Expression Of Stromelysin Research Articles

Cancer associated fibroblasts stimulated by transforming growth factor beta1 (TGF-β1) increase invasion rate of tumor cells: a population study

Cancer associated fibroblasts (CAFs) are believed to promote tumor growth and progression. Our objective was to measure the effect of TGF-beta1 on fibroblasts isolated from invasive breast cancer patients. Fibroblasts were isolated from tissue obtained at surgery from patients with invasive breast cancer (CAF; n = 28) or normal reduction mammoplasty patients (normal; n = 10). Myofibroblast activation was measured by counting cells immunostained for smooth muscle alpha actin (ACTA2) in cultures +/- TGF-beta 1. Conditioned media (CM) was collected for invasion assays and RNA was isolated from cultures incubated in media +/- TGF-beta1 for 24 h. Q-PCR was used to measure expression of cyclin D1, fibronectin, laminin, collagen I, urokinase, stromelysin-1, and ACTA2 genes. Invasion rate was measured in chambers plated with MDA-MB-231 cells and exposed to CM in the bottom chamber; the number of cells that invaded into the bottom chamber was counted. Wilcox Rank Sum tests were used to evaluate differences in CAFs and normal fibroblasts and the effect of TGF-beta 1. There was no difference in percent myofibroblasts or invasion rate between normal and CAF cultures. However, TGF-beta1 significantly increased the percent of myofibroblasts (P < 0.01) and invasion rate (P = 0.02) in CAF cultures. Stromelysin-1 expression was significantly higher in normal versus CAF cultures (P < 0.01). TGF-beta 1 significantly increased ACTA2 expression in both normal and CAF cultures (P < 0.01). Expression of fibronectin and laminin was significantly increased by TGF-beta in CAF cultures (P < 0.01). CAFs were measurably different from normal fibroblasts in response to TGF-beta 1, suggesting that TGF-beta stimulates changes in CAFs that foster tumor invasion.

P38 MAP Kinase Pathway and Stromelysin Regulation in Trabecular Meshwork Cells

Increased expression of stromelysin-1 (matrix metalloproteinase [MMP]-3) by the trabecular meshwork (TM) initiates extracellular matrix turnover and increases aqueous humor outflow facility. Tumor necrosis factor (TNF)alpha and interleukin (IL)-1alpha are efficacious inducers of MMP-3 in TM. To facilitate understanding of the regulation of MMP-3, the authors investigated the involvement of p38 MAP kinase pathway proteins in this process. Western immunoblots were used to determine the effects of these cytokines and p38 MAP kinase pathway inhibitors on MMP-3 protein levels, p38 MAP kinase isoforms, and phosphorylation levels in human and porcine TM cells. The effects of a dominant-negative p38 MAP kinase construct on MMP-3 expression were evaluated. Morphologic changes in the cells were also examined. Both cytokines increased MMP-3 levels. The p38 MAP kinase inhibitor SB202190 diminished MMP-3 induction by TNFalpha at all times and at 24 hours by IL-1alpha but potentiated the IL-1alpha-induced increase in MMP-3 at later times. MMP-3 induction by both cytokines was blocked by dominant-negative p38 MAP kinase constructs. Each cytokine increased phosphorylation of the p38 MAP kinase pathway components and altered TM cell morphology. The p38 inhibitor blocked only the morphologic changes produced by TNFalpha. Human and porcine TM cells expressed p38 alpha, beta, delta, and gamma isoforms, which migrate coincident with bands of specific phosphorylation. The effects of p38 inhibitors and the dominant-negative construct on TNFalpha and IL-1alpha induction of MMP-3 demonstrate an essential role for p38 in this signaling process. Differences between p38 inhibitor effects on TNFalpha and IL-1alpha induction of MMP-3 suggest divergent p38 isoform use, as do the morphologic responses. The anomalous p38 inhibitor effect on IL-1alpha induction of MMP-3 and phosphorylation of p38 delta/gamma suggests complex interactions between p38 MAP kinase isoforms and their differential uses by TNFalpha and IL-1alpha in TM.

