Expression Of Early Growth Response Research Articles

WNT11 is a direct target of early growth response protein 1

WNT11 is a member of the non-canonical Wnt family and plays a crucial role in tumor progression. However, the regulatory mechanisms underlying WNT11 expression are unclear. Tumor necrosis factor-alpha (TNFα) is a major inflammatory cytokine produced in the tumor microenvironment and contributes to processes associated with tumor progression, such as tumor invasion and metastasis. By using site-directed mutagenesis and introducing a serial deletion in the 5’-regulatory region of WNT11, we observed that TNFα activates the early growth response 1 (EGR1)-binding sequence (EBS) in the proximal region of WNT11 and that the transcription factor EGR1 is neces-sary for the TNFα-induced transcription of WNT11. EGR1 bound directly to the EBSs within the proximal 5'-regulatory region of WNT11 and ectopic expression of EGR1 stimulated WNT11 promoter activity, whereas the knockdown of EGR1 expression by RNA interference reduced TNFα-induced WNT11 expression in T47D breast cancer cells. We also observed that mitogen-activated protein kinases (MAPK), extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 kinase mediated TNFα-induced transcription of WNT11 via EGR1. Our results suggest that EGR1 directly targets WNT11 in response to TNFα stimulation in breast cancer cells.

Ring finger protein 2 promotes colorectal cancer progression by suppressing early growth response 1.

Ring finger protein 2 (RNF2) is an important component of polycomb repressive complex 1. RNF2 is upregulated in many kinds of tumors, and elevated RNF2 expression is associated with a poor prognosis in certain cancers. To assess the function of RNF2 in colorectal cancer, we examined RNF2 protein levels in 313 paired colorectal cancer tissues and adjacent normal tissues. We then analyzed the association of RNF2 expression with the patients’ clinicopathologic features and prognoses. RNF2 expression was upregulated in colorectal cancer tissues and was associated with the tumor differentiation status, tumor stage and prognosis. In colorectal cancer cell lines, downregulation of RNF2 inhibited cell proliferation and induced apoptosis. Gene microarray analysis revealed that early growth response 1 (EGR1) was upregulated in RNF2-knockdown cells. Knocking down EGR1 partially reversed the inhibition of cell proliferation and the induction of apoptosis in RNF2-knockdown cells. RNF2 was enriched at the EGR1 promoter, where it mono-ubiquitinated histone H2A, thereby inhibiting EGR1 expression. These results indicate that RNF2 is oncogenic in colorectal cancer and may promote disease progression by inhibiting EGR1 expression. RNF2 is thus a potential prognostic marker and therapeutic target in colorectal cancer.

Early growth response 1 promoted the invasion of glioblastoma multiforme by elevating HMGB1.

Glioblastoma multiforme (GBM) is the most common and deadly glioma subtype. Early growth response 1 (EGR1) participates in the progression of several cancer types, but the expression and function of EGR1 in GBM was rarely investigated. The expressions of EGR1 in GBM were detected with qRT-PCR and immunohistochemistry in 12 pairs of fresh GBM tissues and 116 paraffin-embedded specimens. The patients were divided into high and low EGR1 groups according to the IHC score of EGR1, and the prognostic significances of different groups were evaluated with univariate and multivariate analyses. With in-vitro experiments, we assessed the role of EGR1 in the proliferation and invasion of GBM cells. In our study, EGR1 was up-regulated in GBM tissues compared with tumor-adjacent normal tissues. High expression of EGR1 or HMGB1 were unfavorable prognostic biomarkers of GBM. Coexpression of EGR1 and HMGB1 could predict the prognosis of GBM more sensitively. EGR1 facilitated the proliferation and invasion of GBM cells. Moreover, EGR1 promoted the invasion, instead of proliferation, of GBM cells by elevating the expression of HMGB1. ERG1 was a prognostic biomarker of GBM, and ERG1 and HMGB1 synergistically could predict the GBM prognosis more precisely. ERG1 could promote GBM cell invasion by inducing HMGB1 expression.

