Suppression of heregulin ß signaling by the single N-glycan deletion mutant of soluble ErbB3 protein.

Heregulin signaling is involved in various tumor proliferations and invasions; thus, receptors of heregulin are targets for the cancer therapy. In this study we examined the suppressing effects of extracellular domains of ErbB2, ErbB3, and ErbB4 (soluble ErbB (sErbB)) on heregulin ß signaling in human breast cancer cell line MCF7. It was found that sErbB3 suppresses ligand-induced activation of ErbB receptors, PI3K/Akt and Ras/Erk pathways most effectively; sErbB2 scarcely suppresses ligand-induced signaling, and sErbB4 suppresses receptor activation at ∼10% efficiency of sErbB3. It was revealed that sErbB3 does not decrease the effective ligands but decreases the effective receptors. By using small interfering RNA (siRNA) for ErbB receptors, we determined that sErbB3 suppresses the heregulin ß signaling by interfering ErbB3-containing heterodimers including ErbB2/ErbB3. By introducing the mutation of N418Q to sErbB3, the signaling-inhibitory effects were increased by 2-3-fold. Moreover, the sErbB3 N418Q mutant enhanced anticancer effects of lapatinib more effectively than the wild type. We also determined the structures of N-glycan on Asn-418. Results suggested that the N-glycan-deleted mutant of sErbB3 suppresses heregulin signaling via ErbB3-containing heterodimers more effectively than the wild type. Thus, we demonstrated that the sErbB3 N418Q mutant is a potent inhibitor for heregulin ß signaling.

Implication of antigenic conversion of Helicobacter pylori lipopolysaccharides that involve interaction with surfactant protein D.

We propose two antigenic types of Helicobacter pylori lipopolysaccharides (LPS): highly antigenic epitope-carrying LPS (HA-LPS) and weakly antigenic epitope-carrying LPS (WA-LPS) based on human serum reactivity. Strains carrying WA-LPS are highly prevalent in isolates from gastric cancer patients. WA-LPS exhibits more potent biological activities compared to HA-LPS, namely, upregulation of Toll-like receptor 4 (TLR4) expression and induction of enhanced epithelial cell proliferation. The results of competitive binding assays using monosaccharides and methylglycosides, as well as binding assays using glycosidase-treated LPS, suggested that ß-linked N-acetyl-D-glucosamine and ß-linked D-galactose residues largely contributed to the highly antigenic epitope and the weakly antigenic epitope, respectively. WA-LPS exhibited greater binding activity to surfactant protein D (SP-D) in a Ca(2+)-dependent manner, and this interaction was inhibited by methyl-ß-D-galactoside. The biological activities of WA-LPS were markedly enhanced by the addition of SP-D. Lines of evidence suggested that removal of ß-N-acetyl-D-glucosamine residue, which comprises the highly antigenic epitope, results in exposure of the weakly antigenic epitope. The weakly antigenic epitope interacted preferentially with SP-D, and SP-D enhanced the biological activity of WA-LPS.

Pulmonary surfactant protein A protects lung epithelium from cytotoxicity of human ß-defensin 3.

Defensins are important molecules in the innate immune system that eliminate infectious microbes. They also exhibit cytotoxicity against host cells in higher concentrations. The mechanisms by which hosts protect their own cells from cytotoxicity of defensins have been poorly understood. We found that the cytotoxicity of human ß-defensin 3 (hBD3) against lung epithelial cells was dose-dependently attenuated by pulmonary surfactant protein A (SP-A), a collectin implicated in host defense and regulation of inflammatory responses in the lung. The direct interaction between SP-A and hBD3 may be an important factor in decreasing this cytotoxicity because preincubation of epithelial cells with SP-A did not affect the cytotoxicity. Consistent with in vitro analysis, intratracheal administration of hBD3 to SP-A(-/-) mice resulted in more severe tissue damage compared with that in WT mice. These data indicate that SP-A protects lung epithelium from tissue injury caused by hBD3. Furthermore, we found that the functional region of SP-A lies within Tyr(161)-Lys(201). Synthetic peptide corresponding to this region, tentatively called SP-A Y161-G200, also inhibited cytotoxicity of hBD3 in a dose-dependent manner. The SP-A Y161-G200 is a candidate as a therapeutic reagent that prevents tissue injury during inflammation.

