Matrix Metalloproteinases Inhibitors TIMP-1 Research Articles

#1004 Single-nucleus RNA sequencing identifies upregulated antigen presentation, apoptosis, mTORC1 signaling and EMT in primary FSGS podocytes

Abstract Background and Aims Primary focal segmental glomerulosclerosis (FSGS) is associated with irreversible podocyte damage, but the underlying cause and pathophysiology are incompletely understood. Furthermore, diagnostic markers that allow reliable differentiation between primary and secondary FSGS subtypes are lacking. We applied single-nucleus RNA-sequencing (snRNA-seq) on podocytes of patients with primary or secondary (maladaptive) FSGS, to identify subtype-specific differentially expressed genes and pathways. Method We used droplet-based snRNA-seq (10X Genomics Chromium) on cryopreserved kidney biopsy cores from human patients with newly diagnosed primary FSGS (n = 9, all nephrotic), maladaptive FSGS (n = 9, not nephrotic), proteinuric controls (PLA2R-positive membranous nephropathy (PLA2R+ MN), n = 3), and healthy controls (pre-perfusion biopsies, n = 4). Differential gene expression (DGE) analysis was done with a pseudobulk method provided by the EdgeR R-package to reduce bias and false discoveries. The fGSEA R-package was used for subsequent gene set enrichment analysis. Results We identified a total of 120 751 high-quality nuclei, of which 2 471 were podocytes (Fig. 1A). DGE analysis identified the top 20 differentially expressed genes (DEGs) per diagnosis (Fig. 1B). Remarkably, primary FSGS podocytes showed more transcriptional similarity to PLA2R+ MN podocytes, compared to maladaptive FSGS podocytes. This underscores the heterogeneity of patients with FSGS and shows that heavy proteinuria, regardless of the underlying diagnosis, may be associated with a distinct transcriptional profile in podocytes. Upregulated genes in primary FSGS podocytes included TIMP1, BTG2, ZNF44, EHD4, ZNF250, ECM1, SEMA4G, PHLDB1, and HDAC5. Upregulation of the matrix metalloproteinase inhibitor TIMP1 may possibly contribute to the development of glomerulosclerosis. Upregulation of BTG2 has previously been found to be pathogenic in FSGS by promoting epithelial-mesenchymal transition (EMT)-induced podocyte injury [1]. Next, gene set enrichment analysis on the identified DEGs in primary FSGS podocytes showed upregulation of pathways involved in antigen presentation (driven by upregulation of HLA-A, HLA-B, HLA-C, HLA-E and B2M), mTORC1 signaling, apoptosis and EMT (Fig. 1C). In maladaptive FSGS, upregulated pathways were mainly involved in extracellular matrix interaction. This may correlate with the significantly higher degree of chronic damage on kidney biopsy in the maladaptive FSGS patients (median Mayo Clinic Chronicity Score of 6.0, IQR [4.0-7.0]) vs. primary FSGS (2.0, [1.0-4.0]) and PLA2R+ MN (1, IQR [1.0-1.5]) (Kruskal-Wallis test, P = 0.007). Conclusion Our results corroborate previous studies that implicate apoptosis, mTORC1 signaling, EMT and auto-immunity in primary FSGS. Our future work will focus on cell-cell interactions within the glomerular niche and validation studies of candidate podocyte-specific genes.

Reversing wrinkled skin and hair loss in mice by restoring mitochondrial function

Mitochondrial DNA (mtDNA) depletion is involved in mtDNA depletion syndromes, mitochondrial diseases, aging and aging-associated chronic diseases, and other human pathologies. To evaluate the consequences of depletion of mtDNA in the whole animal, we created an inducible mtDNA-depleter mouse expressing, in the polymerase domain of POLG1, a dominant-negative mutation to induce depletion of mtDNA in various tissues. These mice showed reduced mtDNA content, reduced mitochondrial gene expression, and instability of supercomplexes involved in oxidative phosphorylation (OXPHOS) resulting in reduced OXPHOS enzymatic activities. We demonstrate that ubiquitous depletion of mtDNA in mice leads to predominant and profound effects on the skin resulting in wrinkles and visual hair loss with an increased number of dysfunctional hair follicles and inflammatory responses. Development of skin wrinkle was associated with the significant epidermal hyperplasia, hyperkeratosis, increased expression of matrix metalloproteinases, and decreased expression of matrix metalloproteinase inhibitor TIMP1. We also discovered markedly increased skin inflammation that appears to be a contributing factor in skin pathology. Histopathologic analyses revealed dysfunctional hair follicles. mtDNA-depleter mice also show changes in expression of aging-associated markers including IGF1R, KLOTHO, VEGF, and MRPS5. mtDNA-repleter mice showed that, by turning off the mutant POLG1 transgene expression, mitochondrial function, as well as the skin and hair pathology, is reversed to wild-type level. To our knowledge that restoration of mitochondrial functions can reverse the skin and hair pathology is unprecedented.

