Abbreviations: ARNT, aryl hydrocarbon receptor nuclear translocator; bFGF, basic fibroblast growth factor; bHLH, basic helix-loop-helix; BNIP3, BCL2/adenovirus E1B 19-kDa interacting protein 3; CBP, cyclic AMP response element-binding protein (CREB)-binding protein; CCRC, clear cell renal carcinoma; CSN, constitutive photomorphogenesis (COP9) signalosome; eIF3, eukaryotic translation initiation factor 3; FIH-1, factor inhibiting HIF-1; GLUT1, glucose transporter 1; HDAC, histone deacetylase; HIF, hypoxia-inducible factor; HRE, hypoxia-responsive element; ID, inhibitory domain; IGF-1, insulin-like growth factor-1; Int6, translation initiation factor 6; MCP-1, monocyte chemoattractant protein 1; MMTV, mouse mammary tumor virus; N-CoR, nuclear receptor corepressor; PAS, Per/ARNT/SIM; PINT, proteasome/Int6/Nip1/thyroid receptor-interacting protein 15 (TRIP-15); SMRT, silencing mediator for retinoic acid receptor (RAR) and thyroid hormone receptor (TR); TAD, transactivation domain; VHL, von Hippel-Lindau AbstractThe mechanisms that regulate angiogenesis in hypoxia or hypoxic microenvironment are modulated by several pro-and antiangiogenic factors. Hypoxia-inducible factors (HIFs) have been established as the basic and major inducers of angiogenesis, but understanding the role of interacting proteins is becoming increasingly important to elucidate the angiogenic processes of a hypoxic response. In particular, with regard to wound healing and the novel therapies for vascular disorders such as ischemic brain and heart attack, it is essential to gain insights in the formation and regulation of HIF transcriptional machineries related to angiogenesis. Further, identification of alternative ways of inhibiting tumor growth by disrupting the growth-triggering mechanisms of increasing vascular supply via angiogenesis depends on the knowledge of how tumor cells develop their own vasculature. Here, we review our findings on the interactions of basic HIFs, HIF-1α and HIF-2α, with their regulatory binding proteins, histone deacetylase 7 (HDAC7) and translation initiation factor 6 (Int6), respectively. The present results and discussion revealed new regulatory interactions of HIF-related mechanisms.

The hypoxia-inducible factors HIF-1␣ and HIF-2␣ are structurally similar as regards their DNA-binding and dimerization domains, but differ in their transactivation domains and, as is shown by experiments using hif-1␣؊/؊ and hif-2␣ ؊/؊ mice, in their functions. This implies that HIF-1␣ and HIF-2␣ may have unique target genes. To address this discrepancy and identify HIF-2␣-specific target genes, we performed yeast two-hybrid analysis and identified the tumor suppressor Int6/eIF3e/p48 as a novel target gene product involved in HIF-2␣ regulation. The int6 gene was first identified from a screen in which the mouse mammary tumor virus was employed as an insertional mutagen to identify genes whose functions are critical for breast tumor formation. Here, by using two-hybrid analysis, immunoprecipitation in mammalian cells, and HRE-reporter assays, we report the specific interaction of HIF-2␣ (but not HIF-1␣ or HIF-3␣) with Int6. The results indicate that the direct interaction of Int6 induces proteasome inhibitor-sensitive HIF-2␣ degradation. This degradation was clearly observed in renal cell carcinoma 786-O cells, and was found to be both hypoxia-and pVHL-independent. Furthermore, Int6 protein knockdown by int6-siRNA vectors or the dominant-negative mutant Int6-⌬C increased endogenous HIF-2␣ expression, even under normoxia, and induced sets of critical angiogenic factors comprising vascular endoplasmic growth factor, angiopoietin, and basic fibroblast growth factor mRNA. These results indicate that Int6 is a novel and critical determinant of HIF-2␣-dependent angiogenesis as well as cancer formation, and that int6-siRNA transfer may be an effective therapeutic strategy in pathological conditions such as heart and brain ischemia, hepatic cirrhosis, and obstructive vessel diseases.Hypoxia-responsive genes are involved in glucose transport, glycolysis, erythropoiesis, angiogenesis, vasodilation, and respiratory rate. However, they are also involved in the pathogenesis of many cardiovascular diseases and cancer (1, 2). Central to many molecular and physiological responses to hypoxia in most mammals cells are the hypoxia-inducible factors HIF-1␣ and HIF-2␣ (HIFs), 2 heterodimers with HIF-1␤ (also referred to as aryl hydrocarbon receptor nuclear translocator, ARNT) (3-5). Both HIF-1␣ and ARNT belong to the basic helix loop helix Per-Arnt-Sim (PAS) family of transcription factors, which share several conserved structural domains (6).Under normoxia, HIF-1␣ is ubiquitinated via interaction with the von Hippel-Lindau tumor suppressor protein (pVHL) and is subsequently degraded by the 26 S proteasome (7-12). pVHL, the recognition component of an E3 ubiquitin ligase complex, binds HIF-1␣ when it is hydroxylated at proline residues 402 and 577 (13-15). The proline hydroxylation of HIF-1␣ is catalyzed by prolyl hydroxylase domain-containing proteins, which are members of the 2-oxoglutarate-dependent dioxygenase superfamily whose activity requires O 2 as a cofactor (16,17). During hypoxia, the activity of prolyl hydroxylase domainco...