HCV core and NS3 proteins manipulate human blood-derived dendritic cell development and promote Th 17 differentiation.

Hepatitis C virus (HCV) chronic infection is characterized by low-level or undetectable cellular immune response against HCV antigens. HCV proteins affect various intracellular events and modulate immune responses, although the mechanisms that mediate these effects are not fully understood. In this study, we examined the effect of HCV proteins on the differentiation of human peripheral blood monocytes to dendritic cells (DCs). The HCV core (HCVc) and non-structural 3 (NS3) proteins inhibited the expression of CD1a, CD1b and DC-SIGN during monocyte differentiation to DCs, while increasing some markers characteristic of macrophages (CD14 and HLA-DR) and also PD-L1 expression. Meanwhile, HCVc and NS3 could induce differentiating monocytes to secrete IL-10. However, anti-IL-10 mAb could not reverse HCVc and NS3 inhibition of monocyte differentiation into DCs. The HCVc and NS3 proteins increased IL-6 secretion both in immature and in fully differentiated DCs and also promoted CD4+ T-cell IL-17 production. Since T(h) 17 cells are active in many examples of immunopathology, these effects may contribute to HCV autoimmune responses in chronically infected patients.

Pulmonary collectins play distinct roles in host defense against Mycobacterium avium.

Pulmonary collectins, surfactant protein A (SP-A) and surfactant protein D (SP-D), play important roles in the innate immunity of the lung. Mycobacterium avium is one of the well-known opportunistic pathogens that can replicate within macrophages. We examined the effects of pulmonary collectins in host defense against M. avium infection achieved via direct interaction between bacteria and collectins. Although both pulmonary collectins bound to M. avium in a Ca(2+)-dependent manner, these collectins revealed distinct ligand-binding specificity and biological activities. SP-A and SP-D bound to a methoxy group containing lipid and lipoarabinomannan, respectively. Binding of SP-D but not SP-A resulted in agglutination of M. avium. A chimeric protein with the carbohydrate recognition domain of SP-D, which chimera revealed a bouquet-like arrangement similar to SP-A, also agglutinated M. avium. The ligand specificity of the carbohydrate recognition domain of SP-D seems to be necessary for agglutination activity. The binding of SP-A strongly inhibited the growth of M. avium in culture media. Although pulmonary collectins did not increase membrane permeability of M. avium, they attenuated the metabolic rate of the bacteria. Observations under a scanning electron microscope revealed that SP-A almost completely covers bacterial surfaces, whereas SP-D binds to certain areas like scattered dots. These observations suggest that a distinct binding pattern of collectins correlates with the difference of their biological activities. Furthermore, the number of bacteria phagocytosed by macrophages was significantly increased in the presence of SP-D. These data indicate that pulmonary collectins play critical roles in host defense against M. avium.

HIV-1 Vpr induces TLR4/MyD88-mediated IL-6 production and reactivates viral production from latency.

Vpr, a HIV-1 accessory protein, was believed to be present in the plasma of HIV-1-positive patients, and our previous work demonstrated the presence of plasma Vpr in 20 out of 52 patients. Interestingly, our data revealed that patients' viral titer was correlated with the level of Vpr detected in their plasma. Here, we first show that rVpr, when incubated with human monocytes or MDMs, caused viral production from latently infected cells, and IL-6 was identified as a responsible factor. The induction of IL-6 by rVpr was dependent on signaling through TLR4 and its adaptor molecule, MyD88. We next provide evidence that rVpr induced the formation of OxPC and that a mAb against OxPC blocked rVpr-induced IL-6 production with the concomitant attenuation of MAPK activation. Moreover, the addition of NAC, a scavenger of ROS, abrogated the rVpr-induced formation of OxPC, the phosphorylation of C/EBP-beta, a substrate of MAPK, and IL-6 production. As rIL-6 reactivated viral replication in latently infected cells, our data indicate that rVpr-induced oxidative stress triggers cell-based innate immune responses and reactivates viral production in latently infected cells via IL-6 production. Our results suggest that Vpr should be monitored based on the viral titer, and they provide the rationale for the development of novel, anti-AIDS therapeutics targeting Vpr.

