Carboxypeptidases cathepsins X and B display distinct protein profile in human cells and tissues

Cathepsin X, a recently discovered lysosomal cysteine protease, shares common structural features and activity properties with cysteine protease cathepsin B. Based on its widespread mRNA distribution in primary tumors and tumor cell lines, a redundant function in tumor progression has been proposed. In this study, we have shown that these two related proteases exhibit different profiles with respect to their protein distribution in cells and tissues and to their possible roles in malignancy. Protein level of cathepsin X did not differ significantly between matched pairs of lung tumor and adjacent lung tissue obtained from patients with lung cancer whereas that of cathepsin B was 9.6-fold higher in tumor compared to adjacent lung tissue. Immunohistochemical analysis of lung tumor cathepsin X revealed very faint staining in tumor cells but positive staining in infiltrated histiocytes, alveolar macrophages, bronchial epithelial cells, and alveolar type II cells. Cathepsin X stained positive also in CD68+ cells in germinal centers of secondary follicles in lymph nodes, corresponding to tingible body macrophages. Two cell lines with proven invasive behavior, MCF-10A neoT and MDA-MB 231, showed positive staining for cathepsin B, but negative for cathepsin X. We showed that the invasive potential of MCF-10A neoT cells can be impaired by specific inhibitor of cathepsin B but not by that of cathepsin X. Cathepsin X was found in large amounts in the pro-monocytic U-937 cell line, in monocytes and in dendritic cells, generated from monocytes in vitro. Our results show that cathepsin X is not involved in degradation of extracellular matrix, a proteolytic event leading to tumor cell invasion and metastasis. Its expression, restricted to immune cells suggests a role in phagocytosis and the regulation of immune response.     

Carboxy-monopeptidase substrate specificity of human cathepsin X

Cathepsin X is a papain-like cysteine protease with restricted positional specificity, acting primarily as a carboxy-monopeptidase. We mapped the specificities at the S2, S1, and S1' subsites of human cathepsin X by systematically and independently substituting the P2, P1, and P1' positions of the carboxy-monopeptidase substrate Abz-FRF(4NO(2)) with natural amino acids. Human cathepsin X has broad S2, S1, and S1' specificities within two orders of magnitude in k(cat)/K(M), excluding proline that is not tolerated at these subsites. Glycine is not favored in S2, but is among the preferred residues in S1 and S1', which highlights S2 as the affinity-determinant subsite. The presence of peculiar residues at several binding site positions (Asp76, His234, Asn75, and Glu72) does not translate into a markedly different sequence specificity profile relative to other human cathepsins. These findings suggest that a specific function of human cathepsin X is unlikely to result from sequence specificity, but rather from a combination of its unique positional specificity and the co-localization of enzyme and substrate in a specific cellular environment.     

Determining beta2-integrin and intercellular adhesion molecule 1 binding kinetics in tumor cell adhesion to leukocytes and endothelial cells by a gas-driven micropipette assay

Interactions between polymorphonuclear neutrophils (PMNs) and tumor cells have been reported to facilitate the adhesion and subsequent extravasation of tumor cells through the endothelium under blood flow, both of which are mediated by binding β(2)-integrin to intercellular adhesion molecule 1 (ICAM-1). Here the adhesions between human WM9 metastatic melanoma cells, PMNs, and human pulmonary microvascular endothelial cells (HPMECs) were quantified by a gas-driven micropipette aspiration technique (GDMAT). Our data indicated that the cellular binding affinity of PMN-WM9 pair was 3.9-fold higher than that of the PMN-HPMEC pair. However, the effective binding affinities per molecular pair were comparable between the two cell pairs no matter whether WM9 cells or HPMECs were quiescent or cytokine-activated, indicating that the stronger adhesion between PMN-WM9 pair is mainly attributed to the high expression of ICAM-1 on WM9 cells. These results proposed an alternative mechanism, where WM9 melanoma cells adhere first with PMNs near vessel-wall regions and then bind to endothelial cells via PMNs under blood flow. In contrast, the adhesions between human MDA-MB-231 metastatic breast carcinoma cells and PMNs showed a comparable cellular binding affinity to PMN-HPMEC pair because the ICAM-1 expressions on MDA-MB-231 cells and HPMECs are similar. Furthermore, differences were observed in the intrinsic forward and reverse rates of the β(2)-integrin-ICAM-1 bond between PMN-TC and PMN-EC pairs. This GDMAT assay enables us to quantify the binding kinetics of cell adhesion molecules physiologically expressed on nucleated cells. The findings also further the understanding of leukocyte-facilitated tumor cell adhesion from the viewpoint of molecular binding kinetics.     

