Akemichi Ueno

Buprenorphine hydrochloride induces apoptosis in NG108-15 nerve cells

A morphine alkaloid derivative, buprenorphine hydrochloride, induces apoptosis in NG108-15 cells. Apoptosis was detected mainly by apoptosis-specific DNA fragmentation and morphological changes. This apoptosis was dose-dependent and the time-course experiment indicated that DNA fragmentation occurred within 4 h after administration of buprenorphine hydrochloride. Specific inhibitors of the previously characterized apoptotic signal cascade as well as antagonists for opioid receptors were tested. Zn2+, herbimycin A, caspase inhibitors YVAD (Ac-Tyr-Val-Ala-Asp-CHO) and DEVD (Ac-Asp-Glu-Val-Asp-CHO), naloxone and naltrindole had no effect on apoptosis-specific DNA fragmentation. The serine protease inhibitor TPCK (N-tosyl-L-phenylalanyl chloromethyl ketone) specifically inhibited apoptosis-specific DNA fragmentation induced by buprenorphine hydrochloride; however, cell viability measurements revealed that cell death still occurred in NG108-15 cells. Thus TPCK pretreatment before buprenorphine hydrochloride administration induced apoptosis-independent cell death, presumably necrosis, in NG108-15 cells. This suggests that an unidentified serine protease, presumably functioning in the buprenorphine hydrochloride-specific death-signal cascade, could be pivotal for the rapid apoptosis observed in NG108-15 cells upon treatment with buprenorphine hydrochloride.

MC3T3-E1-conditioned medium-induced mineralization by clonal rat dental pulp cells

Dental pulp is thought to participate in supplementary mineralization, such as reparative dentin and pulp stones, but no direct proof of this has been reported. To study this process at a molecular level, we investigated the matrix mineralization of dental pulp using a clonal cell line (RPC-C2A) derived from rat incisor dental pulp. Mineralized nodules in extracellular matrix were formed by RPC-C2A cells cultured in the presence of conditioned medium (CM) from confluent osteoblastic MC3T3-E1 cells. These nodules were stained by the von Kossa method and with alizarin red S and quantified by the measurement of acid-soluble calcium deposition. This CM was most effective when collected 3-6 days after confluency and added at 50% to the culture medium. The CM-treated RPC-C2A cells showed high alkaline phosphatase activity, a high mRNA level of osteocalcin and decreases in the mRNA levels of osteopontin and osteonectin, but undetectable levels of mRNA of dentin sialophosphoprotein by Northern blot analyses. A pan-specific anti-transforming growth factor (TGF)-beta antibody and a soluble form of receptor for bone morphogenetic protein (BMP)-2/-4 did not neutralize the CM-induced mineralization. These results suggest that some soluble factor(s) other than TGF-beta or BMP-2/-4 in the CM from MC3T3-E1 cells cause differentiation of RPC-C2A cells to osteoblast-like cells.

Sulfated glycosaminoglycan synthesis and its regulation by transforming growth factor-β in rat clonal dental pulp cells

Dental pulps contain sulfated glycosaminoglycans (GAGs), such as chondroitin 4-sulfate (CSA/4CS), dermatan sulfate (CSB/DS), and chondroitin 6-sulfate (CSC/6CS). Sulfated GAGs play important roles in mineralization and collagen fibrillogenesis during primary, secondary, and reparative dentin formations. Transforming growth factor-beta (TGF-beta) is a potent regulator for several extracellular matrix (ECM) components and modulates the proliferation and differentiation. Using rat clonal dental pulp cells (RPC-C2A), we investigated the constituents of GAGs synthesized by the cells and the effect of TGF-beta on their synthesis by measuring the radioactivity of [35S]sulfate incorporated into GAG fractions. Cellulose acetate electrophoresis analysis revealed that RPC-C2A cells synthesized CSA and CSB but not CSC and that 10 ng/ml of TGF-beta increased the production of CSA and CSB in the cell/ECM fraction. Measurement of [35S]sulfate incorporation showed a significant increase in the amount of GAGs by TGF-beta, 1.3-fold CSA, and 1.2-fold CSB in the cell/ECM fraction. In the medium fraction the most secreted GAG was CSA, whereas CSB was stored in the cell/ECM fraction. Secreted CSA in the medium was markedly increased by 10 ng/ml of TGF-beta (1.7-fold). These findings indicate that CSA and CSB are major sulfated GAGs synthesized by RPC-C2A cells and that TGF-beta acts as a stimulator of sulfated GAG synthesis in dental pulp cells.

