FRA1:c-JUN:HDAC1 complex down-regulates filaggrin expression upon TNFα and IFNγ stimulation in keratinocytes.

Filaggrin (FLG), an essential structural protein for skin barrier function, is down-regulated under chronic inflammatory conditions, leading to disruption of the skin barrier. However, the detailed molecular mechanisms of how FLG changes in the context of chronic inflammation are poorly understood. Here, we identified the molecular mechanisms by which inflammatory cytokines inhibit FLG expression in the skin. We found that the AP1 response element within the -343/+25 of the FLG promoter was necessary for TNFα + IFNγ-induced down-regulation of FLG promoter activity. Using DNA affinity precipitation assay, we observed that AP1 subunit composition binding to the FLG promoter was altered from c-FOS:c-JUN (at the early time) to FRA1:c-JUN (at the late time) in response to TNFα + IFNγ stimulation. Knockdown of FRA1 or c-JUN abrogated TNFα + IFNγ-induced FLG suppression. Histone deacetylase (HDAC) 1 interacted with FRA1:c-JUN under TNFα + IFNγ stimulation. Knockdown of HDAC1 abrogated the inhibitory effect of TNFα + IFNγ on FLG expression. The altered expression of FLG, FRA1, c-JUN, and HDAC1 was confirmed in mouse models of 2,4-dinitrochlorobenzene-induced atopic dermatitis and imiquimod-induced psoriasis. Thus, the current study demonstrates that TNFα + IFNγ stimulation suppresses FLG expression by promoting the FRA1:c-JUN:HDAC1 complex. This study provides insight into future therapeutic strategies targeting the FRA1:c-JUN:HDAC1 complex to restore impaired FLG expression in chronic skin inflammation.

Saikosaponin A and Saikosaponin C Reduce TNF-α-Induced TSLP Expression through Inhibition of MAPK-Mediated EGR1 Expression in HaCaT Keratinocytes.

Atopic dermatitis (AD) is one of the most common chronic inflammatory skin diseases worldwide, characterized by intense pruritus and eczematous lesions. Aberrant expression of thymic stromal lymphopoietin (TSLP) in keratinocytes is associated with the pathogenesis of AD and is considered a therapeutic target for the treatment of this disease. Saikosaponin A (SSA) and saikosaponin C (SSC), identified from Radix Bupleuri, exert anti-inflammatory effects. However, the topical effects of SSA and SSC on chronic inflammatory skin diseases are unclear. In this study, we investigated the effects of SSA and SSC on TSLP suppression in an AD-like inflammatory environment. We observed that SSA and SSC suppressed tumor necrosis factor-α-induced TSLP expression by downregulating the expression of the transcription factor early growth response 1 (EGR1) via inhibition of the extracellular signal-regulated kinase 1/2, c-Jun N-terminal kinase 1/2, and p38 mitogen-activated protein kinase pathways. We also confirmed that topical application of SSA or SSC reduced AD-like skin lesions in BALB/c mice challenged with 2,4-dinitrochlorobenzene. Our findings suggest that suppression of EGR1-regulated TSLP expression in keratinocytes might be attributable to the anti-inflammatory effects of SSA and SSC in AD-like skin lesions.

ß-Caryophyllene Ameliorates 2,4-Dinitrochlorobenzene-Induced Atopic Dermatitis through the Downregulation of Mitogen-Activated Protein Kinase/EGR1/TSLP Signaling Axis.

Atopic dermatitis (AD) is one of the most common inflammatory skin diseases accompanied by severe itching. ß-caryophyllene (BCP), which displays anti-inflammatory activity, is a natural agonist of cannabinoid receptor 2. However, the therapeutic effects of BCP on atopic dermatitis (AD) remain poorly understood. The current study aimed to evaluate the topical therapeutic efficacy of BCP in an AD-like mouse model. Thymic Stromal Lymphopoietin (TSLP) is a keratinocyte-derived cytokine that drives AD pathogenesis. This study also investigated the effect of BCP on the interleukin 4 (IL-4)-induced expression of TSLP in HaCaT keratinocytes. We found that the topical application of BCP alleviated AD-like skin inflammation and inhibited the infiltration of proinflammatory cells into skin lesions. Moreover, the topical application of BCP reduced EGR1 (Early Growth Response 1) and TSLP expression in AD-like skin lesions. We also found that BCP inhibited IL-4-induced TSLP expression by downregulating mitogen-activated protein kinase (MAPK)-mediated EGR1 expression in HaCaT keratinocytes. These findings demonstrate that BCP ameliorates DNCB-induced AD-like skin lesions through the downregulation of the MAPK/EGR1/TSLP signaling axis. BCP may be applicable for developing topical therapeutic agents for chronic skin inflammatory diseases, such as AD.