Anionic pulmonary surfactant phospholipids inhibit inflammatory responses from alveolar macrophages and U937 cells by binding the lipopolysaccharide-interacting proteins CD14 and MD-2.

Lipopolysaccharide (LPS), derived from Gram-negative bacteria, is a major cause of acute lung injury and respiratory distress syndrome. Pulmonary surfactant is secreted as a complex mixture of lipids and proteins onto the alveolar surface of the lung. Surfactant phospholipids are essential in reducing surface tension at the air-liquid interface and preventing alveolar collapse at the end of the respiratory cycle. In the present study, we determined that palmitoyl-oleoyl-phosphatidylglycerol and phosphatidylinositol, which are minor components of pulmonary surfactant, and synthetic dimyristoylphosphatidylglycerol regulated the inflammatory response of alveolar macrophages. The anionic lipids significantly inhibited LPS-induced nitric oxide and tumor necrosis factor-alpha production from rat and human alveolar macrophages and a U937 cell line by reducing the LPS-elicited phosphorylation of multiple intracellular protein kinases. The anionic lipids were also effective at attenuating inflammation when administered intratracheally to mice challenged with LPS. Binding studies revealed high affinity interactions between the palmitoyl-oleoyl-phosphatidylglycerol and the Toll-like receptor 4-interacting proteins CD14 and MD-2. Our data clearly identify important anti-inflammatory properties of the minor surfactant phospholipids at the environmental interface of the lung.

Mutational analysis of Cys(88) of Toll-like receptor 4 highlights the critical role of MD-2 in cell surface receptor expression.

The role of MD-2 in cell surface expression of Toll-like receptor (TLR) 4 has been controversial. The purposes of this study were to characterize the N-glycan of TLR4 and to investigate the roles of MD-2 in N-linked glycosylation and cell surface expression of TLR4. Lectin blot and cell surface biotinylation revealed that TLR4 exhibited the 110 kDa protein with high mannose type N-glycans and the 130 kDa protein with complex type N-glycans and that only the 130 kDa TLR4 with complex type N-glycans was expressed on the cell surface. The cells transfected with a mutant TLR4(C88A) alone expressed only the 110 kDa TLR4 with a high mannose type N-glycan, which did not appear on the cell surface. However, TLR4(C88A) acquired complex type N-glycans and was expressed on the cell surface when MD-2 was co-transfected. The amount of the 130 kDa TLR4(C88A) with complex type N-glycans expressed on the cell surface depended on that of MD-2 transfected. alpha-Mannosidase II inhibitor blocked the processing N-glycans to complex type, but TLR4 with high mannose type appeared on the cell surface, suggesting that TLR4 is destined to locate on the cell surface before processing N-glycans from a high mannose type to a complex type. From these results, we conclude that MD-2 is critical for cell surface expression of TLR4(C88A). This study provides evidence that MD-2 possesses potential ability to play an essential role in cell surface expression of TLR4.

Pulmonary surfactant protein D inhibits lipopolysaccharide (LPS)-induced inflammatory cell responses by altering LPS binding to its receptors.

Pulmonary surfactant protein D (SP-D) is a member of the collectin family that plays an important role in regulating innate immunity of the lung. We examined the mechanisms by which SP-D modulates lipopolysaccharide (LPS)-elicited inflammatory cell responses. SP-D bound to a complex of recombinant soluble forms of Toll-like receptor 4 (TLR4) and MD-2 with high affinity and down-regulated tumor necrosis factor-alpha secretion and NF-kappaB activation elicited by rough and smooth LPS, in alveolar macrophages and TLR4/MD-2-transfected HEK293 cells. Cell surface binding of both serotypes of LPS to TLR4/MD-2-expressing cells was attenuated by SP-D. In addition, SP-D significantly reduced MD-2 binding to both serotypes of LPS. A chimera containing the N-terminal region and the collagenous domain of surfactant protein A, and the coiled-coil neck and lectin domains of SP-D, was a weak inhibitor of LPS-induced cell responses and MD-2 binding to LPS, compared with native SP-D. The collagenase-resistant fragment consisting of the neck plus the carbohydrate recognition domain of SP-D also was a very weak inhibitor of LPS activation. This study demonstrates that SP-D down-regulates LPS-elicited inflammatory responses by altering LPS binding to its receptors and reveals the importance of the correct oligomeric structure of the protein in this process.

