Enhanced Matrix Metalloproteinase Research Articles

Wnt5a deficiency in osteocalcin-expressing cells could not alleviate the osteoarthritic phenotype in a mouse model of post-traumatic osteoarthritis.

Wnt5a, which regulates the activities of osteoblasts and osteoclasts, is reportedly overexpressed in osteoarthritis (OA) tissues. The purpose of this study was to elucidate its role in the development of OA by deleting Wnt5a in osteocalcin (OCN)-expressing cells. Knee OA was induced by anterior cruciate ligament transection (ACLT) in OCN-Cre;Wnt5afl/fl knockout (Wnt5a-cKO) mice and control littermates. Eight weeks after surgery, histological changes, cell apoptosis, and matrix metabolism of cartilage were evaluated by toluidine blue, TUNEL staining, and im-immunohistochemistry analyses, respectively. In addition, the subchondral bone microarchitecture of mice was examined by micro-computed tomography (micro-CT). Histological scores show substantial cartilage degeneration occurred in ACLT knees, coupled with decreased collagen type II expression and enhanced matrix metalloproteinase 13 expression, as well as higher proportions of apoptotic cells. Micro-CT results show that ACLT resulted in decreased bone mineral density, bone volume/trabecular volume, trabecular number, and structure model index of subchondral bones in both Wnt5a-cKO and control littermates; although Wnt5a-cKO mice display lower BMD and BV/TV values, no significant difference was observed between Wnt5a-cKO and control mice for any of these values. Our findings indicate that Wnt5a deficiency in OCN-expressing cells could not prevent an osteoarthritic phenotype in a mouse model of post-traumatic OA.

STAT3 Deficiency Alters the Macrophage Activation Pattern and Enhances Matrix Metalloproteinase 9 Expression during Staphylococcal Pneumonia.

Staphylococcus aureus is a significant cause of morbidity and mortality in pulmonary infections. Patients with autosomal-dominant hyper-IgE syndrome due to STAT3 deficiency are particularly susceptible to acquiring staphylococcal pneumonia associated with lung tissue destruction. Because macrophages are involved in both pathogen defense and inflammation, we investigated the impact of murine myeloid STAT3 deficiency on the macrophage phenotype invitro and on pathogen clearance and inflammation during murine staphylococcal pneumonia. Murine bone marrow-derived macrophages (BMDM) from STAT3 LysMCre+ knockout or Cre- wild-type littermate controls were challenged with S. aureus, LPS, IL-4, or vehicle control invitro. Pro- and anti-inflammatory responses as well as polarization and activation markers were analyzed. Mice were infected intratracheally with S. aureus, bronchoalveolar lavage and lungs were harvested, and immunohistofluorescence was performed on lung sections. S. aureus infection of STAT3-deficient BMDM led to an increased proinflammatory cytokine release and to enhanced upregulation of costimulatory MHC class II and CD86. Murine myeloid STAT3 deficiency did not affect pathogen clearance invitro or invivo. Matrix metalloproteinase 9 was upregulated in Staphylococcus-treated STAT3-deficient BMDM and in lung tissues of STAT3 knockout mice infected with S. aureus. Moreover, the expression of miR-155 was increased. The enhanced inflammatory responses and upregulation of matrix metalloproteinase 9 and miR-155 expression in murine STAT3-deficient as compared with wild-type macrophages during S. aureus infections may contribute to tissue damage as observed in STAT3-deficient patients during staphylococcal pneumonia.

Ubiquitin-specific protease TRE17/USP6 promotes tumor cell invasion through the regulation of glycoprotein CD147 intracellular trafficking

Disordered expression and distribution of plasma membrane proteins at the cell surface leads to diverse malignant phenotypes in tumors, including cell invasion. The ubiquitin-specific protease TRE17/USP6, an oncogene identified in Ewing sarcoma, is highly expressed in several cancers and locally aggressive tumor-like lesions. We have previously demonstrated that TRE17 regulates the trafficking of plasma membrane proteins that enter cells via clathrin-independent endocytosis (CIE); TRE17 prevents CIE cargo proteins from being targeted to lysosomes for degradation by deubiquitylating them. However, functional insights into the effects of TRE17-mediated CIE cargo trafficking on cell invasion remain unknown. Here, we show that increased expression of TRE17 enhances invasiveness of the human sarcoma cell line HT-1080 by elevating the cell surface levels of the membrane glycoprotein CD147, which plays a central role in tumor progression. We demonstrate overexpression of TRE17 decreases ubiquitylated CD147, which is accompanied by suppression of CD147 transport to lysosomes, resulting in the stabilization and increase of cell surface-localized CD147. On the other hand, we show knockdown of TRE17 decreases cell surface CD147, which is coupled with reduced production of matrix metalloproteinases, the enzymes responsible for extracellular matrix degradation. Furthermore, we demonstrate that inhibition of CD147 by a specific inhibitor alleviated the TRE17-promoted tumor cell invasion. We therefore propose a model for the pathogenesis of TRE17-driven tumors in which TRE17 increases CD147 at the cell surface by preventing its lysosomal degradation, which in turn enhances matrix metalloproteinase synthesis and matrix degradation, thereby promoting tumor cell invasion.

