Mechanisms of matrix metalloproteinase-2 (mmp-2) transcriptional repression by progesterone in jar choriocarcinoma cells

<p>Abstract</p> <p>Background</p> <p>Although the MMP-2 promoter lacks a canonical progesterone response element (PRE), the hormone inhibits MMP-2 expression and is part of treatment protocols in gynecological invasive pathologies, including endometriosis and endometrial hyperplasia. This study aimed to explore the mechanism by which progesterone inhibits MMP-2 expression.</p> <p>Methods</p> <p>The effect of progesterone on MMP-2 expression in the JAR human choriocarcinoma cell line was analyzed by gelatin zymography. MMP-2 transcript expression was studied using Northern blot and semi-quantitative RT-PCR. Rat promoter deletion analysis, electrophoretic mobility shift and chromatin immuno-precipitation assays were performed in order to locate the DNA binding site and the transcription factors involved in MMP-2 regulation.</p> <p>Results</p> <p>Progesterone significantly decreased secretion of pro-MMP-2 and MMP-2 transcript expression level in a dose-dependent manner. Progesterone (1 microM) significantly decreased both human and rat MMP-2 promoter activity (80.1% +/- 0.3 and 81.3% +/- 0.23, respectively). Progesterone acts through the SP1 family transcription factors-binding site, located between -1433 and -1342 bp region from the transcriptional start site of the rat MMP-2 promoter, which are present in the orthologous human MMP-2 promoter. Progesterone receptor (PR), SP2, SP3 and SP4 proteins are constitutively bound to this consensus sequence.</p> <p>Conclusion</p> <p>Progesterone reducesPR and SP4 binding to the MMP-2 promoter, thereby suppressing transcription. Progesterone also promotes SP4 degradation. These novel mechanisms of MMP-2 regulation by progesterone provide the biological rationale for the use of progesterone in clinical settings associated with increased MMP-2 expression.</p

Differential transcriptional activation of matrix metalloproteinase-2 and membrane type-1 matrix metalloproteinase by experimental deep venous thrombosis and thrombin

ObjectiveResolution of deep venous thrombosis (DVT) is involved in the pathogenesis of postthrombotic syndrome. Matrix metalloproteinases (MMPs) are a family of proteolytic enzymes that are critical in angiogenesis and tissue remodeling. We hypothesized that MMP-2 and its membrane-bound activator membrane type-1 matrix metalloproteinase (MT1-MMP) expression would be expressed and activated during the resolution of DVT.MethodsDVT was generated by caval ligation in wild-type and MMP-2 transgenic reporter mice. Ligated and sham-operated (control) cavae were analyzed for MMP-2 transcription (β–galactosidase activity in MMP-2 reporter mice) and MT1-MMP mRNA by real-time polymerase chain reaction. MMP-2 activity was determined by zymography, and immunohistochemical staining for β–galactosidase and MT1-MMP protein was used to localize expression. Human umbilical vascular endothelial cells (HUVEC) were treated with 10 U/mL thrombin and MMP-2 and MT1-MMP mRNA levels and MMP-2 activity was determined.ResultsMMP-2 activity increased 71% (n = 5, P < .05) at day 8 in ligated vs control cavae by zymography. β-galactosidase activity showed a 1.2-fold (n = 8, P < .05) and 1.7-fold (n = 8, P < .05) induction in MMP-2 transcription at day 3 and day 8, respectively. No significant MT1-MMP gene induction was seen at day 3 in ligated vs control cavae, but MT1-MMP mRNA was upregulated 2.5-fold (n = 8, P < .05) in ligated cavae at day 8. Immunohistochemical staining localized MMP-2 and MT1-MMP expression to the vein wall and cellular infiltrates of the thrombus. Thrombin-treated HUVEC showed differential responses of MMP-2 and MT1-MMP. Zymography of conditioned media and cell lysates illustrated a 220% (152.6 ± 8.6 vs 69.445 ± 5.46 pixels/unit area, n = 5, P < .05) and 150% (74.1 ± 7.3 vs 49.2 ± 5.7 pixels/unit area, n = 5, P < .05) increases in MMP-2 activity respectively. MMP-2 mRNA levels were downregulated 30% (0.48 ± 0.023 vs 0.63 ± 0.035 copies of MMP-2 mRNA/copy GAPDH, n = 5, P < .05), whereas MT1-MMP message was upregulated 250% (0.147 ± 0.009 vs 0.059 ± 0.005 copies of MT1-MMP mRNA/copy GAPDH, n = 5, P < .05).ConclusionsResolution of DVT is associated with increased MMP-2 transcription and activity as well as MT1-MMP expression. Thrombin may mediate the increase in MT1-MMP noted in DVT. This is the first article studying MMP-2 and MT1-MMP transcription in DVT. These findings add DVT resolution to the class of inflammatory and fibrotic disorders in which transcriptional activation of the MT1-MMP/MMP-2 genes occurs and identify a potential therapeutic target to modulate this clinically relevant process.Clinical RelevancePostthrombotic syndrome remains a significant clinical problem after deep venous thrombosis (DVT), but the cellular and molecular mechanisms involved in thrombus resolution and vein wall fibrosis remain undefined. Matrix metalloproteinase (MMP) enzymes are critical to cell migration and matrix breakdown. We identify gene transcription and activity of two MMP isoforms, MMP-2 and MMP-14 (membrane type MMP 1, MT1-MMP) in the resolution phase of experimental DVT and in thrombin-treated endothelial cells. These studies define new proteases potentially important to resolution of DVT and development of postthrombotic syndrome

