Early Growth Response 3 (EGR3)
In 1991, the human EGR3 gene was first described by Patwardhan and colleagues. The EGR3 is an immediate early growth response gene first observed to be induced by mitogenic stimulation of rodent and human fibroblasts and monkey kidney epithelial cell line. The EGR3 cDNA sequence infers a 387 amino acid protein, and the EGR3 gene has a single intron and the cytogenetic location is 8p21.3 (Patwardhan et al. 1991).
The expression of EGR3 has been reported in various tissues, such as lymphocytes, muscle, endothelial cells, and different brain regions. The EGR3 plays important roles, as in cellular growth and in neuronal development in response to many cellular stimuli, including growth, stress, and inflammation (O’Donovan et al. 1999). In addition, studies have shown that EGR3 is essential for normal hippocampal long-term potentiation (LTP) and for hippocampal and amygdala-dependent learning and memory (Li et al. 2007; Gallitano-Mendel et al. 2007).
Pathway of EGR3-Mediated Transcriptional Activation
As other IEG transcription factors, EGR3 is rapidly and transiently induced by a large number of stimuli in the regulation of late response genes. EGR3 is induced in response to growth factors or mitogens. In the brain, EGR3 activation is triggered by neurotransmitter-receptor stimulation or depolarization (O’Donovan et al. 1999), indicating the relevance of this response also to mature neurons in the adult nervous system and not just in differentiating neurons.
At first, changes in the expression of EGR genes were considered as a general neuronal response to natural forms of stimulation involving normal synaptic activity. However, each IEG can be differently regulated via different stimuli in distinct brain regions. EGR genes are expressed at basal levels throughout the brain, including in the cortex, hippocampus and other limbic areas, and the basal ganglia. EGR3 expression is rapidly induced at high levels in these regions in response to changes in the environment, including stressful stimuli across a range of intensities, such as novelty, handling, restraint, and pain as observed in animals (Gallitano-Mendel et al. 2007). Studies have also been reported that agents that alter dopamine-dependent signaling induce the expression of EGR genes in the brain. Regarding EGR3, cocaine administration and haloperidol (a D2 antagonist) induces a rapid increase in its mRNA levels in the striatum, that is blocked by a selective D1 antagonist (Yamagata et al. 1994), suggesting the involvement of multiple neurotransmitter systems to mediate EGR3 expression.
The neuronal expression of EGR3 is regulated by synaptic activity and is coupled to MAPK-ERK signaling (Li et al. 2007; O’Donovan et al. 1999). Together with EGR1, EGR3 is the most abundant EGR proteins upregulated by synaptic activity in the brain. They may have some overlapping roles in regulating gene expression but not completely redundant since they differ in expression patterns and phenotypes in mutant mice. In contrast to the rapid and transient rise in EGR1 protein levels, EGR3 protein is more stable and remains in neurons for longer periods after activity-mediated activation (Li et al. 2007; O’Donovan et al. 1999). Sequential expression of these EGR members could represent a regulation mechanism of temporal expression pattern of specific target genes.
Once bound to the promoter of target genes, EGR3 participates in regulating their expression by a process in which molecular and cellular mechanisms are still poorly defined (Pérez-Cadahía et al. 2011). Several factors have been described to modulate the activity of EGR genes. One is the presence of activation–repression domains. All EGR-family members present the zinc-finger motifs that represent the DNA-binding domain, and they (except EGR4) also present a repressor domain (RD), the NAB-binding domain (NGFI-A binding) (Fig. 1), where a pair of proteins (NAB1 and NAB2), produced in the brain as well as in other tissues, could bind to and suppress EGR activity. Thus, NAB may represent an endogenous negative-feedback mechanism that regulates EGR-mediated transcription. A second regulatory mechanism, studied in other EGR genes but likely applied also to EGR3, is the EGR posttranslational phosphorylation that increases the half life of the protein and its DNA-binding activity. N-glucosylation in the second zinc finger and EGR crosstalk with AP-1 members has also been reported for EGR-family members as a way of regulating the DNA-binding activity (Pérez-Cadahía et al. 2011).
Physiological Functions of EGR3
Studies evaluated the relationship between EGR3 and circadian rhythms, using rodents maintained in constant darkness. They have searched for additional genes whose expressions are induced in the suprachiasmatic nucleus by light exposure and have identified the gene encoding EGR3 as a candidate transcription factor involved in this form of plasticity. The authors stated that EGR3 probably participates in the transcriptional regulation of genes in response to retinal input in the suprachiasmatic nucleus of the hypothalamus, as had been proposed for FOS, a transcription-regulatory factor that is also rapidly produced in response to growth factors (Morris et al. 1998).
