MMP-9 Promoter Research Articles

Luteolin suppresses androgen receptor-positive triple-negative breast cancer cell proliferation and metastasis by epigenetic regulation of MMP9 expression via the AKT/mTOR signaling pathway

BackgroundTriple-negative breast cancer (TNBC) represents up to 20% of all breast cancers. This cancer lacks the expression of the estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2. The current therapeutic strategy for patients with this subtype is the use of cytotoxic chemotherapy and surgery. Luteolin is a natural herbal flavonoid and a potential therapeutic candidate for multiple diseases. The use of a treatment that combines Chinese herbal medicine and western medicine is rising in Asia. PurposeThe present study evaluates the effects and molecular mechanisms involved with luteolin treatment and evaluates whether this herb affects androgen receptor-positive breast cancer cell proliferation or metastasis. Study DesignIn vitro evaluation of the effect of luteolin on androgen receptor-positive TNBC cell proliferation and metastasis MethodsCell viability analysis was used for the cytotoxicity test. Colony formation and Bromodeoxyuridine (BrdU) staining-based proliferation experiments were used for cell proliferation. Wound healing and transwell assays were used for in vitro migration/invasion. The RT-qPCR analysis was used for gene expression. Furthermore, ChIP-qPCR analysis was used for epigenetic modification of gene promoters. ResultsLuteolin significantly inhibited the proliferation and metastasis of androgen receptor-positive TNBC. Furthermore, luteolin inactivated the AKT/mTOR signaling pathway and reversed the epithelial-mesenchymal transition (EMT). The combination of luteolin and inhibitors of AKT/mTOR synergistically repressed an androgen receptor-positive TNBC cell proliferation and metastasis. Luteolin also downregulated MMP9 expression by decreasing the levels of the AKT/mTOR promoting H3K27Ac and H3K56A on the MMP9 promoter region. ConclusionOur findings indicate that luteolin inhibited the proliferation and metastasis of androgen receptor-positive TNBC by regulating MMP9 expression through a reduction in the levels of AKT/mTOR-inducing H3K27Ac and H3K56Ac.

The virulence factor GroEL promotes gelatinase secretion from cells in the osteoblast lineage: Implication for direct crosstalk between bacteria and adult cells

ObjectiveThe aim of this study was to demonstrate the influence of the virulence factor GroEL on osteoblast behavior by characterizing the changes of secreted gelatinases. DesignELISA was performed to detect GroEL from samples from patients with or without apical periodontitis. An apical periodontitis model was established in rats and the expression of MMP-2, MMP-9 and NF-κB was evaluated by immunofluorescence staining. The primary osteoblasts and osteoblast-like MC3T3 cells were stimulated with recombinant GroEL, and gelatin zymography was used to determine the activity and expression of MMP-2 and MMP-9. Western blot was used to screen signaling pathways, and immunofluorescence staining was performed to confirm the activated signaling. ResultsFirst, we found expression of GroEL to be higher in oral saliva, gingival crevicular fluid and periradicular granulation tissue of patients with apical periodontitis than it was in healthy control patients. We next found that recombinant GroEL could increase the activity of the gelatinases, MMP-2 and MMP-9, which were secreted by both primary osteoblasts and MC3T3 cells. In a rat apical periodontitis model, strong expression of gelatinases was confirmed. Then, we found that GroEL-enhanced gelatinase activity was mediated through activation of NF-κB signaling. Acetylated NF-κB accumulated in the cell nucleus and bound to the promoter of MMP-2 and MMP-9 genes, thus initiating their high expression. ConclusionThis study reveals a direct interaction between oral bacteria and adult cells by demonstrating that gelatinase secretion is induced by GroEL, which partially explains bone resorption through gelatinase activation.

Galangin Inhibits LPS-Induced MMP-9 Expression via Suppressing Protein Kinase-Dependent AP-1 and FoxO1 Activation in Rat Brain Astrocytes.

