Article

Interleukin-1 beta induction of matrix metalloproteinase-1 transcription in chondrocytes requires ERK-dependent activation of CCAAT enhancer-binding protein-beta

June 2006
Journal of Cellular Physiology 207(3):683-8

DOI:10.1002/jcp.20608

Source
PubMed

Authors:

Interleukin-1 beta (IL-1beta) is a central mediator of inflammation and connective tissue destruction in rheumatoid arthritis. IL-1beta activates articular chondrocytes to produce matrix metalloproteinase-1 (MMP-1), an enzyme capable of dismantling the collagen scaffold of articular cartilage. To define the transcription factors and signaling intermediates that activate MMP-1 transcription in chondrocytes, we performed transient transfection of MMP-1 promoter constructs followed by reporter assays. These studies identified an IL-1beta-responsive region of the human MMP-1 promoter that contains a consensus CCAAT enhancer-binding protein (C/EBP) binding site. Deletion of this site reduced overall transcriptional activity of the MMP-1 promoter, as well as decreased fold induction by IL-1beta. IL-1beta stimulation of chondrocytes increased binding of C/EBP-beta to the MMP-1 C/EBP site. Extracellular signal regulated kinase (ERK) pathway-dependent phosphorylation of C/EBP-beta on threonine 235 activates this transcription factor. Here we show that IL-1beta stimulation of chondrocytes induced phosphorylation of C/EBP-beta on threonine 235, and that the ERK pathway inhibitor PD98059 reduced this phosphorylation. We further show that PD98059 reduces IL-1beta-induced MMP-1 mRNA expression in chondrocytes. Moreover, inhibition of the ERK pathway by expression of dominant-negative forms of ERK1 and ERK2 impaired the ability of IL-1beta to transactivate the MMP-1 promoter. Our findings demonstrate a novel role for C/EBP-beta in IL-1beta-induced connective tissue disease and define a new nuclear target for the ERK pathway in MMP-1 gene activation.

The effects of interleukin-1β in modulating osteoclast-conditioned medium’s influence on gelatinases in chondrocytes through mitogen-activated protein kinases

Article

Oct 2015

Osteoarthritis is recognised to be an interactive pathological process involving the cartilage, subchondral bone and synovium. The signals from the synovium play an important role in cartilage metabolism, but little is known regarding the influence of the signalling from bone. Additionally, the collagenases and stromelysin-1 are involved in cartilage catabolism through mitogen-activated protein kinase (MAPK) signalling, but the role of the gelatinases has not been elucidated. Here, we studied the influence of osteoclastic signals on chondrocytes by characterising the expression of interleukin-1β (IL-1β)-induced gelatinases through MAPK signalling. We found that osteoclast-conditioned media attenuated the gelatinase activity in chondrocytes. However, IL-1β induced increased levels of gelatinase activity in the conditioned media group relative to the mono-cultured chondrocyte group. More specifically, IL-1β restored high levels of gelatinase activity in c-Jun N-terminal kinase inhibitor-pretreated chondrocytes in the conditioned media group and led to lower levels of gelatinase activity in extracellular signal-regulated kinase or p38 inhibitor-pretreated chondrocytes. Gene expression generally correlated with protein expression. Taken together, these results show for the first time that signals from osteoclasts can influence gelatinase activity in chondrocytes. Furthermore, these data show that IL-1β restores gelatinase activity through MAPK inhibitors; this information can help to increase the understanding of the gelatinase modulation in articular cartilage.

TiO2 Nanoparticles Induce MMP-1 in Human Pulmonary Fibroblasts, Partly via an Interleukin-1β-Dependent Mechanism.

Article

Full-text available

Dec 2012
AM J RESP CELL MOL

Exposure to titanium dioxide (TiO2) nanoparticles (NP) can be associated to lung remodeling, but the underlying mechanisms are yet unknown. Matrix metalloprotease (MMP)-1 is an important actor of matrix homeostasis and could therefore participate in TiO2 NP effects. Our aim was to evaluate the effects of TiO2 NP on MMP-1 expression and activity in lung pulmonary fibroblasts, in addition to understand the underlying mechanisms and assess the importance of the physicochemical characteristics of the particles in these effects. Human pulmonary fibroblasts (MRC-5 cell line and primary cells) were exposed to 10 or 100 µg/cm² TiO2 (2 anatases, 2 anatase/rutile mix, 1 rutile NP, and 1 micrometric), and carbon black (CB) NP for 6 to 48h. We examined cell viability, MMP-1 expression and activity, as well as the implication of oxidative stress, transforming growth factor (TGF)-β, extracellular MMP inducer (EMMPRIN) and interleukin (IL)-1β in MMP-1 expression. All TiO2 NP induce MMP-1 (mRNA and protein expression), repression of procollagen-1 and α-actin expression, but only the 2 anatase/rutile mix induced MMP-1 activity. Micrometric TiO2 had smaller effects than TiO2 NP, and CB NP didn't induce MMP-1. MMP-1 induction by TiO2 NP was not related to TGF-ß, oxidative stress or EMPRIN expression, but was related to IL-1ß expression, which partly drives MMP-1 induction by 2 TiO2 NP (1 anatase/rutile mix and the rutile one). Taken together, our results show that TiO2 NP are potent inducers and regulators of MMP-1 expression and activity, partly via an IL-1β-dependent mechanism. This may explain TiO2 lung remodeling effects.

CCAAT/Enhancer Binding Protein beta Mediates Expression of Matrix Metalloproteinase 13 in Human Articular Chondrocytes in Inflammatory Arthritis

Article

Mar 2009

To determine the function of CCAAT/enhancer binding protein beta (C/EBPbeta) in the expression of matrix metalloproteinase 13 (MMP-13) in chondrocytes in inflammatory arthritis. Cartilage obtained from patients with rheumatoid arthritis and osteoarthritis was immunostained for expression of C/EBPbeta or MMP-13. Interleukin-1beta- or tumor necrosis factor alpha (TNFalpha)-stimulated chondrocytes were subjected to Western blotting and real-time reverse transcriptase-polymerase chain reaction (RT-PCR). MMP-13 promoter assays were conducted, and the C/EBPbeta response element was characterized by deletion and mutation analysis. C-28/I2 cells were treated with TNFalpha and subjected to chromatin immunoprecipitation (ChIP) assays. Finally, C/EBPbeta-liver-enriched activator protein (LAP) was overexpressed in C-28/I2 cells or cartilage tissues, and MMP-13 expression was analyzed. C/EBPbeta and MMP-13 expression was colocalized in chondrocytes in arthritic cartilage. MMP-13 promoter activity was stimulated by C/EBPbeta overexpression in a dose-dependent manner. Luciferase assays revealed that a -981-bp promoter had the greatest activity, while deletion to -936 bp strongly diminished promoter activity. Luciferase activity was repressed to basal levels by mutations in potential C/EBP binding sites. The stimulatory effects of C/EBPbeta overexpression were diminished by mutation. ChIP assays revealed that TNFalpha treatment enhanced the binding of C/EBPbeta to the MMP-13 promoter. When C/EBPbeta-LAP was overexpressed in C-28/I2 cells, endogenous MMP-13 expression was stimulated up to 32-fold as detected by real-time RT-PCR. Furthermore, following adenoviral overexpression of C/EBPbeta-LAP in organ culture of articular cartilage, stimulation of MMP-13 was also detected by immunohistochemistry. C/EBPbeta directly binds to the MMP-13 promoter region and stimulates the expression of MMP-13 in chondrocytes in inflammatory arthritis.

Role of Urokinase in the Fibrogenic Response of the Lung to Mineral Particles

Article

Full-text available

Mar 1998

The lung plasminogen activator (PA) response was examined in four different models of particle-induced pulmonary lesions in NMRI mice (single intratracheal administration, 0.75 to 5 mg/mouse). Sequential changes in cellular (total and differential counts) and biochemical markers of alveolitis (lactate dehydrogenase [LDH], total proteins) were monitored in bronchoalveolar fluid (BALF) and the fibrotic lung response was assessed histologically. An intense but spontaneously resolving alveolitis was produced by manganese dioxide (MnO2) and a fibrosing alveolitis was elicited by crystalline silica (DQ12). Minimal and noninflammatory responses were obtained after instillation of titanium dioxide (TiO2) and tungsten carbide (WC), respectively. The comparison between the resolving and the fibrosing alveolitis model was especially taken into consideration in an attempt to identify fibrinolytic changes associated with the development of fibrosis. At the alveolitis stage, similarly increased BALF PA activities were measured in both the resolving and the fibrosing alveolitis models whereas only slight and no PA modifications were noted after administration of TiO2 and WC, respectively. Persistently (up to 120 d) increased BALF PA activity was selectively associated with the progression to fibrosis (DQ12), suggesting that PA is involved in the fibrotic process. ELISA measurements demonstrated that the changes in BALF PA activity were exclusively related to changes in urokinase (uPA), not tissue-type PA. A rapid and persisting (up to Day 30) upregulation of cell-associated PA activity occurred after DQ12, MnO2, and TiO2 treatment only. Cellular PA activity was however significantly higher in fibrogenic inflammatory cells recovered from DQ12 than from MnO2-treated mice suggesting that the intensity of cellular PA upregulation may represent an early indicator of the progression to fibrosis. The implication of urokinase in the pathogenesis of silica-induced fibrosis was demonstrated by the use of a uPA knockout mice. The acceleration of the fibrotic process in uPA-deficient compared with the wild type animals demonstrated the contribution of uPA to limit the fibrotic process.

Hemorrhagic and ischemic stroke in patients with coronavirus disease 2019: Incidence, risk factors, and pathogenesis - a systematic review and meta-analysis

Article

Full-text available

Jan 2021

Background : In this study, we aimed to determine the global prevalence, chronological order of symptom appearance, and mortality rates with regard to hemorrhagic and ischemic stroke in patients with coronavirus disease 2019 (COVID-19) and to discuss possible pathogeneses of hemorrhagic and ischemic stroke in individuals with the disease. Methods : We searched the PubMed, Scopus, and Web of Science databases for relevant articles published up to November 8, 2020. Data regarding study characteristics, hemorrhagic stroke, ischemic stroke, and COVID-19 were retrieved in accordance with the PRISMA guidelines. The Newcastle-Ottawa scale was used to assess the quality of the eligible studies. The pooled prevalence and mortality rate of hemorrhagic and ischemic stroke were calculated. Results : The pooled estimate of prevalence of hemorrhagic stroke was 0.46% (95% CI 0.40%–0.53%; I ² =89.81%) among 67,155 COVID-19 patients and that of ischemic stroke was 1.11% (95% CI 1.03%–1.22%; I ² =94.07%) among 58,104 COVID-19 patients. Ischemic stroke was more predominant (incidence: 71.58%) than hemorrhagic stroke (incidence: 28.42%) in COVID-19 patients who experienced a stroke. In COVID-19 patients who experienced a stroke, hospital admission with respiratory symptoms was more commonly reported than that with neurological symptoms (20.83% for hemorrhagic stroke and 5.51% for ischemic stroke versus 6.94% for hemorrhagic stroke and 5.33% for ischemic stroke, respectively). The pooled mortality rate of COVID-19 patients who experienced a hemorrhagic and ischemic stroke was 44.72% (95% CI 36.73%–52.98%) and 36.23% (95% CI 30.63%–42.24%), respectively. Conclusions : Although the occurrence of hemorrhagic and ischemic stroke is low, the mortality rates of both stroke types in patients with COVID-19 are concerning, and therefore, despite several potential pathogeneses that have been proposed, studies aimed at definitively elucidating the mechanisms of hemorrhagic and ischemic stroke in individuals with COVID-19 are warranted. PROSPERO registration: CRD42020224470 (04/12/20)

Insights on biology and pathology of HIF-1α/-2α, TGFβ/BMP, Wnt/β-catenin, and NF-κB pathways in osteoarthritis

Article

May 2012
CURR PHARM DESIGN

Osteoarthritis (OA) constitutes a major health problem. Different signaling pathways are involved that impair homeostasis, but the cross-talk between them (although well investigated and partly understood), remains unclear. HIF-1α promotes chondrocyte differentiation and survival, while HIF-2α coactivates with β-catenin and NF-κB pathways to promote chondrocyte apoptosis and endochondral ossification. Depending on the ALK1/ALK5 ratio in chondrocytes, the TGFβ pathway can play an anabolic or catabolic role. TGFβ1 can activate the β-catenin signaling pathway via ALK5, Smad3, PI3K, and PKA pathways. The mediator Axins balance TGF-β and Wnt/β-catenin signaling during chondrocyte proliferation and maturation. However, the biological functions of Wnt/β-catenin signaling are still controversial. Both excessive and insufficient β-catenin levels may impair the homeostasis of articular chondrocytes by enhancing pathological maturation and apoptosis, respectively; loss- and gain-of-functions of β-catenin cause apoptosis at the center of the joint and chondrocyte maturation at the periphery, depending on the vascularity. The NF-κB transcription factor can be triggered by a host of stress-related stimuli including pro-inflammatory cytokines. The recent discovery of functional cross-regulation between these pathways has shown complex roles for HIF-1α/HIF-2α, TGFβ/BMP, Wnt/β-catenin, and NF-κB signaling pathways in the pathogenesis of OA. This has important implications for potential therapeutic agents directed at these pathways. This review attempts to cover the literature of the past three years dealing with the biology and pathology of the HIF-1α/-2α, TGFβ/BMP, Wnt/β-catenin, and NF-κB/cytokines signaling pathways in OA.

CCAAT Enhancer Binding Protein- Regulates Matrix Metalloproteinase-1 Expression in Interleukin-1 -Stimulated A549 Lung Carcinoma Cells

Article

Full-text available

Oct 2009
MOL CANCER RES

Matrix metalloproteinase-1 (MMP-1) is an inflammation-inducible neutral protease that mediates extracellular matrix remodeling and promotes tumor invasion. In this study, we examined the activation of MMP-1 gene expression in A549 lung carcinoma cells stimulated with the inflammatory cytokine interleukin-1beta (IL-1beta). We found that MMP-1 mRNA levels were maximal following 16 hours of IL-1beta stimulation and that this correlated with the expression of the transcription factor CCAAT enhancer-binding protein-beta (CEBPB). Knockdown of CEBPB expression with short hairpin RNA abrogated the expression of MMP-1, MMP-3, and MMP-10 in IL-1beta-stimulated A549 cells. An established CEBP element in the MMP-1 promoter was found to be required for basal and IL-1beta-induced transcription. Electrophoresis mobility shift assays showed that CEBPB binds to this promoter element maximally 16 hours after IL-1beta stimulation. DNA affinity chromatography studies showed that the LAP1, LAP2, and LIP isoforms of CEBPB bind to the IL-1beta-responsive CEBPB site in the MMP-1 promoter. Exogenous expression of the LAP1 and LAP2 isoforms stimulated the MMP-1 promoter, whereas LIP had no effect. Phosphorylation of CEBPB at Thr(235) peaked at 16 hours in IL-1beta-stimulated cells. The MEK inhibitor U0126 inhibited this phosphorylation and reduced MMP-1 gene induction. These studies establish CEBPB as an important mediator of metalloproteinase gene activation during inflammatory responses in lung cancer cells and highlight the different regulatory roles of CEBPB isoforms.

