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

<p>Abstract</p> <p>Background</p> <p>Angiogenesis 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.</p> <p>Results</p> <p>We 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.</p> <p>Conclusion</p> <p>Altogether, MMP-19 exhibits an anti-angiogenic effect on endothelial cells via generation of angiostatin-like fragments.</p

Myopia disease mouse models: a missense point mutation (S673G) and a protein-truncating mutation of the Zfp644 mimic human disease phenotype.

Zinc finger 644 (Zfp644 in mouse, ZNF644 in human) gene is a transcription factor whose mutation S672G is considered a potential genetic factor of inherited high myopia. ZNF644 interacts with G9a/GLP complex, which functions as a H3K9 methyltransferase to silence transcription. In this study, we generated mouse models to unravel the mechanisms leading to symptoms associated with high myopia. Employing TALEN technology, two mice mutants were generated, either with the disease-carrying mutation (Zfp644 S673G ) or with a truncated form of Zfp644 (Zfp644 Δ8 ). Eye morphology and visual functions were analysed in both mutants, revealing a significant difference in a vitreous chamber depth and lens diameter, however the physiological function of retina was preserved as found under the high-myopia conditions. Our findings prove that ZNF644/Zfp644 is involved in the development of high-myopia, indicating that mutations such as, Zfp644 S673G and Zfp644 Δ8 are causative for changes connected with the disease. The developed models represent a valuable tool to investigate the molecular basis of myopia pathogenesis and its potential treatment

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 expression in keratinocytes is repressed by transcription factors Tst-1 and Skn-1a: Implications for keratinocyte differentiation

Matrix metalloproteinase-19 (MMP-19), unlike other members of the MMP family, is expressed in basal keratinocytes of intact epidermis whereas keratinocytes in suprabasal and higher epidermal layers express this enzyme only during cutaneous disorders. As the activity of MMP-19 effects proliferation, migration, and adhesion of keratinocytes we examined whether transcription factors involved in keratinocyte differentiation repress the expression of MMP-19. Using luciferase reporter assays, POU transcription factors Tst-1 (Oct-6) and Skn-1a (Oct-11) markedly downregulated the activity of MMP-19 promoter in COS-7 cells and HaCaT keratinocytes. Tst-1 alone was able to inhibit 85% of the promoter activity. Skn-1a exhibited a weak inhibitory effect although it synergistically increased effects of Tst-1. HaCaT cells stably transfected with Tst-1 showed a strong decrease of activity of MMP-19 promoter that correlated with suppression of MMP-19, cytokeratin 14 and 5, decreased cell proliferation, and altered expression of involucrin and loricrin. The expression of MMP-9 was also significantly reduced in Tst-1 expressing keratinocytes. MMP-2 was substantially affected during its activation whereas the expression of MMP-28 was unchanged. Our results suggest that Tst-1 and Skn-1a regulate expression of MMPs in keratinocytes and effect both the expression and activation of these proteolytic enzymes

KLK5 and KLK7 Ablation Fully Rescues Lethality of Netherton Syndrome-Like Phenotype

Netherton syndrome (NS) is a severe skin disease caused by the loss of protease inhibitor LEKTI, which leads to the dysregulation of epidermal proteases and severe skin-barrier defects. KLK5 was proposed as a major protease in NS pathology, however its inactivation is not sufficient to rescue the lethal phenotype of LEKTI-deficient mice. In this study, we further elucidated the in vivo roles of the epidermal proteases in NS using a set of mouse models individually or simultaneously deficient for KLK5 and KLK7 on the genetic background of a novel NS-mouse model. We show that although the ablation of KLK5 or KLK7 is not sufficient to rescue the lethal effect of LEKTI-deficiency simultaneous deficiency of both KLKs completely rescues the epidermal barrier and the postnatal lethality allowing mice to reach adulthood with fully functional skin and normal hair growth. We report that not only KLK5 but also KLK7 plays an important role in the inflammation and defective differentiation in NS and KLK7 activity is not solely dependent on activation by KLK5. Altogether, these findings show that unregulated activities of KLK5 and KLK7 are responsible for NS development and both proteases should become targets for NS therapy

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

Analysis of skin-barrier abnormalities.

<p><b>(A)</b> Newborn (P0) pups were analysed by barrier penetration assay using toluidine blue (TB). Remaining barrier-defect in the area of nostrils of Klk5^-/-Klk7^-/-Sp5^A135X/A135X pups in the vicinity of nostrils is marked by black arrowhead <b>(B)</b> TEWL analysis of P0 pups as a reduction of body-weight over time, n≥4 for each genotype. Error bars represent standard errors of mean; Klk/Spink5 mutant lines were compared with the wt line using Mann-Whitney U-test at 3h and 4h, ns means “not significant“; * p < 0.05 <b>(C)</b> Epidermal barrier in P5 Klk5^-/-Sp5^A135X/A135X pups was compromised in the proximity of hair follicles <b>(D)</b> Vibrissae hair (upper panel) and dorsal skin (lower panel) obtained from P5 pups analysed using scanning electron microscopy. Defective separation of hair shafts from the root sheath in Klk5^-/-Sp5^A135X/A135X is marked by white arrowhead. Dorsal skin of Klk5^-/-Sp5^A135X/A135X mice showed complete absence of hair shafts and exposed upper parts of hair follicles (yellow arrowheads); Scale bar, 300 μm.</p

Gross phenotype of Spink5- Klk- deficient mutant lines.