PurposeNeuroinflammation, characterized by the increased expression of inflammatory proteins such as matrix metalloproteinases (MMPs), plays a critical role in neurodegenerative disorders. Lipopolysaccharide (LPS) has been shown to upregulate MMP-9 expression through the activation of various transcription factors, including activator protein 1 (AP-1) and forkhead box protein O1 (FoxO1). The flavonoid 3,5,7-trihydroxy-2-phenyl-4H-1-benzopyran-4-one (galangin) has been demonstrated to possess antioxidant and anti-inflammatory properties in various types of cells. Here, we investigated the mechanisms underlying the inhibitory effect of galangin on LPS-induced MMP-9 expression in rat brain astrocytes (RBA-1 cells).MethodsPharmacological inhibitors and siRNAs were employed to explore the effects of galangin on LPS-challenged RBA-1 cells. Gelatin zymography, Western blotting, real-time PCR, and a luciferase reporter assay were used to detect MMP-9 activity, protein expression, mRNA levels, and promoter activity, respectively. The protein kinases involved in the LPS-induced MMP-9 expression were determined by Western blot. A chromatin immunoprecipitation (ChIP) assay was employed to evaluate the activity of c-Jun at the MMP-9 promoter.ResultsGalangin treatment attenuated the LPS-mediated induction of MMP-9 protein and mRNA expression, as well as the activity at the MMP-9 promoter. In addition, galangin exerted its inhibitory effects on MMP-9 expression through suppressing the LPS-stimulated activation of proline-rich tyrosine kinase (Pyk2), platelet-derived growth factor receptor beta (PDGFRβ), phosphoinositide 3-kinase (PI3K), protein kinase B (Akt), mammalian target of rapamycin (mTOR), and mitogen-activated protein kinases (MAPKs). Pretreatment with galangin attenuated the LPS-induced phosphorylation of c-Jun and FoxO1. LPS-induced cell migration was also suppressed by galangin pretreatment.ConclusionGalangin attenuates the LPS-induced inflammatory responses, including the induction of MMP-9 expression and cell migration, via inhibiting Pyk2/PDGFRβ/PI3K/Akt/mTOR/JNK1/JNK2 and p44/p42 MAPK cascade-dependent AP-1 and FoxO1 activities. These results provide new insights into the mechanisms through which galangin mitigates LPS-induced inflammatory responses, and suggest novel strategies for the management of LPS-related brain diseases.

Association of MMP2 and MMP9 gene polymorphisms with the recurrent spontaneous abortion: A meta-analysis.

Recurrent spontaneous abortion (RSA) accounts for the most common complication of early pregnancy in humans. Matrix metalloproteinases (MMPs) play important regulatory roles in implantation and placentation to ensure a successful pregnancy. Single nucleotide polymorphisms (SNPs) have been identified in the promoters of MMP2 and MMP9 genes. However, the associations between MMP2 and MMP9 SNPs and the RSA risk remain unclear. The aim of this meta-analysis was to investigate whether MMP2 (-735C>T) and MMP9 (-1562C>T) SNPs are associated with the risk of RSA. Literatures published before 17th April 2020 were screened to identify the eligible studies. Heterogeneity, sensitivity and publication bias analysis were analyzed by the STATA software. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated by the Review Manager software with fixed effects model. After screening, 2 studies for MMP2 (-735C>T) (278 RSA cases and 265 controls) and 4 studies for MMP9 (-1562C>T) (520 RSA cases and 512 controls) were enrolled in this meta-analysis. Results showed that MMP2 (-735C>T) presented a statistically significant association with the risk of RSA under allelic (T vs C: OR=1.50, 95% CI=1.14-1.98, P=0.004, I2=31%), heterozygote (CT vs CC: OR=1.74, 95% CI=1.22-2.50, P=0.003, I2=41%) and dominant (TT+CT vs CC: OR=1.74, 95% CI=1.23-2.45, P=0.002, I2=40%) genetic models. MMP9 (-1562C>T) in allelic (T vs C: OR=1.34, 95% CI=1.08-1.65, P=0.007, I2=0%), heterozygote (CT vs CC: OR=1.38, 95% CI=1.06-1.79, P=0.02, I2=0%) and dominant (TT+CT vs CC: OR=1.41, 95% CI=1.10-1.82, P=0.008, I2=0%) genetic models were significantly correlated with the RSA risk. Our meta-analysis results suggest that MMP2 -735T allele and MMP9 -1562T allele have significant association with the risk of RSA.

