Production Of Matrix Metalloproteases Research Articles

L-Wnk1 Deletion in Smooth Muscle Cells Causes Aortitis and Inflammatory Shift.

The long isoform of the Wnk1 (with-no-lysine [K] kinase 1) is a ubiquitous serine/threonine kinase, but its role in vascular smooth muscle cells (VSMCs) pathophysiology remains unknown. AngII (angiotensin II) was infused in Apoe-/- to induce experimental aortic aneurysm. Mice carrying an Sm22-Cre allele were cross-bred with mice carrying a floxed Wnk1 allele to specifically investigate the functional role of Wnk1 in VSMCs. Single-cell RNA-sequencing of the aneurysmal abdominal aorta from AngII-infused Apoe-/- mice revealed that VSMCs that did not express Wnk1 showed lower expression of contractile phenotype markers and increased inflammatory activity. Interestingly, WNK1 gene expression in VSMCs was decreased in human abdominal aortic aneurysm. Wnk1-deficient VSMCs lost their contractile function and exhibited a proinflammatory phenotype, characterized by the production of matrix metalloproteases, as well as cytokines and chemokines, which contributed to local accumulation of inflammatory macrophages, Ly6Chi monocytes, and γδ T cells. Sm22Cre+Wnk1lox/lox mice spontaneously developed aortitis in the infrarenal abdominal aorta, which extended to the thoracic area over time without any negative effect on long-term survival. AngII infusion in Sm22Cre+Wnk1lox/lox mice aggravated the aortic disease, with the formation of lethal abdominal aortic aneurysms. Pharmacological blockade of γδ T-cell recruitment using neutralizing anti-CXCL9 antibody treatment, or of monocyte/macrophage using Ki20227, a selective inhibitor of CSF1 receptor, attenuated aortitis. Wnk1 deletion in VSMCs led to aortic wall remodeling with destruction of elastin layers, increased collagen content, and enhanced local TGF-β (transforming growth factor-beta) 1 expression. Finally, in vivo TGF-β blockade using neutralizing anti-TGF-β antibody promoted saccular aneurysm formation and aorta rupture in Sm22 Cre+ Wnk1lox/lox mice but not in control animals. Wnk1 is a key regulator of VSMC function. Wnk1 deletion promotes VSMC phenotype switch toward a pathogenic proinflammatory phenotype, orchestrating deleterious vascular remodeling and spontaneous severe aortitis in mice.

The Epidermal Environment’s Influence on the Dermal Environment in Response to External Stress

Introduction: The outermost layer of the skin, the epidermis, is directly exposed to external stress (e.g., irradiation, allergens, and chemicals). Changes in epidermal conditions/environment in response to this stress could also influence conditions of the dermis, located directly beneath the epidermis. Yet, whether/how any epidermal environment changes in response to external stress affect dermal functions has not been completely clarified. Methods: We employed ultraviolet irradiation B (UVB) (which hardly reaches the dermis) as a model of external stress. Human keratinocytes and human dermal fibroblasts were treated with UVB and conditioned medium of keratinocytes exposed to UVB (UVB-keratinocyte-M), respectively. We assessed (1) inflammatory cytokines and lipid mediators in keratinocytes; (2) matrix metalloprotease (MMP) levels and collagen degradation in fibroblasts; (3) ex vivo organ-cultured human skin was treated with UVB. MMP levels and collagen degradation were examined; (4) test whether the mixture of agent (agent cocktail) consisting of dihydroceramide, niacin amide, resveratrol, glucosyl hesperidin, and phytosterol ester that has been shown to improve skin barrier integrity can mitigate influence of UVB in skin; and (5) a pilot one-arm human clinical test to assess efficacy of formulation containing agent cocktail on stratum corneum hydration, skin elasticity, and wrinkle index. Results: Inflammatory-cytokine and -lipid mediator production were increased in cultured keratinocytes treated with UVB, while matrix MMP-1, -3, and -9 production and collagen degradation were increased in fibroblasts incubated with UVB-keratinocyte-M. mRNA expression of COL1A1 (that codes type 1 collagen) levels was decreased in fibroblasts incubated with UVB-keratinocyte-M. The study using ex vivo organ-cultured human skin showed both MMP-1 and MMP-9 expression were increased in both epidermis and dermis and increased dermal collagen degradation following UVB irradiation. Increased MMP production and collagen degradation were attenuated by application of an agent cocktail. Finally, a pilot clinical study demonstrated that the formulation containing our agent cocktail likely has the ability to improve skin hydration, increase skin elasticity, and reduce the appearance of wrinkles. Conclusion: Epidermal changes in epidermal environment and conditions in response to external stress affect dermal conditions, and these negative effects of external stress on various skin layers can be pharmacologically mitigated.

