The Uptake of Heparanase into Mast Cells Is Regulated by Its Enzymatic Activity to Degrade Heparan Sulfate.

Mast cells take up extracellular latent heparanase and store it in secretory granules. The present study examined whether the enzymatic activity of heparanase regulates its uptake efficiency. Recombinant mouse heparanase mimicking both the latent and mature forms (L-Hpse and M-Hpse, respectively) was internalized into mastocytoma MST cells, peritoneal cell-derived mast cells, and bone marrow-derived mast cells. The internalized amount of L-Hpse was significantly higher than that of M-Hpse. In MST cells, L-Hpse was continuously internalized for up to 8 h, while the uptake of M-Hpse was saturated after 2 h of incubation. L-Hpse and M-Hpse are similarly bound to the MST cell surface. The expression level of cell surface heparan sulfate was reduced in MST cells incubated with M-Hpse. The internalized amount of M-Hpse into mast cells was significantly increased in the presence of heparastatin (SF4), a small molecule heparanase inhibitor that does not affect the binding of heparanase to immobilized heparin. Enzymatically quiescent M-Hpse was prepared with a point mutation at Glu335. The internalized amount of mutated M-Hpse was significantly higher than that of wild-type M-Hpse but similar to that of wild-type and mutated L-Hpse. These results suggest that the enzymatic activity of heparanase negatively regulates the mast cell-mediated uptake of heparanase, possibly via the downregulation of cell surface heparan sulfate expression.

Sulfated Hyaluronan Binds to Heparanase and Blocks Its Enzymatic and Cellular Actions in Carcinoma Cells.

We examined whether sulfated hyaluronan exerts inhibitory effects on enzymatic and biological actions of heparanase, a sole endo-beta-glucuronidase implicated in cancer malignancy and inflammation. Degradation of heparan sulfate by human and mouse heparanase was inhibited by sulfated hyaluronan. In particular, high-sulfated hyaluronan modified with approximately 2.5 sulfate groups per disaccharide unit effectively inhibited the enzymatic activity at a lower concentration than heparin. Human and mouse heparanase bound to immobilized sulfated hyaluronan. Invasion of heparanase-positive colon-26 cells and 4T1 cells under 3D culture conditions was significantly suppressed in the presence of high-sulfated hyaluronan. Heparanase-induced release of CCL2 from colon-26 cells was suppressed in the presence of sulfated hyaluronan via blocking of cell surface binding and subsequent intracellular NF-κB-dependent signaling. The inhibitory effect of sulfated hyaluronan is likely due to competitive binding to the heparanase molecule, which antagonizes the heparanase-substrate interaction. Fragment molecular orbital calculation revealed a strong binding of sulfated hyaluronan tetrasaccharide to the heparanase molecule based on electrostatic interactions, particularly characterized by interactions of (-1)- and (-2)-positioned sulfated sugar residues with basic amino acid residues composing the heparin-binding domain-1 of heparanase. These results propose a relevance for sulfated hyaluronan in the blocking of heparanase-mediated enzymatic and cellular actions.

COL8A1 facilitates the growth of triple-negative breast cancer via FAK/Src activation.

PURPOSE: Triple-negative breast cancer (TNBC) is one of the most aggressive breast cancer subtypes, and treatment options are limited because of the lack of signature molecules and heterogeneous properties of cancer. COL8A1 expression is higher in breast cancer than in normal tissues and is strongly correlated with worse overall survival in patients with breast cancer. However, the biological function of COL8A1 on cancer progression is not fully understood. In this study, we investigated the biological function of COL8A1 on TNBC progression. METHODS: COL8A1-deficient cells were generated using the CRISPR-Cas9 system. The tumor growth and metastasis of TNBC cells were evaluated using three-dimensional culture (3D) methods and xenograft mouse models. The activation of focal adhesion kinase (FAK)/Src by COL8A1 in TNBC cells was evaluated by immunoblotting. RESULTS: COL8A1 expression was primarily distributed into TNBC cell lines. Further, relapse-free survival in TNBC patients with the MSL subtype was strongly associated with the COL8A1 expression. MDA-MB-231 and Hs578T cells, classified as the MSL subtype, strongly express COL8A1, and COL8A1 protein expression was induced by hypoxia in both cell lines. Loss of COL8A1 expression inhibited spheroid /tumor growth and metastasis in vitro and in vivo. Further, exogenous COL8A1 promoted TNBC growth via the FAK/Src activation. Finally, the spheroid growth of MDA-MB-231 and Hs578T cells was inhibited by defactinib, a FAK inhibitor, without cytotoxicity. CONCLUSION: These results indicate that COL8A1-mediated FAK/Src activation produces a more aggressive phenotype in TNBC, and its target inhibition may be an efficacious treatment for TNBC.