CEBP-β and PLK1 as Potential Mediators of the Breast Cancer/Obesity Crosstalk: In Vitro and In Silico Analyses

Article

Full-text available

Jun 2023

Over the last two decades, obesity has reached pandemic proportions in several countries, and expanding evidence is showing its contribution to several types of malignancies, including breast cancer (BC). The conditioned medium (CM) from mature adipocytes contains a complex of secretes that may mimic the obesity condition in studies on BC cell lines conducted in vitro. Here, we report a transcriptomic analysis on MCF-7 BC cells exposed to adipocyte-derived CM and focus on the predictive functional relevance that CM-affected pathways/processes and related biomarkers (BMs) may have in BC response to obesity. CM was demonstrated to increase cell proliferation, motility and invasion as well as broadly alter the transcript profiles of MCF-7 cells by significantly modulating 364 genes. Bioinformatic functional analyses unraveled the presence of five highly relevant central hubs in the direct interaction networks (DIN), and Kaplan–Meier analysis sorted the CCAAT/enhancer binding protein beta (CEBP-β) and serine/threonine-protein kinase PLK1 (PLK1) as clinically significant biomarkers in BC. Indeed, CEBP-β and PLK1 negatively correlated with BC overall survival and were up-regulated by adipocyte-derived CM. In addition to their known involvement in cell proliferation and tumor progression, our work suggests them as a possible “deus ex machina” in BC response to fat tissue humoral products in obese women.

Fundamentals of Osteoarthritis: Inflammatory mediators in Osteoarthritis

Article

Jun 2023
OSTEOARTHR CARTILAGE

Objectives: As more has become known of the pathophysiology of osteoarthritis (OA), evidence that inflammation plays a critical role in its development and progression has accumulated. Here, we aim to review current knowledge of the complex inflammatory network in the OA joint. Design: This narrative review is presented in three main sections: local inflammation, systemic inflammation, and therapeutic implications. We focused on inflammatory mediators and their link to OA structural changes in the joint. Results: OA is characterized by chronic and low-grade inflammation mediated mostly by the innate immune system, which results in cartilage degradation, bone remodeling and synovial changes. Synovitis is regarded as an OA characteristic and associated with increased severity of symptoms and joint dysfunction. However, the articular cartilage and the subchondral bone also produce several pro-inflammatory mediators thus establishing a complex interplay between the different tissues of the joint. In addition, systemic low-grade inflammation induced by aging, obesity and metabolic syndrome can contribute to OA development and progression. The main inflammatory mediators associated with OA include cytokines, chemokines, growth factors, adipokines, and neuropeptides. Conclusions: Future research is needed to deeper understand the molecular pathways mediating the inflammation in OA to provide new therapeutics that target these pathways, or to repurpose existing drugs.

Plasma Glycosaminoglycans in Children with Juvenile Idiopathic Arthritis Being Treated with Etanercept as Potential Biomarkers of Joint Dysfunction

Article

Full-text available

Jul 2022

We assessed the effect of two-year etanercept (ETA) therapy on the metabolism of the cartilage extracellular matrix (ECM) in patients with juvenile idiopathic arthritis (JIA). Methods: We performed a quantitative evaluation of glycosaminoglycans (GAGs) (performed by the multistage extraction and purification method) in blood obtained from patients before and during 24 months of ETA treatment, as potential biomarker of joint dysfunction and indicators of biological effectiveness of therapy. Since the metabolism of GAGs is related to the activity of proteolytic enzymes and prooxidant-antioxidant factors, we decided to evaluate the relationship between GAGs and the levels of metalloproteinases (MMP), i.e., MMP-1 and MMP-3 (using immunoenzymatic methods), as well as the total antioxidative status (TAS) (using the colorimetric method) in blood of the JIA patients. Results: When compared to the controls, GAGs and TAS concentrations were significantly lower in patients with an aggressive course of JIA qualified for ETA treatment. MMP-1 and MMP-3 levels were significantly higher versus control values. An anti-cytokine therapy leading to clinical improvement does not lead to the normalization of any of the assessed parameters. GAGs concentration is significantly related to MMP-1, MMP-3, TAS, TOS, and CRP levels. Conclusion: The results of the present study indicate the necessity of constant monitoring of the dynamics of destructive processes of articular cartilage in children with JIA. We suggest that GAGs may be a useful biomarker to assess the clinical status of the extracellular matrix of joints.

Global prevalence of prolonged gastrointestinal symptoms in COVID-19 survivors and potential pathogenesis: A systematic review and meta-analysis

Article

Full-text available

Apr 2021

Background: This study aimed to determine the cumulative prevalence of prolonged gastrointestinal (GI) symptoms, including nausea, vomiting, diarrhea, lack of appetite, abdominal pain, and dysgeusia, in survivors of both mild and severe COVID-19 worldwide and to discuss the potential pathogenesis. Methods: Three databases (PubMed, Scopus, and Web of Science) were searched for relevant articles up to January 30, 2021. Data on study characteristics, clinical characteristics during follow-up, the number of patients with prolonged GI symptoms, and total number of COVID-19 survivors were retrieved according to PRISMA guidelines. The quality of eligible studies was assessed using the Newcastle-Ottawa scale. The pooled prevalence of specific prolonged GI symptoms was calculated and the association between COVID-19 severity and the occurrence of prolonged GI symptoms was assessed if appropriate. Results: The global prevalence of prolonged nausea was 3.23% (95% CI: 0.54%–16.53%) among 527 COVID-19 survivors. Vomiting persisted in 93 of 2,238 COVID-19 survivors (3.19%, 95% CI: 1.62%–6.17%) and prolonged diarrhea was found in 34 of 1,073 survivors (4.12%, 95% CI: 1.07%–14.64%). A total of 156 patients among 2,238 COVID-19 survivors (4.41%, 95% CI: 1.91%–9.94%) complained of persistent decreased or loss of appetite. The cumulative prevalence of prolonged abdominal pain was 1.68% (95% CI: 0.84%–3.32%), whereas persistent dysgeusia was identified in 130 cases among 1,887 COVID-19 survivors (7.04%, 95% CI: 5.96%–8.30%). Data was insufficient to assess the relationship between COVID-19 severity and the occurrence of all prolonged GI symptoms. Conclusion: Persistent GI symptoms among COVID-19 survivors after discharge or recovery raises a concern regarding the long-term impact of the COVID-19 infection on the quality of life of the survivors. Despite several potential explanations proposed, studies that aim to follow patients after recovery from COVID-19 and determine the pathogenesis of the prolonged symptoms of COVID-19 survivors are warranted. PROSPERO registration: CRD42021239187.

Preparation and characterization of an injectable dexamethasone-cyclodextrin complexes-loaded gellan gum hydrogel for cartilage tissue engineering

Article

Sep 2020

In this study, 6-(6-aminohexyl) amino-6-deoxy-β-cyclodextrin-gellan gum complex hydrogel (HCD-GG) was developed to enhance the affinity of anti-inflammatory drug dexamethasone (Dx) and to improve chondrogenesis and decrease the inflammatory response. The modified chemical structure was confirmed by NMR and FTIR. Mechanical and physicochemical properties were characterized by performing viscosity study, compression test, injection force test, swelling kinetic, weight loss, and morphological study. The release profile of the drug-loaded hydrogels was analyzed to confirm the affinity of the hydrophobic drugs and the matrix and characterize cumulative release. In vitro test was carried out with MTT assay, live/dead staining, glycosaminoglycan (GAGs) content, double-stranded DNA (dsDNA) content, morphological analysis, histology, and gene expression. In vivo experiment was conducted by implanting the samples under a subcutaneous area of SPD rat and cartilage defected rabbit model. The results displayed successfully synthesized HCD-GG. The gelation temperature of the modified hydrogels was decreased while the mechanical property was improved when the drug was loaded in the modified hydrogel. Swelling and degradation kinetics resulted in a higher level compared to the pristine GG but was a sufficient level to support drugs and cells. The affinity and release rate of the drug was higher in the HCD-GG group which shows an improved drug delivery system of the GG-based material. The microenvironment provided a suitable environment for cells to grow. Also, chondrogenesis was affected by the existence of Dx and microenvironment, resulting in higher expression levels of cartilage-related genes while the expression of the inflammation mediators decreased when the Dx was loaded. In vivo study showed an improved anti-inflammatory response in the drug-loaded hydrogel. Furthermore, the cartilage defected rabbit model showed an enhanced regenerative effect when the Dx@HCD-GG was implanted. These results suggest that HCD-GG and Dx@HCD-GG have the potential for cartilage regeneration along with multiple applications in tissue engineering and regenerative medicine.

Genetic and Epigenetic Mechanisms Underlying Vascular Smooth Muscle Cell Phenotypic Modulation in Abdominal Aortic Aneurysm

Article

Full-text available

Aug 2020
INT J MOL SCI

Abdominal aortic aneurysm (AAA) refers to the localized dilatation of the infra-renal aorta, in which the diameter exceeds 3.0 cm. Loss of vascular smooth muscle cells, degradation of the extracellular matrix (ECM), vascular inflammation, and oxidative stress are hallmarks of AAA pathogenesis and contribute to the progressive thinning of the media and adventitia of the aortic wall. With increasing AAA diameter, and left untreated, aortic rupture ensues with high mortality. Collective evidence of recent genetic and epigenetic studies has shown that phenotypic modulation of smooth muscle cells (SMCs) towards dedifferentiation and proliferative state, which associate with the ECM remodeling of the vascular wall and accompanied with increased cell senescence and inflammation, is seen in in vitro and in vivo models of the disease. This review critically analyses existing publications on the genetic and epigenetic mechanisms implicated in the complex role of SMCs within the aortic wall in AAA formation and reflects the importance of SMCs plasticity in AAA formation. Although evidence from the wide variety of mouse models is convincing, how this knowledge is applied to human biology needs to be addressed urgently leveraging modern in vitro and in vivo experimental technology.

HIGH expression of OSM and IL-6 are associated with decreased breast cancer survival: synergistic induction of IL-6 secretion by OSM and IL-1β

Article

Full-text available

Mar 2019

Chronic inflammation has been recognized as a risk factor for the development and maintenance of malignant disease. Cytokines such as interleukin-6 (IL-6), oncostatin M (OSM), and interleukin-1 beta (IL-1β) promote the development of both acute and chronic inflammation while promoting in vitro metrics of breast cancer metastasis. However, anti-IL-6 and anti-IL-1β therapeutics have not yielded significant results against solid tumors in clinical trials. Here we show that these three cytokines are interrelated in expression. Using the Curtis TCGA™ dataset, we have determined that there is a correlation between expression levels of OSM, IL-6, and IL-1β and reduced breast cancer patient survival (r = 0.6, p = 2.2 x 10 −23). Importantly, we confirm that OSM induces at least a 4-fold increase in IL-6 production from estrogen receptor-negative (ER−) breast cancer cells in a manner that is dependent on STAT3 signaling. Furthermore, OSM induces STAT3 phosphorylation and IL-1β promotes p65 phosphorylation to synergistically induce IL-6 secretion in ER− MDA-MB-231 and to a lesser extent in ER+ MCF7 human breast cancer cells. Induction may be reduced in the ER+ MCF7 cells due to a previously known suppressive interaction between ER and STAT3. Interestingly, we show in MCF7 cells that ER's interaction with STAT3 is reduced by 50% through both OSM and IL-1β treatment, suggesting a role for ER in mitigating STAT3-mediated inflammatory cascades. Here, we provide a rationale for a breast cancer treatment regime that simultaneously suppresses multiple targets, as these cytokines possess many overlapping functions that increase metastasis and worsen patient survival.

ITGBL1 modulates integrin activity to promote cartilage formation and protect against arthritis

Article

Oct 2018

Developing and mature chondrocytes constantly interact with and remodel the surrounding extracellular matrix (ECM). Recent research indicates that integrin-ECM interaction is differentially regulated during cartilage formation (chondrogenesis). Integrin signaling is also a key source of the catabolic reactions responsible for joint destruction in both rheumatoid arthritis and osteoarthritis. However, we do not understand how chondrocytes dynamically regulate integrin signaling in such an ECM-rich environment. Here, we found that developing chondrocytes express integrin-β–like 1 ( Itgbl1 ) at specific stages, inhibiting integrin signaling and promoting chondrogenesis. Unlike cytosolic integrin inhibitors, ITGBL1 is secreted and physically interacts with integrins to down-regulate activity. We observed that Itgbl1 expression was strongly reduced in the damaged articular cartilage of patients with osteoarthritis (OA). Ectopic expression of Itgbl1 protected joint cartilage against OA development in the destabilization of the medial meniscus–induced OA mouse model. Our results reveal ITGBL1 signaling as an underlying mechanism of protection against destructive cartilage disorders and suggest the potential therapeutic utility of targeting ITGBL1 to modulate integrin signaling in human disease.

IL-1 β -Induced Matrix Metalloprotease-1 Promotes Mesenchymal Stem Cell Migration via PAR1 and G-Protein-Coupled Signaling Pathway

Article

Full-text available

Apr 2018

Mesenchymal stem cells (MSCs) are known for homing to sites of injury in response to signals of cellular damage. However, the mechanisms of how cytokines recruit stem cells to target tissue are still unclear. In this study, we found that the proinflammation cytokine interleukin-1 β (IL-1 β ) promotes mesenchymal stem cell migration. The cDNA microarray data show that IL-1 β induces matrix metalloproteinase-1 (MMP-1) expression. We then used quantitative real-time PCR and MMP-1 ELISA to verify the results. MMP-1 siRNA transfected MSCs, and MSC pretreatment with IL-1 β inhibitor interleukin-1 receptor antagonist (IL-1RA), MMP tissue inhibitor of metalloproteinase 1 (TIMP1), tissue inhibitor of metalloproteinase 2 (TIMP2), MMP-1 inhibitor GM6001, and protease-activated receptor 1 (PAR1) inhibitor SCH79797 confirms that PAR1 protein signaling pathway leads to IL-1 β -induced cell migration. In conclusion, IL-1 β promotes the secretion of MMP-1, which then activates the PAR1 and G-protein-coupled signal pathways to promote mesenchymal stem cell migration.

Mechanistic understanding of nanoparticles' interactions with extracellular matrix: The cell and immune system

Article

Full-text available

Jun 2017

Extracellular matrix (ECM) is an extraordinarily complex and unique meshwork composed of structural proteins and glycosaminoglycans. The ECM provides essential physical scaffolding for the cellular constituents, as well as contributes to crucial biochemical signaling. Importantly, ECM is an indispensable part of all biological barriers and substantially modulates the interchange of the nanotechnology products through these barriers. The interactions of the ECM with nanoparticles (NPs) depend on the morphological characteristics of intercellular matrix and on the physical characteristics of the NPs and may be either deleterious or beneficial. Importantly, an altered expression of ECM molecules ultimately affects all biological processes including inflammation. This review critically discusses the specific behavior of NPs that are within the ECM domain, and passing through the biological barriers. Furthermore, regenerative and toxicological aspects of nanomaterials are debated in terms of the immune cells-NPs interactions.