Constitutive expression of thrombospondin 1 in MC3T3‐E1 osteoblastic cells inhibits mineralization

Thrombospondin 1 (TSP1) is a multifunctional extracellular glycoprotein present mainly in the fetal and adult skeleton. Although an inhibitory effect of TSP1 against pathological mineralization in cultured vascular pericytes has been shown, its involvement in physiological mineralization by osteoblasts is still unknown. To determine the role of TSP1 in biomineralization, mouse osteoblastic MC3T3‐E1 cells were cultured in the presence of antisense phosphorothioate oligodeoxynucleotides complementary to the TSP1 sequence. The 18‐ and 24‐mer antisense oligonucleotides caused concentration‐dependent increases in the number of mineralized nodules, acid‐soluble calcium deposition in the cell/matrix layer, and alkaline phosphatase activity within 9 days, without affecting cell proliferation. The corresponding sense or scrambled oligonucleotides did not affect these parameters. In the antisense oligonucleotide‐treated MC3T3‐E1 cells, thickened extracellular matrix, well‐developed cell processes, increased intracellular organelles, and collagen fibril bundles were observed. On the other hand, the addition of TSP1 to the culture decreased the production of a mineralized matrix by MC3T3‐E1 cells. Furthermore, MC3T3‐E1 clones overexpressing mouse TSP1 were established and assayed for TSP1 protein and their capacity to mineralize. TSP1 dose‐dependently inhibited mineralization by these cells both in vitro and in vivo. These results indicate that TSP1 functions as an inhibitory regulator of bone mineralization and matrix production by osteoblasts to sustain bone homeostasis. J. Cell. Physiol. 209: 322–332, 2006.

cDNA cloning of bovine thrombospondin 1 and its expression in odontoblasts and predentin

The extracellular matrix protein thrombospondin 1 (TSP1) was cloned from odontoblasts of bovine mandibular teeth which participate in dentinogenesis. The 5289 bp cDNA contains a complete open reading frame of 1170 amino acids. Bovine TSP1 has high homologies to its human and mouse counterparts. In immunohistochemical analyses of bovine anterior teeth with anti-TSP1 monoclonal antibody, TSP1 was only detectable at the position of predentin, located between dentin and unmineralized dental pulp. Northern blot analysis showed high levels of two sizes of TSP1 mRNAs in odontoblasts but not dental pulp and gingiva. Previously we found that osteotropic factors such as calcitriol and TGF-beta induce TSP1 at the transcriptional level in clonal rat dental pulp cells. These results suggest a role of TSP1 in dentinogenesis and/or maintenance of dentin and dental pulp.

Inhibition of osteoblastic cell differentiation by conditioned medium derived from the human prostatic cancer cell line PC-3 in vitro.

Human prostatic carcinoma frequently metastasizes to bone tissue and activates bone metabolism, especially bone formation, at the site of metastasis. It has been reported that an extract of prostatic carcinoma and conditioned medium (CM) of a human prostatic carcinoma cell line, PC‐3, established from a bone metastastic lesion, stimulate osteoblastic cell proliferation. However, there is little information about the effect of PC‐3 CM on the differentiation of osteoblastic cells. In this study, we investigated the effect of PC‐3 CM on the differentiation of two types of osteoblastic cells, primary fetal rat calvaria (RC) cells containing many undifferentiated osteoprogenitor cells, and ROS 17/2.8, a well‐differentiated rat osteosarcoma cell line. PC‐3 CM inhibited bone nodule formation and the activity of alkaline phosphatase (ALPase), an osteoblastic marker enzyme, on days 7, 14, and 21 (RC cells) or 3, 6, and 9 (ROS 17/2.8 cells) in a dose‐dependent manner (5–30% CM). However, the CM did not affect cell proliferation or cell viability. PC‐3 CM was found to markedly block the gene expression of ALPase and osteocalcin (OCN) mRNAs but had no effect on the mRNA expression of osteopontin (OPN), the latter two being noncollagenous proteins related to bone matrix mineralization. These findings suggest that PC‐3 CM contains a factor that inhibits osteoblastic cell differentiation and that this factor may be involved in the process of bone metastasis from prostatic carcinoma. J. Cell. Biochem. 67:248–256, 1997.