Background-We previously identified INT6/eIF3e as a novel regulator of hypoxia-inducible factor 2␣ (HIF2␣) activity. Small interfering RNA (siRNA)-Int6 adequately stabilized HIF2␣, even under normoxic conditions, and thereby enhanced the expression of several angiogenic factors in vitro, suggesting that siRNA-Int6 may induce angiogenesis in vivo. Methods and Results-We demonstrated a 6-to 8-fold enhanced formation of normal arteries and veins in the subcutaneous regions of adult mice 5 days after a single siRNA-Int6 application. Subcutaneous fibroblasts were identified as the major source of secreted angiogenic factors that led to the formation of functional vessels during Int6 silencing. Fibroblasts transfected ex vivo with siRNA-Int6 induced potent neoangiogenesis when transplanted into a subcutaneous region of nude mice. Application of siRNA-Int6 promoted neoangiogenesis in the area surrounding the injury in wound healing models, including genetically diabetic mice, thereby accelerating the closure of the injury. HIF2␣ accumulation caused by siRNA-Int6 was confirmed as the unequivocal cause of the angiogenesis by an in vivo angiogenesis assay. Further analysis of the Int6 silencing-induced neoangiogenesis revealed that a negative feedback regulation of HIF2␣ stability was caused by HIF2␣-induced transcription of Int6 via hypoxia-response elements in its promoter. Thus, siRNA-Int6 temporarily facilitates an accumulation of HIF2␣ protein, leading to hypoxia-independent transcription of angiogenic factors and concomitant neoangiogenesis. Conclusions-We suggest that the pathway involving INT6/HIF2␣ acts as a hypoxia-independent master switch of functional angiogenesis; therefore, siRNA-Int6 application might be of clinical value in treating ischemic diseases such as heart and brain ischemia, skin injury, and diseases involving obstructed vessels. (Circulation. 2010;122:910-919.)Key Words: angiogenesis Ⅲ hypoxia Ⅲ molecular biology Ⅲ signal transduction Ⅲ siRNA T he process of vessel formation is complex but well coordinated, involving the combined action of numerous growth factors and related signaling pathways. 1 Nevertheless, a single angiogenic factor such as vascular endothelial growth factor (VEGF), 2 fibroblast growth factor (FGF), 3,4 or plateletderived growth factor 5 can induce neoangiogenesis, 6 -8 albeit with incomplete and leaky vessels. 6,9 The transgenic expression of angiopoietin-1 (ANG-1) and VEGF significantly increases both the size and number of blood vessels. 8 These results suggest that several angiogenic factors are essential for the formation of functional vessels and that they must be expressed in a complementary and coordinated manner 10 to strike a balance among many stimulatory and inhibitory signals. 11 Clinical Perspective on p 919The expression of various angiogenic factors such as VEGF, ANG-1, and pleiotrophin 12 is triggered by hypoxia through the action of hypoxia-inducible factors (HIFs); these angiogenic factors play important roles in blood vessel formation and oncogenesis. 13 ...

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