Characteristics of U-937 and HL-60 leukemia cells differentiated by spinach extract

Some characteristics of U-937 and HL-60 leukemia cell lines treated with a fraction of non-dialyzable extract of spinach are reported. The absorbed fraction separated by a DEAE-Tyopearl 650 column chromatography of the non-dialyzable extract induced NBT reducing activity of U-937 and HL-60 cells. This fraction also induced substrate adhesion of U-937 cells, and the non-specific esterase activity of HL-60 cells. The expression of CD11b, CD11c and CD36 antigens on the U-937 cell surface was enhanced by the treatment with the fraction, whereas CD24 antigen was not. The treatment of HL-60 cells with the fraction also induced the expression of CD11b and CD11c antigens, but CD24 and CD36 were not expressed. These results indicated that the non-dialyzable extract of spinach induced immature differentiation of U-937 and HL-60 cells into monocyte/macrophages.Abbreviations NBT nitroblue tetrazolium - TPA 12-O-tetradecanoyl-phorbol-13-acerate - PBS phosphate buffered saline - FITC fluorescein isothiocyanate     

Identification of beta1 integrin as mediator of melanoma cell adhesion to lumican

Lumican is a small leucine-rich proteoglycan (SLRP) present in the dermal extracellular matrix. Previous data from our laboratory demonstrated that lumican decreases melanoma progression in vivo. Here, we show that melanoma cell migration is decreased by lumican and that this effect is due to an enhanced cell adhesion. The adhesion of A375 human melanoma cells on lumican was dose-dependent and required Mg²⁺ and Mn²⁺ divalent cations. Using a panel of monoclonal antibodies directed against integrin subunits, we showed that A375 cells can bind to recombinant lumican through β1 type integrins. Moreover, the use of rhodocetin, an inhibitor of α2 integrin, suggested that this particular subunit might also be involved in the interaction with lumican. The increased β1 integrin-mediated adhesion of melanoma cells to lumican might explain, at least in part, the anti-invasive effect of this SLRP.     

S100A9 mediates neutrophil adhesion to fibronectin through activation of beta2 integrins

Neutrophil migration from the blood to inflammatory sites follows a cascade of events, in which adhesion to endothelial cells and extracellular matrix proteins is essential. S100A8, S100A9, and S100A12 are small abundant proteins found in human neutrophil cytosol and presumed to be involved in leukocyte migration. Here we investigated the S100 proteins' activities in neutrophil tissue migration by evaluating their effects on neutrophil adhesion to certain extracellular matrix proteins. S100A9 induced adhesion only to fibronectin and was the only S100 protein that stimulated neutrophil adhesion to this extracellular matrix protein. Experiments with blocking antibodies revealed that neither beta1 nor beta3 integrins were strongly involved in neutrophil adhesion to fibronectin, contrary to what the literature predicted. In contrast, neutrophil adhesion to fibronectin was completely inhibited by anti-beta2 integrins, suggesting that S100A9-induced specific activation of beta2 integrin is essential to neutrophil adhesion.     

Maspin, the molecular bridge between the plasminogen activator system and beta1 integrin that facilitates cell adhesion

Maspin is a non-inhibitory serine protease inhibitor (serpin) that influences many cellular functions including adhesion, migration, and invasion. The underlying molecular mechanisms that facilitate these actions are still being elucidated. In this study we determined the mechanism by which maspin mediates increased MCF10A cell adhesion. Utilizing competition peptides and mutation analyses, we discovered two unique regions (amino acid residues 190-202 and 260-275) involved in facilitating the increased adhesion function of maspin. In addition, we demonstrate that the urokinase-type plasminogen activator (uPA)/uPA receptor (uPAR) complex is required for the localization and adhesion function of maspin. Finally, we showed that maspin, uPAR, and β1 integrin co-immunoprecipitate, suggesting a novel maspin-uPA-uPAR-β1 integrin mega-complex that regulates mammary epithelial cell adhesion.     