Interleukin-17 and Tumor Necrosis Factor Show a Functional Hierarchy to Regulate the Production of Matrix Metalloproteases by Monocytes from Patients with Psoriasis.

Interleukin-17 (IL-17) and tumor necrosis factor (TNF) regulate tissue remodeling through matrix metalloproteinases (MMPs). It is not yet clear whether these cytokines have a functional hierarchy in psoriasis. Serum levels of TNF (1,403 versus 1,058 pg/mL), IL-17 (1,528 versus 820 pg/mL), MMP-1 (1,999 versus 1,039 pg/mL), and MMP-9 (1,950 versus 1,561 pg/mL) were higher in psoriasis subjects (n = 60) than in control subjects (n = 60). Tissue inhibitor of MMPs (TIMP-1; 1,374 versus 1,218 pg/mL) was lower in psoriasis subjects. Serum IL-17 was correlated with MMP-2 (rs = 0.40) and TIMP-1 (rs = -0.26) levels. Unstimulated production of MMP-1, MMP-2, and MMP-9 by monocytes was higher in psoriasis subjects, whereas TIMP-1 production was lower. TNF stimulation increased all MMPs, whereas TIMP-1 production was unchanged. IL-17 stimulation increased all MMPs, whereas TIMP-1 production was decreased in psoriasis subjects. MMP-9 production was higher in monocytes stimulated with IL-17 compared with TNF. TIMP-1 production was decreased more by IL-17 than by TNF, but only in psoriasis cells. MMP-1/TIMP-1, MMP-2/TIMP-1, and MMP-9/TIMP-1 ratios were higher after IL-17 stimulation (compared with TNF stimulation) in psoriasis subjects; this occurred in controls only for the MMP-2/TIMP-1 ratio. IL-17 has a greater ability than TNF to dysregulate the MMPs/TIMP-1 balance, supporting IL-17 blockade as first-line treatment in cutaneous psoriasis.

Therapeutic potential of a novel semi-synthetic-sulfated-polysaccharide to suppress inflammatory mediators in P. gingivalis LPS stimulated human monocytes/macrophages

BackgroundChronic periodontitis is associated with an increased risk for systemic conditions such as cardiovascular disease, diabetes, and osteoporosis. During chronic periodontitis, endotoxin (lipopolysaccharide, LPS) produced by P. gingivalis provokes monocyte accumulation and differentiation into macrophages and increased secretion of pro-inflammatory cytokines and matrix metalloproteases (MMPs). While normal levels of MMPs are important in cellular function, increased levels of cytokines and MMPs can cause connective tissue destruction.ResultsIn the current study, we investigated the therapeutic capability of a novel semi-synthetic sulfated polysaccharide (SAGE) on the production of cytokines and MMPs by cultured human mononuclear cells and macrophages stimulated with endotoxin LPS produced by P. gingivalis, a periodontally-relevant cell culture model. Our research demonstrated SAGE inhibited the LPS induced synthesis of inflammatory mediators including TNF-α, IL-1β, PGE2, and MMP-9 in this periodontal-relevant cell culture model. In addition, TLR-2 and TLR-4 levels were also reduced with the SAGE treatment.ConclusionsThe therapeutic potential of this novel semi-synthetic sulfated polysaccharide compound may help to prevent tissue damage and bone loss in patients with periodontal disease or other inflammatory diseases.

