthermore, reverse-transcriptase PCR and immunoblotting demonstrated that VEGF was abundantly expressed in both rat RCC cell lines and primary tumors. The 120-, 164-, and 188-amino-acid isoforms of VEGF were expressed at the RNA and protein levels in RCC-derived cell lines, although only a single band was observed in primary tumors. Taken together, these data suggest that RCC caused by loss of the Tsc-2 tumor suppressor gene (which retain wild-type Vhl) up-regulate VEGF via a HIF2alpha-mediated mechanism. Thus, loss of Tsc-2 and VHL tumor suppressor gene function appears to have similar consequences in Eker rats and humans respectively, identifying dysregulation of HIFalpha and VEGF expression as a common pathway for the development of RCC in different species and in tumors with different molecular etiologies.

Animals↗

PubMed2003

A constitutively active aryl hydrocarbon receptor causes loss of peritoneal B1 cells.

The dioxin/aryl hydrocarbon (Ah) receptor functions as a ligand-activated transcription factor that mediates toxicity of dioxins and related environmental pollutants. We have developed a transgenic mouse model that expresses a constitutively active Ah receptor. The immune system is one of the most sensitive target organs for dioxin toxicity and we have therefore investigated alterations of different lymphocyte populations in these mice. The population of mature bone-marrow derived B cells was enlarged, consistent with previous findings in dioxin exposed mice. In contrast, the peritoneal population of CD5-expressing B cells (B1 cells) was significantly diminished. This is the first study that demonstrates the effect of an activated Ah receptor on B1 cells. Since these cells are important mediators of innate immunity against pathogens such as Influenza virus, these results may explain the decreased resistance against infections that has been documented after dioxin exposure.

Animals↗

PubMed2002

Identification of residues critical for regulation of protein stability and the transactivation function of the hypoxia-inducible factor-1alpha by the von Hippel-Lindau tumor suppressor gene product.

Under normoxic conditions the hypoxia-inducible factor-1alpha (HIF-1alpha) protein is targeted for degradation by the von Hippel-Lindau (pVHL) tumor suppressor protein acting as an E3 ubiquitin ligase. Binding of pVHL to HIF-1alpha is dependent on hydroxylation of specific proline residues by O(2)-dependent prolyl 4-hydroxylases. Upon exposure to hypoxia the hydroxylase activity is inhibited, resulting in stabilization of HIF-1alpha protein levels and activation of transcription of target genes. One of the two critical proline residues, Pro(563) in mouse HIF-1alpha, is located within a bifunctional domain, the N-terminal transactivation domain (N-TAD), which mediates both pVHL-dependent degradation at normoxia and transcriptional activation at hypoxia. Here we have identified two N-TAD residues, Tyr(564) and Ile(565), which, in addition to Pro(563), were critical for pVHL-mediated degradation at normoxia. We have also identified D568A/D569A/D570A, F571A, and L573A as mutations of the N-TAD that abrogated binding to pVHL both in vitro and in vivo, and constitutively stabilized N-TAD against degradation. Moreover, the mutations Y564G, L556A/L558A, and F571A/L573A drastically reduced the transactivation function of either the isolated N-TAD or full-length HIF-1alpha in hypoxic cells. Interestingly, the P563A mutant exhibited a constitutively active and potent transactivation function that was enhanced by functional interaction with the transcriptional coactivator protein CREB-binding protein. In conclusion, we have identified by mutation analysis several residues that are critical for either one or both of the interdigitated and conditionally regulated degradation and transactivation functions of the N-TAD of HIF-1alpha.

Amino Acid Motifs↗

PubMed2002

Functional analysis of hypoxia-inducible factor-1 alpha-mediated transactivation. Identification of amino acid residues critical for transcriptional activation and/or interaction with CREB-binding protein.

