kD Type Research Articles

Matrix Metalloproteinase 9 Promoter Activity Is Induced Coincident with Invasion during Tumor Progression

Matrix metalloproteinase 9 (MMP-9, also known as gelatinase B or 92-kd Type IV collagenase) is overexpressed in many human and murine cancers. We induced carcinomas in mice carrying a transgene that links the MMP-9 promoter to the reporter ss-galactosidase so that activation of the MMP-9 promoter would be indicated by ss-galactosidase. Mammary carcinomas were induced by mating the MMP-9 promoter reporter transgenic mice with mice carrying a transgene for murine mammary tumor virus promoter linked to polyoma middle T antigen, a transgene that leads to rapid development of mammary tumors in female mice. None of the hyperplastic mammary glands and none of the carcinomas in situ expressed ss-galactosidase. However, all invasive tumors had evidence of ss-galactosidase expression. In addition to the breast carcinomas, a malignant teratoma in a female and a papillary adenocarcinoma in the pelvic region of a male arose and were also ss-galactosidase positive. We also induced skin tumors in the mice with the MMP-9 reporter transgene with 7, 12-dimethylbenz[a]anthracene (DMBA) treatment followed by phorbol 12 myristate 13-acetate (TPA). None of the papillomas or in situ carcinomas showed any ss-galactosidase expression, but expression was seen in invasive carcinoma. Although normal skin epithelial cells did not express ss-galactosidase, we did find staining in a few cells at the duct of the sebaceous gland at the base of the hair follicles. The MMP-9 reporter transgene did not lead to expression in the alveolar macrophages, confirming that additional upstream sequences are required for expression in macrophages. These experiments have revealed that MMP-9 promoter activity is induced coincident with invasion during tumor progression. Furthermore, this indicates that the more proximal upstream elements of the promoter are sufficient for MMP-9 transcription during tumor progression.

Matrix metalloproteinase-9 is increased and correlates with severity in Guillain-Barré syndrome.

To study the expression and activity of matrix metalloproteinases (MMPs) MMP-2 (72-kd type IV collagenase, gelatinase A), MMP-3 (58-kd stromelysin-1), and MMP-9 (92-kd type IV collagenase, gelatinase B) and tissue inhibitors of MPs (TIMP) in patients with Guillain-Barre syndrome (GBS). MMPs are able to proteolysis of basement membranes and other matrix components, promoting transmigration of inflammatory cells from circulation to nerve tissue. Twenty-five patients with GBS were analyzed according to the phase of the disease, i.e., progression, plateau, early recovery, and late recovery. Determinations of MMP-2, MMP-3, MMP-9, and TIMP-1 were performed using ELISA, zymography, and immunocytochemistry in circulation or peripheral nerve. MMP-9 plasma levels were increased in 67% of patients on admission and decreased from progression to late recovery (p < 0.002). During the course of GBS, MMP-9 was progressively balanced by its inhibitor TIMP-1, as assessed by the MMP-9/TIMP-1 ratio. MMP-9 and TIMP-1 plasma levels and the MMP-9/TIMP-1 ratio correlated positively with disability. MMP-2 expression was similar to controls. MMP-3 activity was not detected, and plasma levels were not different from those in controls. Positive MMP-9 immunolabeling was 51 +/- 11% of circulating lymphocytes. It was observed in some endothelial cells and mononuclear cells adherent to the endothelium and close to myelinated fibers. Circulating matrix metalloproteinases (MMP-9) correlates with disease severity in Guillain-Barré syndrome (GBS). MMP-9 likely represents an important molecule in the pathogenesis of GBS and therefore could represent an interesting therapeutic target.