ENAMPT Is Localised to Areas of Cartilage Damage in Patients with Hip Osteoarthritis and Promotes Cartilage Catabolism and Inflammation.

Obesity increases the risk of hip osteoarthritis (OA). Recent studies have shown that adipokine extracellular nicotinamide phosphoribosyltransferase (eNAMPT or visfatin) induces the production of IL-6 and matrix metalloproteases (MMPs) in chondrocytes, suggesting it may promote articular cartilage degradation. However, neither the functional effects of extracellular visfatin on human articular cartilage tissue, nor its expression in the joint of hip OA patients of varying BMI, have been reported. Hip OA joint tissues were collected from patients undergoing joint replacement surgery. Cartilage explants were stimulated with recombinant human visfatin. Pro-inflammatory cytokines and MMPs were measured by ELISA and Luminex. Localisation of visfatin expression in cartilage tissue was determined by immunohistochemistry. Cartilage matrix degradation was determined by quantifying proteoglycan release. Expression of visfatin was elevated in the synovial tissue of hip OA patients who were obese, and was co-localised with MMP-13 in areas of cartilage damage. Visfatin promoted the degradation of hip OA cartilage proteoglycan and induced the production of pro-inflammatory cytokines (IL-6, MCP-1, CCL20, and CCL4) and MMPs. The elevated expression of visfatin in the obese hip OA joint, and its functional effects on hip cartilage tissue, suggests it plays a central role in the loss of cartilage integrity in obese patients with hip OA.

Long-term exposure to butyric acid induces excessive production of matrix metalloproteases in human gingival fibroblasts

ObjectiveThe purpose of this study was to clarify the relationship between bacteria-induced butyric acid and periodontal disease progression. DesignNormal human gingival fibroblasts were exposed to butyric acid (0, 1, 5, 10, and 15 mM) adjusted to a pH of 7.2–7.4 using sodium hydroxide for 0–96 h and cell viability was evaluated. In addition, the effects of butyric acid on the production of matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs) in gingival fibroblasts were analyzed by real-time RT-PCR, ELISA, western blotting, and stromelysin zymography. ResultsButyric acid reduced the viability of gingival fibroblasts in a concentration- and time-dependent manner. Furthermore, butyric acid promoted production of MMP-1, MMP-3, and MMP-10 in gingival fibroblasts and suppressed TIMP-2 protein production. ConclusionsButyric acid promoted overproduction of MMPs, resulting in a disruption of the balance between MMPs and TIMPs expression in gingival fibroblasts. Our study suggests that the butyric acid produced by causative bacteria stimulates excessive MMP expression in periodontal tissue, leading to destruction of the tissue.

Macrophage production of matrix metalloproteases in the setting of chorioamnionitis

Maternal responses to bacterial chorioamnionitis (CAM) include production of matrix metalloproteases (MMPs), extracellular membrane-degrading enzymes thought to play a central role in the weakening of fetal membranes during CAM leading to preterm prelabor rupture of membranes (PPROM). Macrophages, the primary innate immune phagocyte of fetal membranes, secrete MMPs when provoked by microbes such as Group B Streptococcus (GBS). The objective of this project is to determine whether maternal cytotrophoblasts (CTBs) decrease macrophage expression of tissue-remodeling MMPs in the setting of GBS infection.

Characterization of Collagen Metabolism in Normal and Chronic Obstructive Pulmonary Diseased Human Lung Fibroblasts Reared in Serum‐free Medium

