key: cord-103625-p55ew8w7
authors: Ramana, Chilakamarti V.
title: Regulation of early growth response-1 (Egr-1) gene expression by Stat1-independent type I interferon signaling and respiratory viruses
date: 2020-08-14
journal: bioRxiv
DOI: 10.1101/2020.08.14.244897
sha: 
doc_id: 103625
cord_uid: p55ew8w7

Respiratory virus infection is one of the leading causes of death in the world. Activation of the Jak-Stat pathway by Interferon-alpha/beta (IFN-α/β) in lung epithelial cells is critical for innate immunity to respiratory viruses. Genetic and biochemical studies have shown that transcriptional regulation by IFN-α/β required the formation of Interferon-stimulated gene factor-3 (ISGF-3) complex consisting of Stat1, Stat2, and Irf9 transcription factors. Furthermore, IFN α/β receptor activates multiple signal transduction pathways in parallel to the Jak-Stat pathway and induces several transcription factors at mRNA levels resulting in the secondary and tertiary rounds of transcription. Transcriptional factor profiling in the transcriptome and RNA analysis revealed that Early growth response-1 (Egr-1) was rapidly induced by IFN-α/β and Toll-like receptor (TLR) ligands in multiple cell types. Studies in mutant cell lines lacking components of the ISGF-3 complex revealed that IFN-β induction of Egr-1 was independent of Stat1, Stat2, or Irf9. Activation of the Mek/Erk-1/2 pathway was implicated in the rapid induction of Egr-1 by IFN-β in serum-starved mouse lung epithelial cells. Interrogation of multiple microarray datasets revealed that respiratory viruses including coronaviruses regulated Egr-1 expression in human lung cell lines. Furthermore, Egr-1 inducible genes including transcription factors, mediators of cell growth, and chemokines were differentially regulated in the human lung cell lines after coronavirus infection, and in the lung biopsies of COVID-19 patients. Rapid induction by interferons, TLR ligands, and respiratory viruses suggests a critical role for Egr-1 in antiviral response and inflammation with potential implications for human health and disease.

Interferons (IFNs) are pleiotropic cytokines that play a central role in innate and adaptive immunity (1, 2) . There are 3 major types of interferons. The type I interferons consists of IFN-alpha/beta (IFN-α/β ). type II interferon represented by interferon-gamma (IFN-γ), and type III interferons represented by interferon lambda (IFN-λ ) . The biological effects of IFN are mediated mainly by the rapid and dramatic changes in gene expression (3)). Type I IFN signaling involves the binding of IFN-α/β to its receptor (IFNAR) and activation of receptor-associated Janus protein tyrosine kinases Jak1 and Tyk2 and the phosphorylation of Stat1 and Stat2 to form a heterodimer. This heterodimer associate with the Interferon responsive factor 9 (Irf9) to form the interferon-stimulated gene factor-3 (ISGF-3) complex on interferon-stimulated response elements (ISRE) in the promoters of type I IFN responsive genes to regulate transcription (1, 2, 4) .

Stat1 homodimer or heterodimer of Stat1/Stat2 can also bind to Gamma activated sequence (GAS) in the promoter and regulate transcription of some type I IFN responsive genes (1, 4) . In addition to this classical canonical Jak-Stat pathway, non-canonical pathways involving Stat2, Irf9, and unphosphorylated ISGF-3 components in the regulation of gene expression have been described (5) . Furthermore, several signal transduction pathways are activated by IFN receptors in parallel to Jak-Stat including extracellular signal-regulated kinases (Erk-1/2) and phosphoinositide 3'-kinase/Akt pathways (6, 7) . The first wave of Interferon signaling is followed by induction of transcription factors such as Interferon regulatory factors (IRFs) that sustain the secondary and tertiary transcriptional responses. TLR recognition of pathogens by immune cells results in the production of multiple cytokines such as IFN-α/β , Tumor Necrosis Factor-α (TNF-α ), and Interleukin-β (IL-1 β ) in innate immunity (8) . Activation of multiple signal transduction pathways and cross-talk between the pathways enables fine-tuning of gene expression in innate immunity (9) . Cross-talk between TNF-α and IFN signaling pathways regulate inflammatory gene expression to influence the immune responses (10, 11 ) . Clinical significance of the balance between immune modulation and inflammation in signal transduction pathways has been demonstrated in a variety of autoimmune diseases (12, 13) .

