A biochemical model of matrix metalloproteinase 9 activation and inhibition

Matrix metalloproteinases (MMPs) are a class of extracellular and membrane-bound proteases involved in an array of physiological processes, including angiogenesis. We present a detailed computational model of MMP9 activation and inhibition. Our model is validated to existing biochemical experimental data. We determine kinetic rate constants for the processes of MMP9 activation by MMP3, MMP10, MMP13, and trypsin; inhibition by the tissue inhibitors of metalloproteinases (TIMPs) 1 and 2; and MMP9 deactivation. This computational approach allows us to investigate discrepancies in our understanding of the interaction of MMP9 with TIMP1. Specifically, we find that inhibition due to a single binding event cannot describe MMP9 inhibition by TIMP1. Temporally accurate biphasic inhibition requires either an additional isomerization step or a second lower affinity isoform of MMP9. We also theoretically characterize the MMP3/TIMP2/pro-MMP9 and MMP3/TIMP1/pro-MMP9 systems. We speculate that these systems differ significantly in their time scales of activation and inhibition such that MMP9 is able to temporarily overshoot its final equilibrium value in the latter. Our numerical simulations suggest that the ability of pro-MMP9 to complex TIMP1 increases this overshoot. In all, our analysis serves as a summary of existing kinetic data for MMP9 and a foundation for future models utilizing MMP9 or other MMPs under physiologically well defined microenvironments.     

Distinct modes of collagen type I proteolysis by matrix metalloproteinase (MMP) 2 and membrane type I MMP during the migration of a tip endothelial cell: insights from a computational model

Matrix metalloproteinases (MMPs) are a family of enzymes responsible for the proteolytic processing of extracellular matrix (ECM) structural proteins under physiological and pathological conditions. During sprouting angiogenesis, the MMPs expressed by a single "tip" endothelial cell exhibit proteolytic activity that allows the cells of the sprouting vessel bud to migrate into the ECM. Membrane type I matrix metalloproteinase (MT1-MMP) and the diffusible matrix metalloproteinase MMP2, in the presence of the tissue inhibitor of metalloproteinases TIMP2, constitute a system of proteins that play an important role during the proteolysis of collagen type I matrices. Here, we have formulated a computational model to investigate the proteolytic potential of such a tip endothelial cell. The cell expresses MMP2 in its proenzyme form, pro-MMP2, as well as MT1-MMP and TIMP2. The interactions of the proteins are described by a biochemically detailed reaction network. Assuming that the rate-limiting step of the migration is the ability of the tip cell to carry out proteolysis, we have estimated cell velocities for matrices of different collagen content. The estimated velocities of a few microns per hour are in agreement with experimental data. At high collagen content, proteolysis was carried out primarily by MT1-MMP and localized to the cell leading edge, whereas at lower concentrations, MT1-MMP and MMP2 were found to act in parallel, causing proteolysis in the vicinity of the leading edge. TIMP2 is a regulator of the proteolysis localization because it can shift the activity of MT1-MMP from its enzymatic toward its activatory mode, suggesting a tight mechanosensitive regulation of the enzymes and inhibitor expression. The model described here provides a foundation for quantitative studies of angiogenesis in extracellular matrices of different compositions, both in vitro and in vivo. It also identifies critical parameters whose values are not presently available and which should be determined in future experiments.     