The structural integrity of the human lung is maintained by a healthy extracellular matrix (ECM). One of the main components, collagen type I is produced by fibroblasts and provides strength and structure to the cells and the function of the lung. A careful balance of collagen degradation and production is controlled by matrix metalloproteases (MMPs). In diseased states such as Chronic Obstructive Pulmonary Disease (COPD), two collagenases, MMP‐8 and MMP‐12, are upregulated. In this study we measure collagen metabolism of human lung fibroblasts (HLFs) reared in serum‐free media (subconfluent normal (NHLF) and COPD diseased (DHLF)). Commercially available early passage human lung fibroblasts established from lung tissue, obtained during biopsy, were cultured and passaged in our laboratory through P4 and P5 for both the NHLF and DHLF. Both normal and diseased HLF cells were viable when cultured at subconfluence for 24 hours in serum free medium. They produced detectable quantities of intact collagen type I secreted and incorporated in the ECM. The amount of intact collagen decreased between passage 4 and 5 (p<0.01) in both cell types indicating there is a limit to the passages that can be utilized to measure ECM alterations. Degraded collagen type I was present in the cell layer fractions of both cell types, but was 10 fold higher in the cell layer of diseased HLF cells. This indicates that the expected ECM profile of diseased HLF can be demonstrated in serum‐free culture. Both cell types produced detectable levels of MMP‐8 and MMP‐12 collagenases. In this study we have demonstrated that normal and diseased HLF can be cultured in serum‐free conditions while maintaining their metabolic profile. The production of MMPs and collagens provides an experimental system in which we can test potential therapeutics that may alter the fibrotic response during COPD.Support or Funding InformationThis work is supported by a grant from the PCOM Alumni and the Division of Research at PCOM.

Cell surface vimentin presentation on articular chondrocytes - a novel marker of cellular senescence or in vitro artefact

Purpose: Cellular senescence has been considered as a major process in osteoarthritic (OA) disease, causing the loss of the chondrogenic phenotype. In fact, senescent cells display a senescence-associated secretory phenotype (SASP) characterized by excessive cytokine, chemokine and matrix metalloprotease production. Identification of senescent cells is largely limited to either these secreted or intracellular (ß-galactosidase activity) markers. Therefore, we investigated one of the rare membrane-associated biomarkers, cell surface vimentin (CSV), on human articular chondrocytes (hAC).

The influence of matrix metalloproteases and biofilm on chronic wound healing: a discussion.

Chronicity in wound healing is a challenge for health services financially and scientifically, with negative consequences on patients' lives. This paper seeks to explore why chronic wounds fail to heal in relation to the inflammatory cellular dysfunction associated with biofilm development. Findings demonstrate an association between chronic wounds failing to heal, the presence of devitalised tissue and abnormal immune cell activity with a consequential excessive release of harmful matrix metalloproteases (MMPs). This process perpetuates the cycle of wound chronicity and extracellular matrix destruction, which prolongs the inflammatory response, fuelling biofilm formation. Evidence suggests that 'trapping' MMPs may increase new tissue growth but, while devitalised tissue is present, phagocytic cells continue to secrete MMPs and chronicity persists. Consequently, by removing the trigger and implementing effective, sustained debridement of devitalised tissue, both MMP and biofilm production will be diminished, with positive healing outcomes.

Knock out of the BASIGIN/CD147 chaperone of lactate/H+ symporters disproves its pro-tumour action via extracellular matrix metalloproteases (MMPs) induction.

BASIGIN/CD147/EMMPRIN is a multifunctional transmembrane glycoprotein strongly expressed in tumours. BASIGIN controls tumour metabolism, particularly glycolysis by facilitating lactic acid export through the two monocarboxylate transporters MCT1 and hypoxia-inducible MCT4. However, before being recognized as a co-carrier of MCTs, BASIGIN was described as an inducer of extracellular matrix metalloproteases (MMPs). Early on, a model emerged in which, tumour cells use the extracellular domain of BASIGIN to recognize and stimulate neighbouring fibroblasts to produce MMPs. However, this model has remained hypothetical since a direct link between BASIGIN and MMPs production has not yet been clearly established. To validate the BASIGIN/MMP hypothesis, we developed BASIGIN knockouts in three human tumour cell lines derived from glioma, colon, and lung adenocarcinoma. By using co-culture experiments of either human or mouse fibroblasts and tumour cell lines we showed, contrary to what has been abundantly published, that the disruption of BASIGIN in tumour cells and in MEFs has no action on the production of MMPs. Our findings do not support the notion that the pro-tumoural action of BASIGIN is mediated via induction of MMPs. Therefore, we propose that to date, the strongest pro-tumoural action of BASIGIN is mediated through the control of fermentative glycolysis.

