All IEGs Are Not Created Equal-Molecular Sorting Within the Memory Engram.

When neurons are recruited to form the memory engram, they are driven to activate the expression of a series of immediate-early genes (IEGs). While these IEGs have been used relatively indiscriminately to identify the so-called engram neurons, recent research has demonstrated that different IEG ensembles can be physically and functionally distinct within the memory engram. This inherent heterogeneity of the memory engram is driven by the diversity in the functions and distributions of different IEGs. This process, which we call molecular sorting, is analogous to sorting the entire population of engram neurons into different sub-engrams molecularly defined by different IEGs. In this chapter, we will describe the molecular sorting process by systematically reviewing published work on engram ensemble cells defined by the following four major IEGs: Fos, Npas4, Arc, and Egr1. By comparing and contrasting these likely different components of the memory engram, we hope to gain a better understanding of the logic and significance behind the molecular sorting process for memory functions.

KAP1 negatively regulates RNA polymerase II elongation kinetics to activate signal-induced transcription.

Signal-induced transcriptional programs regulate critical biological processes through the precise spatiotemporal activation of Immediate Early Genes (IEGs); however, the mechanisms of transcription induction remain poorly understood. By combining an acute depletion system with several genomics approaches to interrogate synchronized, temporal transcription, we reveal that KAP1/TRIM28 is a first responder that fulfills the temporal and heightened transcriptional demand of IEGs. Acute KAP1 loss triggers an increase in RNA polymerase II elongation kinetics during early stimulation time points. This elongation defect derails the normal progression through the transcriptional cycle during late stimulation time points, ultimately leading to decreased recruitment of the transcription apparatus for re-initiation thereby dampening IEGs transcriptional output. Collectively, KAP1 plays a counterintuitive role by negatively regulating transcription elongation to support full activation across multiple transcription cycles of genes critical for cell physiology and organismal functions.

Neural correlates of learning and memory are altered by early-life stress.

The ability to learn and remember, which is fundamental for behavioral adaptation, is susceptible to stressful experiences during the early postnatal period, such as abnormal levels of maternal care. The exact mechanisms underlying these effects still remain elusive. This study examined whether early life stress (ELS) alters memory and brain activation patterns in male mice. Therefore, we examined the expression of the immediate early genes (IEGs) c-Fos and Arc in the dentate gyrus (DG) and basolateral amygdala (BLA) after training and memory retrieval in a fear conditioning task. Furthermore, we examined the potential of RU38486 (RU486), a glucocorticoid receptor antagonist, to mitigate ELS-induced memory deficits by blocking stress signalling during adolescence. Arc::dVenus reporter mice, which allow investigating experience-dependent expression of the immediate early gene Arc also at more remote time points, were exposed to ELS by housing dams and offspring with limited bedding and nesting material (LBN) between postnatal days (PND) 2-9 and trained in a fear conditioning task at adult age. We found that ELS reduced both fear acquisition and contextual memory retrieval. RU486 did not prevent these effects. ELS reduced the number of Arc::dVenus+ cells in DG and BLA after training, while the number of c-Fos+ cells were left unaffected. After memory retrieval, ELS decreased c-Fos+ cells in the ventral DG and BLA. ELS also altered the colocalization of c-Fos+ cells with Arc::dVenus+ cells in the ventral DG, possibly indicating impaired engram allocation in the ventral DG after memory retrieval. In conclusion, this study shows that ELS alters neuronal activation patterns after fear acquisition and retrieval, which may provide mechanistic insights into enduring impact of ELS on the processing of fear memories, possibly via changes in cell (co-) activation and engram cell allocation.

Parental developmental experience affects vocal learning in offspring.

Cultural and genetic inheritance combine to enable rapid changes in trait expression, but their relative importance in determining trait expression across generations is not clear. Birdsong is a socially learned cognitive trait that is subject to both cultural and genetic inheritance, as well as being affected by early developmental conditions. We sought to test whether early-life conditions in one generation can affect song acquisition in the next generation. We exposed one generation (F1) of nestlings to elevated corticosterone (CORT) levels, allowed them to breed freely as adults, and quantified their son's (F2) ability to copy the song of their social father. We also quantified the neurogenetic response to song playback through immediate early gene (IEG) expression in the auditory forebrain. F2 males with only one corticosterone-treated parent copied their social father's song less accurately than males with two control parents. Expression of ARC in caudomedial nidopallium (NCM) correlated with father-son song similarity, and patterns of expression levels of several IEGs in caudomedial mesopallium (CMM) in response to father song playback differed between control F2 sons and those with a CORT-treated father only. This is the first study to demonstrate that developmental conditions can affect social learning and neurogenetic responses in a subsequent generation.

cGAS-STING-TBK1 Signaling Promotes Valproic Acid-Responsive Human Cytomegalovirus Immediate-Early Transcription during Infection of Incompletely Differentiated Myeloid Cells.

