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Supplementary Figure 1 from Matrix Metalloproteinase-8 Functions as a Metastasis Suppressor through Modulation of Tumor Cell Adhesion and Invasion

Collagenase-2 (matrix metalloproteinase-8, MMP-8) is an MMP mainly produced by neutrophils and associated with many inflammatory conditions. We have previously described that MMP-8 plays a protective role in cancer through its ability to regulate the inflammatory response induced by carcinogens. Moreover, it has been reported that experimental manipulation of the expression levels of this enzyme alters the metastatic behavior of human breast cancer cells. In this work, we have used mutant mice deficient in MMP-8 and syngenic melanoma and lung carcinoma tumor cells lines overexpressing this enzyme to further explore the putative antimetastatic potential of MMP-8. We report herein that MMP-8 prevents metastasis formation through the modulation of tumor cell adhesion and invasion. Thus, tumor cells overexpressing MMP-8 have an increased adhesion to extracellular matrix proteins, whereas their invasive ability through Matrigel is substantially reduced when compared with control cells. Analysis of MMP-8 in breast cancer patients revealed that the expression of this metalloproteinase by breast tumors correlates with a lower incidence of lymph node metastasis and confers good prognosis to these patients. On this basis, we propose that MMP-8 is a tumor protective factor, which also has the ability to reduce the metastatic potential of malignant cells in both mice and human. [Cancer Res 2008;68(8):2755–63]

Current somatic mutation callers are biased against repetitive regions, preventing the identification of potential driver alterations in these loci. We developed a mutation caller for repetitive regions, and applied it to study repetitive non protein-coding genes in more than 2200 whole-genome cases. We identified a recurrent mutation at position c.28 in the gene encoding the snRNA U2. This mutation is present in B-cell derived tumors, as well as in prostate and pancreatic cancer, suggesting U2 c.28 constitutes a driver candidate associated with worse prognosis. We showed that the GRCh37 reference genome is incomplete, lacking the U2 cluster in chromosome 17, preventing the identification of mutations in this gene. Furthermore, the 5′-flanking region of WDR74, previously described as frequently mutated in cancer, constitutes a functional copy of U2. These data reinforce the relevance of non-coding mutations in cancer, and highlight current challenges of cancer genomic research in characterizing mutations affecting repetitive genes.

As recently described, the administration of extremely low doses (pg/kg) of CCL4 (Macrophage inflammatory protein 1β, MIP-1β) can induce antinociceptive effects in mice (Garcia-Dominguez et al., 2019b). We describe here that hydrodynamic delivery of a plasmid containing CCL4 cDNA provokes a biphasic response consisting in an initial thermal hyperalgesic reaction for 8 days followed by analgesia at days 10–12, being both responses blocked after the administration of the CCR5 antagonist DAPTA. Both the luminiscence evoked in liver after the administration of a plasmid containing CCL4 and luciferase cDNAs and the hepatic concentration of CCL4 measured by ELISA were maximal 4 days after plasmid administration and markedly diminished at day 10. A dose–effect curve including a wide dose range of exogenous CCL4 revealed thermal analgesia after the administration of 10–100 pg/kg whereas 1000 times higher doses (30–100 ng/kg) induced, instead, thermal hyperalgesia inhibited by DAPTA. This hyperalgesia was absent in mice with reduced white blood cells after cyclophosphamide treatment, thus supporting the involvement of circulating leukocytes. A multiarray bioluminescent assay revealed increased plasma levels of IL-1α, CCL2, CXCL1, CXCL13, IL-16 and TIMP-1 in mice treated with 100 ng/kg of CCL4. The hyperalgesic response evoked by CCL4 was prevented by IL-1R, CXCR2 or CCR2 antagonists or by the neutralization of CXCL13 or IL-16, but not TIMP-1, with selective antibodies. The administration of the anti-IL-16 antibody was the unique treatment able to convert hyperalgesia evoked by 100 ng/kg of CCL4 in an analgesic effect. The ability of IL-16 to evoke hypernociception was confirmed by studying the response to its exogenous administration (10–30 ng/kg). In summary, the present results demonstrate that CCL4 induces a dual modulation of nociception and describe some mechanisms involved in the hyperalgesic response evoked by this chemokine.

Matrix metalloproteinases can modulate the inflammatory response through processing of cyto- and chemokines. Among them, MMP-14 is a non-dispensable collagenase responsible for the activation of other enzymes, triggering a proteolytic cascade. To identify the role of MMP-14 during the pro-inflammatory response, wildtype and Mmp14 −/− mice were challenged with lipopolysaccharide. MMP-14 levels decreased after endotoxemia. Mutant animals showed 100% mortality, compared to 50% in wildtype mice. The increased mortality was related to a more severe lung injury, an impaired lung MMP-2 activation, and increased levels of the alarmin S100A9. There were no differences in the expression of other mediators including Il6, Cxcl2, Tgfb, Il10, or S100a8. A similar result was observed in lung explants of both genotypes cultured in presence of lipopolysaccharide. In this ex vivo model, exogenous activated MMP-2 ameliorated the observed increase in alarmins. Samples from septic patients showed a decrease in serum MMP-14 and activated MMP-2 compared to non-septic critically ill patients. These results demonstrate that the MMP-14-MMP-2 axis is downregulated during sepsis, leading to a proinflammatory response involving S100A9 and a more severe lung injury. This anti-inflammatory role of MMP-14 could have a therapeutic value in sepsis. • MMP-14 levels decrease in lungs from endotoxemic mice and serum from septic patients. • Mmp14 −/− mice show increased lung injury and mortality following endotoxemia. • Absence of Mmp14 decreases activated MMP-2 and increases S100A9 levels in lung tissue. • MMP-14 ameliorates inflammation by promoting S100A9 cleavage by activated MMP-2.

