Fraternal twins: Swiprosin-1/EFhd2 and Swiprosin-2/EFhd1, two homologous EF-hand containing calcium binding adaptor proteins with distinct functions

Expression of EFhd2

An enhancer trap screen has identified a Drosophila strain with an intriguing expression pattern in muscle cells and somatic muscle precursor cells. The insertion is in the Drosophila efhd homologue, CG10641 [19]. Furlong et al. [20] revealed expression of CG10641 by microarray analyses and in situ hybridization during mesoderm development. Estrada et al. show expression of CG10641 in the visceral and somatic mesoderm, stage 9 ventral head mesoderm, stage 10 head mesoderm and in hemocytes but did not follow up the expression patter of CG10641 during mesoderm differentiation [21]. However, based on these expression data Kiefer et al. [22] propose a function for all Drosophila CG10641-related genes in muscle. Indeed, Renate Renkawitz-Pohl's group (C. Hornbruch and R. Renkawitz-Pohl, personal communication) shows expression of Drosophila EFhd (CG10641 encoded) in hematocytes and during myoblast fusion. The EFhd2 orthologue in C. elegans is expressed in pharynx, body wall muscle, the nervous system and the ventral nerve cord. In zebrafish the associated ESTs are expressed in brain, eye, genitourinary tissue, gills, muscle, and olfactory rosettes. In C. elegans, zebrafish and mouse the respective orthologues may have additional functions, including a role in the nervous system [22]. The latter appears to be true in human and mouse, at least under pathological conditions such as neurodegeneration, schizophrenia and alcohol addiction [15, 23–25].

Muscle is derived from mesoderm but the mesoderm gives also rise for instance to bone (osteoblasts and osteoclasts), connective tissue and hematopoietic cells. In humans, EFhd2 becomes transiently up-regulated during osteoblast differentiation from human mesenchymal stem cells [26]. EFhd2 is also up-regulated during differentiation of the murine RAW264 macrophage cell line into osteoclasts after treatment with RANK-L (receptor activator of NF-κB ligand) [27]. Other cell types of mesodermal origin expressing EFhd2 are particularly immune cells: EFhd2 is expressed in human and primary murine mast cells [[28, 29]; our unpublished data], human and murine B cells [30–32], in CD4⁺ and CD8⁺ T cells [32–34], natural killer cells (NK cells) [35] and human peripheral blood mononuclear cells (PBMC) [36, 37]. PBMC are a heterogeneous cell population of lymphocytes (~75% CD4⁺ and CD8⁺ T cells and ~25% NK and B cells), monocytes and macrophages. In accordance, murine efhd2 mRNA is present at relatively high abundance in total spleen by Northern Blot, at similar levels in liver and lung, and at relatively low amounts in kidney [31]. The highest expression of efhd2 has, however, been detected in ectodermal tissue, namely brain [31]. Likewise, the murine EFhd2 protein was identified in mouse brain [38], specifically in brain stem, cerebellum, amygdala, striatum, cortex and frontal lobe [15] as well as in the hippocampus [15, 23]. With the exception of microglia cells [39] it remains to be established which cell type(s) in the brain express EFhd2. Other cells of ectodermal origin expressing EFhd2 are primary immortalized human keratinocytes [40]. Finally, during mouse development EFhd2 is detected strongly in ectodermal tissue such as the most rostral foregut and the caudal hindgut at E8.5 [41]. In addition, EFhd2 is also down-regulated in embryos deficient for the transcription factor Foxa2 and is a marker of definitive endoderm [41]. Interestingly, EFhd2 is already present in murine embryonic stem cells (our unpublished data). Cell lines expressing EFhd2 are all murine B cell lines we analyzed (our unpublished data), HeLa cells [42] and NIH3T3 cells (our unpublished data). In summary, in worm, zebrafish and higher vertebrates EFhd2 is not only expressed during mesoderm development but also in endodermal and ectodermal tissues. Whether this is also the case in D. melanogaster remains to be determined.