Novel Feline Autoimmune Blistering Disease Resembling Bullous Pemphigoid in Humans: IgG Autoantibodies Target the NC16A Ectodomain of Type XVII Collagen (BP180/BPAG2)

In humans and dogs, bullous pemphigoid (BP) is an autoimmune blistering disease associated with the production of basement membrane autoantibodies that target the 180-kd type XVII collagen (BP180, BPAG2) and/or the 230-kd plakin epidermal isoform BPAG1e (BP230). In two adult cats, an acquired dermatosis and stomatitis was diagnosed as BP subsequent to the fulfillment of the following criteria: 1) presence of cutaneous vesicles, erosions, and ulcers; 2) histologic demonstration of subepidermal vesiculation with inflammatory cells, including eosinophils; 3) in vivo deposition of IgG autoantibodies at the epidermal basement membrane zone; and 4) serum IgG autoantibodies targeting a 180-kd epidermal protein identified as type XVII collagen. In both cats, the antigenic epitopes targeted by IgG autoantibodies were shown to be situated in the NC16A ectodomain of type XVII collagen, a situation similar to that of humans and dogs with BP. Feline BP therefore can be considered a clinical, histopathologic, and immunologic homologue of BP in humans and dogs.

Gelatinase activity in keloids and hypertrophic scars.

Keloids and hypertrophic scars are characterized by excessive deposition of collagen, which may result from insufficient protein degradation. Little is known about the levels of two gelatinases, matrix metalloproteinase-2 (72 kD type IV collagenase) and matrix metalloproteinase-9 (matrix metalloproteinase-9; 92 kD type IV collagenase) in these abnormal scars. The purpose of this study was to determine levels of these proteinases in tissue from hypertrophic scars, keloids, and donor skin. Ten hypertrophic scar samples, 9 keloid samples, and 10 donor skin samples were frozen, pulverized, homogenized, clarified by centrifugation, and analyzed for matrix metalloproteinases by quantitative zymography. Identity of matrix metalloproteinases was determined using a conditioned media reference standard, molecular weight ladders, and Western blotting. Levels of matrix metalloproteinase-9 activity were very low or undetectable in all samples. However, matrix metalloproteinase-2 activity was significantly elevated in keloids and hypertrophic scars vs. donor samples: 2.6 and 3.9-fold increases for latent matrix metalloproteinase-2, 7.8 and 6.9-fold increases for active matrix metalloproteinase-2, respectively. We conclude that little matrix metalloproteinase-9 activity (the gelatinase involved in early tissue repair) is present in keloids and hypertrophic scars, while matrix metalloproteinase-2 activity (the gelatinase involved in prolonged tissue remodeling) is present in donor skin and is significantly increased in hypertrophic scars and keloids.

Overexpression of human homologs of the bacterial DnaJ chaperone in the synovial tissue of patients with rheumatoid arthritis.

To study the expression of the chaperone family of J proteins in the synovial tissue of patients with rheumatoid arthritis (RA) or osteoarthritis. Rabbit antibodies specific for a synthetic peptide (pHSJ1: EAYEVLSDKHKREIYD), representing the most conserved part of all J domains thus far identified--among them the Drosophila tumor suppressor Tid56--were used in immunohistochemical analyses of frozen sections of synovial tissue and immunoblotting of protein extracts of adherent synovial cells. IgG specific for Tid56 was also used. Both antisera predominantly and intensely stained synovial lining cells from RA patients; other cells did not stain or stained only faintly. In immunoblots, anti-pHSJ1 specifically detected several bands with molecular weights of >74 kd (type I), 57-64 kd (type II), 41-48 kd (type III), and < or =36 kd (type IV). The strongest band detected in RA adherent synovial cells was the type II band, whereas in a B cell line, a type I band was prominent. Several potentially new members of the J family are described. The type II band represents the human homolog of the Drosophila Tid56 protein and is strongly expressed in RA synovial tissue.

Integrin α2β1 Recognizes Laminin-2 and Induces C-erb B2 Tyrosine Phosphorylation in Metastatic Human Melanoma Cells