Anti-inflammatory effects of an injectable copolymer of fatty acids (Ara 3000 beta®) in joint diseases.

BackgroundIn inflammatory joint disease, such as osteoarthritis or arthritis, there is an increased level of pro-inflammatory cytokines, such as interleukin-1β. These cytokines stimulate the expression and release of matrix metalloproteases (MMP), leading to the degradation of cartilage extracellular matrix and subsequently mobility difficulty and suffering for patients. The aim of this study was to examine the therapeutic potential of a fatty acid copolymer in in vitro and in vivo models of cartilage inflammation.MethodsInflammation was mimicked in vitro by treatment of human articular chondrocytes with interleukin-1β. Effects of a co-treatment with a copolymer of fatty acids (Ara 3000 beta®) were determined by evaluating MMP production by RT-PCR and ELISA, NO release by Griess assay, and PGE2 expression by ELISA. In addition, in vivo analysis (evolution of weight and edema) were also performed after injection of Freund adjuvant in rats treated or not with the copolymer of fatty acids.ResultsThe copolymer of fatty acids clearly reduces inflammation in joint. In vitro, it impairs IL1 stimulated-MMP production and release, as well as the release of NO and PGE2 and the activation of NFκB. Furthermore, in vivo experiments using adjuvant induced-arthritis corroborates the anti-inflammatory effects of the copolymer of fatty acids, with a reduction of edemas, erythemas and ankylosis in arthritic rats.ConclusionsThe results support the hypothesis that a copolymer of fatty acids, such as Ara 3000 beta®, is a powerful anti-inflammatory compounds, suggesting that it has a potential for preventing cartilage degradation associated with chronic inflammatory joint disease.

Abstract 1160: Novel CSF-1 receptor ligand IL-34 modulates macrophage-breast cancer cell crosstalk

Abstract Crosstalk between stromal cells and malignant cells within the tumor microenvironment is important for tumorigenesis. According to current clinical and experimental evidence, tumor associated macrophages (TAMs) play a major role in tumor progression to metastasis in different cancers. In breast cancer, increased number of TAMs is associated with poor prognosis. Previous work in our laboratory has shown that depletion of TAMs by blocking colony-stimulating factor-1 (CSF-1) suppresses tumor growth, matrix metalloprotease production and further recruitment of macrophages in a MCF-7 breast cancer xenograft model. In this study, we aim to investigate Interleukin-34 (IL-34), a novel ligand for the colony stimulating factor-1 receptor (CSF-1R). IL-34 functions as a specific and independent ligand and as an agonist to the CSF-1R. Furthermore it is known to induce proliferation through mitogen activated protein kinase (MAPK) activation in macrophages and cancer cells. To date, nothing is known about the role of IL-34 in breast cancer and its effects on downstream signalling pathway modulation in cancer cell-macrophage crosstalk. Our real-time PCR data shows that in co-cultures with murine macrophages, human MCF-7 and MDA-MB-231 breast cancer cells express increased levels of IL-34. In addition, in vitro migration assays demonstrated that recombinant human and murine IL-34 significantly increases the migration of murine macrophages, MCF-7 and MDA-MB-231 breast cancer cells. IL-34 also increased the proliferation of murine macrophages and breast cancer cells. Experiments on the protein level demonstrated the activation of MAPK/ERK in murine macrophages and breast cancer cells by recombinant IL-34. These preliminary data indicate that TAM-breast cancer cell interactions induce IL-34 production associated with induction of proliferation and the migratory capacity of tumor and stromal cells, which together may influence the invasiveness of the developing tumor. Here we propose IL-34 as a target for the treatment of breast cancer which may affect the accumulation of TAMs and improve breast cancer prognosis. Citation Format: Sandun Gunawardhana, Karin Zins, Trevor Lucas, Dietmar Abraham. Novel CSF-1 receptor ligand IL-34 modulates macrophage-breast cancer cell crosstalk. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1160. doi:10.1158/1538-7445.AM2014-1160

