Induction Of Proteases Research Articles

Selective up-regulation of matrix metalloproteinase-9 expression in human erythema migrans skin lesions of acute lyme disease.

Despite the absence of enzymes that digest extracellular matrix, Borrelia burgdorferi spreads in the skin to form erythema migrans (EM) lesions and then disseminates to other organs. We studied the induction by bacteria of host matrix metalloproteinases (MMPs) in EM skin lesions of patients with acute Lyme disease. In blister fluid from the EM lesions, the expression of MMP-9 was selectively increased by 1900%+/-1037%, compared with blister fluid from the surrounding normal-appearing skin. The expression of all other MMP messenger RNAs was similar in the EM lesions and normal-appearing skin. Selective up-regulation of MMP-9 in the EM lesions was found. Fibroblasts and, to a lesser degree, mononuclear cells were the sources of local MMP-9 production. These results demonstrate specific up-regulation of MMP-9 in the EM skin lesions of patients with acute Lyme disease. Bacterial induction of host proteases may play a role in the dissemination of B. burgdorferi.

Dietary Amino Acids Promote Pancreatic Protease Synthesis at the Translation Stage in Rats

In some tissues, amino acids (AA) stimulate translation initiation via interactions between eukaryote initiation factor (eIF) 4E-binding protein 1 (4E-BP1), eIF4E and eIF4G. Dietary AA have been shown to induce pancreatic proteases independently of cholecystokinin in rats, the mechanism of which has not yet been clarified. In the present study, we examined the mechanism in rats for protease induction by dietary AA and determined the involvement of translation initiation. Male Wistar/ST rats were fed a 20 or 60% casein or AA mixture diet for 7 d and were intravenously injected with [35S] methionine (Met) 30 min before killing on d 7 (expt. 1). In expt. 2, rats were fed a 20 or 60% AA diet for 7 d and after food deprivation and refeeding with the respective diet on d 7 were killed at 0, 1 or 3 h. We measured mRNA and [35S] Met incorporation into chymotrypsinogen, phosphorylation status of 4E-BP1 and the association of eIF4E with 4E-BP1 or eIF4G. In expt. 1, chymotrypsin activity and synthesis were higher in both of the 60% diet groups than in the 20% diet groups, but the mRNA level and 4E-BP1 status did not differ. In expt. 2, chymotrypsin activity increased in the 60% AA diet group in a time-dependent manner. The translation initiation activity via the mTOR pathway indicated an increase similar to chymotrypsin activity. There were no differences in chymotrypsin mRNA level at any point. These results indicate that dietary AA induce chymotrypsin synthesis by promoting translation, and transient activation of translation initiation via mTOR may be associated with this induction.

Effect of ethylene producer ethrel and antioxidant ionol (BHT) on the proteolytic apparatus in coleoptiles of wheat seedlings during apoptosis.

It was established that total proteolytic activity in etiolated wheat seedlings changes in ontogenesis in cycles: peaks of proteolytic activity correspond to the 3rd, 5th, and 8th days of seedling growth, respectively. The maximum of proteolytic activity preceded the maximum of nuclease activity, which may be due to activation of nucleases by proteolytic enzymes. According to inhibitory analysis the cysteine and serine proteases play the main role in apoptosis in wheat coleoptiles. Growing of seedlings in the presence of ethrel stimulated apoptosis in the coleoptile, and it increased (almost 6-fold) the proteolytic activity in its cells. On the other hand, the antioxidant ionol (BHT) suppressed the induction of proteases, particularly at the second stage of coleoptile development, and it slowed down the increase in the nuclease activity after 6th day of the seedling life. It is suggested that phytohormones and antioxidants participate in regulation of apoptosis in the ageing coleoptile, directly or indirectly effecting the proteolytic apparatus in the coleoptile cells.