Coronin-1 is phosphorylated at Thr-412 by protein kinase Cα in human phagocytic cells.

Coronin-1, a hematopoietic cell-specific actin-binding protein, is thought to be involved in the phagocytic process through its interaction with actin filaments. The dissociation of coronin-1 from phagosomes after its transient accumulation on the phagosome surface is associated with lysosomal fusion. We previously reported that 1) coronin-1 is phosphorylated by protein kinase C (PKC), 2) coronin-1 has two phosphorylation sites, Ser-2 and Thr-412, and 3) Thr-412 of coronin-1 is phosphorylated during phagocytosis. In this study, we examined which PKC isoform is responsible for the phosphorylation of coronin-1 at Thr-412 by using isotype-specific PKC inhibitors and small interfering RNAs (siRNAs). Thr-412 phosphorylation of coronin-1 was suppressed by Gö6976, an inhibitor of PKCα and PKCßI. This phosphorylation was attenuated by siRNA for PKCα, but not by siRNA for PKCß. Furthermore, Thr-412 of coronin-1 was phosphorylated by recombinant PKCα in vitro, but not by recombinant PKCß. We next examined the effects of Gö6976 on the intracellular distribution of coronin-1 in HL60 cells during phagocytosis. The confocal fluorescence microscopic observation showed that coronin-1 was not dissociated from phagosomes in Gö6976-treated cells. These results indicate that phosphorylation of coronin-1 at Thr-412 by PKCα regulates intracellular distribution during phagocytosis.

Effects of sterol derivatives in cationic liposomes on biodistribution and gene-knockdown in the lungs of mice systemically injected with siRNA lipoplexes.

Cationic liposomes can be intravenously injected to deliver short interfering (si)RNAs into the lungs. The present study investigated the effects of sterol derivatives in systemically injected siRNA/cationic liposome complexes (siRNA lipoplexes) on gene­knockdown in the lungs of mice. Cationic liposomes composed of 1,2­dioleoyl­3­trimethylammonium­propane or dimethyldioctadecylammonium bromide (DDAB) were prepared as a cationic lipid, with sterol derivatives such as cholesterol (Chol), ß­sitosterol, ergosterol (Ergo) or stigmasterol as a neutral helper lipid. Transfected liposomal formulations composed of DDAB/Chol or DDAB/Ergo did not suppress the expression of the luciferase gene in LLC­Luc and Colon 26­Luc cells in vitro, whereas other formulations induced moderate gene­silencing. The systemic injection of siRNA lipoplexes formulated with Chol or Ergo into mice resulted in abundant siRNA accumulation in the lungs. In comparison, systemically injected DDAB/Chol or DDAB/Ergo lipoplexes of Tie2 siRNA effectively increased the suppression of the Tie2 mRNA expression in the lungs of mice. These findings indicated that DDAB/Chol and DDAB/Ergo liposomes could function as vectors for siRNA delivery to the lungs.

Chondroitin sulfate E blocks enzymatic action of heparanase and heparanase-induced cellular responses.

