The skin is the most extensive tissue of the body and consists of three layers (epidermis, dermis, and subcutaneous tissue). The epidermis is a stratified squamous epithelium of the outermost layer, which provides a mechanical barrier against invasion of foreign substances and loss of body water. The epidermis mechanically maintains a skin structure against environmental stresses, including mechanical injury, UV radiation, and infection. Maintaining a constant skin structure requires dynamically coordinated degradation and synthesis of extracellular matrix (ECM) components.
Matrix metalloproteinases (MMPs) are a family of endopeptidases responsible for the proteolytic degradation of structural components of ECM. MMPs are associated with various physiological and pathological cellular functions, including cell migration, wound healing, angiogenesis, immune responses, cell growth and differentiation, apoptosis, and skin aging (1, 2). To date, at least 24 different MMPs have been identified in humans (1) and classified into four groups based on their domain organization; archetypal MMPs, gelatinases, matrilysins, and furin-activatable MMPs (3). Of archetypal MMPs, MMP-1 is a sub-group of collagenase involved in the initial cleavage of native fibrillar collagens (3). It functions in tissue remodeling, inflammation, and skin photoaging (4) and has been implicated in various pathological processes, such as tumor cell invasion, arthritis, and atherosclerosis (5). Like other MMPs, MMP-1 is expressed at a low level in most normal cells but rapidly induced in response to exogenous signals, including growth factors, inflammatory cytokines, and oncogenic transformation. MMP-1 expression largely depends on transcriptional activity.
Wounds and other inflammatory responses in the skin lead to tissue remodeling characterized by elevated inflammatory cytokines. Tissue necrosis factor α (TNFα) is one of potent pro-inflammatory cytokine produced by dermal fibroblasts, monocytes/macrophages, and keratinocytes in response to ultraviolet irradiation and inflammatory signals (6). TNFα promotes tissue remodeling by increasing the secretion of multiple cytokines and inflammatory mediators in keratinocytes and dermal fibroblasts (6). MMP-1 is also induced by TNFα and contributes to the remodeling of damaged tissues (4). The role of mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinase (ERK), c-Jun-N-terminal kinase (JNK), and p38 kinase, is well established in the regulation of TNFα-induced MMP-1 expression via AP1 upregulation (3).
Early Growth Response (EGR)-1 is an inducible zinc-finger transcription factor that binds to GC-rich sequences (7). Generally, EGR-1 is expressed at the base level but is rapidly and transiently induced by various growth factors and inflammatory cytokines in a variety of cell types (8, 9). In turn, activated EGR-1 regulates the expression of many inflammation-related genes (10-12). Indeed, EGR-1 regulates interleukin (IL)-13-induced lung inflammation by expressing CC and CXC chemokines (13) and TNFα-induced CXCL1 expression (8). Furthermore, EGR-1 induces many pro-inflammatory cytokines in keratinocytes during skin inflammation; TNFα autoregulation (14), IL-17A-induced psorian expression (15), and IL-33-induced thymic stromal lymphopoietin (TSLP) expression (16). We previously showed that EGR-1 transactivates the
We aimed to investigate the role of EGR-1 in the regulation of MMP-1 expression. The objective of this study is to identify the EGR-1-binding sites in the 5’-regulatory region of the
The 5’-regulatory region of the
To determine the role of the putative EBS in TNFα-induced MMP-1 expression, we disrupted EBS by site-specific deletion of core nucleotide (CTC). Disruption of the EGR-1-binding core sequence within the −152/+87 construct (mtEBS) resulted in a near-complete loss of TNFα-stimulated promoter-reporter activity (P < 0.001,
Next, we assessed whether EGR-1 binds to the putative EBS motif within the proximal
EGR-1 protein accumulated in a pattern similar to AP1 components (c-FOS and c-JUN) following TNFα stimulation in HaCaT cells (Fig. 2C). To investigate whether EGR-1 alone could transactivate the
Upon TNFα stimulation, MMP-1 mRNA and protein levels were upregulated in a time-dependent manner in HaCaT cells (Fig. 3A). To verify the role of EGR-1 in TNFα-induced MMP-1 expression, we established HaCaT variant cells expressing shRNAs against scrambled control (shCT) and EGR-1 (shEGR-1). shRNA-mediated knockdown of EGR-1 expression was confirmed by immunoblotting (Fig. 3B). EGR-1 silencing reduced both basal and TNFα-induced MMP-1 mRNA and protein levels (Fig. 3C). Thus, EGR-1 is necessary for TNFα-induced MMP-1 expression.
MAPK pathways regulate EGR-1 expression in various cell types (8,17, 18,22-25). In serum-starved HaCaT cells, ERK1/2, p38, and JNK1/2 MAPK phosphorylation rapidly increased within 15 min following TNFα stimulation (Fig. 4A), suggesting that TNFα activates three different MAPKs in HaCaT cells. To investigate the possible relationship between MAPK activation and EGR-1 upregulation, we examined the effects of pharmacological MAPK inhibitors on EGR-1 expression. MEK inhibitor U0126, JNK1/2 inhibitor SP600125, and p38 kinase inhibitor SB203580 significantly (P < 0.001,
In summary, EGR-1 is necessary for TNFα-induced MMP-1 expression in HaCaT keratinocytes. This study uncovered evidence of a novel EGR-1-binding cis-acting element located at −137/−119, which is necessary for TNFα-induced
TNFα, U0126, SB203580, and SP600125 were obtained from Calbiochem (San Diego, CA, USA). The Firefly and
HaCaT human keratinocytes were obtained from Cell Lines Service GmbH (Eppelheim, Germany). The cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS; BioWest, Kansas City, MO, USA).