Elevated systemic levels of the matrix metalloproteinase inhibitor TIMP-1 correlate with clinical markers of cachexia in patients with chronic pancreatitis and pancreatic cancer

BackgroundTissue inhibitor of metalloproteinases-1 (TIMP-1) is a candidate diagnostic and prognostic biomarker for pancreatic ductal adenocarcinoma (PDAC). Here, we determined the possible association of systemic TIMP-1 levels with cachexia and jaundice, two common PDAC-associated conditions.MethodsPlasma TIMP-1 was measured by ELISA in patients diagnosed with PDAC (n = 36) and chronic pancreatitis (CP) (n = 25). Patients without pancreatic pathologies and known malignancies of other origin served as controls (n = 13). TIMP-1 levels in these patients were tested for asscociation with jaundice and chachexia, and furthermore correlated with cachexia-related clinical parameters such as weight loss and ferritin, parameters of lung function, hemoglobin and liver synthesis parameters.ResultsTIMP-1 plasma levels were mostly higher in CP and PDAC patients with concomitant jaundice or cachexia. Elevated plasma TIMP-1 levels were also associated with clinical cachexia markers, including absolute and relative values of weight loss and lung function, as well as ferritin, hemoglobin, and cholinesterase levels. TIMP-1 levels significantly correlated with cachexia only in patients without jaundice. Jaundice also impaired the use of TIMP-1 as a prognostic marker in cancer patients. Relating to cachexia status alone, a slightly improved association of TIMP-1 levels with survival of PDAC patients was observed.ConclusionThis retrospective study reports for the first time that plasma levels of TIMP-1 are associated with pancreatic lesion-induced cachexia in patients without jaundice. TIMP-1 is counterindicated as a survival marker in patients with jaundice.

Portal fibroblasts marked by the surface antigen Thy1 contribute to fibrosis in mouse models of cholestatic liver injury.

Liver fibrosis, a condition that is characterized by excessive production and accumulation of extracellular matrix, including collagen, is the most common outcome of chronic liver injuries of different etiologies. Vitamin A‐storing hepatic stellate cells (HSCs) are considered to be the main source of this collagen production, with activation in response to liver injury. In contrast, the contribution of other cell types to this fibrogenic response remains largely elusive due to the lack of specific surface markers to identify and isolate these cells for detailed analysis. Here, we identify a mesenchymal population of thymus cell antigen 1 (Thy1)+ CD45− cells (Thy1 MCs) in the mouse liver; these cells reside near the portal vein in vivo and indicate profibrogenic characteristics in vitro, shown by their expression of collagen and α‐smooth muscle actin. Flow cytometric analysis of mouse liver nonparenchymal cells revealed that vitamin A storage and Thy1 expression were mutually exclusive, indicating that Thy1 MCs are distinct from HSCs. Importantly, Thy1 MCs reacted and contributed to the development of liver fibrosis specifically in mouse models of cholestatic liver injury. With the occurrence of cholestatic liver injury, collagen‐producing Thy1 MCs expanded in cell number and inhibited collagen degradation through up‐regulation of matrix metalloproteinase inhibitor Timp1 expression, thereby promoting the accumulation of extracellular matrix in the periportal area. Conclusion: This study establishes Thy1 as a useful cell surface marker to prospectively identify and isolate periportal fibroblasts and further highlights a significant contribution of these cells to the pathogenesis of liver fibrosis caused by cholestatic liver injuries. We suggest that Thy1 MCs may be an interesting therapeutic target for treating liver fibrosis in addition to the well‐characterized HSCs. (Hepatology Communications 2017;1:198‐214)