Characterization and isolation of an extracellular serine protease from the tomato pathogen Colletotrichum coccodes, and it's role in pathogenicity

Extracellular enzymes play an important role in the pathogenicity and virulence of phytopathogenic fungi. Several isolates of Colletotrichum coccodes causal agent of anthracnose on tomato, were screened to determine the relationship between protease activity and virulence. A direct relationship was observed between extracellular protease activity and the induction of disease symptoms of fruit and mortality in plants. Isolate Cc155 exhibited the highest protease activity after five days of growth in protease induction medium and produced an extracellular serine protease (sp78) that was 78 kDa, auto-degradative, glucose repressible, and non-glycosylated. To determine the role of sp78 in pathogenicity, a UV-induced extracellular protease deficient mutant (np155) was generated from the wildtype isolate Cc155. Np155 maintained growth rates comparable to Cc155 and produced wildtype levels of extracellular cellulase but did not produce extracellular protease. Unlike Cc155, np155 caused no disease symptoms on tomato fruit and 0% mortality on tomato seedlings. These results suggest that extracellular protease activity is required for pathogenicity and virulence of C. coccodes and that the elimination of protease activity transforms a virulent pathogen to a non-pathogenic endophyte.

Erythropoietin is a novel vascular protectant through activation of Akt1 and mitochondrial modulation of cysteine proteases.

Erythropoietin (EPO) is a critical regulator for the proliferation of immature erythroid precursors, but its role as a potential cytoprotectant in the cerebrovasculature system has not been defined. We examined the ability of EPO to regulate a cascade of apoptotic death-related cellular pathways during anoxia-induced vascular injury in endothelial cells (ECs). EC injury was evaluated by trypan blue, DNA fragmentation, membrane phosphatidylserine (PS) exposure, protein kinase B activity, mitochondrial membrane potential, and cysteine protease induction. Exposure to anoxia alone rapidly increased genomic DNA fragmentation from 2+/-1% to 40+/-5% and membrane PS exposure from 3+/-2% to 56+/-5% over 24 hours. Administration of a cytoprotective concentration of EPO (10 ng/mL) prevented DNA destruction and PS exposure. Cytoprotection by EPO was completely abolished by cotreatment with anti-EPO neutralizing antibody, which suggests that EPO was necessary and sufficient for the prevention of apoptosis. Protection by EPO was intimately dependent on the activation of protein kinase B (Akt1) and the maintenance of mitochondrial membrane potential. Subsequently, EPO inhibited caspase 8-, caspase 1-, and caspase 3-like activities that were linked to mitochondrial cytochrome c release. The present work serves to illustrate that EPO can offer novel cytoprotection during ischemic vascular injury through direct modulation of Akt1 phosphorylation, mitochondrial membrane potential, and cysteine protease activity.

An inducible Ku86-degrading serine protease in human cells

The Ku autoantigen has been implicated in a number of cellular functions including growth control, immunoglobulin gene rearrangement and DNA repair. A variant truncated form of Ku86, with an apparent molecular weight of 70 kDa, has been reported to be present in many human cell types. We have previously shown that the amount of variant Ku86 is strongly increased in human peripheral blood mononuclear cells (PBMC) by storage of blood prior to isolation of the PBMC. In this study we report that formation of variant Ku86 in protein extracts is mediated by an inducible trypsin-like serine protease with a higher concentration in the nuclear compartment, as compared with the cytoplasm. However, experiments with SDS-PAGE assay of whole cells yielded no evidence of truncated Ku86, suggesting that the protease is not active in intact cells, but is exerting a marked activity during the protein extraction procedure. Interestingly, the protease level became markedly reduced upon transfer of the cells to growth medium. Protease induction did not correlate with apoptosis, necrotic cell death or with signs of general proteolysis or cytotoxicity. Our findings have methodological implications for the interpretation of experimental Ku86 data, and suggest that this protease may play a role for cellular regulation of Ku function.

The chemokine system: tuning and shaping by regulation of receptor expression and coupling in polarized responses.

The chemokine system: tuning and shaping by regulation of receptor expression and coupling in polarized responses.