Muscle spindles are skeletal muscle sensory organs that provide axial and limb position information (proprioception) to the central nervous system. Spindles consist of encapsulated muscle fibers (intrafusal fibers) that are innervated by specialized motor and sensory axons. O’Donovan and colleagues (1999) found the absence of muscle spindles in EGR3-deficient mice, which displayed severe motor abnormalities (as sensory ataxia, scoliosis, resting tremor and ptosis). It is known that innervation of myotubes by proprioceptive Ia afferent fibers is responsible for triggering the differentiation of these fibers into muscle spindles during muscle development. The authors suggest that EGR3 most probably has a key role in the differentiation process that occurs within the postsynaptic muscle cell, as the Ia afferents fibers appear to develop normally in EGR3-deficient mice. These results indicated that type I myotubes are dependent upon EGR3-mediated transcription for proper spindle development (O’Donovan et al. 1999).
Regulation of Immune Response
A study identified the EGR3 as a key negative regulator of T cell activation. Overexpression of EGR3 was associated with an increase in the E3 ubiquitin ligase Cbl-b and inhibition of T cell activation. Conversely, T cells from EGR3-deficient mice had lower expression of Cbl-b and were resistant to in vivo peptide-induced immunologic tolerance. Together, these data indicate that EGR3 is involved in promoting a T cell receptor–induced negative regulatory genetic program (Safford et al. 2005).
Learning, Memory, and Behavior
Considering that the expression of EGR family is extremely sensitive to environmental stimuli capable of inducing plasticity, it is expected that members of the EGR family are involved in learning, memory, and behavior. As MAPK-ERK effector genes, they may regulate target neuronal gene expression required for long-term synaptic changes associated with these process (Li et al. 2007). In fact, numerous behavioral and electrophysiologic studies in animals have shown that the EGR family plays a role in memory acquisition, consolidation, and hippocampal synaptic plasticity (Gallitano-Mendel et al. 2007; Li et al. 2007). EGR3, in particular, is essential for the normal response to stress as well as in the neuroplasticity induced by this responsivity since EGR3 regulates the expression of important plasticity-associated genes in a physiologically relevant manner (Gallitano-Mendel et al. 2007), such as Arc gene involved in synaptic plasticity and memory formation.
EGR3-deficient (EGR3−/−) mice appear to have normal brains and basal synaptic transmission in CA3-CA1 hippocampal neurons where EGR3 is highly expressed; however, they have abnormal LTP in CA1 neurons and present impairments in context of associative learning/memory and in short-term and long-term object recognition memory (Li et al. 2007). Other study with EGR3-deficient mice showed accentuated behavioral responses to the mild stress of handling and increased release of the stress hormone corticosterone. Moreover, these animals presented abnormal responses to novel environments and failure to habituate to social cues or acoustic stimuli (Gallitano-Mendel et al. 2007). Since stress and novelty stimulate hippocampal long-term depression (LTD), this form of synaptic plasticity in EGR3−/− mice was evaluated, showing that these animals failed to establish hippocampal LTD in response to low-frequency stimulation and presented dysfunction of an ifenprodil-sensitive (NR1/NR2B) NMDA receptor subclass. This work demonstrated the requirement for EGR3 in mediating the response to stress and novelty and in the establishment of LTD (Gallitano-Mendel et al. 2007).
Regulation of Endothelial Cell Growth
EGR3 is upregulated by VEGF in endothelial cells, which indicates that EGR3 has a critical downstream role in VEGF-mediated endothelial functions leading to angiogenesis and could be important in adult angiogenic processes involved in vascular repair and disease (Liu et al. 2008). Its role in tissue repair and fibrosis has been poorly studied.
Sympathetic Neuron Autonomous Role
EGR3 is regulated by NGF signaling in sympathetic neurons during sympathetic nervous system development when they depend upon NGF for survival and target tissue innervation. EGR3-deficient mice have severe sympathetic target tissue innervation abnormalities and profound physiological dysautonomia. EGR3 modulates downstream target genes affecting the outgrowth and branching of sympathetic neuron dendrites and axons. The results indicate that EGR3 is a novel NGF signaling effector that regulates sympathetic neuron gene expression required for normal target tissue innervation and function (Eldredge et al. 2008).
EGR3 and Pathologies
EGR3 in Psychiatry
EGR genes translate environmental events into long-term changes in neural gene expression. This has led to the hypothesis that dysfunction in EGRs may contribute for both the genetic and environmental influences on risk for psychiatric disorders. As mentioned before, mice lacking functional EGR3 show behavioral and physiologic changes consistent with models of mental illness. These include a heightened response to stress (observed by elevated release of corticosterone and behavior alterations), hyperactivity, and difficulty to habituate to environmental stimuli and social cues (Gallitano-Mendel et al. 2007). Furthermore, several proteins associated with risk for psychotic illness induce EGR3, including NRG1, CaN, NMDA receptors, and BDNF (Gallitano-Mendel et al. 2007; Hippenmeyer et al. 2002; Yamada et al. 2007; Roberts et al. 2006); the last has been proposed as a critical factor in the pathophysiology of bipolar disorder and schizophrenia.