Up-regulation of MMP-2 by histone H3K9 β-hydroxybutyrylation to antagonize glomerulosclerosis in diabetic rat.

Besides energy supply, β-hydroxybutyrate (BHB) acts as a bioactive molecule to play multiple protective roles, even in diabetes and its complications. The aim of this study was to investigate the antagonizing effects of BHB against diabetic glomerulosclerosis and the underlying mechanism. Male Sprague-Dawley rats were intraperitoneally injected with streptozotocin to induce diabetes and then treated with different concentrations of β-hydroxybutyrate. After 10weeks, body weight, blood glucose, serum creatinine and 24-h urine protein were examined. Glomerular morphological changes and the contents of collagen type IV (COL IV) were evaluated. Then, transforming growth factor (TGF)-β/Smad3 contents and matrix metalloproteinase-2 (MMP-2) generation were detected. Moreover, the total contents of trans-activating histone H3K9 β-hydroxybutyrylation (H3K9bhb) and the contents of H3K9bhb in the Mmp-2 promoter were measured. It was firstly confirmed that BHB treatments reduced renal biochemical indicators and attenuated glomerular morphological changes of the diabetic rats, with COL IV content decreased in a concentration-dependent manner. Then, BHB treatments were found to up-regulate renal MMP-2 generation of the diabetic rats significantly, while not affecting the increased TGF-β/Smad3 contents. Furthermore, the contents of H3K9bhb in the Mmp-2 promoter were elevated significantly for the middle and high concentrations of BHB treatments, up-regulating MMP-2 generation. BHB treatments could up-regulate MMP-2 generation via causing elevated H3K9bhb in its promoter to antagonize glomerulosclerosis in the diabetic rats.

Sesquiterpene lactone Zaluzanin D alters MMP-9 promoter methylation in differentiated THP-1 monocytic cells and down regulates inflammatory cytokines IL-1β and TNF-α

Zaluzanin D (ZD) is a sesquiterpene lactone isolated from the leaves of Vernonia arborea. Earlier studies have highlighted the Sesquiterpene lactones (SLs) as molecules of medicinal value. The current study investigates the anti-inflammatory potential of ZD and its biotransformed derivatives in PMA differentiated human monocytic THP-1 cells. ZD and its fungal biotransformed derivatives Zaluzanin C (ZC) and 11,13- dihydrozaluzanin C (DZC) were screened for anti-inflammatory activity using their IC50 concentration. ZD showed significant ability to reduce PMA mediated THP-1 activation, while both ZC and DZC did not show any anti-inflammatory activity. Further studies revealed that ZD had ability to attenuate intracellular reactive oxygen species production in THP-1 cells as confirmed with FACS and fluorescence microscope experiments. Similarly, Oil red O (ORO) assay showed ability of ZD to inhibit lipid accumulation in monocytes. ZD also significantly reduced the expression of pro-inflammatory markers, tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, MMP (Matrix metalloproteinases)-9 and MMP-2 as observed with RT-PCR and ELISA. Interestingly the molecule ZD also partially reversed DNA methylation levels in the PMA activated THP-1 cells. This indicated the ability of ZD to influence the epigenetic machinery of the cell. Overall the current study indicates that ZD has ability to attenuate inflammation in differentiated human THP-1 cells by regulating genes involved in the atherosclerosis inflammatory pathway. Thus ZD could potentially be used to modulate inflammation in atherosclerosis like disorders wherein monocytes play a key role.

