Abstract-Ischemia is a stimulus for production of angiogenic cytokines that activate local vascular cells and mobilize angiogenic cells to the circulation. These responses are impaired in elderly patients with peripheral arterial disease. Hypoxia-inducible factor (HIF)-1 mediates adaptive responses to ischemia, including production of angiogenic cytokines. In this study, we demonstrate that aging and HIF-1 loss-of-function impair the expression of multiple angiogenic cytokines, mobilization of angiogenic cells, maintenance of tissue viability, and recovery of limb perfusion following femoral artery ligation. We show that HIF-1 directly activates transcription of the gene encoding stem cell factor and that mice lacking the cognate receptor C-KIT have impaired recovery from ischemia. Administration of AdCA5, an adenovirus encoding a constitutively active form of HIF-1␣, improved the recovery of perfusion in older mice to levels similar to those in young mice. Injection of AdCA5 into nonischemic limb was sufficient to increase the number of circulating angiogenic cells. These results indicate that HIF-1 activity is necessary and sufficient for the mobilization of angiogenic cells and that HIF-1␣ gene therapy can counteract the pathological effects of aging in a mouse model of limb ischemia.

Oxygen homeostasis represents an essential organizing principle of metazoan evolution and biology. Hypoxia-inducible factor 1 (HIF-1) is a master regulator of transcriptional responses to changes in O 2 concentration. HIF-1 is a heterodimer of HIF-1␣ and HIF-1␤ subunits. O 2 -dependent degradation of the HIF-1␣ subunit is mediated by prolyl hydroxylase, von HippelLindau protein (VHL)/Elongin-C E3 ubiquitin ligase, and the proteasome. O 2 -independent degradation of HIF-1␣ is regulated by the competition of RACK1 and HSP90 for binding to HIF-1␣. RACK1 binding results in the recruitment of the Elongin-C E3 ubiquitin ligase, leading to VHL-independent ubiquitination and degradation of HIF-1␣. In this report, we show that calcineurin inhibits the ubiquitination and proteasomal degradation of HIF-1␣. Calcineurin is a serine/threonine phosphatase that is activated by calcium and calmodulin. The phosphatase activity of calcineurin is required for its regulation of HIF-1␣. RACK1 binds to the catalytic domain of calcineurin and is required for HIF-1␣ degradation induced by the calcineurin inhibitor cyclosporine A. Elongin-C and HIF-1␣ each bind to RACK1 and dimerization of RACK1 is required to recruit Elongin-C to HIF-1␣. Phosphorylation of RACK1 promotes its dimerization and dephosphorylation by calcineurin inhibits dimerization. Serine 146 within the dimerization domain is phosphorylated and mutation of serine 146 impairs RACK1 dimerization and HIF-1␣ degradation. These results indicate that intracellular calcium levels can regulate HIF-1␣ expression by modulating calcineurin activity and RACK1 dimerization.All metazoan organisms possess physiological systems to regulate the delivery and/or utilization of O 2 . Hypoxia-inducible factor 1 (HIF-1) 3 is a critical mediator of adaptive responses to reduced oxygen availability in many developmental, physiological, and pathological contexts through its transcriptional regulation of genes that encode proteins required for tissue oxygen delivery and energy metabolism (1-4). HIF-1 is required for embryonic vascularization. Mouse embryos with complete HIF-1␣ deficiency or expression of a dominant negative form of HIF-1 in endothelial cells fail to develop proper vascularization and die at embryonic days 10 -11 (4 -6). HIF-1-dependent induction of angiogenic factors promotes vascularization and cardiac function in the initial compensated phase of left ventricular hypertrophy caused by pressure overload (7). In contrast to this adaptive response, HIF-1 contributes to the pathogenesis of hypoxia-induced pulmonary hypertension and right ventricular hypertrophy (8). HIF-1 also plays an important pathogenic role in many critical aspects of cancer biology, including cell immortalization, energy metabolism, vascularization, invasion, metastasis, and resistance to radiation and chemotherapy (9). HIF-1 is a heterodimeric transcription factor composed of a HIF-1␤ subunit, which is constitutively expressed, and a HIF-1␣ subunit, the expression and transcriptional activity of which are regul...

