Prior Research

At the center of our studies is Hypoxia Inducible Factor 2 (HIF-2), the second of three related and highly conserved stress-activated transcription factors in vertebrates. Over the years, we have made novel contributions to the HIF-2 field, which have advanced our overall understanding of stress signaling in mammals. Here are highlights of our past and current research:

Generation of a viable adult HIF-2 knockout mouse model

 
This figure demonstrates multi-organ pathological findings present in HIF-2 knockout mice, which are consistent with apparent mitochondrial dysfunction. The pathological findings include retinal histological and functional abnormalities in panel (A), gross anatomical evidence of hepatosteatosis plus abnormal acyl carnitine serum profiles in panel (B), and gross anatomical changes and echocardiography findings consistent with cardiomegaly in panel (C).

The founding member of the HIF family, HIF-1, was biochemically purified from hypoxic cells by Gregg Semenza at Johns Hopkins University. HIF-2 was identified from the human genome project almost simultaneously by several groups around the world. This was followed by the generation of several global knockout mouse models lacking HIF-2. Due to developmental or early post-natal lethality of conventional HIF knockout mice, the physiological role of HIF-1 and HIF-2 eluded HIF investigators for several years. Using a genetic breeding strategy, we generated viable adult mice lacking HIF-2 in all cell types. Studies of these HIF-2 knockout mice led to an unanticipated finding that HIF-2 regulates Sod2 and Cas1 (Cat), genes encoding major antioxidant enzymes located in mitochondria and peroxisome, respectively. HIF-2 knockout mice are a virtual phenocopy of Sod2 knockout mice and exhibit dysfunction of multiple organs consistent with an apparent mitochondrial defect.

  • Scortegagna M, Ding K, Oktay Y, Gaur A, Thurmond F, Yan LJ, Marck BT, Matsumoto AM, Shelton JM, Richardson JA, Bennett MJ, Garcia JA. Multiple organ pathology, metabolic abnormalities and impaired homeostasis of reactive oxygen species in Epas1-/- mice. Nat Genet 35(4):331-40 (2003), PMID: 14608355.
  • Ding K, Scortegagna M, Seaman R, Birch DG, Garcia JA. Retinal disease in mice lacking hypoxia-inducible transcription factor-2alpha. Invest Ophthalmol Vis Sci 46(3):1010-6 (2005), PMID: 15728559.

Identification of HIF-2 as the regulator of endocrine erythopoitein production

This figure reveals that HIF-2 is a key regulator of erythropoitein and red blood cell homeostasis in mice. In panel (A), HIF-2 knockout mice (black bars) have reduced hematocrit levels relative to wild-type mice (white bars). In panel (B), there is no reduction in kidney size relative to body size for knockout mice (black bars) relative to wild-type mice (white bars). In panel (C), knockout mice (black bars) relative to wild-type mice (white bars) have reduced erythropoietin (Epo) messenger RNA levels under either room air (RA) or following exposure to short-term intermittent hypoxia (STIH).

One characteristic of conventional HIF-2 global knockout mice evident early on was reduced red blood cell levels. While evaluating the molecular basis for this phenotype, we identified HIF-2 as the major regulator of endocrine erythropoietin (Epo) production. This surprising finding led to the recognition of HIF-2, rather than HIF-1, as the major HIF member responsible for transcriptional activation of Epo in late embryonic, juvenile, and adult mice. Other labs using conditional knockout of HIF-2 in mice subsequently confirmed the role of HIF-2 in hepatic and renal endocrine Epo regulation. Findings from multiple groups revealed that mutations impairing or enhancing HIF-2 signaling affect Epo production in cells or mice in a parallel manner. The findings in mice were confirmed in humans by other investigators who found HIF-2 mutations affecting Epo levels and hematocrit levels in affected patients.

  • Scortegagna M, Morris MA, Oktay Y, Bennett M, Garcia JA. The HIF family member EPAS1/HIF-2alpha is required for normal hematopoiesis in mice. Blood 102(5):1634-40 (2003), PMID: 12750163.
  • Scortegagna M, Ding K, Zhang Q, Oktay Y, Bennett MJ, Bennett M, Shelton JM, Richardson JA, Moe O, Garcia JA. HIF-2alpha regulates murine hematopoietic development in an erythropoietin-dependent manner. Blood 105(8):3133-40 (2005), PMID: 15626745.

Delineating the Acss2/Cbp/Sirt1/HIF-2 signaling axis

This figure is our current working model for Acss2/CBP/Sirt1/HIF-2 signaling axis. Endogenous acetate, generated in response to reduced oxygen levels, directs production of a nuclear acetyl CoA pool following ACSS2 translocation. CBP uses this nuclear acetyl CoA pool for HIF-2α acetylation and thereby facilitates CBP/HIF-2α complex formation to augment HIF-2 signaling. When acetylation is complete, CBP is released from HIF-2α and SIRT1 deacetylates HIF-2α, which allows CBP to re-engage HIF-2 as long as the ACSS2-generated acetyl CoA pool is available. In the absence of this acetyl CoA pool, HIF-2α complexes with p300 during hypoxia, but p300/HIF-2α is inefficient at inducing HIF-2 signaling compared to CBP/HIF-2α.