Abstract 6096: The microarchitecture and fibrillar collagen expression of leiomyosarcoma are associated with malignancy

Abstract The expression, abundance, and microarchitecture of fibrillar collagens are associated with tumor development and aggressiveness in various carcinomas. However, the impact of fibrillar collagens on mesenchymal tumors is less understood. While uterine leiomyomas, also known as fibroids, are characterized by high fibrillar collagen deposition and depend on ECM signaling for cell proliferation, the impact of fibrillar collagens on malignant uterine leiomyosarcomas has not been explored. Thus, identifying malignancy and aggressiveness-associated features of the fibrillar collagen-leiomyosarcoma crosstalk may provide novel biomarkers and therapeutic targets for these aggressive tumors. We used publicly available RNAseq data and performed RNAseq and picrosirius red analysis of fiber microarchitecture in a cohort of normal myometrium (MM; n =68 ), leiomyoma (LM; n = 66), and leiomyosarcoma (LMS; n = 67) tissues. Furthermore, we cultured patient-derived primary cells (4 MM, 3 LM, and 4 LMS) on collagen I-functionalized polyacrylamide gels at stiffness ranging from 0.5 to 115 kPa, covering the physiological and pathological stiffness, to investigate distinct behaviors between cell types, including proliferation, migration, and activity of the ECM stiffness molecular rheostat YAP/TAZ. At the protein level, analysis of fibrillar collagen microarchitecture revealed that LMS tumors present reduced fibrillar collagen density and hyphal growth units and enhanced fiber endpoints compared to both MM and LM. At the gene expression level, however, LMS tumors did not show reduced fibrillar collagen expression, instead they exhibited enhanced matrix metalloproteinase expression, particularly of MMP14. Furthermore, COL11A1 was specifically upregulated in LMS tumors and its expression was associated with poor prognosis. Finally, in vitro response of MM, LM, and LMS cells to collagen I at defined stiffness showed that LMS cell migration, proliferation, and subcellular localization of YAP/TAZ are less sensitive to substrate stiffness than in MM and LM cells, although the response varied between distinct donors. In conclusion, we show that LMS tumors typically present low fibrillar collagen protein expression likely due to enhanced degradation. In addition, collagen I adhesion and stiffness have a lower impact on malignant LMS cells than on MM and LM, which may explain their ability to grow in low-collagen microenvironments. Furthermore, this study shows that COL11A1 is a potential biomarker with prognostic value in leiomyosarcoma. Citation Format: Jordi Gonzalez-Molina, Paula Hahn, Raul Maia Falcão, Georgia Kokaraki, Jorge Estefano de Souza, Tirzah Braz Petta Lajus, Kaisa Lehti, Joseph W. Carlson. The microarchitecture and fibrillar collagen expression of leiomyosarcoma are associated with malignancy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6096.

UBE2C promotes the progression of pancreatic cancer and glycolytic activity via EGFR stabilization-mediated PI3K-Akt pathway activation.

Pancreatic cancer (PC) is among the most prevalent and deadliest endocrine tumors, yet the mechanisms governing its pathogenesis remain to be fully clarified. While ubiquitin-conjugating enzyme E2C (UBE2C) has been identified as an important oncogene in several cancers, its importance in PC has yet to be established. UBE2C expression in PC tumor samples and cell lines was examined via quantitative real-time polymerase chain reaction (qRT-PCR), while appropriate commercial kits were used to assess lactate production, ATP generation, and the uptake of glucose. UBE2C was found to be upregulated in PC patient tumors and correlated with poorer survival outcomes. In PC cell lines, the silencing of this gene suppressed the malignant activity of cells, thus supporting its identification as an oncogene in this cancer type. Mechanistically, UBE2C was found to promote enhanced matrix metalloproteinase (MMP) protein expression via activating the PI3K-Akt pathway. Moreover, it was found to bind to the epidermal growth factor receptor (EGFR), stabilizing it and driving additional PI3K-Akt pathway activation. UBE2C knockdown in PC cells impaired their uptake of glucose and their ability to produce lactate and ATP. In conclusion, the results of this study support a role for UBE2C as a driver of metastatic PC progression owing to its ability to bind to EGFR and to induce signaling via the PI3K-Akt pathway.