Control and inhibition analysis of complex formation processes

ABSTRACT: BACKGROUND: Proteolytic degradation of the extracellular matrix (ECM) is a key event in tumour metastasis and invasion. Matrix metalloproteinases (MMPs) are a family of endopeptidases that degrade most of the components of the ECM. Several broad-spectrum MMP inhibitors (MMPIs) have been developed, but have had little success due to side effects. Thus, it is important to develop mathematical methods to provide new drug treatment strategies. Matrix metalloproteinase 2 (MMP2) activation occurs via a mechanism involving complex formation that consists of membrane type 1 MMP (MT1-MMP), tissue inhibitor of matrix metalloproteinase 2 (TIMP2) and MMP2. Here, we focus on developing a method for analysing the complex formation process. RESULTS: We used control analysis to investigate inhibitor responses in complex formation processes. The essence of the analysis is to define the response coefficient which measures the inhibitory efficiency, a small fractional change of concentration of a targeting molecule in response to a small fractional change of concentration of an inhibitor. First, by using the response coefficient, we investigated models for general classes of complex formation processes: chain reaction systems composed of ordered steps, and chain reaction systems and site-binding reaction systems composed of unordered multi-branched steps. By analysing the ordered step models, we showed that parameter-independent inequalities between the response coefficients held. For the unordered multi-branched step models, we showed that independence of the response coefficients with respect to equilibrium constants held. As an application of our analysis, we discuss a mathematical model for the MMP2 activation process. By putting the experimentally derived parameter values into the model, we were able to conclude that the TIMP2 and MMP2 interaction is the most efficient interaction to consider in selecting inhibitors. CONCLUSIONS: Our result identifies a new drug target in the process of the MMP2 activation. Thus, our analysis will provide new insight into the design of more efficient drug strategies for cancer treatment.     

Thioredoxin alters the matrix metalloproteinase/tissue inhibitors of metalloproteinase balance and stimulates human SK-N-SH neuroblastoma cell invasion.

Thioredoxin (Trx) inhibited tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 activity with an approximate IC50 of 0.3 microM, matrix metalloproteinase (MMP)-2 activity with an approximate IC50 of 2 microM but did not inhibit MMP-9 activity. This differential capacity of Trx to inhibit TIMP and MMP activity resulted in the promotion of MMP-2 and MMP-9 activity in the presence of molar TIMP excess. Inhibition of TIMP and MMP-2 activity by Trx was dependent upon thioredoxin reductase (TrxR), was abolished by Trx catalytic site mutation and did not result from TIMP or MMP-2 degradation. HepG2 hepatocellular carcinoma cells induced to secrete Trx inhibited TIMP activity in the presence of TrxR. SK-N-SH neuroblastoma cells secreted TrxR, which inhibited TIMP and MMP-2 activity in the presence of Trx. Trx stimulated SK-N-SH invasive capacity in vitro in the absence of exogenous TrxR. This study therefore identifies a novel extracellular role for the thioredoxin/thioredoxin reductase redox system in the differential inhibition of TIMP and MMP activity and provides a novel mechanism for altering the TIMP/MMP balance that is of potential relevance to tumor invasion.     

uPA and MMP‐2 were involved in self‐assembled network formation in a two dimensional co‐culture model of bone marrow stromal cells and endothelial cells

Two dimensional (2D) co‐cultures of human bone marrow stromal cells (HBMSCs) and human umbilical vein endothelial cells (HUVECs) stimulate osteoblastic differentiation of HBMSCs, induce the formation of self‐assembled network and cell interactions between the two cell types involving many vascular molecules. Because of their strong activities on angiogenesis and tissue remodeling, urokinase plasminogen activator (uPA), plasminogen activator inhibitor‐1 (PAI‐1), matrix metalloproteinase‐2 (MMP‐2) as well tissue inhibitors of matrix metalloproteinase‐2 (TIMP‐2) were investigated in this 2D co‐culture model. We found that the expression of uPA, MMP‐2 in the co‐cultured cells was significantly higher than those in mono‐cultured cells. In opposite, PAI‐1, expressed only by HUVECs is not regulated in the co‐culture. Inhibition assays confirm that uPA played a critical role in the formation of self‐assembled network as neutralization of uPA disturbed this network. In the same context, inhibition of MMP‐2 prevented the formation of self‐assembled network, while the inhibition of uPA abolished the over expression and the activity of MMP‐2. This upregulation could initiate the uPA expression and proteolysis processes through the MMP‐2 activity, and may contribute to endothelial cell migration and the formation of this self‐assembled network observed in these 2D co‐cultured cells. J. Cell. Biochem. 114: 650–657, 2013. © 2012 Wiley Periodicals, Inc.     