In patients with bipolar disorder or schizophrenia, a study have found significantly increased expression of the TREM-1, a EGR3 target expressed in activated monocytes and microglia and important in inflammation process (Weigelt et al. 2011). EGR3 has been more closely studied in patients with schizophrenia; this gene has been significantly associated with this illness and has been considered a potential susceptibility candidate in schizophrenia (Yamada et al. 2007). Regarding a potential role for EGR3 in bipolar disorder patients, a family-based association study identified a nominal association of EGR3 with risk for child with bipolar disorder (Gallitano et al. 2012). And more recently, a study using an innovative approach to analyze transcriptional regulation in bipolar disorder identified the regulatory unit of EGR3 robustly repressed in both of the two bipolar gene expression data sources examined from postmortem prefrontal cortex (Pfaffenseller et al. 2016), indicating the EGR3 as a potential key target in bipolar disorder. Altogether, these findings suggest that EGR3, and its targets, may be a fruitful pathway for future studies to identify mechanisms by which environment and genetic predisposition interact to influence psychiatric disorders.
EGR3 in Cancer
Several studies have shown association between EGR3 gene and cancer. For instance, a study using a whole genome gene expression database evaluated that EGR3 mRNA is significantly overexpressed in prostate cancer compared to normal prostate tissue. Furthermore, EGR3 protein is significantly increased in patients with prostate cancer compared with normal patients. Analysis of EGR3 mRNA expression in relation to the relapse status reveals that EGR3 mRNA expression is increased in tumor cells of nonrelapsed samples compared to normal prostate cells but is significantly lower in relapsed samples compared to nonrelapse. The authors determined a list of genes correlated with this unique expression pattern; these EGR3-correlated genes were enriched with EGR binding sites in their promoters. The gene list contains inflammatory genes, such as IL-6, IL-8, IL-1b, and COX-2, which have extensive connections to prostate cancer (Pio et al. 2013).
Regarding gastric cancer, Liao et al. (2013) suggest that decreased EGR3 expression might play a critical role in the differentiation, proliferation, metastasis, and progression of these cancer cells and may be a potential diagnostic marker for gastric cancer. This study showed that EGR3 expression was significantly lower in gastric cancer tissues compared with matched nontumors tissues and that patients with lower EGR3 expression had a poorer prognosis compared with patients with higher EGR3 expression (Liao et al. 2013). Taken together, these studies suggest the involvement of EGR3 expression in cancer and its potential as a prognostic marker for this disease. More studies are required to verify the relationship between EGR3 and other types of cancer and to evaluate its ability to become a marker that can assist in the prognosis of this condition.
In this chapter, the current knowledge about EGR3 gene and its pathway were revised, with focus in neuronal cells since the major findings are found in these cells. The physiological functions of EGR3 which have been investigated in various different tissues using genetically modified animals also were discussed. In this sense, the roles of the protein encoded by the EGR3 gene in a wide variety of biological processes were explored, including the transcriptional regulation of genes involved in biological rhythms, muscle development, lymphocyte development, endothelial cell growth and migration, neuronal development, learning, memory, and behavior. Furthermore, the association between EGR3 gene and pathologies, such as psychiatric disorders and cancer, were discussed.
As a transcription factor of several genes and pathways that mediate critical biological processes, it is relevant to extend the studies about roles of EGR3 in order to better understand the relationship between environment and the influence of numerous genes in physiological and pathological conditions. In addition, further studies are needed to evaluate EGR3 targets, their role, and the mechanisms of action of drugs associated to this pathway.
- Pfaffenseller B, da Silva Magalhães PV, De Bastiani MA, Castro MA, Gallitano AL, Kapczinski F, et al. Differential expression of transcriptional regulatory units in the prefrontal cortex of patients with bipolar disorder: potential role of early growth response gene 3. Transl Psychiatry. 2016;6:e805. doi: 10.1038/tp.2016.78.PubMedPubMedCentralCrossRefGoogle Scholar
- Pio R, Jia Z, Baron VT, Mercola D. UCI NCI SPECS consortium of the strategic partners for the evaluation of cancer signatures-prostate cancer. Early growth response 3 (Egr3) is highly over-expressed in non-relapsing prostate cancer but not in relapsing prostate cancer. PLoS One. 2013;8:e54096. doi: 10.1371/journal.pone.0054096.PubMedPubMedCentralCrossRefGoogle Scholar
- Weigelt K, Carvalho LA, Drexhage RC, Wijkhuijs A, de Wit H, van Beveren NJ, et al. TREM-1 and DAP12 expression in monocytes of patients with severe psychiatric disorders. EGR3, ATF3 and PU.1 as important transcription factors. Brain Behav Immun. 2011;25:1162–9. doi: 10.1016/j.bbi.2011.03.006.PubMedCrossRefGoogle Scholar
- Yamada K, Gerber DJ, Iwayama Y, Ohnishi T, Ohba H, Toyota T, et al. Genetic analysis of the calcineurin pathway identifies members of the EGR gene family, specifically EGR3, as potential susceptibility candidates in schizophrenia. Proc Natl Acad Sci USA. 2007;104:2815–20.PubMedPubMedCentralCrossRefGoogle Scholar