The Natural Janus Kinase Inhibitor Agerarin Downregulates Interleukin-4-Induced PER2 Expression in HaCaT Keratinocytes.

The circadian clock system is closely associated with inflammatory responses. Dysregulation of the circadian clock genes in the skin impairs the skin barrier function and affects the pathophysiology of atopic dermatitis. Interleukin 4 (IL-4) is a proinflammatory cytokine derived from T-helper type 2 cells; it plays a critical role in the pathogenesis of atopic dermatitis. Agerarin (6,7-dimethoxy-2,2-dimethyl-2H-chromene) is a natural JAK1/2/3 inhibitor isolated from Ageratum houstonianum that has a protective effect on the epidermal skin barrier. However, it remains unclear whether agerarin affects the circadian clock system. The aim of this study is to investigate the effect of agerarin on IL-4-induced PER2 gene expression in human keratinocytes through reverse transcription (RT)-PCR, quantitative real-time PCR (qPCR), immunoblotting, immunofluorescence microscopic analysis, and real-time bioluminescence analysis. We found that agerarin reduced IL-4-induced PER2 mRNA expression by suppressing the JAK-STAT3 pathway. In addition, real-time bioluminescence analysis in PER2:luc2p promoter-reporter cells revealed that agerarin restored the oscillatory rhythmicity of PER2 promoter activity altered by IL-4. These findings suggest that agerarin may be useful as a cosmeceutical agent against inflammatory skin conditions associated with disrupted circadian rhythms, such as atopic dermatitis.

EGR-1 acts as a transcriptional activator of KLK7 under IL-13 stimulation.

Kallikrein-related peptidase 7 (KLK7) is a chymotrypsin-like serine peptidase that plays a crucial role in regulating skin desquamation. KLK7 expression is highly upregulated in atopic dermatitis (AD) skin lesions in both humans and mice. Th2-lymphocyte-derived cytokines, including interleukin (IL)-4 and IL-13, have been shown to promote KLK7 expression in keratinocytes in patients with AD. However, the molecular mechanism underlying KLK7 expression remains poorly understood. Here, we demonstrated that the EGR-1-binding sequence (EBS) in the promoter region of KLK7 played a crucial role in IL-13-induced KLK7 transcription. Disruption of the EBS induced by a point mutation inhibited IL-13-induced KLK7 promoter activity. EGR-1 was shown to directly bind to the EBS, and EGR1 knockdown with shRNA abrogated IL-13-induced KLK7 expression. Using Egr1 knockout mice, we showed that Egr-1 was necessary for KLK7 expression in AD-like lesions induced by the repeated topical application of 2,4-dinitrobenzene on the dorsal skin of mice. We also demonstrated that the ERK1/2 mitogen-activated protein kinase (MAPK) pathway was responsible for EGR-1-dependent KLK7 transcription in response to IL-13 stimulation. Our findings delineate a signaling pathway that contributes to the regulation of KLK7 expression through the IL13-ERK MAPK-EGR1 signaling axis.

Chrysin Inhibits TNFα-Induced TSLP Expression through Downregulation of EGR1 Expression in Keratinocytes.

Thymic stromal lymphopoietin (TSLP) is an epithelial cell-derived cytokine that acts as a critical mediator in the pathogenesis of atopic dermatitis (AD). Various therapeutic agents that prevent TSLP function can efficiently relieve the clinical symptoms of AD. However, the downregulation of TSLP expression by therapeutic agents remains poorly understood. In this study, we investigated the mode of action of chrysin in TSLP suppression in an AD-like inflammatory environment. We observed that the transcription factor early growth response (EGR1) contributed to the tumor necrosis factor alpha (TNFα)-induced transcription of TSLP. Chrysin attenuated TNFα-induced TSLP expression by downregulating EGR1 expression in HaCaT keratinocytes. We also showed that the oral administration of chrysin improved AD-like skin lesions in the ear and neck of BALB/c mice challenged with 2,4-dinitrochlorobenzene. We also showed that chrysin suppressed the expression of EGR1 and TSLP by inhibiting the extracellular signal-regulated kinase (ERK) 1/2 and c-Jun N-terminal kinase (JNK) 1/2 mitogen-activated protein kinase pathways. Collectively, the findings of this study suggest that chrysin improves AD-like skin lesions, at least in part, through the downregulation of the ERK1/2 or JNK1/2-EGR1-TSLP signaling axis in keratinocytes.