The hypoxia-inducible factor-1 alpha (HIF-1 alpha) is a key regulator of adaptive responses to hypoxia. HIF-1 alpha has two independent transactivation domains (TADs). Whereas the N-terminal TAD (N-TAD) also constitutes a degradation box, the C-terminal TAD (C-TAD) functions in a strictly hypoxia-inducible fashion. Oxygen-dependent hydroxylation of an asparagine residue has recently been reported to regulate C-TAD function by disrupting the interaction with the CH1 domain of the p300/CBP coactivator at normoxia. Here we have performed alanine-scanning mutagenesis of a predicted alpha-helix within the C-TAD of mouse HIF-1 alpha to identify residues important for transactivation and interaction of the C-TAD with transcriptional coactivators. We observed that several hydrophobic residues, Ile(802), Leu(808), Leu(814), Leu(815), and Leu(818), were critical for transactivation and binding to the CH1 domain of CBP in hypoxic cells. Moreover, E812A/E813A and D819A mutations impaired hypoxia-dependent transactivation without disrupting binding to CH1. In the context of full-length HIF-1 alpha, mutation of the leucine residues conferred conformational changes to the protein and significantly reduced the transactivation function as well as functional interaction with the transcriptional coactivators CBP and SRC-1. These mutations also affected intranuclear redistribution of HIF-1 alpha in the presence of CBP, indicating that the integrity of the C-TAD is critical for intracellular localization of mouse HIF-1 alpha.

Amino Acid Motifs↗

PubMed2002

Inhibitory PAS domain protein (IPAS) is a hypoxia-inducible splicing variant of the hypoxia-inducible factor-3alpha locus.

The inhibitory PAS (Per/Arnt/Sim) domain protein, IPAS, functions as a dominant negative regulator of hypoxia-inducible transcription factors (HIFs) by forming complexes with those proteins that fail to bind to hypoxia response elements of target genes. We have previously observed that IPAS is predominantly expressed in mice in Purkinje cells of the cerebellum and in corneal epithelium of the eye where it appears to play a role in negative regulation of angiogenesis and maintenance of an avascular phenotype. Sequencing of the mouse IPAS genomic structure revealed that IPAS is a splicing variant of the HIF-3alpha locus. Thus, in addition to three unique exons (1a, 4a, and 16) IPAS shares three exons (2, 4, and 5) with HIF-3alpha as well as alternatively spliced variants of exons 3 and 6. In experiments using normal mice and mice exposed to hypoxia (6% O(2)) for 6 h we observed alternative splicing of the HIF-3alpha transcript in the heart and lung. The alternatively spliced transcript was only observed under hypoxic conditions, thus defining a novel mechanism of hypoxia-dependent regulation of gene expression. Importantly, this mechanism may establish negative feedback loop regulation of adaptive responses to hypoxia/ischemia in these tissues.

Alternative Splicing↗

PubMed2002

A constitutively active dioxin/aryl hydrocarbon receptor induces stomach tumors.

The dioxin/aryl hydrocarbon receptor (AhR) functions as a ligand-activated transcription factor regulating transcription of a battery of genes encoding xenobiotic metabolizing enzymes. Known receptor ligands are environmental pollutants including polycyclic aromatic hydrocarbons and polychlorinated dioxins. Loss-of-function (gene-disruption) studies in mice have demonstrated that the AhR is involved in toxic effects of dioxins but have not yielded unequivocal results concerning the physiological function of the receptor. Gain-of-function studies therefore were performed to unravel the biological functions of the AhR. A constitutively active AhR expressed in transgenic mice reduced the life span of the mice and induced tumors in the glandular part of the stomach, demonstrating the oncogenic potential of the AhR and implicating the receptor in regulation of cell proliferation.

Animals↗

PubMed2002

Conditional expression of a constitutively active aryl hydrocarbon receptor in MCF-7 human breast cancer cells.