Early Growth Response Gene Upregulation in Epstein-Barr Virus (EBV)-Associated Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS).

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a chronic multisystem disease exhibiting a variety of symptoms and affecting multiple systems. Psychological stress and virus infection are important. Virus infection may trigger the onset, and psychological stress may reactivate latent viruses, for example, Epstein–Barr virus (EBV). It has recently been reported that EBV induced gene 2 (EBI2) was upregulated in blood in a subset of ME/CFS patients. The purpose of this study was to determine whether the pattern of expression of early growth response (EGR) genes, important in EBV infection and which have also been found to be upregulated in blood of ME/CFS patients, paralleled that of EBI2. EGR gene upregulation was found to be closely associated with that of EBI2 in ME/CFS, providing further evidence in support of ongoing EBV reactivation in a subset of ME/CFS patients. EGR1, EGR2, and EGR3 are part of the cellular immediate early gene response and are important in EBV transcription, reactivation, and B lymphocyte transformation. EGR1 is a regulator of immune function, and is important in vascular homeostasis, psychological stress, connective tissue disease, mitochondrial function, all of which are relevant to ME/CFS. EGR2 and EGR3 are negative regulators of T lymphocytes and are important in systemic autoimmunity.

EZH2 overexpression dampens tumor-suppressive signals via an EGR1 silencer to drive breast tumorigenesis.

The mechanism underlying EZH2 overexpression in breast cancer and its involvement in tumorigenesis remain poorly understood. In this study, we developed an approach to systematically identify the trans-acting factors regulating the EZH2 expression, and identified more than 20 such factors. We revealed reciprocal regulation of early growth response 1 (EGR1) and EZH2: EGR1 activates the expression of EZH2, and EZH2 represses EGR1 expression. Using CRISPR-mediated genome/epigenome editing, we demonstrated that EHZ2 represses EGR1 expression through a silencer downstream of the EGR1 gene. Deletion of the EGR1 silencer resulted in reduced cell growth, invasion, tumorigenicity of breast cancer cells, and extensive changes in gene expression, such as upregulation of GADD45, DDIT3, and RND1; and downregulation of genes encoding cholesterol biosynthesis pathway enzymes. We hypothesize that EZH2/PRC2 acts as a "brake" for EGR1 expression by targeting the EGR1 silencer, and EZH2 overexpression dampens tumor-suppressive signals mediated by EGR1 to drive breast tumorigenesis.

Regulation of pro-opiomelanocortin (POMC) gene transcription by interleukin-31 via early growth response 1 (EGR-1) in HaCaT keratinocytes.

Pro-opiomelanocortin (POMC) is a large precursor protein of and β-endorphin. POMC expressed in keratinocytes regulates various pathophysiological responses, such as pruritus in atopic dermatitis. Interleukin (IL)-31 is a T helper 2 (Th2)-derived cytokine that functions as a pruritogen, stimulating the sensory neurons in the skin. However, the regulatory mechanism underlying IL-31-induced POMC expression in keratinocytes remains largely unknown. Herein, using a 5'-serial deletion and site-specific mutation constructs of the regulatory region of POMC, we demonstrated that a putative EGR1-binding sequence (EBS) motif in POMC is required for its upregulation by IL-31 in HaCaT keratinocytes. Notably, EGR-1 directly interacted with the EBS motif in POMC. The ectopic expression of EGR-1 stimulated the POMC promoter activity, whereas the knockdown of EGR-1 expression by RNA interference reduced IL-31-induced POMC expression. Furthermore, we observed that three major mitogen-activated protein kinases, ERK, JNK, and p38 kinase, mediated IL-31-induced EGR-1 expression. In summary, our results suggest that EGR-1 trans-activates POMC in response to IL-31 stimulation in HaCaT keratinocytes.

