TGFβ induces proHB-EGF shedding and EGFR transactivation through ADAM activation in gastric cancer cells

Background and aims: Transforming growth factor-beta (TGFβ) is known to potently inhibit cell growth. Loss of responsiveness to TGFβ inhibition on cell growth is a hallmark of many types of cancer, yet its mechanism is not fully understood. Membrane-anchored heparin-binding EGF-like growth factor (proHB-EGF) ectodomain is cleaved by a disintegrin and metalloproteinase (ADAM) members and is implicated in epidermal growth factor receptor (EGFR) transactivation. Recently, nuclear translocation of the C-terminal fragment (CTF) of pro-HB-EGF was found to induce cell growth. We investigated the association between TGFβ and HB-EGF signal transduction via ADAM activation.Materials and methods: The CCK-8 assay in two gastric cancer cell lines was used to determine the effect for cell growth by TGFβ. The effect of two ADAM inhibitors was also evaluated. Induction of EGFR phosphorylation by TGFβ was analyzed and the effect of the ADAM inhibitors was also examined. Nuclear translocation of HB-EGF-CTF by shedding through ADAM activated by TGFβ was also analyzed. EGFR transactivation, HB-EGF-CTF nuclear translocation, and cell growth were examined under the condition of ADAM17 knockdown.Result: TGFβ-induced EGFR phosphorylation of which ADAM inhibitors were able to inhibit. TGFβ induced shedding of proHB-EGF allowing HB-EGF-CTF to translocate to the nucleus. ADAM inhibitors blocked this nuclear translocation. TGFβ enhanced gastric cancer cell growth and ADAM inhibitors suppressed this effect. EGFR phosphorylation, HB-EGF-CTF nuclear translocation, and cell growth were suppressed in ADAM17 knockdown cells.Conclusion: HB-EGF-CTF nuclear translocation and EGFR transactivation from proHB-EGF shedding mediated by ADAM17 activated by TGFβ might be an important pathway of gastric cancer cell proliferation by TGFβ.     

Purification of dihydrofolate reductase amplified in Escherichia coli K-12

The Escherichia coli strain carrying pTP 6-10 which was constructed in our previous work (Iwakura, M., et al. (1983) J. Biochem. 93, 927-930) produces more than 400-fold dihydrofolate reductase as compared with the strain without the plasmid. Dihydrofolate reductase was highly purified from the cell-free extract of the plasmid strain simply by two steps; ammonium sulfate fractionation and ion-exchange chromatography. By 10-fold purification, the enzyme was essentially homogeneous as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The restriction map of pTP 6-10 was also determined and the plasmid was shown to have an Ava I, an EcoR I, a Pst I, a Pvu I, and a Pvu II site. Our results indicate that the plasmid strain is suitable as a source of the enzyme and that plasmid pTP 6-10 is promising as a versatile plasmid vector for efficiently yielding the product of the cloned gene.     

Pitavastatin prevents NMDA-induced retinal ganglion cell death by suppressing leukocyte recruitment

Excitotoxicity is a major cause of retinal ganglion cell (RGC) death during ischemic diseases such as vessel occlusion and diabetic retinopathy. However, the underlying mechanisms are not well understood. Statins, inhibitors of the HMG-CoA reductase, have neuroprotective effects in addition to their original role in lowering cholesterol. We hypothesize that pitavastatin, a recently introduced potent statin, is protective against N-methyl-d-aspartic acid (NMDA)-induced RGC death. Pitavastatin, administered by gavage, abolished NMDA-induced loss of RGCs. To elucidate the mechanisms underlying the neuroprotective effect of pitavastatin, we investigated its impact on inflammation. NMDA increased the expression of interleukin-1beta and TNF-alpha, and endothelial adhesion molecules, including ICAM-1, and induced leukocyte accumulation in the retinal vessels. Pitavastatin significantly reduced NMDA-induced leukocyte accumulation and up-regulation of endothelial adhesion molecules, whereas cytokine expression was unaffected. Systemic blockade of ICAM-1 in wild-type mice or absence of CD18 in gene-deficient (CD18(-/-)) mice significantly suppressed NMDA-induced leukocyte accumulation and RGC death. These findings suggest a novel and causative role for inflammatory leukocyte recruitment in NMDA-induced excitotoxicity. Furthermore, we show the novel neuroprotective effect of statins against excitotoxicity-induced RGC death. Statins or other anti-inflammatory agents may thus have therapeutic benefits in excitotoxicity-associated neuronal diseases through blockade of leukocyte recruitment.     