Novel Signaling Pathways That Control Ovarian Follicular Development, Ovulation, and Luteinization

The interactions of peptide and steroid hormone signaling cascades in the ovary are critical for follicular growth, ovulation, and luteinization. Although the pituitary gonadotropins follicle-stimulating hormone (FSH) and luteinizing hormone (LH) play key regulatory roles, their actions are also dependent on other peptide signaling pathways, including those stimulated by insulin-like growth factor-1 (IGF-1), transforming growth factor-beta (TGF-beta) family members (e.g., inhibin, activin, growth differentiation factor-9, bone morphogenic proteins), fibroblast growth factor, and Wnts (via Frizzled receptors). Each of these factors is expressed and acts in a cell-specific manner at defined stages of follicular growth. IGF-1, estrogen, and FSH comprise one major regulatory system. The Wnt/Frizzled pathways define other aspects relating to ovarian embryogenesis and possibly ovulation and luteinization. Likewise, the steroid receptors as well as orphan nuclear receptors and their ligands impact ovarian cell function. The importance of these multiple signaling cascades has been documented by targeted deletion of specific genes. For example, mice null for the LH-induced genes progesterone receptor (PR) and cyclo-oxygenase-2 (COX-2) fail to ovulate. Whereas PR appears to regulate the induction of novel proteases, COX-2 appears to regulate cumulus expansion. This review summarizes some new aspects of peptide and steroid hormone signaling in the rodent ovary.

Post-translational regulation of nitrate reductase: mechanism, physiological relevance and environmental triggers.

Assimilatory nitrate reductase (NR) of higher plants is a most interesting enzyme, both from its central function in plant primary metabolism and from the complex regulation of its expression and control of catalytic activity and degradation. Here, present knowledge about the mechanism of post-translational regulation of NR is summarized and the properties of the regulatory enzymes involved (protein kinases, protein phosphatases and 14-3-3-binding proteins) are described. It is shown that light and oxygen availability are the major external triggers for the rapid and reversible modulation of NR activity, and that sugars and/or sugar phosphates are the internal signals which regulate the protein kinase(s) and phosphatase. It is also demonstrated that stress factors like nitrate deficiency and salinity have remarkably little direct influence on the NR activation state. Further, changes in NR activity measured in vitro are not always associated with changes in nitrate reduction rates in vivo, suggesting that NR can be under strong substrate limitation. The degradation and half-life of the NR protein also appear to be affected by NR phosphorylation and 14-3-3 binding, as NR activation always correlates positively with its stability. However, it is not known whether the molecular form of NR in vivo affects its susceptibility to proteolytic degradation, or whether factors that affect the NR activation state also independently affect the activity or induction of the NR protease(s). A second and potentially important function of NR, the production of nitric oxide (NO) from nitrite is briefly described, but it remains to be determined whether NR produces NO for pathogen/stress signalling in vivo.

Transforming Growth Factor-β- and Tumor Necrosis Factor-α-mediated Induction and Proteolytic Activation of MMP-9 in Human Skin

Both cytokines and matrix metalloproteinases (MMPs) are active during physiologic and pathologic processes such as cancer metastasis and wound repair. We have systematically studied cytokine-mediated MMP regulation. Cytokine-mediated proteinase induction and activation were initially investigated in organ-cultured human skin followed by determination of underlying cellular and molecular mechanisms using isolated skin cells. In this report we demonstrate that tumor necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta (TGF-beta) synergistically induce pro-MMP-9 in human skin as well as isolated dermal fibroblasts and epidermal keratinocytes. Furthermore, TNF-alpha promotes proteolytic activation of pro-MMP-9 by conversion of the 92-kDa pro-MMP-9 to the 82-kDa active enzyme. This activation occurred only in skin organ culture and not by either isolated fibroblasts or keratinocyte, although the pro-MMP-9 activation could be measured in a cell-free system derived from TNF-alpha-activated skin. The cytokine-mediated induction of pro-MMP-9 in dermal fibroblasts was evident by increased mRNA. At the transcription level, we examined the cytokine-mediated transactivation of the 5'-region promoter of the human MMP-9 in dermal fibroblasts. The results demonstrated that TNF-alpha and TGF-beta could independently stimulate the 5'-flanking 670-base pair promoter. A TGF-beta-response element (-474) and an NF-kappaB-binding site (-601) were identified to be the cis-elements for TGF-beta or TNF-alpha activation, respectively. Taken together, these findings suggest a specific mechanism whereby multiple cytokines can regulate MMP-9 expression/activation in the cells of human skin. These results imply roles for these cytokines in the regulation of MMP-9 in physiologic and pathologic tissue remodeling.