Repression of human cytomegalovirus (HCMV) immediate-early (IE) gene expression is a key regulatory step in the establishment and maintenance of latent reservoirs. Viral IE transcription and protein accumulation can be elevated during latency by treatment with histone deacetylase inhibitors such as valproic acid (VPA), rendering infected cells visible to adaptive immune responses. However, the latency-associated viral protein UL138 inhibits the ability of VPA to enhance IE gene expression during infection of incompletely differentiated myeloid cells that support latency. UL138 also limits the accumulation of IFNß transcripts by inhibiting the cGAS-STING-TBK1 DNA-sensing pathway. Here, we show that, in the absence of UL138, the cGAS-STING-TBK1 pathway promotes both IFNß accumulation and VPA-responsive IE gene expression in incompletely differentiated myeloid cells. Inactivation of this pathway by either genetic or pharmacological inhibition phenocopied UL138 expression and reduced VPA-responsive IE transcript and protein accumulation. This work reveals a link between cytoplasmic pathogen sensing and epigenetic control of viral lytic phase transcription and suggests that manipulation of pattern recognition receptor signaling pathways could aid in the refinement of MIEP regulatory strategies to target latent viral reservoirs.

Perinatal stress modulates glutamatergic functional connectivity: A post-synaptic density immediate early gene-based network analysis.

Early life stress may induce synaptic changes within brain regions associated with behavioral disorders. Here, we investigated glutamatergic functional connectivity by a postsynaptic density immediate-early gene-based network analysis. Pregnant female Sprague-Dawley rats were randomly divided into two experimental groups: one exposed to stress sessions and the other serving as a stress-free control group. Homer1 expression was evaluated by in situ hybridization technique in eighty-eight brain regions of interest of male rat offspring. Differences between the perinatal stress exposed group (PRS) (n = 5) and the control group (CTR) (n = 5) were assessed by performing the Student's t-test via SPSS 28.0.1.0 with Bonferroni correction. Additionally, all possible pairwise Spearman's correlations were computed as well as correlation matrices and networks for each experimental group were generated via RStudio and Cytoscape. Perinatal stress exposure was associated with Homer1a reduction in several cortical, thalamic, and striatal regions. Furthermore, it was found to affect functional connectivity between: the lateral septal nucleus, the central medial thalamic nucleus, the anterior part of the paraventricular thalamic nucleus, and both retrosplenial granular b cortex and hippocampal regions; the orbitofrontal cortex, amygdaloid nuclei, and hippocampal regions; and lastly, among regions involved in limbic system. Finally, the PRS networks showed a significant reduction in multiple connections for the ventrolateral part of the anteroventral thalamic nucleus after perinatal stress exposure, as well as a decrease in the centrality of ventral anterior thalamic and amygdaloid nuclei suggestive of putative reduced cortical control over these regions. Within the present preclinical setting, perinatal stress exposure is a modifier of glutamatergic early gene-based functional connectivity in neuronal circuits involved in behaviors relevant to model neurodevelopmental disorders.

Characterization of the Ictalurid herpesvirus 1 immediate-early gene ORF24 and its potential role in transcriptional regulation in yeast.

Herpesviruses adhere to a precise temporal expression model in which immediate-early (IE) genes play a crucial role in regulating the viral life cycle. However, there is a lack of functional research on the IE genes in Ictalurid herpesvirus 1 (IcHV-1). In this study, we identified the IcHV-1 ORF24 as an IE gene via a metabolic inhibition assay, and subcellular analysis indicated its predominant localisation in the nucleus. To investigate its function, we performed yeast reporter assays using an ORF24 fusion protein containing the Gal4-BD domain and found that BD-ORF24 was able to activate HIS3/lacZ reporter genes without the Gal4-AD domain. Our findings provide concrete evidence that ORF24 is indeed an IE gene that likely functions as a transcriptional regulator during IcHV-1 infection. This work contributes to our understanding of the molecular mechanisms underlying fish herpesvirus IE gene expression.