In the vast majority of pediatric patients with dilated cardiomyopathy, the specific etiology is unknown. Studies on families with dilated cardiomyopathy have exemplified the role of genetic factors in cardiomyopathy etiology. In this study, we applied whole-exome sequencing to members of a non-consanguineous family affected by a previously unreported congenital dilated cardiomyopathy syndrome necessitating early-onset heart transplant. Exome analysis identified compound heterozygous variants in the FLNC gene. Histological analysis of the cardiac muscle demonstrated marked sarcomeric and myofibrillar abnormalities, and immunohistochemical staining demonstrated the presence of Filamin C aggregates in cardiac myocytes. We conclude that biallelic variants in FLNC can cause congenital dilated cardiomyopathy. As the associated clinical features of affected patients are mild, and can be easily overlooked, testing for FLNC should be considered in children presenting with dilated cardiomyopathy.

© The Author(s), under exclusive licence to Springer Nature Limited 2019, In cancer, recurrent somatic single-nucleotide variants-which are rare in most paediatric cancers-are confined largely to protein-coding genes1-3. Here we report highly recurrent hotspot mutations (r.3A>G) of U1 spliceosomal small nuclear RNAs (snRNAs) in about 50% of Sonic hedgehog (SHH) medulloblastomas. These mutations were not present across other subgroups of medulloblastoma, and we identified these hotspot mutations in U1 snRNA in only

Cancers are caused by genomic alterations known as drivers. Hundreds of drivers in coding genes are known but, to date, only a handful of noncoding drivers have been discovered—despite intensive searching1,2. Attention has recently shifted to the role of altered RNA splicing in cancer; driver mutations that lead to transcriptome-wide aberrant splicing have been identified in multiple types of cancer, although these mutations have only been found in protein-coding splicing factors such as splicing factor 3b subunit 1 (SF3B1)3–6. By contrast, cancer-related alterations in the noncoding component of the spliceosome—a series of small nuclear RNAs (snRNAs)—have barely been studied, owing to the combined challenges of characterizing noncoding cancer drivers and the repetitive nature of snRNA genes1,7,8. Here we report a highly recurrent A>C somatic mutation at the third base of U1 snRNA in several types of tumour. The primary function of U1 snRNA is to recognize the 5′ splice site via base-pairing. This mutation changes the preferential A–U base-pairing between U1 snRNA and the 5′ splice site to C–G base-pairing, and thus creates novel splice junctions and alters the splicing pattern of multiple genes—including known drivers of cancer. Clinically, the A>C mutation is associated with heavy alcohol use in patients with hepatocellular carcinoma, and with the aggressive subtype of chronic lymphocytic leukaemia with unmutated immunoglobulin heavy-chain variable regions. The mutation in U1 snRNA also independently confers an adverse prognosis to patients with chronic lymphocytic leukaemia. Our study demonstrates a noncoding driver in spliceosomal RNAs, reveals a mechanism of aberrant splicing in cancer and may represent a new target for treatment. Our findings also suggest that driver discovery should be extended to a wider range of genomic regions. A highly recurrent A>C somatic mutation in U1 small nuclear RNA, which alters the splicing pattern of genes that include known drivers of cancer, is identified in several types of tumour.

Matrix metalloproteinases (MMPs) have been implicated in a variety of human diseases, including neuroimmunological disorders such as multiple sclerosis. However, the recent finding that some MMPs play paradoxical protective roles in these diseases has made necessary the detailed study of the specific function of each family member in their pathogenesis. To determine the relevance of collagenase-2 (MMP-8) in experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis, we have performed two different analyses involving genetic and biochemical approaches. First, we have analyzed the development of EAE in mutant mouse deficient in MMP-8, with the finding that the absence of this proteolytic enzyme is associated with a marked reduction in the clinical symptoms of EAE. We have also found that MMP-8-/- mice exhibit a marked reduction in central nervous system-infiltrating cells and demyelinating lesions. As a second approach, we have carried out a pharmacological inhibition of MMP-8 with a selective inhibitor against this protease (IC50 = 0.4 nm). These studies have revealed that the administration of the MMP-8 selective inhibitor to mice with EAE also reduces the severity of the disease. Based on these findings, we conclude that MMP-8 plays an important role in EAE development and propose that this enzyme may be a novel therapeutic target in human neuro-inflammatory diseases such as multiple sclerosis.

Ageing constitutes a critical impediment to somatic cell reprogramming. We have explored the regulatory mechanisms that constitute age-associated barriers, through derivation of induced pluripotent stem cells (iPSCs) from individuals with premature or physiological ageing. We demonstrate that NF-κB activation blocks the generation of iPSCs in ageing. We also show that NF-κB repression occurs during cell reprogramming towards a pluripotent state. Conversely, ageing-associated NF-κB hyperactivation impairs the generation of iPSCs by eliciting the reprogramming repressor DOT1L, which reinforces senescence signals and downregulates pluripotency genes. Genetic and pharmacological NF-κB inhibitory strategies significantly increase the reprogramming efficiency of fibroblasts from Néstor-Guillermo progeria syndrome and Hutchinson-Gilford progeria syndrome patients, as well as from normal aged donors. Finally, we demonstrate that DOT1L inhibition in vivo extends lifespan and ameliorates the accelerated ageing phenotype of progeroid mice, supporting the interest of studying age-associated molecular impairments to identify targets of rejuvenation strategies.

MT1‐MMP ( MMP14 ) is a collagenolytic enzyme located at the cell surface and implicated in extracellular matrix (ECM) remodeling. Mmp14 −/− mice present dwarfism, bone abnormalities, and premature death. We demonstrate herein that the loss of MT1‐MMP also causes cardiac defects and severe metabolic changes, and alters the cytoskeleton and the nuclear lamina structure. Moreover, the absence of MT1‐MMP induces a senescent phenotype characterized by up‐regulation of p16 INK 4a and p21 CIP 1/ WAF 1 , increased activity of senescence‐associated β‐galactosidase, generation of a senescence‐associated secretory phenotype, and somatotroph axis alterations. Consistent with the role of retinoic acid signaling in nuclear lamina stabilization, treatment of Mmp14 −/− mice with all‐ trans retinoic acid reversed the nuclear lamina alterations, partially rescued the cell senescence phenotypes, ameliorated the pathological defects in bone, skin, and heart, and extended their life span. These results demonstrate that nuclear architecture and cell senescence can be modulated by a membrane protease, in a process involving the ECM as a key regulator of nuclear stiffness under cell stress conditions.

