Jouko Lohi

Proteolytic processing of the 72,000-Da type IV collagenase by urokinase plasminogen activator☆

Regulation of the activity of proteolytic enzymes is of major importance in the turnover of connective tissues. The search for physiologically relevant activation mechanisms of principal tissue-degrading enzymes, e.g., metalloproteinases, has therefore been of wide interest. We have now studied whether the initiating factor of the fibrinolytic system, urokinase plasminogen activator (u-PA), may also function in the early steps of activation of one of the metalloproteinases, the M(r) 72,000 gelatinase/type IV collagenase produced by cultured fibroblasts. Treatment of the secreted M(r) 72,000 proteinase by u-PA yielded a cleavage product of M(r) 62,000 as revealed by fluorography of radioactively labeled proteins as well as by gelatin zymography SDS-PAGE gels. The u-PA-catalyzed cleavage of the M(r) 72,000 proteinase was blocked by anti-u-PA antibodies, but was unaffected by the plasmin inhibitor aprotinin, thus indicating a specific action for the activator. On the contrary, the tissue activator of plasminogen, t-PA, did not cleave the type IV collagenase in similar assays. u-PA-catalyzed cleavage of recombinant type IV collagenase, produced in a baculovirus expression system, yielded a similar M(r) 62,000 activity in gelatinolysis assay. Zymograms of the isolated pericellular matrices of cultured fibroblasts also revealed M(r) 72,000 gelatinolytic polypeptide that was converted to an M(r) 62,000 form by u-PA. Both polypeptides were recognized in immunoblotting by antibodies against the gelatinase/type IV collagenase, suggesting immunological identity with the secreted enzyme. Thus the M(r) 72,000 gelatinase/type IV collagenase is not only secreted, but also deposited into the pericellular fibroblast matrix, and both forms are substrates for u-PA. The results suggest a new potential role for u-PA as a direct regulator of metalloproteinase-mediated extracellular proteolysis via the cleavage of the M(r) 72,000 gelatinase/type IV collagenase to an M(r) 62,000 form.

Epilysin (MMP-28) induces TGF-β mediated epithelial to mesenchymal transition in lung carcinoma cells

Epilysin (MMP-28) is the newest member of the matrix metalloproteinase (MMP) family. Although it is expressed in a number of tissues, no biological substrates or functions for this enzyme have been identified yet. We have expressed recombinant epilysin in A549 lung adenocarcinoma cells and found that this resulted in stable and irreversible epithelial to mesenchymal transition (EMT) accompanied by loss of cell surface E-cadherin, proteolytic processing of latent TGF-β-complexes and increased levels of active TGF-β. The cascade of events leading to the onset of EMT is prevented by the MMP inhibitor GM6001 or antibodies neutralizing the activity of TGF-β. Once EMT had occurred the cell phenotype could, however, not be reversed by the MMP-inhibitor. Importantly, the expression of epilysin also resulted in upregulation of MT1-MMP and gelatinase-B (MMP-9) and in the collagen invasive activity of A549 cells. Further, we found that epilysin and the recombinant hemopexin domain were targeted to the surface of epithelial cells. This cell surface interaction was sensitive to the proteolytic activity of MT1-MMP, and was lost after EMT. Current results indicate that epilysin can induce EMT and cell invasion through a TGF-β-dependent mechanism suggesting novel biological roles for this enzyme in the regulation of epithelial cell function and in the induction of carcinogenesis.

Esophageal Morbidity and Function in Adults With Repaired Esophageal Atresia With Tracheoesophageal Fistula A Population-Based Long-term Follow-up

