Sodium Valproate Enhances Semaphorin 3A-mediated Anti-angiogenesis and Tumor Growth Inhibition in Human Osteosarcoma Cells.

BACKGROUND/AIM: Class 3 semaphorins, including semaphorin 3A (SEMA3A), are known endogenous angiogenesis inhibitors associated with endothelial cell migration and proliferation, and have been identified in many cancer cells. SEMA3A suppresses tumor angiogenesis by competing with VEGF, but tumors are known to have active angiogenesis, suggesting that expression of SEMA3A and its receptors is epigenetically restrained. To overcome this condition, we aimed to use histone deacetylase (HDAC) inhibitors to enhance the SEMA3A expression in osteosarcoma (OS) cells, thereby suppressing angiogenesis and inhibiting their proliferation and metastasis. MATERIALS AND METHODS: OS cell lines and human microvascular endothelial (HMVE) cells were treated with HDAC inhibitors such as sodium valproate (VPA) and Trichostatin A (TSA). Changes in the SEMA3A expression and its related receptors at the mRNA and protein levels, as well as the inhibitory effects on tumor angiogenesis, were investigated. RESULTS: VPA and TSA increased the expression of SEMA3A and its receptor NRP1, without inducing PLXNA1 in OS cells. Similarly, SEMA3A and NRP1 expression was increased in HMVE cells, but no growth inhibition was observed. Furthermore, SEMA3A induced by VPA in OS cell culture medium inhibited vascular tube formation of HMVE cells, and overexpression of SEMA3A enhanced OS cell growth inhibition. This growth-inhibitory effect of SEMA3A induced G1/S cell cycle arrest in OS cells. CONCLUSION: HDAC inhibitors have anti-angiogenic and anti-tumor activities that may be, in part, mediated via the SEMA3A/NRP1/PLXNA1 autocrine and paracrine pathways.

G­CSF delays tooth extraction socket bone healing via the inhibition of bone turnover in mice.

Granulocyte colony-stimulating factor (G-CSF) regulates the survival, proliferation and differentiation of all cells in the neutrophil lineage, and is consequently used for neutropenic conditions. Upon G-CSF administration, osteoblasts and osteocytes are suppressed, and the support system allowing hematopoietic stem cells to remain in the microenvironment is diminished. The present study focused on and investigated G-CSF as a regulatory factor of bone remodeling. The aim of the present study was to investigate the effect of G-CSF administration on the bone healing of tooth extraction sockets. Significant differences in the bone volume fraction, and trabecular separation of the proximal femurs and alveolar septa were observed between the G-CSF and control (saline-treated) groups. The trabecular bone of the femur and alveolar septa was reduced in the G-CSF group compared with that in the control group. In addition, serum procollagen type 1 N-terminal propeptide levels, a marker of bone formation, were lower in the G-CSF group compared with in the control group. Fibrous connective tissues and immature bone were observed in the extraction socket, and bone healing was delayed in the G-CSF group compared with that in the control group. The bone area in the extraction socket 6 days after tooth extraction was significantly smaller in the G-CSF group (23.6%) than that in the control group (45.1%). Furthermore, G-CSF administration reduced the number of canaliculi per osteocyte and inhibited the connection of osteocyte networks. Consequently, osteoblast activation was inhibited and bone remodeling changed to a state of low bone turnover in the G-CSG group. Analysis of bone formation parameters revealed that the G-CSF group exhibited a lower mineral apposition rate compared with in the control group. In conclusion, these findings indicated that G-CSF may delay bone healing of the socket after tooth extraction.

Establishment of an oral squamous cell carcinoma cell line expressing vascular endothelial growth factor a and its two receptors.