IBP regulates epithelial-to-mesenchymal transition and the motility of breast cancer cells via Rac1, RhoA and Cdc42 signaling pathways

Epithelial-to-mesenchymal transition (EMT) is a crucial process for the invasion and metastasis of epithelial tumors. However, the molecular mechanisms underlying this transition are poorly understood. In this study, we demonstrate that interferon regulatory factor 4 binding protein (IBP) regulates EMT and the motility of breast cancer cells through Rac1, RhoA and Cdc42 signaling pathways. We found that increased expression of IBP was associated with the progression of breast cancer and that IBP protein levels were significantly elevated in matched distant metastases. High IBP levels also predict shorter overall survival of breast cancer patients. Furthermore, the forced expression of IBP decreased the expression of the epithelial marker E-cadherin but increased the mesenchymal markers in breast cancer cells. In contrast, silencing IBP in metastatic breast tumor cells promoted a shift toward an epithelial morphology concomitant with increased expression of E-cadherin and decreased expression of mesenchymal markers. IBP silencing also reduced the expression of EMT-inducing transcription factors (Snail, Slug, ZEB1 and ZEB2). Moreover, we identified a role for IBP in endogenous EMT induced by epidermal growth factor (EGF) and deletion of IBP attenuated EGF receptor (EGFR) signaling in breast cancer cells. Furthermore, IBP regulates the migration, invasion and matrix metalloprotease production in breast cancer cells as well as actin cytoskeleton rearrangement and the activation of GTP-Rac1, GTP-RhoA and GTP-Cdc42. Taken together, our findings demonstrate an oncogenic property for IBP in promoting the metastatic potential of breast cancer cells.

Abstract 1424: Cyclophilin A induces head and neck squamous cell carcinoma (HNSCC) invasion by its interaction to EMMPRIN/CD147

Abstract Extracellular matrix (ECM) remodeling that is crucial in tumorigenesis involves proteolytic enzymes, primarily matrix metalloproteases (MMPs). The production of MMPs is stimulated by different factors, including extracellular matrix metalloprotease inducer (EMMPRIN/CD147). Over-expression of EMMPRIN, a member of the immunoglobulin super-family, promotes invasion, metastasis, growth and survival of malignant cells. Cyclophilin A (CypA) is a multifunction protein and promotes cancer progression in various cancer types. It has been reported CypA can interact and activate EMMPRIN, however the role of EMMPRIN-Cyp A interaction in oncogenicity is not fully understood in malignant cells including head and neck squamous cell carcinoma (HNSCC). In order to evaluate tumorigenicity that are induced by EMMPRIN-CypA interaction, EMMPRIN expressing HNSCC cell lines were stimulated with CypA. The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] dye assay revealed that HNSCC cell proliferation was increased, whereas cisplatin induced cell death was declined in the presence of CypA. In addition, CypA induced MMP-9 up-regulation was detected in culture medium by gelatin zymography. Moreover, increment of invaded cells through matrigel-coated membranes was found by CypA stimulation. Interestingly the elevated invasive potential was abrogated by EMMPRIN function blocking antibody. These findings suggest that CypA, by its interaction to EMMPRIN, might contribute to the HNSCC tumorigenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1424. doi:10.1158/1538-7445.AM2011-1424

Peran Lipopolisakarida Helicobacter pylori terhadap Aktivitas Neutrofil pada Penderita Infark Miokard Akut melalui Degradasi Kolagen Tipe IV

