Mitochondria Regulate Hypoxic Stabilization of Hypoxiainducible Factor1

Cells respond to hypoxia by activating a multitude of responses designed to prevent cells from reaching 0% oxygen. The best-characterized cellular response is the activation of the transcription factor HIF-1. HIF-1 is a dimeric transcription factor composed of HIF-1 a and HIF-1p subunits. HIF-1 is a transcriptional activator that is required for the upregulation expression of genes encoding for vascular endothelial growth factor, erythropoietin, glycolytic enzymes, and endothelin-1 in response to low oxygen concentration (27). HIF-1 plays important roles in normal development, physiological responses to hypoxia, and the pathophysiology of common human diseases. In mice, complete HIF-la deficiency results in embryonic lethality at mid-gestation because of cardiac and vascular malformations (17). Mice that are partially HIF-la deficient due to the loss of one allele (heterozygous) develop normally. However, when these mice are subjected to long-term hypoxia (10% 02 for 3 weeks), they have impaired hypoxia-induced pulmonary hypertension as indicated by a diminished medial wall hypertrophy in small pulmonary arterioles (37). Furthermore, chronic hypoxia increases cell capacitance, reduces Kv current density, and depolarizes PASMCs, and these effects are reduced or absent in PASMCs from mice partially deficient for HIF-1 (28). The mechanism underlying hypoxic activation of HIF-1 is important for understanding the pathology of diseases such as pulmonary hypertension.

Figure 3. Prolyl hydroxylases (PHD) catalyze hydroxy lation of proline residues in HIF-la under normal oxygen conditions. Proline hydroxylation requires oxygen as a substrate and iron as a cofactor. Prolyl hydroxylation is recognized by pVHL which targets HIF-la for ubiquitination and subsequent protein degradation by the 26S proteasome. Under hypoxia, HIF-la is not hydroxylated thereby HIF-la protein interacts with HIF-113

HIF-1 is a heterodimer of two basic helix loop-helix/PAS proteins, HIF-la and the aryl hydrocarbon nuclear trans-locator (ARNT orHIF-ip) (33). ARNT protein levels are constitutively expressed and not significantly affected by oxygen. In contrast, HIF-la protein is present only in hypoxic cells. During normoxia (21% 02), HIF-la is polyubiquitinated by an E3 ubiquitin ligase complex that contains the von Hippel-Lindau tumor suppressor protein (pVHL), elongin B, elongin C, Cul2, and Rbx1 (22, 24). The binding of pVHL to the oxygen-dependent degradation (ODD) domain is located in the central region of HIF-la. pVHL binding to HIF-la is dependent on the hydroxylation of proline residues within HIF-1 a (Fig. 3) (16, 18). This hydroxylated prolyl residue forms two critical hydrogen bonds with p VHL side chains present within the (3 domain.

Normoxia

Hypoxia

(HlFta)

Hypoxia

(HlFta)

Hydroxy Hif

Figure 3. Prolyl hydroxylases (PHD) catalyze hydroxy lation of proline residues in HIF-la under normal oxygen conditions. Proline hydroxylation requires oxygen as a substrate and iron as a cofactor. Prolyl hydroxylation is recognized by pVHL which targets HIF-la for ubiquitination and subsequent protein degradation by the 26S proteasome. Under hypoxia, HIF-la is not hydroxylated thereby HIF-la protein interacts with HIF-113

Normoxia

This constitutes the pVHL substrate recognition unit. The enzymatic hydroxylation reaction is inherently oxygen-dependent since the oxygen atom of the hydroxy group is derived from molecular oxygen. In addition, prolyl hydroxylation requires 2-oxoglutarate and iron as cofactors. 2-oxoglutarate is required because the hydroxylation reaction is coupled to the decarboxylation of 2-oxoglutarate to succinate, which accepts the remaining oxygen atom. In mammalian cells, HIF prolyl hydroxylation is carried out by one of three homologs of C. elegans Egl-9 (EGLN1, EGLN2, and EGLN3; also called PHD2, PHD1, and PHD3, respectively, or HPH-2, HPH-3, and HPH-1, respectively) (9). Presumably, the prolyl hydroxylated mediated degradation of HIF-1 a protein is suppressed under hypoxic conditions ranging from 0-5% 02.