Background/purpose: Vascular endothelial growth factor receptor (VEGFR) expression in oral squamous cell carcinoma (OSCC) promotes tumor growth through both autocrine and paracrine signaling. VEGF-positive OSCC cases are associated with a high depth of invasion, increased metastasis, and poor prognosis. In this study we established and then molecularly and functionally analyzed an OSCC cell line that co-expresses VEGF-A, VEGFR-1, and VEGFR-2, termed HCM-SqCC010 cells. Materials and methods: VEGF-A, VEGFR-1, and VEGFR-2 expression in HCM-SqCC010 cells were examined by immunohistochemistry and immunoblotting. Expression and inhibition of VEGF-A, VEGFR-1, and VEGFR-2 in HCM-SqCC010 cells were verified by quantitative real-time PCR. Results: Our analysis of HCM-SqCC010 cells revealed that their proliferation depended on VEGF-A, and selective inhibition of VEGFR-1 or VEGFR-2 resulted in decreased cell growth. Conclusion: We established an OSCC cell line, HCM-SqCC010, that expresses VEGF-A, VEGFR-1, and VEGFR-2. This triple-positive cell line showed no effect from a molecular targeted drug toward VEGF-A, but it did show strong cell growth inhibition in response to a VEGFR inhibitor. Thus, new therapeutic strategies against OSCC should include a VEGFR inhibitor.

Substantial improvement of histopathological diagnosis by whole-slide image-based remote consultation.

Consultation by subspecialty experts is the most common mode of rendering diagnosis in challenging cases in pathological practice. Our study aimed to highlight the diagnostic benefits of whole-slide image (WSI)-based remote consultation. We obtained diagnostically challenging cases from two institutions from the years 2010 and 2013, with histological diagnoses that contained keywords "probable," "suggestive," "suspicious," "inconclusive," and "uncertain." A total of 270 cases were selected for remote consultation using WSIs scanned at 40 × . The consultation process consisted of three rounds: the first and second rounds each with 12 subspecialty experts and the third round with six multi-expertise senior pathologists. The first consultation yielded 44% concordance, and a change in diagnosis occurred in 56% of cases. The most frequent change was from inconclusive to definite diagnosis (30%), followed by minor discordance (14%), and major discordance (12%). Out of the 70 cases which reached the second round, 31 cases showed discrepancy between the two consultants. For these 31 cases, a consensus diagnosis was provided by six multi-expertise senior pathologists. Combining all WSI-based consultation rounds, the original inconclusive diagnosis was changed in 140 (52%) out of 266 cases. Among these cases, 80 cases (30%) upgraded the inconclusive diagnosis to a definite diagnosis, and 60 cases (22%) changed the diagnosis with major or minor discordance, accounting for 28 cases (10%) and 32 cases (12%), respectively. We observed significant improvement in the pathological diagnosis of difficult cases by remote consultation using WSIs, which can further assist in patient healthcare. A post-study survey highlighted various benefits of WSI-based consults.

Establishment of a patient-derived mucoepidermoid carcinoma cell line with the CRTC1-MAML2 fusion gene.

Mucoepidermoid carcinoma (MEC) is the most common malignant tumor of the major and minor salivary glands. Surgical resection is the only curative treatment and there is no effective post-operative therapy for MEC. The present study reports an Institutional Review Board-approved case of a 45-year-old Japanese female diagnosed with low-grade MEC in the hard palate. Radical resection, supraomohyoid neck dissection and antero-lateral thigh flap reconstruction was performed. A MEC cell line was then established from the resected tumor tissue. Short tandem repeat profiling confirmed the origin and authenticity of the cell line, that harbors a CRTC1-MAML2 translocation, which is frequently observed in MEC. Amphiregulin (AREG), identified as one of the targets of the CRTC1-MAML2 fusion gene, was expressed in the cell line. The AREG receptor, epidermal growth factor receptor (EGFR) was also highly phosphorylated. The results predicted that AREG-EGFR signaling, which is required for tumor growth and survival, might be activated in the cell line in a cell-autonomous manner. As AREG expression is associated with EGFR-targeted drug resistance, this cell line might assist with the identification of novel strategies for MEC treatment.

The implicated clinical factors for outcomes in 304 patients with salivary duct carcinoma: Multi-institutional retrospective analysis in Japan.