Introduction: Genomic studies of chronic lymphocytic leukemia (CLL) have uncovered >80 potential driver mutations. The vast majority of these mutations affect coding regions, and just two potential drivers have been identified in non-coding elements. Aim: To describe the biological and clinical impact of a recurrent A>C mutation at the third base of the small nuclear RNA U1, the non-coding component of the spliceosome involved in the recognition of the 5' splice site (5'SS). Methods: Whole-genome sequencing (WGS) and RNA-seq from 318 CLL patients were used to identify and characterize a highly recurrent A>C point mutation occurring at position 3 of the U1 snRNA gene (g.3A>C mutation). The U1 wild-type and mutant forms were introduced into three CLL cell lines (JVM3, HG3, MEC1) to validate in vitro the predicted effect of this alteration. We screened two independent cohorts including a total of 1,314 CLL patients for the presence of the mutation using the rhAmp SNP genotyping assay, and integrated the U1 mutational status with well-known driver alterations, IGHV and epigenetic subgroups, and clinical parameters. Results: The U1 mutation was found in 8/78 (10.3%) CLL cases analyzed by WGS. Given its role in 5'SS recognition by base-pairing, we reasoned that this mutation was likely to alter the splicing and expression patterns of CLL. We were able to confirm widespread specific alterations in the transcriptome by comparing RNA-seq data between wild-type and g.3A>C mutated samples. Applying this knowledge to an algorithm aimed to infer the U1 mutational status from expression data, we were able to identify 4 mutated cases among 240 additional cases that had RNA-seq but no WGS. In total, 12/318 (3.8%) CLL patients analyzed by WGS and/or RNA-seq harbored this mutation. This g.3A>C U1 mutation changes the preferential A-U base-pairing between U1 and 5'SS to C-G base-pairing, creating novel splice junctions and altering the splicing pattern of 3,193 introns in 1,519 genes. In addition to altered splicing, 869 genes were differentially expressed between mutated and wild-type cases. We identified specific cancer genes (e.g. MSI2, POLD1, or CD44) and pathways (B-cell receptor signaling, promotion of apoptosis, telomere maintenance, among others) altered by the U1 mutation. To confirm a causal link between this mutation and splicing changes, we introduced exogenous U1 genes with or without the mutation into three cell lines. Subsequent RNA-seq of these cell lines recapitulated the altered splicing and expression patterns observed in CLL patients. We next screened for the presence of the U1 mutation 1,057 patients (cohort 1) using the rhAmp assay and it was found in 30 (2.8%) cases. The distribution of the mutation was similar in Binet stages and CLL vs monoclonal B-cell lymphocytosis. However, the U1 mutation was almost always found in IGHV unmutated CLL (29/30, p=9.0e-11) and within the naïve-like CLL epigenetic subgroup (p=3.7e-7). None of the U1 mutated cases had mutations in the SF3B1 splicing factor. Considering only pre-treatment CLL samples, U1 mutation was associated with a shorter time to first treatment independently of the Binet stage, IGHV mutational status, epigenetic subgroups, and mutations in the well-known CLL drivers SF3B1, NOTCH1, ATMor TP53. In cohort 2 (n=257), this mutation was found in 13 (5.1%) patients, confirming its enrichment in IGHV unmutated cases, naïve-like epigenetic subgroup, and splicing modulation. Despite the relatively small number of pre-treatment samples carrying the U1 mutation (7/178) and short follow-up of the patients (median 2.6 years), the effect of this mutation on time to first treatment in cohort 2 was compatible with the one observed in cohort 1. Finally, we screened for the U1 mutation a cohort of diffuse large B-cell lymphoma (n=108), mantle cell lymphoma (n=101), follicular lymphoma (n=87), splenic marginal zone lymphoma (n=12), acute myeloid leukemia (n=52), and myelodysplastic syndrome (n=67). The mutation was not present in any of the samples analyzed. Conclusions: Here we have reported that the third base of the small nuclear RNA U1 is recurrently mutated in CLL, proved its effect in splicing and gene expression, and shown that this mutation is independently associated with faster disease progression. The g.3A>C U1 mutation represents a novel non-coding driver alteration in CLL with potential clinical and therapeutic implications. Disclosures Ramirez Payer: GILEAD SCIENCES: Research Funding. Terol:Astra Zeneca: Consultancy; Gilead: Research Funding; Abbvie: Consultancy; Janssen: Consultancy, Research Funding; Roche: Consultancy. Lopez-Guillermo:Celgene: Consultancy, Research Funding; Janssen: Research Funding; Roche: Consultancy, Research Funding; Gilead: Consultancy, Research Funding.

Matrix metalloproteinases (MMPs) have been traditionally implicated in cancer progression because of their ability to degrade the extracellular matrix. However, some members of the MMP family have recently been identified as proteases with antitumor properties. Thus, it has been described that collagenase-2 (MMP-8) has a protective role in tumor and metastasis progression, but the molecular mechanisms underlying these effects are unknown. We show herein that Mmp8 expression causes a decrease in miR-21 levels that in turn leads to a reduction in tumor growth and lung metastasis formation by MDA-MB-231 (4175) breast cancer cells. By using both in vitro and in vivo models, we demonstrate that the mechanism responsible for these MMP-8 beneficial effects involves cleavage of decorin by MMP-8 and a subsequent reduction of transforming growth factor β (TGF-β) signaling that controls miR-21 levels. In addition, miR-21 downregulation induced by MMP-8 increases the levels of tumor suppressors such as programmed cell death 4, which may also contribute to the decrease in tumor formation and metastasis of breast cancer cells overexpressing this metalloproteinase. These findings reveal a new signaling pathway for cancer regulation controlled by MMP-8, and contribute to clarify the molecular mechanisms by which tumor-defying proteases may exert their protective function in cancer and metastasis.