Soluble interleukin-18 receptor complex is a novel biomarker in rheumatoid arthritis

Introduction

There has been no report in the literature of a soluble form of interleukin (IL)-18 receptor α (IL-18Rα). In this study, we evaluated the levels and characteristics of soluble IL-18Rα (sIL-18Rα) in the sera of patients with rheumatoid arthritis (RA) and compared these results to control populations.

Methods

The sIL-18Rα complex was isolated from pooled human blood serum using an anti-IL-18Rα monoclonal antibody affinity column. The purified sIL-18Rα was then examined using Western blot analysis and used in experiments to evaluate the effects on an IL-18-responsive natural killer (NK) human cell line, NK0. An enzyme-linked immunosorbent assay was developed, and sera from 145 patients with RA, 6 patients with adult-onset Still's disease, 31 patients with osteoarthritis (OA), 39 patients with systemic lupus erythematosus (SLE) and 67 controls were tested, along with levels of immunoglobulin M, rheumatoid factor, anticyclic citrullinated peptide antibody, IL-18, IL-13 and interferon (IFN)-γ. Area under the receiver operating characteristic curve (ROC-AUC) analysis was used to evaluate the diagnostic utility of the sIL-18Rα complex.

Results

The isolated sIL-18Rα complex can be associated with IL-18 and the soluble form of the IL-18Rβ chain. The sIL-18Rα complex bound to the surface to the NK0 cell line, antagonized the stimulatory effects of IL-18 and IL-2 on the NK0 cell line and inhibited IFN-γ production by the cells. The serum levels of sIL-18Rα complex in RA (186.0 ± 33.5 ng/mL, n = 145) and adult-onset Still's disease (98.2 ± 8.9 ng/mL, n = 6) were significantly (P < 0.001) higher than those in the healthy controls (52.3 ± 8.5 ng/mL, n = 67), OA (38.6 ± 5.4 ng/mL, n = 31), SLE (44.6 ± 3.2 ng/mL, n = 39). The serum level of sIL-18Rα complex was not significantly different between RA and adult-onset Still's disease patients. The serum levels of IL-18, IL-13 and IFN-γ in the RA patients were significantly (P < 0.01) higher than in OA and SLE patients as well as healthy controls. ROC-AUC analysis of the serum concentration of sIL-18Rα indicated that it was significantly diagnostic of RA. Moreover, a tumor necrosis factor inhibitor, etanercept, significantly (P < 0.0001) decreased levels of sIL-18Rα in the sera of 29 RA patients 6 months after treatment.

Conclusions

The sIL-18Rα complex could be a potentially useful biomarker for the diagnosis of RA.     

Interactions of MCP1 with components of the replication machinery in mammalian cells

Eukaryotic DNA replication starts with the assembly of a pre-replication complex (pre-RC) at replication origins. We have previously demonstrated that Metaphase Chromosome Protein 1 (MCP1) is involved in the early events of DNA replication. Here we show that MCP1 associates with proteins that are required for the establishment of the pre-replication complex. Reciprocal immunoprecipitation analysis showed that MCP1 interacted with Cdc6, ORC2, ORC4, MCM2, MCM3 and MCM7, with Cdc45 and PCNA. Immunofluorescence studies demonstrated the co-localization of MCP1 with some of those proteins. Moreover, biochemical studies utilizing chromatin-immunoprecipitation (ChIP) revealed that MCP1 preferentially binds replication initiation sites in human cells. Interestingly, although members of the pre-RC are known to interact with some hallmarks of heterochromatin, our co-immunoprecipitation and immunofluorescence analyses showed that MCP1 did not interact and did not co-localize with heterochromatic proteins including HP1β and MetH3K9. These observations suggest that MCP1 is associated with replication factors required for the initiation of DNA replication and binds to the initiation sites in loci that replicate early in S-phase. In addition, immunological assays revealed the association of MCP1 forms with histone H1 variants and mass spectrometry analysis confirmed that MCP1 peptides share common sequences with H1.2 and H1.5 subtypes.     

