Mutsuki Kawabe

The promotional effect of IL-22 on mineralization activity of periodontal ligament cells

OBJECTIVES Interleukin (IL)-22 acts on non-immune cells to induce anti-microbial responses, protection from tissue damage, and enhance cell regeneration. However, little is known about the involvement of IL-22 in periodontal biology. This study investigated the biological effects of IL-22 on periodontal ligament (PDL) cells as part of studies to assess the involvement of IL-22 in periodontal disease. MATERIALS AND METHODS Gene expression levels of IL-22 and its receptors in PDL cells and gingival tissue samples were evaluated by real-time PCR. Proliferative responses and mineralized-matrix forming activities of PDL cells were examined in the presence and absence of IL-22. RESULTS In contrast to the expression of IL-22 receptors detected in PDL tissues and their cell lines, gingival tissues showed modest or no gene expressions of IL-22. The production of several cytokines including IL-11, IL-8 and CCL2 was upregulated by IL-22 treatment of PDL cells in a dose-dependent manner. IL-22 treatment had no effect on the proliferative response in PDL cells. Meanwhile, IL-22 precipitated mineralized nodule formation and induced gene expressions of RUNX2, MSX2 and osteocalcin in PDL cells, suggesting that IL-22 enhances the mineralized matrix-forming activities of PDL cells. CONCLUSION IL-22 has the potential to promote mineralizing activity in PDL cells and to develop appropriate regenerative therapy.

Sodium valproate, a histone deacetylase inhibitor, modulates the vascular endothelial growth inhibitor-mediated cell death in human osteosarcoma and vascular endothelial cells

The level of vascular endothelial growth inhibitor (VEGI) has been reported to be negatively associated with neovascularization in malignant tumors. The soluble form of VEGI is a potent anti-angiogenic factor due to its effects in inhibiting endothelial cell proliferation. This inhibition is mediated by death receptor 3 (DR3), which contains a death domain in its cytoplasmic tail capable of inducing apoptosis that can be subsequently blocked by decoy receptor 3 (DcR3). We investigated the effects of sodium valproate (VPA) and trichostatin A (TSA), histone deacetylase inhibitors, on the expression of VEGI and its related receptors in human osteosarcoma (OS) cell lines and human microvascular endothelial (HMVE) cells. Consequently, treatment with VPA and TSA increased the VEGI and DR3 expression levels without inducing DcR3 production in the OS cell lines. In contrast, the effect on the HMVE cells was limited, with no evidence of growth inhibition or an increase in the DR3 and DcR3 expression. However, VPA-induced soluble VEGI in the OS cell culture medium markedly inhibited the vascular tube formation of HMVE cells, while VEGI overexpression resulted in enhanced OS cell death. Taken together, the HDAC inhibitor has anti-angiogenesis and antitumor activities that mediate soluble VEGI/DR3-induced apoptosis via both autocrine and paracrine pathways. This study indicates that the HDAC inhibitor may be exploited as a therapeutic strategy modulating the soluble VEGI/DR3 pathway in osteosarcoma patients.

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.

Annexin A3 plays a role in cytoplasmic calcium oscillation by extracellular calcium in the human promyelocytic leukemia HL-60 cells differentiated by phorbol-12-myristate-13-acetate

The roles of annexin A3 (ANXA3) in macrophages are not fully understood. In contrast to C5a, we have demonstrated that C-terminal ribosomal protein S19 (RP S19)-tagged S-tagged C5a (S-tagged C5a/RP S19) raises an alternative cytoplasmic calcium oscillation by extracellular calcium during macrophage migration into apoptotic cells. We here differentiated human promyelocytic leukemia HL-60 cells bearing with either control sense RNA and shRNA for ANXA3 mRNA or a vector cDNA with or without ANXA3 cDNA into macrophage-like cells by phorbol-12-myristate-13-acetate and found that a fluorescence ratio (340 nm/380 nm) upon the S-tagged C5a/RP S19-induced alternative cytoplasmic calcium oscillation by extracellular calcium was an equilateral association with a dose of ANXA3. Moreover, the ANXA3-dependent modification was partially reflected upon the S-tagged C5a-induced classical cytoplasmic calcium oscillation by both intracellular calcium and extracellular calcium. ANXA3 seems to extend the C5aR-mediated cytoplasmic calcium oscillation by extracellular calcium at least in the HL-60 macrophage-like cells.

Iron-induced calcification in human aortic vascular smooth muscle cells through interleukin-24 (IL-24), with/without TNF-alpha

In CKD patients, arteriosclerotic lesions, including calcification, can occur in vascular smooth muscle cells in a process called Moenckeberg’s medial arteriosclerosis. Iron overload induces several complications, including the acceleration of arteriosclerosis. However, the relationship between Moenckeberg’s arteriosclerosis in vascular smooth muscle cells and iron accumulation has remained unknown. We tested the accelerated effect of iron on calcification in cultured human aortic vascular smooth muscle cells (HASMCs). After establishment of this model, we performed a microarray analysis using mRNA from early stage culture HASMCs after iron stimulation with or without TNF-alpha stimulation. The role of interleukin-24 (IL-24) was confirmed from candidate genes that might contribute to calcification. HASMCs demonstrated calcification induced by iron and TNF-alpha. Calcification of HASMCs was synergistically enhanced by stimulation with both iron and TNF-alpha. In the early phase of calcification, microarray analysis revealed up-regulation of IL-24. Stimulation of HASMCs by IL-24 instead of iron induced calcification. The anti-IL-24 antibody reversed the effect of IL-24, supporting the important role of IL-24 in HASMCs calcification. In conclusion, iron-induced calcification in vascular smooth muscle cells occurred via IL-24, IL-24 was increased during the calcification process induced by iron, and IL-24 itself caused calcification in the absence of iron.

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.

Wernicke's encephalopathy after chemoradiotherapy for tongue cancer: Report of a case

Abstract A case of acute Wernickes encephalopathy induced by thiamine deficiency due to malnutrition subsequent to chemoradiotherapy for tongue cancer is presented. A 63-year-old male patient had severe oral mucositis and mental stress in a long-term hospitalization for chemoradiotherapy of tongue cancer. After discharge from hospital, he had alcoholism and malnutrition. Three months later, disturbance of consciousness and ataxia followed by memory disturbance appeared. Laboratory examination revealed low serum level of thiamine. The symptoms, laboratory data, and magnetic resonance imaging suggested acute Wernickes encephalopathy. The patient was intravenously administered thiamine and received nutritional control, thereby the symptoms were improved.