Radislav Sedlacek

MMP‐19: cellular localization of a novel metalloproteinase within normal breast tissue and mammary gland tumours

Matrix metalloproteinases (MMPs) are instrumental in promoting and facilitating the spread of malignant diseases and in the de novo formation of blood vessels. This study has mapped the immunoreactivity of a novel, angiogenesis‐related metalloproteinase – MMP‐19 – in normal breast tissue and in benign and malignant breast lesions and compared this pattern of expression with that of MMP‐2. In the normal resting mammary gland, MMP‐19 was strongly expressed in the myoepithelial layer of the ductal system; the alveolar and ductal epithelia displayed considerable, but lobule‐specific, variations in labelling intensity. MMP‐19 was also present within the smooth muscle and endothelial layers of large and medium‐sized blood vessels, as well as within capillary walls. In benign lesions, all tumour cells and their surrounding vasculature were uniformly and strongly immunoreactive for MMP‐19. Progression towards an invasive phenotype and neoplastic dedifferentiation led to the disappearance of MMP‐19 from tumour cells and blood vessels and a concomitant rise in the levels of MMP‐2. In vitro experiments conducted with isolated smooth muscle cells cultivated on a solid substratum, or within the interstices of a collagen matrix, indicated that the expression of MMP‐19 is influenced by the architecture of the surrounding extracellular matrix. Copyright

Matrix metalloproteinase-19 is expressed in myeloid cells in an adhesion-dependent manner and associates with the cell surface.

We have previously reported the isolation of the human matrix metalloproteinase (MMP)-19 (also referred to as RASI) from a synovium of a patient suffering from rheumatoid arthritis and its expression at the cell surface of activated PBMC. In this study, we have analyzed the regulation and cell surface expression of human MMP-19 in several human cell lines and blood-derived cells. Among the cell lines analyzed, MMP-19 is largely expressed by lung fibroblasts as well as by myeloid cell lines THP-1 and HL-60. After fractionating PBMC into CD14− and CD14+ populations we found that only the latter one expresses MMP-19. Although the myeloid cell lines as well as CD14+ cells express MMP-19 without stimulation, its production can be up-regulated by phorbol esters (PMA) or by adhesion. The adhesion-dependent expression was down-regulated or even abrogated by blockade of adhesion or interfering with adhesion-controlling signaling using α-tocopherol. We have shown that MMP-19 associates with the cell surface of myeloid cells. This cell surface association was not affected by phospholipase C. However, acidic treatment of the THP-1-derived cell membranes abolished the immunoprecipitation of MMP-19 thereof. Moreover, a high salt treatment of THP-1 cells diminished the MMP-19 detection on the cell surface. This implicates a noncovalent attachment of MMP-19 to the cell surface. Because a truncated form of the MMP-19, in which the hemopexin-like domain was deleted (ΔhpMMP-19), does not associate with the surface, the hemopexin-like domain appears to be critical for the cell surface attachment of human MMP-19.

Horizontal transfer of whole mitochondria restores tumorigenic potential in mitochondrial DNA-deficient cancer cells

Recently, we showed that generation of tumours in syngeneic mice by cells devoid of mitochondrial (mt) DNA (ρ0 cells) is linked to the acquisition of the host mtDNA. However, the mechanism of mtDNA movement between cells remains unresolved. To determine whether the transfer of mtDNA involves whole mitochondria, we injected B16ρ0 mouse melanoma cells into syngeneic C57BL/6Nsu9-DsRed2 mice that express red fluorescent protein in their mitochondria. We document that mtDNA is acquired by transfer of whole mitochondria from the host animal, leading to normalisation of mitochondrial respiration. Additionally, knockdown of key mitochondrial complex I (NDUFV1) and complex II (SDHC) subunits by shRNA in B16ρ0 cells abolished or significantly retarded their ability to form tumours. Collectively, these results show that intact mitochondria with their mtDNA payload are transferred in the developing tumour, and provide functional evidence for an essential role of oxidative phosphorylation in cancer. DOI: http://dx.doi.org/10.7554/eLife.22187.001

MMP19 is upregulated during melanoma progression and increases invasion of melanoma cells.

During the progression of cutaneous melanomas, matrix metalloproteinases (MMPs) facilitate the tumour cells to traverse the basement membrane and invade the dermis. In this study, we analysed the expression of MMP19 in the course of melanoma progression. Although MMP19 was absent in melanocytes and melanoma cells of early stages of melanoma development, its expression was strongly upregulated in the neighbouring keratinocytes that may facilitate the vertical outgrowth of melanoma cells. In contrast to early stages, MMP19 was upregulated during the vertical growth phase of melanoma and in metastases. The upregulation of MMP19 in melanoma of Clark levels IV and V correlates with that of MMP2 and also simultaneously with ceased expression of E-cadherin. To reveal whether MMP19 facilitates the invasion of melanomas, we examined adhesion and migratory capacity of selected melanoma cell lines. Melanoma cell lines with low expression of MMP19 exhibited increased adhesion to various substrates and lower migration in comparison with the cell line with higher expression of MMP19. Moreover, ectopic expression of MMP19 could restore the migratory capacity of melanoma cells with low endogenous level of MMP19. These results suggest that the increase of MMP19 expression hallmarks the progression of cutaneous melanoma and might augment melanoma growth by promoting the invasion of tumour cells.