A fundamental question for understanding HIF-la regulation involves the mechanism by which cells sense the lack of oxygen and initiate a signaling cascade that results in the stabilization of HIF-la protein. Early progress in understanding molecular mechanisms underlying mammalian oxygen sensing came from the observation that erythropoietin mRNA can be induced under normoxic conditions in the human hepatoma Hep3B cell line by incubation with transition metals such as cobalt and iron chelators such as desferrioxamine (DFO) (12). This led to the proposal that a rapidly turning over heme protein capable of interacting with 02 is a putative oxygen sensor. However, studies using heme synthesis inhibitors failed to show any effect on the hypoxic activation of HIF-1, suggesting that rapidly turning over heme proteins are not involved in hypoxia sensing (29). Subsequently, the NADPH oxidase was proposed as a possible oxygen sensor by decreasing reactive oxygen species (ROS) generation during hypoxia (1). The decrease in ROS triggers the stabilization of HIF-1. However, this model is confounded by the observation that DPI, a wide-ranging inhibitor of flavoprotein-containing enzymes including NAD(P)H oxidase, does not trigger HIF-1 stabilization during normoxia. Rather, DPI inhibits the hypoxic induction of HIF-1 dependent genes (11). We propose a model in which the increased generation of reactive oxygen species at complex III of the mitochondrial electron transport chain serves as the oxygen sensor for HIF-la protein stabilization during hypoxia. In support of this model, hypoxia increases ROS generation, HIF-la protein accumulation, and the expression of a luciferase reporter construct under the control of a hypoxic response element in wild-type cells, but not in cells depleted of their mitochondrial DNA (p0 cells) (5, 6). Furthermore, catalase overexpression abolishes the luciferase expression in response to hypoxia. Hydrogen peroxide can stabilize protein levels and activate luciferase expression under normoxic conditions in both wild-type and cells. Thus, ROS are both required and sufficient to trigger HIF-1 activation. The site of ROS generation during hypoxia is localized to complex III within the mitochondrial electron transport chain. Mitochondrial complex I inhibitors, such as rotenone, that prevent electron flux upstream of complex III, ablate ROS generation during hypoxia and subsequently, the hypoxic stabilization of HIF-la protein stabilization. These results have been corroborated by Lamanna and colleagues who have demonstrated that the neurotoxin 1-methyl-4-phenyl-1,2, 3,6-tetrahydropyridine(MPTP), a complex I inhibitor, prevents the hypoxic stabilization of HIF-laproteininPC 12 cells (2). These investigators have also shown that hypoxic stabilization of HIF-la protein is severely reduced in human xenomitochondrial cybrids harboring a partial (40%) complex I deficiency. Further evidence that mitochondria regulate HIF-1 activation comes from Stratford and colleagues, who demonstrated that the complex IV inhibitor cyanide is sufficient to activate HIF-1 dependent transcription in wild-type Chinese hamster ovary (CHO) cells and HT1080 cells under normoxic conditions (36). Cyanide inhibits electron transport chain downstream of complex III, thus eliciting an increase in ROS generation.

Recently, Ratcliffe and colleagues have challenged the role of mitochondria as a potential oxygen sensor. These investigators have demonstrated that p° cells are able to stabilize HIF-la protein levels at oxygen concentration of 0.1% 02 (32). They have proposed that the prolyl hydroxylases are oxygen sensors that regulate hypoxic stabilization of HIF-1 a protein. This model is based on the observation that prolyl hydroxylases require molecular oxygen and iron to catalyze the hydro xylation of proline residues within HIF-la. In the absence of oxygen or iron, HIF-1 a would not undergo proline hydroxylation and subsequent pVHL mediated ubiquitin-targeted degradation. The hydroxylation inhibition due to anoxia or iron chelation would indicate prolyl hydroxylases as the sensor. However, it is not known if prolyl hydroxylase would intrinsically be inhibited at oxygen concentration as low as 1-2%, where HIF-1 is activated. We recently investigated the response to p° cells to both hypoxia and anoxia because Ratcliffe and colleagues used conditions close to anoxia in examining their hypoxic response to p° cells. Our results demonstrate that the stabilization of HIF-la protein at 1-2% 02 did not occur in p° cells. However, p° cells were able to stabilize HIF-la protein at 0% 02 or in the presence of an iron chelator under normoxic conditions (26). This observation is consistent with the requirement of proline hydroxylation as a mechanism for HIF-la protein degradation under normal oxygen conditions. In the absence of oxygen, hydroxylation of proline residues within HIF-la by prolyl hydroxylases cannot occur and intracellular signaling events are not required for the stabilization of HIF-la protein. Thus, prolyl hydroxylases would effectively serve directly as the oxygen sensors during anoxia or during iron chelation under normoxia. Furthermore, if prolyl hydroxylase is, by itself, the oxygen sensor for both hypoxia- and anoxia-induced HIF-1 then there would be no signaling required upstream of prolyl hydroxylase, i.e., kinases/ROS upstream ofprolyl hydroxylase. However, other investigators have shown that hypoxia (1% 02) stimulates Racl activity, and Rac1 is required for the hypoxic stabilization of HIF-la protein (15). Both the hypoxic activation of Rac1 and the stabilization of HIF-la protein were abolished by the complex I inhibitor rotenone. These results indicate that Rac1

is downstream of mitochondrial signaling. Moreover, mitochondrial dependent oxidant signaling has been shown to regulate HIF-la protein accumulation following exposure to TNFa (14). Non-mitochondrial dependent oxidant signaling has also been shown to stabilize HIF-la protein under normoxia. For example, thrombin or angiotensin II stabilizes HIF-la under normoxia through an increase in ROS generation from non-mitochondrial sources (13). Further support for the idea that hypoxic signaling is distinct from anoxia or iron chelation comes from the observation that DPI, an inhibitor of a wide range of flavoproteins including complex I, prevents stabilization ofHIF-la protein and HIF-1 target genes at oxygen levels of 1% (11). However, DPI fails to affect stabilization of HIF-1 in response to the iron chelator desferrioxamine (DFO). This observation is consistent with the notion that iron chelators or lack of oxygen directly inhibit prolyl hydroxylase activity due to substrate limitations and stabilize HIF-la protein (Fig. 3). Interestingly, DPI can prevent a variety of other hypoxic responses such as pulmonary vasoconstriction and carotid body nerve firing (1). We speculate that the ultimate target of the oxidant dependent signaling pathway originating from mitochondria during hypoxia or non-mitochondrial sources such as angiotensin II during normoxia is to inhibit proline hydroxylation (Fig. 4).

Hypoxia

Anoxia j_ROS Prolyl Hydroxylase1 1

HIFloc —Proline —Ubiqui tin-media ted Degradation

Figure 4. Hypoxia requires a functional mitochondrial electron transport chain to initiate oxidant dependent signaling that ultimately inhibits prolyl hydroxylases and stabilizes HIF-la. By contrast, in the absence of oxygen (anoxia), proline hydroxylation cannot occur because oxygen is a required substrate for hydroxylation. Thus, anoxia would directly inhibit prolyl hydroxylases thereby stabilizing HIF-la.

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