CCAAT/enhancer-binding protein β promotes receptor activator of nuclear factor-kappa-B ligand (RANKL) expression and osteoclast formation in the synovium in rheumatoid arthritis

Article

Full-text available

Dec 2015

CCAAT/enhancer-binding protein β (C/EBPβ) is a transcription factor that is activated in the synovium in rheumatoid arthritis (RA) and promotes expression of various matrix metalloproteinases. In this study, we examined whether C/EBPβ mediates the expression of receptor activator of nuclear factor-kappa-B ligand (RANKL) and drives osteoclast formation in primary fibroblast-like synoviocytes (FLS) from RA patients. The cooperation of C/EBPβ and activation transcription factor-4 (ATF4) in the regulation of the RANKL promoter was also investigated. Immunofluorescence staining was performed for C/EBPβ, RANKL, and ATF4 in synovium from RA patients. Adenovirus expression vectors for two major isoforms, C/EBPβ-liver-enriched activator protein (LAP) and - liver-enriched inhibitory protein (LIP), or small interfering RNA for C/EBPβ, were used to manipulate C/EBPβ expression in RA-FLS. RA-FLS over-expressing C/EBPβ were co-cultured with peripheral blood mononuclear cells (PBMCs) to test osteoclast formation by tartrate-resistant acid phosphatase (TRAP) staining. A promoter assay for RANKL, a chromatin immunoprecipitation (ChIP) assay and an immunoprecipitation (IP) assay were also performed. Immunofluorescence staining showed colocalization of C/EBPβ, ATF4 and RANKL in RA synovium. Western blotting revealed the expression of C/EBPβ-LAP and -LIP in RA-FLS. Over-expression of either C/EBPβ-LAP or -LIP significantly increased the expression of RANKL mRNA, while C/EBPβ-LIP down-regulated osteoprotegerin (OPG) mRNA. The RANKL/OPG mRNA ratio was significantly increased by C/EBPβ-LIP over-expression. Knockdown of C/EBPβ with siRNA decreased the expression of RANKL mRNA. The number of TRAP-positive multinucleated cells was increased in co-cultures of PBMCs and FLS over-expressing either C/EBPβ-LAP or -LIP, but was more significant with LIP. C/EBPβ-LIP does not have a transactivation domain. However, promoter assays showed that C/EBPβ-LIP and ATF4 synergistically transactivate the RANKL promoter. ChIP and IP assays revealed the cooperative binding of C/EBPβ and ATF4 on the RANKL promoter. We demonstrated that C/EBPβ, especially C/EBPβ-LIP in cooperation with ATF4, is involved in osteoclast formation by regulating RANKL expression in RA-FLS. These findings suggest that C/EBPβ plays a crucial role in bone destruction in RA joints.

Die Bedeutung von Ceramid und der sauren Sphingomyelinase für die intestinale und hepatische Matrixdegradation

Article

Dissertation

Licochalcone A inhibits the migration and invasion of human lung cancer cells via inactivation of the Akt signaling pathway with downregulation of MMP-1/-3 expression

Article

Full-text available

Aug 2014

Licochalcone A (LicA), a major phenolic constituent of Glycyrrhiza inflata, has been reported to exhibit anti-tumor, anti-inflammatory, and anti-metastatic properties in various cancer cells and animal models. The aim of this study was to determine the anti-tumor effects of LicA on lung cancer cells. The results indicated that LicA exhibited effective inhibition of cell migration and invasion of A549 and H460 cells under non-cytotoxic concentrations. Furthermore, LicA was also found to significantly inhibit the proteins and messenger RNA (mRNA) expression of MMP-1 and MMP-3 in A549 cells. Moreover, treatment of A549 cells with LicA-inhibited activation of the phosphorylation of Akt and inhibition of Akt by LY294002 (PI3K inhibitor) or transfection with the constitutive active-Akt (CA-Akt) expression vector significantly abolished the LicA-inhibited migration and invasion through activation of the Akt pathway. Further mechanistic studies revealed that LicA inhibits Akt signaling pathways and downstream transcription factors Sp1 expression. These findings imply a critical role for Akt inhibition in the LicA-inhibited migration and invasion of lung cancer cells. Thus, LicA might be used as an anti-invasive agent in the treatment of lung cancer.

Extracellular Hsp70 inhibits pro-inflammatory cytokine production by IL-10 driven down-regulation of C/EBPβ and C/EBPδ

Article

Full-text available

Jun 2013
INT J HYPERTHER

Abstract Purpose: Extracellular Hsp70 has anti-inflammatory potential, demonstrated in different models of inflammatory diseases. We investigated probable mechanisms used by Hsp70 to down-regulate pro-inflammatory cytokines. Materials and methods: We analysed cytokine mRNA levels in bone marrow-derived murine dendritic cells treated with Hsp70, lipopolysaccharide (LPS) and peptidoglycan (PGN) or OVA (an irrelevant protein control), hypothesising that this was mediated by C/EBPβ and C/EBPδ transcription factors. We also tested the involvement of TLR2, IL-10, ERK and STAT3, using genetically deficient mice and pharmacological inhibitors. Results: C/EBPβ and C/EBPδ levels were inhibited in bone marrow derived dendritic cells (BMDCs) treated with Hsp70, and that correlated with inhibition of TNF-α, IFN-γ and MCP-1. Such inhibition was not observed in TLR2 or IL-10 knockout mice, and was also abrogated upon pretreatment of cells with ERK and JAK2/STAT3 inhibitors. Conclusions: C/EBPβ and C/EBPδ transcription factors are inhibited by Hsp70 treatment, and their inhibition occurs via the TLR2-ERK-STAT3-IL-10 pathway in BMDCs, mediating the anti-inflammatory effects of Hsp70.

Intratracheally administered titanium dioxide or carbon black nanoparticles do not aggravate elastase-induced pulmonary emphysema in rats

Article

Full-text available

Jul 2012
BMC Pulm Med

Background Titanium dioxide (TiO2) and carbon black (CB) nanoparticles (NPs) have biological effects that could aggravate pulmonary emphysema. The aim of this study was to evaluate whether pulmonary administration of TiO2 or CB NPs in rats could induce and/or aggravate elastase-induced emphysema, and to investigate the underlying molecular mechanisms. Methods On day 1, Sprague-Dawley rats were intratracheally instilled with 25 U kg−1 pancreatic porcine elastase or saline. On day 7, they received an intratracheal instillation of TiO2 or CB (at 100 and 500 μg) dispersed in bovine serum albumin or bovine serum albumin alone. Animals were sacrificed at days 8 or 21, and bronchoalveolar lavage (BAL) cellularity, histological analysis of inflammation and emphysema, and lung mRNA expression of heme oxygenase-1 (HO-1), interleukin-1β (IL-1β), macrophage inflammatory protein-2, monocyte chemotactic protein-1, and matrix metalloprotease (MMP)-1, and -12 were measured. In addition, pulmonary MMP-12 expression was also analyzed at the protein level by immunohistochemistry. Results TiO2 NPs per se did not modify the parameters investigated, but CB NPs increased perivascular/peribronchial infiltration, and macrophage MMP-12 expression, without inducing emphysema. Elastase administration increased BAL cellularity, histological inflammation, HO-1, IL-1β and macrophage MMP-12 expression and induced emphysema. Exposure to TiO2 NPs did not modify pulmonary responses to elastase, but exposure to CB NPs aggravated elastase-induced histological inflammation without aggravating emphysema. Conclusions TiO2 and CB NPs did not aggravate elastase-induced emphysema. However, CB NPs induced histological inflammation and MMP-12 mRNA and protein expression in macrophages.

Interleukin-1 Induction of Aggrecanase Gene Expression in Human Articular Chondrocytes is Mediated by Mitogen-Activated Protein Kinases

Article

Jul 2012
CELL PHYSIOL BIOCHEM

We investigated the unknown molecular mechanisms of Interleukin-1 (IL-1β)-induced cartilage aggrecan degeneration by aggrecanase (ADAMTS-A Disintegrin And Metalloproteinase with ThromboSpondin motifs) in human articular chondrocytes, a model mimicking human arthritis. Chondrocytes were pretreated with various pharmacological inhibitors and then stimulated with IL-1β for 24 h. ADAMTS-4 expression or activity was studied by RT-PCR or ELISA and other proteins measured by Western blotting. MAP kinase kinase-specific inhibitor, U0126 inhibited IL-1-induced phosphorylation of ERK1/2 and down-regulated ADAMTS-4 expression and activity. Protein 38 inhibitor, SB203580 down-regulated the phosphorylation of p38 and its target, activating transcription factor-2 (ATF-2), ADAMTS-4 mRNA and activity. C-Jun N-terminal kinase (JNK) inhibitor, SP600125 diminished IL-1-stimulated JNK phosphorylation, ADAMTS-4 mRNA expression and enzyme activity. A c-fos/lipoxygenase pathway inhibitor and antioxidant, nordihydroguaiaretic acid (NDGA) significantly suppressed ADAMTS-4 mRNA induction and activity. Activating protein (AP-1) and nuclear factor kappa B (NF-ĸB) transcription factor inhibitors, curcumin and pyrrolidine dithiocarbamate (PDTC) partially inhibited ADAMTS-4 induction and activity. These results suggest partial involvement of ERK-, p38-and JNK-MAPKs as well as AP-1, ATF-2 and NF-ĸB transcription factors in IL-1-induced ADAMTS-4 in chondrocytes. Inhibition of these targets by the specific pharmacological agents could be useful for reducing aggrecanase-driven cartilage resorption in arthritis.

Increased invasiveness of MMP-9-deficient tumors in two mouse models of neuroendocrine tumorigenesis

Article

Full-text available

Mar 2012

Despite their apparent success in pre-clinical trials, metalloproteinase (MMP) inhibitors proved to be inefficacious in clinical settings. In an effort to understand the underlying causes of this unanticipated outcome, we modeled the consequences of long-term MMP inhibition by removing one of the major players in tumorigenesis, MMP9, in two complimentary mouse models of pancreatic neuroendocrine carcinogenesis: Myc;BclXl and RIP1-Tag2. By employing gel zymography and a fluoregenic solution assay, we first established that MMP9 is expressed and activated in Myc;BclXl tumors in an interleukin-1β-dependent manner. The genetic deletion of MMP9 in Myc;BclXl mice impairs tumor angiogenesis and growth analogous to its absence in the RIP1-Tag2 model. Notably, tumors that developed in the context of MMP9-deficient backgrounds in both models were markedly more invasive than their typical wild-type counterparts, and expressed elevated levels of pro-invasive cysteine cathepsin B. The increased invasion of MMP9-deficient tumors was associated with a switch in the spectrum of inflammatory cells at the tumor margins, involving homing of previously undetected, cathepsin-B expressing CD11b;Gr1-positive cells to the invasive fronts. Thus, plasticity in the tumor inflammatory compartment is partially responsible for changes in the expression pattern of tumor-associated proteases, and may contribute to the compensatory effects observed on MMP inhibition, hence accounting for the heightened tumor progression described in late stage clinical trials.Oncogene advance online publication, 5 March 2012; doi:10.1038/onc.2012.60.

Semaphorin 3A Is Expressed in Human Osteoarthritic Cartilage and Antagonizes Vascular Endothelial Growth Factor 165-Promoted Chondrocyte Migration An Implication for Chondrocyte Cloning

Article

Oct 2011
ARTHRIT RHEUM-ARTHR

Vascular endothelial growth factor 165 (VEGF165) and its receptors, including neuropilin 1 (NRP-1), are overexpressed in human osteoarthritic (OA) articular cartilage, although their functional roles in the cartilage are not fully understood. An axon-guidance molecule, semaphorin 3A (Sema3A), which binds to NRP-1, acts as an antagonist of VEGF signaling in endothelial cells. The aim of this study was to examine the expression of Sema3A and the functions of the VEGF165/Sema3A/NRP-1 axis in OA cartilage. The expression of Sema3A in OA and normal cartilage samples was examined by real-time polymerase chain reaction and immunohistochemical analyses. Functional analyses of VEGF165 and Sema3A were carried out using OA chondrocytes in culture. The migration activity of chondrocytes was examined in a monolayer wound assay. The effects of Sema3A on VEGF165-induced up-regulation of matrix metalloproteinases (MMPs) and intracellular signaling were also studied in cultured chondrocytes. Sema3A expression was significantly elevated in OA cartilage as compared to normal cartilage. Sema3A immunoreactivity directly correlated with the Mankin score and with chondrocyte cloning. VEGF165 promoted the migration of chondrocytes, and this activity was suppressed by VEGF receptor 2 tyrosine kinase inhibitors. Sema3A antagonized the chondrocyte migration promoted by VEGF165, and the activity was blocked by a selective inhibitor of, or small interfering RNA for, Sema3A. VEGF165-induced overexpression of MMPs and phosphorylation of ERK and focal adhesion kinase in chondrocytes were inhibited by Sema3A. Our findings provide the first evidence that Sema3A is overexpressed, with a direct correlation with cloning, in OA cartilage and that it suppresses the VEGF165-promoted migration of chondrocytes. Our findings also suggest that Sema3A plays a role in chondrocyte cloning through inhibition of cell migration in OA cartilage.

Focal adhesions and Ras are functionally and spatially integrated to mediate IL-1 activation of ERK

Article

Jun 2011
FASEB J

In connective tissue cells, IL-1-induced ERK activation leading to matrix metalloproteinase (MMP)-3 expression is dependent on cooperative interactions between focal adhesions and the endoplasmic reticulum (ER). As Ras can be activated on the ER, we investigated the role of Ras in IL-1 signaling and focal adhesion formation. We found that constitutively active H-Ras, K-Ras or N-Ras enhanced focal adhesion maturation and β1-integrin activation. IL-1 promoted the accumulation of Ras isoforms in ER and focal adhesion fractions, as shown in cells cotransfected with GFP-tagged Ras isoforms and YFP-ER protein and by analysis of subcellular fractions enriched for ER or focal adhesion proteins. Dominant-negative H-Ras or K-Ras reduced accumulation of H-Ras and K-Ras in focal adhesions induced by IL-1 and also blocked ERK activation and focal adhesion maturation. Ras-GRF was enriched constitutively in focal adhesion fractions and was required for Ras recruitment to focal adhesions. We conclude that Ras activation and IL-1 signaling are interactive processes that regulate the maturation of focal adhesions, which, in turn, is required for ERK activation.