Bioactive pulvinones from a marine algicolous fungus Aspergillus terreus NTU243

Marine fungi are regarded as an under-explored source of structurally interesting and bioactive natural products with the potential to provide attractive lead compounds for drug discovery. In this study, several fungal strains were isolated from marine algae collected from the northeastern coast of Taiwan. In the preliminary antimicrobial screening against bacteria and fungi, the ethyl acetate extract of the fermented products of Aspergillus terreus NTU243 derived from a green alga Ulva lactuca was found to exhibit significant antimicrobial activities. Therefore, bioassay-guided separations of the active principle from liquid and solid fermented products of A. terreus NTU243 were undertaken, which resulted in the isolation and purification of 16 compounds. Their structures were elucidated by spectroscopic analysis to be four previously undescribed aspulvinones S–V as well as twelve known compounds. All the isolates were assessed for anti-inflammatory activity by measuring the amount of nitric oxide (NO) production in lipopolysaccharide (LPS)-induced BV-2 cells, and aspulvinone V, butyrolactone I, and (+)-terrein inhibited 45.0%, 34.5%, and 49.2% of NO production, respectively, at 10 μM concentration. Additionally, zymography showed that the conditioned medium of THP-1 cells post-LPS challenged significantly enhanced matrix metalloproteinase (MMP)-9-mediated gelatinolysis, and pretreatment with aspulvinones U and V significantly attenuated MMP-9-mediated gelatinolysis by 56.0% and 67.8%, separately.

Abstract 14103: Loss of Bone Morphogenetic Protein (BMP) 9 Activity Reduces Survival and Increases Collagen Turnover via Enhanced Matrix Metalloproteinase Activity After Acute Myocardial Infarction

Introduction: Acute myocardial infarction (AMI) is a major cause of heart failure and mortality. The extracellular matrix plays a central role in post-infarct remodeling. We recently reported that BMP9 negatively regulates cardiac fibrosis in heart failure. A functional role for BMP9 in post-infarct remodeling remains poorly defined. We hypothesize that BMP9 signaling via activin like kinase 1 (ALK1) promotes survival after AMI by negatively regulating fibrosis and limiting matrix metalloproteinase (MMP) activity. Methods: AMI was induced by left coronary artery ligation in wild type (WT), BMP9-/- and ALK-/+ mice. After 14 days, LV function was assessed using conductance catheter and myocardial BMP9 signaling pathways were studied. In vitro siRNA mediated protein knockdown in human atrial fibroblasts (AtFB) further tested this signaling pathway. Results: BMP9-/- and ALK-/+ mice had significantly reduced survival after AMI compared to WT (p=0.04 and 0.008 respectively) and was associated with an increased incidence of cardiac rupture. LV ejection fraction decreased significantly among BMP9-/- and ALK1+/- survivors compared to sham (p<0.01 for both). pSMAD3 was increased and pSMAD1 decreased in both BMP9-/- and ALK1-/+ mice after AMI. Reduced pSMAD1 expression was directly associated with increased type 1 collagen levels in BMP9-/- (R 2 =0.48, p=0.02) and ALK1-/+ (R 2 =0.42, p=0.01) mice compared to WT. LV protein levels of endoglin increased significantly in both BMP9-/- and ALK+/- compared to WT sham (p<0.01 for both) after AMI. LV levels and activity of MMP9 were increased in BMP9-/-, but not in ALK1+/-, compared to WT after AMI (p=0.02 and 0.8 respectively). Circulating biomarkers of collagen degradation:synthesis (ICTP:PINP ratio) increased significantly (p=0.02) in BMP9-/- compared to WT after AMI. BMP9 but not ALK1 knockdown in AtFBs increased expression and activity of MMP9 (p=0.01 and 0.3 respectively). Conclusions: These findings identify a novel functional role for BMP9 and ALK1 as determinants of post-infarct survival and cardiac remodeling and further suggest that loss of BMP9-ALK1 signaling increases collagen degradation and turnover. BMP9/ALK1 signaling may be a novel target of therapy for post-infarct remodeling and survival.