BACKGROUND: Salivary duct carcinoma (SDC) is a high-grade salivary malignancy that frequently occurs as the carcinomatous component of carcinoma ex pleomorphic adenoma. We herein examined the clinical factors affecting outcomes in a large cohort of SDC. METHODS: We selected 304 SDC cases and investigated clinical characteristics and the factors affecting outcomes. RESULTS: The median age of the cases examined was 68 years, the most common primary site was the parotid gland (238 cases), and there was a male predominance (M/F = 5:1). Outcomes were significantly worse when the primary tumor site was the minor salivary glands (SG) than when it was the major SG. Outcomes were also significantly worse in pN(+) cases (161 cases) than in pN0 cases, particularly those with a metastatic lymph node number ≥11. The cumulative incidence of relapse and distant metastases was significantly higher in stage IV cases than in stage 0-III cases. CONCLUSIONS: The absolute number of lymph node metastases, higher stages, and the minor SG as the primary tumor site were identified as factors affecting the outcome of SDC.

Salivary Duct Carcinoma With Rhabdoid Features-No or Aberrant Expression of E-cadherin and Genetic Changes in CDH1: Immunohistochemical and Genetic Analyses of 17 Cases.

Salivary duct carcinoma is a relatively uncommon malignancy of the salivary glands; however, it frequently occurs as a carcinomatous component of carcinoma ex pleomorphic adenoma. We previously reported salivary duct carcinoma with rhabdoid features (SDCRF) as an extremely rare subtype of salivary duct carcinoma, and that it occurred as a salivary counterpart of pleomorphic lobular carcinoma of the breast (PLCB). We collected new cases of SDCRF for this study, in which we examined a total of 17 cases immunohistochemically and genetically. As it is known that PLCB exhibits loss of or aberrant E-cadherin expression and carries nonsense/missense mutations in or deletion of the CDH1 gene, we examined the CDH1 gene status of our SDCRF cases. All of the examined SDCRF cases involved the diffuse proliferation of large ovoid cells with eosinophilic cytoplasm and eccentric nuclei, which displayed reduced cell-cell adhesion. Most cases were positive for pan-cytokeratin, androgen receptor, gross cystic disease fluid protein-15, SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily B member 1, and WI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily A member 4, whereas they were negative for vimentin. No and decreased/cytoplasmic E-cadherin expression was observed in 11 and 4 of 17 cases, respectively, whereas no and decreased/cytoplasmic ß-catenin expression were observed in 10 and 5 of 17 cases, respectively. Among the 11 cases that could be genetically analyzed, a nonsense mutation (1 case), missense mutations (6 cases), and insertions (1 case) were detected in the CDH1 gene. In conclusion, we propose that SDCRF is the salivary counterpart of PLCB due to its morphology and immunophenotype, and the genetic status of CDH1.

Programmed cell death 1 positive lymphocytes at palate tonsils in the elder patients with chronic tonsillitis.

Circulating lymphocytes infiltrate into local foci at the inflammatory phase of acute wound healing for activation of the immune system and express an immune checkpoint protein programmed cell death 1 (PD-1) at the resolution phase for inactivation of the immune system. Conversely, the PD-1 expression was still found even on circulating lymphocytes of the elder patients with chronic tonsillitis at the palliative stage. Recently, an adhesion G protein coupled receptor 56 (GPR56) was reported to at least work as a proliferation factor for infiltrated lymphocytes into local foci at the resolution phase of acute wound healing. To preliminary examine a similar role of PD-1 and GPR56 at local foci at chronic inflammation, palate tonsils were prepared from small amounts of patients with chronic tonsillitis and tonsillar hypertrophy. A positive relationship of RNA expression might be observed between PD-1 and GPR56 in the elder patients with chronic tonsillitis. In regard to immunohistopathological findings, there were huge and small amounts of PD-1 and GPR56 expression at the marginal zone of lymphoid follicles of palate tonsils with chronic tonsillitis. Moreover, the positive relationship of RNA expression between PD-1 and GPR56 confirmed in large numbers of the elder patients with chronic tonsillitis. Probably, GPR56 participates in a supplement of PD-1+ lymphocytes to circulating bloods of the elder patients with chronic tonsillitis through a lymphocyte cell maintenance system at the marginal zone of the lymphoid follicles of palate tonsils.