Host-derived diagnostic markers related to soft tissue destruction and bone degradation in periodontitis

Article

Mar 2011
J CLIN PERIODONTOL

A major challenge in clinical periodontics is to find a reliable molecular marker of periodontal tissue destruction with high sensitivity, specificity and utility. The aim of this systematic review is to evaluate available literature on 'the utility of molecular markers of soft and hard periodontal tissue destruction'. Based on the focused question, 'What is the utility of molecular markers of soft and hard periodontal tissue destruction', an electronic and manual search was conducted for human studies presenting clinical data for the potential of molecular markers of tissue destruction in biofluids; gingival crevicular fluid (GCF), saliva, and serum. Papers fulfilling the inclusion criteria were selected. All relevant data from the selected papers were extracted and recorded in separate tables for molecules in GCF, saliva, and serum. Within the defined limits of the Problem/Population, Intervention, Comparison, Outcome, the present analysis reveals that (a) no single or combination of markers exists that can disclose periodontal tissue destruction adequately; (b) while the most fruitful source of biomarkers for periodontal destruction appears to be in molecules tightly related to bone and soft tissue destruction, this remains to be objectively demonstrated. Currently, clinical measurements are still the most reliable.

Toll-like receptor 2-dependent inhibition of interferon gamma signaling by Mycobacterium tuberculosis [electronic resource]: /

Article

Full-text available

Meghan E Pennini

[School of Medicine] Department of Pathology. Thesis (Ph. D.)--Case Western Reserve University, 2006. Includes bibliographical references. Requires Adobe Acrobat Reader for viewing.

Matrix metalloproteinase-1 expression induced by IL-1β requires acid sphingomyelinase

Article

Apr 2009
FEBS LETT

Matrix metalloproteinase-1 (MMP-1) is increased in inflammatory conditions leading to destruction of extracellular matrix. Many inflammatory stimuli activate sphingomyelinases (SMases), which generate ceramide. We aimed to define the relevance and type of SMase responsible for the regulation of MMP-1. Acid sphingomyelinase (ASM)-deficient human fibroblasts failed to phosphorylate extracellular signal-regulated kinase (ERK), or upregulate MMP-1 mRNA and protein expression upon stimulation with interleukin-1 beta (IL-1beta), whereas phosphorylation of p38 mitogen-activated protein kinase and IL-8 production remained unaffected. Transfection of ASM restored MMP-1 production. Addition of exogenous SMase was sufficient to restore activation of ERK and increase MMP-1 mRNA. Inhibition of ASM with imipramine completely abrogated MMP-1 induction. The results suggest that IL-1beta-induced expression of MMP-1 is dependent on ASM.

C/EBPβ Promotes Transition from Proliferation to Hypertrophic Differentiation of Chondrocytes through Transactivation of p57Kip2

Article

Full-text available

Feb 2009
PLOS ONE

Although transition from proliferation to hypertrophic differentiation of chondrocytes is a crucial step for endochondral ossification in physiological skeletal growth and pathological disorders like osteoarthritis, the underlying mechanism remains an enigma. This study investigated the role of the transcription factor CCAAT/enhancer-binding protein beta (C/EBPbeta) in chondrocytes during endochondral ossification. Mouse embryos with homozygous deficiency in C/EBPbeta (C/EBPbeta-/-) exhibited dwarfism with elongated proliferative zone and delayed chondrocyte hypertrophy in the growth plate cartilage. In the cultures of primary C/EBPbeta-/- chondrocytes, cell proliferation was enhanced while hypertrophic differentiation was suppressed. Contrarily, retroviral overexpression of C/EBPbeta in chondrocytes suppressed the proliferation and enhanced the hypertrophy, suggesting the cell cycle arrest by C/EBPbeta. In fact, a DNA cell cycle histogram revealed that the C/EBPbeta overexpression caused accumulation of cells in the G0/G1 fraction. Among cell cycle factors, microarray and real-time RT-PCR analyses have identified the cyclin-dependent kinase inhibitor p57(Kip2) as the transcriptional target of C/EBPbeta. p57(Kip2) was co-localized with C/EBPbeta in late proliferative and pre-hypertrophic chondrocytes of the mouse growth plate, which was decreased by the C/EBPbeta deficiency. Luciferase-reporter and electrophoretic mobility shift assays identified the core responsive element of C/EBPbeta in the p57(Kip2) promoter between -150 and -130 bp region containing a putative C/EBP motif. The knockdown of p57(Kip2) by the siRNA inhibited the C/EBPbeta-induced chondrocyte hypertrophy. Finally, when we created the experimental osteoarthritis model by inducing instability in the knee joints of adult mice of wild-type and C/EBPbeta+/- littermates, the C/EBPbeta insufficiency caused resistance to joint cartilage destruction. C/EBPbeta transactivates p57(Kip2) to promote transition from proliferation to hypertrophic differentiation of chondrocytes during endochondral ossification, suggesting that the C/EBPbeta-p57(Kip2) signal would be a therapeutic target of skeletal disorders like growth retardation and osteoarthritis.

Myeloid Differentiation Factor 88 Regulates Basal and UV-Induced Expressions of IL-6 and MMP-1 in Human Epidermal Keratinocytes

Article

Full-text available

Sep 2008
J INVEST DERMATOL

Myeloid differentiation factor 88 (MyD88) is known as an adaptor protein for the Toll-like receptor (TLR) family and participates in signal transduction by binding to the cytoplasmic Toll/IL-1 receptor (TIR) domains of activated TLR. In this study, we demonstrated that expression of MyD88 is increased in photoaged skin compared with intrinsic aged human skin of the same elderly individuals, and that acute UV irradiation increases MyD88 expression in human skin in vivo. To investigate the effects of these high levels of MyD88 in photoaged skin and acutely UV-irradiated skin, human epidermal keratinocytes were infected with adenovirus expressing wild-type (MyD88wt), dominant-positive (MyD88DeltaC), and dominant-negative (MyD88DeltaN) MyD88 forms. Overexpression of MyD88wt and MyD88DeltaC, but not of MyD88DeltaN, increased the basal expressions of IL-6 and matrix metalloproteinase-1 (MMP-1) in human epidermal keratinocytes. Moreover, overexpression of MyD88DeltaN prevented UV-induced expressions of IL-6 and MMP-1 by inhibiting UV-induced activation of NF-kappaB and activating protein-1. These results suggest that MyD88 is important in IL-6 and MMP-1 expressions in both acutely UV-irradiated skin and in chronically sun-exposed human skin.

Evaluation of t-PA, PAI-2, IL-1 β and PGE 2 in gingival crevicular fluid of rheumatoid arthritis patients with periodontal disease

Article

Sep 2006

This study was undertaken to compare periodontal conditions, gingival crevicular fluid (GCF) levels of tissue-type plasminogen activator (t-PA), its inhibitor plasminogen activator inhibitor-2 (PAI-2), interleukin-1beta (IL-1beta), prostaglandin E(2) (PGE(2)) in rheumatoid arthritis (RA) patients and control groups. Twenty-three RA patients, 17 systemically healthy patients with periodontal disease (PD), and 17 systemically and periodontally healthy subjects were recruited. GCF samples were obtained from two single-rooted teeth. Full-mouth clinical periodontal measurements were recorded at six sites/tooth. GCF samples were analysed using relevant ELISA kits. Data were tested statistically by appropriate tests. Total amounts of t-PA, PAI-2 and PGE(2) in GCF samples of the healthy control group were significantly lower than the other groups (p<0.05). The RA group exhibited a higher total amount of t-PA in GCF samples than the PD group (p<0.05). PAI-2, IL-1beta and PGE(2) total amounts were similar in RA and PD groups (p>0.05). The coexistence of RA and periodontitis does not seem to affect clinical periodontal findings or systemic markers of RA. Similar inflammatory mediator levels in RA and PD groups, despite the long-term usage of corticosteroids, non-steroidal anti-inflammatory drugs, suggest that RA patients may have a propensity to overproduce these inflammatory mediators.

A RecA-mediated exon profiling method

Article

Full-text available

Jul 2006
NUCLEIC ACIDS RES

We have developed a RecA-mediated simple, rapid and scalable method for identifying novel alternatively spliced full-length cDNA candidates. This method is based on the principle that RecA proteins allow to carry radioisotope-labeled probe DNAs to their homologous sequences, resulting in forming triplexes. The resulting complex is easily detected by mobility difference on electrophoresis. We applied this exon profiling method to four selected mouse genes as a feasibility study. To design probes for detection, the information on known exonic regions was extracted from public database, RefSeq. Concerning the potentially transcribed novel exonic regions, RNA mapping experiment using Affymetrix tiling array was performed. As a result, we were able to identify alternative splice variants of Thioredoxin domain containing 5, Interleukin1β, Interleukin 1 family 6 and glutamine-rich hypothetical protein. In addition, full-length sequencing demonstrated that our method could profile exon structures with >90% accuracy. This reliable method can allow us to screen novel splice variants from a huge number of cDNA clone set effectively.

The effect of antioxidant supplementation on dysmenorrhea and endometriosis-associated painful symptoms: a systematic review and meta-analysis of randomized clinical trials

Article

Jan 2024

This study aimed to review randomized controlled trials (RCTs) investigating the effects of dietary antioxidant supplements on the severity of endometriosis-related pain symptoms. The PubMed/Medline, Scopus, and Web of Science databases were searched until April 2022. Additionally, we manually searched the reference lists. Endpoints were summarized as standardized mean difference (SMD) with 95% confidence intervals (CIs) in a random-effects model. The I2 statistic was used to assess heterogeneity. Ten RCTs were included in this meta-analysis. Overall, 10 studies were related to dysmenorrhea, four to dyspareunia, and four to pelvic pain. Antioxidants significantly reduced dysmenorrhea (SMD, -0.48; 95% CI, -0.82 to -0.13; I2=75.14%). In a subgroup analysis, a significant reduction of dysmenorrhea was observed only in a subset of trials that administered vitamin D (SMD, -0.59; 95% CI, -1.13 to -0.06; I2=69.59%) and melatonin (SMD, -1.40; 95% CI, -2.47 to -0.32; I2=79.15%). Meta-analysis results also suggested that antioxidant supplementation significantly improved pelvic pain (SMD, -1.51; 95% CI, -2.74 to -0.29; I2=93.96%), although they seem not to have a significant beneficial impact on the severity of dyspareunia. Dietary antioxidant supplementation seems to beneficially impact the severity of endometriosis-related dysmenorrhea (with an emphasis on vitamin D and melatonin) and pelvic pain. However, due to the relatively small sample size and high heterogeneity, the findings should be interpreted cautiously, and the importance of further well-designed clinical studies cannot be overstated.

C/EBPβ: The structure, regulation, and its roles in inflammation-related diseases

Article

Nov 2023

Identifying effector molecules, cells, and cytokines of innate immunity in OA

Article

Feb 2020

Inflammatory changes are observed in affected joints of osteoarthritis (OA) patients and are thought to be involved in the pathology that develops along OA progression. This narrative review provides an overview of the various cell types that are present in the joint during OA and which alarmins, cytokines, chemokines, growth factors, and other mediators they produce. Moreover, the involvement of more systemic processes like inflammaging and its associated cellular senescence in the context of OA are discussed.

Chondrocytes

Article

Jan 2007

Rheumatoid arthritis (RA) is one of the inflammatory joint diseases in a heterogeneous group of disorders that share features of destruction of the extracellular matrices of articular cartilage and bone. The underlying disturbance in immune regulation that is responsible for the localized joint pathology results in the release of inflammatory mediators in the synovial fluid and synovium that directly and indirectly influence cartilage homeostasis. Analysis of the breakdown products of the matrix components of joint cartilage in body fluids and quantitative imaging techniques have been used to assess the effects of the inflammatory joint disease on the local remodeling of joint structures. The role of the chondrocyte itself in cartilage destruction in the human rheumatoid joint has been difficult to address but has been inferred from studies in vitro and in animal models. This review covers current knowledge about the specific cellular and biochemical mechanisms that account for the disruption of the integrity of the cartilage matrix in RA.

Chondrocytes: Pathogenesis of Rheumatoid Arthritis

Article

Jan 2009

Mary B Goldring

Effecet of titanium dioxide nanoparticles on metalloproteases, influence of physicochemical parameters

Article

Full-text available

Dec 2011

Lucie Armand

Exposure to titanium dioxide (TiO2) nanoparticles (NP) can lead to matrix remodeling, a phenomenon in which Matrix metalloprotease-1 (MMP-1) notably could play an important role. To validate this hypothesis, we 1/ studied MMP-1 expression in human pulmonary fibroblasts after exposure to TiO2 particles of different sizes, shapes and crystal phases and one Carbon Black (CB) NP, and 2/ measured the effect of TiO2 CB NP on an elastase-induced emphysema model in rats.On human lung fibroblasts, some TiO2 NP induced MMP-1 through an interleukin-1β (IL-1β) dependent mechanism, and increased MMP-1 activity. TiO2 micrometric particles induced MMP-1 without IL-1β implication, and CB NP did not induce MMP-1. Rat exposure to TiO2 NP had no effect but exposure to NC NP induced inflammation and MMP-12 expression. TiO2 NP did not modulate elastase effects. NC NP aggravated elastase-induced inflammation.In total, our work shows a role for TiO2 NP in matrix remodeling, via MMP-1 induction and activation in vitro. In vivo, CB NP can aggravate some elastase-induced effects. Particles composition and size seem to be important determinants of their effect.

Interaction of ERK1/2 and Smad2/3 signaling pathways in TGF-β1-induced TIMP-3 expression in rat chondrocytes

Article

Sep 2014

Tissue inhibitor of metalloproteinase-3 (TIMP-3) is an important natural inhibitor of matrix metalloproteinases (MMPs) and of a disintegrin and metalloproteinase with thrombospondin motif (ADAMTs), which can cleave cartilage extracellular matrix components to cause cartilage degradation. In this study, our data suggest TGF-β1 induces TIMP-3 expression through activations of both the ERK1/2 and Smad2/3 signaling pathways. TGF-β1-stimulated TIMP-3 expression was significantly inhibited by SB525334 (TGF-β receptor I kinase inhibitor), accompanied by a reduction in ERK1/2 and Smad3 phosphorylation. We used PD98059 (MEK inhibitor) and SIS3 (inhibitor of Smad3 phosphorylation) to investigate the respective roles of ERK1/2 and Smad2/3 signaling pathways in TGF-β1-induced TIMP-3 expression. The results show PD98059 treatment significantly suppressed TGF-β1-induced ERK1/2 phosphorylation and TIMP-3 expression. Under these conditions, the degree of Smad3 phosphorylation correlated with ERK1/2 activation, which suggests that ERK1/2 may activate Smad3 phosphorylation. SIS3 significantly inhibited TGF-β1-induced Smad3 phosphorylation and TIMP-3 expression. ERK1/2 phosphorylation alone had no effect on TGF-β1-induced TIMP-3 expression, which suggests ERK1/2 via Smad3 phosphorylation regulates TGF-β1-induced TIMP-3 expression. Here, we demonstrate that ERK1/2 may be capable of activating the Smad2/3 signaling pathway to result in TGF-β1-induced TIMP-3 up-regulation.