In Vitro and In Vivo Antitumor Efficacy of Hizikia fusiforme Celluclast Extract against Bladder Cancer

Various physiological benefits have been linked to Hizikia fusiforme (HF), an edible brown seaweed. Here, fucose-containing sulfated polysaccharides were extracted from celluclast-processed HF (SPHF) and their antitumor efficacy against bladder cancer was evaluated in vitro and in vivo. SPHF possesses high sulfated polysaccharide and fucose contents and free radical scavenging activities compared to those of celluclast-processed HF extracts (CHF). SPHF inhibited bladder cancer EJ cell proliferation via G1-phase cell cycle arrest. This was due to the induction of p21WAF1 expression associated with the downregulation of CDKs and cyclins. Moreover, JNK phosphorylation was identified as an SPHF-mediated signaling molecule. SPHF treatment also hindered the migration and invasion of EJ cells by inhibiting MMP-9 expression, which was attributed to the repression of transcriptional binding to NF-κB, AP-1, and Sp-1 in the MMP-9 promoter region. In an animal study, SPHF treatment suppressed EJ tumor growth in xenograft mice similarly to cisplatin. Furthermore, no toxicity signs were found after weight loss assessment, biochemical tests, and organ tissue immunostaining during oral administration of 20–200 mg/kg SPHF for 20 days. Therefore, our study demonstrates the antitumor efficacy of SPHF in vitro and in vivo, thus highlighting its potential for bladder cancer treatment development.

Bromodomain Epigenetic Protein Promotes Metastatic Potential in Melanoma Cells through Increased Invasiveness and Decreased Macrophage-Mediated Phagocytosis

Bromodomain Epigenetic Protein Promotes Metastatic Potential in Melanoma Cells through Increased Invasiveness and Decreased Macrophage-Mediated Phagocytosis

Polymorphisms in the Promoters of MMP-1 and TIMP-1 Genes in Patients with Acne Vulgaris

Grid metalloproteinases (MMPs) and their particular inhibitors (tissue inhibitors of metalloproteinases, TIMPs) are fundamental for the redesigning of the extracellular framework, and incendiary infections, for example, skin inflammation. Skin break out vulgaris is an incessant incendiary illness that influences most of the populace. It is described by comedones, pustules, and papules. In this examination, it was expected to research the possible relationship between MMP-1 quality polymorphism and the hazard for skin inflammation advancement. Polymorphism of MMP-1 Gene was estimated in 60 patients with skin break out vulgaris and 40 solid controls utilizing polymerase chain response (PCR). A noteworthy connection between MMP-1 (1607 A/G) polymorphism and skin inflammation vulgaris was recognized.

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

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

Smooth Muscle Sirtuin 1 Blocks Thoracic Aortic Aneurysm/Dissection Development in Mice.

Advancing age is the major risk factor for thoracic aortic aneurysm/dissection (TAAD). However, the causative link between age-related molecules and TAAD remains elusive. Here, we investigated the role of Sirtuin 1 (SIRT1, also known as class III histone deacetylase), the best studied member of the longevity-related Sirtuin family, in TAAD development in vivo. We used male smooth muscle-specific SIRT1 transgenic (ST-Tg) mice, smooth muscle-specific SIRT1 knockout (ST-KO) mice, and their wild-type (WT) littermates on a C57BL/6J background to establish a TAAD model induced by oral administration of 3-aminopropionitrile fumarate (BAPN). We analyzed the incidence and fatality rates of TAAD in the groups. We examined matrix metallopeptidase 2 (MMP2) and MMP9 expression in aortas or cultured A7r5 cells via western blotting and real-time polymerase chain reaction (PCR). We performed chromatin immunoprecipitation (ChIP) to clarify the epigenetic mechanism of SIRT1-regulated MMP2 expression in vascular smooth muscle cells (VSMCs). BAPN treatment markedly increased the incidence of TAAD in WT mice but caused less disease in ST-Tg mice. Moreover, ST-KO mice had the highest BAPN-induced TAAD fatality rate of all the groups. Mechanistically, SIRT1 overexpression resulted in lower MMP2 and MMP9 expression after BAPN treatment in both mouse aortas and cultured A7r5 cells. The downregulation of BAPN-induced MMP2 expression by SIRT1 was mediated by deacetylation of histone H3 lysine 9 (H3K9) on the Mmp2 promoter in the A7r5 cells. Our findings suggest that SIRT1 expression in SMCs protects against TAAD and could be a novel therapeutic target for TAAD management.