Protease A activity and nitrogen fractions released during alcoholic fermentation and autolysis in enological conditions.

Determination of protease A activity during alcoholic fermentation of a synthetic must (pH 3.5 at 25 degrees C) and during autolysis showed that a sixfold induction of protease A activity occurred after sugar exhaustion, well before 100% cell death occurred. A decrease in protease A activity was observed when yeast cell autolysis started. Extracellular protease A activity was detected late in the autolysis process, which suggests that protease A is not easily released. Evolution of amino acids and peptides was determined during alcoholic fermentation and during autolysis. Amino acids were released in early stationary phase. These amino acids were subsequently assimilated during the fermentation. The same pattern was observed for peptides; this has never been reported previously. During autolysis, the concentration of amino acids and peptides increased to reach a maximum of 20 and 40 mg N l(-1), respectively. This study supports the idea that although protease A activity seemed to be responsible for peptides release, there is no clear correlation among protease A activity, cell death, and autolysis. The amino acid composition of the peptides showed some variations between peptides released during alcoholic fermentation and during autolysis. Depending on aging time on yeast lees, the nature of the peptides present in the medium changed, which could lead to different organoleptic properties.

Overproduction of β-1,6-glucanase in Trichoderma harzianum is controlled by extracellular acidic proteases and pH

To produce high amounts of extracellular endo-β-1,6-glucanase, we overexpressed the gene bgn16.2 from Trichoderma harzianum under the control of the pyruvate kinase gene promoter ( pki) of T. reesei. Transcription of bgn16.2 gene increased under most conditions but not extracellular β-1,6-glucanase levels. Relationship of extracellular BGN16.2 protein and presence of proteases was studied in order to maximize production. After changing the carbon and nitrogen sources and buffering the culture media at different pHs, four major proteases, the acidic ones being pH-regulated, were detected. Overexpression of BGN16.2 at low pH resulted in BGN16.2 degradation, due to the induction of aspartyl proteases and to instability at pH below 3. Maximal overproduction of BGN16.2 albeit pure was achieved in buffered medium, where pH-induced aspartyl proteases were absent or when some nitrogen sources, such as yeast extract, peptone or casein were substrate for these proteases.

Apoptosis and extracellular matrix–cell interactions in kidney disease

Apoptosis and extracellular matrix-cell interactions in kidney disease. Extracellular matrix (ECM)-cell interactions have major effects on phenotypic features such as cell growth, differentiation, and gene expression. Apoptosis is an active form of cell death that is crucial for maintaining an appropriate number of cells as well as tissue organization. Recent reports have implied that ECM can influence survival and apoptosis of several cell lineages including glomerular mesangial cells (MC). Numerous glomerular diseases are associated with the expansion of the mesangial ECM, which may eventually produce glomerular scarring. Glomerular cell apoptosis is associated with the deletion of glomerular cells and the accumulation of ECM in the progression of glomerulosclerosis in rat remnant kidney model induced by 5/6 nephrectomy. Our recent study indicated that basement membrane matrix (a model for normal ECM components) prevented cultured MC from undergoing apoptosis after serum deprivation, thus promoting their survival, compared with type I collagen matrix (a model for abnormal ECM components). Inhibition of matrix-derived signals by antisense oligonucleotides against beta1 integrin increased MC apoptosis. Data suggest that the survival and death of MC are regulated by the surrounding ECM through integrin molecules. The mechanism of regulation of MC apoptosis by ECM requires further in vivo study to gain new insight into the treatment of glomerular diseases as well as the pathophysiology of the mesangium. Diabetic nephropathy is characterized by the abnormal ECM accumulation and the phenotypic change of MC. Some speculations on the possible involvement of apoptosis in diabetic nephropathy are also discussed.