Early growth response 1 (human EGR1/ mouse Egr1; referred in this manuscript as Egr-1) belongs to a family of immediate-early response genes that contain a conserved zinc finger DNA-binding domain and binds to a GC-rich sequence in the promoters of target genes (14) . A variety of signals, including serum, growth factors, cytokines, and hormones stimulate Egr-1 expression (15, 16) . Egr-1 has been shown to play an important role by regulating inflammatory gene expression in a variety of lung diseases and in mouse lung injury models including asthma, emphysema, airway inflammation, and pulmonary fibrosis (17) (18) (19) . Ischemia-mediated activation of Egr-1 triggers the expression of pivotal regulators of inflammation including the chemokine, adhesion receptor, and pro-coagulant gene expression (20) . Egr-1 stimulates chemokine production in interleukin-13 mediated airway inflammation, and remodeling in the lung (21) .

High levels of expression of Egr-1 and Egr-1 inducible genes were reported in atherosclerosis, an inflammatory disease (22) . Egr-1 may have a potential role in liver injury and in acute pancreatitis (23) (24) (25) . Lipopolysaccharide (LPS) induction of Egr-1 was mediated by the activation of Erk-1/2 pathway and serum response elements (26) . In this study, transcription factor profiling in interferon-mediated gene expression data sets and RT-PCR revealed that Egr-1 was rapidly induced by IFN-α/β and TLR ligands in multiple cell types. Studies in mouse and human fibroblast mutant cell lines revealed that Egr-1 induction by type I interferons was independent of 4 transcription factors Stat1, Stat2 or Irf9 . Furthermore, the regulation of Egr-1 by IFN-β was mediated by the activation of the Erk-1/2 pathway in serum-starved mouse lung epithelial cells.

Respiratory pathogens including coronaviruses (SARS-CoV-1 and 2) and influenza viruses regulated the expression of Egr-1 in human lung cell lines and in lung biopsies and peripheral blood cells of COVID-19 patients, These studies suggest that the regulation of Egr-1 may play an important role in the antiviral response and inflammatory disease.

Gene expression in response to Interferon, TNF-α. and TLR agonist treatment in human peripheral blood mononuclear cells (PBMC), human hepatoma cells (Huh-7), mouse bone marrow-derived macrophages (BMDM) were reported previously (27) (28) (29) .

Supplementary data was downloaded from the Journal publisher websites and Geo datasets were analyzed with the GeoR2R method (NCBI). Cluster analysis was performed using gene expression software tools at www.heatmapper.ca. Gene expression datasets representing human lung cell lines infected with respiratory viruses and from COVID-19 patients were reported previously (30, 31) . Gene expression resources from Immgen RNA seq SKYLINE were used ( http://rstats.immgen.org/Skyline_COVID-19/skyline.html ). Outliers of expression were not included in the analysis.

Mouse lung epithelial (MLE-Kd) and macrophage (RAW264.7) cell lines were used (11, 32) . Human fibrosarcoma cell line (2fTGH) and mutant cell lines lacking Stat1 (U3A), Stat2 (U6A), and IRF9 (U2A) were described previously (33) . Wild -type and Stat1-knockout mouse embryo fibroblast (MEF) cell lines were used (34) . Cells were 5 maintained in DMEM supplemented with 10% FBS and 1% penicillin and streptomycin. Cells were plated at 60-70% density and maintained in full medium for a day. Cells were incubated in serum-free medium for another 24 hours. Cells were treated with TNF-α (20 ng/ml) or IFN-β were performed using Ambion Retroscript (Austin, TX), according to the manufacturer's protocol. Primer sequences for Egr-1, Irf1, and Gapdh were obtained from the molecular reagents section of Mouse Genome Informatics (MGI). Human Egr-1 and β− Actin primer sequences were previously described (22) . PCR products were resolved on a 1% agarose gel containing ethidium bromide and visualized with U.V. Light and images were captured on a digital system.

