Emerging Potential of Immediate Early Response Gene X-1 in Cardiovascular and Metabolic Diseases.

Introduction

Immediate early response gene X‐1 (IEX‐1) or immediate early response 3 is an immediate early response gene that is highly expressed in epithelial, endothelial, and immune cells, including macrophages.1, 2, 3, 4 Similar to other immediate early response genes, its induction can be rapidly induced in response to several stimuli, such as UV light, inflammatory cytokines, and mechanical strain.4, 5, 6, 7, 8 Under stress, many immediate early response genes, such as c‐Fos, c‐Jun, c‐Myc, Egr‐1, and other zinc‐finger proteins, usually serve as transcription factors, activators, or repressors to regulate the transcriptional activity of several genes that are critical for a cell to appropriately respond to stress.4, 9, 10, 11, 12 However, IEX‐1 lacks a DNA binding domain and its putative transcription activity remains elusive.4, 13 Investigations from our laboratory show that IEX‐1 controls mitochondrial superoxide production by regulating the electron transport chain and oxidative phosphorylation.13, 14, 15, 16 This action of IEX‐1 might explain the transcriptional activity associated with IEX‐1 because the reactive oxygen species (ROS) at low levels can act as a signaling molecule to alter gene transcription through reduction‐oxidation–sensitive transcription factors or a signaling pathway, such as nuclear factor‐κB (NF‐κB) and nuclear factor erythroid 2‐related factor.17, 18, 19 Consistent with a key role of mitochondria in vascular and metabolic functions, IEX‐1 deficiency impairs vascular function and increases energy expenditure in mice. A key role of IEX‐1 in regulation of mitochondrial respiration and ROS production may also help to explain how IEX‐1 produces both antiapoptotic and proapoptotic actions in different cell types. Macrophages, the central player in the pathogenesis of many chronic inflammatory conditions, express high levels of IEX‐1. In agreement with the putative role of IEX‐1 in macrophage function, we recently identified a crucial role of IEX‐1 in many chronic conditions, including hypertension, sepsis, insulin resistance, obesity, and arthritis,16, 20, 21, 22 revealing previously unknown functions of IEX‐1 in cardiovascular and metabolic diseases.

In this article, we reviewed the findings in IEX‐1 knockout mice from our laboratory and others and primarily focused on the roles of IEX‐1 in cardiovascular and metabolic disorders. We also alluded to the important findings from other laboratories that are relevant for its role in cardiovascular and metabolic diseases. In addition, an attempt is made to underline the involvement of IEX‐1 in inflammation and its possible link with the lipid‐mediated metabolic diseases. However, because of the limitations and scope of this article, we refrained from discussing an important role of IEX‐1 in many types of cancers that has been widely reported before.

IEX‐1 Regulates Mitochondrial F1F0‐ATP Synthase Activity

One of the well‐described functions of IEX‐1 is its ability to regulate apoptosis in various cell types, which is in a good agreement with its role in the control of mitochondrial oxidative phosphorylation, given that mitochondria are key in the initiation of this process.13, 23, 24 The oxidative phosphorylation takes place at the respiratory chain in the inner mitochondrial membrane, which consists of 5 enzyme complexes (I–V) (Figure 1). The first 4 complexes oxidize nicotinamide‐adenine dinucleotide, reduced form, and flavin adenine dinucleotide, reduced form, produced by the Krebs cycle and the β‐oxidation, to generate a proton gradient across the mitochondrial inner membrane. F1F0‐ATP synthase (complex V) uses this electrochemical gradient to produce ATP, the major source of energy in a cell. When mitochondrial respiration is compromised (eg, during oxygen deprivation [hypoxia]), the F1F0‐ATP synthase reverses its action and begins to hydrolyze ATP (ATPase) to maintain the mitochondrial membrane potential. This ATPase activity can be repressed by an inhibitory protein F1F0‐ATPase inhibitor (IF1) to preserve energy.25, 26 IF1 binds to subunit of F1 complex through its inhibitory domain and inhibits ATP hydrolysis.26 Increased IF1 activity during diminished mitochondrial respiration conserves ATP by inhibiting F1F0‐ATPase at the expense of membrane potential and protects against ischemia.26 Conceivably, increasing IF1 expression under normal mitochondrial function may inhibit oxidative phosphorylation and decrease ATP synthesis.27 This scenario, thus, would promote a switch from oxidative phosphorylation to glycolysis. Indeed, tumor cells in which glycolysis is putatively high (Warburg effect) exhibit increased IF1 expression concomitant with reduced F1F0‐ATP synthase activity.27

Figure 1

Proposed mechanism of immediate early response gene X‐1 (IEX‐1) in the regulation of reactive oxygen species (ROS) production in mitochondria. IEX‐1 targets F1F0‐ATPase inhibitor (IF1) for degradation and maintains a high level of F1F0‐ATPase activity and more efficient electron transport along the respiratory chain, resulting in reduced ROS production. In contrast, a deficiency of IEX‐1 prevents the degradation of IF1, which, in turn, reduces F1F0‐ATPase activity, thereby hindering the electron transport. This phenomenon causes an increased ROS production as a result of proton (H+) retardation along the respiratory chain. Co Q indicates coenzyme Q; Cyt c, cytochrome c; O2, oxygen; , superoxide radicals.