Immune Aging and How It Works for Inflammation and Fibrosis.

Almost all mature cells that undergo apoptosis in an age-dependent or an accidental manner are completely recovered in tissue-specific microenvironments without any physiological changes. After peripheral blood leukocytes are released into the local region, fibroblast cells and new blood vessels commonly proliferate during wound healing. Inducible repair tools mainly supplied from blood vessels are cleared by peripheral blood phagocytic macrophages. Finally, hematopoietic stem cell (HSC)-derived precursor cells migrate from bone marrow (BM) to the microenvironment to rebuild damaged tissues (the mature immune system). In contrast to the mature immune system, the effects of aging on HSCs (long-term HSCs) and peripheral blood lymphocytes (long-term PBLs) are not clearly understood in the BM and thymus niches with tissue-specific microenvironments with some physiological changes (the aged BM niche) for incomplete rebuilding of damaged tissues (the aged immune system). In this review, the roles of the aged immune system in both a delay of acute inflammation and the development of chronic inflammation or fibrosis are discussed.

Epigenetic modulators hydralazine and sodium valproate act synergistically in VEGI-mediated anti-angiogenesis and VEGF interference in human osteosarcoma and vascular endothelial cells.

Vascular endothelial growth inhibitor (VEGI; also referred to as TNFSF15 or TL1A) is involved in the modulation of vascular homeostasis. VEGI is known to operate via two receptors: Death receptor­3 (DR3) and decoy receptor­3 (DcR3). DR3, which is thus far the only known functional receptor for VEGI, contains a death domain and induces cell apoptosis. DcR3 is secreted as a soluble protein and antagonizes VEGI/DR3 interaction. Overexpression of DcR3 and downregulation of VEGI have been detected in a number of cancers. The aim of the present study was to investigate the effects of sodium valproate (VPA), a histone deacetylase inhibitor, in combination with hydralazine hydrochloride (Hy), a DNA methylation inhibitor, on the expression of VEGI and its related receptors in human osteosarcoma (OS) cell lines and human microvascular endothelial (HMVE) cells. Combination treatment with Hy and VPA synergistically induced the expression of VEGI and DR3 in both OS and HMVE cells, without inducing DcR3 secretion. In addition, it was observed that the combination of VPA and Hy significantly enhanced the inhibitory effect on vascular tube formation by VEGI/DR3 autocrine and paracrine pathways. Furthermore, the VEGI/VEGF­A immune complex was pulled down by immunoprecipitation. Taken together, these findings suggest that DNA methyltransferase and histone deacetylase inhibitors not only have the potential to induce the re­expression of tumor suppressor genes in cancer cells, but also exert anti­angiogenic effects, via enhancement of the VEGI/DR3 pathway and VEGI/VEGF­A interference.

Osteonecrosis of the jaws caused by bisphosphonate treatment and oxidative stress in mice.

Aging is a significant risk factor for the development of bisphosphonate-related osteonecrosis of the jaws (BRONJ). Accumulating evidence suggests that bone aging is associated with oxidative stress (OS), and OS is associated with osteonecrosis. To elucidate the mechanisms of the onset of BRONJ, the present study focused on OS and the effects of treatment with the pro-oxidant DL-buthionine-(S,R)-sulfoximine (BSO), an oxidative stressor, on healing of a surgically induced penetrating injury of the palate. Six-week-old C57BL/6J mice were randomly divided into four groups (n=5 each) and treated with or without zoledronic acid (ZOL) and with or without BSO (experimental groups: ZOL, BSO, and ZOL+BSO; control group: saline solution). A penetrating injury of the midline palate was surgically created using a root elevator. ZOL (250 µg/kg/day) was injected intraperitoneally every day from 7 days prior to the surgical treatment to 4 days following the surgical treatment. BSO (500 µg/kg/day) was administered 7 days prior to the surgical treatment as a single intraperitoneal injection. The maxillae were harvested at 5 days following the surgical treatment for histological and histochemical studies. The presence of empty osteocyte lacunae in the palatal bone was increased by ZOL and BSO treatment. The highest number of empty osteocyte lacunae was observed in the ZOL+BSO group. The number of tartrate-resistant acid phosphatase-positive cells was decreased by ZOL treatment and increased by BSO treatment. The number of canaliculi per osteocyte lacuna was significantly decreased by BSO treatment. The mineral apposition rate was significantly lower in the treatment groups than the control group. Bisphosphonates and OS suppressed bone turnover. The present study has demonstrated that BSO treatment affects osteocytes, and OS in osteocytes exacerbates impairment of the osteocytic canalicular networks. As a result, bisphosphonates and OS may induce osteonecrosis following invasive dentoalveolar surgery. OS has been identified as an additional risk factor for the development of BRONJ.