CCAAT/Enhancer-binding Protein (C/EBP) Is Acetylated at Multiple Lysines ACETYLATIONOFC/EBPATLYSINE39MODULATESITSABILITYTO ACTIVATETRANSCRIPTION

Article

Transcription factor function can be modulated by post- translational modifications. Because the transcription factor CCAAT/enhancer-binding protein (C/EBP) associates with the nuclear coactivator p300, which contains acetyltransferase activity, acetylation of C/EBP was examined to understand its regulation and function. C/EBP is acetylated by acetyltrans- ferases p300 and p300/CREB-binding protein associated factor. Endogenous C/EBP in 3T3-F442A preadipocytes is also recog- nized by an acetyl-lysine-specific antibody. Analysis of trunca- tions of C/EBP and peptides based on C/EBP sequences iden- tified multiple lysines within C/EBP that can be acetylated. Among these, a novel acetylation site at lysine 39 of C/EBP was identified. Mutation of Lys-39 to arginine or alanine impairs its acetylation and the ability of C/EBP to activate transcription at the promoters for C/EBP and c-fos. Different C/EBP-respon- sive promoters require different patterns of acetylated lysines in C/EBP for transcription activation. Furthermore, C/EBP acetylation was increased by growth hormone, and mutation of Lys-39 impaired growth hormone-stimulated c-fos promoter activation. These data suggest that acetylation of Lys-39 of C/EBP, alone or in combination with acetylation at other lysines, may play a role in C/EBP-mediated transcriptional activation.

Interleukin-1 beta and transforming growth factor-beta 3 cooperate to activate matrix metalloproteinase expression and invasiveness in A549 lung adenocarcinoma cells

Article

Jul 2012

Cytokines present in the tumor microenvironment can promote the invasiveness and metastatic potential of cancer cells. We therefore investigated the effects of interleukin-1 beta (IL-1B) and transforming growth factor beta-3 (TGFB3) on the non-small cell lung carcinoma (NSCLC) cell line A549. We found that these cytokines synergistically activated matrix metalloproteinase (MMP)-1, MMP-3, and MMP-10 gene expression in these cells through mitogen-activated protein kinase (MAPK)-dependent pathways. Consistent with this, both cytokines stimulated epithelial to mesenchymal transition and MAPK-dependent invasion through Matrigel™. These studies identify IL-1B and TGFB3 as pro-invasive factors in NSCLC and potential therapeutic targets for tumor progression.

Increased Expression of Matrix Metalloproteinases Mediates Thromboxane A2-Induced Invasion in Lung Cancer Cells

Article

Apr 2012
CURR CANCER DRUG TAR

Thromboxane A(2) receptor (TP) has been shown to play an important role in multiple aspects of cancer development including regulation of tumor growth, survival and metastasis. Here we report that TP mediates cancer cell invasion by inducing expression of matrix metalloproteinases (MMPs). TP agonist, I-BOP, significantly elevated MMP-1, MMP-3, MMP-9 and MMP-10 mRNA levels in A549 human lung adenocarcinoma cells overexpressing TPα or TPβ. The secretion of MMP-1 and MMP-9 in conditioned media was determined using Western blot analysis and zymographic assay. Signaling pathways of I-BOP-induced MMP-1 expression were examined in further detail as a model system for MMPs induction. Signaling molecules involved in I-BOP-induced MMP-1 expression were identified by using specific inhibitors including small interfering (si)-RNAs of signaling molecules and promoter reporter assay. The results indicate that I-BOP-induced MMP-1 expression is mediated by protein kinase C (PKC), extracellular signal-regulated kinase (ERK)-activator protein-1(AP-1) and ERK-CCAAT/enhancer-binding protein β (C/EBPβ) pathways. I-BOP-induced cellular invasiveness of A549 cells expressing TPα or TPβ was determined by invasion assay. GM6001, a general inhibitor of MMPs, decreased basal and I-BOP-induced cell invasion. Knockdown of MMP-1 and MMP-9 by their respective siRNA partially reduced I-BOP-stimulated cell invasion suggesting that other MMPs induced by I-BOP were also involved. Our studies establish the relationship between TP and MMPs in cancer cell invasion and suggest that the thromboxane A(2) (TXA(2))-TP signaling is a potential therapeutic target for cancer invasion and metastasis.

CCAAT/enhancer binding protein regulates expression of matrix metalloproteinase-3 in arthritis

Article

Full-text available

Sep 2011
ANN RHEUM DIS

To investigate whether CCAAT/enhancer binding protein β (C/EBPβ) mediates the expression of matrix metalloproteinase-3 (MMP-3) and aggrecanases in arthritis. Localisation of C/EBPβ and MMP-3 in synovium and cartilage from patients with rheumatoid arthritis and osteoarthritis was determined by immunohistochemistry. Cell lines SW982, C28/I2 and human fibroblast-like synoviocytes stimulated by interleukin 1β (IL-1β) were subjected to western blotting and quantitative PCR. Overexpression of C/EBPβ by adenovirus was performed in cells and organ culture of normal cartilage. Knockdown of C/EBPβ by small interference RNA was performed in cells. Activity of the human MMP-3 and aggrecanase-2 ADAMTS-5 (a disintegrin and metalloproteinase with thrombospondin motifs) promoters was analysed by a luciferase assay. To determine whether C/EBPβ directly binds to the MMP-3 or ADAMTS-5 promoter,a chromatin immunoprecipitation assay was performed. Immunohistochemistry showed that C/EBPβ and MMP-3 were co-localised in arthritic synovium and cartilage. Western blots revealed increased C/EBPβ expression in cells treated with IL-1β. Expression of MMP-3, MMP-13 and ADAMTS-5 mRNA was significantly increased by the overexpression of C/EBPβ. C/EBPβ stimulated MMP-3 expression and induced matrix degradation in cartilage explants. C/EBPβ knockdown reduced MMP-3 and ADAMTS-5 expression. C/EBPβ stimulated the 2011 bp MMP-3 promoter and the 1768 bp ADAMTS-5 promoter in a dose-dependent manner. Deletion and mutation analysis of the MMP-3 promoter showed that the C/EBPβ core responsive element was located between -108 bp and -100 bp. The chromatin immunoprecipitation assay showed that C/EBPβ was directly bound to MMP-3 and ADAMTS-5 promoters. These data demonstrate that C/EBPβ is involved in expression of MMP-3 and ADAMTS-5 in arthritic synovium and cartilage.

Roles of inflammatory and anabolic cytokines in cartilage metabolism

Article

Dec 2008
ANN RHEUM DIS

In osteoarthritis (OA), adult articular chondrocytes undergo phenotypic modulation in response to alterations in the environment owing to mechanical injury and inflammation. These processes not only stimulate the production of enzymes that degrade the cartilage matrix but also inhibit repair. With the use of in vitro and in vivo models, new genes, not known previously to act in cartilage, have been identified and their roles in chondrocyte differentiation during development and in dysregulated chondrocyte function in OA have been examined. These new genes include growth arrest and DNA damage (GADD)45beta and the epithelial-specific ETS (ESE)-1 transcription factor, induced by bone morphogenetic protein (BMP)-2 and inflammatory cytokines, respectively. Both genes are induced by NF-kappaB, suppress COL2A1 and upregulate matrix meatalloproteinase-13 (MMP-13) expression. These genes have also been examined in mouse models of OA, in which discoidin domain receptor 2 is associated with MMP-13-mediated remodelling, in order to understand their roles in physiological cartilage homoeostasis and joint disease.

Epigenetic Repression of Matrix Metalloproteinases in Myofibroblastic Hepatic Stellate Cells through Histone Deacetylases 4 Implication in Tissue Fibrosis

Article

Oct 2010
Am J Pathol

Matrix metalloproteinases (MMPs), which are highly expressed in acute injury, are progressively repressed or silenced in fibrotic liver, favoring extracellular matrix accumulation, while the underlying mechanism is largely unknown. Similarly, normal/quiescent hepatic stellate cells (HSCs) express high levels of MMPs in response to injury signals, such as interleukin-1. After transdifferentiation, the myofibroblastic HSCs are incapable of expressing many MMPs; however, the major signaling pathways required for MMP expression are intact, indicating that repression is at the level of the chromatin. Indeed, both the MMP9 and MMP13 genes are inaccessible to transcription factors and RNA polymerase II, in association with impaired histone acetylation in their promoters. In accordance with impaired histone acetylation at the cellular level, histone deacetylase-4 is accumulated during HSC transdifferentiation. Furthermore, ectopic expression of histone deacetylase-4 in quiescent HSCs results in repression of MMP promoter activities as well as endogenous MMP9 protein expression. Thus, our findings suggest that a histone deacetylase-4-dependent mechanism underlies the epigenetic silencing of MMP genes during tissue fibrogenesis.

Differential down-regulation of COX-2 and MMP-13 in human skin fibroblasts by glucosamine-hydrochloride

Article

Sep 2009

Evidence suggests anti-inflammatory effects of glucosamine (GS) on inflammatory diseases. COX-2 is an enzyme to produce prostaglandins. MMPs are the family of matrix metalloproteinases degradable of ECM. Excess expression of COX-2 or MMPs involves in skin inflammation. We evaluated whether GS-HCl modulates expression of COX-2 and/or MMPs by IL-1beta or PMA in human skin fibroblasts (HSF) or keratinocytes (HaCaT). HSF or HaCaT cells were exposed to IL-1beta or PMA without or with GS-HCl. COX-2 or MMPs protein and mRNA expression, respectively, were analyzed by Western blot and RT-PCR. MTS assay was utilized to assess the cytotoxicity of GS-HCl on HSF cells. In HSF cells, IL-1beta treatment induced COX-2 and MMP-13 expressions in association with activation of ERKs, p38 MAPK, JNKs, and NF-kappaB. PMA treatment also induced COX-2 and MMP-13 expressions in association with p38 MAPK activation. Of interest, treatment with GS-HCl (10mM) led to blockage of p38 MAPK activation, accumulation of 66kDa COX-2 protein variant (without affecting COX-2 mRNA expression), and transcriptional down-regulation of MMP-13 in the IL-1beta- or PMA-treated HSF cells. Distinctly, pharmacological inhibition of p38 MAPK with SB203580 was associated with transcriptional down-regulation of COX-2 and MMP-13 in the IL-1beta- or PMA-treated HSF cells. In addition, the GS-HCl-mediated COX-2 protein modification was observed in both endogenous and PMA-induced COX-2 in HaCaT cells. GS-HCl differentially down-regulates COX-2 and MMP-13 expression in the IL-1beta- or PMA-treated human skin fibroblasts via the p38 MAPK-independent COX-2 translational inhibition and the p38 MAPK-dependent MMP-13 transcriptional suppression, respectively.

Site controlled transgenic mice validating increased expression from human matrix metalloproteinase (MMP-1) promoter due to a naturally occurring SNP

Article

Aug 2009

Matrix metalloproteinases (MMPs) comprise a family of more than 20 members, each with the ability to degrade components of the extracellular matrix. The interstitial collagenases have the unique capacity to degrade the stromal collagens, types I, II and III, the body's most abundant proteins. These collagenases include MMP-1, MMP-8, MMP-13 and MMP-14. MMP-1, with a very broad expression pattern, has major roles in mediating matrix destruction in many diseases. We have described a single nucleotide polymorphism (SNP) in the MMP-1 promoter that augments transcription. This SNP is the presence or absence of an extra guanine (G) at -1607 bp, which creates the sequence 5'-GGAA-3'(2G allele), and which is an ETS binding site. Compared to the 1G allele (5'-GAA-3'), the 2G SNP is associated with enhanced transcription of MMP-1 and increased enzymatic activity. Although murine systems are often used to model human diseases, mice have only distant homologues of human MMP-1. Therefore, we used a technique for the targeted insertion of a single copy of a gene at the HPRT locus to compare expression of the 1G and 2G alleles. We generated transgenic mice with -4372 bp of the human MMP-1 promoter containing either the 1G or 2G SNP in front of the lac Z (E.coli ss-galactosidase) gene. We measured the relative expression of the transgenes in vitro in embryonic stem (ES) cells and in fibroblasts derived from embryonic mice. Our data show modest constitutive expression of ss-galactosidase mRNA and protein from these alleles, with the 2G allele more transcriptionally active than the 1G allele. We conclude that these mice represent a model for integration of a single copy of the human MMP-1 promoter into the murine genome, and could be used to study MMP-1 gene expression in a murine system.

RelA is Required for IL-1β Stimulation of Matrix Metalloproteinase-1 Expression In Chondrocytes

Article

May 2007

Interleukin-1beta (IL-1beta) stimulates collagenase-1 (Matrix Metalloproteinase-1 (MMP-1)) expression in articular chondrocytes, leading to cleavage of type II collagen and irreversible cartilage degradation. The nuclear factor-kappa B (NF-kappaB) pathway is potently activated in IL-1beta-stimulated cells and has been implicated as an intermediate in MMP-1 gene expression. However, the roles of individual NF-kappaB family members during IL-1beta-induced MMP-1 gene expression have not been defined. To address the relationship between the NF-kappaB pathway and MMP-1 gene activation in chondrocytes, primary cultured human articular chondrocyte cultures (HAC) and SW-1353 cells were stimulated with IL-1beta over a 24-h time course and MMP-1, NF-kappaB1, NF-kappaB2 and RelA gene expression was assayed. IL-1beta-induced MMP-1 expression was comparable in HAC and SW-1353 cells both temporally and quantitatively. MMP-1 gene expression was mirrored by increases in NF-kappaB gene expression, and inhibition of NF-kappaB nuclear translocation with dominant-negative IkappaBalpha reduced IL-1beta-dependent MMP-1 gene expression. IL-1beta activated the NF-kappaB pathway in chondrocytes, both through phosphorylation and transient degradation of IkappaBalpha, as well as through sustained phosphorylation of RelA. Small inhibitory RNAs (siRNA) specific for RelA resulted in significant reduction of MMP-1 mRNA, whereas siRNA for NF-kappaB1 and NF-kappaB2 augmented IL-1beta-induced MMP-1 expression. Our data demonstrate that IL-1beta activation of the NF-kappaB pathway is required for IL-1beta induction of MMP-1 in chondrocytes and that RelA can work independently of NF-kappaB1 or NF-kappaB2 to activate this gene expression program.

CCAAT/enhancer-binding protein (C/EBP) beta is acetylated at multiple lysines - Acetylation of C/EBP beta at lysine 39 modulates its ability to activate transcription

Article

Full-text available

Feb 2007

Transcription factor function can be modulated by post-translational modifications. Because the transcription factor CCAAT/enhancer-binding protein (C/EBP) β associates with the nuclear coactivator p300, which contains acetyltransferase activity, acetylation of C/EBPβ was examined to understand its regulation and function. C/EBPβ is acetylated by acetyltransferases p300 and p300/CREB-binding protein associated factor. Endogenous C/EBPβ in 3T3-F442A preadipocytes is also recognized by an acetyl-lysine-specific antibody. Analysis of truncations of C/EBPβ and peptides based on C/EBPβ sequences identified multiple lysines within C/EBPβ that can be acetylated. Among these, a novel acetylation site at lysine 39 of C/EBPβ was identified. Mutation of Lys-39 to arginine or alanine impairs its acetylation and the ability of C/EBPβ to activate transcription at the promoters for C/EBPα and c-fos. Different C/EBPβ-responsive promoters require different patterns of acetylated lysines in C/EBPβ for transcription activation. Furthermore, C/EBPβ acetylation was increased by growth hormone, and mutation of Lys-39 impaired growth hormone-stimulated c-fos promoter activation. These data suggest that acetylation of Lys-39 of C/EBPβ, alone or in combination with acetylation at other lysines, may play a role in C/EBPβ-mediated transcriptional activation.