Investigation from our laboratory has provided crucial evidence that IF1 activity is tightly regulated by IEX‐1 in the mitochondria.15 By using the yeast 2‐hybrid assay and coimmunoprecipitation techniques, we demonstrated that IEX‐1 directly interacts with the C‐terminus of IF1. This renders IF1 sensitive to degradation by a mitochondrial protease.15 Thus, an increase in IEX‐1 expression reduced IF1 level by promoting its degradation. Consequently, IEX‐1–mediated IF1 degradation caused an increase in ATP hydrolysis by augmenting ATPase activity and prevented an increase in mitochondrial membrane potential induced by apoptotic stimuli.13, 15 In agreement with its stimulatory action in mitochondria, overexpression of IEX‐1 inhibited both basal and apoptotic stimuli‐induced ROS production in multiple cell types by facilitating electron transfer along the respiratory chain.13, 15 Furthermore, gene silencing of IF1 produced the similar effects to IEX‐1 overexpression on ATP hydrolysis and ROS production, corroborating a key role of IF1 degradation in the effects produced by IEX‐1.15 These observations are in close agreement and correlate well with the IEX‐1–mediated antiapoptotic effect in many cell types, revealing an important role of IEX‐1 in regulation of cellular reduction‐oxidation homeostasis. In contrast to the aforementioned observations, silencing of IEX‐1 expectedly increased IF1 levels that, in turn, inhibited F1F0‐ATP synthase activity. Reduced F1F0‐ATP synthase activity consequently perturbed proton transport along the respiratory chain and impaired mitochondrial membrane potential, resulting in excessive mitochondrial ROS (mROS) production.13, 15, 20 Moreover, because of the inhibition of F1F0‐ATP synthase activity, oxidative phosphorylation was downregulated in IEX‐1–deficient cells, as a result of which these cells switched their cellular metabolism from oxidative phosphorylation to glycolysis.15, 21 Accordingly, IEX‐1–deficient mouse embryonic fibroblasts used more glucose to compensate energy depletion and proliferate better in complete media. However, they produced more lactate under hypoxia and were susceptible to glucose deprivation.13, 15 These findings argue that interaction of IEX‐1 and IF1 may have an important role in regulation of mitochondrial respiration and ROS production in aerobic conditions in addition to their putative role during hypoxia. This notion may have greater implications than were previously thought because IEX‐1 is abundantly expressed and is highly inducible in different cell types, such as small and large arteries, keratinocytes, immune cells (including macrophages and T cells), and white adipose tissue (WAT).1, 15, 20, 21 This pattern of IEX‐1 expression provided first key evidence supporting a potential physiological importance of this novel protein in cardiovascular and chronic inflammatory conditions.

Antiapoptotic and Proapoptotic Actions of IEX‐1

Apoptosis is a tightly regulated process wherein the mitochondria take a central part. IEX‐1 is highly expressed in mitochondria and has been reported to produce both antiapoptotic and proapoptotic actions.28 However, the survival action is usually observed when the basal cellular ROS level is relatively low, as observed under in vivo physiological conditions.15, 29, 30 For example, overexpression of IEX‐1 in T cells inhibited apoptosis and caused a lupuslike syndrome and T‐cell lymphoma in mice.29, 30 Likewise, IEX‐1 protected T cells from apoptosis induced by ligation of Fas and the T‐cell receptor–CD3 complex.31 Similarly, in nontumor cell lines, such as megakaryoblastic cells UT7, or in CHO‐ER cells, IEX‐1 prevented staurosporin‐induced apoptosis.32, 33 In contrast, the lack of IEX‐1 in CHO or mouse embryonic fibroblasts cells increased apoptosis and reduced their survival under a low glucose condition.15 These observations strongly support a survival function of IEX‐1 under physiological conditions.