EGR2 is elevated and positively regulates inflammatory IFN\u03b3 production in lupus CD4+ T cells

BackgroundRecent studies have shown that early growth response 2 (EGR2) is highly induced in activated T cells and regulates T cell functions. In normal C57BL/6 (B6) mice, deletion of EGR2 in lymphocytes results in the development of lupus-like systemic autoimmune disease, which implies indirectly an autoimmune protective role of EGR2. Conversely, increased EGR2 gene expression is suggested to link with high risk of human lupus. In the present studies we sought to clarify the expression and inflammation regulatory role of EGR2 in murine lupus T cells directly.ResultsWe performed RT-qPCR analysis and found a significant increase of EGR2 mRNA expression in human lupus PBMCs and in CD4+ T cells from three different murine lupus models including MRL-lpr, B6-lpr, and B6.sle123 mice at diseased stage when compared to age-matched control MRL or B6 mice. By performing intracellular flow cytometry analysis, we found that EGR2 protein expression was significantly increased in resting lupus (either MRL-lpr or B6.sle123) CD4+ T cells when compared to CD4+ T cells from their respective non-autoimmune controls. However, there was no difference of EGR2 protein expression in anti-CD3 and anti-CD28 stimulated control and lupus CD4+ T cells since there was a stronger induction of EGR2 in activated control CD4+ T cells. EGR2 expression was significantly increased in MRL-lpr mice at an age when lupus is manifested. To understand further the function of elevated EGR2 in lupus CD4+ T cells, we inhibited EGR2 with a specific siRNA in vitro in splenocytes from MRL-lpr and control MRL mice at 15 weeks-of-age. We found that EGR2 inhibition significantly reduced IFNγ production in PMA and ionomycin activated MRL-lpr lupus CD4+ T cells, but not control MRL CD4+ T cells. We also found that inhibition of EGR2 in vitro suppressed the Th1 differentiation in both MRL and MRL-lpr naïve CD4+ T cells.ConclusionsEGR2 is highly upregulated in human and murine lupus cells. Our in vitro data suggest a positive role of EGR2 in the regulation of Th1 differentiation and IFNγ production in lupus effector CD4+ T cells.

UHRF1 Controls Thymocyte Fate Decisions through the Epigenetic Regulation of EGR1 Expression.

Thymocyte differentiation is a highly complex process that is accompanied by epigenetic changes. Ubiquitin-like containing PHD ring finger 1 (UHRF1) is a critical epigenetic modifier involved in various cellular processes. In this study, we demonstrated that it is highly expressed in T cell precursors of the thymus. Further, its deficiency results in significantly reduced thymocyte cellularity and thymus size in mice. Through systematic analysis based on single-cell RNA sequencing, we found that UHRF1 deficiency thwarts αβ T cell lineage development, whereas biasing γδ T lineage differentiation dampens the progression of immature single-positive cells. UHRF1 deficiency promotes the IL-17 secreting and RORγt expression in γδ T cell, indicating a Tγδ17 phenotype. Further, the analysis of gene-regulatory networks demonstrated that UHRF1 controls the expression of early growth response 1 (EGR1). UHRF1 interacts with DNA methyltransferase 1 (DNMT1) at the CpG promoter region of Egr1 loci and affects the nearby chromatin modifications of H3K9me3 and H3K4me3. Taken together, our results demonstrate that UHRF1 is a key factor that mediates the epigenetic regulation of EGR1 and, consequently, thymocyte fate decisions.

A novel regulatory loop miR-101/ANXA2/EGR1 mediates malignant characteristics of liver cancer stem cells.