Inhibition of Asparagine-linked Glycosylation Participates in Hypoxia-induced Down-regulation of Cell-surface MICA Expression.

BACKGROUND/AIM: Hypoxia down-regulates the expression of cell surface major histocompatibility class I-related chain molecule A (MICA) without increasing its shedding. Recently, the inhibition of N-linked glycosylation was also shown to reduce the cell-surface expression of MICA. We investigated the participation of asparagine (Asn)-linked glycosylation in hypoxia-induced down-regulation of cell-surface MICA using osteosarcoma cells. MATERIALS AND METHODS: The cell-surface expression and Asn-N-glycosylation of MICA were estimated by flow cytometry, and western blot analyses, respectively. RESULTS: Hypoxia reduced the expression of N-linked glycosylated MICA, as well as the ratio of N-linked glycosylated to non-glycosylated MICA. 2-Deoxy-D-glucose, which inhibits N-linked glycosylation, reduced the cell-surface expression of MICA under normoxia, while D-Mannose increased N-glycosylated MICA, increasing cell-surface MICA under hypoxia. Cells transfected with wild-type MICA expression vector expressed cell surface MICA more than those transfected with mutant MICA expression vectors designed for abrogation of N-linked glycosylation. CONCLUSION: The inhibition of Asn-N-linked glycosylation participates in hypoxia-induced down-regulation of cell-surface expression of MICA.

Participation of delta annexin A3 in the ribosomal protein S19 C-terminus-dependent inhibitory mechanism of the neutrophil C5a receptor through delta lactoferrin.

Although C5a receptor (C5aR) interacting with its agonist C5a promotes acute inflammation during the initiation phase, the roles of the recycling C5aR during the resolution phase are still unclear. We found that C5aR interacted with its antagonist/agonist ribosomal protein S19 (RP S19) polymer or a RP S19 polymer functional analogue S-tagged C5a/RP S19, which connects an RP S19 C-terminus (IAGQVAAANKKH) to the S-tagged C5a C-terminus, promoted acute inflammation at the resolution phase via an activation of the apoptosis-inducing transcription factor delta lactoferrin (δLf) in neutrophils and the membrane mobilizing factor full-length annexin A3 (ANXA3) in macrophages. To confirm the antagonistic system of the recycling C5aR, S-tagged δLf-coupled BrCN-activated Sepharose 4B beads were incubated with cytoplasmic proteins and identified a neutrophil-specific δANXA3 via pull-down experiments. The S-tagged C5a/RP S19-induced agonistic functions in macrophage-like cells that were differentiated from human promyelocytic leukemia HL-60 cells by phorbol-12-myristate-13-acetate were suppressed by δLf and δANXA3 co-overexpression. δANXA3 seems to participate in the antagonistic system of the neutrophil C5aR involving IAGQVAAANKKH and δLf. Most likely, δANXA3 works as antagonist for the recycling C5aR on neutrophils during the resolution phase of acute inflammation.

Erythroblast differentiation at spleen in Q137E mutant ribosomal protein S19 gene knock-in C57BL/6J mice.