Replication forks encounter impediments as they move through the genome, including natural barriers due to stable protein complexes and highly transcribed genes. Unlike lesions generated by exogenous damage, natural barriers are encountered in every S phase. Like humans, Schizosaccharomyces pombe encodes a single Pif1 family DNA helicase, Pfh1. Here, we show that Pfh1 is required for efficient fork movement in the ribosomal DNA, the mating type locus, tRNA, 5S ribosomal RNA genes, and genes that are highly transcribed by RNA polymerase II. In addition, converged replication forks accumulated at all of these sites in the absence of Pfh1. The effects of Pfh1 on DNA replication are likely direct, as it had high binding to sites whose replication was impaired in its absence. Replication in the absence of Pfh1 resulted in DNA damage specifically at those sites that bound high levels of Pfh1 in wild-type cells and whose replication was slowed in its absence. Cells depleted of Pfh1 were inviable if they also lacked the human TIMELESS homolog Swi1, a replisome component that stabilizes stalled forks. Thus, Pfh1 promotes DNA replication and separation of converged replication forks and suppresses DNA damage at hard-to-replicate sites.

a b s t r a c tTelomeres protect the ends of eukaryotic chromosomes from being recognized and processed as double strand breaks. In most organisms, telomeric DNA is highly repetitive with a high GC-content. Moreover, the G residues are concentrated in the strand running 3 0 -5 0 from the end of the chromosome towards its center. This G-rich strand is extended to form a 3 0 single-stranded tail that can form unusual secondary structures such as T-loops and G-quadruplex DNA. Both the duplex repeats and the single-stranded G-tail are assembled into stable protein-DNA complexes. The unique architecture, high GC content, and multi-protein association create particularly stable protein-DNA complexes that are a challenge for replication, recombination, and transcription. Helicases utilize the energy of nucleotide hydrolysis to unwind base paired nucleic acids and, in some cases, to displace proteins from them. The telomeric functions of helicases from the RecQ, Pifl, FANCJ, and DNA2 families are reviewed in this article. We summarize data showing that perturbation of their telomere activities can lead to telomere dysfunction and genome instability and in some cases human disease.

Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric transcription factor that functions as a master regulator of oxygen homeostasis. The HIF-1␣ subunit is subjected to O 2 -dependent prolyl hydroxylation leading to ubiquitination by the von Hippel-Lindau protein (VHL)-Elongin C ubiquitin-ligase complex and degradation by the 26 S proteasome. In this study, we demonstrate that spermidine/spermine-N 1 -acetyltransferase (SSAT) 2 plays an essential role in this process. SSAT2 binds to HIF-1␣, VHL, and Elongin C and promotes ubiquitination of hydroxylated HIF-1␣ by stabilizing the interaction of VHL and Elongin C. Multivalent interactions by SSAT2 provide a mechanism to ensure efficient complex formation, which is necessary for the extremely rapid ubiquitination and degradation of HIF-1␣ that is observed in oxygenated cells.Oxygen homeostasis represents a critical organizing principle of metazoan evolution and biology (1, 2). Hypoxia-inducible factor 1 (HIF-1) 2 functions as a master regulator of oxygen homeostasis in metazoan species as diverse as Caenorhabditis elegans, an organism of Ͻ10 3 cells with no specialized systems for O 2 delivery, to Homo sapiens, an organism with complex respiratory and circulatory systems to capture O 2 and deliver it to each of Ͼ10 13 cells (1, 3-7). Precise moment-to-moment matching of O 2 supply and demand is required for maintenance of cellular energetics and redox equilibrium that in turn is necessary for the survival of individual cells and of the organism (2, 8). To achieve this essential task, HIF-1 regulates the transcription of hundreds of human genes (9, 10) encoding proteins that are required for proper development of the respiratory and circulatory systems (3,11,12) and play essential roles in adaptive physiological responses to hypoxia and ischemia in postnatal life (13)(14)(15).Delineation of the complex network of interacting proteins that regulates HIF-1 activity has progressed rapidly since the discovery (16), biochemical purification (17), and determination of the nucleic acid sequences encoding HIF-1 (18). HIF-1 is a heterodimer composed of a constitutively expressed HIF-1␤ subunit and a HIF-1␣ subunit, the expression of which increases dramatically as cellular O 2 concentration declines (17-19). HIF-1␣ levels are low in oxygenated cells because of the rapid proteasomal degradation of the protein in response to its modification by an E3 ubiquitin-protein ligase complex containing the von Hippel-Lindau (VHL) protein (20,21). VHL binds to HIF-1␣ and to Elongin C, which recruits Elongin B, Cullin 2, Rbx 1, and other components of the ubiquitin ligase complex (22).VHL only binds after the hydroxylation of HIF-1␣ on Pro 402 and/or Pro 564 (4, 23-26) by HIF-1␣ prolyl hydroxylase domain proteins (PHDs) 1-3, which are dioxygenases that utilize O 2 to hydroxylate HIF-1␣ in a reaction that also consumes ␣-ketoglutarate and generates succinate and CO 2 as by-products (27). Hydroxylase activity is inhibited as cellular O 2 concentration declines, either as a result of substr...