Increasing evidence suggests that liver cancer stem cells (LCSCs) are the cellular determinants that promote tumor recurrence and metastases. Aberrantly expressed miRNAs were identified in LCSCs and found to play a significant role in modulating biological characteristics of LCSCs. In this study, we implemented miRNA microarrays in CD133+ LCSCs and found miR-101 expression was downregulated. Increasing miR-101 expression repressed the metastasis and tumorigenic potential in LCSCs. Further investigations showed that ANXA2 was a novel target of miR-101. And we revealed that ANXA2 plays a critical role in acceleration of cell cycle and enhancing the migration and invasion abilities of LCSCs. Elevated ANXA2 increased activation of extracellular signal-regulated kinase (ERK) which regulated SOX2 and cell cycle-related kinases. Moreover, ERK phosphorylation inhibited the expression of early growth response 1 (EGR1) which in turn restrained the transcription of miR-101. In vivo experiments, overexpression of miR-101 produced potent inhibitory effects on the growth of LCSCs xenograft tumors as well as ANXA2 knockdown. Taken together, our findings suggest a novel regulatory loop miR-101/ANXA2/EGR1 in LCSCs and may serve as potential therapeutic targets in liver cancer.

Increased expression of EGR1 and KLF4 by polysulfide via activation of the ERK1/2 and ERK5 pathways in cultured intestinal epithelial cells.

Sodium trisulfide (Na2S3) releases hydrogen polysulfide (H2Sn) and is useful for the investigation of the effects of H2Sn on the cell functions. In the present study, we first examined the effects of Na2S3 on the gene expression of IEC-6 cells, a rat intestinal epithelial cell line. Microarray analysis and reverse transcription-polymerase chain reaction analysis revealed that Na2S3 increased the gene expression of early growth response 1 (EGR1) and Kruppel-like transcription factor 4 (KLF4). It was interesting that U0126, an inhibitor of the activation of extracellular signal-regulated kinase 1 (ERK1), ERK2, and ERK5, inhibited the Na2S3-induced gene expression of EGR1 and KLF4. Na2S3 activated ERK1 and ERK2 (ERK1/2) within 15 min. In addition to ERK1/2, Na2S3 activated ERK5. We noticed that the electrophoretic mobility of ERK5 was decreased after Na2S3 treatment. Phos-tag analysis and in vitro dephosphorylation of the cell extracts indicated that the gel-shift of ERK5 was due to its phosphorylation. The gel-shift of ERK5 was inhibited completely by both U0126 and ERK5-IN-1, a specific inhibitor of ERK5. From these results, we concluded that the gel-shift of ERK5 was induced through autophosphorylation by activated ERK5 after Na2S3 treatment. The present study suggested that H2Sn affected various functions of intestinal epithelial cells through the activation of the ERK1/2 and ERK5 pathways.

UHRF1 Controls Thymocyte Fate Decisions Through the Epigenetic Regulation of EGR1 Expression

Abstract Thymocyte differentiation is a highly complex process that is accompanied by epigenetic changes. Ubiquitin-like containing PHD ring finger 1 (UHRF1) is a critical epigenetic modifier involved in various cellular processes. Here, we demonstrated that it is highly expressed in T-cell precursors of the thymus. Further, its deficiency results in significantly-reduced thymocyte cellularity and thymus size. Through systematic analysis based on single-cell RNA-sequencing, we found that UHRF1 deficiency thwarts αβ T cell lineage development while biasing γδ lineage differentiation, dampens the progression of immature single-positive cells (ISPs). Further, the analysis of gene-regulatory networks demonstrated that UHRF1 controls the expression of early growth response 1 (EGR1). UHRF1 interacts with DNA methyltransferase 1 (DNMT1) at the CpG promoter region of Egr1 loci and affects the nearby chromatin modifications of H3K9me3 and H3K4me3. Taken together, our results demonstrate that UHRF1 is a key factor that mediates the epigenetic regulation of EGR1 and consequently thymocyte fate decisions.