Short term doxycycline treatment induces sustained improvement in myocardial infarction border zone contractility.

Decreased contractility in the non-ischemic border zone surrounding a MI is in part due to degradation of cardiomyocyte sarcomeric components by intracellular matrix metalloproteinase-2 (MMP-2). We recently reported that MMP-2 levels were increased in the border zone after a MI and that treatment with doxycycline for two weeks after MI was associated with normalization of MMP-2 levels and improvement in ex-vivo contractile protein developed force in the myocardial border zone. The purpose of the current study was to determine if there is a sustained effect of short term treatment with doxycycline (Dox) on border zone function in a large animal model of antero-apical myocardial infarction (MI). Antero-apical MI was created in 14 sheep. Seven sheep received doxycycline 0.8 mg/kg/hr IV for two weeks. Cardiac MRI was performed two weeks before, and then two and six weeks after MI. Two sheep died prior to MRI at six weeks from surgical/anesthesia-related causes. The remaining 12 sheep completed the protocol. Doxycycline induced a sustained reduction in intracellular MMP-2 by Western blot (3649±643 MI+Dox vs 9236±114 MI relative intensity; p = 0.0009), an improvement in ex-vivo contractility (65.3±2.0 MI+Dox vs 39.7±0.8 MI mN/mm2; p&lt;0.0001) and an increase in ventricular wall thickness at end-systole 1.0 cm from the infarct edge (12.4±0.6 MI+Dox vs 10.0±0.5 MI mm; p = 0.0095). Administration of doxycycline for a limited two week period is associated with a sustained improvement in ex-vivo contractility and an increase in wall thickness at end-systole in the border zone six weeks after MI. These findings were associated with a reduction in intracellular MMP-2 activity

Evaluation of the pilot of domestic violence protection orders

In 2011/12, a 15 month pilot took place in three police force areas (Greater Manchester, West Mercia and Wiltshire) to test a new civil provision, Domestic Violence Protection Orders (DVPOs). DVPOs were designed to provide immediate protection for victim-survivors following a domestic violence incident in circumstances where, in the view of the police, there are no other enforceable restrictions that can be placed upon the perpetrator. DVPOs aim to give victim-survivors time, space and support to consider their options by placing conditions on perpetrators, including restricting/removing perpetrators from households, and preventing contact with, or molestation of, victim-survivors. The approach, introduced by the Crime and Security Act 2010, comprises an initial temporary notice (Domestic Violence Protection Notice, DVPN), authorised by a senior police officer and issued to the perpetrator by the police, followed by a DVPO that can last from 14 to 28 days, imposed at the magistrates’ court

Evaluation of the pilot of domestic violence protection orders

In 2011/12, a 15 month pilot took place in three police force areas (Greater Manchester, West Mercia and Wiltshire) to test a new civil provision, Domestic Violence Protection Orders (DVPOs). DVPOs were designed to provide immediate protection for victim-survivors following a domestic violence incident in circumstances where, in the view of the police, there are no other enforceable restrictions that can be placed upon the perpetrator. DVPOs aim to give victim-survivors time, space and support to consider their options by placing conditions on perpetrators, including restricting/removing perpetrators from households, and preventing contact with, or molestation of, victim-survivors. The approach, introduced by the Crime and Security Act 2010, comprises an initial temporary notice (Domestic Violence Protection Notice, DVPN), authorised by a senior police officer and issued to the perpetrator by the police, followed by a DVPO that can last from 14 to 28 days, imposed at the magistrates’ court