The impact of pubertal stress and adult hormone exposure on the transcriptome of the developing hypothalamus.

Why individuals suffer negative consequences following stress is a complex phenomenon that is dictated by individual factors, the timing of stress within the lifespan, and when in the lifespan the consequences are measured. Women who undergo adverse childhood experiences are at risk for lasting biological consequences, including affective and stress dysregulation. We have shown that pubertal adversity is associated with a blunted hypothalamic-pituitary-adrenal axis glucocorticoid response in peripartum humans and mice. In mice, our prior examination of the paraventricular nucleus (PVN) of the hypothalamus showed that pubertal stress led to an upregulation of baseline mRNA expression of six immediate early genes (IEGs) in the PVN of adult, pregnant mice. Separately, we showed that the pregnancy-associated hormone allopregnanolone is necessary and sufficient to produce the blunted stress response phenotype in pubertally stressed mice. In the current study, we further examined a potential mechanistic role for the IEGs in the PVN. We found that in pubertally stressed adult female, but not male, mice, intra-PVN allopregnanolone was sufficient to recapitulate the baseline IEG mRNA expression profile previously observed in pubertally stressed, pregnant mice. We also examined baseline IEG mRNA expression during adolescence, where we found that IEGs have developmental trajectories that showed sex-specific disruption by pubertal stress. Altogether, these data establish that IEGs may act as a key molecular switch involved in increased vulnerability to negative outcomes in adult, pubertally stressed animals. How the factors that produce vulnerability combine throughout the lifespan is key to our understanding of the etiology of stress-related disorders.

Hemispheric dominance in HVC is experience-dependent in juvenile male zebra finches.

Juvenile male zebra finches (Taeniopygia guttata) must be exposed to an adult tutor during a sensitive period to develop normal adult song. The pre-motor nucleus HVC (acronym used as a proper name), plays a critical role in song learning and production (cf. Broca's area in humans). In the human brain, left-side hemispheric dominance in some language regions is positively correlated with proficiency in linguistic skills. However, it is unclear whether this pattern depends upon language learning, develops with normal maturation of the brain, or is the result of pre-existing functional asymmetries. In juvenile zebra finches, even though both left and right HVC contribute to song production, baseline molecular activity in HVC is left-dominant. To test if HVC exhibits hemispheric dominance prior to song learning, we raised juvenile males in isolation from adult song and measured neuronal activity in the left and right HVC upon first exposure to an auditory stimulus. Activity in the HVC was measured using the immediate early gene (IEG) zenk (acronym for zif-268, egr-1, NGFI-a, and krox-24) as a marker for neuronal activity. We found that neuronal activity in the HVC of juvenile male zebra finches is not lateralized when raised in the absence of adult song, while normally-reared juvenile birds are left-dominant. These findings show that there is no pre-existing asymmetry in the HVC prior to song exposure, suggesting that lateralization of the song system depends on learning through early exposure to adult song and subsequent song-imitation practice.

Investigating the role of an immediate early gene FOS as a potential regulator of autophagic response to hypoglycemia in embryonic hypothalamic neurons.

Hypoglycemia-associated autonomic failure (HAAF) is a well-established complication of diabetes. Although HAAF has serious outcomes such as recurrent morbidity, coma, and death, the mechanisms of HAAF and its pathological components are largely unknown. Our previous studies have revealed that hypoglycemia is associated with the upregulation of an immediate early gene - FOS. In addition, it is documented that glucose deprivation activates neuronal autophagic activities. Therefore, the present study aimed to identify the role of FOS and one of the core components of the autophagy pathway, Beclin-1 (encoded by the BECN1 gene), in the regulation of autophagic mechanisms in embryonic hypothalamic neurons in response to hypoglycemic conditions. Embryonic Mouse Hypothalamic Cell Line N39 (mHypoE-N39 or N39) was cultured in reduced concentrations of glucose (2000, 900, 500, and 200 mg/L). Gene and protein expression, as well as immunofluorescence studies on autophagy were conducted under different reduced glucose concentrations in N39 hypothalamic neurons with and without FOS and BECN1 gene knockdowns (KD). The outcomes of the present study have demonstrated a significant increase in autophagosome formation and subsequent lysosomal degradation in the hypothalamic neurons in response to reduced glucose concentrations. This hypoglycemic response appears to be lowered to a similar extent in the FOS KD and BECN1 KD cells, albeit insignificantly from the negative control, is indicative of the involvement of FOS in the autophagic response of hypothalamic neurons to hypoglycemia. Moreover, the KD cells exhibited a change in morphology and reduced cell viability compared with the control cells. Our findings suggest that reduced FOS expression could potentially be associated with impaired autophagic activities that are dependent on BECN1, which could lead to decreased or blunted hypothalamic activation in response to hypoglycemia, and this, in turn, may contribute to the development of HAAF.

