Hypoxia-inducible factor (HIF) transcription factors respond to multiple environmental stressors, including hypoxia and hypoglycemia. We report that mice lacking the HIF family member HIF-2alpha (encoded by Epas1) have a syndrome of multiple-organ pathology, biochemical abnormalities and altered gene expression patterns. Histological and ultrastructural analyses showed retinopathy, hepatic steatosis, cardiac hypertrophy, skeletal myopathy, hypocellular bone marrow, azoospermia and mitochondrial abnormalities in these mice. Serum and urine metabolite studies showed hypoglycemia, lactic acidosis, altered Krebs cycle function and dysregulated fatty acid oxidation. Biochemical assays showed enhanced generation of reactive oxygen species (ROS), whereas molecular analyses indicated reduced expression of genes encoding the primary antioxidant enzymes (AOEs). Transfection analyses showed that HIF-2alpha could efficiently transactivate the promoters of the primary AOEs. Prenatal or postnatal treatment of Epas1-/- mice with a superoxide dismutase (SOD) mimetic reversed several aspects of the null phenotype. We propose a rheostat role for HIF-2alpha that allows for the maintenance of ROS as well as mitochondrial homeostasis.

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Erythropoiesis in the adult mammal depends critically on erythropoietin, an inducible cytokine with pluripotent effects. Erythropoietin gene expression increases under conditions associated with lowered oxygen content such as anemia and hypoxia. HIF-1␣, the founding member of the hypoxia-inducible factor (HIF) alpha class, was identified by its ability to bind and activate the hypoxia-responsive enhancer in the erythropoietin regulatory region in vitro. The existence of multiple HIF alpha members raises the question of which HIF alpha member or members regulates erythropoietin expression in vivo. We previously reported that mice lacking wild-type HIF-2␣, encoded by the EPAS1 gene, exhibit pancytopenia. In this study, we have characterized the etiology of this hematopoietic phenotype. Molecular studies of EPAS1-null kidneys reveal dramatically decreased erythropoietin gene expression. EPAS1-null as well as heterozygous mice have impaired renal erythropoietin induction in response to hypoxia. Treatment of EPAS1-null mice with exogenous erythropoietin reverses the hematopoietic and other defects. We propose that HIF-2␣ is an essential regulator of murine erythropoietin production. IntroductionHematopoiesis involves complex interactions of specialized cell types and molecular signaling events that vary according to development. 1 The major sites of murine hematopoietic development depend on the particular developmental or postnatal stage. 2,3 Erythropoiesis, that aspect of hematopoiesis concerned with generation of erythrocytes, occurs in 2 distinct developmental phases characterized by primitive erythropoiesis in the yolk sac blood islands and definitive erythropoiesis in the fetal liver or bone marrow later in development. 1,4,5 Besides location, primitive and definitive erythropoiesis also differ in their growth factor or cytokine requirements.Erythropoietin was first described as an endocrine regulator of erythropoiesis produced in the kidneys; later studies revealed a paracrine role of this cytokine in global hematopoiesis and other aspects of mammalian physiology. [6][7][8][9] Primitive erythropoiesis in the yolk sac is erythropoietin-independent, but requires other cytokines such as vascular endothelial growth factor (VEGF) and c-Kit. [10][11][12] In contrast, definitive erythropoiesis in the fetal liver and in the adult bone marrow is erythropoietin-dependent as evident by gene disruption studies of the erythropoietin 13 or erythropoietin receptor 14 gene. The temporal distinction of primitive and definitive erythropoiesis is in part due to developmental timing of erythropoietin and erythropoietin receptor gene expression. 15 The sites of definitive erythropoiesis are also sites of erythropoietin production. 16 These sites include resident macrophages in the adult bone marrow as well as in the fetal liver blood islands, 17 the predominant sites of definitive erythropoiesis in the adult and neonate, respectively. Erythropoietin produced in these locations may function as a paracrine growth factor for global ...

The Siah ubiquitin ligases are members of the RING finger E3 ligases. The Siah E3s are conserved from fly to mammals. Primarily implicated in cellular stress responses, Siah ligases play a key role in hypoxia, through the regulation of HIF-1α transcription stability and activity. Concomitantly, physiological conditions associated with varying oxygen tension often highlight the importance of Siah, as seen in cancer and neurodegenerative disorders. Notably, recent studies also point to the role of these ligases in fundamental processes including DNA damage response, cellular organization and polarity. This review summarizes the current understanding of upstream regulators and downstream effectors of Siah2.

