The transcription factor hypoxia-inducible factor (HIF) plays a critical role in the mammalian response to oxygen (O2
) levels. HIF1, the first characterized member of the HIF family, transcriptionally activates hundreds of genes associated with angiogenesis in cancer, exercise, and ischemia, as well as energy metabolism, nutrient transport, cell cycle, and cell migration (85
HIF1α and HIF1β make up the HIF1 heterodimer. The β-subunit is constitutively expressed in cells. Expression of the α-subunit may be induced by a number of pathways, and its degradation is highly sensitive to O2
levels. Called a master switch for hypoxic gene expression (76
), intracellular HIF1α in normoxia is experimentally undetectable; during hypoxia, it rapidly accumulates in the cell nucleus and triggers gene expression. Molecular players involved in this process have come to light over the past 6 years; research has begun to define roles for prolyl hydroxylases, iron, ascorbate, hydrogen peroxide, 2-oxoglutarate, succinate, and von Hippel-Lindau protein in the HIF1 pathway.
Concomitantly, the study of reactive oxygen species (ROS) and the interest in antioxidants as potential dietary supplements for prevention of cancer, cardiac dysfunction, and neurodegeneration has grown rapidly. Ongoing debate surrounds the role of these compounds in hypoxic responses and the utility in pursuing them as preventative therapeutics. Some studies have shown increased ROS expression in hypoxia (10
), while others show a decrease (33
). Increased HIF1α expression has been found to contribute to mitochondrial activity (1
), and specifically ROS formation, during hypoxia (26
). However, other studies have demonstrated a decrease in HIF1α with increasing ROS (22
). Finally, some studies have shown no effects of H2
) or mitochondrial ROS in general (96
). Related observations seem nearly as conflicting. Under hypoxic conditions, mitochondrial complex III may produce ROS, and the presence of high ROS concentrations generated from the mitochondria has been shown to stabilize HIF1α (8
). On the other hand, ROS may be produced in the cytosol, derived from NADPH oxidases (17
), and ROS may play a larger role in HIF1α expression during normoxia than hypoxia (43
There are several hypotheses as to how ROS interact with the HIF1 pathway and alter HIF1α expression (recent related reviews include references 41
). One possibility is that hydrogen peroxide oxidizes ferrous iron (Fe2+
) to its ferric form (Fe3+
), prohibiting the necessary binding of ferrous iron to the HIF1α hydroxylation enzymes, prolyl hydroxylases (PHDs) (71
). Another change could be in the recruitment of ascorbate as a free radical scavenger, preventing ascorbate from reducing ferric iron and/or preventing ascorbate from binding directly to the PHDs. If ROS increased rather than decreased free Fe2+
, as suggested by some experiments, HIF1α hydroxylation would instead increase (56
). Additionally, 2-oxoglutarate (2OG) and succinate (SC) are also compounds involved in HIF1α hydroxylation whose concentrations could be altered by free radicals and mitochondrial dysfunction (38
). A fourth mechanism by which ROS could influence the HIF1 pathway is through changing the availability of oxygen to bind directly to the PHDs or changing PHD phosphorylation.
To address these alternate mechanisms and analyze possible competing factors involved in pro- and antioxidant therapy in cancer and ischemia, we developed a computational model describing the in vivo system and used it to observe dynamics currently inaccessible at the molecular level in vivo. Experimentally, ROS have been shown to affect the HIF1 pathway through changes in H2
, Asc, 2OG, or SC levels (61
), and mechanisms involving these compounds were the focus of this study.
The model consists of kinetic equations mapping the molecular steps in HIF1α degradation in normoxia, HIF1α synthesis in chronic hypoxia, and effects of the enzyme and cofactors involved in the HIF hydroxylation pathway. Kinetic values were estimated from in vitro studies, and results were validated by comparison to a series of independent experimental data. The input is cellular oxygen level, and the output is HIF1α levels in the nucleus in relation to necessary intermediate reactions, including reactions with prolyl hydroxlylase, iron, 2-oxoglutarate, ascorbate, succinate, and von Hippel-Lindau (VHL) ligase. The model was expanded to represent two possible mechanisms of how ROS interact in the HIF1 pathway: (i) at high concentrations, ROS induce HIF1α by decreasing the activity of prolyl hydroxylases, and ROS effects can be silenced by antioxidants; (ii) in some cells with damaged mitochondria, the opposite effect (ROS decreasing HIF1 activity) is possible through increased iron and 2-oxoglutarate, cofactors in HIF1α degradation.
Using this model, we demonstrate how ascorbate, iron, hydrogen peroxide, 2-oxoglutarate, and succinate would alter HIF1α expression in two representative hypoxic microenvironments: cancer cells and cells during ischemia. We show how these compounds affect adaptation to chronic hypoxia, taking into account possible changes in succinate and reactive oxygen species levels associated with increased anaerobic metabolism and oxidative phosphorylation, such as those found in cancer. Results offer insight into the pro- and antioxidant effects of five compounds present in the HIF1 pathway and how they differ in a tumor microenviroment compared to ischemia. The model demonstrates temporal-specific molecular mechanisms that could be harnessed for use in cancer prevention, recovery from ischemic injury, and repression of angiogenesis and inflammatory signaling regulated by HIF1α expression.