PDGF-BB enhances expression of,and reduces adhesion to,laminin-5 in vascular smooth muscle cells

The laminin family of extracellular matrix (ECM) proteins plays crucial roles in regulating cellular growth, migration, and differentiation. We report here that laminin-5 is expressed in the tunica media of the rat aorta and pulmonary arteries. Using indirect immunofluorescence microscopy, Western blots, and RT-PCR analysis, we found that primary cultures of rat arterial smooth muscle cells express laminin-5 and deposit it into their insoluble ECM. These cells also attach strongly to laminin-5 via beta1 integrin receptors in 30 min adhesion assays. Laminin-5 expression in these cells is upregulated by growth factors in vitro and platelet-derived growth factor (PDGF-BB) stimulation reduces adhesion to laminin-5. As laminin-5 promotes enhanced migration of other cell types, the production of and adhesion to laminin-5 by vascular smooth muscle cells may play a role in the pathological growth and migration of these cells associated with restenosis following vascular injury.     

SNARE-mediated trafficking of alpha5beta1 integrin is required for spreading in CHO cells

In this study, the role of SNARE-mediated membrane traffic in regulating integrin localization was examined and the requirement for SNARE function in cellular spreading was quantitatively assessed. Membrane traffic was inhibited with the VAMP-specific catalytic light chain from tetanus toxin (TeTx-LC), a dominant-negative form (E329Q) of N-ethylmaleimide-sensitive fusion protein (NSF), and brefeldin A (BfA). Inhibition of membrane traffic with either E329Q-NSF or TeTx-LC, but not BfA, significantly inhibited spreading of CHO cells on fibronectin. Spreading was rescued in TeTx-LC-expressing cells by co-transfection with a TeTx-resistant cellubrevin/VAMP3. E329Q-NSF, a general inhibitor of SNARE function, was a more potent inhibitor of cell spreading than TeTx-LC, suggesting that tetanus toxin-insensitive SNAREs contribute to adhesion. It was found that E329Q-NSF prevented trafficking of alpha5beta1 integrins from a central Rab11-containing compartment to sites of protrusion during cell adhesion, while TeTx-LC delayed this trafficking. These results are consistent with a model of cellular adhesion that implicates SNARE function as an important component of integrin trafficking during the process of cell spreading.     

Protogenin mediates cell adhesion for ingression and re-epithelialization of paraxial mesodermal cells

In the avian embryo, precursor cells of the paraxial mesoderm that reside in the epiblast ingress through the primitive streak and migrate bilaterally in an anterolateral direction. Herein, we report on the roles of Protogenin (PRTG), an immunoglobulin superfamily protein expressed on the surface of the ingressing and migrating cells that give rise to the paraxial mesoderm, in paraxial mesoderm development. An aggregation assay using L-cells showed that PRTG mediates homophilic cell adhesion. Overexpression of PRTG in the presumptive paraxial mesoderm delayed mesodermal cell migration due to augmented adhesiveness. In contrast, siRNA knockdown of PRTG impaired successive ingression of epiblast cells and disorganized the epithelial structure of the somites. These results suggest that PRTG mediates cell adhesion to regulate continuous ingression of cells giving rise to the paraxial mesodermal lineage, as well as tissue integrity.     

Trophoblast interactions with endothelial cells are increased by interleukin-1beta and tumour necrosis factor alpha and involve vascular cell adhesion molecule-1 and alpha4beta1

Interactions between fetal extravillous trophoblast cells and maternal uterine cells are of critical importance in successful placentation. In the first trimester, trophoblasts invade the uterine environment and reach the spiral arteries where they interact with vascular cells; however, little is known of the nature of these interactions. We have developed a fluorescent binding assay to investigate the contact between trophoblasts and endothelial cells and to determine its regulation by cytokines and adhesion molecules. Stimulation of an endothelial cell line (SGHEC-7) with interleukin-1beta or tumour necrosis factor-alpha significantly increased adhesion of the first-trimester extravillous trophoblast-derived cell line, SGHPL-4. Using blocking antibodies, vascular cell adhesion molecule-1 (VCAM-1) and integrin alpha4beta1 (VLA-4), but not intercellular adhesion molecule-1 (ICAM-1), were shown to be important in trophoblast binding to activated endothelial cells. SGHPL-4 cells were shown to express HLA-G, alpha4beta1 and ICAM-1 at high levels and LFA-1 and VCAM-1 at lower levels. ICAM-1 and VCAM-1 are expressed on SGHEC-7 cells and their expression was confirmed on primary decidual endothelial cells. In conclusion, we have demonstrated the importance of VCAM-1 and alpha4beta1 in trophoblasts-endothelial interactions. Improved knowledge of the nature of these fetal-maternal interactions will have implications for understanding situations when placentation is compromised.     

