Key Role for Matrix Metalloproteinases -19 and -28 in Maintenance of Intestinal Homeostasis

The present study investigates the role of two matrix metalloproteinases, MMP-19 and MMP-28, in intestinal homeostasis and inflammation by means of a mouse model for inflammatory bowel disease (IBD). MMP-19 and -28 are abundantly expressed in epithelial tissues. However, their functions in this compartment and their functional overlap have not been elucidated so far. MMP-19 contributes to IGF-signaling, cell proliferation, and migration and is able to cleave several soluble proteins and components of extracellular matrix. MMP-28 was shown to be important in wound healing. Comparison of wildtype and MMP-19-deficient mice revealed an increased susceptibility to acute and chronic DSS-induced colitis accompanied by impaired healing process compared to the wildtype animals. Survival rates during acute and chronic colitis were significantly reduced. In contrast, the MMP-28-deficient mice show only moderate signs of disease and behaved similarly to the DSS-treated wildtype group. Strikingly, the MMP-19/-28 double-deficient mice showed the most severe phenotype in acute colitis and during the recovery, which was reflected in very poor survival rates and impaired wound healing in the surviving animals. In conclusion, this study provides several lines of evidence for a pivotal role of MMP-19 and MMP-28 in maintenance of intestinal tissue homeostasis. The data suggest that loss-of-function of MMP-19 results in an increased susceptibility to colonic inflammation, mediated by an exacerbated innate immune response and inability of colonic tissue to heal

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

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

Thymocytes of MMP19^−/− mice exhibit strongly reduced proliferation.

<p>(A) Immature MMP19^−/− mice exhibit low numbers of Ki-67⁺ medullar cells in the thymus. Scale bars: 50 µm. (B and C). To quantify proliferating thymocytes mice were injected i.p. with BrdU. After 14 h, CD3⁺, CD4⁺, and CD8⁺ thymocytes were analyzed for BrdU incorporation using flow cytometry. Grey, isotype control; light red, MMP19^−/− (n = 4); red, MMP19^+/+ (n = 4). (C) Quantification of BrdU positive thymocytes. CD3⁺, CD4⁺, and CD8⁺ thymocytes from MMP19^−/− mice exhibit markedly diminished proliferation. Black bars: MMP19^+/+, white bars: MMP19^−/−. (D) Quantitative RT-PCR analysis of MMP19 in the thymus shows expression in WT mice and confirmed the absence in MMP19^−/− mice. The expression of MMP19 in the thymus is lower compared to that in the liver. C_T, cycle of threshold. Significances with **p<0,01 (student's t-test).</p

MMP19^−/− mice show impaired ear swelling and inflammatory reaction in CHS.

<p>(A) Five days after abdominal sensitization with the hapten (FITC), mouse ears were challenged with FITC and ear thickness was measured after 24, 48, and 72 h. Mean values are given in mm as difference to time point 0 h. (B) Hematoxylin-eosin staining of ear sections 24 h after challenge shows reduced influx of neutrophils and eosinophils in MMP19^−/− mice. (C) Staining with anti-Ki-67 antibody revealed that proliferation of keratinocytes in MMP19-deficient mice was strongly reduced in basal and suprabasal layers. Ki-67-positive cells were counted and calculated as percentage of basal and total keratinocytes. Six images (magnification 400x) per mice were analyzed. (D) Decreased processing of IGFBP-3 in MMP19-deficient mice. Primary keratinocytes from MMP19^+/− and MMP19^−/− mice were grown for 72 h and conditioned media were analyzed for IGFBP-3 proteolysis by western blotting. The arrowhead indicates the position of intact IGFBP-3, whereas the arrow points to 30 kD IGFBP-3 proteolytic fragment. (E) Anti-CD8 staining (red) of ear sections. MMP19^−/− mice show low numbers of CD8⁺ T cells in CHS (upper panel) as well as in a T cell-independent model of inflammation, i.e. irritant dermatitis, induced by croton oil (middle panel). Ears of unsensitized mice (lower panel) exhibit low numbers of CD8⁺ cells; no difference was observed between MMP19^+/+ and MMP19^−/− mice. CHS and irritant dermatitis were carried out in four independent experiments each with 4 wild-type and 4 MMP19^−/− mice. (F) 24 h after FITC challenge ear lysates were analyzed for cytokine expression that was generally reduced in MMP19-deficient mice compared to wild-type animals. (G) Reduced levels of lymphotactin and I-TAC from three independent experiments are shown. Bars in F and G represent values of MMP19^−/− mice given as fold decrease to wild-type mice. Significant values with *p<0.05 and **p<0.01; student's t-test. Scale bars: B, 50 µm; C, 20 µm; D, 50 µm.</p

T cell population in blood of immature MMP19^−/− mice is distorted.

<p>(A) Blood samples of 3-weeks old MMP19^+/+ and MMP19^−/− mice were analyzed for monocytes, granulocytes (CD11b and Ly-6), B cells (B220), and T cell populations by flow cytometry. MMP19^−/− mice exhibit significantly reduced numbers of CD4⁺ and CD8⁺ T cells. (B) A typical dot plot analysis of CD4⁺ and CD8⁺ populations gated on CD3⁺ cells in individual mice is shown. (C) No differences in T cell subpopulations were measured in blood of adult mice (12 weeks old). CD4⁺ and CD8⁺ T cells shown in A and C are also positive for CD3. Analyses were carried out in five independent experiments with MMP19^+/+ (n = 4) and MMP19^−/− mice (n = 4). Numbers of B and T lymphocytes were analyzed by gating the lymphocyte region. Significances with *p<0.05; student's t-test.</p

Generation of MMP19^−/− mice.

<p>(A) Schematic representation of the murine <i>mmp19</i> gene and its exon/intron-organization as previously described by Mueller et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002343#pone.0002343-Mueller1" target="_blank">[7]</a>; pPNT targeting vector construct, and the resulting deleted active site locus of the mouse <i>mmp19</i> gene are depicted. The targeting construct based on the pPNT vector <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002343#pone.0002343-Tybulewicz1" target="_blank">[70]</a>, was generated by replacement of 1088 bp region of <i>mmp19</i> gene spanning the end of exon 3 and the whole exon 4 encoding the catalytic domain by the neomycin resistance cassette. Homologous recombination led to introduction of the PGK-Neo cassette and allowed selection of homologous recombinants. The 3′-probe used for detection of replacement events is indicated by thick bars. Also shown are restriction sites used for southern hybridization screening as well as primer binding sites used for diagnostic PCR. Screening for replacement mutants employed restriction digestion of genomic DNA with <i>Stu</i>I and <i>Eco</i>RV and southern hybridization with the described 3′-probe. (B) For screening of MMP19-deficient mice Southern blot analyses were performed: mouse DNA digested with <i>Stu</i>I and <i>Eco</i>RV was probed with the diagnostic 3′-probe. Probing led to identification of either a 7.3 kb band (wild-type allele, +/+) or a 5 kb band for the targeted allele (−/−). In heterozygous mice (+/−) both alleles are present. (C) Genotyping of targeted alleles using PCR. Wild-type alleles are detected by an 800 bp band, while PCR for the MMP19-deficient allele results in a 600 bp product. (D) Primary keratinocytes isolated from wild-type, heterozygous, and homozygous MMP19-deficient mice were analyzed for MMP19 expression by western blotting using anti-MMP19 antibodies purified against a peptide derived from the hinge region of murine MMP19, that is deteced in size of 59 kD. (E) Immunohistochemical analysis of murine skin with anti-MMP19 antibodies described above. Scale bars: 50 µm.</p