We examined whether chondroitin sulfates (CSs) exert inhibitory effects on heparanase (Hpse), the sole endoglycosidase that cleaves heparan sulfate (HS) and heparin, which also stimulates chemokine production. Hpse-mediated degradation of HS was suppressed in the presence of glycosaminoglycans derived from a squid cartilage and mouse bone marrow-derived mast cells, including the E unit of CS. Pretreatment of the chondroitin sulfate E (CS-E) with chondroitinase ABC abolished the inhibitory effect. Recombinant proteins that mimic pro-form and mature-form Hpse bound to the immobilized CS-E. Cellular responses as a result of Hpse-mediated binding, namely, uptake of Hpse by mast cells and Hpse-induced release of chemokine CCL2 from colon carcinoma cells, were also blocked by the CS-E. CS-E may regulate endogenous Hpse-mediated cellular functions by inhibiting enzymatic activity and binding to the cell surface.

Stereoisomer-Specific Induction of G2/M Phase Arrest and Apoptosis by 9-(E,Z)-Hydroxyoctadecadienoic Acid in Mouse Lymphoma Cells.

Hydroxyoctadecadienoic acids (HODEs) are generated by oxidation of linoleic acid in vivo and thought to mediate various pathophysiological responses. In this study, we examined the effects of HODEs on EL4 mouse lymphoma cell growth and found that 9-(E,Z)-HODE inhibited EL4 cell growth in a dose-dependent manner, whereas no such growth inhibition was observed with other isomers (9-(E,E)-, 13-(Z,E)-, or 13-(E,E)-HODE), suggesting that the growth-inhibitory effect of HODEs was stereospecific. Analysis by flow cytometry (FACS) with annexin V and propidium iodide (PI) staining showed that 9-(E,Z)-HODE induced apoptosis with G2/M phase arrest. We next examined the growth inhibition profile of 9-(E,Z)-HODE against a panel of 39 human cancer cell lines (JFCR39). The fingerprint of growth inhibition by 9-(E,Z)-HODE exhibited a high degree of similarity to that by MLN4924, an inhibitor of NEDD8-activating enzyme. The intracellular NEDD8 (ubiquitin-like protein) expression in EL4 cells was decreased by the treatment with 9-(E,Z)-HODE as assessed by immunoblotting and flow cytometry. In conclusion, 9-(E,Z)-HODE specifically induced G2/M phase arrest and apoptosis, and the decrease of NEDD8 expression might be involved in this effect.

Selective Cytotoxicity of Staphylococcal α-Hemolysin (α-Toxin) against Human Leukocyte Populations.

Staphylococcus aureus produces a variety of exoproteins that interfere with host immune systems. We attempted to purify cytotoxins against human leukocytic cells from the culture supernatant of S. aureus by a combination of ammonium sulfate precipitation, ion-exchange chromatography on a CM-cellulose column and HPLC on a Mono S 5/50 column. A major protein possessing cytotoxicity to HL60 human promyelocytic leukemia cells was purified, and the protein was identified as α-hemolysin (Hla, α-toxin) based on its molecular weight (34 kDa) and N-terminal amino acid sequence. Flow cytometric analysis suggested differential cytotoxicity of Hla against different human peripheral blood leukocyte populations. After cell fractionation with density-gradient centrifugation, we found that peripheral blood mononuclear cells (PBMCs) were more susceptible to Hla than polymorphonuclear leukocytes. Moreover, cell surface marker analysis suggested that Hla exhibited slightly higher cytotoxicity against CD14-positive PBMCs (mainly monocytes) than CD3- or CD19-positive cells (T or B lymphocytes). From these results, we conclude that human leukocytes have different susceptibility to Hla depending on their cell lineages, and thereby the toxin may modulate the host immune response.

Identification of human plasma C1 inhibitor as a target protein for staphylococcal superantigen-like protein 5 (SSL5).