Hypoxia activates beta(1)-integrin via ERK 1/2 and p38 MAP kinase in human vascular smooth muscle cells

The progress in the use of HAART for the treatment of HIV-infected individuals has been limited by the development of viral resistance and the maintenance of viral latency. New therapeutic strategies geared toward improvement in the host's immune response are now being considered. We found that IFN-gamma induces CIITA through the JAK-STAT pathway and inhibits HIV-1 replication in latently infected cells. Its effect appears to be mediated through the reciprocal action of Tat and CIITA. With this beneficial effect, IFN-gamma and its inducers can be considered as an adjunct to the currently available therapy. We also addressed the safety of using simvastatin, an HMG-CoA reductase inhibitor, to treat dyslipidemia often associated with the use of protease inhibitors. Simvastatin did not show any unfavorable effects on HIV replication, thus could be used safely unless there are any drug interactions when administered.     

Identification of a unique gene cluster of Brucella spp. that mediates adhesion to host cells

Brucella, the causative agent of brucellosis, a major zoonotic disease affecting a broad range of mammals, is a gram-negative bacterium whose virulence is dependent on the capacity to attach and invade different cells of the host. The bacterium is able to infect through a diverse repertoire of epitheliums: skin, airways or gastric. Although much has been studied on the mechanisms Brucella uses to establish an intracellular replication niche, almost none is known on how the bacterium adheres and invades host cells. We report here the identification of a pathogenicity island that harbors a gene homologous to proteins with bacterial immunoglobulin-like domains present in other pathogens that play a role in attachment and invasion. Deletion of the entire island results in a mutant with a reduced attachment capacity measured by intracellular replication and adhesion assays. Intraperitoneal and oral experimental infection of mice strongly suggests that this island plays a role during the oral infection probably mediating attachment and trespassing of the gastric epithelium to establish a systemic infection.     

A NPxY-independent beta5 integrin activation signal regulates phagocytosis of apoptotic cells

Integrin receptors are heterodimeric transmembrane receptors with critical functions in cell adhesion and migration, cell cycle progression, differentiation, apoptosis, and phagocytosis of apoptotic cells. Integrins are activated by intracellular signaling that alter the binding affinity for extracellular ligands, so-called inside to outside signaling. A common element for integrin activation involves binding of the cytoskeletal protein talin, via its FERM domain, to a highly conserved NPxY motif in the β chain cytoplasmic tails, which is involved in long-range conformation changes to the extracellular domain that impinges on ligand affinity. When the human beta-5 (β5) integrin cDNA was expressed in αv positive, β5 and β3 negative hamster CS-1 cells, it promoted NPxY-dependent adhesion to VTN-coated surfaces, phosphorylation of FAK, and concomitantly, β5 integrin-EGFP protein was recruited into talin and paxillin-containing focal adhesions. Expression of a NPxY destabilizing β5 mutant (Y750A) abrogated adhesion and β5-Y750A-EGFP was excluded from focal adhesions at the tips of stress fibers. Surprisingly, expression of β5 Y750A integrin had a potent gain-of-function effect on apoptotic cell phagocytosis, and further, a β5-Y750A-EGFP fusion integrin readily bound MFG-E8-coated 10 μm diameter microspheres developed as apoptotic cell mimetics. The critical sequences in β5 integrin were mapped to a YEMAS motif just proximal to the NPxY motif. Our studies suggest that the phagocytic function of β5 integrin is regulated by an unconventional NPxY-talin-independent activation signal and argue for the existence of molecular switches in the β5 cytoplasmic tail for adhesion and phagocytosis.     

Inhibitors of protein kinase C selectively enhanced leukotriene D4-induced calcium mobilization in differentiated U-937 cells