Nicotine Exacerbates TAAD Formation Induced by Smooth Muscle-Specific Deletion of the TGF-β Receptor 2.

Tobacco smoke is an established risk factor for thoracic aortic aneurysms and dissections (TAAD). However, little is known about its underlying mechanisms due to the lack of validated animal models. The present study developed a mouse model that may be utilized to investigate exacerbation of TAAD formation by mimetics of tobacco smoke. TAADs were created via inducible deletion of smooth muscle cell-specific Tgfbr2 receptors. Using this model, the first set of experiments evaluated the efficacy of nicotine salt (34.0 mg/kg/day), nicotine free base (NFB, 5.0 mg 90-day pellets), and cigarette smoke extract (0.1 ml/mouse/day). Compared with their respective control groups, only NFB pellets promoted TAAD dilation (23 ± 3% vs. 12 ± 2%, P = 0.014), and this efficacy was achieved at a cost of >50% acute mortality. Infusion of NFB with osmotic minipumps at extremely high, but nonlethal, doses (15.0 or 45.0 mg/kg/day) failed to accelerate TAAD dilation. Interestingly, costimulation with β-aminopropionitrile (BAPN) promoted TAAD dilation and aortic rupture at dosages of 3.0 and 45.0 mg/kg/day, respectively, indicating that BAPN sensitizes the response of TAADs to NFB. In subsequent analyses, the detrimental effects of NFB were associated with clustering of macrophages, neutrophils, and T-cells in areas with structural destruction, enhanced matrix metalloproteinase- (MMP-) 2 production, and pathological angiogenesis with attenuated fibrosis in the adventitia. In conclusion, modeling nicotine exacerbation of TAAD formation requires optimization of chemical form, route of delivery, and dosage of the drug as well as the pathologic complexity of TAADs. Under the optimized conditions of the present study, chronic inflammation and adventitial mal-remodeling serve as critical pathways through which NFB exacerbates TAAD formation.

Anxa1 in smooth muscle cells protects against acute aortic dissection

Acute aortic dissection (AAD) is a life-threatening disease with high morbidity and mortality. Previous studies have showed that vascular smooth muscle cell (VSMC) phenotype switching modulates vascular function and AAD progression. However, whether an endogenous signalling system that protects AAD progression exists remains unknown. Our aim is to investigate the role of Anxa1 in VSMC phenotype switching and the pathogenesis of AAD. We first assessed Anxa1 expression levels by immunohistochemical staining in control aorta and AAD tissue from mice. A strong increase of Anxa1 expression was seen in the mouse AAD tissues. In line with these findings, micro-CT scan results indicated that Anxa1 plays a role in the development of AAD in our murine model, with systemic deficiency of Anxa1 markedly progressing AAD. Conversely, administration of Anxa1 mimetic peptide, Ac2-26, rescued the AAD phenotype in Anxa1-/- mice. Transcriptomic studies revealed a novel role for Anxa1 in VSMC phenotype switching, with Anxa1 deficiency triggering the synthetic phenotype of VSMCs via down-regulation of the JunB/MYL9 pathway. The resultant VSMC synthetic phenotype rendered elevated inflammation and enhanced matrix metalloproteinases (MMPs) production, leading to augmented elastin degradation. VSMC-restricted deficiency of Anxa1 in mice phenocopied VSMC phenotype switching and the consequent exacerbation of AAD. Finally, our studies in human AAD aortic specimens recapitulated key findings in murine AAD, specifically that the decrease of Anxa1 is associated with VSMC phenotype switch, heightened inflammation, and enhanced MMP production in human aortas. Our findings demonstrated that Anxa1 is a novel endogenous defender that prevents AAD by inhibiting VSMC phenotype switching, suggesting that Anxa1 signalling may be a potential target for AAD pharmacological therapy.

Ectopic Expression of Ankrd2 Affects Proliferation, Motility and Clonogenic Potential of Human Osteosarcoma Cells.