With an intent to delineate the signal transduction pathway for HIF-2 signaling, we sought potentially relevant findings from other stress signal transducers. The major antioxidant enzyme-encoding Sod2 and Cas1 (Cat) genes are also regulated by another important stress-activated transciptional regulator, FoxO. Because HIF-2 knockout mice closely resemble Sod2 knockout mice, we reasoned that HIF-2 functions in a parallel pathway to FoxO signaling and shares some common target genes including Sod2. Based on these similarities, we hypothesized and subsequently confirmed that HIF-2 is acetylated by the same cellular factors that regulate FoxO acetylation/deacetylation, the acetyltransferase Creb-binding protein (Cbp) and the genetic regulator/deacetylase Sirtuin 1 (Sirt1).

 
  • Dioum EM, Chen R, Alexander MS, Zhang Q, Hogg RT, Gerard RD, Garcia JA. Regulation of Hypoxia Inducible Factor 2 alpha signaling by the stress-responsive deacetylase Sirtuin 1. Science, 324(5932):1289-93 (2009), PMID: 19498162.
  • Chen R, Xu M, Hogg RT, Li J, Little B, Gerard RD, Garcia JA. The acetylase/deacetylase couple Creb Binding Protein/Sirtuin 1 controls Hypoxia Inducible Factor 2 signaling. J Biol Chem, 287(36):30800-11 (2012), PMID: 22807441.

Identifying beneficial physiological roles for Acss2/HIF-2 signaling

This figure demonstrates that acetate supplementation increases hematocrit and erythropoietin levels in chronically anemic mice. Partial nephrectomy induces chronic anemia as a result of renal failure. Before (Pre) partial nephrectomy, wild-type mice had normal hematocrit and low serum erythropoietin (Epo) levels as plotted in panels (A) and (B), respectively. Following (Post) partial nephrectomy, these same mice had markedly reduced hematocrit levels and increased Epo levels. When treated thrice-weekly with acetate (Ac) injections, hematocrits and Epo levels increased significantly with acetate, but not vehicle (Veh), treatment.

We subsequently identified the rate-limiting step for Cbp-mediated acetylation of HIF-2 as availability of a specific acetyl CoA pool produced by an acetate-dependent acetyl CoA generator, Acss2. Although predominantly cytosolic, we showed that Acss2 translocates into, or is enriched in, the nucleus during oxygen deprivation (hypoxia). In translational studies employing cell and mouse models, we found that Acss2 is required for maximal HIF-2 signaling and Epo induction during hypoxia or with anemia. Supplementation of cells or mice with acetate also augmented Acss2/HIF-2 signaling. Induction of Acss2/HIF-2 signaling by acetate in mice resulted in faster recovery from an acute anemia model and raised steady-state hematocrit levels in chronic anemia models.

  • Xu M, Nagati JS, Xie J, Li J, Walters H, Moon Y-A, Gerard RD, Huang C-L, Comerford SA, Hammer RE, Horton JD, Chen R, Garcia JA. A mammalian acetate switch regulates stress erythropoiesis. Nature Medicine, 20(9):1018-26 (2014), PMID: 25108527.

Identifying detrimental pathophysiological roles for Acss2/HIF-2 signaling

This figure uses immunofluorescence microscopy to demonstrate that wild-type Acss2 translocates from the cytosol into the nucleus in mammalian cells following exposure to reduced oxygen, reduced glucose, or increased acetate conditions. Under ambient conditions in panel (A), wild-type (WT) and cytosol-restricted mutant (CYT) Acss2 reside mainly in the cytosol. Under reduced oxygen (hypoxia), reduced glucose (low glucose), or acetate-supplemented conditions in panels (B), (C), and (D), respectively, only WT Acss2 translocates from the cytosol into the nucleus.

As with other important pro-survival signal transducers, HIF signaling can be usurped by cancer cells to provide a growth advantage in solid tumors, which also experience oxygen and glucose deprivation. Accordingly, augmenting Acss2/HIF-2 signaling promotes whereas impeding Acss2/HIF-2 signaling impairs the growth and development of tumors in a mouse model. Similar to its behavior during oxygen deprivation (hypoxia), we found that Acss2 translocates into the nucleus during glucose deprivation (hypoglycemia) as well as following addition of acetate to the cell culture media. Enzymatically active Acss2 that is restricted to the cytosol cannot induce HIF-2 signaling, which supports a nuclear-specific role for Acss2 in addition to its role in the cytosol.

  • Chen R, Xu M, Nagati JS, Hogg RT, Das A, Gerard RD, Garcia JA. The acetate/ACSS2 switch regulates HIF-2 stress signaling in the tumor cell microenvironment. PLoS ONE, 10(2):e0116515 (2015), PMID: 25689462.
  • Chen R, Xu M, Nagati JS, Garcia JA. Coordinate regulation of stress signaling and epigenetic events by Acss2 and HIF-2 in cancer cells. PLoS ONE, 12(12):e0190241 (2017), PMID: 29281714.