68 research outputs found
The GTPase RAB20 is a HIF target with mitochondrial localization mediating apoptosis in hypoxia
AbstractHypoxia is a common pathogenic stress, which requires adaptive activation of the Hypoxia-inducible transcription factor (HIF). In concert transcriptional HIF targets enhance oxygen availability and simultaneously reduce oxygen demand, enabling survival in a hypoxic microenvironment. Here, we describe the characterization of a new HIF-1 target gene, Rab20, which is a member of the Rab family of small GTP-binding proteins, regulating intracellular trafficking and vesicle formation. Rab20 is directly regulated by HIF-1, resulting in rapid upregulation of Rab20 mRNA as well as protein under hypoxia. Furthermore, exogenous as well as endogenous Rab20 protein colocalizes with mitochondria. Knockdown studies reveal that Rab20 is involved in hypoxia induced apoptosis. Since mitochondria play a key role in the control of cell death, we suggest that regulating mitochondrial homeostasis in hypoxia is a key function of Rab20. Furthermore, our study implicates that cellular transport pathways play a role in oxygen homeostasis. Hypoxia-induced Rab20 may influence tissue homeostasis and repair during and after hypoxic stress
Renal tubular HIF-2α expression requires VHL inactivation and causes fibrosis and cysts.
The Hypoxia-inducible transcription Factor (HIF) represents an important adaptive mechanism under hypoxia, whereas sustained activation may also have deleterious effects. HIF activity is determined by the oxygen regulated α-subunits HIF-1α or HIF-2α. Both are regulated by oxygen dependent degradation, which is controlled by the tumor suppressor "von Hippel-Lindau" (VHL), the gatekeeper of renal tubular growth control. HIF appears to play a particular role for the kidney, where renal EPO production, organ preservation from ischemia-reperfusion injury and renal tumorigenesis are prominent examples. Whereas HIF-1α is inducible in physiological renal mouse, rat and human tubular epithelia, HIF-2α is never detected in these cells, in any species. In contrast, distinct early lesions of biallelic VHL inactivation in kidneys of the hereditary VHL syndrome show strong HIF-2α expression. Furthermore, knockout of VHL in the mouse tubular apparatus enables HIF-2α expression. Continuous transgenic expression of HIF-2α by the Ksp-Cadherin promotor leads to renal fibrosis and insufficiency, next to multiple renal cysts. In conclusion, VHL appears to specifically repress HIF-2α in renal epithelia. Unphysiological expression of HIF-2α in tubular epithelia has deleterious effects. Our data are compatible with dedifferentiation of renal epithelial cells by sustained HIF-2α expression. However, HIF-2α overexpression alone is insufficient to induce tumors. Thus, our data bear implications for renal tumorigenesis, epithelial differentiation and renal repair mechanisms
In vivo Identification and Specificity assessment of mRNA markers of hypoxia in human and mouse tumors
<p>Abstract</p> <p>Background</p> <p>Tumor hypoxia is linked to poor prognosis, but identification and quantification of tissue hypoxia remains a challenge. The hypoxia-specificity of HIF-1α target genes in vivo has been questioned due to the confounding influence of other microenvironmental abnormalities known to affect gene expression (e.g., low pH). Here we describe a new technique that by exploiting intratumoral oxygenation heterogeneity allows us to identify and objectively rank the most robust mRNA hypoxia biomarkers.</p> <p>Methods</p> <p>Mice carrying human (FaDu<sub>dd</sub>) or murine (SCCVII) tumors were injected with the PET hypoxia tracer FAZA. Four hours post-injection tumors were removed, frozen, and crushed into milligram-sized fragments, which were transferred individually to pre-weighed tubes containing RNAlater and then weighed. For each fragment radioactivity per tissue mass and expression patterns of selected mRNA biomarkers were analyzed and compared.