DNA methylation and mRNA expression of IGF-1 and MMP-2 after form-deprivation myopia in guinea pigs.

The molecular mechanism of form-deprivation myopia is unclear. This study was aimed to investigate the roles of scleral DNA methylation and mRNA expression of IGF-1 and MMP-2 in a guinea pig model of form-deprivation myopia. Seventy 2-week-old male guinea pigs were assigned to three groups: (1) zero week group that was used to collect baseline data; (2) monocular deprivation treatment (MDT) group, in which a thin slice of opaque latex glove was placed over the right eyes of the animals for four weeks, and the left eyes were untreated and served as the monocular contralateral control (MCC) group; (3) control group (CG), in which the animals grew four weeks, but received no manipulation. Animals in each group were evenly divided for DNA methylation assay and quantitative PCR (qPCR). After eye enucleation, the sclerae were harvested for DNA methylation assay and qPCR. The DNA methylation pattern in the promoter and exon regions of IGF-1 and MMP-2, along with the mRNA expression level of them, were determined by base-specific cleavage and mass spectrometry and qPCR, respectively. After four weeks of form-deprivation, DNA methylation at 4/8 cytosine-guanine sites in the IGF-1 promoter was significantly lower in the MDT eyes than in the MCC or CG eyes. In addition, the level of IGF-1 mRNA was moderately higher in MDT eyes compared to the MCC eyes and CG eyes. DNA methylation at 4/14 cytosine-guanine sites in the MMP-2 gene was very low, and no significant change was observed between the MDT eyes and the MCC or CG ones. However, the level of MMP-2 mRNA in MDT eyes was significant higher compared with MCC eyes and CG eyes, with an increase of 217% and 222%, respectively. In our guinea pig model of form-deprivation myopia, the methylation of four cytosine-guanine sites in the IGF-1 gene promoter was significantly lower in the sclera after four weeks of MDT, and the transcription level of scleral IGF-1 was moderately higher. Hence, the IGF-1 gene methylation might play a role in the pathogenesis of form-deprivation myopia in guinea pigs. The level of MMP-2 mRNA in the sclera of MDT eyes was significantly higher, but not regulated by the methylation pathway, as the methylation status of MMP-2 was unchanged.

Cervical transverse MRI in ALS diagnosis and possible link to VEGF and MMP9 single nucleotide polymorphisms. Case Report

We report the case of a patient diagnosed with ALS and presenting with a “snake eyes” sign on axial cervical MRI. Given the peculiar radiological presentation and our patient’s young age, we decided to perform a genetic analysis to better investigate the molecular mechanisms underlying the pathogenesis of his clinical phenotype. In our study, we identified different polymorphisms of interest at the level of VEGF and MMP9 promoters that we suggest could contribute to neuronal sufferance by promoting vascular abnormalities and tissue hypoxia, in accordance with previous published papers. When combined with repetitive mechanical stress at cervical level, as evident in our patient’s case, the resultant radiological image appears to be the “snake eyes” sign in the anterior horns of the cervical spinal cord, while the clinical phenotype is that of an atypical ALS case, possibly an ALS-mimic. To correctly identify and diagnose such patients, in particular when disease course and neuroradiological data do not correspond to those of a classical ALS case, we underline the importance of axial cervical MRI studies and suggest the possibility of their implementation into routine clinical practice.