This work was funded by the Spanish Ministry of Economy and Competitiveness (grant SAF2013-45836-R). J.G-A. is supported by a fellowship from the Spanish Ministry of Education. We thank Fundación Caja Rural de Asturias for financial collaborative support to Laboratorio de Oncología Molecular (HUCA). The Instituto Universitario de Oncología is supported by Fundación Bancaria Caja de Ahorros de Asturias, Spain.

Proteases are defined as enzymes that have the ability to perform the hydrolysis of peptide bonds. Owing to this characteristic, proteases were initially described as nonspecific enzymes of protein catabolism, participating in processes such as tissue destruction or degradation of dietary proteins. More recently, a better understanding of their functions has allowed consideration of proteases as enzymes that perform highly specific reactions and take part in multiple biological processes such as DNA replication and transcription, cell proliferation, differentiation and migration, tissuemorphogenesis and remodeling, heat shock and unfolded protein responses, neurogenesis, angiogenesis, ovulation, fertilization, wound repair, stem cell mobilization, coagulation, immunity, inflammation, senescence, autophagy, apoptosis, and necrosis [1]. According to the essential roles performed by proteases in all living organisms, alterations in their proteolytic activities may lead to important pathologies such as arthritis, cardiovascular alterations, neurodegenerative disorders, progeroid syndromes, and cancer [2, 3]. The biochemical reaction catalyzed by all proteases consists in the hydrolysis of a peptide bond through the nucleophilic attack at the carbonyl group. However, the way this reaction is performed differs between specific proteases. This characteristic feature has allowed the establishment of six different catalytic classes of proteases according to the group performing the nucleophilic attack: aspartic, metallo, cysteine, serine, and threonine proteases, as well as the most recently described group of glutamic proteases, which has only been found in some species of fungi and bacteria. In the case of aspartic, glutamic, and metalloproteases, a polarized water molecule located in the active center acts directly as a nucleophile, while in the other three classes, the reactive element is a hydroxyl (serine and threonine) or sulfhydryl (cysteine) group from the corresponding catalytic core [4]. Within each class, proteases can be further subdivided into different families and clans according to sequence conservation and three-dimensional structure similarities. The diversity and complexity of proteases have made necessary the introduction of concepts and tools for their global analysis and characterization. Thus, the term

Chemokines are central in the activation and direction of leukocyte subsets to target tissues. However, the monocyte chemoattractant protein-3 (MCP-3) has not been associated with chronic periodontitis. Chronic periodontitis is an infection showing episodic supporting tissue destruction. The aim of this study is to determine the levels and expression of MCP-3 in periodontal sites characterized by active periodontal connective tissue destruction.The study population consisted of 15 patients with a progression of periodontitis (15 of 56 patients), 18 patients with chronic periodontitis, and 10 healthy subjects without periodontal disease. As determined by the tolerance method, the 15 patients with moderate to advanced chronic periodontitis showed a progression of periodontitis over a 4-month period. Periodontitis was characterized by at least six sites with a probing depthor=5 mm, clinical attachment levelor=3 mm, and radiographic bone loss. Gingival crevicular fluid was collected using a paper strip. The total protein concentration was determined. An enzyme-linked immunosorbent assay was performed to determine the total amount of MCP-3, and an immunoWestern blot was conducted to assess molecular MCP-3 forms. To determine the MCP-3 expression by immunohistochemistry, gingival biopsies were obtained from patients with chronic periodontitis and healthy subjects during third-molar extraction surgery. Statistical analyses were performed using statistical software. Data were expressed as subject means +/- SD, using the chi(2) and Student t tests.The total amount and concentration of chemokine MCP-3 were significantly higher in patients with chronic periodontitis than in healthy subjects (8.25 pg versus 0.53 pg, P = 0.006 and 2.95 pg/microl versus 0.45 pg/microl, P = 0.04, respectively). Active sites showed a significantly higher total amount and concentration of MCP-3 than inactive sites (11.12 versus 2.88 pg, P value = 0.005 and 3.95 versus 1.02, P value = 0.005, respectively). Western blot and immunohistochemical staining confirmed the presence of MCP-3 in periodontal disease, with observable differences between patients with chronic periodontitis and healthy subjects.MCP-3 was highly expressed in patients with chronic periodontitis, particularly in those with progressive periodontal lesions. MCP-3 could be involved in the recruitment of inflammatory cells toward periodontal tissues during the progression of the disease.

Proteins of the plasminogen activation system are broadly expressed throughout the nervous system, and key roles for these proteins in neuronal function have been demonstrated. Recent reports have established that plasminogen is synthesized in neuroendocrine tissues, making this protein and the proteolytic activity of the product of its activation, plasmin, available at sites separated anatomically from circulating, hepatocyte-derived plasminogen. Results with plasminogen-deficient humans and mice suggest a role for plasminogen in neuritogenesis. To elucidate the role of the plasminogen activation system in these processes, the function of plasminogen during neuritogenesis and neurite outgrowth was studied. It is shown here that plasminogen participates in neuritogenesis, as plasmin inhibitors reduced both neurite outgrowth and neurite length in PC-12 cells. The addition of exogenous plasminogen enhanced neurite outgrowth and neurite length in both PC-12 cells and primary cortical neurons. The proteolytic activity of plasmin was required, since mutation of the catalytic serine residue completely abolished the stimulatory activity. Furthermore, mutation of the lysine binding site within kringle 5 of the plasminogen molecule also reduced the neuritogenic activity of plasminogen. Additionally, we demonstrate that plasminogen specifically bound to laminin-1, the interaction resulted in increased plasminogen activation by tissue-type plasminogen activator, and was dependent on a functional lysine binding site within plasminogen kringle 5. Moreover, during NGF-induced neuritogenesis, laminin-1 was degraded, and this cleavage was catalyzed by plasmin. This study provides the first direct evidence that plasminogen participates in neurite outgrowth and also suggests that laminin-1 degradation by plasmin contributes to the process of neuritogenesis.