We recently found that erythroblast-like cells derived from human leukaemia K562 cells express C5a receptor (C5aR) and produce its antagonistic and agonistic ligand ribosomal protein S19 (RP S19) polymer, which is cross-linked between K122 and Q137 by tissue transglutaminases. RP S19 polymer binds to the reciprocal C5aRs on erythroblast-like cells and macrophage-like cells derived from human monocytic THP-1 cells and promotes differentiation into reticulocyte-like cells through enucleation in vitro. To examine the roles of RP S19 polymer in mouse erythropoiesis, we prepared Q137E mutant RP S19 gene knock-in C57BL/6J mice. In contrast to wild-type mice, erythroblast numbers at the preliminary stage (CD71high/TER119low) in spleen based on transferrin receptor (CD71) and glycophorin A (TER119) values and erythrocyte numbers in orbital artery bloods were not largely changed in knock-in mice. Conversely, erythroblast numbers at the early stage (CD71high/TER119high) were significantly decreased in spleen by knock-in mice. The reduction of early erythroblast numbers in spleen was enhanced by the phenylhydrazine-induced pernicious anemia model knock-in mice and was rescued by a functional analogue of RP S19 dimer S-tagged C5a/RP S19. These data indicated that RP S19 polymer plays the roles in the early erythroblast differentiation of C57BL/6J mouse spleen.

The roles of a ribosomal protein S19 polymer in a mouse model of carrageenan-induced acute pleurisy.

C5-deficient mice usually present moderate neutrophil activation during the initiation phase of acute inflammation. Conversely, C5a receptor (C5aR)-deficient mice show unusually excessive activation of neutrophils. We identified the ribosomal protein S19 (RP S19) polymer, which is cross-linked at Lys122 and Gln137 by transglutaminases in apoptotic neutrophils, as a second C5aR ligand during the resolution phase of acute inflammation. The RP S19 polymer promotes apoptosis via the neutrophil C5aR and phagocytosis via the macrophage C5aR. To confirm the roles of the RP S19 polymer, we employed a carrageenan-induced acute pleurisy mouse model using C57BL/6J mice with a knock-in of the Gln137Glu mutant RP S19 gene and replaced the RP S19 polymer with either an S-tagged C5a/RP S19 recombinant protein or the RP S19122-145 peptide monomer and dimer (as functional C5aR agonists/antagonists) and the RP S19122-145 peptide trimer (as a functional C5aR antagonist). Neutrophils and macrophages were still present in the thoracic cavities of the knock-in mice at 24h and 7days after carrageenan injection, respectively. Knock-in mice showed structural organization and severe hemorrhaging from the surrounding small vessels of the alveolar walls in the lung parenchyma. In contrast to the RP S19122-145 peptide monomer and trimer, the simultaneous presence of S-tagged C5a/RP S19 and the RP S19122-145 peptide dimer completely improved the physiological and pathological acute inflammatory cues. The RP S19 polymer, especially the dimer, appears to play a role at the resolution phase of carrageenan-induced acute pleurisy in C57BL/6J model mice.

Salivary duct carcinoma with rhabdoid features: a salivary counterpart of pleomorphic lobular carcinoma of the breast.

AIM: To analyse the clinicopathological features and immunohistochemical characteristics of nine cases of salivary duct carcinoma (SDC) with rhabdoid features (SDCRF), representing a new, extremely rare type of salivary gland malignancy. METHODS AND RESULTS: We analysed 2511 cases of salivary gland tumour, clinicopathologically and immunohistochemically. The incidence of SDCRF was 0.4%. Eight patients were male. The age of patients ranged from 36 years to 85 years (mean, 61 years). SDC arose from the parotid glands and submandibular gland in six and three cases, respectively. Seven cases appeared as a carcinoma component of carcinoma ex pleomorphic adenoma cases. Six patients died of disease. Histologically, diffuse proliferations of non-coherent large ovoid or polygonal carcinoma cells with eosinophilic cytoplasm and eccentric nuclei were observed in all cases; such cytological characteristics were defined as 'rhabdoid features'. Immunohistochemically, all cases were positive for cytokeratin, gross cystic disease fluid protein-15, androgen receptor, and SMARCB1, seven cases were positive for HER2, and two cases were positive for epidermal growth factor receptor. However, all cases were negative for vimentin and myoepithelial markers. Eight cases showed no or aberrant expression of E-cadherin and ß-catenin. The results suggest that SDCRF is an extremely rare subtype of SDC, and not a sarcomatoid variant of SDC. SDCRF is histologically unique, and is positive for SDC markers but negative for vimentin, unlike rhabdoid-type carcinomas arising from other organs. CONCLUSIONS: The morphogenesis of SDCRF is related to no or aberrant expression of cell-cell adhesion molecules. Therefore, SDCRF could be a salivary counterpart to pleomorphic lobular breast carcinoma.