Osteotropic factor‐stimulated synthesis of thrombospondin in rat dental pulp cells

The amount of thrombospondin (TSP) mRNA in confluent clonal rat dental pulp cells was increased by transforming growth factor‐β (TGF‐β), 1α,25‐dihydroxyvitamin D3 (1,25(OH)2D3), and phorbol 12,13‐didecanoate (PDD), with maximal levels 6, 36 and 2 h, respectively, after stimulation. These increases were accompanied by enhanced syntheses of TSP proteins which were found in the different forms in cell layer/matrix fraction (198 and 165 kDa TSP) and the culture medium (180 kDa TSP). These three factors also raised the mRNA level of osteopontin, which is thought to play an important role in mineralization in dentin and bone. The order of effectiveness of these factors was PDD > TGF‐β > 1,25(OH)2D3 for all the stimulations described above. These results suggest that the osteotropic factors enhance TSP synthesis at the pretranslational level and that TSP produced by dental pulp cells participates in formation of reparative dentin.

Inhibition of tyrosine autophosphorylation of the solubilized insulin receptor by an insulin-stimulating peptide derived from bovine serum albumin

A polypeptide from a tryptic digest of bovine serum albumin potentiates glucose oxidation stimulated by insulin in isolated rat adipocytes. We studied whether this effect is related to a modification of the insulin receptor kinase. In a solubilized rat adipocytes receptor system, the peptide caused dose-dependent inhibition of the stimulation by insulin of phosphorylation of the 95,000 dalton subunit of insulin receptor. The peptide also inhibited stimulation by vanadate of tyrosine autophosphorylation of the beta subunit of the receptor, though it enhanced vanadate-stimulated glucose oxidation. During the phosphorylation reaction, no phosphorylated forms of the peptide could be detected. The peptide had no effect on dephosphorylation of the phosphorylated beta subunit of the insulin receptor. These results strongly suggest that the inhibition of phosphorylation by the peptide is due not to either simple substrate competition or activation of phosphoprotein phosphatase, but to specific inhibition of tyrosine-specific protein kinase.

Insulin-stimulating peptide from tryptic digest of bovine serum albumin.

Insulin-stimulating peptide was isolated from a tryptic digest of bovine serum albumin by gel permeation, SP Sephadex column chromatography, reversed phase HPLC and cation-exchange HPLC. This peptide, with a molecular weight of about 8,400, had no insulin-like activity by itself, but enhanced fatty acid synthesis from glucose in rat adipose tissue explants in the presence of suboptimal concentrations of insulin. It also stimulated the effect of insulin on CO2 production from glucose in rat adipocytes, without affecting insulin binding. These stimulations were dose-dependent and were observed at concentrations of more than 2 X 10(-7) M peptide only in the presence of a suboptimal concentration of insulin.

Putrescine-stimulated intracellular Ca2+ release for invasiveness of rat ascites hepatoma cells

Our previous study showed that treatment of highly invasive rat ascites hepatoma (LC‐AH) cells with α‐difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase, decreased both their intracellular level of putrescine and their in vitro invasion of a monolayer of calf pulmonary arterial endothelial (CPAE) cells, and that both these decreases were completely reversed by exogenous putrescine, but not spermidine or spermine. Here we show that all adhering control (DFMO‐untreated) cells migrated beneath CPAE monolayer with morphological change from round to cauliflower‐shaped cells (migratory cells). DFMO treatment increased the number of cells that remained round without migration (nonmigratory cells). Exogenous putrescine, but not spermidine or spermine, induced transformation of all nonmigratory cells to migratory cells with a concomitant increase in their intracellular Ca2+ level, [Ca2+]i. The putrescine‐induced increase in their [Ca2+]i preceded their transformation and these effects of putrescine were not affected by antagonists of the voltage‐gated Ca2+ channel, but were completely suppressed by ryanodine, which also suppressed the invasiveness of the control cells. The DFMO‐induced decreases in both [Ca2+]i and the invasiveness of the cells were restored by thapsigargin, which elevated [Ca2+]i by inhibiting endoplasmic Ca2+‐ATPase, indicating that thapsigargin mimics the effects of putrescine. These results support the idea that putrescine is a cofactor for Ca2+ release through the Ca2+ channel in the endoplasmic reticulum that is inhibited by ryanodine, this release being initiated by cell adhesion and being a prerequisite for tumor cell invasion.