influenza-virus-propagation-in-embryonated-chicken-eggs

Influenza Virus Propagation in Embryonated Chicken Eggs

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous ...

Cedars-Sinai Medical Center

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 alpha 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.

MMP19 is essential for T cell development and T cell-mediated cutaneous immune responses.

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.

Matrix metalloproteinase-19 is highly expressed in astroglial tumors and promotes invasion of glioma cells.

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.

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

A balanced proteolytic activity in the epidermis is vital to maintain epidermal homoeostasis and barrier function. Distinct protease-inhibitor systems are operating in different epidermal layers. In the uppermost layer, the stratum corneum, kallikrein-like proteases and their inhibitors are responsible for desquamation of the cornified keratinocytes, thus regulating the integrity of the epidermal barrier. Following discovery and characterisation of the human multidomain inhibitor LEKTI (lympho-epithelial Kazal-type-related inhibitor, encoded by hspink5), several new members of the Kazal-type inhibitor family have been identified. Here we describe expression and regulation of murine SPINK12, a potential orthologue of human LEKTI2. Its expression was analysed by RT-PCR and immunohistochemistry revealing organ-specific pattern with high level of expression in the epidermis and several epithelia including the stomach, kidney and uterus. In addition, mSPINK12 expression in the epidermis of skin at footpads, where stratification is markedly pronounced, was several folds higher than in the abdominal epidermis. mSPINK12 mRNA levels were not affected by any cytokines tested while treatment of primary murine keratinocytes with the combination of calcium and sorbitol resulted in a strong increase in its mRNA. It appears that mspink12 is especially expressed in the epidermal areas with thick skin and that its regulation generally responds to differentiation signals. mrSPINK12 shows an inhibitory activity against murine keratinocyte-derived trypsin-like proteolytic activity, thus, the protein does appear orthologous to human LEKTI2 and may play an role in the regulation of epithelial cell functions.

Expression and regulation of murine SPINK12, a potential orthologue of human LEKTI2.

Syndecan-1 is a cell surface proteoglycan that can organize co-receptors into a multimeric complex to transduce intracellular signals. The syndecan-1 core protein has multiple domains that confer distinct cell- and tissue-specific functions. Indeed, the extracellular, transmembrane, and cytoplasmic domains have all been found to regulate specific cellular processes. Our previous work demonstrated that syndecan-1 controls lung epithelial migration and adhesion. Here, we identified the necessary domains of the syndecan-1 core protein that modulate its function in lung epithelial repair. We found that the syndecan-1 transmembrane domain has a regulatory function in controlling focal adhesion disassembly, which in turn controls cell migration speed. In contrast, the extracellular domain facilitates cell adhesion through affinity modulation of α(2)β(1) integrin. These findings highlight the fact that syndecan-1 is a multidimensional cell surface receptor that has several regulatory domains to control various biological processes. In particular, the lung epithelium requires the syndecan-1 transmembrane domain to govern cell migration and is independent from its ability to control cell adhesion via the extracellular domain.

Transmembrane and extracellular domains of syndecan-1 have distinct functions in regulating lung epithelial migration and adhesion.

Matrix metalloproteinase-7 (MMP7) expression is quickly up-regulated after injury, and functions to regulate wound repair and various mucosal immune processes. We evaluated the global transcriptional response of airway epithelial cells from wild-type and Mmp7-null mice cultured at an air-liquid interface. The analysis of differentially expressed genes between genotypes after injury revealed an enrichment of functional categories associated with inflammation, cilia, and differentiation. Because these analyses suggested that MMP7 regulated ciliated cell formation, we evaluated the recovery of the airway epithelium in wild-type and Mmp7-null mice in vivo after naphthalene injury, which revealed augmented ciliated cell formation in the absence of MMP7. Moreover, in vitro studies evaluating cell differentiation in air-liquid interface cultures also showed faster ciliated cell production under Mmp7-null conditions compared with wild-type conditions. These studies identified a new role for MMP7 in attenuating ciliated cell differentiation during wound repair.

Article	Year
MMP19 is essential for T cell development and T cell-mediated cutaneous immune responses. PloS one\| PubMed ID: 18523579	2008
Matrix metalloproteinase-19 is highly expressed in astroglial tumors and promotes invasion of glioma cells. Journal of neuropathology and experimental neurology\| PubMed ID: 20142769	2010
Matrix metalloproteinase-19 inhibits growth of endothelial cells by generating angiostatin-like fragments from plasminogen. BMC biochemistry\| PubMed ID: 21787393	2011
Expression and regulation of murine SPINK12, a potential orthologue of human LEKTI2. Experimental dermatology\| PubMed ID: 21899598	2011
Transmembrane and extracellular domains of syndecan-1 have distinct functions in regulating lung epithelial migration and adhesion. The Journal of biological chemistry\| PubMed ID: 22936802	2012
Matrix metalloproteinase-7 coordinates airway epithelial injury response and differentiation of ciliated cells. American journal of respiratory cell and molecular biology\| PubMed ID: 23258229	2013

Rena Brauer

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