Previous studies have shown that tumor cells with metastatic propensity secrete more of the laminin α2 chain than non-metastatic tumor cells do, and that laminin-2, which contains the α2 chain, promotes cell adhesion better than laminin-1 (Jenq et al. (1994). Differentiation, 58, 29-36). The current studies were designed to determine whether a correlation exists between the expression of the laminin-2 isoform and the metastatic phenotype in melanoma cells. We found that expression of the laminin-2 isoform was upregulated in the metastatic melanoma cell lines tested. Cell attachment studies showed that metastatic melanoma cells attached more efficiently to laminin-2 substrates. Studies on integrin expression revealed that the presence of α2β1 integrin correlated with expression of the laminin-2 isoform in metastatic melanoma cells; anti-integrin α2 antibody prevented cell attachment to laminin-2 substrates. The data suggest that the α2β1 integrin is the receptor mediating cell attachment to the laminin-2 isoform. This interaction, mediated by the α2β1 integrin, stimulates secretion of the 72 kD type IV collagenase and induces a specific 185 kD protein tyrosine phosphorylation. The 185 kD tyrosine-phosphorylated protein was identified as the p185/C-erb B2 oncoprotein by immunoprecipitation. These studies suggest that upregulation of expression of the laminin-2 chain correlates with the metastatic phenotype of melanoma cells and provides evidence that the specific p185/C-erb B2 tyrosine phosphorylation may be involved in integrin-mediated signaling during tumor cell invasion and metastasis.

Prognostic value of MMP‐2 immunoreactive protein (72 kD type IV collagenase) in primary skin melanoma

The penetration of the subepithelial basement membrane is the first critical step in the dissemination of melanoma. In vitro studies have suggested that the 72 kD type IV collagenase (MMP-2) may be important in melanoma invasion. It has recently been demonstrated that the expression of MMP-2 immunoreactive protein increased with increasing atypia in melanocytic tumours and was associated with later haematogenous metastases in melanoma. This paper investigates the value of MMP-2 as a possible prognostic marker in melanoma. The expression of MMP-2 immunoreactive protein was studied with immunoperoxidase staining in paraffin-embedded sections of 50 cases of primary skin melanoma by using specific, affinity purified antibodies. Positive immunostaining was quantified by counting the percentage of positive cancer cells and was compared with clinical patient characteristics and survival. Sixty-four per cent of the primary melanoma cases displayed positive cytoplasmic immunostaining for MMP-2 in tumour cells. Marked overexpression of MMP-2 protein (≥34 per cent of melanoma cells positive) correlated with the 5-year survival of the patients when compared with patients with lower MMP-2 positivity, 55 per cent vs. 85 per cent, respectively (P<0·05). Male patients displayed positive staining more often than females (75 per cent vs. 54 per cent, respectively). There was no correlation between MMP-2 positivity and Clark level or Breslow classification. A distinct group with unfavourable prognosis was identified. The 10-year survival for MMP-2-positive male melanoma patients was 39 per cent as opposed to 79 per cent with the other melanoma patients (P<0·05). In the hierarchic Cox regression model for survival, MMP-2 immunoreactive protein was found to be independent of Clark level and Breslow classification. Overexpression of MMP-2 protein indicated a 4·5-fold relative risk of dying from melanoma. It is concluded that MMP-2 immunoreactive protein in melanoma cells is an independent prognostic factor for survival. High MMP-2 expression in male melanoma patients indicates an unfavourable prognosis. © 1998 John Wiley & Sons, Ltd.

Prognostic value of MMP-2 immunoreactive protein (72 kD type IV collagenase) in primary skin melanoma.