Stromelysin-3 expression in the differential diagnosis of dermatofibroma and dermatofibrosarcoma protuberans: comparison with factor XIIIa and CD34

The distinction between dermatofibroma (DF) and dermatofibrosarcoma protuberans (DFSP) is a well-known challenge for dermatopathologists. Immunohistochemical stains have been used to augment routine histological examination to aid in differentiating DF from DFSP. Stromelysin-3 (ST3) is a member of the matrix metalloproteinase (MMP) family, MMP-11, which is expressed in the skin during wound healing and in the stroma of basal cell carcinoma. Recent studies demonstrated that DFs expressed ST3, whereas DFSPs were only rarely ST3 positive. To assess the expression of ST3 in DF and DFSP and to ascertain whether ST3 is superior to factor XIIIa or CD34 in differentiating DF from DFSP, by comparison with factor XIIIa and CD34 expression. Immunohistochemical staining was performed on 23 cases of DF and 17 cases of DFSP, using antibodies to ST3, factor XIIIa and CD34. ST3 was expressed in all cases of DF (23 of 23) but only one case showed weakly positive staining in DFSP (one of 17). The mean +/- SD ST3 immunohistochemistry (IHC) score in DF was 4.52 +/- 0.67. The sensitivity of ST3 was 100% and the specificity was 94%. Factor XIIIa was expressed in all cases of DF (23 of 23) and in five of the 17 DFSPs. The mean +/- SD factor XIIIa IHC score in the DFs was 4.43 +/- 0.73. The sensitivity of factor XIIIa was 100% and the specificity was 71%. CD34 was expressed in four of the 23 DFs and 16 of the 17 DFSPs. The mean +/- SD CD34 IHC score in the DFSPs was 4.41 +/- 1.37. The sensitivity of CD34 was 94% and the specificity was 83%. Immunohistochemical staining with a commercial anti-ST3 antibody can be successfully carried out in routine dermatopathology. We confirmed that ST3 is a positive marker for DF and that ST3 staining might be more reliable than factor XIIIa staining in differential diagnosis of DF and DFSP. As the present study showed that ST3 was not absolutely negative in all cases of DFSP, the combination with CD34 immunostaining could make the distinction more reliable.

Stromelysin-3 over-expression enhances tumourigenesis in MCF-7 and MDA-MB-231 breast cancer cell lines: involvement of the IGF-1 signalling pathway

BackgroundStromelysin-3 (ST-3) is over-expressed in the majority of human carcinomas including breast carcinoma. Due to its known effect in promoting tumour formation, but its impeding effect on metastasis, a dual role of ST-3 in tumour progression, depending on the cellular grade of dedifferentiation, was hypothesized.MethodsThe present study was designed to investigate the influence of ST-3 in vivo and in vitro on the oestrogen-dependent, non-invasive MCF-7 breast carcinoma cell line as well as on the oestrogen-independent, invasive MDA-MB-231 breast carcinoma cell line. Therefore an orthotopic human xenograft tumour model in nude mice, as well as a 3D matrigel cell culture system, were employed.ResultsUsing both in vitro and in vivo techniques, we have demonstrated that over-expression of ST-3 in MCF-7 and MDA-MB-231 cells leads to both increased cell numbers and tumour volumes. This observation was dependent upon the presence of growth factors. In particular, the enhanced proliferative capacity was in MCF-7/ST-3 completely and in MDA-MB-231/ST-3 cells partially dependent on the IGF-1 signalling pathway. Microarray analysis of ST-3 over-expressing cells revealed that in addition to cell proliferation, further biological processes seemed to be affected, such as cell motility and stress response. The MAPK-pathway as well as the Wnt and PI3-kinase pathways, appear to also play a potential role. Furthermore, we have demonstrated that breast cancer cell lines of different differentiation status, as well as the non-tumourigenic cell line MCF-10A, have a comparable capability to induce endogenous ST-3 expression in fibroblasts.ConclusionThese data reveal that ST-3 is capable of enhancing tumourigenesis in highly differentiated "early stage" breast cancer cell lines as well as in further progressed breast cancer cell lines that have already undergone epithelial-mesenchymal transition. We propose that ST-3 induction in tumour fibroblasts leads to the stimulation of the IGF-1R pathway in carcinoma cells, thus enhancing their proliferative capacity. In addition, further different cellular processes seem to be activated by ST-3, possibly accounting for the dual role of ST-3 in tumour progression and metastasis.