</p> <p>Results</p> <p>In both tumour models, fragmentation into pieces weighing 10 to 60 mg resulted in tissue fragments with highly variable relative content of hypoxic cells as evidenced by an up to 13-fold variation in FAZA radioactivity per mass of tissue. Linear regression analysis comparing FAZA retention with patterns of gene expression in individual tissue fragments revealed that CA9, GLUT1 and LOX mRNA levels were equally and strongly correlated to hypoxic extent in FaDu<sub>dd</sub>. The same link between hypoxia and gene expression profile was observed for CA9 and GLUT1, but not LOX, in SCCVII tumors. Apparent in vivo hypoxia-specificity for other putative molecular markers of tissue hypoxia was considerably weaker.</p> <p>Conclusions</p> <p>The portrayed technique allows multiple pairwise measurements of mRNA transcript levels and extent of hypoxia in individual tumors at a smallest possible volumetric scale which (by limiting averaging effects inherent to whole-tumor analysis) strengthen the conclusiveness on true hypoxia-specificity of candidate genes while limiting the required number of tumors. Among tested genes, our study identified CA9, GLUT1 and possibly LOX as highly specific biomarkers of tumor hypoxia in vivo.</p
Diverse molecular causes of unsolved autosomal dominant tubulointerstitial kidney diseases
Autosomal Dominant Tubulointerstitial Kidney Disease (ADTKD) is caused by mutations in one of at least five genes and leads to kidney failure usually in mid adulthood. Throughout the literature, variable numbers of families have been reported, where no mutation can be found and therefore termed ADTKD-not otherwise specified. Here, we aim to clarify the genetic cause of their diseases in our ADTKD registry. Sequencing for all known ADTKD genes was performed, followed by SNaPshot minisequencing for the dupC (an additional cytosine within a stretch of seven cytosines) mutation of MUC1. A virtual panel containing 560 genes reported in the context of kidney disease (nephrome) and exome sequencing were then analyzed sequentially. Variants were validated and tested for segregation. In 29 of the 45 registry families, mutations in known ADTKD genes were found, mostly in MUC1. Sixteen families could then be termed ADTKD-not otherwise specified, of which nine showed diagnostic variants in the nephrome (four in COL4A5, two in INF2 and one each in COL4A4, PAX2, SALL1 and PKD2). In the other seven families, exome sequencing analysis yielded potential disease associated variants in novel candidate genes for ADTKD; evaluated by database analyses and genome-wide association studies. For the great majority of our ADTKD registry we were able to reach a molecular genetic diagnosis. However, a small number of families are indeed affected by diseases classically described as a glomerular entity. Thus, incomplete clinical phenotyping and atypical clinical presentation may have led to the classification of ADTKD. The identified novel candidate genes by exome sequencing will require further functional validation
Gene Expression Programs in Response to Hypoxia: Cell Type Specificity and Prognostic Significance in Human Cancers
BACKGROUND: Inadequate oxygen (hypoxia) triggers a multifaceted cellular response that has important roles in normal physiology and in many human diseases. A transcription factor, hypoxia-inducible factor (HIF), plays a central role in the hypoxia response; its activity is regulated by the oxygen-dependent degradation of the HIF-1α protein. Despite the ubiquity and importance of hypoxia responses, little is known about the variation in the global transcriptional response to hypoxia among different cell types or how this variation might relate to tissue- and cell-specific diseases. METHODS AND FINDINGS: We analyzed the temporal changes in global transcript levels in response to hypoxia in primary renal proximal tubule epithelial cells, breast epithelial cells, smooth muscle cells, and endothelial cells with DNA microarrays. The extent of the transcriptional response to hypoxia was greatest in the renal tubule cells. This heightened response was associated with a uniquely high level of HIF-1α RNA in renal cells, and it could be diminished by reducing HIF-1α expression via RNA interference. A gene-expression signature of the hypoxia response, derived from our studies of cultured mammary and renal tubular epithelial cells, showed coordinated variation in several human cancers, and was a strong predictor of clinical