Involvement of EFhd2 in immune cell activation

Innate immune cells respond to pathogen associated molecular patterns (PAMP) and to danger signals released by necrotic cells. Many signaling elements that are important in adaptive immunity have recently also been shown to control innate immunity [43]. Interestingly, signaling molecules of innate immune cells in higher organisms are shared even in Drosophila where they control for instance removal of necrotic tissue [43]. Although insects do not possess an adaptive immune system and rely solely on their innate immune system, they utilize ITAMs (immunoreceptor tyrosine based activation motifs). The ITAM is a tyrosine phosphorylation consensus motif (YxxLx_(7-12)YxxL) [44] found in many receptors and co-receptors of the adaptive and innate immune system as well as in ERM (ezrin, radixin, moesin) proteins [43]. When phosphorylated on their tyrosine residues by Src kinases or Syk (splenic tyrosine kinase) [45], ITAMs bind the SH2 domains of Syk, relieving Syk from autoinhibition and eventually activating it, resulting in a positive feedback loop [45]. Syk is key to signaling from adaptive as well as many innate immune receptors [43, 46]. We have recently shown that murine EFhd2 is a positive regulator of Syk activity in response to BCR stimulation in the murine B cell line WEHI231 [14].

Hemocytes of insects are crucial players of immune surveillance by migrating through the body, phagocyting invading pathogens and secreting antimicrobial peptides [47]. One type of hemocytes are the plasmatocytes which resemble mammalian monocytic and macrophage cells [47]. Differentiated macrophage-like hemocytes of Drosophila express the Drosophila EFhd protein (C. Hornbruch and R. Renkawitz-Pohl, personal communication). This fits to other reports revealing Drosophila EFhd expression in hemocytes [48] and in the Drosophila S2 cell line which exhibits phagocytotic activity [47]. Many of the hematopoietic factors have been conserved across taxonomic groups [43]. Likewise, EFhd2 is expressed in the murine monocyte cell line RAW264 [27], in human PBMC [36], in microglia cells [39] and in NK-like cells [35]. EFhd2 becomes up-regulated - together with actin - in response to stimulation of the human monocyte cell line THP-1 with a recombinant Mycobacterium bovis strain [49]. This stimulation improved the antigen-presenting capacity of THP-1 cells, the CD8⁺ immune response and TNF-α production [49]. To sum up, EFhd2 is expressed across species in many cell types of the innate and adaptive immune system. It is tempting to speculate that EFhd2 is involved in Syk activation downstream of other immune receptors than the BCR and plays a role in antigen presentation.

Connection of EFhd2 to the cytoskeleton

Migration and phagocytotic activity of macrophages require a dynamic cytoskeleton. For instance, the small GTPase Cdc42 is required for cellular polarity, formation of filopodia and Fcγ-receptor mediated phagocytosis [50, 51]. Rac is required for cell migration and Fcγ-receptor mediated phagocytosis [50, 52, 53] and Rho is required to retract the trailing edge of migrating cells and for uptake of apoptotic cell fragments [50, 54]. EFhd2 is associated with the cytoskeleton in the human mast cell line HMC-1 and in NK-like cells [29, 35]. More specifically, EFhd2 is associated in a caspase-9-containing complex with the cytoskeletal protein ezrin [55]. In the brain of a mouse model (JNPL3 mouse) [56] for neurodegeneration (frontotemporal dementia and parkinsonism linked to chromosome 17; FTDP-17), where neurodegeneration and neuroinflammation are induced through transgenic expression of a mutated human tau gene (htau P301L), EFhd2 was identified in a complex with the transgenic human tau protein [15]. In addition, endogenous EFhd2 and tau associate in brain lysates of humans with Alzheimer's disease and FTDP-17 [15]. Finally, EFhd2 becomes dephosphorylated after epidermal growth factor (EGF) stimulation of HeLa cells with the same kinetics as the actin binding protein gelsolin [42]. Taken together, these data demonstrate that EFhd2 is associated with the cytoskeleton but the exact connection is unknown at present. However, the related AIF-1 is associated with the cytoskeleton as well and involved in macrophage survival, migration and activation [57]. Specifically, it augments macrophage phagocytotic activity [58]. Calcium binding to AIF-1 by means of its functional EF-hand is required for its ability to activate Rac or G-CSF expression in vascular smooth muscle cells [59]. Similarly, EFhd2 also binds calcium directly [15] which may lead to interactions with cytoskeletal regulators to modulate functions of innate immune cells, such as cell migration, phagocytosis and antigen presentation. In fact, in the human mast cell line HMC-1 EFhd2 co-localizes with, and modulates F-actin [29].