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 ß-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.

Chrysoeriol Prevents TNFα-Induced CYP19 Gene Expression via EGR-1 Downregulation in MCF7 Breast Cancer Cells.

Estrogen overproduction is closely associated with the development of estrogen receptor-positive breast cancer. Aromatase, encoded by the cytochrome P450 19 (CYP19) gene, regulates estrogen biosynthesis. This study aimed to identify active flavones that inhibit CYP19 expression and to explore the underlying mechanisms. CYP19 expression was evaluated using reverse transcription PCR, quantitative real-time PCR, and immunoblot analysis. The role of transcription factor early growth response gene 1 (EGR-1) in CYP19 expression was assessed using the short-hairpin RNA (shRNA)-mediated knockdown of EGR-1 expression in estrogen receptor-positive MCF-7 breast cancer cells. We screened 39 flavonoids containing 26 flavones and 13 flavanones using the EGR1 promoter reporter activity assay and observed that chrysoeriol exerted the highest inhibitory activity on tumor necrosis factor alpha (TNFα)-induced EGR-1 expression. We further characterized and demonstrated that chrysoeriol inhibits TNFα-induced CYP19 expression through inhibition of extracellular signal-regulated kinase 1/2 (ERK1/2)-mediated EGR-1 expression. Chrysoeriol may be beneficial as a dietary supplement for the prevention of estrogen receptor-positive breast cancer, or as a chemotherapeutic adjuvant in the treatment of this condition.

Inhibitory Effect of Alisma canaliculatum Ethanolic Extract on NF-κB-Dependent CXCR3 and CXCL10 Expression in TNFα-Exposed MDA-MB-231 Breast Cancer Cells.

CXC motif chemokine ligand 10 (CXCL10) and its receptor CXC motif chemokine receptor 3 (CXCR3), play important roles in the motility of breast cancer cells. Alisma canaliculatum is a herb that has been used as a traditional medicine for thousands of years in Korea and China. Whether A. canaliculatum inhibits the motility of metastatic breast cancer cells is not clear yet. In this study, we show that A. canaliculatum ethanolic extract (ACE) prevented tumor necrosis factor-alpha (TNFα)-induced migration of MDA-MB-231 cells. ACE significantly attenuated TNFα-induced upregulation of CXCL10 and CXCR3 expression at the gene promoter level. Mechanistically, ACE inhibits TNFα-induced phosphorylation of inhibitor of κB (IκB) kinase (IKK), IκB and p65/RelA, leading to the suppression of nuclear translocation of p65/RelA nuclear factor kappa-B (NF-κB). Also, ACE inhibited NF-κB-dependent CXCR3 and CXCL10 promoter activities. These results suggest that ACE abrogates TNFα-induced migration of MDA-MB-231 breast cancer cells through down-regulation of IKK-NF-κB-dependent CXCR3 and CXCL10 expression. Our results suggest that ACE has potential as a herbal supplement for the inhibition of breast cancer metastasis.

Downregulation of α-Melanocyte-Stimulating Hormone-Induced Activation of the Pax3-MITF-Tyrosinase Axis by Sorghum Ethanolic Extract in B16F10 Melanoma Cells.

Ultraviolet irradiation-induced hyperpigmentation of the skin is associated with excessive melanin production in melanocytes. Tyrosinase (TYR) is a key enzyme catalyzing the rate-limiting step in melanogenesis. TYR expression is controlled by microphthalmia-associated transcription factor (MITF) expression. Sorghum is a cereal crop widely used in a variety of foods worldwide. Sorghum contains many bioactive compounds and is beneficial to human health. However, the effects of sorghum in anti-melanogenesis have not been well characterized. In this study, the biological activity of sorghum ethanolic extract (SEE) on α-melanocyte-stimulating hormone (α-MSH)-induced TYR expression was evaluated in B16F10 melanoma cells. SEE attenuated α-MSH-induced TYR gene promoter activity through the downregulation of the transcription factor MITF. We found that paired box gene 3 (Pax3) contributes to the maximal induction of MITF gene promoter activity. Further analysis demonstrated that SEE inhibited α-MSH-induced Pax3 expression. The collective results indicate that SEE attenuates α-MSH-induced TYR expression through the suppression of Pax3-mediated MITF gene promoter activity. Targeting the Pax3-MITF axis pathway could be considered a potential strategy to increase the efficacy of anti-melanogenesis.