IntroductionInflammation assumes many forms, such as the response to acute epithelial barrier injury, acute microbial infection, recurrent tissue nondestructive lesions as in psoriasis, chronic tissue destruction as seen in a myriad of autoimmune or innate inflammatory diseases, or chronic microbial/viral infestations and infections. Chronic inflammation with attendant microenvironmental alterations, resulting from growth factor, chemokine, cytokine secretion, and reactive oxygen species enrichment, also provides a fertile soil for de novo development of epithelial cancer. 1,2 Elucidation of the mechanisms of inflammatory modulation by epithelial cells is an emergent focus of investigation. Models of epithelial elaboration of specific inflammatory chemokines, cytokines, or growth factors have shed light on the roles of specific signaling networks in disease. In contrast, the concept that transcription factors regulate production of a repertoire of inflammatory chemokines and cytokines by epithelial cells is relatively new. 3,4 Here we demonstrate that gain of function of the transcription factor hypoxia inducible factor-1 (HIF-1), signaling indirectly through nuclear factor [kappa2]B (NFB), can "prime" and remodel the local stromal environment to affect an augmented, enhanced responsiveness "hyper-responsiveness" to an inflammatory stimulus. This may be yet another mechanism, broadcast from the epithelial cells themselves, by which both HIF-1 and NFB conspire to increase the severity of inflammatory diseases and carcinogenic progression.HIF-1 is a fundamental mediator of cellular adaptation to hypoxia, activating metabolic and signaling pathways promoting cell survival. HIF-1 consists of the oxygen-sensitive subunit HIF-1␣ and the constitutively expressed HIF-1␤ subunit. In normoxia, the alpha subunit is hydroxylated in position 402 and 564 by 3 prolyl hydroxylase enzymes (PHD) 1 to 3. These modifications allow interaction between HIF-1␣ and the von Hippel-Lindau protein, targeting HIF-1␣ for proteasomal degradation. In hypoxia, PHD activity is decreased, preventing HIF-1␣ degradation. 5,6 When HIF-1␣ levels increase, functional HIF-1 regulates transcription at hypoxia response elements of target gene enhancers, up-regulating genes involved in energy metabolism and angiogenesis. 7 HIF-1␣ protein synthesis can be regulated in an O 2 -independent manner by activation of the phosphatidylinositol 3-kinase and ERK mitogen-activated kinase pathways that are either physiologically stimulated by cognate growth factors or cytokines or activated by mutation. Thus, proinflammatory cytokines, interleukin-1␤ and tumor necrosis factor␣ (TNF␣) have each been shown to stabilize HIF-1␣ protein, suggesting that HIF-1␣ functions can be recruited by tissue inflammation. [8][9][10] The concept The online version of this article contains a data supplement.The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked ''advertisement'' in a...

Phosphoinositide-dependent kinase-1 (PDK-1) is a serine/threonine protein kinase that phosphorylates members of the conserved AGC kinase superfamily, including AKT and PKC, and is implicated in important cellular processes including survival, metabolism and tumorigenesis. In large cohorts of nevi and melanoma samples, PDK1 expression was significantly higher in primary melanoma, compared with nevi, and was further increased in metastatic melanoma. PDK1 expression suffices for its activity, due to auto-activation, or elevated phosphorylation by phosphoinositide 3'-OH-kinase (PI 3-K). Selective inactivation of Pdk1 in the melanocytes of BrafV600E::Pten−/− or BrafV600E::Cdkn2a−/−::Pten−/− mice delayed the development of pigmented lesions and melanoma induced by systemic or local administration of 4-HT. Melanoma invasion and metastasis were significantly reduced or completely prevented by Pdk1 deletion. Administration of the PDK1 inhibitor GSK2334470 (PDKi) effectively delayed melanomagenesis and metastasis in BrafV600E::Pten−/− mice. Pdk1−/− melanomas exhibit a marked decrease in the activity of AKT, P70S6K and PKC. Notably, PDKi was as effective in inhibiting AGC kinases and colony forming efficiency of melanoma with Pten WT genotypes. Gene expression analyses identified Pdk1-dependent changes in FOXO3a-regulated genes and inhibition of FOXO3a restored proliferation and colony formation of Pdk1−/− melanoma cells. Our studies provide direct genetic evidence for the importance of PDK1, in part through FOXO3a-dependent pathway, in melanoma development and progression.