Pulmonary collectins in innate immunity of the lung.

Pulmonary collectins, hydrophilic surfactant proteins A and D (SP-A and SP-D), have been implicated in the regulation of pulmonary host defence and inflammation. SP-A and SP-D directly interact with a variety of microorganisms including bacteria and viruses, and attenuate the growth of Gram-negative bacteria, Histoplasma capsulatum and Mycoplasma pneumoniae. The collectins are thought to contribute to bacterial clearance. These lectins augment the phagocytosis of the bacteria by macrophages. SP-A serves as an opsonin and stimulates the uptake of bacteria and bacillus Calmette-Guérin through a C1q receptor- and an SP-R210-mediated processes. The collectin also stimulates FcR- and CR1-mediated phagocytosis by activating the macrophages. In addition, SP-A and SP-D directly interact with macrophages and enhance the phagocytosis of Streptococcus pneumoniae and Mycobacterium by increasing cell surface localization of the phagocytic receptors, scavenger receptor A and mannose receptor. The collectins also modulate pulmonary inflammation. SP-A and SP-D bind to cell surface receptors including Toll-like receptors, SIRPalpha and calreticulin/CD91, and attenuate or enhance inflammation in a microbial ligand-specific manner. In this article we review the immunomodulatory functions of SP-A and SP-D and their possible mechanisms in direct actions on microbes, macrophage phagocytosis and modulation of inflammation.

The 6-fluoro-8-methoxy quinolone gatifloxacin down-regulates interleukin-8 production in prostate cell line PC-3.

Fluoroquinolones exhibit immunomodulatory effects on monocytes and macrophages, in addition to their bactericidal activities. It remains unknown even whether the quinolones act directly on the prostate. This study was based on the understanding of the molecular mechanisms of the actions of the fluoroquinolones that can be used for the treatment of chronic prostatitis/chronic pelvic pain syndrome. We investigated whether the 6-fluroro-8-methoxy quinolone gatifloxacin (GFLX) affected the production and secretion of interleukin-8 (IL-8) in the prostate cell line PC-3. GFLX decreased the level of IL-8 release from unstimulated PC-3 cells. GFLX also attenuated IL-8 secretion from PC-3 cells stimulated with peptidoglycan, Mycoplasma hominis, phorbol ester, and tumor necrosis factor alpha (TNF-alpha), indicating that GFLX exhibits an anti-inflammatory effect on the prostate cell line. However, GFLX failed to alter activation of the NF-kappaB and AP-1 elicited by these stimulants. GFLX significantly attenuated the expression of IL-8 mRNA in TNF-alpha-stimulated PC-3 cells and down-regulated the transcriptional activity of the 5'-flanking region of the IL-8 gene from -1481 to +44 bp. The deletion construct without the 5'-flanking region from -1481 to -170 bp but not the construct without the region from -1481 to -188 bp reversed the suppressive effect of GFLX on IL-8 promoter activity. These results demonstrate that GFLX suppresses IL-8 expression in the prostate cell line by decreasing the promoter activity of the IL-8 gene.

Toll-like receptor 4 region Glu24-Lys47 is a site for MD-2 binding: importance of CYS29 and CYS40.