MMP‐2 and MMP‐9 as prognostic markers for the early detection of urinary bladder cancer

The present study assessed protein and gene expression levels of tissue inhibitor of metalloproteinase‐2 (TIMP‐2), matrix metalloproteinase‐2 (MMP‐2), and MMP‐9 in urine and blood samples of 50 patients with bladder carcinoma. The expression of TIMP‐2, MMP‐2, and MMP‐9 levels with tumor stage and grade was also assessed. Results showed that the expression levels of MMP‐2 and MMP‐9 in both blood and urine were significantly elevated in group 1 when compared with groups 2 and 3 healthy subjects. The discriminatory ability in the diagnosis of bladder carcinoma of MMP‐2 and MMP‐9 expression was confirmed by receiver operating characteristic curve analysis that revealed a sensitivity and specificity of 100%. MMP‐2 and MMP‐9 levels were not correlated with grade or stage of the tumor. With respect to TIMP‐2 blood and urine levels, results showed a significant decrease in gene expression levels in bladder carcinoma group, whereas, TIMP‐2 protein showed a significant increase in bladder carcinoma.     

Membrane type 1 matrix metalloproteinase and gelatinase A synergistically degrade type 1 collagen in a cell model

A fibrosarcoma cell line transfected with the matrix metalloproteinase MT1 MMP showed an enhanced ability to degrade 14C-labelled collagen films. As previously shown for proMMP 2 activation, TIMP 1 was an ineffective inhibitor of the process of collagenolysis whereas TIMP 2 was efficient and completely prevented collagen degradation. In the presence of the calcium ionophore, ionomycin, proteolytic processing of MT1 MMP was restricted and collagenolysis did not occur indicating that the 63 kDa form of the enzyme is not a functional collagenase. The collagenolytic activity of MT1 MMP was shown to be enhanced by the addition of proMMP 2, but TIMP 1 inhibition remained poor relative to that of TIMP 2. The study demonstrated that synergy between two non-conventional collagenases effectively degrades insoluble pericellular collagen. Due to the membrane localisation of MT1 MMP, this could potentially occur in a highly localised manner.     

Simvastatin decreases invasiveness of human endometrial stromal cells

Recently we reported that statins, the competitive inhibitors of the key enzyme regulating the mevalonate pathway, 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR), decrease proliferation of human endometrial stromal (HES) cells. Furthermore, we found that simvastatin treatment reduces the number and the size of endometrial implants in a nude mouse model of endometriosis. The present study was undertaken to investigate the effect of simvastatin on HES cell invasiveness and on expression of selected genes relevant to invasiveness: matrix metalloproteinase 2 (MMP2), MMP3, tissue inhibitor of matrix metalloproteinase 2 (TIMP2), and CD44. Because statin-induced inhibition of HMGCR reduces the production of substrates for isoprenylation-geranylgeranyl pyrophosphate (GGPP) and farnesyl pyrophosphate (FPP)-the effects of GGPP and FPP were also evaluated. Simvastatin induced a concentration-dependent reduction of invasiveness of HES cells. This effect of simvastatin was abrogated by GGPP but not by FPP. Simvastatin also reduced the mRNA levels of MMP2, MMP3, and CD44, but increased TIMP2 mRNA; all these effects of simvastatin were partly or entirely reversed in the presence of GGPP. The present findings provide a novel mechanism of action of simvastatin on endometrial stroma that may explain reduction of endometriosis in animal models of this disease. Furthermore, the presently described effects of simvastatin are likely mediated, at least in part, by inhibition of geranylgeranylation.     