Simple SummaryOsteosarcoma is a rare malignancy of bone, primarily affecting children and young adults. The main objective of this study was to identify novel therapeutic targets to fight the progression of this insidious disease. To this aim, the role of Ankrd2, a stress- and mechano- sensor protein known for being mostly expressed in muscle fibers, was analyzed in the modulation of osteosarcoma progression. By subjecting human osteosarcoma cell lines expressing or silencing Ankrd2 to several functional assays, our results demonstrated that Ankrd2 is involved in the pathogenesis of this cancer. Nonetheless, due to observations obtained by other studies in other model systems, our findings also suggest that Ankrd2 might behave as a “double-faced” cancer driver gene.Ankrd2 is a protein known for being mainly expressed in muscle fibers, where it participates in the mechanical stress response. Since both myocytes and osteoblasts are mesenchymal-derived cells, we were interested in examining the role of Ankrd2 in the progression of osteosarcoma which features a mechano-stress component. Although having been identified in many tumor-derived cell lines and -tissues, no study has yet described nor hypothesized any involvement for this protein in osteosarcoma tumorigenesis. In this paper, we report that Ankrd2 is expressed in cell lines obtained from human osteosarcoma and demonstrate a contribution by this protein in the pathogenesis of this insidious disease. Ankrd2 involvement in osteosarcoma development was evaluated in clones of Saos2, U2OS, HOS and MG63 cells stably expressing Ankrd2, through the investigation of hallmark processes of cancer cells. Interestingly, we found that exogenous expression of Ankrd2 influenced cellular growth, migration and clonogenicity in a cell line-dependent manner, whereas it was able to improve the formation of 3D spheroids in three out of four cellular models and enhanced matrix metalloproteinase (MMP) activity in all tested cell lines. Conversely, downregulation of Ankrd2 expression remarkably reduced proliferation and clonogenic potential of parental cells. As a whole, our data present Ankrd2 as a novel player in osteosarcoma development, opening up new therapeutic perspectives.

Myeloid-specific blockade of Notch signaling alleviates murine pulmonary fibrosis through regulating monocyte-derived Ly6clo MHCIIhi alveolar macrophages recruitment and TGF-β secretion.

Macrophages in lung, including resident alveolar macrophages (AMs) and interstitial macrophages (IMs), and monocyte-derived macrophages, play important roles in pulmonary fibrosis (PF), but mechanisms underlying their differential regulation remain unclear. Recombination signal-binding protein Jκ (RBP-J)-mediated Notch signaling regulates macrophage development and phenotype. Here, using bleomycin-induced fibrosis model combined with myeloid-specific RBP-J disruption (RBP-JcKO ) mouse, we investigated the role of Notch signaling in macrophages during PF. Compared with the control, RBP-JcKO mice exhibited alleviated lung fibrosis as manifested by reduced collagen deposition and inflammation, and decreased TGF-β production. FACS analysis suggested that decreased Ly6clo MHCIIhi AMs might make the major contribution to attenuated fibrogenesis in RBP-JcKO mice, probably by reduced inflammatory factor release and enhanced matrix metalloproteinases expression. Using clodronate-mediated macrophage depletion in RBP-JckO mice, we demonstrated that embryonic-derived AMs play negligible role in lung fibrosis, which was further supported by adoptive transfer experiments. Moreover, on CCR2 knockout background, the effect of RBP-J deficiency on fibrogenesis was not elicited, suggesting that Notch regulated monocyte-derived AMs. Co-culture experiment showed that monocyte-derived AMs from RBP-JcKO mice exhibit reduced myofibroblast activation due to decreased TGF-β secretion. In conclusion, monocyte-derived Ly6clo MHCIIhi AMs, which are regulated by RBP-J-mediated Notch signaling, play an essential role in lung fibrosis.

A circular RNA derived from MMP9 facilitates oral squamous cell carcinoma metastasis through regulation of MMP9 mRNA stability