Toll-like receptor 4 (TLR4) is a signaling receptor for lipopolysaccharide (LPS), but its interaction with MD-2 is required for efficient responses to LPS. Previous studies with deletion mutants indicate a critical role of the amino-terminal TLR4 region in interaction with MD-2. However, it is uncertain which region in the TLR4 molecule directly binds to MD-2. The purpose of this study was to determine a critical stretch of primary sequence in the TLR4 region that directly binds MD-2 and is critical for LPS signaling. The synthetic TLR4 peptide corresponding to the TLR4 region Glu(24)-Lys(47) directly binds to recombinant soluble MD-2 (sMD-2). The TLR4 peptide inhibited the binding of a recombinant soluble form of the extracellular TLR4 domain (sTLR4) to sMD-2 and significantly attenuated LPS-induced NF-kappaB activation and IL-8 secretion in wild type TLR4-transfected cells. Reduction and S-carboxymethylation of sTLR4 abrogated its association with sMD-2. The TLR4 mutants, TLR4(C29A), TLR4(C40A), and TLR4(C29A,C40A), were neither co-precipitated with MD-2 nor expressed on the cell surface and failed to transmit LPS signaling. These results demonstrate that the TLR4 region Glu(24)-Lys(47) is a site for MD-2 binding and that Cys(29) and Cys(40) within this region are critical residues for MD-2 binding and LPS signaling.

Surfactant protein A directly interacts with TLR4 and MD-2 and regulates inflammatory cellular response. Importance of supratrimeric oligomerization.

The purpose of the current study was to examine the binding of pulmonary surfactant protein A (SP-A) to TLR4 and MD-2, which are critical signaling receptors for lipopolysaccharides (LPSs). The direct binding of SP-A to the recombinant soluble form of extracellular TLR4 domain (sTLR4) and MD-2 was detected using solid-phase binding, immunoprecipitation, and BIAcore. SP-A bound to sTLR4 and MD-2 in a Ca2+-dependent manner, and an anti-SP-A monoclonal antibody whose epitope lies in the region Thr184-Gly194 blocked the SP-A binding to sTLR4 and MD-2, indicating the involvement of the carbohydrate recognition domain (CRD) in the binding. SP-A avidly bound to the deglycosylated forms of sTLR4 and MD-2, suggesting a protein/protein interaction. In addition, SP-A attenuated cell surface binding of smooth LPS and smooth LPS-induced NF-kappaB activation in TLR4/MD-2-expressing cells. To know the role of oligomerization in the interaction of SP-A with TLR4 and MD-2, the collagenase-resistant fragment (CRF), which consisted of CRD plus neck domain of SP-A, was isolated. CRF assembled as a trimer, whereas SP-A assembled as a higher order oligomer. Although CRD was suggested to be involved in the binding, CRF exhibited approximately 600- and 155-fold higher KD for the binding to TLR4 and MD-2, respectively, when compared with SP-A. Consistently significantly higher molar concentrations of CRF were required to inhibit smooth LPS-induced NF-kappaB activation and tumor necrosis factor-alpha secretion. These results demonstrate for the first time the direct interaction between SP-A and TLR4/MD-2 and suggest the importance of supratrimeric oligomerization in the immunomodulatory function of SP-A.

Prostate cell lines secrete IL-8 in response to Mycoplasma hominis through Toll-like receptor 2-mediated mechanism.

BACKGROUND: The roles of Toll-like receptor 2 (TLR2) and Mycoplasma in prostate inflammation remain unclear. We investigated whether Mycoplasma induced inflammatory cytokine secretion through TLR2-mediated mechanism in prostate cancer cell line. METHODS: Cell surface expression of TLR2 on PC-3 cells were examined by flowcytometry. PC-3 cells were stimulated with Mycoplasma hominis (M. hominis), and IL-8 secretion and NF-kappaB activation were examined. RESULTS: PC-3 cells expressed TLR2 mRNA and cell surface TLR2 protein. The membrane fraction of M. hominis induced IL-8 secretion from PC-3 cells and NF-kappaB activation in a concentration-dependent manner. Transient transfection of the dominant negative mutant TLR2(P681H) into PC-3 cells attenuated M. hominis-induced IL-8 secretion and NF-kappaB activation. Antibody against the extracellular TLR2 domain significantly suppressed M. homnis-induced IL-8 secretion from the prostate cell lines including PC-3, PrEC, and transformed myofibroblasts. CONCLUSIONS: These results clearly demonstrate that the prostate cell line can secrete inflammatory cytokine in response to M. hominis through a TLR2-mediated mechanism.