The EGR1-Artemin Axis in Keratinocytes Enhances the Innervation of Epidermal Sensory Neurons during Skin Inflammation Induced by House Dust Mite Extract from Dermatophagoidesfarinae.

Epidermal hyperinnervation is a critical feature of pruritus during skin inflammation. However, the mechanisms underlying epidermal hyperinnervation are unclear. This study investigates the role of the transcription factor EGR1 in epidermal innervation by utilizing wild-type (Egr1+/+) and Egr1-null (Egr1‒/‒) mice topically applied Dermatophagoides farinae extract from dust mite. Our findings revealed that Egr1‒/‒ mice exhibited reduced scratching behaviors and decreased density of epidermal innervation compared with Egr1+/+ mice. Furthermore, we identified artemin, a neurotrophic factor, as an EGR1 target responsible for Dermatophagoides farinae extract-induced hyperinnervation. It has been demonstrated that Dermatophagoides farinae extract stimulates toll-like receptors in keratinocytes. To elucidate the cellular mechanism, we stimulated keratinocytes with Pam3CSK4, a toll-like receptor 1/2 ligand. Pam3CSK4 triggered a toll-like receptor 1/2-mediated signaling cascade involving IRAK4, IκB kinase, MAPKs, ELK1, EGR1, and artemin, leading to increased neurite outgrowth and neuronal migration. In addition, increased expression of EGR1 and artemin was observed in the skin tissues of patients with atopic dermatitis. These findings highlight the significance of the EGR1-artemin axis in keratinocytes, promoting the process of epidermal innervation and suggesting it as a potential therapeutic target for alleviating itch and pain associated with house dust mite-induced skin inflammation.

The JNK-EGR1 signaling axis promotes TNF-α-induced endothelial differentiation of human mesenchymal stem cells via VEGFR2 expression.

Mesenchymal stem cells (MSCs) can differentiate into endothelial cells; however, the mechanisms underlying this process in the tumor microenvironment (TME) remain elusive. This study shows that tumor necrosis factor alpha (TNF-α), a key cytokine present in the TME, promotes the endothelial differentiation of MSCs by inducing vascular endothelial growth factor receptor 2 (VEGFR2) gene expression. EGR1 is a member of the zinc-finger transcription factor family induced by TNF-α. Our findings indicate that EGR1 directly binds to the VEGFR2 promoter and transactivates VEGFR2 expression. We also demonstrate that EGR1 forms a complex with c-JUN activated by c-JUN N-terminal kinase (JNK) to promote VEGFR2 transcription and endothelial differentiation in MSCs in response to TNF-α stimulation. The shRNA-mediated silencing of EGR1 or c-JUN abrogates TNF-α-induced VEGFR2 transcription and the endothelial differentiation of MSCs. To further evaluated the role of EGR1 in the endothelial differentiation of BM-MSCs, we used a syngenic tumor implantation model. 4T1 mouse mammary tumor cells were injected subcutaneously into BALB/c mice with primary mBM-MSCs isolated from wild-type (Egr1+/+) or Egr1-null (Egr1-/-) mice. CD31-positive cells were predominantly observed at the border of the tumor in the 4T1 plus wild-type MSC group, while staining less in the 4T1 alone or 4T1 plus Egr1-null MSC group. Collectively, these findings demonstrate that the JNK-EGR1 signaling axis plays a crucial role in the TNF-α-induced endothelial differentiation of MSCs in the TME, which could be a potential therapeutic target for solid tumors vasculatures.

Transcription Factor EGR1 Regulates the Expression of the Clock Gene PER2 under IL-4 Stimulation in Human Keratinocytes.