Matrix metalloproteinase-induced epithelial–mesenchymal transition: Tumor progression at Snail's pace

Matrix metalloproteinases (MMPs) are enzymes that digest components of the extracellular matrix (ECM) as well as cell surface receptors for soluble factors and junctional proteins involved in cell–cell and cell–ECM interactions. MMPs are involved in many physiological processes that require tissue remodeling but are also expressed in nearly all tumors, where they stimulate tumor growth, invasion, and metastasis. Previous studies have shown that expression of stromelysin-1/MMP-3 in the mammary glands of transgenic mice causes the production of invasive carcinomas; our recent investigations provide insight into how MMPs can promote the development of such tumors. We show that exposing mouse mammary epithelial cells to MMP-3 stimulates epithelial–mesenchymal transition (EMT), a phenotypic alteration in which epithelial cells acquire invasive mesenchymal characteristics. EMT is a necessary component of embryonic development, but acquisition of EMT characteristics by cancer cells facilitates invasion and metastasis. MMP-3-induced EMT involves induction of Snail, a transcription factor long studied for its role in physiological EMT but which is increasingly recognized as a factor involved in tumor progression and malignancy. In this review, we examine how MMPs and Snail function in tumor progression and how identification of an MMP-Snail signaling axis provides insight into new anticancer therapeutic strategies.

Activation of synovial fibroblasts by laminin-1 and transforming growth factor beta induces expression of stromelysins independently of TNFα, IL-1β or NF-κB

Recently it was shown that attachment of synovial fibroblasts (SF) from rheumatoid arthritis patients to laminin-111 (LM-111) induced an elevated expression of stromelysin-1 (MMP-3). We therefore investigated the regulation of additional matrix metalloproteinases (MMPs) and their specific tissue inhibitors of matrix metalloproteinases (TIMPs) by attachment to LM-111 in the presence of transforming growth factor beta (TGFβ). Changes in steady-state mRNA levels encoding TIMPs and MMPs were investigated by quantitative RT-PCR. Production of MMPs and cytokines was monitored by a multiplexed immunoarray or by ELISA. Signal transduction pathways were studied by immunoblotting. Attachment of SF to LM-111 in the presence of TGFβ induced significant increases in stromelysin-1 mRNA (12.35-fold, P < 0.001) and protein (mean 62 ng/ml, sixfold, P < 0.008). Expression of stromelysin-2 (MMP-10) mRNA (11.68-fold, P < 0.05) and protein (54 ng/ml, 20-fold, P ≥ 0.02) was significantly activated as well. All other TIMPs and MMPs investigated failed to show this LM-111-facilitated TGFβ response. Induction of stromelysin-1 and stromelysin-2 was associated with the activation of transcription factors c-fos and Egr-1, but phosphorylation of NF-κB was not observed. Further, LM-111-activated and TGFβ-activated SF failed to produce remarkable amounts of IL-1β or TNFα. We conclude that costimulation of synovial fibroblasts by LM-111 together with TGFβ suffices to induce significant expression of MMP-3 and MMP-10 by SF and that this induction is independent of phosphorylation of NF-κB.

Matrix metalloproteinase‐3 expression profile differentiates adaptive and maladaptive synaptic plasticity induced by traumatic brain injury

The interaction between extracellular matrix (ECM) and regulatory matrix metalloproteinases (MMPs) is important in establishing and maintaining synaptic connectivity. By using fluid percussion traumatic brain injury (TBI) and combined TBI and bilateral entorhinal cortical lesion (TBI + BEC), we previously demonstrated that hippocampal stromelysin-1 (MMP-3) expression and activity increased during synaptic plasticity. We now report a temporal analysis of MMP-3 protein and mRNA response to TBI during both degenerative (2 day) and regenerative (7, 15 day) phases of reactive synaptogenesis. MMP-3 expression during successful synaptic reorganization (following unilateral entorhinal cortical lesion; UEC) was compared with MMP-3 expression when normal synaptogenesis fails (after combined TBI + BEC insult). Increased expression of MMP-3 protein and message was observed in both models at 2 days postinjury, and immuohistochemical (IHC) colocalization suggested that reactive astrocytes contribute to that increase. By 7 days postinjury, model differences in MMP-3 were observed. UEC MMP-3 mRNA was equivalent to control, and MMP-3 protein was reduced within the deafferented region. In contrast, enzyme mRNA remained elevated in the maladaptive TBI + BEC model, accompanied by persistent cellular labeling of MMP-3 protein. At 15 days survival, MMP-3 mRNA was normalized in each model, but enzyme protein remained higher than paired controls. When TBI + BEC recovery was enhanced by the N-methyl-D-aspartate antagonist MK-801, 7-day MMP-3 mRNA was significantly reduced. Similarly, MMP inhibition with FN-439 reduced the persistent spatial learning deficits associated with TBI + BEC insult. These results suggest that MMP-3 might differentially affect the sequential phases of reactive synaptogenesis and exhibit an altered pattern when recovery is perturbed.