Nonperturbative Effects from the Resummation of Perturbation Theory

Using the general argument in Borel resummation of perturbation theory that links the divergent perturbation theory to the nonperturbative effect we argue that the nonperturbative effect associated with the perturbation theory should have a branch cut only along the positive real axis in the complex coupling plane. The component in the weak coupling expansion of the nonperturbative amplitude, which usually includes the leading term in the weak coupling expansion, that gives rise to the branch cut can be calculated in principle from the perturbation theory combined with some exactly calculable properties of the nonperturbative effect. The realization of this mechanism is demonstrated in the double well potential and the two-dimensional O(N) nonlinear sigma model. In these models the leading term in weak coupling of the nonperturbative effect can be obtained with good accuracy from the first terms of the perturbation theory. Applying this mechanism to the infrared renormalon induced nonperturbative effect in QCD, we suggest some of the QCD condensate effects can be calculated in principle from the perturbation theory.Comment: 21 Pages, 1 Figure; To appear in Phys Rev

Methodology supporting cognitive activity in students creative projects

Scrum-described software development methodology small student teams within the discipline of "Creative project" in "Computer Science and Engineering." It is shown that the iterative technology flexible software development projects based on the principles of system analysis and design international standards, ideally support the structure and content of the set active practice of students that will be formed from them in the process of studying this discipline

Sub-Doppler laser cooling of 40K with Raman gray molasses on the D2 line

Gray molasses is a powerful tool for sub-Doppler laser cooling of atoms to low temperatures. For alkaline atoms, this technique is commonly implemented with cooling lasers which are blue-detuned from either the D1 or D2 line. Here we show that efficient gray molasses can be implemented on the D2 line of 40K with red-detuned lasers. We obtained temperatures of 48(2)µK, which enables direct loading of 9.2(3)x106 atoms from a magneto-optical trap into an optical dipole trap. We support our findings by a one-dimensional model and three-dimensional numerical simulations of the optical Bloch equations which qualitatively reproduce the experimentally observed cooling effects.PreprintPublisher PDFPeer reviewe

Enhanced cardiac expression of two isoforms of matrix metalloproteinase-2 in experimental diabetes mellitus.

BackgroundDiabetic cardiomyopathy (DM CMP) is defined as cardiomyocyte damage and ventricular dysfunction directly associated with diabetes independent of concomitant coronary artery disease or hypertension. Matrix metalloproteinases (MMPs), especially MMP-2, have been reported to underlie the pathogenesis of DM CMP by increasing extracellular collagen content.PurposeWe hypothesized that two discrete MMP-2 isoforms (full length MMP-2, FL-MMP-2; N-terminal truncated MMP-2, NTT-MMP-2) are induced by high glucose stimulation in vitro and in an experimental diabetic heart model.MethodsRat cardiomyoblasts (H9C2 cells) were examined to determine whether high glucose can induce the expression of the two isoforms of MMP-2. For the in vivo study, we used the streptozotocin-induced DM mouse heart model and age-matched controls. The changes of each MMP-2 isoform expression in the diabetic mice hearts were determined using quantitative real-time polymerase chain reaction (qRT-PCR). Immunohistochemical stains were conducted to identify the location and patterns of MMP-2 isoform expression. Echocardiography was performed to compare and analyze the changes in cardiac function induced by diabetes.ResultsQuantitative RT-PCR and immunofluorescence staining showed that the two MMP-2 isoforms were strongly induced by high glucose stimulation in H9C2 cells. Although no definite histologic features of diabetic cardiomyopathy were observed in diabetic mice hearts, left ventricular systolic dysfunction was determined by echocardiography. Quantitative RT-PCR and IHC staining showed this abnormal cardiac function was accompanied with the increases in the mRNA levels of the two isoforms of MMP-2 and related to intracellular localization.ConclusionTwo isoforms of MMP-2 were induced by high glucose stimulation in vitro and in a Type 1 DM mouse heart model. Further study is required to examine the role of these isoforms in DM CMP