Objective Aberrant matrix metalloproteinase (MMP) and human β-defensin (HBD) functions have been found in inflammatory diseases. The objectives of this study were to investigate the immunolocalisation, mRNA expression and molecular forms of MMP-25, MMP-26, HBD-1 and HBD-2 in chronic and aggressive periodontitis and in peri-implantitis. The expression of MMP-25 by cultured human plasmacytoma cells and macrophages, and the effects of MMP-26 and Porphyromonas gingivalis trypsin-like proteinase on HBD-1 and -2 were also studied. Design Immunohistochemistry, immunofluorescent analysis, reverse transcriptase polymerase chain reaction and immunoblotting were used to assess localisation, mRNA expression and molecular forms of MMP-25, MMP-26, HBD-1 and HBD-2. HBD degradation by MMP-26 and P. gingivalis proteinase was studied by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Results MMP-25 was present in plasma cells and polymorphonuclear leucocytes, and MMP-26 was present in oral and sulcular basement membrane zones. HBD-1 was distributed perivasculary in gingival connective tissue and in oral and sulcular epithelium, and HBD-2 was found to a lesser extent in the perivascular space. Low MMP-25, MMP-26, HBD-1 and HBD-2 mRNA expression was found. Immunoblot revealed 29–57-kDa MMP-25 in myeloma cell lysates, but not in macrophages, and partly activated MMP-25 and -26 in diseased gingival crevicular fluid and peri-implant sulcular fluid. P. gingivalis trypsin-like proteinase degraded HBD-1 and -2. Conclusions Both MMP-25 and -26 were expressed more strongly in extensively inflamed gingiva compared with healthy gingiva. The expression of HBD-1 was stronger than that of HBD-2 in periodontitis and peri-implantitis. De-novo expression of MMP-25 and -26 is associated with periodontal and peri-implant inflammation. Furthermore, P. gingivalis trypsin-like proteinase, but not MMP-26, can degrade HBD-1 and -2, which could lead to a weakened innate immune response.

Matrix metalloproteases (MMPs) regulate innate immunity acting over proinflammatory cytokines, chemokines, and other immune-related proteins. MMP-25 (membrane-type 6-MMP) is a membrane-bound enzyme predominantly expressed in leukocytes whose biological function has remained largely unknown. We have generated Mmp25-deficient mice to elucidate the in vivo function of this protease. These mutant mice are viable and fertile and do not show any spontaneous phenotype. However, Mmp25-null mice exhibit a defective innate immune response characterized by low sensitivity to bacterial LPS, hypergammaglobulinemia, and reduced secretion of proinflammatory molecules. Moreover, these immune defects can be tracked to a defective NF-κB activation observed in Mmp25-deficient leukocytes. Globally, our findings provide new mechanistic insights into innate immunity through the activity of MMP-25, suggesting that this proteinase could be a potential therapeutic target for immune-related diseases.

Proteolytic enzymes are implicated in multiple physiological and pathological processes. The availability of the sequence of the chimpanzee genome has allowed us to determine that the chimpanzee degradome—the repertoire of protease genes from this organism—is composed of at least 559 protease and protease-like genes and is virtually identical to that of human, containing 561 genes. Despite the high degree of conservation between both genomes, we have identified important differences that vary from deletion of whole genes to small insertion/deletion events or single nucleotide changes that lead to the specific gene inactivation in one species, mostly affecting immune system genes. For example, the genes encoding PRSS33/EOS, a macrophage serine protease conserved in most mammals, and GGTLA1 are absent in chimpanzee, while the gene for metalloprotease MMP23A, located in chromosome 1p36, has been specifically duplicated in the human genome together with its neighbor gene CDC2L1. Other differences arise from single nucleotide changes in protease genes, such as NAPSB and CASP12, resulting in the presence of functional genes in chimpanzee and pseudogenes in human. Finally, we have confirmed that the Trypanosoma lytic factor HPR is inactive in chimpanzee, likely contributing to the susceptibility of chimpanzees to T. brucei infection. This study provides the first analysis of the chimpanzee degradome and might contribute to the understanding of the molecular bases underlying variations in host defense mechanisms between human and chimpanzee.

Proteolytic enzymes play an essential role in different physiological processes, including development, reproduction and host defence, as well as in numerous pathologies, like inflammatory diseases, neurological disorders or cancer. The completion of the human genome sequence allowed us to determine that more than 2% of all human genes are proteases or protease inhibitors, reflecting the importance of proteolysis in human biology. To understand better the complexity of proteases in human and other model organisms, we have used the available genome sequences of different mammalian organisms, including mouse, rat and chimpanzee, to identify and compare their degradomes, the complete set of protease genes in these species. Surprisingly, the rodent protease complement is more complex when compared with that of primates, mainly due to the expansion of protease families implicated in reproduction and host defence. Similarly, most differences between human and chimpanzee proteases are found in genes implicated in the immune system, which might explain some of the differences between both organisms. We have also found several genes implicated in reproduction, nutrition and the immune system, which are functional in rat, mouse or chimpanzee, but have been inactivated by mutations in the human lineage. These findings suggest that pseudogenization of specific protease genes has been a mechanism contributing to the evolution of the human genome. Finally, we found that proteases implicated in human hereditary diseases, and especially in neurodegenerative disorders, are highly conserved among mammals.