TIMP1 contributes to ovarian anomalies in both an MMP-dependent and -independent manner in a rat model

Ovulatory dysfunction occurs in women with endometriosis, yet the mechanisms are unknown. We have shown that endometriotic lesions synthesize and secrete tissue inhibitor of metalloproteinase (TIMP) 1 into the peritoneal cavity in humans and a rat model of endometriosis, where excess TIMP1 localizes in the ovarian theca in endometriosis and modulating peritoneal TIMP1 alters ovarian dynamics. Here, we evaluated whether mechanisms whereby excessive peritoneal fluid TIMP1 negatively impacts ovarian function are matrix metalloproteinase (MMP)-dependent and/or MMP-independent actions. Rats were treated with a mutated TIMP1 without MMP inhibitory function (Ala-TIMP1), wild-type TIMP1 (rTIMP1), or PBS. Rats treated with Ala-TIMP1 or rTIMP1 had fewer antral follicles, fewer new corpora lutea, and the presence of luteinized unruptured follicle syndrome compared with PBS rats. Ala-TIMP1 and rTIMP1 differentially caused downstream changes in gene expression and protein localization related to ovulation, as measured by whole-genome microarray with quantitative real-time PCR validation and immunohistochemistry. More vascular endothelial growth factor and FN were expressed and localized in ovaries of Ala-TIMP1-treated rats compared to rTIMP1- and PBS-treated rats inferring MMP-independent functions. Less caspase 3 localized in ovaries of rTIMP1 compared with the other two groups, and was thus dependent on MMP action. Furthermore, after coimmunoprecipitation, more CD63 was bound to TIMP1 in ovaries of rats treated with Ala-TIMP1 than in rTIMP1-treated rats, providing evidence for another MMP-independent mechanism of ovulatory dysfunction. We predict that MMP-dependent and MMP-independent events are involved in improper fortification of the follicular wall through multiple mechanisms, such as apoptosis inhibition, extracellular matrix components and angiogenesis. Collectively, excessive peritoneal TIMP1 causes changes in ovarian dynamics, both dependently and independently of MMP inhibition.     

Robustness,flexibility, and the role of lateral inhibition in the neurogenic network

BACKGROUND: Many gene networks used by developing organisms have been conserved over long periods of evolutionary time. Why is that? We showed previously that a model of the segment polarity network in Drosophila is robust to parameter variation and is likely to act as a semiautonomous patterning module. Is this true of other networks as well? RESULTS: We present a model of the core neurogenic network in Drosophila. Our model exhibits at least three related pattern-resolving behaviors that the real neurogenic network accomplishes during embryogenesis in Drosophila. Furthermore, we find that it exhibits these behaviors across a wide range of parameter values, with most of its parameters able to vary more than an order of magnitude while it still successfully forms our test patterns. With a single set of parameters, different initial conditions (prepatterns) can select between different behaviors in the network's repertoire. We introduce two new measures for quantifying network robustness that mimic recombination and allelic divergence and use these to reveal the shape of the domain in the parameter space in which the model functions. We show that lateral inhibition yields robustness to changes in prepatterns and suggest a reconciliation of two divergent sets of experimental results. Finally, we show that, for this model, robustness confers functional flexibility. CONCLUSIONS: The neurogenic network is robust to changes in parameter values, which gives it the flexibility to make new patterns. Our model also offers a possible resolution of a debate on the role of lateral inhibition in cell fate specification.     

人组织型基质金属蛋白酶抑制剂-1在Pichia pastoris酵母中重组表达研究

 为研究组织型基质金属蛋白酶抑制剂 (TIMPs)的分子作用机制 ,探讨了在 Pichia pastoris酵母中高效表达分泌型人组织型基质金属蛋白酶抑制剂 - 1 (TIMP- 1 )的技术路线 ,并对产物性质进行初步研究 .通过 PCR从含有 TIMP- 1基因的 p BS质粒获得了该基因的全长序列 ,构建了 p PIC9/T1表达载体 ,电击法转化酵母 ,通过表型筛选和 PCR鉴定证实了目的基因已稳定整合入 Pichiapastoris酵母基因组中 .SDS- PAGE表明表达量高达 40 mg/L培养上清 .用免疫印迹法确定了产物的正确性 ;同时 ,反向明胶酶谱法证明了重组蛋白具有抑制基质金属蛋白酶的活性 .     