The penetration of the subepithelial basement membrane is the first critical step in the dissemination of melanoma. In vitro studies have suggested that the 72 kD type IV collagenase (MMP-2) may be important in melanoma invasion. It has recently been demonstrated that the expression of MMP-2 immunoreactive protein increased with increasing atypia in melanocytic tumours and was associated with later haematogenous metastases in melanoma. This paper investigates the value of MMP-2 as a possible prognostic marker in melanoma. The expression of MMP-2 immunoreactive protein was studied with immunoperoxidase staining in paraffin-embedded sections of 50 cases of primary skin melanoma by using specific, affinity purified antibodies. Positive immunostaining was quantified by counting the percentage of positive cancer cells and was compared with clinical patient characteristics and survival. Sixty-four per cent of the primary melanoma cases displayed positive cytoplasmic immunostaining for MMP-2 in tumour cells. Marked overexpression of MMP-2 protein (> or = 34 per cent of melanoma cells positive) correlated with the 5-year survival of the patients when compared with patients with lower MMP-2 positivity, 55 per cent vs. 85 per cent, respectively (P < 0.05). Male patients displayed positive staining more often than females (75 per cent vs. 54 per cent, respectively). There was no correlation between MMP-2 positivity and Clark level or Breslow classification. A distinct group with unfavourable prognosis was identified. The 10-year survival for MMP-2-positive male melanoma patients was 39 per cent as opposed to 79 per cent with the other melanoma patients (P < 0.05). In the hierarchic Cox regression model for survival, MMP-2 immunoreactive protein was found to be independent of Clark level and Breslow classification. Overexpression of MMP-2 protein indicated a 4.5-fold relative risk of dying from melanoma. It is concluded that MMP-2 immunoreactive protein in melanoma cells is an independent prognostic factor for survival. High MMP-2 expression in male melanoma patients indicates an unfavourable prognosis.

Expression of Matrix Metalloproteinases and Their Tissue Inhibitors in Human Brain Tumors

In this study, we investigated the expression patterns of 15 matrix metalloproteinases (MMPs) and three tissue inhibitors of metalloproteinase in gliomas, medulloblastomas, and normal brain tissue. By Northern blot analysis we found increased levels of mRNAs encoding for gelatinase A, gelatinase B, two membrane-type MMPs (mt1- and mt2-MMP), and tissue inhibitors of metalloproteinase-1 in glioblastomas and medulloblastomas. We observed a significant increase of mt1-MMP, gelatinase A, gelatinase B, and tissue inhibitors of metalloproteinase-1 in glioblastomas as compared with low-grade astrocytomas, anaplastic astrocytomas, and normal brain. In medulloblastomas, the expression of mt1-MMP, mt2-MMP, and gelatinase A were also increased, but to a lesser extent than that observed in glioblastomas. These data were confirmed at the protein level by immunostaining analysis. Moreover, substrate gel electrophoresis showed that the activated forms of gelatinases A and B were present in glioblastomas and medulloblastomas. These results suggest that increased expression of mt1-MMP/gelatinase A is closely related to the malignant progression observed in gliomas. Furthermore, the present study demonstrates, to our knowledge for the first time, that medulloblastomas express high levels of MMP.

Constitutive release of metalloproteinase-9 (92-kd type IV collagenase) by Kaposi's sarcoma cells.

Kaposi's sarcoma (KS) is an angioproliferative disease characterized by proliferating spindle-shaped cells, angiogenesis, and inflammatory cell infiltration. Several lines of evidence suggest that KS is a multifocal cytokine-mediated disease of vascular origin. Because metalloproteinases (MMPs) are important enzymes involved in angiogenesis, we studied their activity in five different KS-derived cell lines and compared these data with those obtained with human umbilical vein endothelial cells (HUVEC). We focused on the activity of the 72- and 92-kd type IV collagenases because these enzymes are thought to play an important role in the process of tumoral invasion. Nonstimulated HUVEC released a weak 72-kd collagenase activity and no 92-kd collagenase activity, as determined by zymographic analysis. Stimulation of HUVEC with phorbol myristate acetate (PMA) or TNF-alpha increased the 72-kd collagenase activity and also induced a 92-kd collagenase activity. By contrast, KS-derived cells constitutively released significant 72- and 92-kd collagenase activities. The basal release of these enzymes by KS cells was further enhanced by TNF-alpha or PMA. Conversely after in vivo exposure to chemotherapy, KS-derived cells showed a downregulation of the production of MMPS that could be reversed by the addition of TNF or PMA. These results suggest that KS cells have constitutive features of activated cells that have an invasive and metastasizing potential.