Elevation of Intracellular cAMP Inhibits Growth Factor-Mediated Matrix Metalloproteinase-9 Induction and Keratinocyte Migration

Receptor tyrosine kinases are regulators of diverse cellular functions including cell growth, cell survival, differentiation, locomotion, and morphogenesis. Activation of the cAMP-dependent protein kinase A inhibits receptor tyrosine kinase-stimulated growth responses in a number of cell types. In this study, we investigated the consequences of elevated cAMP on growth factor-mediated keratinocyte migration and matrix metalloproteinase (MMP)-9 induction in a human keratinocyte cell line. We found that elevation of intracellular cAMP by forskolin abolishes epidermal growth factor (EGF)- or scatter factor/hepatocyte growth factor-dependent colony dispersion. Concentrations of forskolin that inhibit growth factor-induced motility also eliminate EGF- or scatter factor/hepatocyte growth factor-dependent induction of the 92-kDa gelatinase/MMP-9. In contrast to findings obtained in fibroblasts, elevated intracellular cAMP did not interfere with growth factor-dependent activation of the p42/44 extracellular signal-regulated kinases, indicating that cAMP-dependent inhibition of migration and MMP-9 induction does not occur through perturbation of the extracellular signal-regulated kinases/mitogen-activated protein kinase pathway. However, forskolin effectively inhibited EGF-dependent activation of c-Jun N-terminal kinase and p38, demonstrating that cAMP selectively interferes with a different subset of growth factor-induced mitogen-activated protein kinase signaling cascades than reported previously in fibroblasts. These findings illustrate that EGF concurrently activates multiple mitogen-activated protein kinase signaling cascades in keratinocytes and suggests that each pathway contributes to maximal EGF-dependent migration and proteinase induction.

Luminal dietary protein, not amino acids, induces pancreatic protease via CCK in pancreaticobiliary-diverted rats.

We determined whether pancreatic adaptation to a high-protein diet depends on ingested protein in the intestinal lumen and whether such adaptation depends on a CCK or capsaicin-sensitive vagal afferent pathway in pancreaticobiliary-diverted (PBD) rats. Feeding a high-casein (60%) diet but not a high-amino acid diet to PBD rats increased pancreatic trypsin and chymotrypsin activities compared with those after feeding a 25% casein diet. In contrast, feeding both the high-nitrogen diets induced pancreatic hypertrophy in PBD rats. These pancreatic changes by the diets were abolished by treatment with devazepide, a CCK-A receptor antagonist. Protease zymogen mRNA abundance in the PBD rat was not increased by feeding the high-casein diet and was decreased by devazepide. Perivagal capsaicin treatment did not influence the values of any pancreatic variables in PBD rats fed the normal or high-casein diet. We concluded that luminal protein or peptides were responsible for the bile pancreatic juice-independent induction of pancreatic proteases on feeding a high-protein diet. The induction was found to be dependent on the direct action of CCK on the pancreas. Pancreatic growth induced by high-protein feeding in PBD rats may depend at least partly on absorbed amino acids.

Vascular endothelial growth factor production by fibroblasts in response to factor VIIa binding to tissue factor involves thrombin and factor Xa.

