MicroRNA-210 Controls Mitochondrial Metabolism during Hypoxia by Repressing the Iron-Sulfur Cluster Assembly Proteins ISCU1/2

“First, by induction of PDK1, HIF-1a decreases the activity of the TCA cycle, increases glycolysis, and reduces mitochondrial electron flux (Kim et al., 2006).”

“280 Cell Metabolism 10, 273–284, October 7, 2009 ª2009 Elsevier Inc. to oxidative stress (Beinert et al., 1997), along with repression of iron-sulfur cluster biogenesis via downregulated ISCU1/2, may provide a real-time redox-sensitive ‘‘switch’’ that is triggered by hypoxia to induce downstream…”

“Second, HIF-1a induces a COX4-1 to COX4-2 subunit switch during hypoxia to improve the efficiency of electron transfer to oxygen at Complex IV (Fukuda et al., 2007).”

“Conditions were based on studies of hypoxic exposure of HPAECs (Manalo et al., 2005) to allow for steady-state adaptation without nonspecific cell death.”

“…with other studies demonstrating the repression of apoptosis by miR-210 during hypoxia in other cellular contexts (Fasanaro et al., 2008; Kulshreshtha et al., 2007), these data lend credence to a model in which miR-210 carries a maladaptive role during normoxia, causing a mismatch of…”

“In particular, this response may be especially important to the pulmonary vascular endothelium, which directly responds to hypoxia by controlling the release of vasoactive effectors, thereby critically influencing normal vascular physiology and the development of pulmonary hypertension (Stenmark et al., 2006).”

“Some, but not all, of these miRNA have been identified in other transformed (Kulshreshtha et al., 2007) or primary endothelial cells (Fasanaro et al., 2008) exposed to hypoxia.”

“Additionally, miR-210 is specifically induced by HIF-1a but not HIF-2a (Camps et al., 2008), a second transcriptional regulator of the hypoxic response, which carries partial but not full redundancy in function with HIF-1a (Wang et al., 2005).”

“The elucidation of the miR-210-ISCU1/2 regulatory pathway substantially clarifies the nature of the complex crosstalk between HIF-1a and mitochondria (Seagroves et al., 2001) and complements other HIF-dependent processes proposed to regulate the mismatch of reduced oxygen tension and mitochondrial electron transport.”

“Thus, these coordinated changes allow HIF-1a to manipulate multiple targets and ensure precise, fail-safe control over energy production and cellular survival during hypoxic stress (Semenza, 2007).”

“The cellcycle regulator E2F3 (Giannakakis et al., 2008), the receptor tyrosine kinase ligand ephrin A3 (Fasanaro et al., 2008), and the DNA repair protein RAD52 (Crosby et al., 2009) have been studied as targets for repression by miR-210; however, the complete functions of this miRNA have yet to be delineated.”

“To demonstrate downregulation of ISCU1/2 during HIF-1a expression in vivo, tissue was analyzed from mice carrying tamoxifen-dependent, conditionally inactivated von Hippel Lindau alleles (VHL / ), an established model of sustained stabilization of HIF proteins during nonhypoxic, or ‘‘normoxic,’’ conditions (Haase et a…”

“The degree of alteration in these parameters is consistent with the dependence of cultured endothelial cells on OXPHOS for a minority of total ATP production (Mann et al., 2003).”

“, rotenone) (Wolvetang et al., 1994) or by disruption of iron-sulfur clusters in the mitochondrial complexes themselves (Napoli et al.”

“ISCU1/2 facilitate the assembly of [4Fe-4S] and [2Fe-2S] ironsulfur clusters, prosthetic groups that promote electron transport and oxidation-reduction reactions integral to numerous cellular processes, including ribosome biogenesis, purine catabolism, heme biosynthesis, DNA repair, and iron metabolism, among others (…”

“The cellcycle regulator E2F3 (Giannakakis et al., 2008), the receptor tyrosine kinase ligand ephrin A3 (Fasanaro et al., 2008), and the DNA repair protein RAD52 (Crosby et al., 2009) have been studied as targets for repression by miR-210; however, the complete functions of this miRNA have yet to be delineated.”