The family of staphylococcal superantigen-like proteins (SSLs) have a structure similar to bacterial superantigens but exhibit no superantigenic activity. These exoproteins have recently been shown to disturb the host immune defense system. One family member, SSL5, was reported to bind to human leukocyte P-selectin glycoprotein ligand-1 (PSGL-1) and matrix metalloproteinase-9 (MMP-9) and to interfere with leukocyte trafficking. In the present study, we explored human plasma proteins bound by glutathione S-transferase (GST)-tagged recombinant SSL5 (GST-SSL5) and identified plasma protease C1 inhibitor (C1Inh) as a major SSL5-binding protein based on the results of peptide mass fingerprinting analysis with MALDI-TOFMS. GST-SSL5 was found to attenuate the inhibitory activity of recombinant histidine-tagged C1Inh (C1Inh-His) toward complement C1s. We also observed that the treatment of C1Inh-His with neuraminidase markedly decreased its binding to GST-SSL5. Moreover, C1Inh-His produced by Lec2 mutant cells (deficient in sialic acid biosynthesis) showed much lower binding affinity for SSL5 than that produced by the wild-type CHO-K1 cells, as assessed by pull-down assay. These results suggest that SSL5 binds to C1Inh in a sialic acid-dependent fashion and modulates the host immune defense through perturbation of the complement system in association with S. aureus infection.

Stimulation of Peritoneal Mesothelial Cells to Secrete Matrix Metalloproteinase-9 (MMP-9) by TNF-α: A Role in the Invasion of Gastric Carcinoma Cells.

It has recently been recognized that inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), upregulate the secretion of matrix metalloproteinase-9 (MMP-9) from cancer cells and thereby promote peritoneal dissemination. In this study, we found that TNF-α also stimulated peritoneal mesothelial cells to secrete MMP-9 as assessed by zymography. MMP-9 gene expression in mesothelial cells induced by TNF-α was confirmed by quantitative RT-PCR analysis. We then utilized the reconstituted artificial mesothelium, which was composed of a monolayer of mesothelial cells cultured on a Matrigel layer in a Boyden chamber system, to examine the effects of TNF-α on carcinoma cell invasion. The transmigration of MKN1 human gastric carcinoma cells through the reconstituted mesothelium was promoted by TNF-α in a dose-dependent manner. The increased MKN1 cell migration was partially inhibited by the anti-α3 integrin antibody, indicating that the invasion process involves an integrin-dependent mechanism. Finally, we observed that the invasion of MMP-9-knockdown MKN1 cells into Matrigel membranes was potentiated by the exogenous addition of purified proMMP-9. These results suggest that TNF-α-induced MMP-9 secretion from mesothelial cells plays an important role in the metastatic dissemination of gastric cancer.

Generation of a Monoclonal Antibody Against Staphylococcal Superantigen-Like Protein 5 (SSL5) That Discriminates SSL5 from Other SSL Proteins.

Staphylococcus aureus secretes a family of exoproteins structurally homologous to bacterial superantigens, such as toxic shock syndrome toxin-1 (TSST-1), and those exoproteins are thus called staphylococcal superantigen-like proteins (SSLs). Recent studies have revealed that SSLs play roles in evasion of the host defense by disturbing host immune responses. We previously reported that staphylococcal superantigen-like protein 5 (SSL5; a member of the SSL family) inhibited matrix metalloproteinase-9 (MMP-9), which is crucial for leukocyte recruitment to sites of infection. In this study, we established a mouse hybridoma clone (30G5C) producing a monoclonal antibody specific for SSL5. In immunoblotting analysis using recombinant His-tagged SSL1 to SSL14 (His-SSLs), the antibody was found to specifically recognize SSL5 without crossreactivity with other His-SSLs. The antibody bound to the C-terminal region of SSL5 (ß-grasp domain), but did not interfere with the binding of SSL5 to MMP-9, suggesting that this antibody is useful for identification of SSL5-producing S. aureus and screening for inhibitors of the SSL5/MMP-9 complex formation.

Incorporation, intracellular trafficking and processing of extracellular heparanase by mast cells: Involvement of syndecan-4-dependent pathway.