Enhancement of autophagy is a potential modality for tumors refractory to radiotherapy

Radiotherapy is a well-established treatment for cancer. However, the existence of radioresistant cells is one of the major obstacles in radiotherapy. In order to understand the mechanism of cellular radioresistance and develop more effective radiotherapy, we have established clinically relevant radioresistant (CRR) cell lines, which continue to proliferate under daily exposure to 2 Gray (Gy) of X-rays for >30 days. X-ray irradiation significantly induced autophagic cells in parental cells, which was exiguous in CRR cells, suggesting that autophagic cell death is involved in cellular radiosensitivity. An autophagy inducer, rapamycin sensitized CRR cells to the level of parental cells and suppressed cell growth. An autophagy inhibitor, 3-methyladenine induced radioresistance of parental cells. Furthermore, inhibition of autophagy by knockdown of Beclin-1 made parental cells radioresistant to acute radiation. These suggest that the suppression of autophagic cell death but not apoptosis is mainly involved in cellular radioresistance. Therefore, the enhancement of autophagy may have a considerable impact on the treatment of radioresistant tumor.     

A novel type of immunity protein, NukH, for the lantibiotic nukacin ISK-1 produced by Staphylococcus warneri ISK-1

Staphylococcus warneri ISK-1 produces a lantibiotic, nukacin ISK-1. The nukacin ISK-1 gene cluster consists of at least six genes, nukA, -M, -T, -F, -E, and -G, and two open reading frames, ORF1 and ORF7 (designated nukH). Sequence comparisons suggested that NukF, -E, -G, and -H contribute to immunity to nukacin ISK-1. We investigated the immunity levels of recombinant Lactococcus lactis expressing nukFEG and nukH against nukacin ISK-1. The co-expression of nukFEG and nukH resulted in a high degree of immunity. The expression of either nukFEG or nukH conferred partial immunity against nukacin ISK-1. These results suggest that NukH contributes cooperatively to self-protection with NukFEG. The nukacin ISK-1 immunity system might function against another lantibiotic, lacticin 481. Western blot analysis showed that NukH expressed in Staphylococcus carnosus was localized in the membrane. Peptide release/bind assays indicated that the recombinant L. lactis expressing nukH interacted with nukacin ISK-1 and lacticin 481 but not with nisin A. These findings suggest that NukH contributes cooperatively to host immunity as a novel type of lantibiotic-binding immunity protein with NukFEG.     

Shigella effector IpaH9.8 binds to a splicing factor U2AF(35) to modulate host immune responses

Shigella effectors injected into the host cell via the type III secretion system are involved in various aspects of infection. Here, we show that one of the effectors, IpaH9.8, plays a role in modulating inflammatory responses to Shigella infection. In murine lung infection model, DeltaipaH9.8 mutant caused more severe inflammatory responses with increased pro-inflammatory cytokine production levels than did wild-type Shigella, which resulted in a 30-fold decrease in bacterial colonization. Binding assays revealed that IpaH9.8 has a specific affinity to U2AF(35), a mammalian splicing factor, which interferes with U2AF(35)-dependent splicing as assayed for IgM pre-mRNA. Reducing the U2AF(35) level in HeLa cells and infecting HeLa cells with wild-type caused a decrease in the expression of the il-8, RANTES, GM-CSF, and il-1beta genes as examined by RT-PCR. The results indicate that IpaH9.8 plays a role in Shigella infection to optimize the host inflammatory responses, thus facilitating bacterial colonization within the host epithelial cells.     

Relative biological effectiveness of fission neutrons for induction of micronucleus formation in mouse reticulocytes in vivo

Following whole-body irradiation of ICR mice with various doses of fission neutrons or X-rays, the frequency of micronuclei (MNs) in peripheral blood reticulocytes was measured at 12 h intervals beginning immediately after irradiation and ending at 72 h after irradiation. The resulting time-course curve of MN frequency had a clear peak 36 h after irradiation, irrespective of the type of radiation applied and the dose used. The MN frequency, averaged as the unweighted mean over the experimental time course, showed a linear increase with increasing dose of either fission neutrons or X-rays. The linear response to X-rays supports reported conclusion that induction of MN formation in reticulocytes is a dose-rate independent phenomenon. The relative biological effectiveness (RBE) of fission neutrons to X-rays for MN induction was estimated to be 1.9 +/- 0.3. This value is considerably lower than the RBE value of 4.6 +/- 0.5 reported for the same fission neutrons for induction of lymphocyte apoptosis in the thymus of ICR mice that represents dose-rate independent, one-track event. Based on these results, we propose that MNs increased in reticulocytes after irradiation mostly represent acentric fragments caused by single chromosome breaks, and that some confounding factor is operating in erythroblasts for the formation of aberrations from non-rejoining DNA double-strand breaks more severely after high-LET radiation than after low-LET radiation.