The RING finger E3 ubiquitin ligase Siah2 is implicated in control of diverse cellular biological events, including MAPK signaling and hypoxia. Here we demonstrate that Siah2 is subject to regulation by the deubiquitinating enzyme USP13. Overexpression of USP13 increases Siah2 stability by attenuating its autodegradation. Consequently, the ability of Siah2 to target its substrates prolyl hydroxylase 3 and Spry2 (Sprouty2) for ubiquitin-mediated proteasomal degradation is attenuated. Conversely, inhibition of USP13 expression with corresponding shRNA decreases the stability of both Siah2 and its substrate Spry2. Thus, USP13 limits Siah2 autodegradation and its ubiquitin ligase activity against its target substrates. Strikingly, the effect of USP13 on Siah2 is not mediated by its isopeptidase activity: mutations in its ubiquitin-binding sequences positioned within the ubiquitin-specific processing protease and ubiquitin-binding domains, but not within putative catalytic sites, abolish USP13 binding to and effect on Siah2 autodegradation and targeted ubiquitination. Notably, USP13 expression is attenuated in melanoma cells maintained under hypoxia, thereby relieving Siah2 inhibition and increasing its activity under low oxygen levels. Significantly, on melanoma tissue microarray, high nuclear expression of USP13 coincided with high nuclear expression of Siah2. Overall, this study identifies a new layer of Siah2 regulation mediated by USP13 binding to ubiquitinated Siah2 protein with a concomitant inhibitory effect on its activity under normoxia.Siah proteins are RING finger E3 ubiquitin ligases implicated in the ubiquitination and proteasome-dependent degradation of substrate molecules, which also limit their own availability through self-ubiquitination and degradation (1-3). Siah was first identified in Drosophila melanogaster as seven in absentia (sina), which regulates formation of the R7 photoreceptor through control of tramtrack stability (4, 5). Three murine Siah genes (Siah1a, Siah1b, and Siah2) share significant homology with the two human (SIAH1 and SIAH2) orthologues (6, 7).Siah2 is an important regulator of pathways activated under stress and hypoxia. Among substrates reportedly regulated by Siah under these conditions are TRAF2, ␣-ketoglutarate dehydrogenase, Spry2 (Sprouty2), and two of the three prolyl hydroxylases (8 -10). Given the role of PHD proteins as oxygen sensors and regulators of HIF1␣, the master transcription factor responding to hypoxia, Siah is implicated in the control of hypoxia, particularly within the range of 2-6% oxygen at which PHD proteins retain sufficient activity (11,12). The role of Siah2 in tumor development has been extensively studied. Inhibition of Siah reportedly attenuates breast, pancreatic, lung, prostate, and melanoma tumors (13-16). Mechanistically, Siah2 contributes to tumor development and metastasis via its control of diverse substrates, as shown in a melanoma model (14).A search for novel Siah2 interacting factors led to identification of the isopeptidase USP13 ...

Hypoxia-inducible factor-1 is a known cancer progression factor, promoting growth, spread, and metastasis. However, in selected contexts HIF-1 is a tumor suppressor coordinating hypoxic cell cycle suppression and apoptosis. Prior studies focused on HIF-1 function in established malignancy, however little is known about its role during the entire process of carcinogenesis from neoplasia induction to malignancy. Here we tested HIF-1 gain of function during multistage murine skin chemical carcinogenesis in K14-HIF-1αPro402A564G (K14-HIF-1αDPM) transgenic mice. Transgenic papillomas appeared earlier and were more numerous, 6±3 transgenic versus 2±1.5 nontransgenic papillomas per mouse, yet they were more differentiated, their proliferation was lower, and their malignant conversion was profoundly inhibited, 7% in transgenic versus 40% in non transgenic mice. Moreover, transgenic cancers maintained squamous differentiation whereas epithelial mesenchymal transformation was frequent in nontransgenic malignancies. Transgenic basal keratinocytes upregulated the HIF-1 target N-myc downstream regulated gene-1, a known tumor suppressor gene in human malignancy, and its expression was maintained in transgenic papillomas and cancer. We also discovered a novel HIF-1 target gene, selenium binding protein-1 (selenbp1), a gene of unknown function whose expression is lost in human cancer. Thus, HIF-1 can function as a tumor suppressor through transactivation of genes that are themselves targets for negative selection in human cancers.