Functional analysis of differences in transcriptional activity conferred by genetic variants in the 5' flanking region of the IL12RB2 gene.

Interleukin 12 receptor ß chain (IL12RB2) is a crucial regulatory factor involved in cell-mediated immune responses, and genetic variants of the gene encoding IL12RB2 are associated with susceptibility to various immune-related diseases. We previously demonstrated that haplotypes with single nucleotide polymorphisms (SNPs) in the 5' flanking region of IL12RB2, including -1035A>G (rs3762315) and -1023A>G (rs3762316), affect the expression of IL12RB2, thereby altering susceptibility to leprosy and periodontal diseases. In the present study, we identified transcription factors associated with the haplotype-specific transcriptional activity of IL12RB2 in T cells and NK cells. The -1023G polymorphism was found to create a consensus binding site for the transcription factor activating protein (AP)-1, and enzyme-linked immunosorbent assay (ELISA)-based binding assays showed that these SNPs enhanced AP-1 binding to this region. In reporter assays, suppression of JunB expression using siRNA eliminated differences in the -1035G/-1023G and -1035A/-1023A regions containing IL12RB2 promoter activity in Jurkat T cells and NK3.3 cells. These results suggested that the -1035/-1023 polymorphisms created differential binding affinities for JunB that could lead to differential IL12RB2 expression. Moreover, the -1035G and -1035A alleles formed binding sites for GATA-3 and myocyte enhancer factor-2 (MEF-2), respectively. Our data indicated that in addition to JunB, the SNP at -1035/-1023 influenced GATA-3 and MEF-2 binding affinity, potentially altering IL12RB2 transcriptional activity. These findings confirm the effects of rs3762315 and rs3762316 on IL12RB2 transcription. These genetic variants may alter cellular activation of T cells and NK cells and modify cell-mediated immune responses.

RP S19 C-terminal peptide trimer acts as a C5a receptor antagonist.

We have demonstrated that ribosomal protein S19 (RP S19) polymer, when crosslinked between Lys122 and Gln137 by activated coagulation factor XIII, acts as a C5a receptor (C5aR) antagonist/agonist. Based on experimental data obtained using RP S19 analog peptide and recombinant protein monomer, we suggested that L131DR, I134AGQVAAAN and K143KH moieties in the RP S19 C-terminus act in, respectively, C5aR binding, penetration of the plasma membrane, and interaction with either an apoptosis-inducing molecule in neutrophils (delta lactoferrin) or a calcium channel-activating molecule (annexin A3) to induce the p38 MAPK pathway in macrophages. Recently, we observed RP S19 trimer in serum. To study the effects of this RP S19 trimer on C5aR, we prepared mutant RP S19 C-terminal peptide (RP S19122-145) dimer and trimer, and examined their chemotactic activities and signal transduction pathways in human C5aR-overexpressing squamous cell carcinoma HSC-1 (HSC-1C5aR) cells using 24 trans-well chamber and western blotting assays, respectively. HSC-1C5aR cells were attracted by RP S19122-145 dimer and vice versa by RP S19122-145 trimer. The RP S19122-145 dimer-induced attraction was competitively blocked by pre-treatment with RP S19122-145 trimer. Moreover, RP S19122-145 trimer-induced p38 MAPK phosphorylation was stronger than RP S19122-145 dimer-induced p38 MAPK phosphorylation. RP S19122-145 trimer appeared to act as a C5aR antagonist. The agonistic and antagonistic effects of RP S19122-145 dimers and trimers were reflected by monocytic, THP-1-derived macrophage-like cells. Unlike the C5aR agonist C5a, which acts at the inflammation phase of acute inflammation, RP S19 trimer might act as a C5aR antagonist at the resolution phase.