outcomes in breast and ovarian cancers. In an analysis of a large, published gene-expression dataset from breast cancers, we found that the prognostic information in the hypoxia signature was virtually independent of that provided by the previously reported wound signature and more predictive of outcomes than any of the clinical parameters in current use. CONCLUSIONS: The transcriptional response to hypoxia varies among human cells. Some of this variation is traceable to variation in expression of the HIF1A gene. A gene-expression signature of the cellular response to hypoxia is associated with a significantly poorer prognosis in breast and ovarian cancer
An investigation in the correlation between Ayurvedic body-constitution and food-taste preference
Mechanismen und Konsequenzen sauerstoffabhÀngiger Genregulation
Die stĂ€ndige VerfĂŒgbarkeit von molekularem Sauerstoff (O2) ist ein elementarer Bestandteil multizellulĂ€rer Lebensformen. Zur Aufrechterhaltung der Homöostase sind diese auf die Bildung des Energiesubstrates ATP durch oxidative Phosphorylierung angewiesen. Aus diesem Grunde muĂten höhere Organismen wĂ€hrend der Evolution komplexe Systeme entwickeln, die die Aufnahme und Verteilung von O2 in jede Zelle sicherstellen, sowie eine Adaptation in Phasen der Hypoxie erlauben. Mit der Identifikation des Transkriptionsfaktors "Hypoxia-inducible Factor-1" (HIF-1, 1995) wurde ein entscheidender Regulator der hypoxischen Adaptation gefunden. Unter anderem werden Prozesse wie die Erythropoiese, die Angiogenese, die Modulation des GefĂ€Ătonus, des Glukosetransportes und der Glykolyse wesentlich durch HIF reguliert. HIF ist ein Heterodimer bestehend aus zwei Untereinheiten; einer konstitutiven beta- und einer regulativen alpha-Untereinheit. Letztere zeigt ein inverses Expressionsmuster zur perizellulĂ€ren O2-Konzentration. Unter normoxischen Bedingungen ist HIFalpha instabil und wird mit einer Halbwertzeit von nur wenigen Minuten degradiert. Erst unter Hypoxie wird HIFalpha stabilisiert und ist transkriptionell aktiv. Es konnten bisher zwei funktionell relevante O2-abhĂ€ngige alpha-Untereinheiten identifiziert werden: HIF-1 und HIF-2alpha. Die Bedeutung dieser beiden Systeme, der unterliegenden Regulationsmechanismen sowie die Relevanz dieses Systems in vivo waren weitgehend ungeklĂ€rt und sind wesentlicher Teil der hier zusammengefaĂten Arbeiten. In den vorgelegten Studien ist es gelungen, die Expression und Regulation der beiden unterschiedlichen HIFalpha Isoformen sowohl in der Zellkultur, als auch in gesunden Geweben zu charakterisieren. In Zellkulturen zeigte sich ein sehr Ă€hnliches Regulationsmuster hinsichtlich der O2-abhĂ€ngigen Degradation, bzw. dem Induktionsverhalten unter Hypoxie, sowie der chemisch/pharmakologischen Modulation, so dass offensichtlich beide Isoformen ĂŒber den gleichen O2-Sensing- und Transduktionsapparat reguliert werden. An Geweben von gesunden Ratten fĂŒhrten wir eine systematische Analyse der Expression und Regulation der beiden HIFalpha Isoformen durch. Nur unter systemischer Hypoxie konnten deutliche Signale fĂŒr beide Isoformen gesehen werden. Interessanterweise zeigte sich, daĂ beide nur von spezifischen Zellpopulationen exprimiert werden. In vivo lassen sich also klare Unterschiede im Expressionsmuster der beiden Systeme feststellen. Ăber die unterschiedlichen zellulĂ€ren Funktionen und different exprimierten Zielgene vermuten wir einen funktionell relevanten Unterschied. Mit der Identifikation des "von Hippel Lindau" Tumor Suppressor Gens als der bindende Anteil der E3 Ubiquitin Ligase, die fĂŒr die HIFalpha Destruktion verantwortlich ist, konnte ein wichtiger Beitrag zu der spĂ€teren KlĂ€rung des O2-Sensing-Mechanismus geleistet werden. Diese Befunde wurden initial anhand von Zellkultur-Linien erhoben, liessen sich aber auf Nierenzellkarzinome aus einer klinischen Sammlung ĂŒbertragen. Letzterer Befund ist daher fĂŒr das VerstĂ€ndnis der Rolle von HIF fĂŒr die Tumorbiologie, eventuell aber auch fĂŒr die Entwicklung therapeutischer AnsĂ€tze von Bedeutung.The permanent availability of molecular oxygen (O2) is an elemental need of multicellular life. For the maintenance of hemeostasis these are dependent on generation of the energy substrate ATP by oxidative