Imbalance Between Matrix Metalloproteinase (MMPs) and their Inhibitors in BALs of Mechanically Ventilated Neonates † 1704

Background: Neutrophil-derived proteinases (mainly serine proteinases) play an important role in neonatal lung tissue damage. However the exent to which MMPs (92 kD Type IV and 72 kD (Interstitial collagenases) participate in the remodelling of the extracellutar matrix (ECM) is unknown. Intrapulmonary leucocytes (neutrophils and alveolar macrophages) are the main secretors of MMPs gelatinases, induced through a prostaglandin-E2 (PG-E2) dependent pathway involving cyclooxygenase-2 (COX-2) and balanced by the natural tissue inhibitors of MMPs (TIMPs). Aims: 1) To evaluate the gelatinolytic activity of MMPs isoforms and their inhibitors in BAL fluids from mechanically ventilated infants; 2) To study the effect of bacterial lipopolysaccaride (LPS) on MMPs activity and inhibition in conditioned media (CM) of cultured BAL cells, in the presence and absence of Ibuprofen (Ibu) or Dexamethasone (Dex). Methods: BAL samples were obtained from 13 intubated and mechanically ventilated neonates(GA>27 629<4039 gr) age 1-30 days with RDS, perinatal asphyxia, diaphragmatic hernia, BPD) and from 3 pediatric patients with chronic pulmonary disease (dysmotile cilia syndrome, cystic fibrosis). After separation of cellular components from fluid phase (FP) by centrifugation, washed pellets were resuspended in RPMI medium, (supplemented with 0,1% FCS and antibiotics), allowed to adhere to Petri dishes at 37°C and 5% CO2 for 1 hour (to select the monocytic-macrophagic population) and then tested for induction of MMPs and production of PG-E2, under basal condition and after 24 hours of treatment with diluent (PBS) versus LPS (10 mg/ml). +/- 50mM Ibu or 1mM Dex. Radioimmunoassays for PG-E2 quantitation, zymograms and reversezymograms for MMPs gelatinolytic activity and inhibition, were of CM of cultured adherent cells and FPs from BALs. Results: Zymograms performed on samples from FPs and CM of cultured BAL cells, in basal conditions, show an appreciable gelatinolytic activity (mainly type IV collagenase MMP-9), whereas reverse zymograms show less evident bands of inhibition (TIMPs). A marked induction of MMP-9 (more evident in the neonates than in the pediatric patients) and a consistent reduction of TIMPs activity, associated with a 10-100 fold increase of PG-E2 production, were observed after 24 hours of LPS stimulus. The patterns of gelatinolytic activity and inhibition were almost totally reversed in the presence of Ibu, with only minimal changes induced by Dex. Conclusions An imbalance between ECM degradative activity (particularly of MMP-9) and inhibition is observed in FPs and cells from BALs, under basal conditions and after LPS induction. Sub-epithelial basal membrane ECM components (particular type IV collagen) are more susceptible to damage since MMP-9 specifically degrades type IV collagen and can significantly contribute to the sustained increase in pulmonary permeability characteristic of the flogistic reaction that follows acute neonatal lung injury. The role of the MMPs/TIMPs imbalance in the evolution of neonatal lung disease and its possible modulation by acting on the COX-2-mediated PG-E2 inducible synthesis, warrants further studies.

Expression of gelatinase B by trophoblast cells: Down-regulation by progesterone

Objective: It is now accepted that gelatinase B (92 kd type IV collagenase) is involved in blastocyst implantation and trophoblast invasion. However, little is known about the regulation of this enzyme at the fetomaternal interface. Progesterone has been demonstrated to inhibit gelatinase B secretion from endometrial cells, myometrium, and cervical fibroblasts. Interestingly, the promotor of gelatinase B contains a progesterone-responsive element that may explain transcriptional activation of this metalloproteinase by progesterone. It may be hypothesized that progesterone secreted from trophoblast cells, representing the fetal part of the fetomaternal interface, may have a role in the regulation of gelatinase secretion and blastocyst implantation. Study Design: To this end, use was made of first-trimester trophoblast cells obtained from first-trimester pregnancy terminations. The trophoblast cells were separated by trypsin degradation and fractionation on Percoll gradients. Metalloproteinase activity was measured by zymography, and the expression of the gelatinase B messenger ribonucleic acid was determined by the solution hybridization/ribonuclease protection assay. Results: Primary cell cultures of trophoblasts from first trimesters of pregnancy constitutively elaborated two species of type IV collagenases (gelatinase A and B) as assessed on a gelatin matrix. Treatment with progesterone decreased the accumulation of a gelatinase B species in a dose-dependent fashion. Administration of a progesterone receptor antagonist onapristone (ZK-98.299) neutralized the progesterone inhibitory effect on the gelatinase B in a dose-dependent fashion, thus supporting the presumption that the progesterone effect is receptor mediated. Progesterone significantly attenuated the expression of gelatinase B by trophoblast cells, an effect that was neutralized by ZK-98.299. Conclusion: These observations provide strong indirect support for the participation of progesterone in the regulation of gelatinase B in trophoblast cells. It may be an important regulator of gelatinase production at the fetomaternal interface. (Am J Obstet Gynecol 1998;178:457-61.)