PER2 is a core circadian clock gene that regulates circadian rhythms. IL-4 plays a critical role in the pathogenesis of skin inflammation, including atopic dermatitis. IL-4 enhances PER2 expression, suggesting a relationship between inflammation and the circadian clock. However, little is known about the molecular and cellular mechanisms regulating PER2 expression by inflammatory cytokines. This study showed that transcription factor EGR1 interacted with the PER2 promoter and promoted IL-4‒induced transcriptional activation of the PER2, as revealed by promoter‒reporter assay, electrophoretic mobility shift assay, DNA affinity precipitation assay, and chromatin immunoprecipitation analysis. We also found that IL-4 can use both MAPK and Jak signaling pathways to induce EGR1-mediated PER2 expression, and c-Jun N-terminal kinase 1/2 can augment IL-4‒induced activation of the Jak‒signal transducer and activator of transcription 3 pathway. Consistently, Per2 expression was reduced in dinitrochlorobenzene-induced atopic dermatitis‒like skin lesions in Egr1‒/‒ mice compared with that in Egr1+/+ mice. In addition, using a real-time bioluminescence assay, we observed that EGR1 is required for rhythmic oscillation of PER2 expression under IL-4 exposure. These findings provide further insight into the role of EGR1 in regulating PER2 expression in impaired circadian rhythm in skin inflammation.

Effect of 6,7-dimethoxy-2,2-dimethyl-2H-chromene (agerarin) on the recovery of filaggrin expression through targeting of Janus kinases in the inflammatory skin.

Filaggrin (FLG) is a structural component of the stratum corneum that is essential for maintaining the barrier function of the skin and for the formation of natural moisturizing factors. 6,7-Dimethoxy-2,2-dimethyl-2H-chromene (Agerarin) is a bioactive compound derived from Ageratum houstonianum, a plant that is used as a traditional medicine to treat skin diseases. This study aimed to evaluate the effect of agerarin on skin inflammation in a dinitrochlorobenzene (DNCB)-induced atopic dermatitis mouse model. We found that the topical administration of agerarin ameliorates atopic dermatitis-like skin lesions. We also showed that agerarin restores the reduced filaggrin (FLG) expression in DNCB-applied skin sections. Moreover, agerarin decreased phosphorylation of JAK1 and JAK2 kinases to enhance FLG expression, which was reduced by TNFα+IFNγ and IL4+IL13 treatment, in HaCaT keratinocytes. These results demonstrate the feasibility of agerarin as a possible therapeutic against conditions of skin inflammation, such as atopic dermatitis, by improving the upregulation of FLG expression.

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 necessary 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 signalregulated 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. [BMB Reports 2020; 53(12): 628-633].

Transcription factor EGR-1 transactivates the MMP1 gene promoter in response to TNFα in HaCaT keratinocytes.

Matrix metalloproteinase 1 (MMP-1), a calcium-dependent zinccontaining collagenase, is involved in the initial degradation of native fibrillar collagen. Tissue necrosis factor-alpha (TNFα) is a pro-inflammatory cytokine that is rapidly produced by dermal fibroblasts, monocytes/macrophages, and keratinocytes and regulates inflammation and damaged-tissue remodeling. MMP-1 is induced by TNFα and plays a critical role in tissue remodeling and skin aging processes. However, the regulation of the MMP1 gene by TNFα is not fully understood. We aimed to find additional cis-acting elements involved in the regulation of TNFα-induced MMP1 gene transcription in addition to the nuclear factor-kappa B (NF-κB) and activator protein 1 (AP1) sites. Assessments of the 5'-regulatory region of the MMP1 gene, using a series of deletion constructs, revealed the requirement of the early growth response protein 1 (EGR-1)-binding sequence (EBS) in the proximal region for proper transcription by TNFα. Ectopic expression of EGR-1, a zinc-finger transcription factor that binds to G-C rich sequences, stimulated MMP1 promoter activity. The silencing of EGR-1 by RNA interference reduced TNFα-induced MMP-1 expression. EGR-1 directly binds to the proximal region and transactivates the MMP1 gene promoter. Mutation of the EBS within the MMP1 promoter abolished EGR-1-mediated MMP-1 promoter activation. These data suggest that EGR-1 is required for TNFα-induced MMP1 transcriptional activation. In addition, we found that all three MAPKs, ERK1/2, JNK, and p38 kinase, mediate TNFα-induced MMP-1 expression via EGR-1 upregulation. These results suggest that EGR-1 may represent a good target for the development of pharmaceutical agents to reduce inflammation-induced MMP-1 expression. [BMB Reports 2020; 53(6): 323-328].