DNA methylation in the promoters of PD-L1, MMP9, ARG1, galectin-9, TIM-3, VISTA and TGF-β genes in HLA-DR– myeloid cells, compared with HLA-DR+ antigen-presenting cells

ABSTRACT Myeloid cells, including antigen-presenting cells (APCs) and myeloid-derived suppressor cells (MDSCs) play opposing roles to orchestrate innate and adaptive immune responses during physiological and pathological conditions. We investigated the role of DNA methylation in regulating the transcription of inhibitory/suppressive molecules in myeloid suppressive cells (identified as CD33+HLA-DR–) in comparison to APCs. We selected a number of immune checkpoints (ICs), IC ligands, and immunosuppressive molecules that have been implicated in MDSC function, including PD-L1, TIM-3, VISTA, galectin-9, TGF-β, ARG1 and MMP9. We examined their mRNA expression levels, and investigated whether DNA methylation regulates their transcription in sorted myeloid cell subpopulations. We found that mRNA levels of PD-L1, TIM-3, TGF-β, ARG1 and MMP9 in CD33+HLA-DR– cells were higher than APCs. However, VISTA and galectin-9 mRNA levels were relatively similar in both myeloid subpopulations. CpG islands in the promoter regions of TGF-β1, TIM-3 and ARG1 were highly unmethylated in CD33+HLA-DR–cells, compared with APCs, suggesting that DNA methylation is one of the key mechanisms, which regulate their expression. However, we did not find differences in the methylation status of PD-L1 and MMP9 between CD33+HLA-DR– and APCs, suggesting that their transcription could be regulated via other genetic and epigenetic mechanisms. The promoter methylation status of VISTA was relatively similar in both myeloid subpopulations. This study provides novel insights into the epigenetic mechanisms, which control the expression of inhibitory/suppressive molecules in circulating CD33+HLA-DR– cells in a steady-state condition, possibly to maintain immune tolerance and haemostasis.

Erratum to "Polymorphism of MMP1 and MMP3 promoter regions and HR-HPV infection in women from Burkina Faso and Côte d'Ivoire".

The single nucleotide polymorphism (SNP) of the promoter region of MMP-1 (at 1607 bp) and MMP-3 (at 1171 bp) create Ets binding sites. Correlations between these SNPs and sensitivity to several biological processes such as metastasis and recurrence of cancer have been reported in several studies. In this case-control study, we looked for these SNPs in women infected with or not with high-risk human papillomaviruses (HR-HPV). The frequency, distribution and correlation of these SNPs with the presence or absence of HR-HPV infection were evaluated. Genotypes 1G1G, 1G2G and 2G2G for MMP1 and genotypes 5A5A, 5A6A, 6A6A for MMP3 were found in our study population. In general, we noted that the 1G (40.8%) and 2G (64.8%) alleles were more frequent in non-infected women and infected women, respectively, and more specifically this difference was significant in women from Cote d'Ivoire. These results, although yet to be reaffirmed with assays for quantifying the mRNA of these genes, suggest that the SNP of the MMP-1 promoter could promote infection with HR-HPV.

Polymorphism of MMP1 and MMP3 promoter regions and HR-HPV infection in women from Burkina Faso and Côte d'Ivoire.

The single nucleotide polymorphism (SNP) of the promoter region of MMP-1 (at 1607 bp) and MMP-3 (at 1171 bp) create Ets binding sites. Correlations between these SNPs and sensitivity to several biological processes such as metastasis and recurrence of cancer have been reported in several studies. In this case-control study, we looked for these SNPs in women infected with or not with high-risk human papillomaviruses (HR-HPV). The frequency, distribution and correlation of these SNPs with the presence or absence of HR-HPV infection were evaluated. Genotypes 1G1G, 1G2G and 2G2G for MMP1 and genotypes 5A5A, 5A6A, 6A6A for MMP3 were found in our study population. In general, we noted that the 1G (40.8%) and 2G (64.8%) alleles were more frequent in non-infected women and infected women, respectively, and more specifically this difference was significant in women from Côte d'Ivoire. These results, although yet to be reaffirmed with assays for quantifying the mRNA of these genes, suggest that the SNP of the MMP-1 promoter could promote infection with HR-HPV.