In addition to inducing transcription of a battery of target genes encoding drug-metabolizing enzymes, the environmental pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is known to induce antiestrogenic responses. However, the mechanisms underlying such complex biologic responses affecting growth and differentiation remain unclear. In the present study we have investigated biological effects of a constitutively active mutant of the aryl hydrocarbon (Ah) receptor (CA-AhR), in particular whether it modulates estrogen receptor function in human MCF-7 breast cancer cells. To this end, the CA-AhR protein was conditionally expressed using the tet repressor. Expression of CA-AhR resulted in constitutive formation of a DNA-binding AhR-aryl hydrocarbon receptor nuclear translocator heterodimeric complex and enhanced expression of the Ah receptor target gene CYP1A1 in the absence of TCDD. Moreover, expression of CA-AhR inhibited estrogen-dependent cathepsin D expression and growth of these cells. Thus, the present model system conditionally expressing the CA-AhR protein provides a novel tool for the investigation of AhR-mediated signaling pathways.

Breast Neoplasms↗

PubMed2002

Hypoxia alters gene expression in human neuroblastoma cells toward an immature and neural crest-like phenotype.

Insufficient oxygen and nutrient supply often restrain solid tumor growth, and the hypoxia-inducible factors (HIF) 1 alpha and HIF-2 alpha are key transcription regulators of phenotypic adaptation to low oxygen levels. Moreover, mouse gene disruption studies have implicated HIF-2 alpha in embryonic regulation of tyrosine hydroxylase, a hallmark gene of the sympathetic nervous system. Neuroblastoma tumors originate from immature sympathetic cells, and therefore we investigated the effect of hypoxia on the differentiation status of human neuroblastoma cells. Hypoxia stabilized HIF-1 alpha and HIF-2 alpha proteins and activated the expression of known hypoxia-induced genes, such as vascular endothelial growth factor and tyrosine hydroxylase. These changes in gene expression also occurred in hypoxic regions of experimental neuroblastoma xenografts grown in mice. In contrast, hypoxia decreased the expression of several neuronal/neuroendocrine marker genes but induced genes expressed in neural crest sympathetic progenitors, for instance c-kit and Notch-1. Thus, hypoxia apparently causes dedifferentiation both in vitro and in vivo. These findings suggest a novel mechanism for selection of highly malignant tumor cells with stem-cell characteristics.

Animals↗

PubMed2002

The dietary flavonoid quercetin modulates HIF-1 alpha activity in endothelial cells.

The mechanism of activation of the transcription factor hypoxia-inducible factor-1 (HIF-1) has been studied extensively. Under normal cellular oxygen conditions protein levels of the alpha subunit (HIF-1 alpha) are kept low due to massive ubiquitination and subsequent proteosomal degradation. However, during hypoxia ubiquitination is inhibited, causing stabilisation of the HIF-1 alpha protein. HIF-1 alpha can then translocate to the nucleus and facilitate transcription of numerous target genes, the majority of which are involved in glycolysis and angiogenesis via heterodimerisation with the beta subunit (HIF-1 beta/ARNT). Until now hypoxia has been the only naturally occurring signal shown to activate this transcription factor. We report here that the dietary flavonoid quercetin also activates HIF-1 alpha in all steps of its activation pathway, in a manner similar to hypoxia. We found that quercetin, an inhibitor of Ser/Thr kinases, stabilises HIF-1 alpha and causes nuclear localisation of the protein in a transcriptionally active state. Taken together these results strongly indicate that the dietary flavonoid quercetin regulates HIF-1 function at normal oxygen concentrations.

Animals↗

PubMed2002

Identification of a tightly regulated hypoxia-response element in the promoter of human plasminogen activator inhibitor-1.

Plasminogen activator inhibitor-1 (PAI-1) plays a key role in control of coagulation and tissue remodeling and has been shown to be regulated by a number of cell stimuli, among those hypoxia. In this study we characterize the hypoxia-mediated induction of PAI-1 in human hepatoma cell line HepG2. We found that PAI-1 is tightly regulated in a narrow oxygen gradient. After incubation at oxygen concentrations of 1% to 2%, a 60-fold increase in PAI-1 messenger RNA levels was observed, whereas mild hypoxic conditions of more than 3.5% did not appear to induce transcription. Moreover, increased levels of PAI-1 protein were observed after incubation at low oxygen tensions. Through sequence analysis, several putative hypoxia-response elements (HREs 1-5) were identified in the human PAI-I promoter. Reporter gene assays showed that the HRE-2 (-194 to -187) was necessary and sufficient for the hypoxia-mediated response. By electrophoretic mobility assay we observed hypoxia-dependent binding of a protein complex to the HRE-2 motif. Further analysis demonstrated that HRE-2 was specifically recognized by the hypoxia-inducible transcription factor 1alpha-arylhydrocarbon nuclear translocator complex. Taken together, our data demonstrate that hypoxia-induced transcription is mediated through HIF-1 interaction with the HRE-2 site of the human PAI-1 promoter.