Image files were processed with ImageJ (NIH) software. Specific gene expression was normalized to GAPDH or β -Actin and fold changes in the treated samples were calculated with respect to controls. MLE-Kd cell extracts were prepared and proteins were separated by electrophoresis using 8%-10% SDS-PAGE gels. Proteins in the gel were electrophoretically transferred to polyvinylidene difluoride membranes (Bio-Rad, CA) and subjected to immunoblotting with the antibodies for phosphorylated Erk-1/2 (Thr202/Tyr 204) or total Erk-1/2 from Cell Signaling Technology (Beverly, MA). Blots were visualized by enhanced chemiluminescence western detection system (Pierce, IL). (Figure 2A and 2B). These results were consistent with previous studies in human fibroblasts (15) . TNF-α induction of Egr-1 was much higher than IFN α/β in 2fTGH cells ( Figure 2B ). Interestingly, TNF-α but not IFN-α induced Egr-1 mRNA in Hela cells suggesting differential pathway regulation (15) . Furthermore, IFN-λ induction of Egr-1 was higher than with IFN-α/β in Huh7 cells ( Figure 2C ) Irf9 (U2A) demonstrated that IFN-β induction of Egr-1 was independent of ISGF-3 components ( Figure 4B ). TNF-α and TLR ligands activate several Mitogen-activated protein kinase (MAPK) pathways such as Extracellular signal-regulated kinase (ERK), Jun N-terminal kinase (JNK) and p38 (37) . Activation of the Erk-1/2 pathway leading to the phosphorylation of transcription factors such as Elk1 and Srf1 was implicated in the rapid induction of Egr-1 in response to multiple stimuli (26, 38) . (42) . Detailed promoter analysis revealed that multiple distal and proximal cis-elements were involved in Egr-1 induction (43).

The generation of an inflammatory response is a complex process involving multiple cytokines acting in parallel and in concert in innate and adaptive immunity (36, 44) . Influenza virus-infected dendritic cells and macrophages, which reside in close proximity to lung epithelium, can produce significant amounts of TNF-α and type I IFN in response to virus infection (45) . IFN-α / β is involved in signaling cross-talk with TNF −α that enhance or dampen the severity of inflammatory response (9, 10) . Interaction between Interferon-α/β and TNF-α signaling was reported in autoimmune diseases. (12, 13) . A select list of genes was induced by both TNF-α and Interferon-a /β in PBMC and BMDM gene expression datasets ( Figure 6 ). TNF-α induction of these genes was much higher than IFN α/β in the mouse BMDM cells. Interestingly, Egr-1 was induced by both cytokines in PBMC and BMDM. These genes are involved in transcriptional regulation and integrating signal transduction pathways that are likely to play an important role in the cytokine storm and shift to hyperinflammatory gene expression in response to coronavirus infections (30,65).

Egr-1 is a zinc finger transcription factor that binds to a GC-rich sequence in the promoter regions of target genes. A shortlist of Egr-1 regulated genes was generated from the TRUUST transcription factor database and published studies in different cell types and under different stimuli (20) (21) (22) 46) .