APPLICATION IMMUNOHISTOCHEMICALLY RESEARCH METHODS IN THE DIAGNOSIS OF PROSTATE CANCER

Introduction. The actual problem of modern urology remains differential diagnostics of various diseases of the prostate gland. The purpose of the study. Increasing the effectiveness of differential diagnosis of diseases of the prostate. Materials and methods. In the biopsy specimens of patients with benign prostatic hyperplasia (BPH) and prostate cancer (PCa), an immunohistochemical study of the production of the Ki-67 proliferation marker, matrix metalloproteinase-9, the matrix metalloproteinase inhibitor TIMP-1, and the distribution of collagen type IV was performed. Results. A moderate positive correlation was found between Gleason gradation and the cell proliferation index for Ki 67 (rs = 0.674) and a moderate negative correlation of Gleason gradation with the level of production of matrix metalloproteinase-9 (rs = -0.660). A weak significant negative correlation was established between the level of proliferative cell activity and the production of MMP-9 tumor cells (rs = -0.369). A significant decrease in the level of MMP-9 and TIMP-1 in adenocarcinoma of different grades was revealed. The invasive properties of tumor cells, expressed in the destruction of collagen of the IV type of the basal membrane and connective tissue prostatic stroma, are mediated by the imbalance between MMP-9 and the protein blocking this enzyme - TIMP-1, whose production decreases in adenocarcinomas of different grades compared with BPH. Conclusions: 1. BPH is characterized by high production of MMP-9 type, which destroys the collagen of the basal membranes and stroma, the proteolytic action of which is blocked by the high content of TIMP-1.

Endothelial cell TIMP-1 is upregulated by shear stress via Sp-1 and the TGFβ1 signaling pathways

Laminar shear stress promotes vascular integrity by inhibiting proteolysis of the extracellular matrix (ECM) surrounding the microvasculature. We hypothesized that the matrix metalloproteinase inhibitor TIMP-1 would be upregulated in endothelial cells exposed to shear stress. Microvascular endothelial cells isolated from rat or mouse skeletal muscles were exposed to laminar shear stress for 2, 4, or 24 h. A biphasic increase in TIMP-1 protein was observed at 2 and 24 h of shear stress exposure. Sp-1 siRNA prevented the increase in TIMP-1 after 2, but not 24, hours of shear exposure. TGFβ production and Smad2/3 phosphorylation are increased by shear stress. Inhibition of TGFβ signaling, either by use of the TGFβ receptor 1 inhibitor SB-431542 or with Smad 2/3 siRNA, abrogated the shear stress-induced increase in TIMP-1 mRNA after 24 h of shear stress exposure. These results suggest that both acute and chronic elevated laminar shear stress act to maintain vessel integrity through increasing TIMP-1 production, but that the TGFβ signaling pathway is essential to maintain TIMP-1 expression during chronic shear stress.

Endothelial cell microparticles act as centers of matrix metalloproteinsase‐2 (MMP‐2) activation and vascular matrix remodeling

Endothelial cell (EC)-derived microparticles (MPs) are small membrane vesicles associated with various vascular pathologies. Here, we investigated the role of MPs in matrix remodeling by analyzing their interactions with the extracellular matrix. MPs were shown to bind preferentially to surfaces coated with matrix molecules, and MPs bound fibronectin via integrin α(V) . MPs isolated from EC-conditioned medium (Sup) were significantly enriched for matrix-altering proteases, including matrix metalloproteinases (MMPs). MPs lacked the MMP inhibitors TIMP-1 and TIMP-2 found in the Sup and, while Sup strongly inhibited MMP activities but MPs did not. In fact, MPs were shown to bind and activate both endogenous and exogenous proMMP-2. Taken together, these results indicate that MPs interact with extracellular matrices, where they localize and activate MMP-2 to modify the surrounding matrix molecules. These findings provide insights into the cellular mechanisms of vascular matrix remodeling and identify new targets of vascular pathologies.

The role of collagen receptors Endo180 and DDR-2 in the foreign body reaction against non-crosslinked collagen and gelatin

Despite the use of collagen-derived scaffolds in regenerative medicine, little is known about the degradation mechanisms of these scaffolds in vivo. Non-crosslinked dermal sheep (NDSC) and gelatin disks were implanted subcutaneously in mice. NDSC disks showed a very low degradation rate, despite the presence of high numbers of macrophages and the influx of neutrophils. This was attributed to the presence of the matrix metalloproteinase inhibitor TIMP-1. The limited degradation occurred mainly in the later stages of the foreign body reaction, and could be attributed to (1) phagocytosis by macrophages due to a co-expression of Endo180 and MT1-MMP on these cells (intracellular degradation) and (2) the presence of MMP-13 due to an upregulation of the expression of the DDR-2 receptor (extracellular degradation). In contrast, gelatin disks degraded quickly, due to the efficient formation of large giant cells as well as the presence of MMP-13; the inhibitor TIMP-1 was absent. The DDR-2 receptor was not expressed in the gelatin disks. Endo180 and MT1-MMP were expressed, but at most times no co-expression was seen. We conclude that the physical state of collagen (native or denatured) had a dramatic outcome on the degradation rate and provoked a completely different foreign body reaction.