It is well documented that the amount of ROS generated and the extent of oxidative stress in a cell determine the fate of the cell to die or survive.34, 35 At lower intracellular concentrations, ROS may induce growth and proliferation by its action as a secondary messenger to activate transcription of various survival genes that involve mitogen‐activated protein kinase and proangiogenic factors, including vascular endothelial growth factor, interleukin‐8, and NF‐κB.36, 37, 38 Interestingly, the antiapoptotic activity of IEX‐1 was strongly correlated with its ability to suppress intracellular ROS production.13 Ectopic expression of IEX‐1 suppressed intracellular ROS production induced by apoptosis inducers.13 Perhaps, by maintaining intracellular ROS concentrations at a low level under apoptotic conditions, IEX‐1 activates oxidant‐sensitive signaling to promote cell proliferation and survival, as previously mentioned15, 21 (Figure 2A). Furthermore, an antiapoptotic activity of IEX‐1 may also be contributed by its putative action on prosurvival factor myeloid cell leukemia‐1 (Mcl‐1).39 Mcl‐1 is an antiapoptotic member of the Bcl‐2 family, which translocates into the nucleus to induce a survival effect in the presence of DNA damage or genomic instability that occurs, for example, after treatment with anticancer drug alkylating agents. In the nucleus, Mcl‐1 triggers Chk1 activation, causes G2 checkpoint arrest, and repairs DNA lesions, promoting cell survival.39 IEX‐1 is essential for Mcl‐1 nuclear translocation during stress.39 Its expression rapidly increases under stress as during γ‐irradiation or treatment with the cytotoxic agents4 (Figure 2A). Thus, by promoting Mcl‐1 migration into the nucleus, IEX‐1 may induce a survival action in cells under stress. Given a critical role of Mcl‐1 in tumor growth, understanding how IEX‐1 expression is regulated under stress may provide a novel therapeutic strategy for cancer chemotherapy. Collectively, these studies support the antiapoptotic action of IEX‐1 under physiological conditions.

Figure 2

A, Schematic representation of the proposed mechanisms of survival and apoptotic actions of immediate early response gene X‐1 (IEX‐1). IEX‐1 induces a survival action by reducing cellular reactive oxygen species (ROS) to a moderate level at which they act as signaling molecules to activate the transcription of various survival genes. In addition, IEX‐1 promotes a survival action by facilitating nuclear translocation of antiapoptotic protein myeloid cell leukemia 1 (Mcl‐1). In ATP compromised cells, such as tumor cells, IEX‐1 overexpression immediately deprives the cells of their energy source by further accelerating ATP degradation as a result of increased F1F0‐ATPase activity, producing a potent apoptotic action. B, Counterregulatory mechanism of IEX‐1 in regulation of apoptosis via extracellular signal‐regulated kinase (ERK) 1/2 and nuclear factor‐κB (NF‐κB) pathways. Phosphorylation/activation of ERK1/2 by thrombopoeitin (TPO) or erythropoietin (EPO) targets and augments the phosphorylation of IEX‐1 that, in turn, interacts and inactivates protein phosphatase A 2 (PPA2). This effect inhibits PPA2‐mediated dephosphorylation of ERK1/2, resulting in prolonged activation of ERK1/2 and activation of survival proteins, such as protein kinase B (Akt) 1. On the other hand, IEX‐1 directly inhibits activation of NF‐κB by several stimuli, such as tumor necrosis factor‐α, and thereby promotes apoptosis by suppressing NF‐κB–mediated antiapoptosis and activation of survival genes, such as Akt1/protein kinase B. IF1 indicates F1F0‐ATPase inhibitor; , superoxide radicals; p‐, phosphorylated.

In addition to its antiapoptotic action, IEX‐1 has also been reported to promote apoptosis in various tumor cell lines. Tumor cells lines, such as HeLa, HEK‐293, HaCaT, Colo320 colorectal cancer, NB4 leukemia, U87‐MG glioma, T47D breast cancer, and PT45‐P1 pancreatic cancer, exhibit high levels of ROS, even at the basal level.2, 28, 40, 41, 42, 43, 44, 45, 46, 47, 48 Tumor cells also generally exhibit the Warburg effect, an increased dependency on glycolysis for ATP production attributable in part to a defective mitochondria and hypoxia.49, 50, 51 Glycolysis is an inefficient source of ATP production, which produces far less ATP molecules than oxidative phosphorylation from a given amount of glucose. We have previously reported that overexpression of IEX‐1 reduces IF1 level in mitochondria by promoting its degradation.15 This results in an increase in F1FO‐ATPase activity, causing acceleration of ATP degradation. Thus, forced overexpression of IEX‐1 in tumor cells or in the cells where ATP generation is already compromised would further dramatically deplete the ATP levels (Figure 2A), thereby inducing apoptosis as a result of severe deprivation of energy.52 Interestingly, exposure of cells to UVB, tumor necrosis factor‐α, or metabolic stress by glucose deprivation has been demonstrated to target prosurvival protein Mcl‐1 for proteasomal degradation, thereby reducing its cellular level.53, 54, 55, 56 This would not only make less Mcl‐1 available to sequester the proapoptotic member of Bcl‐2 family, but also reduce its translocation to nucleus. As a result, the proapoptotic member, such as Bcl‐2 interacting mediator of cell death (BIM), would then be released and migrate into mitochondria to induce apoptosis. IEX‐1 is required to interact with Mcl‐1 for its nuclear translocation.39 Conceivably, Mcl‐1 degradation would result in increased IEX‐1 bioavailability, which may, in turn, allow more efficient interaction of IEX‐1 with IF1 at mitochondria, leading to a further increase in ATP hydrolysis and apoptosis. However, no study to date has been conducted to confirm this notion.