Abnormal Hypermethylation of CpG Dinucleotides in Promoter Regions of Matrix Metalloproteinases Genes in Breast Cancer and its Relation to Epigenomic Subtypes and HER2 Overexpression

Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) substantially contribute to the regulation of intercellular interactions and thereby play a role in maintaining the tissue structure and function. We examined methylation of a subset of 5’-cytosine-phosphate-guanine-3’ (CpG) dinucleotides in promoter regions of the MMP2, MMP11, MMP14, MMP15, MMP16, MMP17, MMP21, MMP23B, MMP24, MMP25, MMP28, TIMP1, TIMP2, TIMP3, and TIMP4 genes by methylation-sensitive restriction enzyme digestion PCR. In our collection of 183 breast cancer samples, abnormal hypermethylation was observed for CpGs in MMP2, MMP23B, MMP24, MMP25, and MMP28 promoter regions. The non-methylated status of the examined CpGs in promoter regions of MMP2, MMP23B, MMP24, MMP25, and MMP28 in tumors was associated with low HER2 expression, while the group of samples with abnormal hypermethylation of at least two of these MMP genes was significantly enriched with HER2-positive tumors. Abnormal methylation of MMP24 and MMP25 was significantly associated with a CpG island hypermethylated breast cancer subtype discovered by genome-wide DNA bisulfite sequencing. Our results indicate that abnormal hypermethylation of at least several MMP genes promoters is a secondary event not directly functional in breast cancer (BC) pathogenesis. We suggest that it is elevated and/or ectopic expression, rather than methylation-driven silencing, that might link MMPs to tumorigenesis.

E2F5 promotes prostate cancer cell migration and invasion through regulation of TFPI2, MMP-2 and MMP-9.

Previously, our laboratory demonstrated that a deregulated E2F5/p38/SMAD3 axis was associated with uncontrolled cellular proliferation in prostate cancer (PCa). Here, we investigate the role of E2F5 in PCa in further details. RNAi-mediated E2F5 knockdown and pathway-focused gene expression profiling in PC3 cells identified TFPI2 as a downstream target of E2F5. Manipulation of E2F5 expression was also found to alter MMP-2 and MMP-9 levels as detected by Proteome Profiler array, western blot and reverse transcription coupled quantitative polymerase chain reaction Site-directed mutagenesis, dual-luciferase assays and chromatin immunoprecipitation with anti-E2F5-IgG coupled with qPCR confirmed recruitment of E2F5 on TFPI2, MMP-2 and MMP-9 promoters. RNAi-mediated knockdown of E2F5 expression in PC3 caused a significant alteration of cell migration while that of TFFI2 resulted in a modest change. Abrogation of E2F5 and TFPI2 expression was associated with significant changes in the gelatinolytic activity of active forms of MMP-2 and MMP-9. Moreover, E2F5, MMP-2 and MMP-9 levels were elevated in biopsies of PCa patients relative to that of benign hyperplasia, while TFPI2 expression was reduced. MMP-9 was coimmunoprecipitated with anti-TFPI2-IgG in PCa tissue samples suggesting a direct interaction between the proteins. Finally, artemisinin treatment in PC3 cells repressed E2F5 along with MMP-2/MMP-9 while triggering TFPI2 expression which alleviated PC3 aggressiveness possibly through inhibition of MMP activities. Together, our study reinstates an oncogenic role of E2F5 which operates as a dual-function transcription factor for its targets TFPI2, MMP-2 and MMP-9 and promotes cellular invasiveness. This study also indicates a therapeutic potential of artemisinin, a natural compound which acts by correcting dysfunctional E2F5/TFPI2/MMP axis in PCa.