Activity of MMP-19 inhibits capillary-like formation due to processing of nidogen-1

AbstractMatrix metalloproteinase 19 (MMP-19) is able to process various proteins of the basement membrane. To investigate the impact of MMP-19 activity on endothelial cells in the context of tumor extracellular matrix (ECM), we treated Matrigel matrix with an active recombinant MMP-19 and analyzed its effect on capillary-like formation. Human microvascular endothelial cells (HMEC-1) could not form capillary-like formation on Matrigel treated with recombinant MMP-19. Analyzing the Matrigel proteins, we found that MMP-19 preferentially cleaved nidogen-1. The cleavage site of nidogen-1 was mapped to Thr867-Leu868. This cleavage separates the G3 globular domain containing the binding site for the γ1 chain of laminin-1 and collagen IV and thus abolishes the capacity of nidogen-1 to cross-link ECM proteins. Anti-nidogen antibodies directed against the G3 domain of nidogen-1 inhibited the capillary-like structure formation to a similar extent as MMP-19. Since nidogen-1 is thought to stabilize microvessels, MMP-19 might be one of the enzymes that interferes with stabilization or maturation of nascent vasculature.

Matrix Metalloproteinase-19 is Highly Expressed in Astroglial Tumors and Promotes Invasion of Glioma Cells

Glial tumors exhibit a high morbidity and mortality because of their invasive nature. Matrix metalloproteinase 19 (MMP19) is a secreted protease that together with epilysin (MMP28) forms a structural subgroup of MMPs. We analyzed their expression by quantitative reverse transcription polymerase chain reaction, Western blot, and immunohistochemistry in tumor and normal control brain tissues and in glioblastoma (GB) cells and performed MMP19 silencing functional assays. Matrix metalloproteinase 28 was transcribed to the same extent in normal brain samples and gliomas but was undetectable in GB cell lines. In contrast, MMP19 was detected by immunohistochemistry in normal brain samples only in endothelial cells but was found at high levels in astrocytomas of different World Health Organization grades in situ and in GB cells in vitro. Matrix metalloproteinase 19 was upregulated in GB cells after exposure to proinflammatory cytokines. In Transwell invasion assays, MMP19-silenced cells migrated more slowly through laminin-, basal lamina-, and brevican-coated membranes than controls. Matrix metalloproteinase 19-silenced GB cells also migrated into brain tissue slices compared with control cells. Brevican, a brain-specific proteoglycan and major component of brain extracellular matrix, was degraded by recombinant human MMP19. Taken together, these results indicate that MMP19 is highly expressed in proliferating astrocytoma/glioma cells, and that its expression may facilitate their invasion through brain extracellular matrix components.

MMP19 Is Essential for T Cell Development and T Cell-Mediated Cutaneous Immune Responses

Matrix metalloproteinase-19 (MMP19) affects cell proliferation, adhesion, and migration in vitro but its physiological role in vivo is poorly understood. To determine the function of MMP19, we generated mice deficient for MMP19 by disrupting the catalytic domain of mmp19 gene. Although MMP19-deficient mice do not show overt developmental and morphological abnormalities they display a distinct physiological phenotype. In a model of contact hypersensitivity (CHS) MMP19-deficient mice showed impaired T cell-mediated immune reaction that was characterized by limited influx of inflammatory cells, low proliferation of keratinocytes, and reduced number of activated CD8+ T cells in draining lymph nodes. In the inflamed tissue, the low number of CD8+ T cells in MMP19-deficient mice correlated with low amounts of proinflammatory cytokines, especially lymphotactin and interferon-inducible T cell α chemoattractant (I-TAC). Further analyses showed that T cell populations in the blood of immature, unsensitized mice were diminished and that this alteration originated from an altered maturation of thymocytes. In the thymus, thymocytes exhibited low proliferation rates and the number of CD4+CD8+ double-positive cells was remarkably augmented. Based on the phenotype of MMP19-deficient mice we propose that MMP19 is an important factor in cutaneous immune responses and influences the development of T cells.

