To survive in hostile environments, organisms activate stress-responsive transcriptional regulators that coordinately increase production of protective factors. Hypoxia changes cellular metabolism and thus activates redox-sensitive as well as oxygen-dependent signal transducers. We demonstrate that Sirtuin 1 (Sirt1), a redox-sensing deacetylase, selectively stimulates activity of the transcription factor hypoxia-inducible factor 2 alpha (HIF-2alpha) during hypoxia. The effect of Sirt1 on HIF-2alpha required direct interaction of the proteins and intact deacetylase activity of Sirt1. Select lysine residues in HIF-2alpha that are acetylated during hypoxia confer repression of Sirt1 augmentation by small-molecule inhibitors. In cultured cells and mice, decreasing or increasing Sirt1 activity or levels affected expression of the HIF-2alpha target gene erythropoietin accordingly. Thus, Sirt1 promotes HIF-2 signaling during hypoxia and likely other environmental stresses.
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 mammalian sex determining gene, SRY, is the founding member of the new growing family of Sox (SRY-like HMG-box gene) genes. Sox genes encode transcription factors with diverse roles in development, and a few of them are involved in sex determination and differentiation. We report here the existence of Sox genes in the rice field eel, Monopterus albus, and DNA sequence information of the HMG box region of five Sox genes. The Sox1, Sox4 and Sox14 genes do not have introns in the HMG box region. The Sox9 gene and Sox17 gene, which each have an intron in the conserved region, show strong identity at the amino acid level with the corresponding genes of mammals and chickens. Similar structure and identity of the Sox9 and Sox17 genes among mammals, chickens and fish suggest that these genes have evolutionarily conserved roles, potentially including sex determination and differentiation.
We previously found increased expression of erythropoietin receptor (EPO-R) in peripheral dog lung during postnatal and postpneumonectomy (PNX) lung growth. To study the upstream regulation of EPO-R, we analyzed the expression of hypoxia-inducible factors (HIF)-1␣, -2␣, and -3␣ during postnatal lung growth in immature and mature (2.5 and 12 mo old, respectively) dogs and during compensatory lung growth 3 wk and 10 mo after right PNX. Relative to their respective controls, HIF-1␣ transcript was 52-95% higher in immature lungs and 284% higher in the remaining lung 3 wk post-PNX. HIF-2␣ transcript did not change during maturation but was 42% lower 3 wk post-PNX. HIF-3␣ transcript was 53-65% lower in both the immature lung and 3 wk post-PNX. Changes were no longer detectable 10 mo post-PNX. No change in HIF transcripts was observed in kidney and liver post-PNX. Consistent with the mRNA changes, HIF-1␣ protein was 120 and 196% higher in growing lungs and 3 wk post-PNX relative to their respective controls. Overexpression of HIF-1␣ in cultured HEK-293 cells increased endogenous expression of EPO-R protein. These results demonstrate regulated expression of the HIF system and parallel changes in HIF-1␣ and EPO-R expression during two types of lung growth. Because the normal growing lung is not hypoxic, the HIF system likely responds to other signals encountered during sustained lung strain.hypoxia-inducible factors; pneumonectomy; postnatal development; lung growth; ribonucleic acid blot; real-time polymerase chain reaction; immunoblot WE HAVE PREVIOUSLY SHOWN increased erythropoietin receptor (EPO-R) protein in peripheral dog lung during the following two types of lung growth: postnatal maturation and postpneumonectomy (PNX) compensatory growth (10). These findings provide evidence that the EPO axis regulates oxygen transport via more than one mechanism. In addition to endocrine EPO signaling that stimulates bone marrow erythropoiesis to enhance convective oxygen delivery, paracrine or autocrine EPO signaling in the lung may modulate growth of gas exchange tissue to enhance alveolar oxygen uptake. Because the EPO axis is one of many oxygen-sensitive pathways responsive to the hypoxia-inducible factors (HIF; see Refs. 3 and 33), activation of the HIF system may be responsible for the elevated EPO-R protein level during lung growth.HIF is a heterodimer composed of ␣-and -subunits. The -subunit, also known as aryl hydrocarbon receptor nuclear translocator (ARNT), is constitutively expressed independently of oxygen availability. The ␣-subunit is stabilized by hypoxia, which allows the HIF dimer to interact with transcriptional coactivator proteins (20). HIF-1␣ is expressed widely in a hypoxia-dependent manner in the bronchial epithelium, bronchial smooth muscle cells, and alveolar epithelium (39). HIF-2␣ and HIF-3␣ are two other members of the basic-loophelix (bHLH) PER/ARNT/SIM (PAS) domain protein superfamily. HIF-2␣, also known as endothelial PAS domain protein-1 or HIF-1␣-like factor, exhibits hypoxic stabilization a...
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