Egr-1 regulates genes involved in cell growth (Egr2, Atf3, Pdgfrb, Cdkna1, Ccnd1, Tnfsf10, and chemokines involved in inflammation such as (Cxcl10, Cxcl2, Ccl2, Ccl3). It is important to note that many of the Egr-1 target genes were also known targets of NF-KB, AP1, and Stat1 in cytokine signaling (46) . Furthermore, Egr-1 interacts with several other transcription factors such as Ets1, Ets2, Elk1 that are common in cytokine responsive genes (46) . Previous studies have shown that transcription factors of innate and adaptive immunity show functional connectivity involving shared interacting protein partners and target genes. (47) . Cluster analysis revealed that Egr-1 induction was correlated with the expression of Egr-1 target genes by IFN-α/β in PBMC and in BMDM cells ( Figure 7 ).

Severe acute respiratory syndrome coronaviruses (SARS-CoV) and influenza viruses are major respiratory pathogens in humans with seasonal epidemics and potential pandemic threats (48, 49) . Gene expression profiling studies of human lung epithelial cell lines such as Calu-3, A549, and NHBE1 in response to respiratory viruses were reported recently (30, 31) . Interrogation of infection also induced Egr-1 mRNA in A549 cells, 24 hours post-infection ( Figure 12C ). These studies suggest that Egr-1 expression is a common host response to many respiratory viruses

Gene expression profiling studies of a limited number of human lung biopsies and PBMC of healthy controls and COVID-19 patients were reported recently (30, 31) . Interrogation of the gene expression data in lung biopsies revealed that Egr-1 expression levels were significantly lower in COVID-19

patients compared with healthy controls. Expression of Egr-1 target genes related to cell growth such as CCND1, EGR2, and PDGFRB were also down-regulated ( Figure 13 and data not shown). In contrast, expression of Egr-1 target genes involved in inflammatory responses such as CXCL10, CCL2, CCL3, CCL4, and TNFSF10 were dramatically enhanced in COVID-19 patients compared with healthy controls (Figure 14 and data not shown). Interestingly, STAT1 expression levels were significantly increased in COVID-19 patients compared with healthy controls (Figure 14 ). There are several limiting factors to interpreting the data. The lung is a complex tissue consisting of more than thirty cell types with differential contributions with respect to cell mass and gene expression. For example, type I and type II cells in mouse lungs constitute the major and minor cell types and express distinct cell markers (35) . Without information on the expression levels in distinct cell types, it is difficult to correlate Egr-1 expression with target genes in the whole lung tissue. In support of this view, in a mouse model of CD8 + T cell -mediated lung injury, chemokine expression was dependent on Egr-1 activation in alveolar type II cells (63) . Another intriguing possibility is that enhanced STAT1 expression compensates for decreased Egr-1 expression in some cell types in the lung. It is important to consider that in addition to changes in mRNA levels, the phosphorylation status of STAT1 and Egr-1 may play an important role in the transcriptional regulation of chemokine target genes. It is likely that differential expression in distinct cell types may account for the disparity of 

Egr-1 has emerged as a key regulator of cell growth, reproduction, and response to tissue injury (16, (61) (62) (63) . Egr-1 was rapidly induced by interferons and pro-inflammatory cytokines such as TNF-α, and IL-1 β (15, 63, 64) . Recent studies have demonstrated dramatic changes in type I interferon, TNF-α, and IL-1 β production by immune cells and cytokine-mediated lung inflammation in COVID-19 patients (30, 57, 65, 66) . However, the role of Egr-1 in innate immunity and antiviral response to respiratory viruses in general and SARS-CoV-2, in particular, remains to be investigated.

Transcriptional factor profiling in the transcriptome revealed that Egr-1was induced by IFN-α/β , TLR ligands, and TNF-α in human and mouse cells. Studies in mutant cell lines lacking Stat1, Stat2, and Irf9 revealed that IFN-α/β induction of Egr-1 was independent of ISGF-3 components and mediated by the activation of Erk-1/2 pathway. Furthermore, respiratory viruses such as SARS-CoV-2

induced Egr-1 and its target genes in several lung epithelial cell lines and in COVID-19 patients.

Activation of Egr-1 by IFN-α/β and TNF-α and cross-talk between the pathways modulates signal transduction and inflammatory response in innate immunity. 

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