RESUMEN
Tree-ring δ15N may depict site-specific, long-term patterns in nitrogen (N) dynamics under N2-fixing species, but field trials with N2-fixing tree species are lacking and the relationship of temporal patterns in tree-ring δ15N to soil N dynamics is controversial. We examined whether the tree-ring δ15N of N2-fixing red alder (Alnus rubra Bong.) would mirror N accretion rates and δ15N of soils and whether the influence of alder-fixed N could be observed in the wood of a neighboring conifer. We sampled a 27-year-old replacement series trial on south-eastern Vancouver Island, with red alder and coastal Douglas-fir (Pseudotsuga menziesii [Mirb.] Franco) planted in five proportions (0/100, 11/89, 25/75, 50/50 and 100/0) at a uniform stem density. An escalation in forest floor N content was evident with an increasing proportion of red alder, equivalent to a difference of ~750 kg N ha-1 between 100% Douglas-fir versus 100% alder. The forest floor horizon also had high δ15N values in treatments with more red alder. Red alder had a consistent quadratic fit in tree-ring δ15N over time, with a net increase of $\sim$1.5, on average, from initial values, followed by a plateau or slight decline. Douglas-fir tree-ring δ15N, in contrast, was largely unchanged over time (in three of four plots) but was significantly higher in the 50/50 mix. The minor differences in current leaf litter N content and δ15N between alder and Douglas-fir, coupled with declining growth in red alder, suggests the plateau or declining trend in alder tree-ring δ15N could coincide with lower N2-fixation rates, potentially by loss in alder vigor at canopy closure, or down-regulation via nitrate availability.
Asunto(s)
Alnus , Pseudotsuga , Nitrógeno , Árboles/fisiología , Bosques , Plantas , Pseudotsuga/fisiologíaRESUMEN
The serine proteinases plasmin and thrombin convert proenzyme matrix metalloproteinases (MMPs) into catalytically active forms. In addition, we demonstrate that plasmin(ogen) and thrombin induce a significant increase in secretion of activated murine macrophage elastase (MMP-12) protein. Active serine protease is responsible for induction, as demonstrated by the absence of MMP-12 induction in plasminogen(Plg)-treated urokinase-type plasminogen activator-deficient macrophages. Since increased MMP-12 protein secretion was not accompanied by an increase in MMP-12 mRNA, we examined post-translational mechanisms. Protein synthesis was not required for early release of MMP-12 but was required for later secretion of activated enzyme. Immunofluorescent microscopy demonstrated basal expression in macrophages that increased following serine proteinase exposure. Inhibition of MMP-12 secretion by hirudin and pertussis toxin demonstrated a role for the thrombin G protein-coupled receptor (protease-activated receptor 1 (PAR-1)). PAR-1-activating peptides were able to induce MMP-12 release. Investigation of signal transduction pathways involved in this response demonstrate the requirement for protein kinase C, but not tyrosine kinase, activity. These data demonstrate that plasmin and thrombin regulate MMP-12 activity through distinct mechanisms: post-translational secretion of preformed MMP-12 protein, induction of protein secretion that is protein kinase C-mediated, and extracellular enzyme activation. Most importantly, we show that serine proteinase MMP-12 regulation in macrophages occurs via the protein kinase C-activating G protein-coupled receptor PAR-1.
Asunto(s)
Proteínas de Caenorhabditis elegans , Macrófagos/enzimología , Elastasa Pancreática/metabolismo , Proteínas Serina-Treonina Quinasas/fisiología , Serina Endopeptidasas/fisiología , Animales , Células Cultivadas , Cicloheximida/farmacología , Activación Enzimática , Fibrinolisina/farmacología , Proteínas de Unión al GTP/fisiología , Ratones , Elastasa Pancreática/genética , Plasminógeno/farmacología , Receptores de Trombina/fisiología , Trombina/farmacología , Transcripción Genética/efectos de los fármacosRESUMEN
Angiostatin, a cleavage product of plasminogen, has been shown to inhibit endothelial cell proliferation and metastatic tumor cell growth. Recently, the production of angiostatin has been correlated with tumor-associated macrophage production of elastolytic metalloproteinases in a murine model of Lewis lung cell carcinoma. In this report we demonstrate that purified murine and human matrix metalloproteinases generate biologically functional angiostatin from plasminogen. Macrophage elastase (MMP-12 or MME) proved to be the most efficient angiostatin-producing MMP. MME was followed by gelatinases and then the stomelysins in catalytic efficiency; interstitial collagenases had little capacity to generate angiostatin. Both recombinant angiostatin and angiostatin generated from recombinant MME-treated plasminogen inhibited human microvascular endothelial cell proliferation and differentiation in vitro. Finally, employing macrophages isolated from MME-deficient mice and their wild-type littermates, we demonstrate that MME is required for the generation of angiostatin that inhibits the proliferation of human microvascular endothelial cells.