U-937 cells differentiated with dimethylsulphoxide for 3-4 days express receptors for leukotriene D4 (LTD4), which are coupled to Ca2+ mobilization and phosphatidylinositol (PI) metabolism. Treatment of U-937 cells with an inhibitor of protein kinase C (PKC) [staurosporine (100 nM)] augmented the Ca2+ mobilized by LTD4. The peak concentration of the LTD4-induced increase in [Ca2+]i was 1500 nM in untreated cells and 3000 nM in cells treated with staurosporine for 30 s. Maximal mobilization responses were observed at 1-10 microM LTD4 in both control and staurosporine-treated cells. The increased Ca2+ response to LTD4 after staurosporine treatment occurred within 30 s and was attributable to both intracellular and extracellular stores. Additionally, a second phase of Ca2+ mobilization occurred after stimulation with LTD4, which was elevated by pretreatment with staurosporine--this effect was maximal after 5-10 min of treatment. Staurosporine either had no effect or decreased the Ca2+ mobilization response of differentiated U-937 cells to other agonists, such as LTB4, platelet activating factor, ATP or the chemotactic peptide f-Met-Leu-Phe. Although staurosporine alone had no effect on basal PI metabolism it increased LTD4-induced PI metabolism. Staurosporine did not prevent the tachyphylaxis observed upon second challenge with LTD4, nor did it prevent LTD4-induced homologous densensitization. Other compounds which inhibit PKC (sphingosine and 1-O-hexadecyl-2-O-methylglycerol), also enhanced the Ca2+ response of U-937 cells to LTD4, but not to other agonists. These data show that inhibition of PKC enhanced responses of LTD4, suggesting that PKC plays a role in determining the responsiveness of LTD4 receptors.     

Sulfite induces adherence of polymorphonuclear neutrophils to immobilized fibrinogen through activation of Mac-1 beta2-integrin (CD11b/CD18)

Sulfite is a major air pollutant which can cause respiratory tract inflammation characterized by an influx of polymorphonuclear neutrophils (PMN). We have previously shown that human PMN can produce sulfite either spontaneously or in response to stimulation with lipopolysaccharide. We now demonstrate that sulfite activates PMN to adhere to immobilized fibrinogen via the beta2-integrin Mac-1 (CD11b/CD18). Mac-1 expression is not altered by treatment with this agent. Although unaffected by pertussis toxin, sulfite-triggered PMN adhesion was abrogated by pretreating cells with the membrane-impermeant sulfhydryl reagent 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), a modifier of thiol groups on the cell surface. These results suggest that sulfite-induced PMN adhesion is dependent on a modification of thiols at the cell surface. Given its potent antioxidant and antimicrobial activities, sulfite may act as an endogenous mediator in host defense and/or inflammation.     

Azepanone-based inhibitors of human cathepsin S: optimization of selectivity via the P2 substituent

A series of azepanone inhibitors of cathepsin S is described. Selectivity over both cathepsin K and cathepsin L was achieved by varying the P2 substituent. Ultimately, a balanced potency and selectivity profile was achieved in compound 39 possessing a 1-methylcyclohexyl alanine at P2 and nicotinamide as the P′ substituent. The cellular potency of selected analogs is also described.     

Integrin alphavbeta3 mediates platelet-derived growth factor-BB-stimulated collagen gel contraction in cells expressing signaling deficient integrin alpha2beta1

The interplay between the collagen-binding integrin, alpha2beta1, and platelet-derived growth factor (PDGF) receptors in the context of functional interactions with collagen was studied. We expressed either wild-type alpha2beta1 (alpha2beta1A) or alpha2beta1 with a Y783/795F mutation in the cytoplasmic tail of the beta1 subunit (alpha2beta1Amut) in the beta1-null fibroblastic cell line, GD25. GD25 cells lack endogenous expression of the alpha1 and alpha2 integrin subunits and do not adhere to collagen even after transfection with beta1A. Cells expressing alpha2beta1Amut contracted three-dimensional collagen lattices less efficiently than those expressing alpha2beta1A. PDGF-BB significantly stimulated lattice contraction by GD25-alpha2beta1Amut cells. Both cell types responded chemotactically to PDGF-BB. Focal adhesion kinase (FAK) and p130(Cas) were phosphorylated when GD25-alpha2beta1A cells, but not GD25-alpha2beta1Amut cells were seeded on collagen-coated dishes. Subsequent treatment with PDGF-BB further increased phosphorylation of FAK and p130(Cas) only in GD25-alpha2beta1A cells. However, when cultured within collagen lattices, FAK and p130(Cas) phosphorylation were stimulated in both alpha2beta1A- and alpha2beta1Amut-expressing cells but further phosphorylation, in response to subsequent treatment with PDGF-BB, was seen only in GD25-alpha2beta1A cells. We show that the stimulatory effects of PDGF-BB on collagen gel contraction and chemotaxis by GD25-alpha2beta1Amut cells were mediated by the alphavbeta3 integrin. Phosphorylation of p130(Cas), but not FAK, in GD25-alpha2beta1Amut cells seeded in collagen lattices also depended on alphavbeta3. Our results show that PDGF-BB stimulation of fibroblast-collagen interactions is mediated by the alphavbeta3 integrin when beta1 integrin function is impaired.