Tissue factor (TF) assembled with activated factor VII (FVIIa) initiates the coagulation cascade. We recently showed that TF was essential for FVIIa-induced vascular endothelial growth factor (VEGF) production by human fibroblasts. We investigated whether this production resulted from TF activation by its binding to FVIIa or from the production of clotting factors activated downstream. Incubation of fibroblasts with a plasma-derived FVIIa concentrate induced the generation of activated factor X (FXa) and thrombin and the secretion of VEGF, which was inhibited by hirudin and FXa inhibitors. By contrast, the addition of recombinant FVIIa to fibroblasts did not induce VEGF secretion unless factor X was present. Moreover, thrombin and FXa induced VEGF secretion and VEGF mRNA accumulation, which were blocked by hirudin and FXa inhibitors, respectively. The effect of thrombin was mediated by its specific receptor, protease-activated receptor-1; in contrast, the effect of FXa did not appear to involve effector cell protease receptor-1, because it was not affected by an anti-effector cell protease receptor-1 antibody. An increase in intracellular calcium with the calcium ionophore A23187 or intracellular calcium chelation by BAPTA-AM had no effect on either basal or FXa-induced VEGF secretion, suggesting that the calcium signaling pathway was not sufficient to induce VEGF secretion. Finally, FVIIa, by itself, had no effect on mitogen-activated protein (MAP) kinase activation, contrary to thrombin and FXa, which activate the p44/p42 MAP kinase pathway, as shown by the blocking effect of PD 98059 and by Western blotting of activated MAP kinases. These findings indicate that FVIIa protease induction of VEGF expression is mediated by thrombin and FXa generated in response to FVIIa binding to TF-expressing fibroblasts; they also exclude a direct signaling involving MAP kinase activation via the intracellular domain of TF when expressed by these cells.

A stable HeLa cell line that inducibly expresses poliovirus 2A(pro): effects on cellular and viral gene expression.

A HeLa cell clone (2A7d) that inducibly expresses the gene for poliovirus protease 2A (2A(pro)) under the control of tetracycline has been obtained. Synthesis of 2A(pro) induces severe morphological changes in 2A7d cells. One day after tetracycline removal, cells round up and a few hours later die. Poliovirus 2A(pro) cleaves both forms of initiation factor eIF4G, causing extensive inhibition of capped-mRNA translation a few hours after protease induction. Methoxysuccinyl-Ala-Ala-Pro-Val-chloromethylketone, a selective inhibitor of 2A(pro), prevents both eIF4G cleavage and inhibition of translation but not cellular death. Expression of 2A(pro) still allows both the replication of poliovirus and the translation of mRNAs containing a picornavirus leader sequence, while vaccinia virus replication is drastically inhibited. Translation of transfected capped mRNA is blocked in 2A7d-On cells, while luciferase synthesis from a mRNA bearing a picornavirus internal ribosome entry site (IRES) sequence is enhanced by the presence of 2A(pro). Moreover, synthesis of 2A(pro) in 2A7d cells complements the translational defect of a poliovirus 2A(pro)-defective variant. These results show that poliovirus 2A(pro) expression mimics some phenotypical characteristics of poliovirus-infected cells, such as cell rounding, inhibition of protein synthesis and enhancement of IRES-driven translation. This cell line constitutes a useful tool to further analyze 2A(pro) functions, to complement poliovirus 2A(pro) mutants, and to test antiviral compounds.

Induction of the putative protective protein ferritin by infrared radiation: implications in skin repair.