phoshorylation. For this reason higher organisms had to develop complex systems during evolution that ensure the uptake and distribution of O2 into each cell, as well as permit adaptation to phases of hypoxia. With the identification of the transcription factor "Hypoxia-inducible Factor-1" (HIF-1, 1995) a master regulator of hypoxic adaptation has been found. Amongst others processes like erythropoiesis, angiogenesis, modulation of vascular tone, glucose transport and glycolysis are largely regulated by HIF. HIF is a heterodimer consisting of two subunits, a constitutive beta and a regulative alpha subunit. The latter shows an inverse relationship to the pericellular O2 concentration. HIFalpha is instable under normoxic conditions and degrades with a half life of only a few minutes. Under hypoxia the HIFalpha subunits are stabilised and are transcriptionally active. To date two functionally relevant O2-dependent alpha subunits have been identified: HIF-1 and HIF-2alpha. The importance of these two systems, the underlying regulatory mechanisms, as well as the relevance of this system in vivo were largely unknown and are a major part of the summarised studies. The presented work succeeded in characterising the expression and regulation of both HIFalpha isoforms in cell culture as well as healthy tissues. In tissue culture a very similar pattern of regulation was seen for oxygen dependent degradation, induction under hypoxia and chemical/pharmacological modulation, indicating that both subunits are regulated by the same O2-sensing and transduction apparatus. We undertook a systematic analysis of expression and regulation of both HIFalpha subunits in tissues of healthy rats. Signals for HIFalpha could only be seen under systemic hypoxia. Interestingly, both subunits were expressed by specific and different cell populations. Therefore, clear differences can be seen in expression pattern of both systems in vivo. We suspect that these differences will be functionally relevant through differing cellular functions and gene expression profile. With the identification of the "von Hippel Lindau" tumor suppressor gene as the binding part of the E3 ubiquitin ligase, which is responsible for HIF degradation, an important contribution to the clarification of the oxygen sensing mechanism was provided. Initially this data was generated in tissue culture lines, but could also be confirmed in renal cell carcinomas of a clinical collection. The latter finding is of importance for the understanding of the role of HIF in tumor biology, possibly also for the development of therapeutic strategies
Discordant Clinical Course of Vitamin-D-Hydroxylase (CYP24A1) Associated Hypercalcemia in Two Adult Brothers With Nephrocalcinosis
Background/Aims: Hypercalcemia can result in nephrocalcinosis/nephrolithiasis and may lead to renal failure. Idiopathic infantile hypercalcemia is caused by mutations of the CYP24A1 gene, which regulates vitamin D activity. Classically infants present with hypercalcemia. Recently, a number of individuals have been reported with late onset clinical manifestation or late diagnosis in adulthood. All these patients are believed to show hypercalciuria. Methods: We report a 24 year old patient of healthy consanguine parents. Genetic analysis was performed by Sanger sequencing of the CYP24A1 gene in the index patient and targeted exon 2 analysis of all other family members. Results: The patient was hospitalized with severe malaise during an acute EBV-infection. He showed hypercalcemia > 3mmol/l and acute, hypovolemic renal failure with profound nephrocalcinosis, but no hypercalciuria. Genetic workup revealed a homozygous loss-of-function mutation p.E143del in the CYP24A1 gene. His clinically asymptomatic brother showed nephrocalcinosis of lesser degree. Repeatedly, low parathyroid hormone levels were detected in both brothers. Conclusion: This family displays the highly variable phenotype of CYP24A1 biallelic mutation carriers. CYP24A1 associated disease is an important differential diagnosis for the workup and counseling of infants as well as adults with hypercalcemia since a proper genetic diagnosis may result in therapeutic consequences
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