An emerging area of research has demonstrated that plasminogen functions in the acute-phase response to tissue injury, neoplastic growth or infection. We have previously shown that the acute-phase mediator, interleukin (IL)-6, increases circulating plasminogen levels via upregulation of plasminogen promoter activity. We also identified a putative IL-6 responsive element (nt -791 to -783; IL6-RE) in the plasminogen gene that is required for maximal stimulation of promoter activity by IL-6. For the present study, we investigated the transcription factors and signaling pathway mediating the response of the plasminogen gene to IL-6. In electrophoretic mobility shift assays (EMSAs), a radiolabeled oligonucleotide IL6-RE probe formed specific complexes with nuclear proteins from untreated hepatocytic cells. The extent of complex formation was markedly increased using nuclear proteins from IL-6-treated cells. Complex formation was abolished by an oligonucleotide with the consensus CCAAT/enhancer binding protein (C/EBP) sequence. Furthermore, complexes were supershifted by antibodies to C/EBPbeta. Treatment of Hepa 1-6 cells with the mitogen-activated protein kinase (MAPK) inhibitor, PD-98059, inhibited IL-6-stimulated plasminogen promoter activity. These results suggest that transcription factor C/EBPbeta and the MAPK pathway play key roles in the response of the plasminogen gene to IL-6, thus elucidating a major mechanism by which the plasminogen system is upregulated to perform its crucial functions in the acute-phase response.

We have identified and cloned 22 human cDNAs encoding novel members of the ubiquitin-specific protease (USP) family. Eighteen of the identified proteins contain all structural features characteristic of these cysteine proteinases, whereas four of them have been classified as non-peptidase homologues. Northern blot analysis demonstrated that the identified USPs are broadly and differentially distributed in human tissues, some of them being especially abundant in skeletal muscle or testis. Enzymatic studies performed with the identified USPs revealed that at least twelve of them are deubiquitylating enzymes based on their ability to cleave ubiquitin from a ubiquitin-beta-galactosidase fusion protein. These results provide additional evidence of the extreme complexity and diversity of the USP proteolytic system in human tissues and open the possibility to explore the relevance of their multiple components in the regulation of ubiquitin-mediated pathways in normal and pathological functions.

Introduction Matrix metalloproteinase-8 (MMP-8; neutrophil collagenase) is an important regulator of innate immunity that has oncosuppressive actions in numerous tumor types. Methods We have intercrossed Mmp8-null mice with the Polyoma virus middle T oncogene-driven (MMTV-PyMT) mouse model of mammary cancer to explore the effects of loss of MMP-8 on the incidence and progression of mammary carcinomas. Results In this aggressive mouse model of breast cancer, loss of MMP-8 accelerated tumor onset even further, such that 90% of MMTV-PyMT; Mmp8-null female mice were tumor-bearing at the time of weaning. Throughout the 14 weeks of the model, tumor burden increased in homozygous Mmp8-null mice compared to Mmp8-wild-type and -heterozygote animals. Likewise, lung metastasis dramatically increased in the MMTV-PyMT; Mmp8-null mice. Immunohistochemistry revealed that tumors in wild-type, Mmp8-heterozygotes and -null animals had similar vascular density at 8 weeks, but at 10 weeks Mmp8-wild-type tumors had a lower vascularity than their heterozygote and null counterparts. No differences in macrophage infiltration were apparent throughout primary tumor development, though at 10 weeks a drop in neutrophil infiltrates was observed in Mmp8-wild-type tumors. Using quantitative real-time RT-PCR, we tracked the expression of the entire Mmp and Timp gene families, observing a significant decrease in Mmp3 expression in Mmp8-null tumors compared to wild-type and heterozygotes throughout the time course of the model, which was confirmed at the protein level. Conclusions These findings provide novel insight into the suppressive action of MMP-8 on mammary tumorigenesis and metastasis, and indicate that the loss of MMP-8 likely has pleiotropic effects on innate immunity and angiogenesis that are reflected in changes in the protease web.

Matrix metalloprotease-9 (MMP9) plays a critical role in acute myeloid leukemia (AML) by increasing the invasive properties of malignant myeloblasts. The role of this enzyme in high-risk myelodysplastic diseases (MDS) and the effect of azacitidine on its expression in MDS and AML have not been studied in detail. In this work, we have analyzed the effect of different concentrations of azacitidine in two well-established, MDS-derived, acute myeloid leukemic cell lines: MOLM-13 and SKM-1. We have demonstrated that 1 μmol/L azacitidine decreases MMP9 DNA methylation levels and that this is correlated with a significant increase in messenger RNA expression in both cell lines. Surprisingly, changes in protein levels were minor. This paradoxic effect is explained by the drug-dependent induction of apoptosis that reduces the amount of active secreting cells. A balance between induced expression and apoptosis was established at an azacitidine concentration of 0.2 μmol/L in MOLM-13 cells. This dose significantly increased the invasive capacity of viable cells, as measured in the Matrigel assay. To evaluate the clinical relevance of this observation, we have examined the effect of azacitidine on MMP9 expression in bone marrow from five patients with MDS, with the finding that this drug significantly increased MMP9 protein levels in all analyzed patients after six cycles of treatment. Based on these results, we conclude that azacitidine increases MMP9 expression and may enhance invasiveness in vitro. Because all five patients relapsed, these findings might explain, at least partially, the clinical failure of the drug and the progression to a more aggressive disease.

