Kaya Yoshida

PTEN expression elicited by EGR-1 transcription factor in calyculin A-induced apoptotic cells

PTEN is a tumor suppressor gene encoding a phosphatase that negatively regulates cell survival mediated by the PI3‐kinase‐Akt pathway. The gene for transcription factor EGR‐1 is an early response gene essential for cellular growth, proliferation, and differentiation. Protein phosphatase inhibitors including calyculin A and okadaic acid are potent inducers of apoptosis in several cell lines; however, the molecular mechanisms underlying their action are unknown. The purpose of this study was to examine the expression of PTEN and EGR‐1 and the phosphorylation status of EGR‐1 and Akt in calyculin A‐treated human squamous carcinoma cells (SCCTF). Phosphorylation of EGR‐1 and upregulation of PTEN expression were observed to occur in SCCTF cells treated with calyculin A in time‐ and dose‐dependent fashions. The level of phosphorylated Akt decreased as the expression of PTEN protein increased in the calyculin A‐treated SCCTF cells. Calyculin A‐stimulated expression of EGR‐1 and PTEN might be p53 independent, because the expression of them was also detected in p53‐null Saos‐2 cells. RNA interference using double‐stranded RNA specific for the EGR‐1 gene inhibited not only EGR‐1 expression but also PTEN expression in SCCTF cells treated or not with calyculin A. Calyculin A induced nuclear fragmentation and chromatin condensation in SCCTF cells. The present results suggest that the level of PTEN expression and the phosphorylation status of Akt were associated with apoptosis induced by calyculin A. These observations also support the view that EGR‐1 regulates PTEN expression in the initial steps of the apoptotic pathway.

Oral Porphyromonas gingivalis translocates to the liver and regulates hepatic glycogen synthesis through the Akt/GSK-3β signaling pathway

Periodontal diseases are common chronic inflammatory disorders that result in the destruction of tissues around teeth. Many clinical studies suggest that periodontal diseases are risk factors for insulin resistance and diabetic mellitus development. However, the molecular mechanisms by which periodontal diseases regulate the progress of diabetes mellitus remain unknown. In this study, we investigated whether Porphyromonas gingivalis (P.g.), a major pathogen of periodontal diseases, present in the oral cavity, moves to the liver and affects hepatic glycogen synthesis. SNAP26b-tagged P.g. (SNAP-P.g.) was introduced into the oral cavity to induce periodontal disease in 4-week old female Balb/c mice. SNAP-P.g. was detected in the liver extracted from SNAP-P.g.-treated mice using nested PCR analysis. High blood glucose levels tended to promote SNAP-P.g. translocation from the oral cavity to the liver in mice. Periodic acid-Schiff staining suggested that hepatic glycogen synthesis decreased in SNAP-P.g.-treated mice. SNAP-P.g. was also internalized into the human hepatoma cell line HepG2, and this attenuated the phosphorylation of insulin receptor substrate (IRS)-1, Akt and glycogen synthase kinase-3β induced by insulin. Insulin-induced glycogen synthesis was suppressed by SNAP-P.g. in HepG2 cells. Our results suggest that P.g. translocation from the oral cavity to the liver may contribute to the progress of diabetes mellitus by influencing hepatic glycogenesis.

Histone H1.2 is translocated to mitochondria and associates with bak in bleomycin‐induced apoptotic cells

Bleomycin induces single‐ and double‐stranded breaks in DNA, with consequent mitochondrial membrane aberrations that lead to the apoptotic cell death. It is poorly understood how DNA damage‐inducing apoptotic signals are transmitted to mitochondria, from which apoptotic factors are released into the cytoplasm. Here, we investigated the localization of histone H1.2 in the bleomycin‐treated human squamous carcinoma SCCTF cells. The presence of DNA double‐strand breaks in the bleomycin‐treated cells was examined by Western analysis using antibody against phosphorylated histone H2AX (γ‐H2AX). Incubation of SCCTF cells for 48 h with 10 µM bleomycin induced apoptosis, as determined by cleavage of lamin B1 to 28 kDa fragment and DNA ladder formation. The mitochondrial permeabilization causing apoptotic feature was also detected with MitoCapture in the bleomycin‐treated cells. Histone H1.2 was translocated from the nucleus to the mitochondria after treatment with bleomycin and co‐localized with Bak in mitochondria. Our present results suggest that histone H1.2 plays an important role in transmitting apoptotic signals from the nucleus to the mitochondria following double‐stranded breaks of DNA by bleomycin. J. Cell. Biochem. 103: 1488–1496, 2008.