Suppressive effect of membrane-permeable peptides derived from autophosphorylation sites of the IGF-1 receptor on breast cancer cells.

Insulin-like growth factor-1 (IGF-1) receptors play a crucial role in the biology of human cancer, making them an attractive target for anti-cancer agents. We previously designed oligopeptides containing the amino-acid sequences surrounding the autophosphorylation sites of the insulin receptor and found that two of them, namely, Ac-DIYET-NH2 and Ac-DYYRK-NH2, suppressed phosphorylation of purified insulin receptors in a non-ATP-competitive manner, whereas Ac-NIYQT-NH2 and Ac-NYYRK-NH2 suppressed in an ATP-competitive manner. Because the IGF-1 receptor is closely related to the insulin receptor, the aim of this study was to observe the effects of these peptides, which correspond to the amino-acid sequences of the autophosphorylation sites of the IGF-1 receptor, on the activity of the human breast cancer cell lines MCF-7, T47D, MDA-MB-231, and MDA-MB-453. To facilitate peptide delivery into breast cancer cells, the cell-penetrating peptide, human immunodeficiency virus type 1-transactivator of transcription (Tat), was linked to these peptides. When breast cancer cells were treated with each of these synthetic Tat-conjugated peptides, the conjugated peptides penetrated into the cells and suppressed cell proliferation. An inhibitory effect of Tat-conjugated peptides against IGF-1-stimulated phosphorylation of IGF-1 receptors was observed. In addition, we found that combinations of these peptides suppressed phosphorylation of IGF-1 receptors to a greater extent than the peptides did individually. In conclusion, IGF-1 receptor autophosphorylation site-derived membrane-permeable peptides have the potential to suppress IGF-1 receptor function in breast cancer cells and to be developed into novel and useful agents for cancer therapy.

The roles of ribosomal protein S19 C-terminus in a shortened neutrophil lifespan through delta lactoferrin.

Cell lifespan is partially regulated by a balance between survival signals via constitutively active G protein-coupled receptors (GPCRs) and death signals via death receptors. We have demonstrated that neutrophils produce a mimic ligand of G protein-coupled C5a receptor (C5aR), ribosomal protein S19 (RP S19) polymer. In contrast to an original ligand C5a, RP S19 polymer induces not only inhibition of the guanine nucleotide exchange factor activity but also initiation of the regulator of G protein signaling 3 (RGS3) promoter in a RP S19 C-terminus dependent manner. To examine an antagonistic effect of the RP S19 C-terminus on G proteins, His-S-tagged C5a or C5a/RP S19, in which an RP S19 C-terminus is bound to the C5a C-terminus, was incubated with neutrophils, and a transcription factor delta lactoferrin (δLf) was identified as a specific binding protein via pull-down experiments. The S-tagged C5a-induced agonistic effects on chemotaxis, cytoplasmic Ca(2+) influx and p38 mitogen-activated protein kinase phosphorylation were not changed by Lf knockdown and δLf overexpression in neutrophil-like or macrophage-like cells, which were differentiated into mature cells from human promyelocytic leukemia HL-60 cells by dimethyl sulfoxide and phorbol-12-myristate-13-acetate, respectively. While, the S-tagged C5a/RP S19-induced antagonistic or agonistic effects on mature HL-60 neutrophil-like or macrophage-like cells were reversed by Lf knockdown and δLf overexpression, respectively. Moreover, RGS3 expression was increased in another HL-60 neutrophil-like cells under spontaneous apoptosis induced by an apoptotic inducer MnCl2. The RGS3 expression in apoptotic neutrophil-like cells was delayed not only by Lf knockdown but also by neutralization of the RP S19 polymer or C5aR. The inhibitory extension from G protein of C5aR to Gα subsets of constitutively active GPCRs along with the RP S19 polymer-induced translocation of δLf from the cytoplasmic face of the plasma membrane to the nucleus seems to shorten the neutrophil cell lifespan.