The Pattern of EGR1 Expression During Chick Limb Development Suggests a Role in Regulating SHH Expression and/or Termination

Limb development is governed by a complex interplay between three signaling centers that mediate growth along three coordinate axes. The apical ectodermal ridge (AER), zone of polarizing activity (ZPA) and the dorsal ectoderm regulate the proximal‐distal, anterior‐posterior and dorsal‐ventral axes, respectively. Development occurs along these three axes to create an asymmetrical limb and is highly dependent on crosstalk between fibroblast growth factors (FGFs) from the AER and sonic hedgehog (SHH) from the ZPA. The mechanisms and intermediate players between FGF and SHH are poorly characterized. Previous work in our lab identified a number of FGF‐responsive candidate genes. Most notably early growth response 1 (EGR1), encoding a zinc finger type protein, was identified as the gene with the most robust induction in response to FGF. EGR1 has been shown to play a role in tendon development but to date its expression in the developing limb has not been well characterized. We found EGR1 to have low endogenous expression in the chick limb by whole mount in situ hybridization (WMISH). We highlighted the WMISH pattern of EGR1 using a 3 hr cycloheximide pretreatment to enhance RNA accumulation. This approach revealed that EGR1 is expressed subjacent to the AER and overlaps the ZPA during robust SHH expression (Hamburger‐Hamilton stage 17 (HH17) through HH24). In contrast, as SHH expression wanes (HH25–26), EGR1 expression recedes from the posterior ZPA, and persists in the anterior progress zone underlying the AER. In addition, EGR1 expression extends proximally from the anterior border of the progress zone. The reduction of EGR1 within the ZPA coincident with the decline in SHH may indicate a role for EGR1 in optimizing SHH expression and triggering termination.

Role of Histone Deacetylases 5 (HDAC5) in Ang II‐Induced the Early Growth Response Protein‐1 (Egr‐1) Expression and Vascular Hypertrophy

Angiotensin II (Ang II), a key vasoactive peptide, is known to play an important role in the pathophysiology of vascular diseases. A heightened activation of Ang II‐induced signaling pathways that promote proliferation, hypertrophy and migration of vascular smooth muscle cells (VSMCs) has been suggested to contribute to vascular dysfunctions. We have shown earlier that Ang II enhances the expression of early growth response protein‐1 (Egr‐1), a zinc transcription factor, which is upregulated in animal models of vascular diseases. Histone deacetylases (HDACs) remove acetyl groups of lysine residues from histone and non‐histone proteins and elevated activation of class IIa HDACs is reported in several vascular pathologies. Recent studies have demonstrated that the phosphorylation and the nuclear export of HDAC5, a member of class IIa HDACs, is associated with vascular remodeling through their ability to interact with transcription factors linked to cell proliferation, migration and hypertrophy. However, the involvement of HDAC5 in Ang II‐induced Egr‐1 expression remain unexplored. Here, we showed that the specific pharmacological inhibition of class IIa HDACs by MC1568 or TMP‐195 as well as siRNA‐mediated silencing of HDAC5 attenuated Ang II induced Egr‐1 expression in VSMCs. In addition, the blockade of the nuclear export of HDAC5 by using nuclear transport inhibitors leptomycin B or KPT‐330 decreased Ang II‐induced Egr‐1 expression. We also demonstrated that siRNA‐induced silencing of HDAC5 or Egr‐1 prevented VSMC hypertrophy in response to Ang II stimulation. In summary, our results show that Ang II‐induced Egr‐1 expression is regulated by the activation of HDAC5 and Ang II‐induced cell hypertrophy is mediated by HDAC5 and Egr‐1.Support or Funding InformationSupported by the Canadian Institutes of Health Research

Lnc003875/miR-363/EGR1 regulatory network in the carcinoma -associated fibroblasts controls the angiogenesis of human placental site trophoblastic tumor (PSTT)