Aryl Hydrocarbon Receptor Nuclear Translocator↗

PubMed2002

Two distinct regions of the immunophilin-like protein XAP2 regulate dioxin receptor function and interaction with hsp90.

The dioxin (aryl hydrocarbon) receptor is a ligand inducible transcription factor, which mediates cellular responses to a variety of xenobiotic compounds such as dioxins. In the absence of ligand the receptor is associated with the molecular chaperone hsp90 and the tetratricopeptide repeat (TPR-) containing immunophilin-like protein XAP2. XAP2 has been implicated in regulation of the intracellular localization of the dioxin receptor and protection of the receptor against degradation. In this study a series of XAP2 mutants has been generated in order to identify the structural motif(s) mediating interaction with the dioxin receptor-hsp90 complex and modulation of receptor function. Immunoprecipitation experiments demonstrated that the C-terminal part of XAP2, including the TPR motifs and the region outside the TPR motifs, was required to directly contact hsp90. The N-terminal part of XAP2 was required for the stability of the ternary dioxin receptor-hsp90-XAP2 complex. In addition, the integrity of the N-terminal region of XAP2 was essential for XAP2 to regulate the intracellular localization of the dioxin receptor. In conclusion, these data demonstrate that two distinct regions of XAP2 modulate dioxin receptor function and interaction with hps90, illustrating the complexity in regulation of dioxin receptor signaling by the hsp90 molecular chaperone machinery.

Amino Acid Motifs↗

PubMedDate not supplied

Hypoxia-dependent activation of HIF into a transcriptional regulator.

Adaptation to conditions of limited oxygen availability (hypoxia) is a critical determinant of cell and tissue viability in several physiological and pathophysiological conditions. The hypoxia-inducible factor (HIF) is an oxygen-sensitive transcriptional activator that, under hypoxia, upregulates the expression of genes involved in the control of glucose metabolism, angiogenesis and cellular proliferation, among others. Activation of HIF to a fully competent transcriptional regulatory protein complex is a multi-step process that involves control of protein stability, subcellular localization, DNA-binding and interaction with transcriptional coregulators. The identity, regulation and hierarchy of interactions between several components of the HIF signal transduction pathway has been the object of intense study over the past decade and will be the subject of this review. Particular emphasis is given to the process of coordinated coactivator recruitment within the cell nucleus. The implications for future development of angiogenic/antiangiogenic therapeutic strategies of HIF activation/inactivation are discussed.

Animals↗

PubMedDate not supplied

Gastric hamartomatous tumours in a transgenic mouse model expressing an activated dioxin/Ah receptor.

BACKGROUND: The dioxin/Aryl hydrocarbon (Ah) receptor is a ligand-activated transcription factor known to mediate the toxic effects of the environmental pollutants dioxins. MATERIALS AND METHODS: Transgenic mice expressing a constitutively active Ah receptor were found to develop gastric tumours. RESULTS: Segments of the normal gastric glands of the corpus region (lined by foveolar, parietal and cardio-pyloral cells) developed cystic tumours. Tumours in the submucosa and muscularis propria consisted of large cysts surrounded by organized lymphocytic, connective, squamous and adipose tissues and vessels. Those tumours were obviously hamartomatous in nature. Despite the fact that the tumours had an inward growth and penetrated all stomach layers, no metastases developed. CONCLUSION: The model here described may open new vistas into the investigation of the role of environmental pollutants in the development of gastric hamartomas.

Animals↗