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

The influence of HIF on tumor-relevant cellular functions

Ziel dieser Arbeit war die Aufklärung, wie sich die Stabilisierung von HIF auf wichtige zellbiologische und vor allem für die Entstehung von Tumoren relevante Prozesse auswirkt. Durch die Etablierung eines transgenen Mausmodels mit renaler, Tubulus spezifischer und Sauerstoff unabhängiger Stabilisierung von HIF-1alpha, sollte das tumorigene Potential von HIF 1 auf gesunde Nierenzellen untersucht werden. In dem zweiten Teilabschnitt dieser Arbeit konnte mit Rab20 ein neues, zuvor unbekanntes Zielgen von HIF-1 charakterisiert werden. Im letzten Teilabschnitt dieser Arbeit wurden erfolgreich Zelllinien generiert, in denen die Expression von HIF-1alpha oder HIF-2alpha durch Gabe eines Antibiotikums gezielt aktiviert werden kann. Die vorgelegten neuen Erkenntnisse demonstrieren, dass vielschichtige Aspekte zellulärer Funktion durch den Transkriptionsfaktor HIF direkt beeinträchtigt werden. So zeigen die dargestellten Daten einen eindeutigen Einfluss von HIF auf die Apoptose und Zellzyklusregulation. Beide Prozesse sind unweigerlich mit der Entwicklung von Tumoren verknüpft. Mit dieser Arbeit konnten somit wichtige Aspekte zum Einfluss von HIF auf die Tumorbiologie näher beleuchtet werden.The purpose of this thesis was to find out, how the stabilisation of HIF affects cellular functions which could contribute to the development of tumours. The aim of the first part of this work was the establishment of a transgenic mouse model with renal, tubular specific and oxygen independent stabilisation of HIF-1alpha in order to analyse the tumourigenic potential of HIF-1 on kidney cells. The second part of this work describes the characterisation of a new HIF-1 target gene Rab20. The last part comprises the successful generation of tetracycline inducible, HIF-1alpha and HIF-2alpha expressing cell lines. All together, the results clearly demonstrate, that HIF targets many different aspects of cellular function. The data show that activation of HIF directly influences apoptosis and cell cycle regulation. Both processes are inevitably linked to the development of tumours. Therefore, this work highlights important aspects of how HIF could influence tumour behaviour

Hypoxia-inducible protein 2 is a novel lipid droplet protein and a specific target gene of hypoxia-inducible factor-1

Hypoxia-inducible protein 2 (HIG2) has been implicated in canonical Wnt signaling, both as target and activator. The potential link between hypoxia and an oncogenic signaling pathway might play a pivotal role in renal clear-cell carcinoma characterized by constitutive activation of hypoxia-inducible factors (HIFs), and hence prompted us to analyze HIG2 regulation and function in detail. HIG2 was up-regulated by hypoxia and HIF inducers in all cell types and mouse organs investigated and abundantly expressed in renal clear-cell carcinomas. Promoter analyses, gel shifts, and siRNA studies revealed that HIG2 is a direct and specific target of HIF-1, but not responsive to HIF-2. Surprisingly, HIG2 was not secreted, and HIG2 overexpression neither stimulated proliferation nor activated Wnt signaling. Instead, we show that HIG2 decorates the hemimembrane of lipid droplets, whose number and size increase on hypoxic inhibition of fatty acid beta-oxidation, and colocalizes with the lipid droplet proteins adipophilin and TIP47. Normoxic overexpression of HIG2 was sufficient to increase neutral lipid deposition in HeLa cells and stimulated cytokine expression. HIG2 could be detected in atherosclerotic arteries and fatty liver disease, suggesting that this ubiquitously inducible HIF-1 target gene may play an important functional role in diseases associated with pathological lipid accumulation.-Gimm, T., Wiese, M., Teschemacher, B., Deggerich, A., Schodel, J., Knaup, K. X., Hackenbeck, T., Hellerbrand, C., Amann, K., Wiesener, M. S., Honing, S., Eckardt, K.-U., Warnecke, C. Hypoxia-inducible protein 2 is a novel lipid droplet protein and a specific target gene of hypoxia-inducible factor-1. FASEB J. 24, 4443-4458 (2010). www.fasebj.or

Biallelic Expression of Mucin-1 in Autosomal Dominant Tubulointerstitial Kidney Disease: Implications for Nongenetic Disease Recognition