Involvement of EFhd2 in NF-κB regulation

In the human mast cell line HMC-1 (which lacks the Fcε receptor) EFhd2 augments expression of pro-inflammatory cytokines after phorbol myristyl acetate (PMA)/ionomycin stimulation. Similarly, AIF-1 augments expression of IL-6, IL-12 but also IL-10 after lipopolysaccharide (LPS) stimulation of macrophages [60]. Therefore, in macrophages and a mast cell line, perhaps also in primary mast cells (where EFhd2 is expressed; our unpublished data), EFhd2 appears to reveal pro-inflammatory activities, perhaps through positive regulation of the protein kinase C β/nuclear factor κB (PKCβ/NF-κB) signaling pathway [29]. The PKCβ pathway is also activated by the BCR downstream of PLCγ2/Ca²⁺ signaling and induces eventually NF-κB activation through the Carma1/Bcl10/MALT1 complex [61]. A positive regulation of PKCβ/NF-κB through EFhd2 would therefore be expected as we showed recently that EFhd2 augments BCR-induced Syk/PLCγ2/Ca²⁺ signaling in the murine B cell line WEHI231 [14]. Unexpectedly, however, we showed already that EFhd2 rather blocks expression of the anti-apoptotic NF-κB target gene bclxL[31]. An explanation could be that EFhd2 binds calcium directly [15] and is therefore part of a negative feedback loop preventing NF-κB activation after BCR stimulation (Figure 3). Apoptosis in lymphocytes is effectively controlled by the transcription factor NF-κB [62]. In the murine B cell line WEHI231 that is susceptible to BCR-induced apoptosis, EFhd2 positively regulates spontaneous and BCR-induced apoptosis [31]. Silencing of EFhd2 by shRNA augments survival of WEHI231 cells spontaneously and in response to BCR stimulation. In contrast, G1 cell cycle arrest and p27Kip up-regulation are still induced by BCR ligation in the absence of EFhd2 [31]. To conclude, EFhd2 regulates specifically apoptosis, but not cell cycle arrest in WEHI231 cells. We propose that this is due to suppression of the NF-κB pathway in this cell line. The pro-apoptotic activity of EFhd2 was suppressed by three stimuli that activate NF-κB (CD40 ligation, LPS, and B cell activating factor of the TNF family [BAFF]) [31]. These data seem to oppose data from the mast cell line HMC-1 where EFhd2 augmented up-regulation of NF-kB target genes after PMA/ionomycin stimulation [29], many of which are anti-apoptotic. However, PMA/ionomycin stimulation as used in HMC1 cells [29] and BCR ligation in WEHI231 cells are different stimuli. EFhd2 may enhance BCR-mediated cell death in WEHI231 cells through inhibition of the NF-κB pathway but, through an unknown molecular switch, enhance likewise the NF-κB activating and anti-apoptotic activities of CD40, LPS, and BAFF in combination with BCR stimulation.