72-kD (MMP-2) and 92-kD (MMP-9) Type IV Collagenase Production and Activity in Different Histologic Types of Lung Cancer Cells

In this study we examined the production of gelatinases A and B (MMP-2 and MMP-9), and their natural inhibitors TIMP-1 and TIMP-2 in cell lines derived from different histologic types of lung cancer. Gelatinolytic activity was measured by zymography and radiolabeled gelatin degradation. Immunocytochemistry and Western blot analysis were performed to corroborate the presence of immunoreactive MMP-2, MMP-9, TIMP-1 and TIMP-2 proteins. The highest gelatinolytic activity was identified in the cell extracts from a small-cell carcinoma cell line. MMP-9 was observed in all samples as a proenzyme, while MMP-2 was present as zymogen in the squamous-cell and in the small-cell carcinomas, and in its active form in one squamous-cell carcinoma cell line. TIMPs were also present in the neoplastic lung cell lines. TIMP-1 was observed in the media of all cells as a 21-kD band, and as TIMP-1 polymers with the exception of the small-cell carcinoma samples. TIMP-2 was found as higher-order molecular immunoreactive complexes that may correspond to proMMP-2/TIMP-2 complexes. These results demonstrate that lung neoplastic cells produce both MMP-2 and MMP-9 and their inhibitors, with the small-cell carcinoma cell extracts showing the highest enzymatic activity. This gelatinolytic activity fits well with the clinical metastatic behavior of this type of lung cancer.

Cytokine mediated regulation of 92-kD Type IV collagenase, tissue inhibitor of metalloproteinase-1 (TIMP-1) and Tissue Inhibitor Of Metalloproteinase-3 (TIMP-3) expression in human endometrial stromal cells

Cytokine mediated regulation of 92-kD Type IV collagenase, tissue inhibitor of metalloproteinase-1 (TIMP-1) and Tissue Inhibitor Of Metalloproteinase-3 (TIMP-3) expression in human endometrial stromal cells

Hypoxia increases human keratinocyte motility on connective tissue.

Re-epithelialization of skin wounds depends upon the migration of keratinocytes from the cut margins of the wound and is enhanced when human keratinocytes are covered with occlusive dressings that induce hypoxia. In this study, two independent migration assays were used to compare cellular motility on connective tissue components under normoxic or hypoxic conditions. Human keratinocytes apposed to collagens or fibronectin exhibited increased motility when subjected to hypoxic (0.2 or 2% oxygen) conditions compared with normoxic (9 or 20% oxygen) conditions. When compared with normoxic cells, hypoxic keratinocytes exhibited increased expression and redistribution of the lamellipodia-associated proteins (ezrin, radixin, and moesin). Furthermore, hypoxic keratinocytes demonstrated decreased secretion of laminin-5, a laminin isoform known to inhibit keratinocyte motility. Hypoxia did not alter the number of integrin receptors on the cell surface, but did induce enhanced secretion of the 92-kD type IV collagenase. These data demonstrate that hypoxia promotes human keratinocyte motility on connective tissue. Hypoxia-driven motility is associated with increased expression of lamellipodia proteins, increased expression of collagenase and decreased expression of laminin-5, the locomotion brake for keratinocytes.