Background Providing the correct diagnosis for patients with tubulointerstitial kidney disease and secondary degenerative disorders, such as hypertension, remains a challenge. The autosomal dominant tubulointerstitial kidney disease (ADTKD) subtype caused by MUC1 mutations (ADTKD-MUC1) is particularly difficult to diagnose, because the mutational hotspot is a complex repeat domain, inaccessible with routine sequencing techniques. Here, we further evaluated SNaPshot minisequencing as a technique for diagnosing ADTKD-MUC1 and assessed immunodetection of the disease-associated mucin 1 frameshift protein (MUC1-fs) as a nongenetic technique. Methods We re-evaluated detection of MUC1 mutations by targeted repeat enrichment and SNaPshot minisequencing by haplotype reconstruction via microsatellite analysis in three independent ADTKD-MUC1 families. Additionally, we generated rabbit polyclonal antibodies against MUC1-fs and evaluated immunodetection of wild-type and mutated allele products in human kidney biopsy specimens. Results The detection of MUC1 mutations by SNaPshot minisequencing was robust. Immunostaining with our MUC1-fs antibodies and an MUC1 antibody showed that both proteins are readily detectable in human ADTKD-MUC1 kidneys, with mucin 1 localized to the apical membrane and MUC1-fs abundantly distributed throughout the cytoplasm. Notably, immunohistochemical analysis of MUC1-fs expression in clinical kidney samples facilitated reliable prediction of the disease status of individual patients. Conclusions Diagnosing ADTKD-MUC1 by molecular genetics is possible, but it is technically demanding and labor intensive. However, immunohistochemistry on kidney biopsy specimens is feasible for nongenetic diagnosis of ADTKD-MUC1 and therefore, a valid method to select families for further diagnostics. Our data are compatible with the hypothesis that specific molecular effects of MUC1-fs underlie the pathogenesis of this disease

Renal fibrosis is the common feature of autosomal dominant tubulointerstitial kidney diseases caused by mutations in mucin 1 or uromodulin

For decades, ill-defined autosomal dominant renal diseases have been reported, which originate from tubular cells and lead to tubular atrophy and interstitial fibrosis. These diseases are clinically indistinguishable, but caused by mutations in at least four different genes: UMOD, HNF1B, REN, and, as recently described, MUC1. Affected family members show renal fibrosis in the biopsy and gradually declining renal function, with renal failure usually occurring between the third and sixth decade of life. Here we describe 10 families and define eligibility criteria to consider this type of inherited disease, as well as propose a practicable approach for diagnosis. In contrast to what the frequently used term 'Medullary Cystic Kidney Disease' implies, development of (medullary) cysts is neither an early nor a typical feature, as determined by MRI. In addition to Sanger and gene panel sequencing of the four genes, we established SNaPshot minisequencing for the predescribed cytosine duplication within a distinct repeat region of MUC1 causing a frameshift. A mutation was found in 7 of 9 families (3 in UMOD and 4 in MUC1), with one indeterminate (UMOD p.T62P). On the basis of clinical and pathological characteristics we propose the term 'Autosomal Dominant Tubulointerstitial Kidney Disease' as an improved terminology. This should enhance recognition and correct diagnosis of affected individuals, facilitate genetic counseling, and stimulate research into the underlying pathophysiology

Expression of HIF-1α and HIF-2α in the human kidney.

<p>HIF-1α and HIF-2α expression was analyzed by immunohistochemistry in human kidneys. After carbon monoxide (CO) intoxication HIF-1α accumulation was detected in tubular epithelial cells (A) and HIF-2α was detected in interstitial cells and in the glomeruli, indicated by arrows (B). In kidneys of RCC patients HIF-1α and HIF-2α were detected in the tumor tissue as well as in the adjacent kidney (C and D). In the latter HIF-1α was found in tubular epithelial cells (E) and HIF-2α restricted to interstitial cells (F).</p

HIF-1α and HIF-2α expression in mouse kidney.

<p>The physiological expression pattern of HIF-1α and HIF-2α was analyzed by immunohistochemistry on kidney sections of normoxic and hypoxic (6 h, 7% O₂) mice. HIF-1α expression was detected in the nuclei of renal tubular epithelial cells after hypoxic stimulation, whereas HIF-2α accumulated in interstitial and glomerular cells.</p