Sprouty-4 Inhibits Transformed Cell Growth, Migration and Invasion, and Epithelial-Mesenchymal Transition, and Is Regulated by Wnt7A through PPARγ in Non–Small Cell Lung Cancer

Sprouty proteins are potent receptor tyrosine kinase inhibitors that antagonize growth factor signaling and are involved in lung development. However, little is known about the regulation or targets of Sprouty-4 (Spry4) in lung cancer. Our study aimed to determine the role of Spry4 in non-small cell lung cancer (NSCLC). We found that Spry4 mRNA expression was decreased in NSCLC cell lines and in dysplastic lung cell lines compared with a nontransformed cell line, suggesting that Spry4 has tumor-suppressing activity. When Spry4 was stably transfected into H157 and H2122 NSCLC cell lines, decreased migration and invasion were observed. Matrix metalloproteinase-9 activity was decreased, and the expression of matrix metalloproteinase inhibitors TIMP1 and CD82 were increased. Stable expression of Spry4 led to reduced cell growth and reduced anchorage-independent growth in NSCLC cell lines, along with upregulation of tumor suppressors p53 and p21. Changes in epithelial and mesenchymal markers indicated that Spry4 expression induces a reversal of the epithelial to mesenchymal transition characteristic of tumor cells. Treatment of a nontransformed lung epithelial cell line with short hairpin RNA to Spry4 led to the decreased expression of epithelial markers and increased cell growth, supporting the concept of Spry4 acting as a tumor suppressor. We showed that the activity of the Spry4 promoter is increased by Wnt7A/Fzd9 signaling through peroxisome proliferator-activated receptor gamma. These data present previously undescribed targets of Spry4 and suggest that Spry4 is a downstream target of Wnt7A/Fzd 9 signaling. Spry4 may have efficacy in the treatment of NSCLC.

Multiple Biological Predictors for Vulnerable Carotid Lesions

Background: In this study a multiscore analysis of various biomarkers including matrix metalloproteinases (MMPs), inflammatory factors and other clinical parameters was performed to establish a set of reliable biomarkers for improved detection of plaque instability in patients with advanced carotid stenosis. Methods: Study patients (n = 101) were classified as histologically stable (n = 37) or unstable (n = 64). Serum levels of MMP-1, -2, -3, -7, -8, -9, MMP inhibitors TIMP-1, -2, and inflammatory factors such as tumor necrosis factor (TNF-α), interleukin (IL)-1β, -6, -8, -10, and -12 were measured by ELISA assays. Multiscore analysis was performed using multiple receiver operating characteristics analysis and determination of appropriate cutoff values. Results: Circulating levels of MMP-1, -7, TIMP-1, TNF-α, and IL-8 were significantly enhanced in patients with unstable plaques compared to individuals with stable lesions, mean differences being 1.2 (p = 0.032), 2.5 (p = 0.004), 30.0 (p = 0.014), 1.3 (p = 0.047), and 2.2 (p = 0.033), respectively. The combination of MMP-1, -7, TIMP-1 and IL-8 demonstrated the highest positive predictive value of 89.4% and negative predictive value of 60.1% for patients correctly classified as individuals with unstable and stable carotid lesions by means of blood sample analysis. Conclusions: Multiple relevant biomarkers that play a decisive role in plaque instability can improve the correct determination of vulnerable carotid plaques in patients with advanced carotid artery stenosis.

MMP-2 Induced Vein Relaxation via Inhibition of [Ca 2+] e-Dependent Mechanisms of Venous Smooth Muscle Contraction. Role of RGD Peptides