<p><b>(A)</b> Phenotype of wt, Sp5^A135X/A135X, Klk5^-/-Sp5^A135X/A135X, Klk7^-/-Sp5^A135X/A135X and Klk5^-/-Klk7^-/-Sp5^A135X/A135X mice 12 hours after birth. Peeling skin was observed in Sp5^A135X/A135X and Klk7^-/-Sp5^A135X/A135X and to lesser extent in Klk5^-/-Sp5^A135X/A135X pups (black arrowheads). <b>(B)</b> wt, Klk5^-/-Sp5^A135X/A135X, and Klk5^-/-Klk7^-/-Sp5^A135X/A135X mice at P5. Klk5^-/-Sp5^A135X/A135X pups showed dry, scaly skin while Klk5^-/-Klk7^-/-Sp5^A135X/A135X mice had stretched and shiny epidermis, with no visual signs of dehydration. <b>(C)</b> wt and Klk5^-/-Klk7^-/-Sp5^A135X/A135X mice at 3 weeks. Klk5^-/-Klk7^-/-Sp5^A135X/A135X mice showed alopecia and growth retardation. <b>(D)</b> wt and Klk5^-/-Klk7^-/-Sp5^A135X/A135X mice at 6 weeks. <b>(E)</b> Vibrissae hair obtained from P5 pups analysed by scanning electron microscopy. Klk5^-/-Klk7^-/-Sp5^A135X/A135X showed hair shaft abnormalities similar to bamboo hair; Scale bar, 30 μm. <b>(F)</b> Progression of body weight of wt, Klk5^-/-Sp5^A135X/A135X, Klk5^-/-Klk7^-/-Sp5^A135X/A135X and Klk5^-/-Klk7^-/- mice, n>5, error bars represent standard errors of mean.</p

Histological analysis of epidermis structure at E18.5 dpc and P0.

<p><b>(A)</b> Hematoxylin and eosin stained skin sections from newborn mice showed a reduced granular layer and acanthosis in epidermis of Sp5^A135X/A135X pups; no obvious defects were observed in the other groups, Scale bar, 100 μm. <b>(B)</b> Analysis of epidermal differentiation in the skin from E18.5 dpc embryos. Sections were stained with antibodies against keratin14 (Krt14), keratin6 (Krt6) and fillagrin (Flg). Increased expression of Krt14 was observed in Sp5^A135X/A135X embryos, which also strongly express the stress marker Krt6. Expression of both Krt6 and Krt14 was not altered in Klk5^-/-Sp5^A135X/A135X, Klk7^-/-Sp5^A135X/A135X and Klk5^-/-Klk7^-/-Sp5^A135X/A135X. Flg staining revealed absence of profilaggrin granules in Sp5^A135X/A135X and Klk7^-/-Sp5^A135X/A135X embryos whereas these granules were present in wt, Klk5^-/-Sp5^A135X/A135X and Klk5^-/-Klk7^-/-Sp5^A135X/A135X mice (white arrowheads). Scale bar, 50 μm.</p

Generation of Spink5/Klk5/Klk7 mutant lines.

<p><b>(A)</b> Klk5^-/- line (depicted as Klk5^-/-Klk7^+/+Spink5^+/+), was used for preparation of Klk5^-/-Klk7^-/- mice (depicted as Klk5^-/-Klk7^-/-Spink5^+/+) by TALEN mutagenesis. Obtained Klk5^-/-Klk7^-/- mice were further crossed to a Flippase (FLPe) expressing mouse line to allow conditionally expressed Klk5, thus generating Klk7^-/- mice (depicted as Klk5^+/+(loxP)Klk7^-/-Spink5^+/+). Klk5^-/-, Klk7^-/- and Klk5^-/-Klk7^-/- lines were subsequently crossed with Spink5^+/- line to obtain Klk5^-/-Sp5^A135X/A135X, Klk7^-/-Sp5^A135X/A135X, and Klk5^-/-Klk7^-/-Sp5^A135X/A135X, i.e. double and triple KO lines. <b>(B)</b> Expression of Klk5, Klk7 and Spink5 at the mRNA level was quantified using qRT-PCR, n≥4 for each genotype, error bars represent standard deviations from mean.</p