Asunto(s)
Regulación de la Expresión Génica/efectos de los fármacos , Metaloendopeptidasas/farmacología , Neovascularización Fisiológica/efectos de los fármacos , Fragmentos de Péptidos/biosíntesis , Plasminógeno/biosíntesis , Amidas/farmacología , Angiostatinas , Animales , Células Cultivadas , Medios de Cultivo Condicionados/farmacología , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Factor 2 de Crecimiento de Fibroblastos/farmacología , Gelatinasas/farmacología , Humanos , Macrófagos Peritoneales/metabolismo , Metaloproteinasa 12 de la Matriz , Metaloproteinasa 3 de la Matriz/farmacología , Metaloendopeptidasas/antagonistas & inhibidores , Metaloendopeptidasas/deficiencia , Metaloendopeptidasas/genética , Ratones , Ratones Noqueados , Fragmentos de Péptidos/genética , Plasminógeno/efectos de los fármacos , Plasminógeno/genética , Plasminógeno/metabolismo , Plasminógeno/farmacología , Inhibidores de Proteasas/farmacología , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Tirosina/análogos & derivados , Tirosina/farmacologíaRESUMEN
Matrix metalloproteinases (MMPs) are a family of inducible enzymes that degrade extracellular matrix components, allowing cells to traverse connective tissue structures efficiently. Specific tissue inhibitors (TIMPs) function as physiologic inhibitors of MMP activity. Because neovascularization may require various proteinases, we characterized the profile of metalloenzyme production by microvascular endothelial cells (MEC) and the modulation of expression by phorbol esters (PMA) and by the physiologically relevant cytokines tumor necrosis factor-alpha (TNF-alpha), basic fibroblast growth factor, and interferon-gamma. MMP expression by MEC and large-vessel human umbilical vein endothelial cells (HUVEC) was determined by enzyme-linked immunosorbent assay, immunoprecipitation, Northern hybridization, and transfection assays. Constitutive expression of MMPs by endothelial cells was low. PMA stimulated the production of collagenase, stromelysin, 92-kDa gelatinase, and TIMP-1 in both endothelial cell types. TIMP-2 was constitutively expressed by MEC and HUVEC, but was down-regulated by PMA. TNF-alpha induced an endothelial-cell-specific up-regulation of collagenase with a concomitant inhibition of PMA-induced TIMP-1 up-regulation, a response that is distinct from that of fibroblasts. Interferon-gamma up-regulated TIMP-1 production by MEC and blocked PMA and TNF-induced up-regulation of collagenase. Northern hybridization assays showed pretranslational control of PMA-, basic fibroblast growth factor-, and TNF-alpha-induced MMP expression. Collagenase-promoter CAT constructs containing 2.28 kb of the 5' region of the collagenase gene demonstrated transcriptional regulation. The potential physiologic relevance of such regulation was shown in an in vitro migration assay. MEC were stimulated to migrate by wounding and exposure to TNF-alpha. Collagenase mRNA was prominently expressed by the migrating cells, as shown by in situ hybridization. In sum, MEC have a unique profile of MMP expression and regulation compared with other cell types, which may be important for wound healing and angiogenesis, particularly during the early phase of migration.
Asunto(s)
Endotelio Vascular/metabolismo , Matriz Extracelular/metabolismo , Sustancias de Crecimiento/farmacología , Metaloendopeptidasas/metabolismo , Piel/irrigación sanguínea , Movimiento Celular , Colagenasas/genética , Endotelio Vascular/citología , Glicoproteínas/metabolismo , Humanos , Interferón gamma/farmacología , Microcirculación , Neovascularización Patológica , Procesamiento Proteico-Postraduccional , ARN Mensajero/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Inhibidores Tisulares de Metaloproteinasas , Transcripción Genética , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Matrilysin is a recently described metalloproteinase with strong catalytic activity against a variety of extracellular matrix substrates including proteoglycans, elastin, laminin, fibronectin, gelatin, and entactin. Production of this metalloproteinase appears to be limited only to a few normal human cell types including glandular epithelium, mononuclear phagocytes, and renal mesangial cells. Furthermore, matrilysin expression in vivo has been demonstrated only in glandular epithelium, especially the endometrium. In the process of examining various cutaneous and lung inflammatory disorders for matrilysin expression by immunohistochemistry and in situ hybridization, we occasionally found monocytes within blood vessels and newly extravasated tissue-associated macrophages that exhibited matrilysin production. In specimens characterized by severe inflammation and, in particular, cystic fibrosis, this feature was commonly observed. We therefore studied the production of matrilysin by monocyte-derived macrophages in vitro in response to various physiologic signals such as endotoxin, phagocytosable material, cytokines, and hormones. We found that matrilysin expression was stimulated by LPS and opsonized zymosan. Up-regulation of matrilysin by LPS was PGE2-dependent, because indomethacin blocked production, an effect at least partially reversed by the addition of exogenous prostaglandin. LPS stimulated matrilysin production pretranslationally and, furthermore, when cultured cells were subjected to in situ hybridization after LPS exposure, considerable variability in matrilysin mRNA expression was observed on an individual cell basis, with some cells having strong signal and others being completely negative. We also found that matrilysin biosynthesis was inhibited by the lymphokines IL-4, IL-10, and IFN-gamma. Other cytokines such as IL-1, TNF-alpha, and IL-6 failed to modulate the production of matrilysin. Finally, matrilysin biosynthesis was suppressed by glucocorticoids and retinoids. Our studies indicate that matrilysin is produced in vivo by mononuclear phagocytes and is a highly regulated metalloproteinase whose production can be modified by a variety of physiologic and pharmacologic signals.