How IEX‐1 switches its action from antiapoptotic to proapoptotic remains elusive. Several mechanisms may be involved for this disparate effect. IEX‐1 plays a pivotal role in the regulation of extracellular signal‐regulated kinase (ERK) activation by controlling its phosphorylation. IEX‐1 contains 3 ERK‐sensitive phosphorylation sites at threonine 18 and 123 and at serine 126. On activation, such as by treatment with thrombopoietin or erythropoietin, ERK1/2 phosphorylates IEX‐1 at threonine residue.32, 33, 57, 58 ERK‐mediated phosphorylation activates IEX‐1 that, in turn, increases ERK phosphorylation back and prolongs ERK signaling, resulting in the induction of phosphorylation of ERK substrates, such as protein kinase B (alias protein kinase B 1), a prosurvival factor.32 This effect is mediated by the direct interaction of IEX‐1 with the protein phosphatase 2A via its subunit B56. Interaction of protein phosphatase 2A with IEX‐1 results in its inactivation and, therefore, it would no longer be available to dephosphorylate ERK1/2. This produces sustained ERK‐dependent phosphorylation of its prosurvival substrates, resulting in the increased survival of the cells, especially during growth‐favoring conditions (Figure 2B).57, 58 In sharp contrast, IEX‐1 has also been reported to reduce the expression of prosurvival factors phosphatidylinositol 3‐kinase and protein kinase B via suppressing NF‐κB expression. Although IEX‐1 is a known direct downstream target of NF‐κB, IEX‐1 decreases the activation of NF‐κB observed under inflammatory conditions (Figure 2B).40, 41 In this way, IEX‐1 exerts a proapoptotic action via downregulating prosurvival proteins. Taken together, these findings demonstrate that IEX‐1 is a negative feedback regulator of NF‐κB and ERK pathways that provides highly effective counterregulatory mechanisms initiated by these factors to regulate apoptosis. Despite its varying, sometimes conflicting effects on cell survival and growth, IEX‐1 appears to be essential for regulating cell survival and death, mainly through its action on mitochondria.13, 15 The key role of IEX‐1 in regulation of mitochondrial function may also be the mechanism whereby deregulation of IEX‐1 contributes to various cardiovascular disorders, such as hypertension,21 atherosclerosis, and cardiac hypertrophy,6, 7 because of the importance of mitochondrial function in the control of cardiovascular activity and metabolism.

Role in Cardiovascular System

IEX‐1 Counteracts Hypertrophy in Cardiovascular Tissues

A deregulated expression level of IEX‐1 has been reported in vasculature, cardiac tissue, or blood cells in several pathological conditions, such as atherosclerosis, pressure overload, and myelodysplastic syndrome (MDS).6, 7, 59 IEX‐1 expression is rapidly induced in vascular smooth muscle cells (VSMCs), cardiac myocytes, and monocytes in response to mechanical stress or pressure overload.6, 7 Its expression is induced not only in response to mechanical strain but also on exposure to various neurohumoral factors, such as inflammatory cytokines interleukin 1‐β, platelet‐derived growth factor, phenylephrine, and phorbol 12‐myristate 13‐acetate.6, 7 This universal induction of IEX‐1 in response to many nonspecific stimuli suggests a broader effect of this protein in the cardiovascular system, in addition to its ability to regulate apoptosis.

In an attempt to study the potential function of this gene in cardiovascular cells, Schulze et al and De Keulenaer et al found that overexpression of IEX‐1 in VSMCs and in cardiac myocytes prevented the hypertrophic response induced by exposure to mechanical strain or various neurohumoral factors, suggestive of its antihypertrophic effect in these experimental settings.6, 7 In addition, in vivo vascular specific overexpression of IEX‐1 in low‐density lipoprotein receptor–deficient mice prevented the neointima formation in response to mechanical injury.6 The observation that mechanical strain, a stress in association with hypertension, induces IEX‐1 expression not only in VSMCs but also in macrophages is consistent with an importance of this gene in vascular pathological conditions, where these cell types are critically involved, such as atherosclerosis and systemic hypertension.6, 60