[Molecular biology of iron in nutritional science].

Iron is regarded as one of the most important nutriments, and many diseases are related to iron deficiency or its overload. Approximately 70% of iron in the body is located in heme, functioning as hemoglobin, myoglobin, and cytochrome P450. Iron itself also has many catalytic functions through the iron-sulfa cluster. It is believed that iron and/or heme plays significant roles in regulation of genes, however, little about the mechanism has been elucidated. Recently, not only iron but also heme has been identified as important regulators of gene activation via oxygen sensing. For example, iron controls the oxygen response of HIF-1 activity by two mechanisms; in cytosol, the half life of HIF-1 alpha is determined by hydroxylation of Pro, and transcriptional activity of HIF1 alpha in nuclei is disturbed by hydroxylation of Asn. Hemoproteins in prokaryotes such as FixL, Dos, and HemAT were found to be oxygen sensors, however, little has been reported in eukaryotes. Our finding on Bach1 seems to be the first report of heme and oxygen-mediated regulation of genes in vertebrates. Understanding of these newly identified mechanisms in iron- and heme-controlled genes is essential in the field of nutritional science. We therefore summarize here the recent findings indicating mechanisms of iron as transcriptional regulators.

Late-onset porphyrias: what are they?

Porphyrias are inherited disorders of heme biosynthesis. ALA dehydratase porphyria (ADP) and congenital erythropoietic porphyria (CEP) are autosomal recessive porphyrias, and are typically expressed at birth or in childhood. However, a few cases of late-onset recessive porphyrias have been reported. Recently we encountered a late-onset ADP patient who developed symptoms of acute porphyria when he was 63 years old. This was accompanied by polycythemia vera. It was concluded that he developed the porphyria because an abnormal ALAD allele was clonally expanded by polycythemia vera. Upon reviewing the literature, a few cases of late-onset CEP were found to be also associated with hematologic abnormalities suggestive of myelodysplastic syndrome (MDS), another clonal disorder. These findings suggest that these late-onset porphyrias may be heterozygous for their gene defects, but clinical expression may be elicited if there is a loss of heterozygosity, either by a clonal expansion of the porphyric allele or by a loss of function mutation in the other allele.

Regulatory heme and trichloroethylene intoxication: A possible explanation of the case of "A Civil Action".

In 1998, a amovie entitled "A Civil Action" was released. The movie described the Woburn case, begun in 1982 and concluded in 1990, one of the most famous cases of trichloroethylene pollution. In a small town near Boston, twelve children died of leukemia, which seemed attributable to trichloroethylene contamination of the drinking water. The victims, however, could not win the case, since evidence that the identified chemicals could cause leukemia and other human illnesses was rather sketchy. There have been many cases of trichloroethylene pollution in industrial nations including Japan, therefore, we reconsidered the missing link. Our conclusion is that the disease occurred not by a direct effect of the chemical hazard on biological macromolecules but by an indirect effect through the physiological system such as signal transduction and transcriptional regulation. In 1984, we reported a marked reduction in the regulatory heme pool by trichloroethylene exposure, however, the biological significance was not well understood. Recently, we found that the DNA binding activity of Bach1, a negative regulator of genes, is controlled by heme, the regulation of which seems to explain how leukemia develops. The heterodimer of Bach1 with MafK recognizes Maf recognition elements (MAREs) competing with the erythroid type positive regulator, a complex of NF-E2 with MafK. Bach1/MafK occupies MAREs under lower heme conditions, whereas MAREs are open to NF-E2/MafK along with increasing heme concentration. Since the NF-E2/MafK function is closely related to normal erythroid differentiation, of which disorders such as sideroblastic anemia are often related to neoplasia; i.e., a clonal disorder that can progress to leukemia. Thus, a marked decline in regulatory heme by trichloroethylene intoxication could be one of the pathways to leukemia.