Emerging evidence demonstrates that dysregulation of circular RNA is linked to tumorigenesis and aggressive progression. However, its role in oral squamous cell carcinoma remains largely uncharacterized. In this study, we identified a novel metastasis-associated circular RNA, circular matrix metalloproteinase 9 (hsa_circ_0001162, a circular RNA derived from matrix metalloproteinase 9), which was remarkably upregulated in oral squamous cell carcinoma and positively correlated with matrix metalloproteinase 9 expression. Patients with high circular matrix metalloproteinase 9 expression were prone to lymph node metastasis and an advanced TNM stage. Importantly, circular matrix metalloproteinase 9 was identified as an efficacious diagnostic and prognostic biomarker for oral squamous cell carcinoma patients. Functional experiments showed that depletion of circular matrix metalloproteinase 9 weakened the migratory and invasive capabilities of oral squamous cell carcinoma cells in vitro as well as inhibited lung metastasis in vivo. Regarding the mechanism, circular matrix metalloproteinase 9 could simultaneously interact with AUF1 and miR-149 to block the inhibitory effect of AUF1 and miR-149 on matrix metalloproteinase 9 3′-untranslated region, resulting in enhanced matrix metalloproteinase 9 messenger RNA stability, thereby facilitating oral squamous cell carcinoma metastasis. Collectively, our data indicate that circular matrix metalloproteinase 9 acts as a metastasis-promoting gene in oral squamous cell carcinoma through regulating the messenger RNA stability of its parental gene. Therapeutic targeting of circular matrix metalloproteinase 9 may be a promising treatment intervention for metastatic oral squamous cell carcinoma patients.

Betulinic acid inhibits cell proliferation, migration, and inflammatory response in rheumatoid arthritis fibroblast-like synoviocytes.

Betulinic acid (BA), a pentacyclic triterpene derived from the bark of the white birch tree, has been reported to have a variety of pharmacological effects, including antioxidant, anti-inflammatory, antitumor, immunomodulatory, and antiarthritis properties. However, the role of BA in rheumatoid arthritis (RA) remains unclear. Thus, the objective of this study was to examine the effects of BA on RA fibroblast-like synoviocytes (RA-FLS) proliferation, migration, and inflammatory response, and further explore the potential underlying mechanisms. Our results showed that BA inhibited the proliferation, migration, and invasion of RA-FLSs. BA also attenuated tumor necrosis factor-α (TNF-α), enhanced matrix metalloproteinases (MMPs) expression, and inflammatory cytokines production in RA-FLS. Furthermore, BA prevented the activation of Akt/NF-κB pathway in RA-FLS exposed to TNF-α. In conclusion, these findings indicated that BA inhibits cell proliferation, migration, and inflammatory response in RA-FLS; and the Akt/NF-κB signaling pathway was involved in the protective effect of BA on RA-FLS. Thus, BA might be a potential therapeutic agent for the treatment of RA.

All-Trans Retinoic Acid Enhances Matrix Metalloproteinase 2 Expression and Secretion in Human Myeloid Leukemia THP-1 Cells.

All-trans retinoic acid (ATRA) is an effective drug for the induction therapy of acute promyelocytic leukemia. However, the treatment is associated with adverse events such as retinoic acid syndrome (RAS) in some patients, whose histologic characteristics included organ infiltration by leukemic cells. Matrix metalloproteinase 2 (MMP-2) is often upregulated in tumor cells and plays a role in tumor cell migration and invasion by degrading the extracellular matrix. In this study, we examined the possible modulatory effects of ATRA on MMP-2 expression and secretion in human myeloid leukemia cell line THP-1. The cells were treated with various concentrations of ATRA, and MMP-2 expression and secretion were examined. MMP-2 expression and secretion started to increase with ATRA concentration as low as 0.1 nM and gradually increased thereafter. Agonists of retinoic acid receptor (RAR) or retinoid X receptor (RXR) alone could enhance MMP-2 secretion, and RAR or RXR antagonists alone could reverse ATRA-induced MMP-2 secretion. ATRA increased intracellular calcium ion levels, and a calcium-channel blocker inhibited ATRA-induced MMP-2 secretion. Dexamethasone suppressed ATRA-induced MMP-2 secretion. Our results suggest that ATRA enhances MMP-2 expression and secretion in human myeloid leukemia THP-1 cells in a calcium ion dependent manner through RAR/RXR signaling pathways, and this enhanced expression and secretion may be associated with the possible mechanisms of RAS.

Matrix metalloproteinase-2-induced epidermal growth factor receptor transactivation impairs redox balance in vascular smooth muscle cells and facilitates vascular contraction