The rare gestational trophoblastic neoplasia placental site trophoblastic tumor (PSTT) frequently demonstrates a high degree of vascularization, which may facilitate the tumor metastasis. However, the underlying mechanisms remain largely unknown. In the present study, we found that early growth response 1 (EGR1) was highly expressed in the carcinoma-associated fibroblasts (CAFs) of PSTT tissues. Further data showed that miR-363 down-regulated EGR1 expression whereas long non-coding RNA NONHSAT003875 (lnc003875) up-regulated EGR1 expression in PSTT derived CAFs. lnc003875 exerted no effect on miR-363 expression, but it recovered the decrease of EGR1 caused by miR-363 mimic. The conditioned media from PSTT CAFs treated with miR-363 mimic abrogated the tube formation capacity of human umbilical vein endothelial cells (HUVECs), which can be partially restored by lnc003875 over-expression. Moreover, over-expression of EGR1 promoted the secretion of Angiopoietin-1 (Ang-1) in PSTT derived CAFs and improved the tube formation of HUVECs, which could be effectively abrogated by Ang-1 siRNAs. In vivo vasculogenesis assay demonstrated that lnc003875/EGR1 in PSTT derived CAFs promoted the vasculogenesis of HUVECs in C57BL/6 mice. Collectively, these findings indicated that lnc003875/miR-363/EGR1/Ang-1 in CAFs may be crucial for the angiogenesis of PSTT.

Transcription factor early growth response-1 plays an oncogenic role in salivary gland pleomorphic adenoma.

Although abnormal expression of early growth response-1 (Egr1) has been revealed in various human solid tumors, the functions and potential mechanisms of Egr1 in the progression of salivary gland pleomorphic adenoma (SGPA) are not entirely understood. An elevated expression of Egr1 was observed both in the human salivary gland pleomorphic adenoma tissues and tumor-initiating cell (TIC) cells, when compared with control group. By loss-of-function assay, the proliferation and invasion capacities of TICs were inhibited, while the cell apoptosis was promoted, which were further evidenced by the protein expression analysis of several key apoptosis-related regulators. Furthermore, TICs with Mithramycin A (an Egr1 inhibitor) treatment achieved the same effects of endogenous Egr1 knockdown. All these data collectively suggest that Egr1 act as an oncogenic factor in salivary gland pleomorphic adenoma, which may be a potential target for the treatment of SGPA.

Acute neuroestrogen blockade attenuates song-induced immediate early gene expression in auditory regions of male and female zebra finches.

Neuron-derived estrogens are synthesized by aromatase and act through membrane receptors to modulate neuronal physiology. In many systems, long-lasting hormone treatments can alter sensory-evoked neuronal activation. However, the significance of acute neuroestrogen production is less understood. Both sexes of zebra finches can synthesize estrogens rapidly in the auditory cortex, yet it is unclear how this modulates neuronal cell signaling. We examined whether acute estrogen synthesis blockade attenuates auditory-induced expression of early growth response 1 (Egr-1) in the auditory cortex of both sexes. cAMP response element-binding protein phosphorylation (pCREB) induction by song stimuli and acute estrogen synthesis was also examined. We administered the aromatase inhibitor fadrozole prior to song exposure and measured Egr-1 across several auditory regions. Fadrozole attenuated Egr-1 in the auditory cortex greater in males than females. Females had greater expression and clustering of aromatase cells than males in high vocal center (HVC) shelf. Auditory-induced Egr-1 expression exhibited a large sex difference following fadrozole treatment. We did not observe changes in pCREB expression with song presentation or aromatase blockade. These findings are consistent with the hypothesis that acute neuroestrogen synthesis can drive downstream transcriptional responses in several cortical auditory regions, and that this mechanism is more prominent in males.

Emodin and AZT synergistically inhibit the proliferation and induce the apoptosis of leukemia K562 cells through the EGR1 and the Wnt/β‑catenin pathway.