Matrix metalloproteinases (MMPs) are implicated in the pathogenesis of varicose veins. We have shown that MMP-2 causes relaxation of venous segments and suggested a role of venous smooth muscle (VSM) hyperpolarization; however, the downstream mechanisms are unclear. We tested whether MMP-2 induced venous relaxation involves inhibition of the Ca(2+) mobilization mechanisms of VSM contraction due to generation of Arg-Gly-Asp (RGD)-containing peptides. Circular segments of inferior vena cava (IVC) were isolated from male Sprague-Dawley rats, suspended between two wires in a tissue bath, and isometric contraction was measured. Contraction data in mg/mg tissue were presented as means +/- SEM. In IVC incubated in normal Krebs (2.5 mM Ca(2+)), the alpha-adrenergic agonist phenylephrine (Phe, 10(-5) M) caused initial peak (133.2 +/- 17.5) followed by a maintained contraction (73.4 +/- 11.6), that was inhibited by MMP-2 (1 microg/mL) to 32.4 +/- 12.8 in 30 min. The inhibitory effects of MMP-2 were reversible by washing the tissue with Krebs or in the presence of the MMP inhibitors TIMP-1 (1 microg/mL), Ro 28-2653, and BB-94 (10(-6) M), and were not associated with changes in IVC structure, demonstrating specificity. Angiotensin II (AngII, 10(-6) M) caused a monophasic contraction (114.2 +/- 12.2), that was also inhibited by MMP-2 (66.0 +/- 7.4), suggesting a post-receptor effect on the downstream mechanisms of VSM contraction. To test the role of Ca(2+) release from the sarcoplasmic reticulum, IVC was incubated in Ca(2+)-free 2 mM ethylene glycol-bis(2-aminoethyl ether-N,N,N',N'-tetra-acetic acid (EGTA) Krebs with or without MMP-2. In Ca(2+)-free Krebs, caffeine did not cause contraction, suggesting a limited role of the Ca(2+)-induced Ca(2+)-release mechanism, and Phe and AngII caused a small contraction (7.2 +/- 1.7 and 14.9 +/- 2.8) that was slightly increased by MMP-2 (10.4 +/- 3.0 and 33.8 +/- 10.0), suggesting little effect on IP(3)-induced Ca(2+) release. To test the role of Ca(2+) entry through membrane channels, after eliciting a transient Phe contraction in nominally 0 Ca(2+) Krebs, increasing concentrations of CaCl(2) (0.1, 0.3, 0.6, 1, 2.5 mM) were added and the extracellular Ca(2+) concentration [Ca(2+)](e)-contraction relationship was constructed. The [Ca(2+)](e)-contraction relation was reduced in MMP-2 treated IVC, suggesting inhibition of Ca(2+) entry. In IVC treated with MMP-2, the Ca(2+) channel blocker diltiazem (10(-5)M) did not cause any further inhibition of Phe contraction, suggesting that Ca(2+) entry is already inhibited by MMP-2. To test whether MMP-2 actions involve generation of RGD and modulation of integrin receptors, experiments where repeated in IVC segments saturated with RGD (10(-5) M), or pretreated with the alpha(v)beta(3) integrin blocker cyclo(Ala-Arg-Gly-Asp-3-aminomethylbenzoyl) (cyclo-RGD). RGD-peptide caused only small relaxation of Phe contracted IVC (6.4 +/- 3.4%), and addition of MMP-2 to RGD-treated IVC caused further relaxation (69.7 +/- 3.0%). Pretreatment of IVC with cyclo-RGD did not significantly affect MMP-2 induced relaxation (55.0 +/- 5.0%). In rat IVC, MMP-2 attenuates [Ca(2+)](e)-dependent VSM contraction without affecting Ca(2+) release from intracellular Ca(2+) stores. MMP-2 induced VSM relaxation may not involve RGD generation or activation of alpha(v)beta(3) integrin receptor. MMP-2 induced inhibition of the Ca(2+) entry mechanism of VSM contraction may play a role in the venous dilation associated with varicose vein formation.

Matrix metalloproteinases and their inhibitors in human traumatic spinal cord injury

BackgroundMatrix metalloproteinases (MMPs) are a family of extracellular endopeptidases that degrade the extracellular matrix and other extracellular proteins. Studies in experimental animals demonstrate that MMPs play a number of roles in the detrimental as well as in the beneficial events after spinal cord injury (SCI). In the present correlative investigation, the expression pattern of several MMPs and their inhibitors has been investigated in the human spinal cord.MethodsAn immunohistochemical investigation in post mortem samples of control and lesioned human spinal cords was performed. All patients with traumatic SCI had been clinically diagnosed as having "complete" injuries and presented lesions of the maceration type.ResultsIn the unlesioned human spinal cord, MMP and TIMP immunoreactivity was scarce. After traumatic SCI, a lesion-induced bi-phasic pattern of raised MMP-1 levels could be found with an early up-regulation in macrophages within the lesion epicentre and a later induction in peri-lesional activated astrocytes. There was an early and brief induction of MMP-2 at the lesion core in macrophages. MMP-9 and -12 expression peaked at 24 days after injury and both molecules were mostly expressed in macrophages at the lesion epicentre. Whereas MMP-9 levels rose progressively from 1 week to 3 weeks, there was an isolated peak of MMP-12 expression at 24 days. The post-traumatic distribution of the MMP inhibitors TIMP-1, -2 and -3 was limited. Only occasional TIMP immuno-positive macrophages could be detected at short survival times. The only clear induction was detected for TIMP-3 at survival times of 8 months and 1 year in peri-lesional activated astrocytes.ConclusionThe involvement of MMP-1, -2, -9 and -12 has been demonstrated in the post-traumatic events after human SCI. With an expression pattern corresponding largely to prior experimental studies, they were mainly expressed during the first weeks after injury and were most likely involved in the destructive inflammatory events of protein breakdown and phagocytosis carried out by infiltrating neutrophils and macrophages, as well as being involved in enhanced permeability of the blood spinal cord barrier. Similar to animal investigations, the strong induction of MMPs was not accompanied by an expression of their inhibitors, allowing these proteins to exert their effects in the lesioned spinal cord.