Transcription factors NF-IL6 and NF-??B synergistically activate transcription of the inflammatory cytokines, interleukin 6 and interleukin 8

Article

Full-text available

Dec 1993

Single binding sites for transcription factors NF-IL6 and NF-kappa B are present in the promoter of the interleukin (IL) 6 gene. Previous studies of internally deleted promoter mutants demonstrated that these two sites are important for the transcriptional regulation of this gene. In this report, we describe the synergistic activation of the IL-6 promoter by transcription factors NF-IL6 and NF-kappa B. Cotransfection of NF-IL6 with the NF-kappa B p65 subunit resulted in strong synergistic activation of an IL-6 promoter-reporter construct. Both the NF-IL6 and NF-kappa B binding sites in the IL-6 promoter were required for synergistic activation. Similar synergistic activation was observed in the IL-8 promoter, which also contains both NF-IL6 and NF-kappa B binding sites. Furthermore, we demonstrated that NF-IL6 and the NF-kappa B p65 subunit directly associated via the basic leucine-zipper domain of NF-IL6 and the Rel homology domain of p65. Since the promoters of many other genes involved in the inflammatory and acute-phase responses also contain binding sites for NF-IL6 and NF-kappa B, the cooperation between these two factors may have an important role in these responses. We also discuss the possible interplay between various viral gene products and these two factors in the process of viral infection and constitutive cytokine production.

A Single Nucleotide Polymorphism in the Matrix Metalloproteinase-1 Promoter Creates an Ets Binding Site and Augments Transcription1

Article

Full-text available

Jan 1999

Matrix metalloproteinases (MMPs) facilitate cellular invasion by degrading the extracellular matrix, and their regulation is partially dependent on transcription. Binding sites for members of the Ets family of transcription factors are present within MMP promoters and are potent positive regulators. We report a single nucleotide polymorphism at -1607 bp in the MMP-1 promoter, where an additional guanine (G) creates an Ets binding site, 5'-GGA-3'. This polymorphism displays significantly higher transcription in normal fibroblasts and in melanoma cells than the 1 G polymorphism, and it binds substantially more nuclear extract and recombinant ETS-1. Analysis of control DNAs from the Center d'Etude du Polymorphisme Humain pedigrees reveals that this polymorphism is not a mutation, with a frequency of the 2 G polymorphism at 30%. In contrast, in eight tumor cell lines, this frequency increased to 62.5% (P < 0.0001). Thus, this MMP-1 polymorphism contributes to increased transcription, and cells expressing the 2 G polymorphism may provide a mechanism for more aggressive matrix degradation, thereby facilitating cancer progression.

ERK1 and ERK2 Activate CCAAAT/Enhancer-binding Protein-beta -dependent Gene Transcription in Response to Interferon-gamma

Article

Full-text available

Feb 2001

Interferons (IFNs) regulate the expression of a number of cellular genes by activating the JAK-STAT pathway. We have recently discovered that CCAAAT/enhancer-binding protein-beta (C/EBP-beta) induces gene transcription through a novel IFN response element called the gamma-IFN-activated transcriptional element (Roy, S. K., Wachira, S. J., Weihua, X., Hu, J., and Kalvakolanu, D. V. (2000) J. Biol. Chem. 275, 12626-12632. Here, we describe a new IFN-gamma-stimulated pathway that operates C/EBP-beta-regulated gene expression independent of JAK1. We show that ERKs are activated by IFN-gamma to stimulate C/EBP-beta-dependent expression. Sustained ERK activation directly correlated with C/EBP-beta-dependent gene expression in response to IFN-gamma. Mutant MKK1, its inhibitors, and mutant ERK suppressed IFN-gamma-stimulated gene induction through the gamma-IFN-activated transcriptional element. Ras and Raf activation was not required for this process. Furthermore, Raf-1 phosphorylation negatively correlated with its activity. Interestingly, C/EBP-beta-induced gene expression required STAT1, but not JAK1. A C/EBP-beta mutant lacking the ERK phosphorylation site failed to promote IFN-stimulated gene expression. Thus, our data link C/EBP-beta to IFN-gamma signaling through ERKs.

ERK2- and p90Rsk2-dependent Pathways Regulate the CCAAT/Enhancer-binding Protein- Interaction with Serum Response Factor

Article

Full-text available

Nov 2001

The serum response element (SRE) of the c-fos promoter is a convergence point for mitogenic signaling pathways. Several transcription factors regulate SRE, including serum response factor (SRF), ternary complex factors, and CCAAT/enhancer-binding protein-β (C/EBPβ). C/EBPβ can interact with both SRF and the ternary complex factor family member Elk-1, but only in response to activated Ras. Transactivation of the SRE by C/EBPβ is also greatly stimulated by Ras. The Ras effectors that signal to C/EBPβ are unknown. In this report, we demonstrate that a consensus MAPK site in C/EBPβ is necessary for Ras stimulation of both C/EBPβ-SRF interaction and transactivation of the SRE by C/EBPβ. To dissect signaling pathways activated downstream of Ras, different Ras effector constructs were analyzed. We show that activated forms of Raf and phosphatidylinositol 3-kinase stimulate C/EBPβ-SRF interaction. We also show a novel selectivity for the MAPK family member ERK2, where dominant-negative ERK2, but not dominant-negative ERK1, blocks Ras stimulation of C/EBPβ-SRF interaction. In addition, recombinant C/EBPβ protein is phosphorylated by ERK2, but not by ERK1, in vitro. Finally, we demonstrate a requirement for p90Rsk2 in regulation of C/EBPβ-SRF interaction. These data show that multiple Ras effectors are required to regulate C/EBPβ and SRF association.

The Induction of Cyclooxygenase-2 mRNA in Macrophages Is Biphasic and Requires both CCAAT Enhancer-binding protein β (C/EBPβ) and C/EBPδ Transcription Factors

Article

Full-text available

Jan 2002

Prostaglandins are important mediators of activated macrophage functions, and their inducible synthesis is mediated by cyclooxygenase-2 (COX-2). Here, we make use of the murine macrophage cells RAW264 as well as of immortalized macrophages derived from mice deficient for the transcription factor CCAAT enhancer-binding protein β (C/EBPβ) to explore the molecular mechanisms regulating COX-2 induction in activated macrophages. We demonstrate that lipopolysaccharide-mediated COX-2 mRNA induction is biphasic. The initial phase is independent of de novo protein synthesis, correlates with cAMP-response element-binding protein (CREB) activation, is inhibited by treatments that abolish CREB phosphorylation and reduce NF-κB-mediated gene activation, and requires the presence of the transcription factor C/EBPβ. On the other hand, C/EBPδ appears to be essential in addition to C/EBPβ to effect the second phase of COX-2 gene transcription, which is important for maintaining the induced state and requires de novo protein synthesis. Indeed, both phases of COX-2 induction were defective in C/EBPβ−/− macrophages. Moreover, the synthesis of C/EBPδ was increased dramatically by treatment with lipopolysaccharide and, like COX-2 induction, repressed by combined inhibition of the MAPK and of the SAPK2/p38 cascades. Taken together, these data identify CREB, NF-κB, and both C/EBPβ and -δ as key factors in coordinately orchestrating transcription from the COX-2 promoter in activated macrophages.

Transcriptional regulation of collagenase (MMP-1, MMP-13) genes in arthritis: Integration of complex signaling pathways for the recruitment of gene-specific transcription factors

Article

Full-text available

Feb 2002
Arthritis Res

Matrix metalloproteinase (MMP)-1, MMP-8 and MMP-13 are interstitial collagenases that degrade type II collagen in cartilage; this is a committed step in the progression of rheumatoid arthritis and osteoarthritis. Of these enzymes, the expression of MMP-1 and MMP-13 is substantially increased in response to IL-1 and tumor necrosis factor-alpha, and elevated levels of these collagenases are observed in arthritic tissues. Therefore, cytokine-mediated MMP-1 and MMP-13 gene regulation is an important issue in arthritis research. In this review, we discuss current models of MMP-1 and MMP-13 transcriptional regulation, with a focus on signaling intermediates and transcription factors that may be future targets for the development of new arthritis drugs.

CCAAT/Enhancer-binding Proteins beta and delta Mediate the Repression of Gene Transcription of Cartilage-derived Retinoic Acid-sensitive Protein Induced by Interleukin-1beta

Article

Full-text available

Sep 2002

Cartilage-derived retinoic acid-sensitive protein (CD-RAP) is a secreted protein expressed by chondrocytes; the expression is repressed by interleukin 1 beta (IL-1 beta). To investigate the transcriptional mechanism, by which CD-RAP expression is suppressed by IL-1 beta, deletion constructs of the mouse CD-RAP promoter were transfected into rat chondrocytes treated with or without IL-1 beta. The results revealed an IL-1 beta-responsive element located between -2138 and -2068 bp. As this element contains a CAAT/enhancer-binding protein (C/EBP) motif, the function of C/EBP beta and C/EBP delta was examined. IL-1 beta stimulated the expression of C/EBP beta and -delta, and the direct binding of C/EBP beta to the C/EBP motif was confirmed. The -2251-bp CD-RAP promoter activity was down-regulated by co-transfection with C/EBP expression vectors. Mutation of the C/EBP motif abolished the inhibitory response to IL-1 beta. Additionally, C/EBP expression vectors were found to down-regulate the construct containing the promoter and enhancer of the type II collagen gene. Finally, the enhancer factor, Sox9, was shown to bind adjacent to the C/EBP site competing with C/EBP binding. Taken together, these results suggest that C/EBP beta and -delta may play an important role in the IL-1 beta-induced repression of cartilage-specific proteins and that expression of matrix proteins will be influenced by the availability of positive and negative trans-acting factors.

Matrix metalloproteinases in arthritic disease

Article

Full-text available

Feb 2002
Arthritis Res

Chapter summary The role of matrix metalloproteinases in the degradative events invoked in the cartilage and bone of arthritic joints has long been appreciated and attempts at the development of proteinase inhibitors as potential therapeutic agents have been made. However, the spectrum of these enzymes orchestrating connective tissue turnover and general biology is much larger than anticipated. Biochemical studies of the individual members of the matrix metalloproteinase family are now underway, ultimately leading to a more detailed understanding of the function of their domain structures and to defining their specific role in cellular systems and the way that they are regulated. Coupled with a more comprehensive and detailed study of proteinase expression in different cells of joint tissues during the progress of arthritic diseases, it will be possible for the future development and application of highly specific proteinase inhibitors to be directed at specific key cellular events.

Dual Regulation of Phosphorylation and Dephosphorylation of C/EBPβ Modulate Its Transcriptional Activation and DNA Binding in Response to Growth Hormone

Article

Full-text available

Dec 2002

The phosphorylation state of transcription factors is a critical determinant of their function. C/EBPbeta occurs in cells as the transcriptional activator liver-enriched activating protein (LAP) and in the truncated form liver-enriched inhibitory protein (LIP) that inhibits transcription. Analysis of C/EBPbeta phosphorylation by isoelectric focusing (IEF) shows that LAP is present in multiple forms, each with a different degree of phosphorylation in 3T3-F442A fibroblasts. Growth hormone (GH) treatment induces a new band near the negative pole, consistent with GH-promoted dephosphorylation of LAP. In addition, bands near the positive pole are rapidly and transiently induced, suggesting that GH also stimulates phosphorylation at some site(s) on LAP. C/EBPbeta contains a highly conserved MAPK consensus site that corresponds to Thr(188) in murine (m) LAP and Thr(37) in mLIP. Immunoblotting with antiphosphopeptide antibodies specific for Thr(188/37) of C/EBPbeta (anti-P-C/EBPbeta) shows that GH rapidly and transiently promotes phosphorylation of mLAP and mLIP on the MAPK site. MEK inhibitors prevent this GH-promoted phosphorylation of LAP and LIP, suggesting that such phosphorylation depends on GH-activated MAPK signaling. Mutation of Thr(235) to Ala in the homologous MAPK site of human (h) LAP (hLAPT235A) inhibits transcription mediated by the c-fos promoter in response to GH, indicating that phosphorylation at the MAPK site is required for LAP to be transcriptionally active in the context of GH-stimulated activation of the c-fos promoter. Complexes bound to the c-fos C/EBP site transiently contain C/EBPbeta phosphorylated at the MAPK site. As phosphorylation subsides, the binding of less transcriptionally active forms of LAP increases, consistent with the transient nature of c-fos stimulation by GH and other growth factors. Thus, both phosphorylation and dephosphorylation of C/EBPbeta, in response to a single physiological stimulus such as GH, coordinately modulate the ability of C/EBPbeta to activate transcription by modulating its DNA binding activity and its transactivation capacity.

The triterpenoid CDDO inhibits expression of matrix metalloproteinase-1, matrix metalloproteinase-13 and Bcl-3 in primary human chondrocytes

Article

Full-text available

Feb 2003
ARTHRITIS RES THER

A synthetic triterpenoid, 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid (CDDO), has been reported to have anti-inflammatory properties and to decrease the interleukin-1 (IL-1)-induced expression of matrix metalloproteinase-1 (MMP-1) and MMP-13. We have shown previously that IL-1 induces expression of the inhibitor of NF-kappaB (IkappaB) family member Bcl-3, and that this contributes to MMP-1 expression. To quantify the effects of CDDO on IL-1-induced MMP-1, MMP-13 and Bcl-3 expression, we stimulated the chondrosarcoma cell line SW-1353 and human primary chondrocytes with IL-1, in the presence or absence of CDDO. Harvested RNA was subjected to quantitative real-time reverse-transcriptase polymerase chain reaction. In SW-1353 cells, 300 nM CDDO significantly decreased the induction of MMP-1 and MMP-13 by IL-1. In human primary chondrocytes, 300 nM CDDO inhibited the induction of these genes by IL-1 to an even greater extent. In both cell types, inhibition of MMP-1 required 24 hours of pretreatment with CDDO, whereas MMP-13 could be inhibited when CDDO and IL-1 were added simultaneously to culture. In human primary chondrocytes, IL-1-induced Bcl-3 expression was inhibited when cells were pretreated with CDDO. To determine whether the inhibitory effect of CDDO on MMP worked through inhibition of Bcl-3 gene expression, SW-1353 cells stably transfected with a Bcl-3 expression plasmid were treated with IL-1 and/or CDDO, and MMP gene expression was assayed. Overexpression of Bcl-3 increased MMP-1, but not MMP-13, mRNA levels. Furthermore, overexpressed Bcl-3 could sustain the CDDO-dependent inhibition of IL-1-induced MMP-1 expression. Our data demonstrate that CDDO inhibits IL-1-induced MMP-1 and MMP-13 expression in human chondrocytes. CDDO also inhibits the expression of Bcl-3, an IL-1-responsive gene that preferentially contributes to MMP-1 gene expression.