Background Matrix metalloproteinases (MMPs) may have pro and antifibrotic roles within the lungs, due to its ability to modulate collagen turnover and immune mediators. MMP-8 is a collagenase that also cleaves a number of cytokines and chemokines. Methodology and Principal Findings To evaluate its relevance in lung fibrosis, wildtype and Mmp8−/− mice were treated with either intratracheal bleomycin or saline, and lungs were harvested at different time points. Fibrosis, collagen, collagenases, gelatinases, TGFβ and IL-10 were measured in lung tissue. Mmp8−/− mice developed less fibrosis than their wildtype counterparts. This was related to an increase in lung inflammatory cells, MMP-9 and IL-10 levels in these mutant animals. In vitro experiments showed that MMP-8 cleaves murine and human IL-10, and tissue from knockout animals showed decreased IL-10 processing. Additionally, lung fibroblasts from these mice were cultured in the presence of bleomycin and collagen, IL-10 and STAT3 activation (downstream signal in response to IL-10) measured by western blotting. In cell cultures, bleomycin increased collagen synthesis only in wildtype mice. Fibroblasts from knockout mice did not show increased collagen synthesis, but increased levels of unprocessed IL-10 and STAT3 phosphorylation. Blockade of IL-10 reverted this phenotype, increasing collagen in cultures. Conclusions According to these results, we conclude that the absence of MMP-8 has an antifibrotic effect by increasing IL-10 and propose that this metalloprotease could be a relevant modulator of IL-10 metabolism in vivo.

Mechanical ventilation is a life-saving therapy that can also damage the lungs. Ventilator-induced lung injury (VILI) promotes inflammation and up-regulates matrix metalloproteinases (MMPs). Among these enzymes, MMP-8 is involved in the onset of inflammation by processing different immune mediators. To clarify the role of MMP-8 in a model of VILI and their relevance as a therapeutic target, we ventilated wild-type and MMP-8-deficient mice with low or high pressures for 2 hours. There were no significant differences after low-pressure ventilation between wild-type and knockout animals. However, lack of MMP-8 results in better gas exchange, decreased lung edema and permeability, and diminished histological injury after high-pressure ventilation. Mmp8(-/-) mice had a different immune response to injurious ventilation, with decreased neutrophilic infiltration, lower levels of IFN-γ and chemokines (LPS-induced CXC chemokine, macrophage inflammatory protein-2), and significant increases in anti-inflammatory cytokines (IL-4, IL-10) in lung tissue and bronchoalveolar lavage fluid. There were no differences in MMP-2, MMP-9, or tissue inhibitor of metalloproteinase-1 between wild-type and knockout mice. These results were confirmed by showing a similar protective effect in wild-type mice treated with a selective MMP-8 inhibitor. We conclude that MMP-8 promotes acute inflammation after ventilation with high pressures, and its short-term inhibition could be a therapeutic goal to limit VILI.

Matrix metalloproteinase-9 (MMP-9) is released by neutrophils at the sites of acute inflammation. This enzyme modulates matrix turnover and inflammatory response, and its activity has been found to be increased after ventilator-induced lung injury. To clarify the role of MMP-9, mice lacking this enzyme and their wild-type counterparts were ventilated for 2 h with high- or low-peak inspiratory pressures (25 and 15 cmH2O, respectively). Lung injury was evaluated by gas exchange, respiratory mechanics, wet-to-dry weight ratio, and histological analysis. The activity of MMP-9 and levels of IL-1β, IL-4, and macrophage inflammatory protein (MIP-2) were measured in lung tissue and bronchoalveolar lavage fluid (BALF). Cell count and myeloperoxidase activity were measured in BALF. There were no differences between wild-type and Mmp9−/− animals after low-pressure ventilation. After high-pressure ventilation, wild-type mice exhibited an increase in MMP-9 in tissue and BALF. Mice lacking MMP-9 developed more severe lung injury than wild-type mice, in terms of impaired oxygenation and lung mechanics, and higher damage in the histological study. These effects correlated with an increase in both cell count and myeloperoxidase activity in the BALF, suggesting an increased neutrophilic influx in response to ventilation. An increase in IL-1β and IL-4 in the BALF only in knockout mice could be responsible for the differences. There were no differences between genotypes in MMP-2, MMP-8, or tissue inhibitors of metalloproteinases. These results show that MMP-9 protects against ventilator-induced lung injury by decreasing alveolar neutrophilic infiltration, probably by modulation of the cytokine response in the air spaces.

Summary. Background: Studies have documented a requirement for an intact plasminogen (Plg) activation system in neurite outgrowth induced by nerve growth factor (NGF). Objective: In this study we addressed the effect of NGF on Plg synthesis in model NGF-responsive PC-12 cells. Methods: The effect of NGF on Plg gene expression was assessed using Western blotting, quantitative polymerase chain reaction, luciferase reporter assays, site directed mutagenesis, electrophoretic mobility shift assays and chromatin immunoprecipitation. Results: NGF treatment increased Plg expression 3-fold and steady state levels of Plg mRNA were increased 6.82-fold. This effect also was observed in cortical neurons. PC-12 cells transfected with a luciferase reporter gene under the control of a 2400 bp fragment of the murine Plg promoter exhibited a 5-fold increase in luciferase activity following treatment with NGF. This response was dependent on Ras/ERK and PI3 K signaling because treatment with PD98059 together with wortmannin decreased promoter activity, in response to NGF, to the level exhibited by untreated cells. Furthermore, co-transfection with a dominant-negative mutant Ha-Ras completely blocked NGF-induced luciferase activity. In deletional and mutational studies we identified two Sp1 binding sites located between nucleotides -255 and -106 of the Plg promoter that were required for the full response of the Plg promoter to NGF. In chromatin immunoprecipitation assays the Sp1 transcription factor bound to the endogenous Plg promoter. Conclusions: These results suggest that Plg gene expression is up-regulated by neurotrophins that may provide a previously unrecognized mechanism for enhancing the effects of neurotrophins via the proteolytic activity of plasmin.