IEX‐1 Mediates Ischemic Preconditioning of the Heart

Brief episodes of ischemia‐reperfusion elicit strong cardioprotection against the subsequently occurring prolonged and sustained ischemia‐induced cardiac injury. This phenomenon is termed as ischemic preconditioning (IPC) because it preconditions the heart against the sustained myocardial ischemia‐reperfusion injury. The expression of several immediate early‐response genes, such as c‐Fos, c‐Myc, c‐Jun, and Egr‐1, has been reported to rapidly increase (within minutes) in the heart in response to IPC.9, 10, 11 Consistent with this, Xu et al recently reported that IEX‐1 expression in rat heart markedly increased within 5 minutes of induction of episodes of IPC, consistent with the observation with other immediate early response genes.61 In contrast, IEX‐1 expression markedly reduced in the heart during sustained ischemia‐reperfusion injury.61 As a result, IPC‐primed hearts were resistant to the sustained ischemic injury‐induced downregulation of IEX‐1. Furthermore, using the small‐interfering RNA–mediated gene silencing approach, Xu et al61 demonstrated that IEX‐1 is required for the full protection conferred by IPC against ischemia‐reperfusion injury. Conversely, they showed that cardiac overexpression of IEX‐1 attenuated ischemia‐reperfusion injury, as evident by reduction in infarct size and improvement in systolic function.61 The authors concluded that IEX‐1–dependent cardiac protection was mediated by phosphorylation and translocation of protein kinase Cε, a key molecule required for IPC, which reduced ROS production in mitochondria.61 These effects inhibited cardiomyocyte apoptosis and necrosis and attenuated myocardial infarction during ischemic injury. These findings are in agreement with our previous reports where we demonstrated that IEX‐1 deficiency specifically increased ROS production in mitochondria in VSMCs,21 whereas its overexpression in T cells inhibited apoptosis, leading to T‐cell lymphoma in mice.30 Nevertheless, this new finding provides another strong evidence for a cardioprotective role of IEX‐1 and reveals a new therapeutic strategy for the ischemic heart diseases.

IEX‐1 Regulates Vascular Tone to Maintain Systemic Blood Pressure

In a quest to study the physiological role of IEX‐1, we generated mice lacking a functional IEX‐1 gene. These mice do not express a functional IEX‐1 protein; however, they grow and breed normally under steady‐state conditions.21 These IEX‐1–deficient mice also weigh similar to their wild‐type littermates and appear devoid of any gross developmental abnormality. There was no significant difference in fasting blood glucose, insulin, and total cholesterol levels between IEX‐1–deficient and wild‐type mice under normal diet‐fed conditions.20, 21 We also failed to detect any significant alteration in food intake and total motor activity in these mice, suggesting no major role of IEX‐1 under normal physiological conditions. However, IEX‐1 deficiency in mice was associated with elevated systemic blood pressure and cardiac hypertrophy as they aged.3, 21 In addition, IEX‐1 paucity also increased ROS production in the vasculature of the mice.3, 15, 21 Hypertension in IEX‐1–deficient mice was not associated with salt sensitivity or abnormality in kidney function and did not progress with age.3, 21 Various vasoregulatory factors, such as angiotensin II, nitrites and nitrates, cAMP, and cGMP, that are implicated in the pathogenesis of hypertension appeared to be normal in these mice.3 Thus, hypertension in IEX‐1–deficient mice may represent an early stage or a unique category of hypertension where its cause remains subtle. We asked why hypertension in IEX‐1–deficient mice did not progress over time and what was the underlying mechanism of this unique form of hypertension. Because kidney function and histological features appeared to be normal in IEX‐1–deficient mice, we next investigated the potential impact of IEX‐1 deficiency on the vascular function that may contribute to elevated blood pressure if altered. We hypothesized that aggravated mROS production attributable to IEX‐1 deficiency would enhance vascular tone by inactivating freely available vasodilator NO, causing an increase in blood pressure. To our surprise, even in the presence of increased mROS levels in aortic tissue and in isolated VSMCs, we failed to observe any significant alteration in NO‐dependent signaling and vasodilation (acetylcholine‐mediated vasodilation),3, 21 consistent with the previous observation of no difference in nitrite and nitrate levels.3 On the contrary, we observed that cAMP‐dependent vasorelaxation, as determined by vascular responses to β2 adrenergic receptor agonist isoproterenol and adenylyl cyclase activator forskolin, was significantly impaired in the vasculature of these mice. This defect remained intact in endothelium‐denuded aortic vessel, suggesting a potential defect in VSMCs.21 The cAMP production required for vascular relaxation is dependent on net adenylyl cyclase activity, which is tightly regulated by G proteins. Although stimulatory G proteins activate the enzyme, inhibitory proteins (G‐protein α subunit (inhibitory) 2 or 3) inhibit its activity to reduce cAMP production. In this regard, ROS has been shown to specifically upregulate G‐protein α subunit (inhibitory) transcription in spontaneously hypertensive rats by increasing its promoter activity via the reduction‐oxidation–sensitive NF‐κB and nuclear factor erythroid 2–related factor transcription factors.17, 62, 63 Thus, to delineate the underlying mechanism of reduced cAMP‐dependent vasorelaxation in the absence of IEX‐1, we determined the expression levels of G‐protein α subunit (inhibitory) in the vasculature of IEX‐1–deficient mice. The molecular, cellular, and vascular analysis revealed that the IEX‐1 deficiency markedly increased G‐protein α subunit (inhibitory) 2 expression level in an mROS‐dependent manner. Thus, inhibition of G‐protein α subunit (inhibitory) 2 with pertussis toxin not only restored cAMP signaling and cAMP‐dependent vasodilation, but also attenuated established hypertension in IEX‐1–deficient mice (Figure 3).21 The lack of vascular inflammation and intact NO signaling in these mice might explain why the hypertension in IEX‐1 deficiency does not deteriorate over time. Collectively, these studies provide novel insight into how a defective mitochondrial function can induce hypertension, helping uncover an inflammation‐independent mechanism in the onset of high blood pressure. Further study of the hypertension in IEX‐1–deficient mice may help us understand how inflammation interacts with and aggravates systemic hypertension. Taken together, the aforementioned studies provide strong evidence for a protective role of IEX‐1 in cardiovascular tissues and identify IEX‐1 as a novel therapeutic and diagnostic target for vascular and ischemic heart diseases.