Cell cycle-dependent phosphorylation of C/EBPbeta mediates oncogenic cooperativity between C/EBPbeta and H-RasV12

Article

Full-text available

Oct 2004

CCAAT/enhancer binding protein β (C/EBPβ) is a widely expressed transcription factor whose activity is regulated by oncogenic Ha-RasV12 signaling. C/EBPβ is essential for the development of mouse skin tumors containing Ras mutations and can cooperate with RasV12 to transform NIH 3T3 cells. Here we have investigated Ras-induced phosphorylation of C/EBPβ in fibroblasts and report a novel proline-directed phosphoacceptor site at Ser64 within the transactivation domain. Ser64 phosphorylation was induced by activated Ras and Raf but was not blocked by chemical inhibitors of MEK1/2, phosphatidylinositol 3-kinase, JNK, or p38 mitogen-activated protein kinases. Ser64 was efficiently phosphorylated in vitro by the cyclin-dependent kinases Cdk2 and Cdc2. Thr189, previously identified as an ERK1/2 phosphorylation site that regulates C/EBPβ activity, was also a substrate for Cdk phosphorylation. Ser64 and Thr189 phosphorylation was low in serum-starved (G0) cells but was strongly increased in mid-G1 cells and in cells arrested in S or M phase. In addition, phosphorylation on both sites was blocked by treating cells with the Cdk inhibitor roscovitine. In contrast to wild-type C/EBPβ, which enhances transformation of NIH 3T3 cells, mutants bearing alanine substitutions at Ser64 and/or Thr189 inhibited RasV12-induced focus formation. Our findings support a role for C/EBPβ as a nuclear effector of Ras signaling and transformation, and they indicate that cell cycle-dependent phosphorylation of C/EBPβ on Ser64 and Thr189 is required to promote Ras-induced transformation of NIH 3T3 cells.

Matrix metalloproteinase-1 up-regulation by hepatocyte growth factor in human dermal fibroblasts via ERK signaling pathway involves Ets1 and Fli1

Article

Full-text available

Feb 2005
NUCLEIC ACIDS RES

In this study, we clarified the molecular mechanism(s) underlying the regulation of matrix metalloproteinase (MMP)-1 gene by hepatocyte growth factor (HGF) in cultured human dermal fibroblasts. HGF induced MMP-1 protein as well as mRNA at a transcriptional level via extracellular signal-regulated kinase (ERK) signaling pathway. The region in the MMP-1 promoter mediating the inducible responsiveness to HGF, defined by the transient transfection analysis of the serial 5′ deletion constructs, contained an Ets binding site. Mutation of this Ets binding site abrogated the HGF-inducible promoter activity. Ets1 up-regulated the expression of MMP-1 promoter activity, whereas Fli1 had antagonistic effects on them. After HGF treatment, the protein level and the binding activity of Ets1 was increased and those of Fli1 was decreased, which were canceled by PD98059. These results suggest that HGF up-regulates MMP-1 expression via ERK signaling pathway through the balance of Ets1 and Fli1, which may be a novel mechanism of regulating MMP-1 gene expression.

Cell-type specific regulation of human interstitial collagenase-1 gene expression by interleukin-1β (IL-1β) in human fibroblasts and BC-8701 breast cancer cells

Article

Sep 1997
J CELL BIOCHEM

Interleukin-1β (IL-1β) is a potent cytokine that stimulates interstitial collagenase-1 (matrix metalloproteinase-1; MMP-1). In this study, we compared the mechanism(s) by which IL-1β induces collagenase gene expression in two very different cells, normal human foreskin fibroblasts (HFFs) and an aggressive breast cancer cell line, BC-8701 cells. Northern analysis showed that the time course of collagenase induction was distinct in the two cells: although both cells expressed low levels of MMP-1 constitutively, addition of IL-1β increased MMP-1 mRNA in HFFs by 1 h and levels remained high over a 24-h period. In contrast, MMP-1 levels in IL-1β-treated BC-8701 cells did not increase until 4 h, peaked by 12 h and then declined. To analyze the transcriptional response, we cloned and sequenced more than 4,300 bp of the human MMP-1 promoter, and from this promoter clone, we prepared a series of 5′-deletion constructs linked to the luciferase reporter and transiently transfected these constructs into both cell types to measure both basal and IL-1β induced transcription. When both cell types were uninduced, promoter fragments containing less than 2,900 bp gave only a minimal transcriptional response, while larger fragments showed increased transcriptional activity. With IL-1β treatment, significant responsiveness (P < 0.001) in HFFs was seen only with the larger fragments, while in the BC-8701 cells, all fragments were significantly induced with IL-1β. Finally, we found that IL-1β stabilized MMP-1 mRNA in normal fibroblasts, but not in BC-8701 breast cancer cells. We conclude that both the transcriptional and post-transcriptional regulation of MMP-1 gene expression by IL-1β is controlled by cell-type specific mechanisms, and we suggest that IL-1 induced MMP-1 expression in tumor cells and in neighboring stromal cells may amplify the invasive ability of tumor cells. J. Cell. Biochem. 66:322–336, 1977. © 1997 Wiley-Liss, Inc.

Phosphorylation of Rat Serine 105 or Mouse Threonine 217 in C/EBPβ Is Required for Hepatocyte Proliferation Induced by TGFα

Article

Dec 1999
MOL CELL

We report that TGFα induces activation of the p90 ribosomal S kinase (RSK), which results in the phosphorylation of rat C/EBPβ on Ser-105 and of mouse C/EBPβ on Thr-217 and concomitantly stimulates proliferation in differentiated hepatocytes. Moreover, C/EBPβ−/− mouse hepatocytes respond to TGFα when wild-type C/EBPβ is reexpressed, whereas they remain refractory to the growth effect of TGFα when expressing phosphoacceptor mutants rat C/EBPβ Ala-105 or mouse C/EBPβ Ala-217. In contrast, C/EBPβ−/− hepatocytes expressing the phosphorylation mimic mutants, rat C/EBPβ Asp-105 or mouse C/EBPβ Glu-217, exhibited marked proliferation in the absence of TGFα. Thus, a site-specific phosphorylation of the transcription factor C/EBPβ is critical for hepatocyte proliferation induced by TGFα and other stimuli that activate RSK.

Cell-Type Specific Regulation of Human Interstitial Collagenase-1 Gene Expression by Interleukin-1?? (IL-1??) in Human Fibroblasts and BC-8701 breast cancer cells

Article

Sep 1997
J CELL BIOCHEM

Critical role of C/EBP?? and C/EBP?? factors in the stimulation of the cyclooxygenase-2 gene transcription by interleukin-1?? in articular chondrocytes

Article

Aug 2003
Eur J Biochem

The activity of the [−831; +103] promoter of the human cyclooxygenase-2 gene in cultured rabbit chondrocytes is stimulated 2.9 ± 0.3-fold by interleukin-1β and this stimulation depends on [−132; −124] C/EBP binding-and [−223; −214] NF-κB binding-sites. The C/EBPβ and C/EBPδ factors bind to the [−132; −124] sequence. The [−61; −53] sequence is also recognized by C/EBPβ and C/EBPδ as well as USF. Mutation of the whole [−61; −53] sequence abolished the stimulation of transcription but single mutations of the C/EBP or USF site did not alter the activity of the promoter, suggesting that the factors bound to the proximal [−61; −53] sequence interact with different members of the general transcription machinery. The [−223; −214] site binds only the p50/p50 homodimer and a non-rel-related protein, but not the transcriptionally active heterodimer p50/p65. The p50/p50 homodimer could interact with the C/EBP family members bound to the [−132; −124] sequence for full stimulation of the COX-2 transcription by interleukin-1β in chondrocytes. By contrast, the [−448; −449] sequence binds with a low affinity both the p50/p50 homodimeric and p50/p65 heterodimeric forms of NF-κB but has no role in the regulation of the human COX-2 promoter in chondrocytes.

Synergistic transcriptional activation of the IL-8 gene by NF-κB p65 (ReIA) and NF-IL-6

Article

Aug 1994

Transcriptional activation of the IL-8 gene by several inflammatory mediators, including the cytokines IL-1 and TNF-alpha, is mediated through sequences located between nucleotide -94 and -71 of the IL-8 promoter. Because adjacent binding sites for the inducible transcription factors NF-kappa B and NF-IL-6 are located within this region, we examined the functional interaction of these two transcription factor families in IL-8 gene regulation. Maximal transcriptional activation by PMA in Jurkat T lymphocytes was shown to require intact binding sites for both NF-kappa B and NF-IL-6. Electrophoretic mobility shift analysis indicates that NF-IL-6, as well as other related members of this family, bind specifically to the NF-IL-6 site in the IL-8 promoter. In addition, NF-kappa B p65 (RelA), but not NF-kappa B p50 (NFKB1), binds specifically to the NF-kappa B site. When incubated together, RelA and NF-IL-6/C/EBP form a ternary complex with this region of the IL-8 promoter; this binding is dependent on intact binding sites for both NF-IL-6 and RelA. Transient cotransfection analyses indicate that the cooperative association of NF-IL-6 and RelA with the IL-8 promoter results in synergistic transcriptional activation. Mutational analyses of RelA demonstrate that the C-terminal transactivation domain and the DNA binding domain are required for synergistic activation with NF-IL-6. In addition, overexpression of the NF-kappa B inhibitor molecule, I kappa B, abolished the RelA- and RelA/NF-IL-6-dependent synergistic activation. These data demonstrate that RelA and members of the C/EBP/NF-IL-6 family can functionally cooperate in transcriptional activation of the IL-8 gene and suggest a common mechanism for inducible regulation of cytokine gene expression.

Anticytokine Therapy in Rheumatoid Arthritis

Article

Aug 1997

Only a few years ago, little was known about the role of cytokines in rheumatoid arthritis. The original view of the cytokine milieu in the inflamed joint as a melange of factors from various cells was clarified by two observations.1 First, in inflammation of rheumatoid arthritis, cytokines from macrophages and fibroblasts dominate, and second, the cytokine profile within the macrophage and fibroblast reflects a hierarchy, with interleukin-1 and tumor necrosis factor α (TNF-α) assuming particular importance. Anticytokine therapy for rheumatoid arthritis has now reached the clinic, and the report in this issue of the Journal by Moreland et al.2 adds . . .

Comparative immunolocalization studies of collagenase 1 and collagenase 3 production in the rheumatoid lesion, and by human chondrocytes and synoviocytes in vitro

Article

Feb 1998

The degradation of fibrillar type II collagen is a major feature of cartilage destruction in rheumatoid arthritis (RA). Since collagenase 3 is produced by chondrocytes and preferentially degrades type II cartilage collagen, it seemed likely that this enzyme would have a prominent role in the destruction of rheumatoid joints. Using immunolocalization techniques, we have examined and compared the production and distributions of collagenase 1 and collagenase 3 in cells and tissues derived from rheumatoid knee arthroplasties. Primary cultures of chondrocytes stimulated with interleukin-1 beta showed that most of the cells produced collagenase 1, whereas only a minority (approximately 5-10%) produced collagenase 3; a few chondrocytes demonstrated the co-ordinate production of both enzymes. Primary cultures of rheumatoid synoviocytes produced collagenase 1, but not collagenase 3. Both enzymes were demonstrated in the rheumatoid lesion. Collagenase 1 was more commonly observed in both synovium and cartilage (22 of the 28 specimens), was especially prominent at cartilage erosion sites, and most of the positive specimens demonstrated extracellular enzyme. By contrast, collagenase 3 was observed less frequently (7/28 specimens) and was produced by relatively few chondrocytes and synovial cells, this usually being much less than that observed for chondrocytes of osteoarthritic cartilage. These observations suggest different regulatory mechanisms for the production of collagenases 1 and 3 in the rheumatoid lesion, and demonstrate that the distribution and production of collagenase 1 are far more prevalent than those for collagenase 3.

Nuclear factor kappaB/p50 activates an element in the distal matrix metalloproteinase 1 promoter in interleukin-1beta-stimulated synovial fibroblasts

Article

Nov 1998

To determine how interleukin-1 (IL-1), through activation of collagenase 1 (matrix metalloproteinase 1 [MMP-1]) transcription in synovial fibroblasts, contributes to cartilage degradation in rheumatoid arthritis. Primary rabbit synovial fibroblasts were transiently transfected with MMP-1 promoter/ luciferase constructs, and promoter activity in response to IL-1 was assessed. A minimal IL-1-response element was defined and used to evaluate DNA binding proteins by electrophoretic mobility shift assay and in situ ultraviolet crosslinking assay. Transcriptional activation of the MMP-1 gene by IL-1 in rabbit synovial fibroblasts required a dorsal-like element, which was located at nucleotide (nt) -3,029, as well as an activator protein 1 site at nt -77. Importantly, an IL-1-induced DNA binding activity that was specific for the dorsal-like element contained the p50 subunit of nuclear factor kappaB (NF-kappaB). These studies demonstrate, for the first time, a role for NF-kappaB in the induction of MMP-1, and suggest a mechanism of NF-kappaB-mediated cartilage degradation in rheumatoid arthritis.

Interleukin-1 induction of collagenase 3 (matrix metalloproteinase 13) gene expression in chondrocytes requires p38, c-Jun N-terminal kinase, and nuclear factor kappa B - Differential regulation of collagenase 1 and collagenase 3

Article

Apr 2000

To examine the mechanism of interleukin-1 (IL-1)-induced collagenase 3 (matrix metalloproteinase 13 [MMP-13]) gene expression in cultured chondrocytes for the purpose of better understanding how the gene is induced in these cells, and how it contributes to cartilage degradation in osteoarthritis. The transcriptional and posttranscriptional responses of the MMP-13 gene to IL-1 were assessed first. Then, direct inhibitors of mitogen-activated protein kinase (MAPK) signaling pathways and a constitutive repressor of nuclear factor kappaB (NF-kappaB) were used to assess the role of each pathway in IL-1-mediated induction of MMP-13. We found that IL-1 induction of MMP-13 requires p38 activity, c-Jun N-terminal kinase (JNK) activity and NF-kappaB translocation. These results suggest that both NF-kappaB and activator protein 1 transcription factors are necessary for IL-1 induction of MMP-13. We also compared the signaling pathways necessary for IL-1 to stimulate collagenase 1 (MMP-1) in articular chondrocytes and chondrosarcoma cells and found that IL-1 induction of MMP-1 requires different pathways from those required by MMP-13. In chondrosarcoma cells, MMP-1 induction depends on p38 and MEK (an MAPK kinase of the extracellular signal-regulated kinase pathway) and does not require JNK or NF-kappaB. In articular chondrocytes, inhibition of MEK had no effect, while inhibition of p38 gave variable results. These studies demonstrate, for the first time, that p38, JNK, and NF-kappaB are required for IL-1 induction of MMP-13. The results also highlight the differential requirements for signaling pathways in the induction of MMP-1 and MMP-13. Additionally, they demonstrate that induction of MMP-1 by IL-1 in chondrocytic cells depends on unique combinations of signaling pathways that are cell type-specific.