Matrix metalloproteinase-19 inhibits growth of endothelial cells by generating angiostatin-like fragments from plasminogen

BackgroundAngiogenesis is the process of forming new blood vessels from existing ones and requires degradation of the vascular basement membrane and remodeling of extracellular matrix (ECM) in order to allow endothelial cells to migrate and invade into the surrounding tissue. Matrix metalloproteinases (MMPs) are considered to play a central role in the remodeling of basement membranes and ECM. However, MMPs contribute to vascular remodeling not only by degrading ECM components. Specific MMPs enhance angiogenesis via several ways; they help pericytes to detach from vessels undergoing angiogenesis, release ECM-bound angiogenic growth factors, expose cryptic pro-angiogenic integrin binding sites in the ECM, generate promigratory ECM component fragments, and cleave endothelial cell-cell adhesions. MMPs can also negatively influence the angiogenic process through generating endogenous angiogenesis inhibitors by proteolytic cleavage. Angiostatin, a proteolytic fragment of plasminogen, is one of the most potent antagonists of angiogenesis that inhibits migration and proliferation of endothelial cells. Reports have shown that metalloelastase, pancreas elastase, plasmin reductase, and plasmin convert plasminogen to angiostatin.ResultsWe report here that MMP-19 processes human plasminogen in a characteristic cleavage pattern to generate three angiostatin-like fragments with a molecular weight of 35, 38, and 42 kDa. These fragments released by MMP-19 significantly inhibited the proliferation of HMEC cells by 27% (p = 0.01) and reduced formation of capillary-like structures by 45% (p = 0.05) compared with control cells. As it is known that angiostatin blocks hepatocyte growth factor (HGF)-induced pro-angiogenic signaling in endothelial cells due to structural similarities to HGF, we have analyzed if the plasminogen fragments generated by MMP-19 interfere with this pathway. As it involves the activation of c-met, the receptor of HGF, we could show that MMP-19-dependent processing of plasminogen decreases the phosphorylation of c-met.ConclusionAltogether, MMP-19 exhibits an anti-angiogenic effect on endothelial cells via generation of angiostatin-like fragments.

The murine ortholog of matrix metalloproteinase 19: its cloning, gene organization, and expression

We have isolated a murine cDNA orthologous to the human matrix metalloproteinase 19 (hMMP-19). The murine MMP-19 cDNA was amplified by RT-PCR using specific primers whose DNA sequences were derived from both murine MMP-19 genomic DNA and partial cDNA sequences. The murine MMP-19 (mMMP-19) is 79% identical to the human ortholog and encodes a protein of 527 amino acids with a deduced molecular mass of 59.1kDa. Analyzing the exon/intron junctions we revealed that the murine MMP-19 gene consists of nine exons and eight introns, and thus differs from the gene organization of other matrix metalloproteinases. Furthermore, a 587bp fragment of the mMMP-19 promoter containing a TATA box and an AP-1 binding motif was cloned, and 3.3kb transcripts of the MMP-19 gene were identified in liver, kidney, spleen, and colon. Finally, immunostaining of murine heart cryosections showed that mMMP-19, like its human counterpart, is expressed in the arterial tunica media of large blood vessels. By cloning mMMP-19 and unraveling its genomic structure, we have obtained valuable information for further study of the function of this MMP in vivo.

Hepatoprotective effect of MMP-19 deficiency in a mouse model of chronic liver fibrosis.

Liver fibrosis is characterized by the deposition and increased turnover of extracellular matrix. This process is controlled by matrix metalloproteinases (MMPs), whose expression and activity dynamically change during injury progression. MMP-19, one of the most widely expressed MMPs, is highly expressed in liver; however, its contribution to liver pathology is unknown. The aim of this study was to elucidate the role of MMP-19 during the development and resolution of fibrosis by comparing the response of MMP-19-deficient (MMP19KO) and wild-type mice upon chronic liver CCl4-intoxication. We show that loss of MMP-19 was beneficial during liver injury, as plasma ALT and AST levels, deposition of fibrillar collagen, and phosphorylation of SMAD3, a TGF-ß1 signaling molecule, were all significantly lower in MMP19KO mice. The ameliorated course of the disease in MMP19KO mice likely results from a slower rate of basement membrane destruction and ECM remodeling as the knockout mice maintained significantly higher levels of type IV collagen and lower expression and activation of MMP-2 after 4 weeks of CCl4-intoxication. Hastened liver regeneration in MMP19KO mice was associated with slightly higher IGF-1 mRNA expression, slightly increased phosphorylation of Akt kinase, decreased TGF-ß1 mRNA levels and significantly reduced SMAD3 phosphorylation. In addition, primary hepatocytes isolated from MMP19KO mice showed impaired responsiveness towards TGF-ß1 stimulation, resulting in lower expression of Snail1 and vimentin mRNA. Thus, MMP-19-deficiency improves the development of hepatic fibrosis through the diminished replacement of physiological extracellular matrix with fibrotic deposits in the beginning of the injury, leading to subsequent changes in TGF-ß and IGF-1 signaling pathways.