Okadaic acid stimulates expression of Fas receptor and Fas ligand by activation of nuclear factor kappa-B in human oral squamous carcinoma cells

In the present study, we used western blot and RT-PCR analysis to examine the expression of proteins and mRNAs of Fas receptor and Fas ligand in human oral squamous carcinoma SCC-25 cells treated with okadaic acid. Treatment with okadaic acid enhanced the expression of proteins and mRNAs of both Fas receptor and Fas ligand in SCC-25 cells. The amount of IkappaB-alpha in whole cell lysates decreased, while the level of NF-kappaB in nucleus increased, in the okadaic acid-treated cells. Okadaic acid-treatment also alters the cellular localization of NF-kappaB, from cytoplasm to nuclei. To investigate the activation of NF-kappaB in okadaic acid-treated SCC-25 cells, we performed electrophoretic mobility gel shift assay using nuclear extracts and the consensus oligonucleotide for NF-kappaB DNA binding site. The binding of nuclear proteins to the oligonucleotide of NF-kappaB increased when the cells had been treated with 20 nM okadaic acid for 4 h. We transfected the cells with pFLF1, which has the promoter region of Fas receptor gene containing NF-kappaB binding site. A luciferase reporter gene assay demonstrated that the activity in the cells transfected with pFLF1 and treated with 20 nM okadaic acid increased in a time-dependent manner and that the activity was more than three-fold over that in the control cells. Our results suggest that NF-kappaB activated at early stages in the okadaic acid-treated SCC-25 cells stimulated the promoter activity of Fas receptor in the cells leading to the apoptotic death of these cells.

PKR-mediated degradation of STAT1 regulates osteoblast differentiation.

The double-stranded RNA-dependent protein kinase (PKR) plays a critical role in various biological responses including antiviral defense, cell differentiation, apoptosis, and tumorigenesis. In this study, we investigated whether PKR could affect the post-translational modifications of STAT1 protein and whether these modifications regulate osteoblast differentiation. We demonstrated that PKR was necessary for the ubiquitination of STAT1 protein. The expressions of bone-related genes such as type I collagen, integrin binding sialoprotein, osteopontin, and osterix were suppressed in osteoblasts lacking PKR activity. In contrast, the expressions of interleukin-6 and matrix metalloproteinases 8 and 13 increased in PKR-mutated osteoblasts. The expression and degradation of STAT1 protein were regulated by PKR in a SLIM-dependent pathway. Inhibition of SLIM by RNA interference resulted in the decreased activity of Runx2 in osteoblasts. Stimulation of interleukin-6 expression and suppression of alkaline phosphatase activity were regulated through by SLIM-dependent pathway. However, expressions of bone-related genes and MMPs were regulated by SLIM-independent pathway. Our present results suggest that the aberrant accumulation of STAT1 protein induced by loss of PKR regulate osteoblast differentiation through both SLIM/STAT1-dependent and -independent pathways.

Cleavage of nucleolin and argyrophilic nucleolar organizer region associated proteins in apoptosis-induced cells

To investigate the behavior of nuclear proteins in apoptotic cells, we examined the changes in nucleolin and proteins of the nucleolar organizing region during apoptosis in human osteoblastic cell lines, Saos-2 and MG63. Apoptosis was induced by treatment of these cells with okadaic acid. Proteins prepared from apoptotic cells were subjected to Western blot analysis and a modified Western blot method using silver nitrate. The anti-nucleolin antibody recognized the 110-kDa band and the staining intensity of this band decreased in the proteins prepared from the okadaic acid-treated apoptotic cells. The additional band of an 80-kDa was also detected in the proteins prepared from the apoptotic cells. Two major silver nitrate-stained bands, 110-kDa and 37-kDa, were detected among the proteins obtained from control cells. Like the Western blot analysis, the intensity of the 110-kDa silver nitrate-staining band decreased; an 80-kDa band appeared and its staining intensity increased in the lysate from the okadaic acid-treated cells. The signal intensity of the 37-kDa protein did not change in the sample from the apoptotic cells. In a cell-free apoptotic system, the 80-kDa protein was also detected and the amount of the 110-kDa protein decreased in the extract of Saos-2 cell nuclei incubated with apoptotic cytosol. The change in nucleolin in Saos-2 cells induced to undergo apoptosis was examined by an immunocytochemical procedure using the anti-nucleolin antibody and Hoechst 33342. Nucleolin was visible as dots in nucleoli in the control cells; however, it was not detected in the cells undergoing apoptosis. The dual-exposure view of Hoechst 33342 and anti-nucleolin staining cells confirmed that nucleolin had disappeared from the apoptotic nuclei of Saos-2.

Protein phosphatase 2A Cα regulates osteoblast differentiation and the expressions of bone sialoprotein and osteocalcin via osterix transcription factor

Serine/threonine protein phosphatase 2A (PP2A) participates in regulating many important physiological processes such as cell cycle, growth, apoptosis, and signal transduction. Osterix is a zinc‐finger‐containing transcription factor that is essential for osteoblast differentiation and regulation of many bone‐related genes. We have recently reported that decrease in α‐isoform of PP2A catalytic subunit (PP2A Cα) accelerates osteoblast differentiation through the expression of bone‐related genes. In this study, we further examined the role of PP2A Cα in osteoblast differentiation by establishing the stable cell lines that overexpress PP2A Cα. Overexpression of PP2A Cα reduced alkaline phosphatase (ALP) activity. Osteoblast differentiation and mineralization were also decreased in PP2A Cα‐overexpressing cells, with reduction of bone‐related genes including osterix, bone sialoprotein (Bsp), and osteocalcin (OCN). Luciferase assay showed that the transcriptional activity of the Osterix promoter region was decreased in PP2A Cα‐overexpressing cells. Introduction of ectopic Osterix rescued the expression of Bsp and OCN in PP2A Cα‐overexpressing cells. These results indicate that PP2A Cα and its activity play a negative role in osteoblast differentiation and Osterix is a key factor responsible for regulating the expressions of Bsp and OCN during PP2A Cα‐mediated osteoblast differentiation. J. Cell. Physiol.