Stromelysin-1 expression is activated in vivo by Ets-1 through palindromic head-to-head Ets binding sites present in the promoter

Regulation of the gene expression of Stromelysin-1 (matrix metalloproteinase-3), a member of the matrix metalloproteinase family, is critical for tissue homeostasis. The Stromelysin-1 promoter is known to be transactivated by Ets proteins through palindromic head-to-head Ets binding sites (EBS), an unusual configuration among metalloproteinase promoters. Patterns of increased co-expression of Stromelysin-1 and Ets-1 genes have been observed in pathological processes such as rheumatoid arthritis, glomerulonephritis and tumor invasion. In this context, we show in a synovial fibroblastic model cell line (HIG-82), which is able to co-express Stromelysin-1 and Ets-1, that the EBS palindrome is essential for the expression of Stromelysin-1. More precisely, using electrophoretic mobility shift assays, DNA affinity purification and chromatin immunoprecipitation, we demonstrate that endogenous Ets-1, but not Ets-2, is present on this palindrome. The use of a dominant-negative form of Ets-1 and the decrease of Ets-1 amount either by fumagillin, an antiangiogenic compound, or by short interfering RNA show that the activation rate of the promoter and the expression of Stromelysin-1 correlate with the level of endogenous Ets-1. Thus, it is the first demonstration, using this cellular model, that endogenously expressed Ets-1 is actually a main activator of the Stromelysin-1 promoter through its effective binding to the EBS palindrome.

The antioxidants curcumin and quercetin inhibit inflammatory processes associated with arthritis

Curcumin and quercetin are antioxidant molecules with anti-proliferative, anti-inflammatory and immunosuppressive activities. The objective of this study was to investigate the inhibitory activity of these agents using four assays of inflammatory aspects of arthritis. Crystal-induced neutrophil activation was measured by luminol-dependent chemiluminescence. Synoviocyte proliferation was measured by an MTS assay using HIG-82 rabbit synoviocytes in cell culture. Chondrocyte (cultured primary cells) expression of the matrix metalloproteinases collagenase and stromelysin was measured by Northern Blot analysis. Angiogenesis was measured using the chorioallantoic membrane of the chick embryo. Both agents inhibited neutrophil activation, synoviocyte proliferation and angiogenesis. Curcumin strongly inhibited collagenase and stromelysin expression at micromolar concentrations whereas quercetin had no effect in this assay. These studies suggest that curcumin and to a lesser extent quercetin may offer therapeutic potential for the treatment of crystal-induced arthritis or rheumatoid arthritis.

Fibroblast extracellular matrix gene expression in response to keratinocyte-releasable stratifin

Termination of wound-healing process requires a fine balance between connective tissue deposition and its hydrolysis. Previously, we have demonstrated that keratinocyte-releasable stratifin, also known as 14-3-3 sigma protein, stimulates collagenase (MMP-1) expression in dermal fibroblasts. However, role of extracellular stratifin in regulation of extracellular matrix (ECM) factors and other matrix metalloproteinases (MMPs) in dermal fibroblast remains unexplored. To address this question, large-scale ECM gene expression profile were analyzed in human dermal fibroblasts co-cultured with keratinocytes or treated with recombinant stratifin. Superarray pathway-specific microarrays were utilized to identify upregulation or downregulation of 96 human ECM and adhesion molecule genes. RT-PCR and Western blot were used to validate microarray expression profiles of selected genes. Comparison of gene profiles with the appropriate controls showed a significant (more than twofold) increase in expression of collagenase-1, stromelysin-1 and -2, neutrophil collagenase, and membrane type 5 MMP in dermal fibroblasts treated with stratifin or co-cultured with keratinocytes. Expression of type I collagen and fibronectin genes decreased in the same fibroblasts. The results of a dose-response experiment showed that stratifin stimulates the expression of stromelysin-1 (MMP-3) mRNA by dermal fibroblasts in a concentration-dependent fashion. Furthermore, Western blot analysis of fibroblast-conditioned medium showed a peak in MMP-3 protein levels 48 h following treatment with recombinant stratifin. In a lasting-effect study, MMP-3 protein was detected in fibroblast-condition medium for up to 72 h post removal of stratifin. In conclusion, our results suggest that keratinocyte-releasable stratifin plays a major role in induction of ECM degradation by dermal fibroblasts through stimulation of key MMPs, such as MMP-1 and MMP-3. Therefore, stratifin protein may prove to be a useful target for clinical intervention in controlling excessive wound healing in fibrotic conditions.