Changes in the balance of the tissue inhibitor of matrix metalloproteinases (TIMPs)-1 and -3 may promote keratocyte apoptosis in keratoconus

Keratoconus is a disease in which the central cornea becomes thinned. This could result from corneal stromal cell apoptosis or be induced or perpetuated by the activation of matrix degrading enzymes, particularly members of the matrix metalloproteinase (MMP) family. In some circumstances, the MMP inhibitors TIMP-1 and TIMP-3 exhibit anti-apoptotic and pro-apoptotic properties, respectively. Because they potentially influence keratoconus progression, the effects of TIMP-1 and TIMP-3 on stromal cell viability were investigated. The TIMP-1 and TIMP-3 proteins were over-expressed in cultured corneal stromal cells by using the adenoviral vectors RAdTIMP-1 and RAdTIMP-3 and quantified by enzyme-linked immunosorbant assay (ELISA). Apoptotic cells were detected by TUNEL and caspase-3 activity. The anti-apoptotic effects of TIMP-1 were investigated by co-infecting it with RAdTIMP-1 and RAdTIMP-3 and by adding TIMP-1 protein to stromal cell cultures prior to infecting them with RAdTIMP-3. Immunohistochemistry was used to localise and determine relative numbers of apoptotic and TIMP producing stromal cells in sections of normal and keratoconic corneas. The results showed that over-expression of TIMP-3 induced apoptosis in corneal stromal cell cultures. Up-regulated TIMP-1 production or the addition of exogenous TIMP-1 protein prevented stromal cell overgrowth, changed stromal cell morphology and reduced the extent of TIMP-3 induced apoptosis. Localised relative concentrations of TIMP-1/TIMP-3 could thus determine whether these cells remain viable or become apoptotic. This may be relevant to the keratoconic condition since significantly more apoptotic cells were identified in the anterior stroma of keratoconic corneas than normal corneas and the majority of theTIMP-1 and TIMP-3 producing stromal cells were also located in this region.

Membrane Type 1 Matrix Metalloproteinase (MT1-MMP/MMP-14) Cleaves and Releases a 22-kDa Extracellular Matrix Metalloproteinase Inducer (EMMPRIN) Fragment from Tumor Cells

Proteolytic shedding is an important step in the functional down-regulation and turnover of most membrane proteins at the cell surface. Extracellular matrix metalloproteinase inducer (EMMPRIN) is a multifunctional glycoprotein that has two Ig-like domains in its extracellular portion and functions in cell adhesion as an inducer of matrix metalloproteinase (MMP) expression in surrounding cells. Although the shedding of EMMPRIN is reportedly because of cleavage by metalloproteinases, the responsible proteases, cleavage sites, and stimulants are not yet known. In this study, we found that human tumor HT1080 and A431 cells shed a 22-kDa EMMPRIN fragment into the culture medium. The shedding was enhanced by phorbol 12-myristate 13-acetate and inhibited by TIMP-2 but not by TIMP-1, suggesting the involvement of membrane-type MMPs (MT-MMPs). Indeed, down-regulation of the MT1-MMP expression in A431 cells using small interfering RNA inhibited the shedding. The 22-kDa fragment was purified, and the C-terminal amino acid was determined. A synthetic peptide spanning the cutting site was cleaved by MT1-MMP in vitro. The cleavage site is located in the linker region connecting the two Ig-like domains. The N-terminal Ig-like domain is important for the MMP inducing activity of EMMPRIN and for cell-cell interactions, presumably through its ability to engage in homophilic interactions, and the 22-kDa fragment retained the ability to augment MMP-2 expression in human fibroblasts. Thus, the MT1-MMP-dependent cleavage eliminates the functional N-terminal domain of EMMPRIN from the cell surface, which is expected to down-regulate its function. At the same time, the released 22-kDa fragment may mediate the expression of MMPs in tumor tissues.