We investigated the fate of proheparanase added to the culture media of mast cells. A recombinant protein mimicking proheparanase was continuously internalized into mastocytoma cells as well as bone marrow- and peritoneal cell-derived mast cells. Internalized heparanase molecules were accumulated in granules and a significant portion was released by stimulation with ionomycin, indicating that the internalized heparanase was sorted into secretory granules. The pro-form heparanase was processed into a mature and an active form inside the cells, in which intracellular heparin was fragmented by the mature enzyme. The internalization was substantially inhibited by addition of heparin and heparan sulfate to the culture medium, suggesting that glycosaminoglycan is involved in the uptake pathway. Out of four syndecans, expression of syndecan-3 and syndecan-4, especially cell surface syndecan-4, was detected in the mastocytoma cells. Two knockdown clones transfected with a shRNA expression vector targeting the syndecan-4 gene took up significantly lower amounts of heparanase than mock cells. We propose that some exogenous substances like proheparanase can be incorporated into mast cell granules via a glycosaminoglycan-mediated, especially syndecan-4-dependent, uptake pathway.

Role of sialic acid-containing glycans of matrix metalloproteinase-9 (MMP-9) in the interaction between MMP-9 and staphylococcal superantigen-like protein 5.

Staphylococcal superantigen-like proteins (SSL) show no superantigenic activity but have recently been considered to act as immune suppressors. It was previously reported that SSL5 bound to P-selectin glycoprotein ligand-1 (PSGL-1) and matrix metalloproteinase (MMP)-9, leading to inhibition of leukocyte adhesion and invasion. These interactions were suggested to depend on sialic acid-containing glycans of MMP-9, but the roles of sialic acids in the interaction between SSL5 and MMP-9 are still controversial. In the present study, we prepared recombinant glutathione S-transferase-tagged SSL5 (GST-SSL5) and analyzed its binding capacity to MMP-9 by pull-down assay after various modifications of its carbohydrate moieties. We observed that GST-SSL5 specifically bound to MMP-9 from a human monocytic leukemia cell line (THP-1 cells) and inhibited its enzymatic activity in a concentration-dependent manner. After MMP-9 was treated with neuraminidase, its binding activity towards GST-SSL5 was markedly decreased. Furthermore, recombinant MMP-9 produced by sialic acid-deficient Lec2 mutant cells showed much lower affinity for SSL5 than that produced by wild-type CHO-K1 cells. Treatment of MMP-9 with PNGase F to remove N-glycan resulted in no significant change in the GST-SSL5/MMP-9 interaction. In contrast, the binding of GST-SSL5 to MMP-9 secreted from THP-1 cells cultured in the presence of an inhibitor for the biosynthesis of O-glycan (benzyl-GalNAc) was weaker than the binding of GST-SSL5 to MMP-9 secreted from untreated cells. These results strongly suggest the importance of the sialic acid-containing O-glycans of MMP-9 for the interaction of MMP-9 with GST-SSL5.

Platelet Supernatant Suppresses LPS-Induced Nitric Oxide Production from Macrophages Accompanied by Inhibition of NF-κB Signaling and Increased Arginase-1 Expression.

We previously reported that mouse bone marrow-derived macrophages (BMDMs) that had been co-cultured with platelets exhibited lower susceptibility to bacterial lipopolysaccharide (LPS) and produced lower levels of nitric oxide (NO) and inflammatory cytokines including TNF-α and IL-6. The suppression of macrophage responses was mediated, at least in part, by platelet supernatant. In the present study, we assessed phenotypic changes of BMDMs induced by incubation with the supernatant from thrombin-activated platelets (PLT-sup) and found that BMDMs cultured with PLT-sup (PLT-BMDMs) expressed a lower level of inducible NO synthase (iNOS) and a higher level of arginase-1, both of which are involved in the L-arginine metabolism, upon stimulation with LPS or zymosan. We also examined possible modulation of the NF-κB signaling pathway and observed suppression of IκBα phosphorylation and a decrease of NF-κB p65 expression in LPS-stimulated PLT-BMDMs. These results suggest that PLT-sup suppresses inflammatory responses of BMDMs via negative regulation of NF-κB signaling leading to lowered expression of iNOS and enhanced L-arginine catabolism by arginase-1.

Development of Splice Variant-Specific Monoclonal Antibodies Against Human α3 Integrin.