Interleukin‐17 receptor deficiency results in impaired synovial expression of interleukin‐1 and matrix metalloproteinases 3, 9, and 13 and prevents cartilage destruction during chronic reactivated streptococcal cell wall–induced arthritis

To examine the role of interleukin-17 receptor (IL-17R) signaling in cartilage destruction and its interrelationship with synovial IL-1 expression during chronic reactivated streptococcal cell wall (SCW)-induced arthritis. SCW arthritis was repeatedly induced in wild-type (WT) and IL-17R-deficient (IL-17R-/-) mice. At different time points, joint inflammation was assessed by using calipers to measure joint swelling. On day 42, mice were killed, and knee joints were removed for histologic analysis. Quantitative polymerase chain reaction (PCR) analyses for different proinflammatory mediators and matrix metalloproteinases (MMPs) were performed on inflamed synovium from WT and IL-17R-/- mice after 5 repeated injections of SCW fragments. IL-17R signaling did not play a significant role in acute joint swelling induced by a single injection of SCW fragments directly into the joint. However, repeated local injections of SCW fragments into the knee joints of IL-17R-/- mice resulted in fewer infiltrating cells in the joint compared with WT mice. Moreover, histologic analysis on day 42 revealed a significant suppression of the degree of chondrocyte death and an absence of cartilage surface erosion in IL-17R-/- mice. Quantitative PCR analysis revealed impaired synovial expression of IL-1, IL-6, cyclooxygenase 2, stromelysin (MMP-3), gelatinase B (MMP-9), and collagenase 3 (MMP-13) in IL-17R-/- mice. These data show a critical role of IL-17R signaling in driving the synovial expression of proinflammatory and catabolic mediators, such as IL-1 and different MMPs, during progression from an acute, macrophage-driven joint inflammation to a chronic, cartilage-destructive, T cell-mediated synovitis. Prevention of IL-17R signaling warrants consideration as a therapeutic target in chronic destructive arthritis.

Isolation, characterization, and expression of stromelysin-1 in primary tumors of dogs

To isolate and characterize the cDNA sequence of canine stromelysin-1 (matrix metalloproteinase [MMPI-3), screen various naturally developing primary tumors of dogs, and assess the effect of stromelysin-1 on survival of dogs with cancer. 3 canine cell lines and biopsy specimens of primary tumors collected from 54 dogs. 3 canine cell lines and biopsy specimens of primary tumors collected from 54 dogs at the University of Illinois Veterinary Teaching Hospital were used in the study. Primer sets based on human stromelysin-1 and consensus sequences were designed for expression, screening, and isolation. Two additional primer sets were designed for screening. Samples were assayed at least in duplicate. Data were analyzed for differences in expression of stromelysin-1 on the basis of sex, age, metastasis, malignant versus nonmalignant tissue origin, and duration of patient survival. A 1,479-bp cDNA nucleotide sequence was amplified from established canine cell lines. The open reading frame encoded a protein consisting of 478 amino acids. This sequence was 70% to 88% homologous with stromelysin-1 of other species at the amino acid level. Fifty-four samples were screened for stromelysin-1. Of these, 34 (63%) had positive results and 20 (37%) had negative results for expression. Stromelysin-1 and metastasis were associated with a poor prognosis for survival. Stromelysin-1 is a potential activator of other members of the MMP family. Additional studies are needed to investigate the relationship between stromelysin-1 production and aggressive biological behavior of tumors in dogs.