High Glucose Levels Increase Osteopontin Production and Pathologic Calcification in Rat Dental Pulp Tissues

INTRODUCTION Pulp stones are frequently formed as a pathologic calcification product in dental pulp tissues, but the pathogenesis is poorly understood. We previously found that osteopontin (OPN) was produced by dental pulp cells, and its expression was associated with formation of the pulp stone matrix. It was reported that amorphous calcification appeared in the dental pulp of diabetic patients. The aim of this study was to determine the relationship between OPN expression and pathologic calcification in rat diabetic pulp. METHODS The effect of glucose on OPN production and alkaline phosphatase activity in cultured rat dental pulp cells (RPC-C2A) was investigated, and then dental pulp calcification and OPN expression in diabetic rats were determined and compared with those in healthy rats by histologic and immunohistochemical analyses. RESULTS In RPC-C2A cells, biochemical analysis showed that a high concentration of glucose (50 mmol/L) increased OPN protein production and alkaline phosphatase activity 1.3-fold and 1.5-fold, respectively. Histologic observations showed more calcified particles in dental pulp tissues in diabetic than in nondiabetic rats. Moreover, a thickened layer of predentin was formed in the radicular pulp of diabetic rats. OPN was more strongly stained around the calcified particles and in the odontoblast zone under the thickened predentin in diabetic rats. CONCLUSIONS OPN might be a key molecule involved in the increase of pathologic pulp calcifications, which are frequently observed in diabetic patients.

Reduction of protein phosphatase 2A Cα enhances bone formation and osteoblast differentiation through the expression of bone-specific transcription factor Osterix

The serine/threonine protein phosphatase 2A (PP2A) participates in regulating many important physiological processes such as control of cell cycle, growth, and division. On the other hand, Osterix is a zinc-finger-containing transcription factor that is essential for the differentiation of osteoblasts and regulation of many bone-related genes. Here we examined the effect of okadaic acid (OA), a specific inhibitor of PP2A, on bone formation in vivo and the molecular mechanism regulated by PP2A Cα in osteoblast differentiation. Administration of 1nM OA to the calvarial region in mice increased bone mineral density, as shown by μCT, while histomorphological analysis showed an increase in mineral apposition and bone thickness in the same region. In addition, treatment with 1nM OA stimulated osteoblast differentiation and the expression of Osterix, bone sialoprotein (Bsp), and osteocalcin (OCN) in mouse osteoblastic MC3T3-E1 cells. Moreover, the expression and phosphatase activity of PP2A Cα was decreased in the initial step of osteoblast differentiation, which was in parallel with an increase in Osterix expression. To further clarify the role of PP2A Cα in osteoblast differentiation, we constructed PP2A knock-down cells by infecting MC3T3-E1 cells with a lentivirus expressing shRNA specific for the PP2A Cα. Accordingly, the silencing of PP2A Cα in MC3T3-E1 cells dramatically increased osteoblast differentiation and mineralization, which were accompanied with expressions of Osterix, Bsp, and OCN. Our data indicate that PP2A Cα plays an important role in the regulation of bone formation and osteoblast differentiation through the bone-related genes.

Okadaic acid induces tyrosine phosphorylation of IκBα that mediated by PKR pathway in human osteoblastic MG63 cells

Treatment of human osteosarcoma cell line MG63 cells with okadaic acid stimulated phosphorylation of IκBα, as judged from the results of Western blot analysis and a λ protein phosphatase dephosphorylation assay. The stimulated phosphorylation of IκBα was both time- and dose-dependent. The phosphorylation sites of IκBα were taken to be tyrosine residues because the anti-phospho-tyrosine antibody bound to the samples immunoprecipitated with the anti-IκBα antibody. In the cells treated with 100 nM okadaic acid consequential translocation of NF-κB p65 from the cytosol to the nucleus occurred. Double-stranded RNA-dependent protein kinase (PKR) is a player in the cellular antiviral response and is involved in transcriptional stimulation through activation of NF-κB. We investigated the functional relationship between PKR and IκBα phosphorylation by constructing MG63 PKR K/R cells that produced a catalytically inactive mutant PKR. NF-κB p65 was detected in the nucleus of these cells, even in the unstimulated cells. Although IκBα was degraded phosphorylation of eIF-2α, a substrate of PKR, did not occur in the mutant cells treated with okadaic acid. Our results suggest that okadaic acid-induced tyrosine phosphorylation of IκBα was mediated by PKR kinase activity, thus indicating the involvement of this kinase in the control mechanism governing the activation of NF-κB. (Mol Cell Biochem 276: 211–217, 2005)