Integration of the NF-κB and mitogen-activated protein kinase/AP-1 pathways at the Collagenase-1 promoter: Divergence of IL-1 and TNF-dependent signal transduction in rabbit primary synovial fibroblasts

Article

Nov 2000

Collagenase-1 (MMP-1) is a protease that is expressed by stromal cells and that is involved in remodeling of the extracellular matrix. IL-1 and TNF-alpha enhance collagenase secretion by stromal cells, and chronic exposure of cells to these cytokines can contribute to connective tissue disease. In this study, we show that the NF-kappaB pathway is required for activation of collagenase-1 transcription in rabbit primary synovial fibroblasts (RSF). Although both IL-1 and TNF activate NF-kappaB in these cells, only IL-1 induces collagenase-1 transcription. We have reported previously that NF-kappaB and AP-1 cooperate to mediate IL-1-induced MMP-1 transcription. Here, we show that IL-1 is superior to TNF at inducing c-Jun synthesis, phosphorylation and binding activity in RSF. Similarly, IL-1 is more effective at activating the mitogen-activated protein kinases (MAPK), including the extracellular signal-regulated kinases (ERK), which are required for IL-1-induced MMP-1 transcription. Thus stimulation of the ERK and AP-1 pathways is an essential component of MMP-1 transcriptional activation, which is deficient in TNF-treated cells. These studies demonstrate cooperation between the MAPK and NF-kappaB signaling pathways for IL-1-dependent collagenase-1 transcription, and they define a dichotomy of IL-1- and TNF-elicited signaling that is relevant to cytokine-mediated connective tissue disease.

Critical role of C/EBPδ and C/EBPβ factors in the stimulation of the cyclooxygenase-2 gene transcription by interleukin-1β in articular chondrocytes

Article

Jan 2001

The activity of the [-831; +103] promoter of the human cyclooxygenase-2 gene in cultured rabbit chondrocytes is stimulated 2.9 +/- 0.3-fold by interleukin-1beta and this stimulation depends on [-132; -124] C/EBP binding-and [-223; -214] NF-kappaB binding-sites. The C/EBPbeta and C/EBPdelta factors bind to the [-132; -124] sequence. The [-61; -53] sequence is also recognized by C/EBPbeta and C/EBPdelta as well as USF. Mutation of the whole [-61; -53] sequence abolished the stimulation of transcription but single mutations of the C/EBP or USF site did not alter the activity of the promoter, suggesting that the factors bound to the proximal [-61; -53] sequence interact with different members of the general transcription machinery. The [-223; -214] site binds only the p50/p50 homodimer and a non-rel-related protein, but not the transcriptionally active heterodimer p50/p65. The p50/p50 homodimer could interact with the C/EBP family members bound to the [-132; -124] sequence for full stimulation of the COX-2 transcription by interleukin-1beta in chondrocytes. By contrast, the [-448; -449] sequence binds with a low affinity both the p50/p50 homodimeric and p50/p65 heterodimeric forms of NF-kappaB but has no role in the regulation of the human COX-2 promoter in chondrocytes.

Targeting interleukin-1 in the treatment of rheumatoid arthritis

Article

Mar 2002

Tower GB, Coon CC, Benbow U, Vincenti MP, Brinckerhoff CEErk 1/2 differentially regulates the expression from the 1G/2G single nucleotide polymorphism in the MMP-1 promoter in melanoma cells. Biochim Biophys Acta 1586: 265-274

Article

May 2002
Biochim Biophys Acta

Matrix metalloproteinase-1 (MMP-1) breaks down interstitial collagens, a major component of stromal tissue and a barrier for invading tumor cells. The degradation of collagen by MMP-1 may, therefore, provide one mechanism for facilitating tumor invasion and metastasis. Because of the potential for excessive matrix degradation, the expression of MMP-1 is tightly regulated, often by the mitogen-activated protein kinase (MAPK) pathway. The MAPK signal cascade consists of three separate pathways, the extracellular response kinase (ERK), p38 and Jun N-terminal kinase, which target proteins of the AP-1 and ETS families transcription of the gene. The MMP-1 promoter contains a single nucleotide polymorphism (SNP) at -1607 bp, which creates an ETS binding site by the addition of a guanine (5'-GGAT-3' or '2G SNP') compared to the 1G SNP (5'-GAT-3'), and enhances MMP-1 transcription. A2058 melanoma cells represent one tumor cell line that is homozygous for the 2G allele and that produces constitutively high levels of MMP-1. Thus, we used these cells to define the mechanism(s) responsible for this high level of expression. We show that inhibition of ERK 1/2 leads to the repression of MMP-1 transcription, and that both the 2G polymorphism and the adjacent AP-1 site at -1602 bp are necessary for high levels of MMP-1 transcription and for the inhibition of MMP-1 expression by PD098059, a specific ERK inhibitor. Furthermore, restoration of MMP-1 levels after ERK 1/2 inhibition requires de novo protein synthesis of a factor necessary for MMP-1 expression. Thus, this study suggests that the ERK 1/2 pathway targets the 2G polymorphism, and that the continuous synthesis of a protein(s) is necessary for the constitutive expression of MMP-1.

Bcl-3 is an interleukin-1-responsive gene in chondrocytes and synovial fibroblasts that activates transcription of the matrix metalloproteinase 1 gene

Article

Dec 2002

To define the role of Bcl-3, a member of the inhibitor of nuclear factor kappaB (NF-kappaB) family and a known regulator of NF-kappaB, in interleukin-1 (IL-1)-induced matrix metalloproteinase 1 (MMP-1) transcription in chondrocytes and synovial fibroblasts. SW-1353 cells, a human chondrosarcoma cell line, were stimulated with IL-1beta, and the harvested RNA was subjected to microarray analysis and quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR). The SW-1353 cells were stimulated with IL-1 or transfected with a plasmid that constitutively expressed Bcl-3, and then MMP-1 messenger RNA (mRNA) expression was assayed by quantitative real-time RT-PCR. SW-1353 cells were transfected with antisense oligonucleotides to Bcl-3, and IL-1-induced MMP-1 mRNA expression was assayed by quantitative RT-PCR. SW-1353 cells and rabbit synovial fibroblasts were transfected with a 4.3-kb human MMP-1 promoter construct along with Bcl-3 and NF-kappaB1 expression constructs, and MMP-1 transcription was assayed. Microarray analysis and real-time RT-PCR showed Bcl-3 to be an IL-1beta-responsive gene in SW-1353 cells. Exogenous expression of Bcl-3 in SW-1353 cells activated MMP-1 transcription. Endogenous Bcl-3 expression was required for IL-1beta induction of MMP-1 gene expression. Bcl-3 also activated MMP-1 transcription in primary synovial fibroblasts. We showed previously that NF-kappaB1 contributes to IL-1beta induction of MMP-1 transcription in stromal cells. We showed here that Bcl-3 can cooperate with NF-kappaB1 to activate MMP-1 transcription in SW-1353 cells. These data define a new role for Bcl-3 in joint cells as an IL-1beta-responsive early gene involved in cell-mediated cartilage remodeling. Our findings implicate Bcl-3 as an important contributor to chronic inflammatory disease states, such as osteoarthritis and rheumatoid arthritis.

Signal Transduction in the Liver: C/Ebpβ Modulates Cell Proliferation and Survival

Article

May 2003

Regulation of elastin gene transcription by interleukin-1 -induced C/EBP isoforms

Article

Jan 2004

We previously showed that interleukin (IL)-1beta decreases elastin gene transcription through activation of the NF-kappaB subunit p65 in neonatal rat lung fibroblasts. The present study was undertaken to further explore the molecular mechanisms responsible for the inhibitory effect of IL-1beta on elastin gene transcription. We found that cycloheximide blocked IL-1beta-induced downregulation of elastin mRNA but did not inhibit IL-1beta-induced translocation of p65 into the nucleus. IL-1beta treatment increased CCAAT/enhancer-binding protein (C/EBP)beta mRNA and protein levels including liver-enriched activating protein (LAP) and liver-enriched inhibitory protein (LIP), which was cycloheximide sensitive. C/EBPbeta isoforms bound a GCAAT-containing sequence in the proximal elastin promoter as determined by electrophoretic gel shift studies and confirmed by using specific anti-C/EBPbeta antibodies and by competition studies with oligonucleotides. Transient transfection of LIP expression vectors strongly decreased the transcriptional activity of the cotransfected elastin promoter and decreased levels of endogenous elastin mRNA. We demonstrated that IL-1beta-induced downregulation of elastin mRNA is dependent on NF-kappaB activation and C/EBPbeta expression. These results indicate that IL-1beta treatment activates NF-kappaB, which subsequently induces LIP expression and inhibition of elastin gene transcription.

In Situ Analysis of Interleukin-1-Induced Transcription of cox-2 and il-8 in Cultured Human Myometrial Cells

Article

Apr 2004

The specific binding of transcription factors to DNA has been shown to be inhibited by chromatin structure and increased by cooperative interactions with other proteins. Consequently, in situ analysis using chromatin immunoprecipitation offers the most accurate view of transcriptional control. Transient transfection studies and in vitro analyses of IL-1-induced cox-2 transcription in a number of cell types have indicated regulation by either nuclear factor kappa B (NF-kappa B) or CCAAT/enhancer binding protein (C/EBP beta), or both acting cooperatively. To determine the mechanisms of COX-2 (cyclooxygenase or prostaglandin endoperoxide synthase) induction in cultured human myometrial cells in situ, we examined the cross-linking of the RelA subunit of NF-kappa B and C/EBP beta to the cox-2 promoter and flanking sequences. As a control, we inspected the interaction of these transcription factors with the IL-8 gene, which has been shown in other cell types to be activated by the cooperative interaction of NF-kappa B and C/EBP beta. Indeed, both transcription factors were cross-linked to the il-8 promoter after IL-1 treatment, but only RelA was cross-linked to cox-2 DNA. The il-8 promoter was also found to physically interact with proteins cross-linked to sites further upstream. IL-1 treatment also increased polymerase II cross-linking to both promoters and increased histone H4 acetylation at specific sites. These results indicate that modification of chromatin structure is part of the response to IL-1 stimulation. Chromatin immunoprecipitation thus provides critical insight into the mechanisms of COX-2 and IL-8 expression in human myometrial cells.

Phosphorylation of C/EBP at a Consensus Extracellular Signal-Regulated Kinase/Glycogen Synthase Kinase 3 Site Is Required for the Induction of Adiponectin Gene Expression during the Differentiation of Mouse Fibroblasts into Adipocytes

Article

Nov 2004

Stimulation of adipogenesis in mouse preadipocytes requires C/EBPbeta as well as activation of the MEK/extracellular signal-regulated kinase (ERK) signaling pathway. In this study, we demonstrate that phosphorylation of C/EBPbeta at a consensus ERK/glycogen synthase kinase 3 (GSK3) site regulates adiponectin gene expression during the C/EBPbeta-facilitated differentiation of mouse fibroblasts into adipocytes. First, we show that exposure of 3T3-L1 preadipocytes to insulin, dexamethasone (DEX), and isobutylmethylxanthine (MIX) leads to the phosphorylation of C/EBPbeta at threonine 188. Pretreating the cells with a MEK1-specific inhibitor (U0126) significantly attenuates this activity. Similarly, these effectors activate the phosphorylation of T188 within an ectopic C/EBPbeta overexpressed in Swiss mouse fibroblasts, and this event involves both MEK1 and GSK3 activity. We further show that expression of C/EBPbeta (p34kD LAP isoform) in Swiss mouse fibroblasts exposed to DEX, MIX, and insulin induces expression of peroxisome proliferator-activated receptor gamma (PPARgamma) and some adiponectin but that it does not activate expression of FABP4/aP2. In fact, complete conversion of these fibroblasts into lipid-laden adipocytes, which includes activation of FABP4 and adiponectin expression, requires their exposure to a potent PPARgamma ligand such as troglitazone. Expression of a mutant C/EBPbeta in which threonine 188 has been modified to alanine (C/EBPbeta T188A) can induce PPARgamma production in the mouse fibroblasts, but it is incapable of stimulating adiponectin expression in the absence or presence of troglitazone. Interestingly, replacement of T188 with aspartic acid creates a C/EBPbeta molecule (C/EBPbeta T188D) that possesses adipogenic activity similar to that of the wild-type molecule. The absence of adiponectin expression correlates with a reduced amount of C/EBPalpha in the adipocytes expressing the T188A mutant suggesting that C/EBPalpha is required for expression of adiponectin. In fact, ectopic expression of PPARgamma in C/EBPalpha-deficient fibroblasts (NIH 3T3 cells) produces a modest amount of adiponectin, whereas expression of both PPARgamma and C/EBPalpha in NIH 3T3 cells facilitates production of abundant quantities of adiponectin. These data demonstrate that phosphorylation of C/EBPbeta at a consensus ERK/GSK3 site is required for both C/EBPalpha and adiponectin gene expression during the differentiation of mouse fibroblasts into adipocytes.

CCAAT/Enhancer-Binding Protein Isoforms and the Regulation of -Smooth Muscle Actin Gene Expression by IL-1

Article

Nov 2004

The role of IL-1beta in inflammation is amply documented, but its ability to inhibit myofibroblast differentiation and, in particular, the suppression of alpha-smooth muscle actin (alpha-SMA) gene expression is less well understood. Because IL-1beta can induce C/EBPbeta expression, the role of C/EBPbeta isoforms in IL-1beta regulation of alpha-SMA gene expression was investigated in rat lung myofibroblasts. The results showed that IL-1beta inhibited alpha-SMA expression in a dose-dependent manner, which was associated with stimulation of the expression of both C/EBPbeta isoforms, liver-enriched activating protein (LAP) and liver-enriched inhibitory protein (LIP). However, a greater increase in LIP relative to LAP expression resulted in a reduced LAP/LIP ratio after IL-1beta treatment. Transfection with an LAP-expressing plasmid stimulated, whereas an LIP-expressing plasmid inhibited, alpha-SMA expression. Cells from C/EBPbeta-deficient mice had reduced levels of alpha-SMA expression and promoter activity, which failed to respond to IL-1beta treatment. Sequence analysis identified the presence of a C/EBPbeta consensus binding sequence in the alpha-SMA promoter, which, when mutated, resulted in diminished promoter activity and abolished its responsiveness to IL-1beta treatment. EMSA revealed binding of C/EBPbeta to this C/EBPbeta consensus binding sequence from the alpha-SMA promoter. Finally, IL-1beta enhanced the expression of eukaryotic initiation factor 4E, a stimulator of LIP expression, which may account for a mechanism by which IL-1beta could alter the LAP/LIP ratio. These data taken together suggest that C/EBPbeta isoforms regulate alpha-SMA gene expression, and that its inhibition by IL-1beta was due to preferential stimulation of LIP expression.

Interleukin-1 beta induction of matrix metalloproteinase-1 transcription in chondrocytes requires ERK-dependent activation of CCAAT enhancer-binding protein-beta

Abstract

No full-text available