Objective:We assessed esophageal morbidity and relationships between surgical complications, symptoms, endoscopic findings, immunohistochemistry, and esophageal motility in adults with repaired esophageal atresia (EA). Summary of Background Data:There exist no previous population-based long-term follow-up studies on EA. Methods:Participants were interviewed, and they underwent esophageal endoscopy and manometry. Matched control subjects (n = 287) served as controls. Results:A total of 101 (42%) individuals representative of the entire study population participated at a mean age of 36 years (range, 21–57). Symptomatic gastroesophageal reflux had occurred in 34% and dysphagia in 85% of the patients and in 8% and 2% of the controls (P < 0.001 for both). Endoscopic findings included hiatal hernia (28%), Barrett′s esophagus (11%), esophagitis (8%), and anastomotic stricture (8%). Immunohistochemistry revealed esophagitis in 25%, and CDX2-positive columnar epithelial metaplasia in 21%, with additional goblet cells and MUC2 positivity in 6%. Gastroesophageal reflux and dysphagia were equally common in individuals with normal histology, esophagitis, or epithelial metaplasia. Manometry demonstrated nonpropagating peristalsis in 80% of the patients, and low distal wave amplitudes of the esophagus in all the changes being significantly worse in those with epithelial metaplasia (P ≤ 0.022 metaplasia vs. esophagitis/normal). Anastomotic complications (odds ratio [OR]: 8.6–24, 95% confidence interval [CI]: 1.7–260, P = 0.011–0.008), age (OR: 20, 95% CI: 1.3–310, P = 0.034), low distal esophageal body pressure (OR: 2.6, 95% CI: 0.7–10, P = 0.002), and defective esophageal peristalsis (OR: 2.2, 95% CI: 0.4–11, P = 0.014) predicted development of epithelial metaplasia. Conclusions:Significant esophageal morbidity associated with EA extends into adulthood. Surgical complications, increasing age, and impaired esophageal motility predict development of epithelial metaplasia after repair of EA.

Differential patterns of stromelysin-2 (MMP-10) and MT1-MMP (MMP-14) expression in epithelial skin cancers

Co-expression of several members of the matrix metalloproteinase (MMP) family is characteristic of human malignant tumours. To investigate the role of stromelysin-2 (MMP-10) in growth and invasion of skin tumours, we studied cutaneous carcinomas with high metastatic capacity (squamous cell carcinomas, SCCs), only locally destructive tumours (basal cell carcinomas, BCCs) and pre-malignant lesions (Bowen’s disease and actinic keratosis) using in situ hybridization. Expression of MMP-10 was compared with that of stromelysin-1 (MMP-3) and of MT1-MMP, the expression of which has been shown to correlate with tumour invasiveness. MMP-10 was expressed in 13/21 SSCs and 11/19 BCCs only in epithelial laminin-5 positive cancer cells, while premalignant lesions were entirely negative. MT1-MMP mRNA was detected in 19/21 SCCs both in epithelial cancer cells and stromal fibroblasts and in 14/18 BCCs only in fibroblasts. The level of MMP-10 was upregulated in a cutaneous SCC cell line (UT-SCC-7) by transforming growth factor-α and keratinocyte growth factor, and by interferon-γ in combination with transforming growth factor-β1 and tumour necrosis factor-α both in UT-SCC-7 and HaCaT cells. Our results show that MMP-10 expression does not correlate with the invasive behaviour of tumours as assessed by their histology and MT1-MMP expression, but may be induced by the wound healing and inflammatory matrix remodelling events associated with skin tumours.

Epilysin (MMP-28) Restrains Early Macrophage Recruitment in Pseudomonas aeruginosa Pneumonia

Several members of the matrix metalloproteinase (MMP) family function in various processes of innate immunity, particularly in controlling leukocyte influx. Epilysin (MMP-28) is expressed in numerous tissues and, in adult mice, it has the highest expression in lung, where it is detected in bronchial epithelial cells (Clara cells). Epilysin is also expressed by bone marrow-derived macrophages, but not by alveolar macrophages, suggesting that its expression by macrophages is dependent on localization and differentiation. To assess the role of this MMP, we generated epilysin-null (Mmp28−/−) mice. Although epilysin is constitutively expressed in normal tissues, Mmp28−/− mice have no overt phenotype. However, using a murine model of Pseudomonas aeruginosa pneumonia, we found that Mmp28−/− mice had an early increase in macrophage recruitment into the lungs, as well as enhanced bacterial clearance and reduced pulmonary neutrophilia, which we predicted were due to accelerated macrophage influx. Macrophage depletion in WT and Mmp28−/− mice confirmed a role for macrophages in clearing P. aeruginosa and regulating neutrophil recruitment. Furthermore, we observed that macrophages derived from Mmp28−/− mice migrated faster than did wild-type cells to bronchoalveolar lavage fluid from P. aeruginosa-treated mice of either genotype. These observations indicate that epilysin functions as an intrinsic negative regulator of macrophage recruitment by retarding the chemotaxis of these cells.