The aim of the present study was to investigate the synergistic antitumor effects of emodin and 3'‑azido‑3'‑deoxythymidine (AZT) on human chronic myeloid leukemia cells and to explore the possible underlying mechanisms. The K562 cells were treated with emodin and AZT, and the rates of cell inhibition and apoptosis were determined by MTT assay and flow cytometry, respectively. The mRNA expression of EGR1 was detected by reverse transcription‑polymerase chain reaction (RT‑PCR) analysis. The expression of EGR1 was silenced using siRNA, and then protein expression of β‑catenin was detected by western blotting. The results demonstrated that AZT enhanced the inhibitory effect of emodin in K562 cells. The IC50 of the emodin/AZT combination at 24, 48 or 72h was 23.6/235.6, 10.2/101.6 or 5.9/58.5µmol/l, respectively, which was significantly lower compared with the IC50 of emodin (all >32µmol/l) or AZT (all >320µmol/l) alone. There was a dose‑dependent response to the combined emodin and AZT treatment, and the calculation of the combination index yielded values <1, demonstrating the synergistic effect of the combined treatment compared with the control (P<0.05). Furthermore, the combination of emodin and AZT increased apoptosis in K562 cells (P<0.05). Apoptosis was higher in the combination group compared with that of either treatment alone or control groups. The expression of early growth response‑1 (EGR1) in K562 cells was upregulated in a time‑dependent manner. The expression of EGR1 was higher in the combination group compared with that in the emodin or AZT alone groups. The expression of the Wnt/β‑catenin signaling pathway in the combination group was lower compared with that in the emodin or AZT alone groups. The expression of the Wnt/β‑catenin signaling pathway was significantly increased following EGR1 siRNA transfection. These data suggest that treating K562 cells with a combination of emodin and AZT exhibits reduced toxicity and improves therapeutic efficacy, and that the growth, inhibition, apoptosis and regulation of the Wnt/β‑catenin signaling pathway in human chronic myeloid leukemia cells by emodin and AZT may be associated with the expression of EGR1.

Early Growth Response Genes Increases Rapidly After Mechanical Overloading and Unloading in Tendon Constructs.

Tendon cells exist in a dense extracellular matrix and mechanical loading is important for the strength development of this matrix. We therefore use a three-dimensional (3D) culture system for tendon formation in vitro. The objectives of this study were to elucidate the temporal expression of tendon-related genes during the formation of artificial tendons in vitro and to investigate if early growth response-1 (EGR1), EGR2, FOS, and cyclooxygenase-1 and -2 (PTGS1 and PTGS2) are sensitive to mechanical loading. First, we studied messenger RNA (mRNA) levels of several tendon-related genes during formation of tendon constructs. Second, we studied the mRNA levels of, for example, EGR1 and EGR2 after different degrees of loading; dynamic physiologic-range loading (2.5% strain), dynamic overloading (approximately 10% strain), or tension release. The gene expression for tendon-related genes (i.e., EGR2, MKX, TNMD, COL3A1) increased with time after seeding into this 3D model. EGR1, EGR2, FOS, PTGS1, and PTGS2 did not respond to physiologic-range loading. But overloading (and tension release) lead to elevated levels of EGR1 and EGR2 (p ≤ 0.006). FOS and PTGS2 were increased after overloading (both p < 0.007) but not after tension release (p = 0.06 and 0.08). In conclusion, the expression of tendon-related genes increases during the formation of artificial tendons in vitro, including EGR2. Furthermore, the gene expression of EGR1 and EGR2 in human tendon cells appear to be sensitive to overloading and unloading but did not respond to the single episode of physiologic-range loading. These findings could be helpful for the understanding of tendon tensional homeostasis. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:173-181, 2020.

Age-related decrease in muscle satellite cells is accompanied with diminished expression of early growth response 3 in mice.