The guanine nucleotide exchange factor RapGEF2 is required for ERK-dependent immediate-early gene (Egr1) activation during fear memory formation.

The MAP kinase ERK is important for neuronal plasticity underlying associative learning, yet specific molecular pathways for neuronal ERK activation are undetermined. RapGEF2 is a neuron-specific cAMP sensor that mediates ERK activation. We investigated whether it is required for cAMP-dependent ERK activation leading to other downstream neuronal signaling events occurring during associative learning, and if RapGEF2-dependent signaling impairments affect learned behavior. Camk2α-cre+/-::RapGEF2fl/fl mice with depletion of RapGEF2 in hippocampus and amygdala exhibit impairments in context- and cue-dependent fear conditioning linked to corresponding impairment in Egr1 induction in these two brain regions. Camk2α-cre+/-::RapGEF2fl/fl mice show decreased RapGEF2 expression in CA1 and dentate gyrus associated with abolition of pERK and Egr1, but not of c-Fos induction, following fear conditioning, impaired freezing to context after fear conditioning, and impaired cAMP-dependent long-term potentiation at perforant pathway and Schaffer collateral synapses in hippocampal slices ex vivo. RapGEF2 expression is largely eliminated in basolateral amygdala, also involved in fear memory, in Camk2α-cre+/-::RapGEF2fl/fl mice. Neither Egr1 nor c-fos induction in BLA after fear conditioning, nor cue-dependent fear learning, are affected by ablation of RapGEF2 in BLA. However, Egr1 induction (but not that of c-fos) in BLA is reduced after restraint stress-augmented fear conditioning, as is freezing to cue after restraint stress-augmented fear conditioning, in Camk2α-cre+/-::RapGEF2fl/fl mice. Cyclic AMP-dependent GEFs have been genetically associated as risk factors for schizophrenia, a disorder associated with cognitive deficits. Here we show a functional link between one of them, RapGEF2, and cognitive processes involved in associative learning in amygdala and hippocampus.

Phosphorylation of pyruvate dehydrogenase inversely associates with neuronal activity.

For decades, the expression of immediate early genes (IEGs) such as FOS has been the most widely used molecular marker representing neuronal activation. However, to date, there is no equivalent surrogate available for the decrease of neuronal activity. Here, we developed an optogenetic-based biochemical screen in which population neural activities can be controlled by light with single action potential precision, followed by unbiased phosphoproteomic profiling. We identified that the phosphorylation of pyruvate dehydrogenase (pPDH) inversely correlated with the intensity of action potential firing in primary neurons. In in vivo mouse models, monoclonal antibody-based pPDH immunostaining detected activity decreases across the brain, which were induced by a wide range of factors including general anesthesia, chemogenetic inhibition, sensory experiences, and natural behaviors. Thus, as an inverse activity marker (IAM) in vivo, pPDH can be used together with IEGs or other cell-type markers to profile and identify bi-directional neural dynamics induced by experiences or behaviors.

Downregulated NPAS4 in multiple brain regions is associated with major depressive disorder.

Major Depressive Disorder (MDD) is a commonly observed psychiatric disorder that affects more than 2% of the world population with a rising trend. However, disease-associated pathways and biomarkers are yet to be fully comprehended. In this study, we analyzed previously generated RNA-seq data across seven different brain regions from three distinct studies to identify differentially and co-expressed genes for patients with MDD. Differential gene expression (DGE) analysis revealed that NPAS4 is the only gene downregulated in three different brain regions. Furthermore, co-expressing gene modules responsible for glutamatergic signaling are negatively enriched in these regions. We used the results of both DGE and co-expression analyses to construct a novel MDD-associated pathway. In our model, we propose that disruption in glutamatergic signaling-related pathways might be associated with the downregulation of NPAS4 and many other immediate-early genes (IEGs) that control synaptic plasticity. In addition to DGE analysis, we identified the relative importance of KEGG pathways in discriminating MDD phenotype using a machine learning-based approach. We anticipate that our study will open doors to developing better therapeutic approaches targeting glutamatergic receptors in the treatment of MDD.