Matrix metalloproteases (MMPs) are proteolytic enzymes that play a critical role in decreasing the stability of atherosclerotic plaque causing Acute Myocardial Infarction (AMI). In this process, degradation of type IV collagen as an important component of endothelial membrane is thought to be important. Several microorganisms and their products including Lipopolysaccharide (LPS) have been suggested to be able to activate MMPs. The aim of this study is to investigate the role of Helicobacter pylori LPS in type IV collagen degradation (in vitro). We used two stages in this study: MMP production stage and degradation of type IV collagen. MMP derived from the neutrophils of AMI and non- AMI patients that had been stimulated with LPS of Helicobacter pylori were used to digest type IV collagen. Analysis of type IV collagen degradation was performed with SDS-PAGE and Western blot. This study showed MMP isolated from neutrophil of AMI patients is an active MMP-9 at Molecular weight of 72 kDa. It can degrade type IV collagen at MW 78.6 kDa, 60 kDa, 50 kDa, 47.8 kDa and 43.8 kDa. Whereas MMP isolated from neutrophil of non-AMI have MW of 91.2 kDa and can only degrade collagen IVat merely 60 kDa. In conclusion, Helicobacter pylori LPS is able to induce n eutrophil of AMIpatients to produce an active MMP-9 at MW 72 kDa and the enzyme may play strategic role in the degradation of type IV collagen.

Divergent and Synergistic Regulation of Matrix Metalloprotease Production by Cytokines in Combination with C-C Chemokines

The chemotactic effects of chemokines on cells has long been known, but it is now clear that chemokines also have much broader activities and are also involved in a number of disease pathologies, such as rheumatoid arthritis, cancer metastasis and other inflammatory processes. This study investigates the effects of four C-C chemokines, CCL2, CCL3, CCL4 and CCL5 either alone or in the presence of two regulatory cytokines TNF-alpha and TGF-beta and their effect on secretion of two matrix metalloproteases MMP, MMP-2 and MMP-9, and the expression of one membrane bound MMP, MMP-14, by a monocytic human cell line, MonoMac6. All four C-C chemokines were shown to be chemotactic, but only CCL2 and CCL4 had any significant stimulatory effect on MMP-9 and MMP-2, respectively. Both TNF-alpha and TGF-beta were found to divergently enhance MMP-9 and MMP-2 secretion respectively, with stimulation indexes of two and five respectively. Simultaneous treatment with TNF-alpha and chemokine resulted in up to a fifteen-fold stimulation of MMP-9 secretion and treatment with TGF-beta and chemokine resulted in up to a fifteen-fold stimulation of MMP-2 secretion, while TNF-alpha in combination with CCL4 stimulated MMP-14 expression five-fold. Chemokine receptor expression was also investigated using a calcium-sensitive dye and FACS analysis. CCL2, CCL3, and CCL5 all resulted in a detectable enhancement of cytoplasmic Ca2+concentration. CCL4 was unable to activate Ca2+ mobilization, despite the presence of CCR5, the receptor for CCL4. There appeared to be no correlation between MMP production and chemotaxis. The strong synergy between chemokines and cytokines and the enhanced production of MMP may signify the differential regulatory mechanisms of the two cytokines and chemokines in disease pathology.

Abstract LB-106: Inhibition of glycolysis with 2-deoxyglucose inhibits spontaneous metastasis in murine osteosarcoma

Abstract We recently reported on a re-analysis of previously published gene expression data from a large set of primary solid and metastatic tumors originating from different tissues using the latest available tools for normalization, identification of differentially expressed genes and pathway analysis (Ptitsyn A., et al, BMC Bioinformatics 2008;9 Suppl: S8). Analysis of multiple independent datasets indicated significantly reduced oxidative phosphorylation in metastases compared to primary solid tumors, implying a potential enhancement of the Warburg phenotype in metastatic progression. We thus sought to determine if inhibition of anaerobic glycolysis with the competitive inhibitor 2-deoxy-D-glucose (2-DG) would reverse this metastatic bioenergetic phenotype in highly metastatic cancer cells. We evaluated the effects of 2-DG on DLM8 osteosarcoma cell migration and Matrigel invasion via Boyden chamber assays, and modulation of in vitro production of matrix metalloproteases, VEGF, and cathepsin L were evaluated by gelatin zymography, ELISA and western analysis respectively. The effects of 2-DG administration in vivo was evaluated in an orthotopic, spontaneously metastatic, luciferase-transfected postsurgical model of murine osteosarcoma utilizing DLM8, which is syngeneic to C3H mice. DLM8 cells were implanted into the distal tibias of C3H mice, followed by amputation of the affected limb 16 days later. 2-DG or saline were administered postoperatively (500 mg/kg IP 3x/week, n=5 per group) until sacrifice. Presence of metastasis was evaluated using biweekly bioluminescence imaging and metastasis-free interval calculated using the Kaplan-Meier method. Concentrations of 2-DG associated with minimal antiproliferative activity dramatically inhibited DLM8 cell migration and invasion. There was a dose-dependent decrease in cathepsin L expression upon 2-DG exposure, and VEGF production was inhibited at high 2-DG concentrations, but there were no repeatable changes in MMP activity within the concentration range associated with inhibition of migration and invasion. Postoperative administration of 2-DG significantly delayed DLM8 metastasis (median metastasis-free interval 8 vs 34 days, p = 0.049). In conclusion, glycolysis inhibition with 2-DG is associated with an inhibition of osteosarcoma migration, invasion and in vivo metastasis, at concentrations associated with minimal antiproliferative activity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-106.