Skeletal muscle regeneration is mostly dependent on muscle satellite cells. Proper muscle regeneration requires enough number of satellite cells. Recent studies have suggested that the number of satellite cells in skeletal muscle declines as we age, leading to the impairment of muscle regeneration in older population. Our earlier study demonstrated that zinc finger transcription factor early growth response 3 (Egr3) plays an important role for maintaining the number of myoblasts, suggesting that age-related decrease in muscle satellite cell should be associated with the expression levels of Egr3. The aim of this study was to investigate whether aging would alter the Egr3 expression in satellite cells. A couple groups of male C57BL/6J mice were examined in this study: young (3 Mo) and old (17 Mo). Immunohistochemical staining showed that the satellite cell number decreased in normal and injured muscles of old mice. In fluorescence-activated cell sorting-isolated muscle satellite cells from normal and injured muscles, the mRNA expression of Egr3 was significantly decreased with age regardless of injury. In harmony with these results, Pax7 mRNA levels also decreased in the satellite cells from old mice. Alternatively, inhibition of Egr3 expression by shRNA decreased Pax7 protein expression in cultured myoblasts. These results suggest that Egr3 is associated with the age-related decline of muscle satellite cells in older population. Also, Egr3 might be implicated in the regulation of Pax7. Therefore, the loss of Egr3 expression may elucidate attenuated MSCs function and muscle regeneration in older age.

Potential roles of lncRNA-Cox2 and EGR1 in regulating epidural fibrosis following laminectomy.

Epidural fibrosis, one of the common complications after spinal surgery, seriously affects the surgical decompression effect. Effectively inhibiting the fibrous tissue hyperplasia is pivotal to reduce the scar adhesion. Previous studies showed that early growth response 1 (EGR1) is associated with the fibroblast reactivity induced by transforming growth factor-beta (TGF-β) and plays a vital regulatory role in scar formation; however, the upstream targets and mechanisms still remain unclear. In this work, it was found that the level of long non-coding ribonucleic acid (lncRNA)-cyclooxygenase-2 (COX2) was significantly negatively correlated with EGR1 expression and the severity of the scar. Therefore, it was conjectured that lncRNA-COX2 may decrease fibroplasia and scar formation by negatively regulating EGR1. TGF-β was used to activate the embryonic and adult rat fibroblasts. Rats underwent laminectomy to establish the epidural fibrosis model. The changes in the levels of fibroplasia-related genes were measured and analyzed through messenger RNA (mRNA), lncRNA, and micro RNA expression profile chips. Quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) was applied to determine the levels of EGR1 and lncRNA-COX2, and Western blotting was adopted to detect the content of EGR1, collagen I (Col-1), Col-3, and alpha-smooth muscle actin (α-SMA). The scar formation was reflected by hematoxylin and eosin (HE) staining and Masson staining, and the expression level of α-SMA in the scar tissues was measured via immunohistochemistry. Finally, micro-magnetic resonance imaging (MRI) was utilized to examine the different degrees of epidural fibroplasia. It was found that the reactivity of embryonic rat fibroblasts to the TGF-β stimulation was different from that of adult rat fibroblasts. LncRNA-COX2 was highly expressed in the embryonic rat fibroblasts, but lowly expressed in the adult rat fibroblasts, which had negative correlations with the EGR1 level in embryonic and adult rat fibroblasts. In addition, it was revealed that the expression of EGR1 in the adult rat fibroblasts was remarkably higher than that in the embryonic rat fibroblasts after the activation with TGF-β. Meanwhile, the level of lncRNA-COX2 was lowered after the activation, especially in the adult rat fibroblasts. It was discovered in the in-vivo model that the degree of fibroplasia was positively associated with EGR1 level and negatively correlated with lncRNA-COX2 level. The results of this research elucidated that the down-regulation of lncRNA-COX2 is involved in the epidural scar formation and related to the elevated EGR1 level which regulates the activation of fibroblasts and secretion of massive extracellular matrixes, suggesting that lncRNA-COX2 may modulate the role of fibroblasts in scar formation as an upstream action target of EGR1.