The influence of transforming growth factor beta 1 on the expression of genes coding for matrix metalloproteinases and tissue inhibitors of metalloproteinases during regeneration from cerulein-induced pancreatitis.

Enhanced synthesis and deposition of extracellular matrix (ECM) components is a characteristic feature during regeneration from acute cerulein-induced pancreatitis in rats. Transforming growth factor beta 1 (TGF beta 1) has been suggested to be an important modulator of the ECM by interfering with a number of essential processes such as the synthesis of ECM components. To study the involvement of the ECM degrading proteases (matrix metalloproteinases; MMPs) and their specific inhibitors in the process of pancreatic regeneration, we examined the expression of these genes on the transcript level and the activation of the corresponding enzymes by use of zymographies. Pancreatic RNA and protein were extracted from rats sacrificed 1, 2, 3, 5, and 7 days after induction of cerulein pancreatitis. To investigate the influence of TGF beta on gene expression of ECM proteases and their specific inhibitors, we blocked the activity of TGF beta 1 during regeneration from acute pancreatitis by use of neutralizing antibodies against TGF beta 1. Steady levels of 72-kD type IV collagenase (MMP-2), stromelysin (MMP-3), and tissue inhibitor of metalloproteinase 2 (TIMP-2) mRNA were significantly increased 2 days after induction of pancreatitis. MMP-9 and MMP-3 enzyme activity was elevated 12 h after induction of pancreatitis, whereas MMP-2 activity increased 12 h later. Inhibition of TGF beta 1 by neutralizing antibodies only reduced the amount of stromelysin transcripts throughout pancreatic regeneration. In summary, ECM degrading proteases, in particular stromelysin, appear to be involved in ECM remodeling during pancreatic regeneration. TGF beta 1 may be responsible for regulation of stromelysin transcription.

Expression of matrix metalloproteinases and their inhibitors in aneurysms and normal aorta

Background. Abdominal aortic aneurysms (AAAs) are characterized by degradation of collagen and elastin resulting from increases in matrix metalloproteinase (MMP) activity. Previous authors have identified isolated increases in expression of specific MMPs in AAAs, but none have compared relative levels of expression of particular MMPs to one another or to those of their inhibitors, the tissue inhibitors of metalloproteinases (TIMPs). This study proposes to quantify relative mRNA levels for interstitial collagenase (MMP-1), 72 kd type IV collagenase (MMP-2), 92 kd type IV collagenase (MMP-9), TIMP-1, and TIMP-2 in normal aorta (NA) and AAA to provide insight as to the relative importance of each in aneurysm formation. Methods. Competitive polymerase chain reactions (PCRs) with gene-specific external standards and cDNA derived from AAAs (n = 8; mean age, 67.4 years) and NA (n = 5; mean age, 40.6 years) were used to quantify mRNA levels. Results were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels, determined by means of competitive PCR, and compared by means of Mann-Whitney statistics. Results. Significant increases in MMP mRNA expression in AAA over NA were observed for MMP-1 (3.64 versus 0.43, p = 0.007), MMP-9 (78.03 versus 3.35, p = 0.003), TIMP-1 (835.32 versus 477.2, p = 0.027), and TIMP-2 (18.09 versus 4.14, p = 0.003). The ratio of MMP to TIMP mRNA levels was higher in AAA than NA (0.135 versus 0.045, p = 0.018). Conclusions. Increases in expression of MMP-1, MMP-9, and MMP/TIMP ratios may result in increased proteolysis and matrix degradation, which characterize AAAs. MMP-9 appears to be the predominant metalloproteinase expressed in AAA, because its mRNA levels were more than 20 times and 2 times higher than those of MMP-1 and MMP-2, respectively. TIMP-1 mRNA levels were in molar excess to those of any of the metalloproteinases studied.

A new prognostic strategy for gastric carcinoma: mRNA expression of tumor growth-related factors in endoscopic biopsy specimens.