Nitric oxide‐mediated regulation of ‐amyloid clearance via alterations of MMP‐9/TIMP‐1

Fibrillar amyloid plaques are largely composed of amyloid‐beta (Aβ) peptides that are metabolized into products, including Aβ1‐16, by proteases including matrix metalloproteinase 9 (MMP‐9). The balance between production and degradation of Aβ proteins is critical to amyloid accumulation and resulting disease. Regulation of MMP‐9 and its endogenous inhibitor tissue inhibitor of metalloproteinase (TIMP)‐1 by nitric oxide (NO) has been shown. We hypothesize that nitric oxide synthase (NOS2) protects against Alzheimer's disease pathology by increasing amyloid clearance through NO regulation of MMP‐9/TIMP‐1 balance. We show NO‐mediated increased MMP‐9/TIMP‐1 ratios enhanced the degradation of fibrillar Aβ in vitro, which was abolished when silenced for MMP‐9 protein translation. The in vivo relationship between MMP‐9, NO and Aβ degradation was examined by comparing an Alzheimer's disease mouse model that expresses NOS2 with a model lacking NOS2. To quantitate MMP‐9 mediated changes, we generated an antibody recognizing the Aβ1‐16 fragment, and used mass spectrometry multi‐reaction monitoring assay for detection of immunoprecipitated Aβ1‐16 peptides. Aβ1‐16 levels decreased in brain lysates lacking NOS2 when compared with strains that express human amyloid precursor protein on the NOS2 background. TIMP‐1 increased in the APPSwDI/NOS2^?/? mice with decreased MMP activity and increased amyloid burden, thereby supporting roles for NO in the regulation of MMP/TIMP balance and plaque clearance.     

Expression of mutant and wild-type TIMP3 in primary gingival fibroblasts from Sorsby's fundus dystrophy patients

Gingival fibroblast cell lines were derived from Sorsby's fundus dystrophy (SFD) patients carrying the S181C TIMP3 and the E139X TIMP3 mutations. These cell lines were grown in culture to study expression of the wild-type and mutant tissue inhibitor of metalloproteinase 3 (TIMP3) alleles from a normal diploid cell type. Firstly, patient cells were found to co-express the wild-type and mutant TIMP3 alleles, S181C TIMP3 or E139X TIMP3, at the mRNA level using restriction fragment length polymorphism (RFLP) analysis. A SpeI RFLP for E139X TIMP3 is described. Low levels of endogenous TIMP3 protein expression were elevated using the natural polysaccharide calcium pentosan polysulfate (CaPPs) in combination with the cytokine IL-1alpha. Immunoblotting detected protein expression from both wild-type and mutant alleles, S181C TIMP3 or E139X TIMP3. S181C TIMP3 from these cells was found to dimerise and retain MMP2 inhibitory activity. To facilitate studies of the E139X TIMP3 protein, the allele was expressed using HighFive insect cells. In this cell type, the E139X TIMP3 was synthesised as a mixture of monomer and dimer. Both monomeric and dimeric E139X TIMP3 protein retained MMP2 inhibitory activity in gelatin zymography. Expression of mutant E139X or S181C TIMP3 protein from a normal diploid patient-derived fibroblast cell had no effect on either MMP2 or MMP9 expression or activation whilst transcribed from their normal promoter context.     

四周尾部悬吊大鼠颈总动脉MMP9和TIMP1的表达及活性改变

目的:探讨四周尾部悬吊模拟失重大鼠颈总动脉基质金属蛋白酶(matrix metalloprotein9,MMP9)和金属蛋白酶组织抑制剂1(tissue inhibitor of metalloproteinase1,TIMP1)的基因、蛋白表达及酶活性变化。方法:采用4周(week,wk)尾部悬吊大鼠模拟失重影响,通过透射电镜检测颈总动脉壁基质含量,实时定量聚合酶链式反应检测MMP9和TIMP1的mRNA表达,Western blot和免疫组织化学染色检测其蛋白表达和分布,明胶酶谱法测定MMP9活性水平。结果:与对照组相比,悬吊组大鼠细胞外基质面积较对照组显著增加(P0.05),胶原蛋白含量显著增加(P0.05);悬吊组大鼠颈总动脉MMP9的m RNA表达量无明显改变,而其蛋白表达量和酶活性均显著降低(P0.05);TIMP1 mRNA和蛋白表达量则显著升高(P0.05)。结论:模拟失重使大鼠颈总动脉MMP9水平降低,TIMP1水平升高,可能与其管壁基质增生和胶原蛋白含量增加有关。     