The pRL-null plasmid encoding
Reporter luciferase activity was measured as described previously (27). In some experiments, pMMP1-Luc (−152/+87) and pMMP1-Luc (−152/+87)mtEGR-1 constructs were co-transfected with mammalian expression vectors for EGR-1 (pcDNA3.1/EGR-1) or c-Jun (pcDNA3.1/Jun). Luminescence was measured using a Centro LB960 dual luminometer (Berthold Tech, Bad Wildbad, Germany). The relative amount of luciferase activity after normalization to the
IPLB-Sf21 insect cells obtained from Clontech (Mountain View, CA, USA) were maintained in Grace’s insect medium (Gibco, Grand Island, NY, USA) supplemented with 10% FBS (Hyclone, Logan, UT, USA), 100 U/ml penicillin, and 100 μg/ml streptomycin at 28°C. The coding DNA sequence of EGR-1 was obtained from the expression plasmid of EGR-1 (pcDNA3.1zeo/EGR-1) by digestion with
Protein-DNA binding was assessed by EMSA using the LightShift Chemiluminescence EMSA kit, according to the manufacturer’s instructions (ThermoFisher Scientific, Waltham, MA, USA). Biotin-labeled deoxyoligonucleotide probes corresponding to the EGR-1-binding sequence (EBS; 5’-biotin-AGA GTG TGT CTC CTT CGC ACA CAT C-3’) and mutated sequence (mtEBS; 5’-biotin-AGA GTG TGT CTT CGC ACA CAT C-3’) were synthesized by Macrogen. Wild-type control or EGR-1-expressing IPLB-Sf21 lysates (3 μg) were mixed with 50 fmol biotin-labeled EBS and mtEBS oligonucleotide probes along with 1 μg poly(dI-dC) (Amersham Pharmacia Biotech Inc., Piscataway, NJ, USA). Samples were electrophoresed in non-denaturing 6% polyacrylamide gels, followed by incubation with horseradish peroxidase (HRP)-conjugated streptavidin. EGR-1-DNA complexes were visualized using an enhanced chemiluminescence (ECL) detection system (ThermoFisher Scientific).
HaCaT cells treated with PBS or 10 ng/ml TNFα were lysed and immunoblot analysis was performed as described previously (8). The blots were developed using LumiGLO peroxidase chemiluminescent substrate (SeraCare, Milford, MA, USA). In some experiments, the relative intensities of the MMP-1 bands were quantified and normalized by GAPDH expression using ImageJ version 1.52a software (National Institutes of Health, Bethesda, MD, USA).
HaCaT cells were stimulated with 10 ng/ml TNFα for 12 h, and nuclear extracts (70 μg) were incubated with streptavidin-conjugated magnetic beads (BIONEER, Daejeon, Korea) and 5 μg of biotinylated EBS (5’-biotin-AGA GTG TGT CTC CTT CGC ACA CAT C-3’) or mtEBS (5’-biotin-AGA GTG TGT CTT CGC ACA CAT C-3’) for 15 min. After washing twice with PBS, the pellets were cooked with Laemmli sample buffer, and the binding proteins were resolved by 10% SDS-PAGE. Immunoblotting was performed using anti-EGR-1 antibodies.
Isolation of total RNA and synthesis of complementary DNA (cDNA) were described elsewhere (27). The resulting cDNA was subjected to PCR analysis using gene-specific primers as follows: forward MMP-1, 5’-CAA AAT CCT GTC CAG CCC ATC G-3’ and reverse MMP-1, 5’-TTC GTA AGC AGC TTC AAG CCC-3’; forward GAPDH, 5’-ACC CAC TCC TCC ACC TTT G-3’ and reverse GAPDH, 5’-CTC TTG TGC TGC TGG G-3’. The PCR conditions included denaturation at 95°C for 30 s, annealing at 62°C for 30 s, and elongation at 72°C for 30 s. The amplicons were electrophoresed on 1% agarose gels and visualized by ethidium bromide staining. In some experiments, relative intensities of MMP-1 PCR bands were quantified and expressed as a ratio to GAPDH intensities using ImageJ version 1.52a software.
HaCaT cells were incubated with lentiviral short hairpin (sh)RNA (TRCN_0000273850; MISSIONⓇ shRNA; Sigma-Aldrich) targeting EGR-1, according to the manufacturer’s instructions. Two weeks after transfection, cells were collected, and the stable knockdown of EGR-1 expression was analyzed by immunoblotting.
Data were analyzed by ANOVA, followed by Sidak’s or Dunnett’s multiple comparisons test, using GraphPad Prism version 8.2.0 software (GraphPad Software Inc., La Jolla, CA, USA). P-values less than 0.05 were considered statistically significant.
This study was supported by the National Research Foundation of Korea (NRF), funded by the Korea government (MSIT) (No. 2018R1A2B2004653). The paper was supported by the KU Research Professor Program of Konkuk University and by the Konkuk University Researcher Fund in 2018. We would like to thank Editage (www.editage.co.kr) for English language editing.
The authors have no conflicting interests.