Integrins are a major family of adhesion molecules, consisting of heterodimers (α and ß subunits). Several reports have suggested the presence of splice variants in the cytoplasmic domain of certain integrin subunits. In the present study, we detected mRNA of integrin α3 splice variants (α3A and α3B) by RT-PCR using total RNA from the human brain as a template. The α3B variant lacks the sequence coded by exon 25 and appears to be generated by alternative splicing. We established mouse hybridomas producing monoclonal antibodies (both of which are of IgG1 class) specific for each variant. Each antibody exhibited specific reactivity towards the corresponding integrin α3 variant in Western blotting and immunoprecipitation experiments, suggesting it to be a useful tool for detection of the respective integrin variant.

Platelets attenuate production of cytokines and nitric oxide by macrophages in response to bacterial endotoxin.

Considerable evidence has been accumulated concerning the roles of platelets in immune responses. In the present study, we examined the functional modulation of macrophages by platelets. When mouse bone marrow-derived macrophages (BMDMs) were co-cultured with platelets, BMDMs produced lower levels of nitric oxide (NO), tumor necrosis factor-α (TNF)-α, and interleukin (IL)-6 in response to a bacterial endotoxin (LPS) and zymosan. The attenuation in the macrophage susceptibility to LPS appeared to be mediated by soluble factors secreted from platelets. The mRNA levels of NOS2 (iNOS), TNF-α, and IL-6 in LPS-stimulated BMDMs that had been cultured with a conditioned medium of platelets were also decreased as analyzed by RT-qPCR. The ability of the platelet-conditioned medium to suppress macrophage NO production was recovered in a high-molecular-weight fraction (>670 kDa) after gel-filtration chromatography on a Superose 6 column. These results suggest that platelets control the susceptibility of macrophages to prevent excessive responses to LPS and provide mechanistic insight into a previous report that experimental thrombocytopenia aggravated organ failure in LPS-induced endotoxemia.

Constitutive turnover of phosphorylation at Thr-412 of human p57/coronin-1 regulates the interaction with actin.

The actin-binding protein p57/coronin-1, a member of the coronin protein family, is selectively expressed in hematopoietic cells and plays crucial roles in the immune response through reorganization of the actin cytoskeleton. We previously reported that p57/coronin-1 is phosphorylated by protein kinase C, and the phosphorylation down-regulates the association of this protein with actin. In this study we analyzed the phosphorylation sites of p57/coronin-1 derived from HL60 human leukemic cells by MALDI-TOF-MS, two-dimensional gel electrophoresis, and Phos-tag® acrylamide gel electrophoresis in combination with site-directed mutagenesis and identified Ser-2 and Thr-412 as major phosphorylation sites. A major part of p57/coronin-1 was found as an unphosphorylated form in HL60 cells, but phosphorylation at Thr-412 of p57/coronin-1 was detected after the cells were treated with calyculin A, a Ser/Thr phosphatase inhibitor, suggesting that p57/coronin-1 undergoes constitutive turnover of phosphorylation/dephosphorylation at Thr-412. A diphosphorylated form of p57/coronin-1 was detected after the cells were treated with phorbol 12-myristate 13-acetate plus calyculin A. We then assessed the effects of phosphorylation at Thr-412 on the association of p57/coronin-1 with actin. A co-immunoprecipitation experiment with anti-p57/coronin-1 antibodies and HL60 cell lysates revealed that ß-actin was co-precipitated with the unphosphorylated form but not with the phosphorylated form at Thr-412 of p57/coronin-1. Furthermore, the phosphorylation mimic (T412D) of p57/coronin-1 expressed in HEK293T cells exhibited lower affinity for actin than the wild-type or the unphosphorylation mimic (T412A) did. These results indicate that the constitutive turnover of phosphorylation at Thr-412 of p57/coronin-1 regulates its interaction with actin.

Involvement of transcription factor Ets-1 in the expression of the α3 integrin subunit gene.