The modification of ferritin in human skin cells in vitro and in vivo following infrared-A irradiation by immunohistochemical analysis and ELISA were evaluated. In addition, we observed that IR-A is not capable of inducing frank damage to DNA (pyrimidine dimers, p53), induction of oxidative stress proteins (heme oxygenase, nitric oxide, superoxide dismutase, heat shock proteins) or proteases (collagenase, stromelysin, gelatinase) involved in carcinogenesis and photoaging of the skin. in vivo, basal levels of ferritin were heterogeneous for all individuals tested but all showed ferritin to stain precisely in the basal layer of unirradiated epidermis. Following IR-A radiation, the ferritin increase was localized to epidermal tissue and showed an increase from 120 to 220%. Parallel to the in vivo analysis, dermal fibroblasts were cultured from six individuals. Quantitative analysis for ferritin in cultured fibroblasts was assessed by ELISA and increases were seen to be dose-dependent and up to 130% of basal levels of ferritin following infrared-A. Our findings indicate that the putative defense system of ferritin that exists in human skin in vivo can be induced by infrared-A radiation and that these wavelengths may prove to be beneficial for human skin. Importantly, following the same doses of IR-A that induced ferritin levels, there was no alteration seen for nuclear DNA type damage, oxidative stress proteins or proteases involved in the degradation of skin. The increased concentrations of this antioxidant in human skin following acute UV radiation could afford increased protection against subsequent oxidative stress.

Glucoamylase::green fluorescent protein fusions to monitor protein secretion in Aspergillus niger.

A glucoamylase::green fluorescent protein fusion (GLA::sGFP) was constructed which allows the green fluorescent protein to be used as an in vivo reporter of protein secretion in Aspergillus niger. Two secretory fusions were designed for secretion of GLA::sGFP which employed slightly different lengths of the glucoamylase protein (GLA499 and GLA514). Expression of GLA::sGFP revealed that fluorescence was localized in the hyphal cell walls and septa, and that fluorescence was most intense at hyphal apices. Extracellular GLA::sGFP was detectable by Western blotting only in the supernatant of young cultures grown in soya milk medium. In older cultures, acidification of the medium and induction of proteases were probably responsible for the loss of extracellular and cell wall fluorescence and the inability to detect GLA::sGFP by Western analysis. A strain containing the GLA::sGFP construct was subjected to UV mutagenesis and survivors screened for mutations in the general secretory pathway. Three mutants were isolated that were unable to form a halo on either starch or gelatin medium. All three mutants grew poorly compared to the parental strain. Fluorescence microscopy revealed that for two of the mutants, GLA::sGFP accumulated intracellularly with no evidence of wall fluorescence, whereas for the third mutant, wall fluorescence was observed with no evidence of intracellular accumulation. These results indicate that the GLA::sGFP fusion constructs can be used as convenient fluorescent markers to study the dynamics of protein secretion in vivo and as a tool in the isolation of mutants in the general secretory pathway.

Cell migration in the immune system: the evolving inter-related roles of adhesion molecules and proteinases.

Leukocyte extravasation into perivascular tissue during inflammation and lymphocyte homing to lymphoid organs involve transient adhesion to the vessel endothelium, followed by transmigration through the endothelial cell (EC) layer and establishment of residency at the tissue site for a period of time. In these processes, leukocytes undergo multiple attachments to, and detachments from, the vessel-lining endothelial cells, prior to transendothelial cell migration. Transmigrating leukocytes must traverse a subendothelial basement membrane en route to perivascular tissues and utilize enzymes known as matrix metalloproteinases to make selective clips in the extracellular matrix components of the basement membrane. This review will focus on the evidence for a link between adhesion of leukocytes to endothelial cells, the induction of matrix metalloproteinases mediated by engagement of adhesion receptors on leukocytes, and the ability to utilize these matrix metalloproteinases to facilitate leukocyte invasion of tissues. Leukocytes with invasive phenotypes express high levels of MMPs, and expression of MMPs enhances the migratory and invasive properties of these cells. Furthermore, MMPs may be used by lymphocytes to proteolytically cleave molecules such as adhesion receptors and membrane bound cytokines, increasing their efficiency in the immune response. Engagement of leukocyte adhesion receptors may modulate adhesive (modulation of integrin affinities and expression), synthetic (proteinase induction and activation), and surface organization (clustering of proteolyric complexes) behaviors of invasive leukocytes. Elucidation of these pathways will lead to better understanding of controlling mechanisms in order to develop rational therapeutic approaches in the areas of inflammation and autoimmunity.