Expression and activation of MMP -2, -3, -9, -14 are induced in rat colon after abdominal X-irradiation

Objective. Colonic response to single-dose irradiation is characterized by epithelial denudation followed by restitution. Extracellular matrix (ECM) remodeling is involved in both of these phases. The aim of this study was to characterize the contribution of matrix metalloproteinases (MMPs) and of their stimulatory and inhibitory pathways in radiation-induced ECM remodeling in colonic tissue. Material and methods. Rats were irradiated with single-dose 10 Gy X-rays to the abdomen. Activity, localization, and mRNA levels of MMPs and molecules involved in their activation and inhibition (plasmin/plasminogen; TIMPs), of inflammatory mediators (IL-1β, TNF-α) in the distal colon, 1, 3, and 7 days after irradiation were analyzed using a combination of approaches including zymography, immunohistochemistry, and real-time reverse transcriptase-polymerase chain reaction (RT-PCR). Results. The main finding of this study is that radiation-induced alteration of the mucosal structure is concomitant with local increased expression and activation of MMP subtypes involved in basement membrane degradation (MMP-2, -3, and -9). We investigated MMP-2 activation pathways and found an early increase in mRNA levels of soluble inflammatory mediators (TNF-α and IL-1β). Furthermore, transcription and activity of MMP-2 activating molecules, such as MMP-14, and molecules involved in the plasminogen/plasmin system were found to increase during the denudation phase. Interestingly, induction of MMP inhibitors TIMP-1 and PAI-1 was observed during the restitution phase. MMP inhibitors may be able to stop acute wound healing response by inhibiting ECM degradation. Conclusions. This study brings new insights into ECM remodeling in the colon after exposure to ionizing radiation and highlights the role of MMP subtypes specialized in basement membrane degradation.

SEL1L Affects Human Pancreatic Cancer Cell Cycle and Invasiveness through Modulation of PTEN and Genes Related to Cell-Matrix Interactions

Previously, it was reported that SEL1L is able to decrease the aggressive behavior of human pancreatic tumor cells both in vitro and in vivo. To gain insights into the involvement of SEL1L in tumor invasion, we performed gene expression analysis on the pancreatic cancer cell line Suit-2 subjected to two complementary strategies: upregulation and downregulation of SEL1L expression by stable transfection of the entire cDNA under an inducible promoter and by RNA-mediated interference. SuperArray and real-time analysis revealed that SEL1L modulates the expression of the matrix metalloproteinase inhibitors TIMP1 (P < .04–.03) and TIMP2 (P < .03–.05), and the PTEN gene (P < .03―.05). Gene expression modulations correlate with the decrease in invasive ability (P < .05) and in accumulation of SEL1L-expressing cells in G1. Taken together, our data indicate that SEL1L alters the expression of mediators involved in the remodeling of the extracellular matrix by creating a microenvironment that is unfavorable to invasive growth and by affecting cell cycle progression through promotion of G1 accumulation.

Inhibition of intraperitoneal tumor growth of human ovarian cancer cells by bi- and trifunctional inhibitors of tumor-associated proteolytic systems.

Several proteolytic systems are involved in (anti)adhesive, migratory, and proteolytic processes, necessary for tumor progression and metastasis. We analyzed whether multifunctional inhibitors of different tumor-associated proteolytic systems reduce tumor growth and spread of human ovarian cancer cells in vivo. Bifunctional inhibitors are composed of the N-terminal domain of either the human matrix metalloproteinase inhibitors TIMP-1 or TIMP-3 and the cysteine protease inhibitor chicken cystatin (chCysWT); trifunctional inhibitors are composed of N-TIMP-1 or -3 and a chicken cystatin variant harboring the uPAR binding site of uPA, chCys-uPA19-31, which in addition to its inhibitory activity toward cysteine proteases interferes with the interaction of the serine protease uPA with its receptor. OV-MZ-6#8 cancer cells, stably transfected with plasmids expressing the multifunctional inhibitors, displayed similar proliferative and adhesive features as the vector-transfected control, but showed significant reduction in their invasive behavior in vitro. The cell lines expressing the multifunctional inhibitors were inoculated into the peritoneum of nude mice. Expression of three of the four inhibitor variants (N-hTIMP-1-chCysWT, N-hTIMP-1-chCys-uPA19-31, and N-hTIMP-3-chCysWT) resulted in a significant reduction of tumor burden compared to the vector-control cell line. These compact and small inhibitors may represent promising agents for gene therapy of solid malignant tumors.

Expression of MMP2, MMP9, MT1-MMP, TIMP1, and TIMP2 mRNA in valvular lesions of the heart.

Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) play an important role in several diseases. This study was undertaken to investigate the mRNA synthesis of MMP2, MMP9, membrane-type 1 (MT1)-MMP, and matrix metalloproteinase inhibitors TIMP1 and TIMP2 by in situ hybridization in a set of heart mitral and aortic valves operatively removed due to degenerative or inflammatory valvular diseases. The material consisted of 21 valves, eight with endocarditis and 13 with a degenerative valvular disease. The samples were studied by in situ hybridization with specific probes for MMP2, MMP9, MT1-MMP, TIMP1, and TIMP2. Synthesis of MMP2 mRNA was found in seven valves, five with endocarditis and two with degenerative valvular disease. Signals for MMP9 mRNA were found in two cases with endocarditis and five cases with degenerative valvular disease. No signal for MT1-MMP mRNA was found in the lesions. TIMP1 mRNA, on the other hand, was found in 17 cases, both endocarditis and degenerative valvular disease. TIMP2 mRNA was found in three cases of endocarditis. The signals for MMP2, MMP9, TIMP1, and TIMP2 mRNA were localized in endothelial cells and in fibroblast-like cells expressing alpha-smooth muscle actin, thus showing myofibroblast-type differentiation. The results show that matrix metalloproteinases MMP2 and MMP9, and matrix metalloproteinase inhibitors TIMP1 and TIMP2 mRNAs are synthesized in diseased valves and suggest that they may contribute to matrix remodelling in valvular disease.

CD30 shedding from Karpas 299 lymphoma cells is mediated by TNF-alpha-converting enzyme.

CD30 is a costimulatory receptor on activated lymphocytes and a number of human lymphoma cells. Specific ligation of membrane-bound CD30 or cellular stimulation by PMA results in a metalloproteinase-mediated down-regulation of CD30 and release of its soluble ectodomain (sCD30). In this report, it is demonstrated that PMA-induced CD30 cleavage from Karpas 299 cells was mediated by a membrane-anchored metalloproteinase which was active on intact cells following 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate extraction of membrane preparations. Moreover, CD30 shedding was blocked by the synthetic hydroxamic acid-based metalloproteinase inhibitor BB-2116 (IC(50), 230 nM) and the natural tissue inhibitor of metalloproteinases (TIMP)-3 (IC(50), 30 nM), but not by the matrix metalloproteinase inhibitors TIMP-1 and TIMP-2. This inhibition profile is similar to that of the TNF-alpha- converting enzyme (TACE) and, indeed, mRNA transcripts of the membrane-bound metalloproteinase-disintegrin TACE could be detected in Karpas 299 cells. The ectodomain of TACE was expressed in bacteria as a GST fusion protein (GST-TACE) which cleaved CD30 from the surface of Karpas 299 cells and concomitantly increased the level of sCD30 in the cell supernatants. Hence, TACE does not only control the release of TNF-alpha, but also that of sCD30.

Expression of gelatinase A and its activator MT1-MMP in the inflammatory periprosthetic response to polyethylene.

Wear debris of polyethylene prosthetic components is known to induce a host granulomatous reaction which recruits numerous macrophages and multinucleated giant cells. By releasing cellular mediators of a nonspecific inflammatory reaction, activated phagocytic cells are thought to play a key role in osteolysis leading to aseptic loosening of the prosthesis. Matrix metalloproteinases (MMPs) have been implicated in this destructive process by their ability to degrade extracellular matrix components of bone and adjacent connective tissue. To investigate the roles of gelatinase A, its activator MT1-MMP, and the MMP inhibitors TIMP-1 and TIMP-2 in aseptic loosening of polyethylene prostheses, immunohistochemistry (IHC) and in situ hybridization (ISH) were performed on periprosthetic pseudosynovial interface tissues. Gelatinase A and MT1-MMP were strongly detected immunohistochemically in macrophages and multinucleated giant cells in contact with polyethylene wear debris. In contrast to MT1-MMP, gelatinase A mRNAs were not found in phagocytic cells but in surrounding fibroblasts, thereby suggesting cooperation between macrophages and fibroblasts in this process. While TIMP-1 was expressed essentially in hyperplastic pseudosynoviocytes as assessed by IHC and ISH, TIMP-2, MT1-MMP, and gelatinase A were colocalized in phagocytic cells. These data support the concept of progelatinase A activation involving a trimolecular complex (MT1-MMP-TIMP-2-gelatinase A) mechanism. Thus, this study demonstrated that gelatinase A and its activator might contribute to the aseptic loosening of polyethylene prostheses.