A Causative Role of Stromelysin-3 in Extracellular Matrix Remodeling and Epithelial Apoptosis during Intestinal Metamorphosis in Xenopus laevis

The matrix metalloproteinases are a family of proteases capable of degrading various components of the extracellular matrix. Expression studies have implicated the involvement of the matrix metalloproteinase stromelysin-3 (ST3) in tissue remodeling and pathogenesis. However, the in vivo role of ST3 has been difficult to study because of a lack of good animal models. Here we used intestinal remodeling during thyroid hormone-dependent metamorphosis of Xenopus laevis as a model to investigate in vivo the role of ST3 during postembryonic organ development in vertebrates. We generated transgenic tadpoles expressing ST3 under control of a heat shock-inducible promoter. We showed for the first time in vivo that wild type ST3 but not a catalytically inactive mutant was sufficient to induce larval epithelial cell death and fibroblast activation, events that normally occur only in the presence of thyroid hormone. We further demonstrated that these changes in cell fate are associated with altered gene expression in the intestine and remodeling of the intestinal basal lamina. These results thus suggest that ST3 regulates cell fate and tissue morphogenesis through direct or indirect ECM remodeling.

Expression of stromelysin-3 (matrix metalloproteinase-11) in macrophages of murine thymus following thymocyte apoptosis

High expression of stromelysin-3 (ST-3), also known as matrix metalloproteinase-11, has been implicated in tumor progression and intense tissue remodeling. Nonetheless, details of the cell type(s) expressing ST-3 are less well defined. Here, we report that ST-3 expression was elevated in mouse thymus following thymocyte apoptosis after administration of anti-CD3 Ab. TUNEL analysis revealed that many thymocytes in the cortical region were induced to apoptotic cell death 14 h after the injection. After an additional 2–6 h, ST-3 expression in the thymus was more apparent. Co-staining analysis by anti-ST-3 and F4/80 Abs showed that most F4/80-positive macrophages were also positive for ST-3. Murine peritoneal macrophages were found to constitutively express ST-3, and exposure to apoptotic cells hardly affected ST-3 expression in the macrophages. Taken together, our results indicate that ST-3 is not involved in the execution process of thymocyte apoptosis, and the increased levels of ST-3 in the thymus may be due to the presence of macrophages responsible for clearance of apoptotic cells.

Stromelysin 3, Ets-1, and Vascular Endothelial Growth Factor Expression in Oral Precancerous and Cancerous Lesions: Correlation with Microvessel Density, Progression, and Prognosis

Identification of molecular changes characteristic of development and progression of oral cancer are of paramount importance for effective intervention. Stromelysin 3 (MMP11) is a unique matrix metalloproteinase shown to have dual function during cancer progression. The transcription factor Ets-1 and vascular endothelial growth factor (VEGF) are important proangiogenic factors in cancer. This study was designed to test the hypothesis that concomitant expression of stromelysin 3, Ets-1, and/or VEGF affects the development, progression, and prognosis of oral cancer. Immunohistochemical analysis of stromelysin 3, Ets-1, VEGF, and platelet/endothelial cell adhesion molecule 1 (a marker for intratumoral microvessel density) was carried out in serial paraffin embedded tissue sections of 220 oral squamous cell carcinomas (OSCC), 90 precancerous lesions (59 hyperplasias and 31 dysplasias), and 81 matched histologically normal oral tissues. Ets-1, VEGF, and stromelysin 3 expression independently correlated with increased intratumoral microvessel density in precancerous lesions (P = 0.05, 0.001, and 0.026, respectively) as well as in SCCs (P = 0.005, 0.01, and 0.031, respectively). Logistic regression analysis revealed that concomitant expression of stromelysin 3 and Ets-1 (stromelysin 3(+)/ Ets-1(+) phenotype; odds ratio, 3.7; P = 0.001) was the most significant predictor for transition to precancerous stage, whereas dual expression of stromelysin 3 and VEGF (stromelysin 3(+)/ VEGF(+) phenotype; odds ratio, 2.07; P = 0.004) was the most important predictor for progression from precancerous stage to frank malignancy. Intriguingly, Ets-1 expression was significantly associated with VEGF expression and stromelysin 3 expression in precancerous tissues as well as OSCCs. Follow-up data for 144 patients for a maximum period of 115 months showed that VEGF [hazards ratio (HR), 4.532; P = 0.004] and Ets-1 (HR = 2.182; P = 0.049) expression significantly correlated with reduced disease-free survival in univariate analysis. In bivariate analysis, patients harboring Ets-1(+)/VEGF(+) phenotype had the worst survival (median disease-free survival, 50 months; HR, 2.943; P = 0.003). Multivariate analysis using Cox's proportional hazards model showed that increased VEGF expression was the most significant adverse prognosticator in OSCC patients (HR, 4.470; P = 0.004). In conclusion, this study provides the first evidence of concomitant expression of stromelysin 3, VEGF, and Ets-1 in clinical specimens in different stages of development of oral cancer. In early stages, concomitant expression of stromelysin 3 and Ets-1 favors the development of a precancerous state, whereas dual expression of stromelysin 3 and VEGF is associated with progression from precancerous to cancerous state. VEGF expression is an adverse prognosticator for disease-free survival.