Increased reactive oxygen species (ROS) formation may enhance matrix metalloproteinase (MMP)-2 activity and promote cardiovascular dysfunction. We show for the first time that MMP-2 is upstream of increased ROS formation and activates signaling mechanisms impairing redox balance. Incubation of vascular smooth muscle cells (VSMC) with recombinant MMP-2 increased ROS formation assessed with dihydroethidium (DHE) by flow cytometry. This effect was blocked by the antioxidant apocynin or by polyethylene glycol-catalase (PEG-catalase), and by MMP inhibitors (doxycycline or GM6001). Next, we showed in HEK293 cells that MMP-2 transactivates heparin-binding epidermal growth factor (HB-EGF) leading to EGF receptor (EGFR) activation and increased ROS concentrations. This effect was prevented by the EGFR kinase inhibitor Ag1478, and by phospholipase C (PLC) or protein kinase C (PKC) inhibitors (A778 or chelerythrine, respectively), confirming the involvement of EGFR pathway in MMP-2-induce responses. Next, we showed that intraluminal exposure of aortas to MMP-2 increased vascular MMP-2 levels detected by immunofluorescence and gelatinolytic activity (by in situ zimography) in association with increased ROS formation. This effect was inhibited by MMP inhibitors (phenanthroline or doxycycline) and by apocynin or PEG-catalase. MMP-2 also increased aortic contractility to phenylephrine and this effect was prevented by MMP inhibitor GM6001 and by apocynin or PEG-catalase, showing again that increased ROS formation mediates functional effects of MMP-2. These results show that MMP-2 activates the EGFR and triggers downstream signaling pathways increasing ROS formation and promoting vasoconstriction. These findings may have various implications for cardiovascular diseases.

SPOT-011 Studying the implication of the RAS/MAPK and PI3K/AKT/TOR pathways in prostate cancer through an in vivo model of drosophila accessory glands tumorigenesis

IntroductionClinical studies have demonstrated that, in prostate cancer, Ras/MAPK and PI3K/AKT/TOR signalling pathways are often aberrantly co-activated in tumours, their activation levels increasing again in resistance phases. As these pathways are important for many fundamental processes (proliferation, cell growth or cell cycle), they are likely implicated in prostate tumorigenesis. However, the relative implication of each of these two pathways during prostate tumorigenesis, especially during the early phases, is not fully understood. Thus, we want to define the molecular mechanisms that are implicated in the co-deregulation of these pathways, and their possible implication in prostate cancer initiation and progression.Material and methodsWe propose and alternative in vivo model of prostate tumorigenesis in drosophila, where accessory glands are a functional equivalent of the human prostate. In this model, we can cumulate molecular modifications for both the considered pathways, and so study the impact of one on tumorigenesis.Results and discussionsOur results show that only the hyperactivation of the Ras/MAPK pathway in accessory glands can promote tumorigenesis, with production of cell masses that recapitulate many cancer hallmarks including uncontrolled cell growth and proliferation, enhanced matrix metalloproteinases expression, loss of expression of epithelial markers, neovascularisation-like tracheogenesis. However, both pathways are necessary to tumorigenesis, but they display different roles: the Ras/MAPK pathway is activated earlier and is able to recruit the PI3K/AKT/TOR pathway. Furthermore, we have shown that tumour formation in the accessory glands relies on an overproduction of Epidermal Growth Factor, EGF, which is known to be expressed at abnormally high levels in prostate cancer.ConclusionWe have developed a Drosophila model that displays many phenomena that are described in human prostate tumorigenesis. We now want to decipher which molecular mechanisms are implicated in the PI3K/AKT/TOR pathway activation. Then, we want to evaluate the pertinence of our findings in the human pathology, by the use of human biopsy samples: our aim is to transfer our results to new therapeutic applications, as the discovery of new mechanisms could allow definition of new biomarkers and eventually of new therapeutic targets.

Study design and rationale to assess Doxycycline Efficacy in preventing coronary Artery Lesions in children with Kawasaki disease (DEAL trial) – A phase II clinical trial

BackgroundKawasaki disease (KD) is the most common acquired heart disease in children of the developed world, and triggers progressive coronary artery lesions (CAL) in 30% of cases if left untreated. Despite standard anti-inflammatory treatment for KD, CAL (dilation or aneurysm) still occurs in 5–10% of children, increasing their risk for fatal coronary artery complications. CAL is mediated by enhanced matrix metalloproteinase activity and elastin breakdown induced by the inflammatory process in the coronary artery wall. Doxycycline is an effective inhibitor of matrix metalloproteinases, and has been shown to reduce elastin breakdown and CAL in a mouse model of KD, but has not been evaluated in patients. ObjectiveWe aim to evaluate the efficacy of doxycycline in the prevention of CAL in children during the acute phase of KD. DesignThis is a phase II prospective, randomized, double-blinded, clinical trial in two steps. In Step 1, any child older than 1month with the diagnosis of KD will be included. Children with KD will be included in Step 2 if they develop coronary artery dilation (z-score≥2.5) within 20days from the onset of fever. Study subjects in Step 2 will be randomized to receive a 3-week course of doxycycline or placebo. EvaluationThe efficacy of a 3-week doxycycline course during the acute phase of KD will be evaluated by measuring the decline in coronary artery z-scores from baseline with doxycycline treatment compared to placebo. Clinical trial registrationThis study was registered on clinicaltrials.gov (NCT01917721).