Tissue and serum loss of metalloproteinase inhibitors in high grade soft tissue sarcomas

The activity of matrix metalloproteinases (MMPs) in degrading extracellular matrix is controlled by activation of pro-enzymes and inhibition of MMP tissue inhibitors (TIMPs). To assess proteolytic cascade imbalance in malignancy progression, the enzymatic activity of MMP2 and MMP9 and the expression and serum level of their inhibitors, TIMP2 and TIMP1 respectively, was evaluated in selected patients with high-risk soft tissue sarcoma (STS). Gelatinase activity and inhibitor expression was evaluated on 69 biopsies by zymography and immunohistochemistry. TIMP1 and TIMP2 serum concentration was tested in 53 STS patients and in 56 controls using a sandwich enzyme immunoassay. Clinical and biological variables were related to clinical outcome of the patients. A significant gelatinolytic activity was seen in a high percentage of STS. TIMP expression was weak or negative in the majority of samples. The difference between disease-free (p=0.001) and overall survival (p=0.007) curves based on TIMP2 immunoreactivity was statistically significant. TIMP plasma concentration of 53 STS revealed significantly lower levels compared to those of 56 controls (p=0.0001). In conclusion, low levels of negative regulators of proteolysis may be related to tumor biological aggressiveness and used to select patients with poor prognosis to improve cure.     

Characterization of matrix metalloproteinase expressed by human embryonic kidney cells

There is little information available on the proteases expressed by human embryonic kidney (HEK) cells, which are often used for expression of recombinant proteins and production of adenovirus vector. The expression profile of proteases in HEK cell line was investigated using zymography, mRNA analysis, western blotting and protein array. The major protease was gelatinase A [or matrix metalloproteinase (MMP)-2]. Beside, other MMPs, such as MMP-1, -2, -3, -8, -9, -10, -13 and membrane type (MT) 1- and 3−MMP, as well as tissue inhibitors of metalloproteinase (TIMP)-1, -2 and -3, were also expressed by HEK cells. Characterization of MMP and TIMP profiles expressed by HEK cells provides the basis for degradation control of recombinant protein and adenovirus vector during culture and purification processes.     

Exploring how extracellular electric field modulates neuron activity through dynamical analysis of a two-compartment neuron model

To investigate how extracellular electric field modulates neuron activity, a reduced two-compartment neuron model in the presence of electric field is introduced in this study. Depending on neuronal geometric and internal coupling parameters, the behaviors of the model have been studied extensively. The neuron model can exist in quiescent state or repetitive spiking state in response to electric field stimulus. Negative electric field mainly acts as inhibitory stimulus to the neuron, positive weak electric field could modulate spiking frequency and spike timing when the neuron is already active, and positive electric fields with sufficient intensity could directly trigger neuronal spiking in the absence of other stimulations. By bifurcation analysis, it is observed that there is saddle-node on invariant circle bifurcation, supercritical Hopf bifurcation and subcritical Hopf bifurcation appearing in the obtained two parameter bifurcation diagrams. The bifurcation structures and electric field thresholds for triggering neuron firing are determined by neuronal geometric and coupling parameters. The model predicts that the neurons with a nonsymmetric morphology between soma and dendrite, are more sensitive to electric field stimulus than those with the spherical structure. These findings suggest that neuronal geometric features play a crucial role in electric field effects on the polarization of neuronal compartments. Moreover, by determining the electric field threshold of our biophysical model, we could accurately distinguish between suprathreshold and subthreshold electric fields. Our study highlights the effects of extracellular electric field on neuronal activity from the biophysical modeling point of view. These insights into the dynamical mechanism of electric field may contribute to the investigation and development of electromagnetic therapies, and the model in our study could be further extended to a neuronal network in which the effects of electric fields on network activity may be investigated.