The matrix metalloproteinase stromelysin-3 cleaves laminin receptor at two distinct sites between the transmembrane domain and laminin binding sequence within the extracellular domain

The matrix metalloproteinase (MMP) stromelysin-3 (ST3) has long been implicated to play an important role in extracellular matrix (ECM) remodeling and cell fate determination during normal and pathological processes. However, like other MMPs, the molecular basis of ST3 function in vivo remains unclear due to the lack of information on its physiological substrates. Furthermore, ST3 has only weak activities toward all tested ECM proteins. Using thyroid hormone-dependent Xenopus laevis metamorphosis as a model, we demonstrated previously that ST3 is important for apoptosis and tissue morphogenesis during intestinal remodeling. Here, we used yeast two-hybrid screen with mRNAs from metamorphosing tadpoles to identify potential substrate of ST3 during development. We thus isolated the 37 kd laminin receptor precursor (LR). We showed that LR binds to ST3 in vitro and can be cleaved by ST3 at two sites, distinct from where other MMPs cleave. Through peptide sequencing, we determined that the two cleavage sites are in the extracellular domain between the transmembrane domain and laminin binding sequence. Furthermore, we demonstrated that these cleavage sites are conserved in human LR. These results together with high levels of human LR and ST3 expression in carcinomas suggest that LR is a likely in vivo substrate of ST3 and that its cleavage by ST3 may alter cell-extracellular matrix interaction, thus, playing a role in mediating the effects of ST3 on cell fate and behavior observed during development and pathogenesis.

Elevation of hippocampal MMP-3 expression and activity during trauma-induced synaptogenesis

The matrix metalloproteinase (MMP) enzyme family contributes to the regulation of a variety of brain extracellular matrix molecules. In order to assess their role in synaptic plasticity following traumatic brain injury (TBI), we compared expression of stromelysin-1 (MMP-3) protein and mRNA in two rodent models of TBI exhibiting different levels of recovery: adaptive synaptic plasticity following central fluid percussion injury and maladaptive synaptic plasticity generated by combined TBI and bilateral entorhinal cortical lesion (TBI + BEC). We sampled the hippocampus at 7 days postinjury, targeting a selectively vulnerable brain region and a survival interval exhibiting rapid synaptogenesis. We report elevated expression of hippocampal MMP-3 mRNA and protein after TBI. MMP-3 immunohistochemical staining showed increased protein levels relative to sham-injured controls, primarily localized to cell bodies within the deafferented dendritic laminae. Injury-related differences in MMP-3 protein were also observed. TBI alone elevated MMP-3 immunobinding over the stratum lacunosum moleculare (SLM), inner molecular layer and hilus, while TBI + BEC generated more robust increases in MMP-3 reactivity within the deafferented SLM and dentate molecular layer (DML). Double labeling with GFAP confirmed the presence of MMP-3 within reactive astrocytes induced by each injury model. Semi-quantitative RT-PCR revealed that MMP-3 mRNA also increased after each injury, however, the combined insult induced a much greater elevation than fluid percussion alone: 1.9-fold vs. 79%, respectively. In the TBI + BEC model, MMP-3 up-regulation was spatio-temporally correlated with increased enzyme activity, an effect which was attenuated with the neuroprotective compound MK-801. These results show that distinct pathological conditions elicited by TBI can differentially affect MMP-3 expression during reactive synaptic plasticity. Notably, these effects are both transcriptional and translational and are correlated with functionally active enzyme.