Engagement of EFhd2 in autoimmune disorders

Regulation of the NF-κB pathway by EFhd2, be it positive or negative, together with its expression in innate and adaptive immune cells and its effects on cytokine production [29] argue for involvement of EFhd2 in normal and pathological immune activation. In fact, PBMC of rheumatoid arthritis (RA) patients express less EFhd2 protein than PBMC of healthy patients [36]. Moreover, there is a protease-mediated cleavage of EFhd2 in RA patients [37]. Expression of pro-apoptotic factors in both B and T cells is important to establish tolerance of the adaptive immune system [63]. If EFhd2 was down-regulated in B cells their survival might be prolonged. This might contribute to the persistence of autoimmune diseases such as RA and SLE [64]. Interestingly, the EFhd2-related AIF-1 is involved in RA as well [57]. Moreover, the murine efhd2 gene is Iocated rather centrally in an SLE susceptibility region (LMB-1, close to D4Mit33) on chromosome 4 [17], thereby, representing a putative genetic determinant of SLE. Memory B cells become persistently re-activated in RA and SLE [65]. In memory B cells as well as in CD4⁺ and CD8⁺ memory T cells, efhd2 expression is part of an evolutionary conserved transcriptional signature [32]. Whether efhd2 expression in immune memory cells is important for their activation, to keep them silent, or to help them patrolling through secondary lymphatic organs is unknown at present but certainly deserves further investigation with respect to autoimmune diseases.

EFhd2 and neuropathological disorders

Neurodegenerative disorders associated with tau, so-called tauopathies, are characterized by successive deposition of protein aggregates consisting of hyperphosphorylated tau [66, 67]. In Parkinson's patients, tau and nitrated α-synuclein induce formation of so-called Lewy bodies that are characteristic for Parkinson's disease [68]. Tau aggregates and Lewy bodies cause synapse loss and neuronal cell death. In addition, neurodegenerative diseases reveal an inflammatory component represented mainly by microglial activation [69]. There are several lines of evidence that EFhd2 is involved in normal and pathological brain function: First, in microglia cells, EFhd2 becomes up-regulated and secreted in response to stimulation with nitrated α-synuclein [39]. Very interestingly, the related AIF-1 (Iba1) is a microglial activation marker [70]. Second, the brain of a FTDP-17 mouse model (JNPL3 mouse) [56] where neurodegeneration and neuroinflammation are induced through transgenic expression of a mutated human tau (htau P301L) contains complexes of the transgenic human tau protein and endogenous EFhd2 [15] (Figure 3). In addition, endogenous EFhd2 and tau associate in brain lysates of humans with Alzheimer's disease and FTDP-17 [15]. Third, EFhd2 is present in membrane rafts of mouse spinal cord, but only when mice over-express a mutant, toxic gain of function form of superoxide dismutase 1 (SOD1), namely the G93A mutant [71] (Figure 3). This mutant is responsible for familial amyotrophic lateral sclerosis, a chronic, progressive neuromuscular disorder [72]. Fourth, EFhd2 is down-regulated in Nogo-A knockout mice that reveal increased neurite growth and an increased regeneration potential when compared to wildtype control mice [73]. Fifth, in the frontal cortex of schizophrenic patients EFhd2 becomes up-regulated together with microtubule associated proteins [24]. Whether all these data represent a causative connection of EFhd2 with neurodegenerative diseases on the one hand, with neuronal regeneration on the other hand or are merely secondary effects is not known at present. Given the increasing incidence of neurodegenerative diseases, it would potentially be important to examine EFhd2 expression and function in human inflammatory diseases, including neuroinflammatory processes, and in murine mouse models for these diseases.

Involvement of EFhd2 in membrane associated pathways

The plasma membrane contains cholesterol and sphingolipid-enriched microdomains (detergent resistant membranes, DRM) that are thought to function in many membrane-associated processes, such as immune receptor signaling [74]. In addition, microdomains control vesicular trafficking, signaling of various receptors other than immune receptors, and cytoskeletal rearrangement [75]. Microdomains and their associated scaffold proteins have also been implicated in the pathogenesis of several neurological diseases, such as Parkinson's and Alzheimer's disease [76] (see paragraph above). EFhd2 was discovered by us through a proteomic analysis of DRM of B cell lines that differ in their apoptotic response to BCR stimulation [30]. We found that EFhd2 associates with DRM in cell lines which undergo apoptosis after BCR stimulation [30] and indeed, as mentioned above, EFhd2 augments BCR signaling and BCR-induced apoptosis in one of these cell lines examined [14, 31]. It was tempting to speculate that the DRM association of EFhd2 controls these effects. More recently we could actually show that the BCR-induced increase in intracellular calcium concentration is dependent on the DRM association of EFhd2 in WEHI231 cells [14]. However, EFhd2 was not associated with DRM in the murine B cell line CH27 [30]. We conclude that membrane raft association of EFhd2 is not constitutive but regulated, presumably in a cell type-specific manner. Hence, DRM association of EFhd2, and thereby its function, may be regulated by a specific pathway active in some cell types. There exist two lines of evidence for this hypothesis:

First, the predicted SH3-binding region (aa 70-78) of EFhd2, targeting EFhd2 to DRM, binds specifically to the SH3 domain of the tyrosine kinase Fgr [14] (Figure 3). In B cells, Fgr is only expressed late in B cell development [77] and there is only one study to our knowledge that examined DRM association of Fgr [78]. This study revealed that, in contrast to other Src family kinases, Fgr is not associated with DRM, at least in RAW264.7 cells [78]. Hence, once Fgr is expressed, it could keep EFhd2 out of DRM. The SH3 domain of Lyn that also binds to EFhd2 [14] could conversely maintain DRM association of EFhd2 (Figure 3). While Lyn is expressed throughout B cell development, Fgr is expressed in mature primary B cells, mantle zone B cells and during myelomonocytic differentiation, but not in immature primary B cells [77, 79], which might regulate the subcellular localisation and the function of EFhd2 in mature B cells. Interestingly, we observed a phosphorylation-dependent interaction of EFhd2 with Lyn and Fgr [14]. Phosphorylation of murine and human EFhd2 at serine residues 74 and 76 [80–83] and, in brain lysates, of tyrosine 83 [38], have been described. Lipopolysaccharide stimulation of macrophages does not alter the phosphorylation of EFhd2 significantly [83]. In our hands, EFhd2 was not phosphorylated on tyrosine before or after BCR engagement as judged by western blotting with the anti-pY mAb PY99 (D.M. et al., unpublished data). In conclusion, phosphorylation of either serine 74 or 76, or both, or other sites would appear to mediate binding of EFhd2 to the SH3 domains of Fgr and Lyn. Interestingly, S74 and S76 of EFhd2 are Cdk1 (cyclin dependent kinase 1) phosphorylation sites [80]. Cdk1 is expressed and active during the G2/S phase of the cell cycle [84], that is, in activated B cells, which are present for instance in germinal centers [85]. Hence, EFhd2 could be modified by phosphorylation to modulate BCR signals in cycling B cells where the BCR induces G1 arrest and apoptosis [86], both of which are enhanced by EFhd2 [31].

Second, EFhd2 is present in DRM of mouse spinal cord, but only when mice over-express a mutant, toxic gain of function form of superoxide dismutase 1 (SOD1), namely the G93A mutant [71]. This mutant is responsible for familial amyotrophic lateral sclerosis, a chronic, progressive neuromuscular disorder [72]. Interactions of DRM with the cytoskeleton have been well documented [75]. Interestingly, the G93A mutant of SOD1 also induces DRM association of cytoskeletal proteins, such as ezrin, clathrin, actin depolymerising factor and Arp2/3 [71] (Figure 3). In accordance, EFhd2 has been identified in a complex with ezrin [55]. Under certain yet to be defined circumstances, EFhd2 may target ezrin, clathrin and others to DRM or become targeted to DRM by cytoskeletal proteins. De-phosphorylation of ezrin after BCR-triggering is important to induce DRM association of the BCR at late time points (more than 15 min) [87]. We have also shown that, at least in the murine B cell line WEHI231, EFhd2 induces constitutive association of the BCR, PLCγ2 and Syk with DRMs [14]. Taken together, EFhd2 may function as a DRM - cytoskeleton integrator through as yet not well characterized protein-protein or protein-lipid interactions.