“Additionally, miR-210 is specifically induced by HIF-1a but not HIF-2a (Camps et al., 2008), a second transcriptional regulator of the hypoxic response, which carries partial but not full redundancy in function with HIF-1a (Wang et al., 2005).”

“The characterization of this type of prosthetic group as a key determinant of this metabolic shift during hypoxia correlates with the known function in bacteria of iron-sulfur clusters as intracellular sensors of oxygen (Lazazzera et al., 1996).”

“Variability in the level of downregulation among littermate pairs likely reflects variation in the degree of VHL inactivation after tamoxifen exposure (Minamishima et al., 2008).”

“Therefore, correlating with the known function of ISCU1/2 in maintaining the iron-sulfur clusters in aconitase (Tong and Rouault, 2006) and the disruption of iron-sulfur clusters in miR-210-positive tissue (Figure 3A), this disruption of aconitase activity likely results from miR-210- and ISCU-dependent repression of…”

“274 Cell Metabolism 10, 273–284, October 7, 2009 a2009 Elsevier I assessed if they were previously reported as upregulated by hypoxia (Kulshreshtha et al., 2008) or by hypoxia-associated stimuli (such as inflammation).”

“In contrast to HIF proteins, which increase during acute hypoxia but are inactivated with chronic hypoxia (i.e., >24 hr of hypoxia) (Ginouves et al., 2008), miR-210 and repression of ISCU1/2 display sustained periods of action and may, therefore, reflect a chronic, rather than transient, adaptation…”

“These may include iron metabolism, which is controlled by ISCU1/2 activity (Tong and Rouault, 2006) and hypoxia (Meyron-Holtz et al., 2004) via mechanisms that have only been partially delineated.”

“In comparison to tamoxifen-treated VHL flox/flox littermate control mice (referred to as ‘‘WT’’), VHL / mice demonstrate significantly higher levels of miR210 in kidney, liver, and heart (Figure 2C), corresponding with elevated HIF-1a expression (Young et al., 2008).”

“In correlation, a mutation in ISCU1/2 has recently been linked to a human genetic syndrome leading to myopathy and lactic acidosis (Mochel et al., 2008).”

“Endogenous microRNA molecules (miRNA) have been identified as essential mediators of numerous cellular processes, potentially including the response to hypoxia (Ivan et al., 2008).”

“To ascertain a link between miR-210 and activity of mitochondrial iron-sulfur proteins essential for electron transport, Complex I activity was assessed, given its dependence on multiple ironsulfur clusters for proper function (Hinchliffe and Sazanov, 2005).”

“Both transcripts carry an identical 30UTR, but they differ in their location: ISCU1 is located in the cytosol, whereas Metabolism 10, 273–284, October 7, 2009 ª2009 Elsevier Inc. 273 ISCU2 is located in the mitochondria (Tong and Rouault, 2000).”

“Studies in frataxin-deficient mice (Thierbach et al., 2005) have revealed its critical role in augmenting OXPHOS and altering iron homeostasis.”

“…delivery of electrons in mitochondria, either by pharmacologic inhibitors of electron transport (i.e., rotenone) (Wolvetang et al., 1994) or by disruption of iron-sulfur clusters in the mitochondrial complexes themselves (Napoli et al., 2006), leads to increased apoptosis and increased ROS flux.”

“Hypoxia-inducible factor-1 alpha (HIF-1a), a master regulator of the cellular hypoxic response, has been shown to control mitochondrial function (Chavez et al., 2008) and is essential for this repression of mitochondrial respiration (Seagroves et al.”

“Among the targets of miR-210 that are downregulated in hypoxic cells are mRNAs encoded by the ISCU gene, which encodes an iron-sulfur cluster assembly enzyme that is required for the activity of mitochondrial electron transport complex I (41, 42).”