ERK2-topoisomerase II regulatory axis is important for gene activation in immediate early genes.

The function of the mitogen-activated protein kinase signaling pathway is required for the activation of immediate early genes (IEGs), including EGR1 and FOS, for cell growth and proliferation. Recent studies have identified topoisomerase II (TOP2) as one of the important regulators of the transcriptional activation of IEGs. However, the mechanism underlying transcriptional regulation involving TOP2 in IEG activation has remained unknown. Here, we demonstrate that ERK2, but not ERK1, is important for IEG transcriptional activation and report a critical ELK1 binding sequence for ERK2 function at the EGR1 gene. Our data indicate that both ERK1 and ERK2 extensively phosphorylate the C-terminal domain of TOP2B at mutual and distinctive residues. Although both ERK1 and ERK2 enhance the catalytic rate of TOP2B required to relax positive DNA supercoiling, ERK2 delays TOP2B catalysis of negative DNA supercoiling. In addition, ERK1 may relax DNA supercoiling by itself. ERK2 catalytic inhibition or knock-down interferes with transcription and deregulates TOP2B in IEGs. Furthermore, we present the first cryo-EM structure of the human cell-purified TOP2B and etoposide together with the EGR1 transcriptional start site (-30 to +20) that has the strongest affinity to TOP2B within -423 to +332. The structure shows TOP2B-mediated breakage and dramatic bending of the DNA. Transcription is activated by etoposide, while it is inhibited by ICRF193 at EGR1 and FOS, suggesting that TOP2B-mediated DNA break to favor transcriptional activation. Taken together, this study suggests that activated ERK2 phosphorylates TOP2B to regulate TOP2-DNA interactions and favor transcriptional activation in IEGs. We propose that TOP2B association, catalysis, and dissociation on its substrate DNA are important processes for regulating transcription and that ERK2-mediated TOP2B phosphorylation may be key for the catalysis and dissociation steps.

Stress-induced nuclear speckle reorganization is linked to activation of immediate early gene splicing.

Current models posit that nuclear speckles (NSs) serve as reservoirs of splicing factors and facilitate posttranscriptional mRNA processing. Here, we discovered that ribotoxic stress induces a profound reorganization of NSs with enhanced recruitment of factors required for splice-site recognition, including the RNA-binding protein TIAR, U1 snRNP proteins and U2-associated factor 65, as well as serine 2 phosphorylated RNA polymerase II. NS reorganization relies on the stress-activated p38 mitogen-activated protein kinase (MAPK) pathway and coincides with splicing activation of both pre-existing and newly synthesized pre-mRNAs. In particular, ribotoxic stress causes targeted excision of retained introns from pre-mRNAs of immediate early genes (IEGs), whose transcription is induced during the stress response. Importantly, enhanced splicing of the IEGs ZFP36 and FOS is accompanied by relocalization of the corresponding nuclear mRNA foci to NSs. Our study reveals NSs as a dynamic compartment that is remodeled under stress conditions, whereby NSs appear to become sites of IEG transcription and efficient cotranscriptional splicing.

Silc1 long noncoding RNA is an immediate-early gene promoting efficient memory formation.

Long noncoding RNAs (lncRNAs) are expressed in many brain circuits and types of neurons; nevertheless, their functional significance for normal brain functions remains elusive. Here, we study the functions in the central nervous system of Silc1, an lncRNA we have shown previously to be important for neuronal regeneration in the peripheral nervous system. We found that Silc1 is rapidly and strongly induced in the hippocampus upon exposure to novelty and is required for efficient spatial learning. Silc1 production is important for induction of Sox11 (its cis-regulated target gene) throughout the CA1-CA3 regions and proper expression of key Sox11 target genes. Consistent with its role in neuronal plasticity, Silc1 levels decline during aging and in models of Alzheimer's disease. Overall, we describe a plasticity pathway in which Silc1 acts as an immediate-early gene to activate Sox11 and induce a neuronal growth-associated transcriptional program important for learning.

Repression of the major immediate early promoter of human cytomegalovirus allows transcription from an alternate promoter.

