HIF prolyl hydroxylase-3 mediates alpha-ketoglutarate-induced apoptosis and tumor suppression

Many solid tumors consist of large regions of poorly perfused cells, resulting in areas of low oxygen (hypoxia) throughout the cell mass. Cells subjected to hypoxia turn on a complex set of responses that alter their metabolism, rebalance their survival mechanisms, increase their invasive capacity, and stimulate angiogenesis. This allows them to at least temporarily escape the nutrient starvation and cell death resulting from this hostile environment. Accordingly, the hypoxic regions of tumors are often sources of the most aggressive and therapy-resistant cells, and therefore those cells that drive tumorigenesis. The hypoxia inducible factor (HIF) prolyl hydroxylases (PHDs) are enzymes that are functionally inactivated in hypoxia, as they use both oxygen and α-ketoglutarate as substrates to hydroxylate target prolyl residues. Although HIF1α, the most highly characterized PHD target, orchestrates many of the cellular responses to hypoxia observed in tumors, PHDs themselves have previously been shown to regulate some hypoxia responses, including apoptosis, in a HIF-independent mechanism. We have previously shown that PHDs can be reactivated under hypoxia and that this results in a metabolic defect, both in vitro and in vivo. This led us to investigate whether chronic reactivation of these enzymes may inhibit tumor progression. We show here that esterified α-ketoglutarate given daily will induce apoptosis and inhibit tumor growth, in vivo. The effects are independent of HIF1α but dependent on the presence of PHD3. These data suggest that PHD3 may be a valid target in vivo for anti-tumor therapy.     

DNA methylation analysis of the HIF‐1α prolyl hydroxylase domain genes PHD1, PHD2, PHD3 and the factor inhibiting HIF gene FIH in invasive breast carcinomas

Huang K T, Mikeska T, Dobrovic A & Fox S B
(2010) Histopathology 57, 451–460
DNA methylation analysis of the HIF‐1α prolyl hydroxylase domain genes PHD1, PHD2, PHD3 and the factor inhibiting HIF gene FIH in invasive breast carcinomas Aims: Hypoxia‐inducible factor‐1 (HIF‐1) activity is regulated by prolyl hydroxylase (PHD1, PHD2, PHD3) and factor inhibiting HIF‐1 (FIH) that target the α subunit of HIF‐1 (HIF‐1α) for proteosomal degradation. We hypothesised that the elevated HIF‐1α level is due in some tumours to epigenetic silencing by DNA hypermethylation of the promoter region of one or more of the PHDs and FIH genes. The aims were to define the presence or absence of promoter methylation of PHDs and FIH in cell lines of various sources and breast carcinomas and, if present, determine its effect on mRNA and protein expression. Methods and results: Tumour cell lines (n = 20) and primary invasive breast carcinomas (n = 168) were examined for promoter region DNA methylation using methylation‐sensitive high‐resolution melting. There was evidence of PHD3 but not of PHD1, PHD2 or FIH DNA methylation in breast cancer (SkBr3) and leukaemic (HL60 and CCRF‐CEM) cell lines, but there was no evidence of methylation in any of 168 breast cancers. Only the high‐level PHD3 methylation seen in leukaemic cell lines correlated with absent mRNA and protein expression. Conclusions: Methylation‐induced epigenetic silencing of PHD1, PHD2, PHD3 and FIH is unlikely to underlie up‐regulated HIF‐1α expression in human breast cancer but may play a role in other tumour types.     

缺氧诱导因子家族蛋白的稳定性与转录活性的调节

缺氧诱导因子是调控哺乳动物缺氧适应性反应的一类关键转录因子,能够调节100多种靶基因的表达从而使机体与组织细胞适应外周环境氧浓度的变化。在组织细胞中缺氧诱导因子家族蛋白稳定性与活性受到严密的调节,主要方式有:羟基化、泛素化、乙酰化、磷酸化、小泛素样因子修饰。     

Oxygen-sensing PHDs regulate bone homeostasis through the modulation of osteoprotegerin

The bone microenvironment is composed of niches that house cells across variable oxygen tensions. However, the contribution of oxygen gradients in regulating bone and blood homeostasis remains unknown. Here, we generated mice with either single or combined genetic inactivation of the critical oxygen-sensing prolyl hydroxylase (PHD) enzymes (PHD1–3) in osteoprogenitors. Hypoxia-inducible factor (HIF) activation associated with Phd2 and Phd3 inactivation drove bone accumulation by modulating osteoblastic/osteoclastic cross-talk through the direct regulation of osteoprotegerin (OPG). In contrast, combined inactivation of Phd1, Phd2, and Phd3 resulted in extreme HIF signaling, leading to polycythemia and excessive bone accumulation by overstimulating angiogenic–osteogenic coupling. We also demonstrate that genetic ablation of Phd2 and Phd3 was sufficient to protect ovariectomized mice against bone loss without disrupting hematopoietic homeostasis. Importantly, we identify OPG as a HIF target gene capable of directing osteoblast-mediated osteoclastogenesis to regulate bone homeostasis. Here, we show that coordinated activation of specific PHD isoforms fine-tunes the osteoblastic response to hypoxia, thereby directing two important aspects of bone physiology: cross-talk between osteoblasts and osteoclasts and angiogenic–osteogenic coupling.     

Myeloid hypoxia-inducible factors in inflammatory diseases

Hypoxia inducible factors (HIF1 and HIF2) have emerged as central regulators of the activity of myeloid cells at inflammatory sites where O(2) is frequently limited. Novel insights in the field have revealed that the expression of HIFs by myeloid cells is not exclusively induced by hypoxia but also in response to central inflammatory mediators independently of O(2) shortage. This has substantially elevated the biological significance of HIFs in the context of inflammatory diseases. As a consequence, the loss of HIF1 or HIF2 in myeloid cells specifically compro-mises some of the processes driven by myeloid cells, such as bactericidal activity and myeloid invasion, as well as inflammation-associated detrimental consequences.     

Hypoxia induces expression of connective tissue growth factor in scleroderma skin fibroblasts

Connective tissue growth factor (CTGF) plays a role in the fibrotic process of systemic sclerosis (SSc). Because hypoxia is associated with fibrosis in several profibrogenic conditions, we investigated whether CTGF expression in SSc fibroblasts is regulated by hypoxia. Dermal fibroblasts from patients with SSc and healthy controls were cultured in the presence of hypoxia or cobalt chloride (CoCl(2)), a chemical inducer of hypoxia-inducible factor (HIF)-1alpha. Expression of CTGF was evaluated by Northern and Western blot analyses. Dermal fibroblasts exposed to hypoxia (1% O(2)) or CoCl(2) (1-100 microM) enhanced expression of CTGF mRNA. Skin fibroblasts transfected with HIF-1alpha showed the increased levels of CTGF protein and mRNA, as well as nuclear staining of HIF-1alpha, which was enhanced further by treatment of CoCl(2). Simultaneous treatment of CoCl(2) and transforming growth factor (TGF)-beta additively increased CTGF mRNA in dermal fibroblasts. Interferon-gamma inhibited the TGF-beta-induced CTGF mRNA expression dose-dependently in dermal fibroblasts, but they failed to hamper the CoCl(2)-induced CTGF mRNA expression. In addition, CoCl(2) treatment increased nuclear factor (NF)-kappaB binding activity for CTGF mRNA, while decreasing IkappaBalpha expression in dermal fibroblasts. Our data suggest that hypoxia, caused possibly by microvascular alterations, up-regulates CTGF expression through the activation of HIF-1alpha in dermal fibroblasts of SSc patients, and thereby contributes to the progression of skin fibrosis.