Matrix metalloproteinases (MMPs) have been implicated in numerous tissue-remodeling processes. The finding that mice deficient in collagenase-2 (MMP-8) are more susceptible to develop skin cancer, prompted us to investigate the role of this protease in cutaneous wound healing. We have observed a significant delay in wound closure in MMP8-/- mice and an altered inflammatory response in their wounds, with a delay of neutrophil infiltration during the first days and a persistent inflammation at later time points. These changes were accompanied by alterations in the TGF-beta1 signaling pathway and by an apoptosis defect in MMP8-/- mice. The delay in wound healing observed in MMP8-/- mice was rescued by bone marrow transplantation from wild-type mice. Analysis of other MMPs showed that MMP8-/- mice had a significant increase in the expression of MMP-9, suggesting that both proteases might act coordinately in this process. This possibility was further supported by the novel finding that MMP-8 and MMP-9 form specific complexes in vivo. Taken together, these data indicate that MMP-8 participates in wound repair by contributing to the resolution of inflammation and open the possibility to develop new strategies for treating wound healing defects.

Wound healing involves highly controlled events including reepithelialization, neoangiogenesis, and reformation of the stromal compartment. Matrix metalloproteinases (MMPs) are a family of neutral zinc-dependent endopeptidases known to be essential for the wound-healing process. MMP-8 (collagenase-2) is a neutrophil-derived highly effective type I collagenase, recently indicated to be important for acute wound healing. MMP-26 is a more recent and less well-studied member of the MMP family. Our aim was to study the expression of MMP-8 and MMP-26 in human cutaneous wound repair and chronic wounds using histological methods and cell culture. MMP-8 expression was associated with epithelial cells, neutrophils, and other inflammatory cells in chronic human wounds. MMP-26 was prominently expressed in the extracellular compartment of most chronic wounds in close vicinity to the basement membrane area. MMP-26 was also expressed in acute day 1 wounds with declining expression thereafter. In vitro wound experiments showed that both MMP-8 and MMP-26 were expressed by migrating human mucosal keratinocytes. Inhibiting MMP-26 resulted in aberrant keratinocyte migration and proliferation. We conclude that MMP-8 and MMP-26 are differentially expressed in acute and chronic wounds.

Zmpste24 (also called FACE-1) is a metalloproteinase involved in the maturation of lamin A (Lmna), an essential component of the nuclear envelope. Both Zmpste24- and Lmna-deficient mice exhibit profound nuclear architecture abnormalities and multiple histopathological defects that phenocopy an accelerated ageing process. Similarly, diverse human progeroid syndromes are caused by mutations in ZMPSTE24 or LMNA genes. To elucidate the molecular mechanisms underlying these devastating diseases, we have analysed the transcriptional alterations occurring in tissues from Zmpste24-deficient mice. We demonstrate that Zmpste24 deficiency elicits a stress signalling pathway that is evidenced by a marked upregulation of p53 target genes, and accompanied by a senescence phenotype at the cellular level and accelerated ageing at the organismal level. These phenotypes are largely rescued in Zmpste24-/-Lmna+/- mice and partially reversed in Zmpste24-/-p53-/- mice. These findings provide evidence for the existence of a checkpoint response activated by the nuclear abnormalities caused by prelamin A accumulation, and support the concept that hyperactivation of the tumour suppressor p53 may cause accelerated ageing.

Plasminogen is the zymogen of the active serine protease plasmin. Plasminogen circulates in plasma at a concentration of 2 μM (Collen and Verstraete, 1975) and is also present in the extravascular fluid. Plasmin is the major enzyme responsible for the degradation of fibrin in blood clots within the vascular tree (Carmeliet and Collen, 1996; Petersen et al., 1990; Ploplis et al., 1995). In addition to its established role in intravascular thrombolysis, plasmin has been implicated in such diverse processes as angiogenesis (Pepper et al., 1987; Baker et al., 2000; van Hinsbergh et al., 2001; Pepper, 2001;), tumor progression (Chappuis et al., 2001; van der Pluijm et al., 2001), degradation of extracellular matrices, neurite outgrowth (Tsirka et al., 1995; Wu et al., 2000; Jacovina et al., 2001), and inflammatory reactions important in host defense and wound repair.

This paper is distributed under the terms of the Creative Commons Attribution-Non-Commercial-Share Alike licence.-- et al., Chronic lymphocytic leukaemia (CLL), the most frequent leukaemia in adults in Western countries, is a heterogeneous disease with variable clinical presentation and evolution(1,2). Two major molecular subtypes can be distinguished, characterized respectively by a high or low number of somatic hypermutations in the variable region of immunoglobulin genes(3,4). The molecular changes leading to the pathogenesis of the disease are still poorly understood. Here we performed whole-genome sequencing of four cases of CLL and identified 46 somatic mutations that potentially affect gene function. Further analysis of these mutations in 363 patients with CLL identified four genes that are recurrently mutated: notch 1 (NOTCH1), exportin 1 (XPO1), myeloid differentiation primary response gene 88 (MYD88) and kelch-like 6 (KLHL6). Mutations in MYD88 and KLHL6 are predominant in cases of CLL with mutated immunoglobulin genes, whereas NOTCH1 and XPO1 mutations are mainly detected in patients with unmutated immunoglobulins. The patterns of somatic mutation, supported by functional and clinical analyses, strongly indicate that the recurrent NOTCH1, MYD88 and XPO1 mutations are oncogenic changes that contribute to the clinical evolution of the disease. To our knowledge, this is the first comprehensive analysis of CLL combining whole-genome sequencing with clinical characteristics and clinical outcomes. It highlights the usefulness of this approach for the identification of clinically relevant mutations in cancer., This work was funded by the Spanish Ministry of Science and Innovation (MICINN) through the Instituto de Salud Carlos III (ISCIII) and Red Temática de Investigación del Cáncer (RTICC) del ISCIII. C.L.-O. is an Investigator of the Botin Foundation and D.T., of the ICREA program. We thank E. Santos for his support of this project, A. Carracedo and J. Benítez for genotyping studies, C. Fortuny for the supply of samples and N. Villahoz and M. C. Muro for their work in the coordination of the CLL-ICGC Consortium.