Follicular dendritic cell migration is facilitated by the expression of matrix metalloproteinase 3 through TNFα-ERK1/2-AP-1 signaling

Abstract Folliculardendritic cells (FDCs) are the crucial stromal components in the germinal center and have the pivotal roles in high-affinity antibody production. FDCs are developed and maintained in the germinal centers by lymphotoxin α1/β2 and tumor necrosis factor-α (TNFα). However, many of the features of FDCs are still not identified, particularly its migration. Here, we demonstrate for the first time that the migration of human FDCs is enhanced by TNFα-mediated metalloproteinase 3 (MMP3) expression. MMP3 was highly expressed in the normal human germinal centers. MMP3 expression was markedly increased in human primary FDCs by TNFα. TNFα induced the ERK1/2 phosphorylation and the promoter activity of the AP-1 binding sites in the Mmp3 promoter region. TNFα treatment increased FDC migration and the knockdown of MMP3 significantly reduced the TNFα-induced migration of FDCs. Overall, we identified a novel control mechanism for the expression of MMP3 in FDCs to modulate the migration, which may play an important role in lymphoid follicle formation. Since lymphoid follicles are observed in the lesions of autoimmune diseases and lymphomas, controlling the MMP3/TNFα-mediated migration of FDCs can provide novel insight into therapeutic modalities for aberrant germinal center-associated disorders.

Mapping Connectome in Mammalian Brain: A Novel Approach by Bioengineering Neuro-Glia specific Vectors

The connectome is the comprehensive map of the brain represented by wiring diagram of the full set of neuro-glia and synapses within entire brain of an organism. Some recent scientific efforts have successfully been made to visualize such map at neuro-glial networking level, however, capturing it as one unit of the entire brain have never been elucidated. Moreover, in order to derive structure-function relationship of different brain regions in response to a defined stimulus, there is a need to elucidate the connectome at single neuro-glial ensemble level after brain is challenged with the known memory function. This needs developing molecular approaches to tag neuro-glial activities in response to a conditioned brain function. Such approaches of using specific molecular tags have been tried to visualize independently neuron and glial specific events in response to a memory function, however, they could not tag the connectome together at single neuro-glia ensemble level.Therefore, there is a need to develop new methods for mapping entire connectome up to a single neuro-glial precision and resolution, with a purpose of tagging specific brain region accountable to execute a special memory formation process. The present hypothetical paper aims to propose a novel molecular method to generate the structural connectome at neuro-glial level in mice brain. Herein, we propose to tag the entire connectome at neuro-glia precision by generating a transgenic mice via transposing and recombining engineered novel “Neuro-Glia specific Vectors” (NGVs: specific to excitatory neurons, inhibitory neurons and glial cells) vis a vis “Transcriptional/ Translational Messenger (TMs: specific to metalloproteinases, MMP-9) coupled with different color protein tags, followed by the Clarity. Herein, the NGVs will be translated via Neuro-glia specific promoters, while TMs will be translated via endogenous MMP-9 promoter in all neuro-glial cells. The viability of all constructs will be verified in cortical/ hippocampal culture by inducing them to undergo chemically induced long term potentionation (cLTP) following visualization of different colored pattern. This will be further confirmed by Immunostaning, Western Blot and RT-PCR analysis. Additionally, in this approach, one can decipher the dynamics of molecular and cellular events associated with MMP-9 seretome by monitoring the trafficking of tagged endogenous MMP-9 protein after neuronal stimulation by cLTP in vitro. However, for visualizing complete connectome, the adult transgenic mice will be challenged with fear consolidation (Fear context and contextual cue) tests followed by Clarity coupled Light Sheet Microscopy to analyze neuro-glia ensemble following whole brain imaging.