Crosstalk Between Human Monocytic U937 Cells and Gingival Fibroblasts in Coculturally Enhanced Matrix Metalloproteinase-2 Expression.

Crosstalk Between Human Monocytic U937 Cells and Gingival Fibroblasts in Coculturally Enhanced Matrix Metalloproteinase-2 Expression.

Photoaging protective effects of BIOGF1K, a compound-K-rich fraction prepared from Panax ginseng

BackgroundBIOGF1K, a compound-K-rich fraction, has been shown to display anti-inflammatory activity. Although Panax ginseng is widely used for the prevention of photoaging events induced by UVB irradiation, the effect of BIOGF1K on photoaging has not yet been examined. In this study, we investigated the effects of BIOGF1K on UVB-induced photoaging events. MethodsWe analyzed the ability of BIOGF1K to prevent UVB-induced apoptosis, enhance matrix metalloproteinase (MMP) expression, upregulate anti-inflammatory activity, reduce sirtuin 1 expression, and melanin production using reverse transcription-polymerase chain reaction, melanin content assay, tyrosinase assay, and flow cytometry. We also evaluated the effects of BIOGF1K on the activator protein-1 signaling pathway, which plays an important role in photoaging, by immunoblot analysis and luciferase reporter gene assays. ResultsTreatment of UVB-irradiated NIH3T3 fibroblasts with BIOGF1K prevented UVB-induced cell death, inhibited apoptosis, suppressed morphological changes, reduced melanin secretion, restored the levels of type I procollagen and sirtuin 1, and prevented mRNA upregulation of MMP-1, MMP-2, and cyclo-oxygenase-2; these effects all occurred in a dose-dependent manner. In addition, BIOGF1K markedly reduced activator-protein-1-mediated luciferase activity and decreased the activity of mitogen-activated protein kinases (extracellular response kinase, p38, and C-Jun N-terminal kinase). ConclusionOur results strongly suggest that BIOGF1K has anti-photoaging activity and that BIOGF1K could be used in anti-aging cosmeceutical preparations.

PP11. RNA-SEQ ANALYSIS OF PRIMARY AND RECURRENT GLIOBLASTOMA IDENTIFIES NOVEL THERAPEUTIC STRATEGIES

AbstractGlioblastoma multiforme (GBM) is the most common and aggressive primary brain tumour. High patient mortality arising from a high propensity for tumour recurrence has driven a need to characterize the genetic changes associated with recurrent GBM. Here, we present a comprehensive landscape of transcriptome profiles of 10 primary and recurrent GBM cancers using RNA-seq, revealing extensive recurrent-specific gene expression changes and somatic mutations. Perturbation of the extracellular matrix, and enhanced ECM degradation, emerge as important changes accompanying GBM recurrence; this perturbation is multimodal and implicates diverse functions including enhanced matrix metalloproteinase (MMP) mediated ECM degradation, silencing of collagen encoding genes (e.g. COL1A1, COL1A2, COL3A1, COL6A3) or their mutation (e.g. COL7A1), and enhanced ECM degradation via dysregulation of SAPK/JNK signaling. Mutational signature analysis revealed striking enrichment of C to T transitions, accounting for 40 to 50% of all base-substitution mutations, implicating CT transition enhancing mutagenic processes in the etiology of GBM. Aberrant epigenetic regulation consistent with dysfunctional polycomb-mediated gene repression manifests in inappropriate activation of putative tumour promoter genes in recurrent tumours. Of 188 genes identified as differentially expressed in our GBM cohort, 38 genes were shared in a separate validation cohort of tumours from the TCGA GBM database. This includes seven genes (ADM, HPCAL4, SNCB, SV2B, TMEM130, H19, VSNL1) with correlations between gene expression and patient survival according to Kaplan-Meier plots generated from TCGA data. This study has highlighted new insights into GBM tumour recurrence and revealed potential novel candidates as prognostic biomarkers and rational therapeutic targets.