Anti-invasion and anti-tumor growth effect of doxycycline treatment for human oral squamous-cell carcinoma – In vitro and in vivo studies

Regional lymph node and distant organ metastasis of oral squamous-cell carcinoma (OSCC) has been associated with increased production of matrix metalloproteases (MMPs), and scientific data showed that doxycycline (Dox) could down-regulate the expression of MMPs. The objective of this study was to evaluate the effect of Dox on the expression of MMPs in vitro using the SCC-15 cell line and in vivo SCC-15 xenografted nude mice. SCC-15 cells maintained under distinct culture conditions expressed high levels of pro-MMP-2 and pro-MMP-9; however, as determined by zymography and Western blot analysis, Dox significantly reduced the production of pro-MMP-2 and pro-MMP-9 after 24h of treatment in a dose-dependent manner (2.5-40 microg/ml). Dox (10 microg/ml) decreased the expression of MMP-9 mRNA but did not alter the level of MMP-2 mRNA after 24h of treatment. In addition, this drug significantly inhibited the invasive and migration activities of SCC-15 cells in vitro (>75% inhibition at 10 microg/ml). On the other hand, daily administration of Dox (3mg/mice) restrained tumor growth in SCC-15 xenografted nude mice, with an inhibition rate of 85.6%. Compared with the control group (treated with normal saline), MMP-9 mRNA levels in the fresh tumor tissue decreased upon Dox treatment (P<0.01) while MMP-2 mRNA levels were unchanged. In conclusion, reduced expression of MMP-9 at the transcriptional level and MMP-2 at the post-transcriptional level caused by Dox was found to be associated with decreased invasion of oral SCC in vitro. Moreover, Dox exerted a significant suppressive effect on tumor growth in an in vivo nude mice model. Taken together, these results, to our knowledge, may first imply that Doxycycline has an adjuvant therapeutic effect on OSCC that is associated with inhibition of MMPs expression.

Effects of human papillomavirus type 16 E5 deletion mutants on epithelial morphology: functional characterization of each transmembrane domain

Human papillomavirus type 16 (HPV-16) is the cause of cervical cancer. The HPV genome encodes three transforming proteins, E5, E6 and E7. E6 and E7 are the main transforming proteins of HPV, while the role of E5 is still poorly understood. Using three dimensional organotypic raft cultures we show that HaCaT human keratinocytes expressing HPV-16 E5 form a very perturbed epithelium, with simultaneous hyperkeratinization of some cells and defective differentiation of other cells. The basal layer is disturbed and many cells invade the collagen matrix. Many cells among the differentiated layers show characteristics of basal cells: progression through the cell cycle, expression of cytokeratin 14, lack of cytokeratin 1 and production of matrix metalloproteases (MMP). Using deletion mutants which encompass the three hydrophobic domains of E5, we have assigned the ability to promote invasion of the matrix to the first hydrophobic domain, and the capacity to induce MMP9 to the C-terminal four amino acids. We also show that invasion and production of MMP9 can be dissociated, as mutants that are still capable of invasion do not produce MMP9 and vice versa.