Proposed function(s) of EFhd2

As detailed above, EFhd2 is a cytoskeleton associated adaptor protein involved in immune and normal as well as pathological neuronal functions, and perhaps in calcium homeostasis. This may be important for immune cell signaling and signaling pathways within or arising from neurological synapses. In accordance, EFhd2 is regulated under inflammatory conditions in immune and brain cells. Hence, we propose that EFhd2 is a modulator of immune and brain cell function under both basal and inflammatory conditions.

Expression of EFhd1

EFhd1 was first described as mitocalcin. It was identified in an attempt to isolate genes involved in neuronal differentiation in 2Y-3t cells, a neuronal progenitor cell line established from an adult p53-deficient mouse cerebellum [88]. In 2Y-3t cells EFhd1 is up-regulated during differentiation. In vivo, EFhd1 is expressed both in cerebellum and cerebrum [16, 88]. Its expression increases postnatally. In adult mice, neurons of cerebellum, cerebrum and hippocampus (cornu ammonis and dentate gyrus) reveal efhd1 mRNA, but only low amounts are present in the white matter of cerebellum and cerebrum [88]. The Purkinje-layer, the internal granule cell layer and the molecular layer, however, do express EFhd1. In addition, spermatocytes, interstitial cells of adult testis and granulosa cells of the cumulus oophorus (cumulus cells) as well as mural granulosa cells in adult ovary exhibit EFhd1 expression [16, 88]. EFhd1 is also expressed in IGF-II-deficient murine C2 myoblasts [89]. There, efhd1 mRNA ceases in the absence of the transcriptional co-activator p300 [89]. Healthy human renal tissue expresses efhd1 mRNA [90] and in murine kidney the collecting ducts, but not the glomeruli, reveal EFhd1 [16, 88]. Interestingly, EFhd1 expression is repressed in renal cell carcinoma (RCC) [90]. In contrast, EFhd1 is present in tumour tissues from patients with stage III and IV melanoma and increased expression of EFhd1 is significantly associated with shorter patient survival times [91]. Whereas EFhd1 has been proposed to exhibit tumour suppressing functions in RCC [90], growth promoting functions may be associated with other tumours. Concomitantly, uterine fibroids reveal less efhd1 mRNA than normal myometrium [92]. Under more physiological conditions EFhd1 becomes up-regulated at term when compared with midgestation [93]. In contrast, human endometrial cells down-regulate EFhd1 after trophoblast co-culture [94].

Function of EFhd1 in normal and malignant tissue

Using an SDS-gel shift assay EFhd1 has been shown to bind calcium [88]. It associates with the mitochondrial inner membrane [16] and over-expression results in neurite extension of 2Y-3t cells. Conversely, down-regulation of EFhd1 through siRNA suppresses neurite outgrowth and promotes cell death [16]. Based on these findings Tominaga and co-workers suggested that EFhd1 is involved in neuronal differentiation and operates through control of mitochondrial function [16]. Whether this proposed function correlates with the calcium binding activity of EFhd1 is not known at present. Mitochondria, however, are important reservoirs of calcium. Interestingly, ectopic expression of manganese superoxide dismutase 2 (SOD2), a mitochondrial enzyme that inactivates superoxide to protect cells from oxidative damage, induces down-regulation of EFhd1 in a pancreatic carcinoma cell line. Conversely, reduction of SOD2 abundance causes increased EFhd1 expression in a human pancreatic carcinoma cell line (MIA-PaCa2) [95]. At first glance it is therefore puzzling that reduction of SOD2 increases neuronal cell death by activating cell death pathways [96, 97]. An explanation could be that up-regulation of EFhd1 through reduced SOD2 expression [95] may be part of an anti-apoptotic mechanism antagonizing oxidative stress in neuronal cells (Figure 4). An example of a small calcium binding protein antagonizing oxidative stress is calmodulin: After calcium binding, calmodulin interacts with antioxidant enzymes involved in ROS homeostasis [1]. A similar mechanism may apply to EFhd1 in a tissue-specific manner.