Membrane-Type 1 Matrix Metalloproteinase Expression Is Regulated by Zonula Occludens-1 in Human Breast Cancer Cells

The acquisition of a migratory/invasive phenotype by tumor cells is characterized by the loss of cell-cell adhesion contacts and the expression of degradative properties. In this study, we examined the effect of the disorganization of occludin/zonula occludens (ZO)-1 tight junction (TJ) complexes on the expression of membrane-type 1 matrix metalloproteinase (MT1-MMP). We first compared the expression of MT1-MMP and the localization of occludin/ZO-1 complexes in breast tumor cell lines displaying various degrees of invasiveness. We showed that the expression of MT1-MMP in invasive breast tumor cell lines correlates with the absence of occludin and with a cytoplasmic localization of ZO-1. In contrast, noninvasive cell lines displayed a membrane staining for both ZO-1 and occludin and did not express MT1-MMP. In vivo, cytoplasmic ZO-1 and MT1-MMP could be detected in invasive tumor clusters of human breast carcinomas. We then used RNA interference strategy to inhibit ZO-1 expression in invasive BT549 cells and to evaluate the effect of ZO-1 down-regulation on MT1-MMP expression. We observed that ZO-1 small interfering RNA transfection down-regulates MT1-MMP mRNAs and proteins and subsequently decreases the ability of tumor cells to invade a reconstituted basement membrane in a Boyden chamber assay. Inversely, transfection of expression vectors encoding wild-type ZO-1 or the NH2-terminal fragment of ZO-1 comprising the PSD95/DLG/ZO-1 domains in BT549 activated a human MT1-MMP promoter luciferase reporter construct and increased cell invasiveness. Such transfections concomitantly activated the beta-catenin/TCF/LEF pathway. Our results therefore show that ZO-1, besides its structural role in TJ assembly, can intervene in signaling events promoting tumor cell invasion.

Persistent abnormal liver fibrosis after weaning off parenteral nutrition in pediatric intestinal failure.

The aim of this study was to evaluate the long‐term effects of pediatric intestinal failure (IF) on liver histology. Altogether, 38 IF patients (median age: 7.2 years; range, 0.2‐27) underwent liver biopsy, gastroscopy, abdominal ultrasound, and laboratory tests. Sixteen patients were on parenteral nutrition (PN) after 74 PN months (range, 2.5‐204). Twenty‐two had weaned off PN 8.8 years (range, 0.3‐27) earlier, after 35 PN months (range, 0.7‐250). Fifteen transplant donor livers served as controls. Abnormal liver histology was found in 94% of patients on PN and 77% of patients weaned off PN (P = 0.370). During PN, liver histology weighted with cholestasis (38% of patients on PN versus 0% of patients weaned off PN; P = 0.003) and portal inflammation (38% versus 9%; P = 0.050) were found. Fibrosis (88% versus 64%; P = 0.143; Metavir stage: 1.6 [range, 0‐4] versus 1.1 [range, 0‐2]; P = 0.089) and steatosis (50% versus 45%; P = 1.000) were equally common during and after weaning off PN. Plasma alanine aminotransferase (78 U/L [range, 19‐204] versus 34 [range, 9‐129]; P = 0.009) and conjugated bilirubin (43 μmol/L [range, 1‐215] versus 4 [range, 1‐23]; P = 0.037) were significantly higher during than after weaning off PN. Esophageal varices were encountered in 1 patient after weaning off PN. Metavir stage was associated with small bowel length (r = −0.486; P = 0.002) and number of septic episodes (r = 0.480; P = 0.002). In a multivariate analysis, age‐adjusted small bowel length (ß = −0.533; P = 0.001), portal inflammation (ß = 0.291; P = 0.030), and absence of an ileocecal valve (ß = 0.267; P = 0.048) were predictive for fibrosis stage. Conclusion: Despite resolution of cholestasis and portal inflammation, significant liver fibrosis and steatosis persist after weaning off PN. Extensive small intestinal resection was the major predictor for liver fibrosis stage. (Hepatology 2013;58:729–738)