Following infection, the human cytomegalovirus (HCMV) genome becomes rapidly associated with host histones which can contribute to the regulation of viral gene expression. This can be seen clearly during HCMV latency where silencing of the major immediate early promoter (MIEP), normally responsible for expression of the key lytic proteins IE72 and IE86, is mediated by histone methylation and recruitment of heterochromatin protein 1. Crucially, reversal of these histone modifications coupled with histone acetylation drives viral reactivation which can be blocked with specific histone acetyltransferase inhibitors (HATi). In lytic infection, a role for HATi is less clear despite the well-established enhancement of viral replication observed with histone deacetylase inhibitors. Here we report that a number of different broad-acting HATi have a minor impact on viral infection and replication during lytic infection with the more overt phenotypes observed at lower multiplicities of infection. However, specific analyses of the regulation of major immediate early (MIE) gene expression reveal that the HATi C646, which targets p300/CBP, transiently repressed MIE gene expression via inhibition of the MIEP but by 24 h post-infection MIE gene expression was rescued due to compensatory activation of an alternative IE promoter, ip2. This suggested that silencing of the MIEP promoted alternative ip2 promoter activity in lytic infection and, consistent with this, ip2 transcription is impaired in cells infected with a recombinant HCMV that does not auto-repress the MIEP at late times of infection. Furthermore, inhibition of the histone methyltransferases known to be responsible for auto-repression is similarly inhibitory to ip2 transcription in wild-type infected cells. We also observe that these discrete transcriptional activities of the MIEP and ip2 promoter are also reflected in reactivation; essentially in cells where the MIEP is silenced, ip2 activity is easier to detect at very early times post-reactivation whereas in cells where robust activation of the MIEP is observed ip2 transcription is reduced or delayed. Finally, we observe that inhibition of pathways demonstrated to be important for reactivation of HCMV in dendritic cells, e.g. in response to IL-6, are preferentially important for activation of the MIEP and not the ip2 promoter. Together, these data add to the hypothesis that the existence of multiple promoters within the MIE region of HCMV can drive reactivation in a cell type- and ligand-specific manner and also suggest that inter-dependent regulatory activity between the two promoters exists.

The midnolin-proteasome pathway catches proteins for ubiquitination-independent degradation.

Cells use ubiquitin to mark proteins for proteasomal degradation. Although the proteasome also eliminates proteins that are not ubiquitinated, how this occurs mechanistically is unclear. Here, we found that midnolin promoted the destruction of many nuclear proteins, including transcription factors encoded by the immediate-early genes. Diverse stimuli induced midnolin, and its overexpression was sufficient to cause the degradation of its targets by a mechanism that did not require ubiquitination. Instead, midnolin associated with the proteasome via an α helix, used its Catch domain to bind a region within substrates that can form a ß strand, and used a ubiquitin-like domain to promote substrate destruction. Thus, midnolin contains three regions that function in concert to target a large set of nuclear proteins to the proteasome for degradation.

Catching proteins for degradation.

Aubiquitin-independent pathway targets nuclear proteins to the proteasome.

Streptolysin S induces pronounced calcium-ion influx-dependent expression of immediate early genes encoding transcription factors.

Anginosus group streptococci (AGS) are opportunistic human pathogens of the oral cavity. The ß-hemolytic subgroup of Streptococcus anginosus subsp. anginosus secretes streptolysin S (SLS) and exhibits not only hemolytic activity but also cytotoxicity toward cultured human cell lines. However, the detailed mechanism of action of SLS and the cellular responses of host cells have not yet been fully clarified. To determine the pathogenic potential of SLS-producing ß-hemolytic S. anginosus subsp. anginosus, the SLS-dependent response induced in the human oral squamous cell carcinoma HSC-2 cells was investigated to determine the pathogenic potential of SLS-producing ß-hemolytic S. anginosus subsp. anginosus. This study revealed that the Ca2+ influx and the expression of immediate early genes (IEGs) encoding transcription factors such as early growth responses (EGRs) and activator protein-1 (AP-1) were greatly increased in HSC-2 cells incubated with the culture supernatant of SLS-producing ß-hemolytic S. anginosus subsp. anginosus. Moreover, this SLS-dependent increase in expression was significantly suppressed by Ca2+ chelation, except for jun. These results suggest that SLS caused Ca2+ influx into the cells following greatly enhanced expression of IEG-encoding transcription factors. The results of this study may help in understanding the pathogenicity of SLS-producing AGS.