Sustained activation of the autocrine IGF-II pathway is critical for myoblast viability and differentiation [98]. Gene expression profiling of IGF-II-deficient myoblasts that undergo apoptosis when incubated in differentiation-promoting medium revealed new mediators of CBP/p300 promoted survival. Myoblasts expressing CBP/p300 showed a 17-fold up-regulation of EFhd1 [89]. This indicates that EFhd1 might be a factor causing CBP/p300 mediated survival in muscle cells. Interestingly, together with PCAF, the transcriptional co-activator CBP/p300 is a key player in neuronal outgrowth by acetylating histones and p53 [99] and exhibits neuroprotective functions [100]. It is tempting to speculate that CBP/p300 up-regulates EFhd1 also in neuronal tissue to promote neuronal survival.

As readily mentioned above, EFhd1 expression has also been described in kidney. This is repressed in renal cell carcinoma (RCC) when compared to healthy tissue [90]. The same holds true for the transcription factor HNF4α (hepatocyte nuclear factor 4α). This suggests that HNF4α exhibits tumour suppressing functions [101]. Interestingly, HNF4α increases expression of EFhd1, revealing that efhd1 is a target gene of HNF4α [90, 102]. Very recently, an independent microarray analysis confirmed this initial finding [103]. Ectopic HNF4α-expression reduces proliferation in HEK293 cells (a human embryonic kidney cell line) and therefore, the authors postulate a possible tumour suppressor activity of HNF4α [102]. However, inducible EFhd1 expression in HEK293 cells revealed that EFhd1 rather augments proliferation slightly [102]. Therefore, efhd1 is a HNF4α target gene that appears to oppose the proliferation suppressing effect of HNF4α in HEK293 cells. Additionally, HNF4α up-regulates hepatocyte iNOS (inducible nitric oxide synthetase) in response to the inflammatory redox state [104, 105]. iNOS exhibits anti-oxidant and anti-apoptotic functions. Hence, it is likely that HNF4α senses the inflammatory redox state in tissues where it is expressed. Taken together, the mitochondrial localization of EFhd1, its ability to promote differentiation of 2Y-3t cells and its converse regulation by the redox factors SOD2 and HNF4α suggest that EFhd1 is part of a redox-sensitive network controlling cell survival and/or differentiation (Figure 4). Whether EFhd1 is actually a survival promoting or tumour suppressing factor may depend on the cell type and environmental context. For example, under pathological conditions, such as hypoxia and ischemia/reperfusion injury, mitochondrial dysfunction results in ROS increase. Normal tissue responds to this challenge with calcium overload, followed by mitochondrial depolarization, cytochrome c release and apoptosis [1]. Tumours, however, have developed mechanisms to survive hypoxia and ROS increase. One of these factors might be EFhd1.

A combined genome-wide ChIP sequencing analysis with gain- and loss-of-function transcriptome analyses in early-stage B cells identified promoters of genes with Ebf1 (early B cell factor-1) binding sites. IL-7 cultured pro-B cells, in which Ebf1 was conditionally inactivated, express less EFhd1 [106], suggesting that efhd1 is a target gene of Ebf1. Hnf4α was also described as direct target of the transcription factor Ebf1 in common lymphoid progenitors [107] and common lymphoid progenitors are subject to oxidative stress [108]. Thus, Ebf1 may up-regulate EFhd1 through HNF4α. These data point to a function of EFhd1 during early B cell development, an issue we are currently addressing. In contrast to transcriptional activators of efhd1, Suz12 is a transcriptional repressor as part of the transcriptional repressor complex PRC2 (polycomb repressor complex) in murine terato-carcinoma cells [109]. These complexes are normally highly abundant in embryonic tissues and are essential for development. In adult tissues, expression of Suz12 is very low [110, 111] but it is very abundant in a variety of human tumours. PRC2 catalyzes trimethylation (me3) of histone 3 lysine 27 (H3K27), thereby, mediating transcriptional repression. Consequently, targeting of the efhd1 promoter by Suz12 should lead to decreased or no expression of EFhd1.