The study analyzed the prognostic value of the transcription of several tumor growth-related genes in gastric carcinoma biopsy specimens. The nodal status is one of the most significant prognostic factors in gastric carcinoma. There are, however, no satisfactory parameters for the preoperative assessment of nodal status. A reverse transcriptase-polymerase chain reaction analysis was used to analyze the transcription of several tumor growth-related genes in endoscopic biopsy specimens from 78 gastric carcinomas. The factors examined were cyclin D1, cyclin E, urokinase-type plasminogen activator, 72-kd type IV collagenase, vascular endothelial growth factor, platelet-derived growth factor-A (PDGF-A), transforming growth factor-beta, and interleukin-10. The relation between the mRNA expression and the clinical pathologic parameters was analyzed statistically. The incidence of PDGF-A (p = 0.010) and transforming growth factor-beta (p = 0.009) mRNA expression increased as the pathologic stage advanced. Nodal metastasis correlated with cyclin D1 (p = 0.045), cyclin E (p = 0.037), urokinase-type plasminogen activator (p = 0.047), and PDGF-A (p = 0.003) mRNA. Interestingly, the expression of PDGF-A mRNA showed a positive correlation (p = 0.004) with the early presence of lymph node metastases. Tumor growth-related factor mRNA in biopsy specimens may be a new prognostic tool.

In vivo potential of m152 gene of murine cytomegalovirus to evade control by MHC class I restricted T cells

Recognition and destruction of virus-infected cells by class I major histocompatibility complex (MHC) restricted cytotoxic T lymphocytes (CTL) is a central part of the immune systems attempts to control virus infection. Cytomegaloviruses (CMV) have diverse mechanisms to evade this type of immune response. Murine CMV (MCMV) encodes more than one gene product that interact with MHC class I maturation. The m152 gene encodes a 40 kD type I transmembrane glycoprotein that arrests the export of peptide loaded MHC class I complexes from the ERGIC/cis-Golgi compartment, and inhibits lysis by CTL (Ziegler et al., Immunity 8:57, 1997). There is no evidence, however, for the biological significance of this gene during viral infection in vivo. If the m152 gene effect on antigen presentation in the MHC class I pathway is relevant in vivo, the deletion of the gene should result in an attenuated virus mutant due to the increased susceptibility to CTL control. The attenuation effect should be absent or reduced in a mouse where this presentation pathway plays no role. The results indicate that recombinant MCMV with the deletion of the m152 gene has restricted replication capacity during the acute infection, compared with wild-type MCMV. Reintroduction of the m152 gene restores the wild-type pattern of virus replication and virulence. The attenuating effect of m152 deletion mutant is not observed in mice devoid of MHC class I molecules, and can be abolished in normal mice by depletion of T cells. Adoptive transfer studies revealed that m152 deletion mutant virus is more susceptible to control by both primed and nonprimed T cells. Although m152 is susceptible to CD8+ T cell control, the results of adoptive transfer studies indicate that this virus is also more susceptible to CD8-independent control mechanisms.

Transcriptional activation of the matrix metalloproteinase-9 gene in an H-ras and v-myc transformed rat embryo cell line.

The 92 kd type IV collagenase/gelatinase (MMP-9) is important in mediating basement membrane and extracellular matrix degradation in metastasis. Because MMP-9 is made in tumor cells, but not in quiescent normal cells, we wished to identify the transcriptional elements responsible for its synthesis in tumor cells. We chose to characterize transcriptional regulation of the MMP-9 gene in a highly metastatic H-ras and v-myc transformed rat embryo cell line which overexpresses MMP-9. Using transient transfection of reporter gene constructs containing either 5'-deleted or mutated MMP-9 promoter fragments, as well as electrophoretic mobility shift assays, we have demonstrated that multiple transcription factor consensus binding motifs in the promoter, including those for NFkappaB, SP-1, Ets, AP-1, and a retinoblastoma binding element, participate in transcriptional regulation of MMP-9 expression in this cell line. Also, deletion of an alternating purine-pyrimidine tract in the downstream promoter was found to decrease transcriptional activity, suggesting that promoter conformation may be important in MMP-9 regulation. Thus multiple pathways leading to activation of NFkappaB, SP-1, Ets, AP-1, and retinoblastoma binding factors in tumor cells all may contribute to MMP-9 transcription and hence to metastasis.