Figure 3

Proposed mechanism of hypertension induced by immediate early response gene X‐1 (IEX‐1) deficiency. IEX‐1 deficiency upregulates G‐protein α subunit (inhibitory) 2 (Gαi2) expression in a reactive oxygen species (ROS)–dependent manner at mitochondria. Overproduction of Gαi2 inhibits adenylyl cyclase–mediated synthesis of cAMP, decreasing cAMP‐dependent vascular smooth muscle cell relaxation. This impairs vasodilation, causing an increase in systemic blood pressure (hypertension). Gαs indicates G‐protein α subunit (stimulatory); , superoxide radicals.

IEX‐1 Contributes to Diet‐Induced Obesity and Insulin Resistance

The observation that IEX‐1–deficient mice develop sustained hypertension with little or no sign of vascular or renal inflammation21 led us to speculate the possible involvement of IEX‐1 in inflammation. In addition, IEX‐1 is one of the major direct downstream transcriptional targets of NF‐κB, a central mediator of inflammation.5, 6, 7 IEX‐1 expression and many of its reported functions could be blocked by inhibiting the NF‐κB pathway,5, 6, 7 identifying activation of NF‐κB as a necessary event in the induction of IEX‐1 expression. Like NF‐κB, IEX‐1 is also capable of manipulating mutually exclusive processes, positively and negatively both (eg, cell survival versus cell death or cell division versus cell cycle arrest, depending on the cell types).5 Along this line, our group has previously reported that IEX‐1 paucity impaired the ability of macrophages and T cells to induce inflammation in response to many stimuli, such as Leishmania major infection and dextran sodium sulfate–induced colitis.64, 65 Collectively, these data suggest that IEX‐1 may be an inherent regulator of inflammation. Because NF‐κB is the central inflammatory mediator that plays a critical role in the pathogenesis of lipid‐driven inflammation and diet‐induced insulin resistance, we reasoned that IEX‐1 could be a downstream mediator of NF‐κB activation in metabolic disorder.

In a continued effort to explore the physiological roles of IEX‐1, we discovered a key role of IEX‐1 under metabolic stress. Although mice lacking IEX‐1 exhibited normal metabolism on normal diet, they were remarkably resistant to high‐fat diet (HFD)–induced insulin resistance and were protected against adipose and hepatic inflammation.20 Unexpectedly, IEX‐1–deficient mice were also highly resistant to HFD‐induced weight gain. After 20 weeks of HFD consumption, knockout mice gained only ≈40% in their body weight, whereas their wild‐type littermates increased their body weight by ≈90% on the same diet.20 Indirect calorimetry further revealed that lean phenotype of IEX‐1–deficient mice on HFD was associated with enhanced energy expenditure, suggesting that IEX‐1 paucity inhibits obesity development by increasing energy use. IEX‐1–deficient mice grew and weighed normally when fed a regular diet, indicating that IEX‐1 has little/no impact on energy metabolism under steady‐state condition. To gain an insight into the underlying cellular mechanism in lean phenotype of IEX‐1–deficient mice, we analyzed WAT of these mice, where we observed a significant increase in IEX‐1 expression after HFD feeding. Consistent with other reports,66, 67, 68 HFD strongly suppressed (Uncoupling Protein 1) UCP1 expression in WAT of wild‐type mice, an effect that was not observed in knockout mice. Increased UCP1 expression in knockout mice was concomitant with induction of thermogenic genes and appearance of multilobular adipocytes, a hallmark of adipocyte beiging (browning).66 These data provided initial cues suggesting that IEX‐1 deficiency increases energy expenditure, likely by inducing adipocyte beiging.66, 69