Matrix metalloproteinase induction in fibrosis and fibrotic nodule formation due to silica inhalation

Matrix metalloproteinases (MMPs) are the principle enzymes that initiate degradation of collagen. We examined the role of MMPs during alveolar wall fibrosis and fibrotic nodule formation from silica exposure. Rats were exposed to filtered air or 15 mg/m(3) silica by inhalation for 5 days/wk, 6 h/day. Lungs were preserved by intratracheal instillation of fixative at 20, 40, 60, 79, and 116 days of exposure. Additional groups were fixed after 20, 40, and 60 days of exposure followed by 36 days of recovery. The number of nodules, defined by a collagenous core and a bounding cell layer detached from the alveolar wall, was determined by morphometry. Lungs showed increased alveolar wall collagen and fibrotic nodules at 79 and 116 days of exposure with increased collagenase and gelatinase activity. The number of nodules per lung in exposed groups increased from 619 +/- 447 at 40 days to 13,221 +/- 1,096 at 116 days (means +/- SE, n = 5). No nodules were seen in control lungs. Silica-exposed rats with a 36-day recovery in filtered air showed enhanced MMP activity over exposure to silica for the same duration with no recovery. MMP-2 and MMP-9 were significantly elevated in alveolar macrophages after 40-day exposure. Stromelysin expression was demonstrated in alveolar macrophages and cells within fibrotic nodules. TIMP-1 expression was not significantly altered. In summary, MMP activity was upregulated at 40 days of silica exposure and progressively increased during ensuing fibrotic responses. Early expression of stromelysin was found in fibrosing alveolar walls and fibrotic nodules.

Mechanisms of abruption-induced premature rupture of the fetal membranes: Thrombin enhanced decidual matrix metalloproteinase-3 (stromelysin-1) expression

The aim of the study was to evaluate thrombin and progestin effects on matrix metalloproteinase-3 expression in term decidual cells as a mechanism of abruption-related preterm delivery. Decidual cells were isolated by standard techniques, purified to homogeneity, grown to confluence, and passaged. Cultures were primed with 10 (-8) M estradiol or estradiol plus 10 (-7) progestin and then incubated in a defined medium with corresponding steroid(s) plus or minus thrombin or the protease-activated thrombin receptor-1 agonist for 24 hours. Secreted matrix metalloproteinase-3 levels were assessed by enzyme-linked immunosorbent assay, and immunoblotting and messenger RNA levels were measured by Northern blotting and quantitative reverse transcription-polymerase chain reaction. Immunoreactive matrix metalloproteinase-3 levels were inhibited 66% by estradiol plus progestin versus estradiol ( P < .05). Thrombin elicited a dose-dependent reversal in this progestin inhibition, producing a 2.5-fold increase at 2.5 U/mL ( P < .05) that attained 33% of matrix metalloproteinase-3 levels in parallel incubations with estradiol plus thrombin. Protease-activated thrombin receptor-1 agonist mimicked 60% of thrombin-enhanced matrix metalloproteinase-3 output. Immunoblotting validated the enzyme-linked immunosorbent assay results. Northern blotting and quantitative reverse transcription-polymerase chain reaction demonstrated corresponding effects on steady-state messenger RNA levels. Abruption-generated thrombin promotes preterm delivery by mediating fetal membrane extracellular matrix degradation via enhanced decidual cell matrix metalloproteinase-3 expression, whereas progesterone blunts this thrombin-induced effect.

Clinicopathological Significance of Stromelysin-3 Expression in Colorectal Cancer

Objective: Stromelysin-3 (ST3) is a member of the matrix metalloproteinases and suggested to play a role in tissue remodeling observed in growth and metastasis of tumors. ST3 overexpression in breast cancer is associated with a worse outcome. Our aims were to analyze ST3 expression in primary colorectal tumors and metastases, and further to identify relationships of the expression to clinicopathological factors. Materials and Methods: ST3 expression was immunohistochemically analyzed in 200 primary colorectal adenocarcinomas and 36 corresponding lymph node metastases. Results: Scoring was performed by counting the percentages of positive cells and the percentages of positive areas. One hundred and one (51%) cases showed ≤5% positive cells and 99 (49%) >5% positive cells. One hundred and two (51%) cases showed ≤30% positive area and 98 (49%) >30% positive area. ST3 expression determined by both scoring methods was individually related to females, distally located tumors, infiltrative growth pattern and microsatellite stability. No relationship was found with age, Dukes’ stage, differentiation and survival. Conclusions: These results suggest that ST3 protein was more involved in the pathway of colorectal cancer development in females, distal locations, infiltrative growth patterns and microsatellite stability.