Epilysin (MMP‐28) – structure, expression and potential functions

Abstract:  Epilysin (MMP‐28) is the newest member of the matrix metalloproteinase (MMP) family of extracellular proteases. Together the MMPs can degrade almost all components of the extracellular matrix (ECM). MMPs also regulate cell behaviour by releasing growth factors and biologically active peptides from the ECM by modulating cell surface receptors and adhesion molecules and by regulating the activity of mediators of the inflammatory pathways. Epilysin differs from most other MMPs as it is expressed in a number of normal tissues, suggestive of functions in tissue homeostasis. The epilysin homologue in Xenopus laevis (XMMP‐28) is expressed in neural tissues, where it cleaves the neural cell adhesion molecule. Enhanced expression of epilysin has been observed in basal keratinocytes during wound healing and in different forms of cancer. There are, however, also reports on the downregulation of epilysin in malignant cells. The roles of epilysin in cancer seem to vary based on tumor type and stage of the disease. Importantly, epilysin can induce stable epithelial to mesenchymal transition (EMT) when overexpressed in epithelial lung carcinoma cells. Transforming growth factor β (TGF‐β) is a crucial mediator of this process, which was characterized by the loss of E‐cadherin and increased cell migration and invasion. Current results suggest a plausible interaction between epilysin and TGF‐β also under physiological circumstances, where epilysin activity may not induce EMT but, instead, trigger less permanent changes in TGF‐β signalling and cell motility.

Matrix metalloproteinase-21, the human orthologue for XMMP, is expressed during fetal development and in cancer

We have characterized a novel human matrix metalloproteinase (MMP-21) from human placenta DNA complementary to RNA (cDNA). The 569 amino acid translation of the cDNA includes all the typical features of an MMP family member, namely a signal sequence, a prodomain with a PRCGVPD motif, a zinc-binding catalytic domain with an HEIGHVLGL sequence, and a hemopexin-like domain flanked by two cysteine residues. Furthermore, MMP-21 has a furin activation sequence, but no transmembrane sequence nor a cytoplasmic domain. As in Xenopus laevis and Cynops pyrrhogaster there is an additional insertion of approximately 30 amino acids between the prodomain and the catalytic domain, which is poorly conserved between the species and is in human MMP-21 especially proline rich. The MMP-21 gene has seven exons and is located in chromosome 10. This new MMP is the human orthologue for XMMP and CyMMP expressed during gastrulation of X. laevis and C. pyrrhogaster, respectively. A 2.5 kb messenger RNA was observed in fetal liver by Northern analysis. By reverse transcription-polymerase chain reaction, MMP-21 is expressed in various human fetal and adult tissues as well as in cancer cell lines. MMP-21 protein can also be detected in malignancies such as ovarian and colon carcinomas by immunohistochemical staining. Our findings suggest that MMP-21 functions in embryogenesis and tumor progression.

Matrix Metalloproteinase-21 Is Expressed Epithelially During Development and in Cancer and Is Up-Regulated by Transforming Growth Factor-β1 in Keratinocytes

Human matrix metalloproteinase-21 (MMP-21), the newest member of the MMP gene family, has been suggested to play an important role in embryogenesis and tumor progression and to be a target of the Wnt, Pax, and Notch signaling pathways. Here we report detection of MMP-21 by RT-PCR in mouse embryos aged 10.5, 12.5, 13.5, and 16.5 days, as well as in various adult murine organs. In both humans and mice, MMP-21 protein was detected in the epithelial cells of developing kidney, intestine, neuroectoderm, and skin but not in normal adult skin using immunohistochemistry with two unrelated antibodies. However, it was present in invasive cancer cells of aggressive subtypes of basal and squamous cell carcinomas, although it was not expressed in skin disorders characterized by mere keratinocyte hyperproliferation. Of several cytokines tested, transforming growth factor-β1 induced MMP-21 in vitro in HaCaTs and keratinocytes as judged by real-time quantitative TaqMan PCR. Although suprabasal differentiating keratinocytes expressed MMP-21 in developing skin in vivo, MMP-21–positive keratinocytes were detected by immunohistochemistry in both low and high calcium cultures. MMP-21 expression was not up-regulated by ras transformation in HaCaT cell lines (HaCaT, A5, II-4, and RT3); in skin and colon cancers, its expression did not associate with apoptosis, β-catenin transactivation, or epithelial MMPs-9 and -10. However, MMP-21 protein was found in the same regions as MMP-7 but not in the same cells. Our results suggest that during development, MMP-21 expression is temporally and spatially tightly controlled. Unlike many classical MMPs, it is present in various normal adult tissues. Among epithelial MMPs, MMP-21 has a unique expression pattern in cancer.