How IEX‐1 deficiency induces beiging is not known yet. Our investigation has provided preliminary evidence that it could be linked to adipose tissue macrophages (ATMs). It has been well documented that HFD induces a robust transition in ATM phenotype from M2‐ to M1‐like state, drastically decreasing the number of resident M2 macrophages in adipose tissue. We also observed such M2 to M1 switch in ATM phenotype of wild‐type mice on HFD feeding. In sharp contrast, we failed to observe this phenomenon in IEX‐1–deficient mice even after 20 weeks of HFD feeding. Thus, knockout mice sustained their M2‐like macrophages in the adipose tissue, unlike wild‐type littermates that lost a majority of those cells on HFD.20 Sustenance of M2‐like macrophages in WAT is likely the underlying mechanism by which IEX‐1 paucity induces beiging because M2 macrophages are potent inducers of beiging program.70 As opposed to M1‐like macrophages, M2 polarization increases the production of catecholamines by increasing the expression of catecholamine‐synthesizing enzymes tyrosine hydroxylase, dopa decarboxylase, and dopamine β‐hydroxylase. Catecholamines released by M2‐like cells present in WAT act on the surrounding adipocytes and induce beiging program.70, 71 On the basis of these findings, we propose that macrophage‐specific IEX‐1 is required for HFD‐induced obesity and that preventing M2 polarization should reverse lean phenotype of IEX‐1–deficient mice. This notion is the subject of current investigations in our laboratory.

IEX‐1 exerts a strong survival action in several cell types, particularly under physiological conditions, as observed in vivo.15, 29, 30, 31, 32, 33 The observation that IEX‐1 deficiency prevents HFD‐induced M2 to M1 transition in ATMs suggests that IEX‐1 promotes the polarization and survival of M1 macrophages as opposed to M2 cells.20 This is in agreement with its differential role in T cells in a subset‐specific manner. For example, IEX‐1 deficiency protected mice against dextran sodium sulfate–induced colitis in mice by enhancing apoptosis specifically in proinflammatory type 1 helper T cells while promoting survival of the anti‐inflammatory type 17 helper T cells.65 Collectively, these data suggest that IEX‐1 in general promotes the survival of proinflammatory immune cells and thereby contributes to inflammatory reaction. How IEX‐1 exerts disparate effects in different subsets of T cells and macrophages is not known yet. In our preliminary investigation, we have found that HFD feeding in mice increases IEX‐1 expression selectively in M1 ATMs but not M2 cells. Given a survival action of IEX‐1, its increased expression in M1 as opposed to M2 may be the underlying mechanism by which HFD feeding promotes M1 polarization. As a result, IEX‐1 deficiency prevents HFD‐induced M2 to M1 transition and resists the development of obesity.20 Similar to its action in macrophages, overexpression of IEX‐1 increases the survival of cardiac myocytes by inhibiting ROS production and thereby reduces ischemia‐reperfusion injury in mice.61 Thus, selective expression of IEX‐1 in different subsets of cells in response to stress, such HFD feeding or ischemia, may explain how IEX‐1 exerts disparate effects in different pathological conditions. Our current investigation emphasizes a crucial role of IEX‐1 in metabolic regulation of macrophage function and inflammatory process. Macrophages play a critical role in many lipid‐driven inflammatory conditions, such as atherosclerosis, obesity, and type 2 diabetes mellitus. Understanding how IEX‐1 controls macrophage phenotype to modulate inflammation and metabolism will provide novel insights into the pathogenesis of these diseases and may help develop novel therapeutic targets. This line of investigation becomes even more important when considering that inflammation and inflammatory pathways are increasingly targeted for therapeutics in treatment of cardiovascular disorders.