The α3ß1 integrin is an adhesion receptor for extracellular matrix proteins, and plays crucial roles in cell motility, proliferation, and differentiation. The aberrant expression of this adhesion molecule on tumor cells is frequently associated with their malignant behaviors. We previously reported that the Ets transcription factor-binding consensus sequence 133 bp upstream of the mouse α3 integrin gene is an important element for its expression in various tumor cell lines. In the present study, we attempted to identify a transcription factor bound to the Ets-consensus sequence, and found that Ets-1 bound to this sequence in an electrophoretic mobility shift assay, chromatin immunoprecipitation assay, and pull-down assay with a tandem repeat of the sequence as adsorbent. We next examined the role of Ets-1 in α3 integrin gene expression by use of a luciferase assay with a reporter plasmid containing the 5'-flanking region of the α3 integrin gene. Cotransfection of HEK293T cells with an Ets-1 expression construct and the reporter plasmid increased luciferase activity. By contrast, transfection of HT1080 cells (high α3 integrin expresser) with a dominant-negative mutant of Ets-1 decreased luciferase activity. Overexpression of Ets-1 in HepG2 hepatocellular carcinoma cells (low α3 integrin expresser) upregulated α3 integrin expression as assessed by immunoprecipitation. Finally, the induction of α3 integrin gene expression in HepG2 cells after transforming growth factor-ß1 treatment was abrogated by the dominant-negative mutant of Ets-1. These results suggest that Ets-1 is involved in transcriptional activation of the α3 integrin gene through its binding to the Ets-consensus sequence at -133 bp.

Monocyte differentiation induced by co-culture with tumor cells involves RGD-dependent cell adhesion to extracellular matrix.

Macrophages that infiltrate tumor tissues, or tumor-associated macrophages (TAMs), affect the malignant behaviors of tumor cells. In this study, we attempted to induce monocytes to differentiate into TAM-like cells producing matrix metalloproteinases (MMPs) by co-culture with tumor cells. When human monocytes were co-cultured for 3-7 days with tumor cell lines, monocytes differentiated to produce MMP-9, accompanied by morphological changes. The in vitro cell invasion of MKN1 human gastric carcinoma cells into Matrigel membranes was promoted in the presence of differentiated monocytes, and the enhancement of cell invasion by differentiated monocytes was correlated with their MMP-9 productivity. The addition of an RGD (Arg-Gly-Asp) peptide to the culture significantly inhibited monocyte differentiation. The MMP-9 production from monocytes was diminished by the depletion of fibronectin from the conditioned media with gelatin-Sepharose, and potentiated by culturing them in fibronectin-coated plates. These results suggest that cell adhesion to the extracellular matrix plays a crucial role in monocyte differentiation into TAM-like cells.

Staphylococcal superantigen-like protein 5 inhibits matrix metalloproteinase 9 from human neutrophils.

Staphylococcal superantigen-like proteins (SSLs) constitute a family of exoproteins exhibiting structural similarities to superantigens and enterotoxins but no superantigenic activity. In this article, we present evidence that SSL5 specifically binds to matrix metalloproteinase 9 (MMP-9) and inhibits its enzymatic activity. When human neutrophil cell lysate was applied to recombinant His-tagged SSL5 conjugated to Sepharose, the bound fraction gave a major band of approximately 100 kDa in SDS-polyacrylamide gel electrophoresis. This protein was identified as the proform of MMP-9 (proMMP-9) by peptide mass fingerprinting analysis. The recombinant SSL5-Sepharose also bound to proMMP-9 secreted by interleukin 8 (IL-8)-stimulated neutrophils and HT1080 fibrosarcoma cells. Surface plasmon resonance analysis revealed that recombinant SSL5 bound to proMMP-9 with rather high affinity (dissociation constant [K(D)] = 1.9 nM). Recombinant SSL5 was found to effectively inhibit MMP-9-catalyzed hydrolysis of gelatin and a synthetic fluorogenic peptide in a noncompetitive manner (K(i) = 0.097 nM), as assessed by zymography and the fluorescence quenching method. Finally, the transmigration of neutrophils across Matrigel basement membranes in response to N-formyl-methionyl-leucyl-phenylalanine (FMLP) was suppressed by the presence of recombinant SSL5. We discuss possible roles that SSL5 may play in immune evasion of staphylococci by inhibiting MMP and interfering with leukocyte trafficking.