Potential Roles in Other Cardiovascular System–Related Disease Conditions

Myelodysplastic Syndrome

MDS is a heterogeneous disorder characterized by impaired hematopoiesis and altered peripheral blood cell morphological features that may progress to acute myeloid leukemia. First evidence for a potential role of IEX‐1 in MDS emerged from a pioneer clinical study demonstrating a strong downregulation of IEX‐1 expression in CD34+ stem cells in ≈60% of patients with MDS compared with healthy donors.72, 73 Interestingly, this aberrant IEX‐1 expression was most prominent in the patients with low‐risk or early stage of the disease and was associated with increased intramedullary apoptosis. Subsequently, by using IEX‐1–deficient mice, our group demonstrated that impaired IEX‐1 expression in MDS contributed to, rather than being a consequence of, the disease. Specifically, IEX‐1 deficiency induced apoptosis and a decrease in the proportion of hematopoietic stem cells (HSCs) in mice without affecting the absolute number under basal conditions.74 However, transplantation of IEX‐1–deficient bone marrow cells in wild‐type mice or exposure of IEX‐1–deficient mice to nonmyeloablative irradiation produced changes resembling MDS, such as thrombocytopenia, anemia, and dysplastic bone marrow morphological features. Similar to the observation in patients with MDS, IEX‐1 deficiency was associated with abnormal increase in apoptosis accompanied by excessive proliferation in HSCs,72, 73, 74 providing cues for how restricted hematopoiesis and impaired myeloid progenitor differentiation occur in patients with MDS, despite the enhanced repopulation capability of HSCs. These findings highlighted a previously unknown role of IEX‐1 in maintenance of HSC quiescence and in the differentiation of multiple progenitors, including erythropoiesis and thrombopoiesis. Further mechanistic studies are warranted to raise the potential of IEX‐1 as a promising therapeutic or diagnostic target in MDS. For example, studies should determine how IEX‐1 expression decreases in stem cells in MDS, although no mutation or rearrangements in the gene are documented. Moreover, how IEX‐1 exerted a myeloid‐biased effect without affecting lymphoid lineage remains to be determined.

Lipopolysaccharide‐Induced Sepsis

Sepsis is associated with multiple organ failure as a result of overactive systemic inflammation attributable to severe infection. It is the leading cause of mortality in the intensive care unit. Ramsey and Wu recently reported that mice lacking IEX‐1 were susceptible to lipopolysaccharide‐induced endotoxemia, liver and kidney damage, and subsequent death.16 Because IEX‐1 deficiency increases mROS production, they treated mice with mitochondrial‐specific antioxidant MitoQ to determine a role of ROS in aggravated phenotype of IEX‐1–deficient mice in response to lipopolysaccharide. Treatment with MitoQ protected knockout mice against pancytopenia and multiple organ failure and decreased the mortality rate.16 These data suggest the downregulation of IEX‐1 expression, and the consequent increase in mROS production may contribute to the cause of sepsis. Thus, targeting the IEX‐1–mROS pathway may be a useful strategy for ameliorating sepsis and associated complications.

Summary and Future Direction

Despite a growing consensus supporting the causative role for inflammation in the development of hypertension, it is uncertain whether these 2 disorders are causally related or whether hypertension can develop independently of inflammation. The finding that persistent, but moderate, systemic hypertension developed in the absence of IEX‐1, concurrent with little sign of inflammation, raises an intriguing possibility. It is that vascular inflammation is not required to initiate the pathogenesis of hypertension, but that it is involved in the full development of this disorder. Therefore, further investigation of the underlying molecular mechanisms in IEX‐1 deficiency–induced hypertension may improve our understanding of this highly prevalent disease. Furthermore, besides its crucial role in mitochondrial respiration and regulation of apoptosis, for which it was originally discovered, IEX‐1 appeared to play a significant role in diverse cellular functions in a variety of tissue types, including systemic blood vessels, WAT, macrophages, and HSCs, as we discussed above in this review. Macrophages are a major source of active inflammation associated with various chronic inflammatory diseases, such as obesity, atherosclerosis, hypertension, and bone loss. Although the role of macrophages in inflammatory conditions is undisputed, it is crucial to investigate for the factors that critically regulate their phenotype. Investigation from our laboratory suggests that IEX‐1 could be one such factor that is required for classic activation of macrophages specifically in context of metabolic disorder. Understanding how IEX‐1 regulates macrophage phenotype and modulates inflammatory and metabolic processes may offer promising opportunities for the development of novel therapeutic or diagnostic approaches for these disorders. Furthermore, findings from our laboratory using IEX‐1–deficient mice raised many critical questions. For instance, how does IEX‐1 deficiency render macrophages and T cells susceptible to apoptosis in a subset‐dependent manner? What are the upstream mediators and downstream targets of IEX‐1 in lipid‐driven stress and inflammation? Answers to these fundamental questions will advance our understanding of how IEX‐1 contributes to lipid‐driven inflammatory conditions and metabolic disorder and may help raise the potential of IEX‐1 as a potential therapeutic target for diseases, such as diabetes mellitus, obesity, and atherosclerosis.

Sources of Funding

The work described herein from Wu and Shahid's laboratory is supported by National Institutes of Health grants AI050822 and AI070785 (Wu); DK103955 and the Eleanor and Miles Shore fellowship from Harvard Medical School (Shahid); and a Grant‐in‐Aid 0855762D from the American Heart Association (Wu).

Disclosures

None.