Biomedical subjects

O Ishibashi

Publications and source records attributed to O Ishibashi.

At least 19 recordsLinked to original sources

PubMed2001

Tamoxifen agonism and estrogen antagonism of c-fos gene promoter activity through non-consensus-responsive elements in MC3T3-E1 osteoblasts.

We find that the activity of a 0.4-kb human c-fos gene promoter (-404/+41), which lacks consensus estrogen-responsive elements (EREs), is regulated by estrogen receptor (ER) ligands in MC3T3-E1 osteoblastic cells through ERs in a manner distinct from ERE-mediated regulation. When ERalpha is coexpressed, both estrogens and antiestrogens upregulate promoter activity. When ERbeta is coexpressed, however, three tested antiestrogens affect c-fos promoter activity, with tamoxifen exerting the greatest effect, while estrogens have no such effect. The tamoxifen agonism through ERbeta is antagonized by 17beta-estradiol, while the 17beta-estradiol agonism through ERalpha is canceled by excess-level coexpression of ERbeta. Deletion analysis revealed that the sequence -206/-110 plays a crucial role in the ERbeta-mediated tamoxifen agonism. Interestingly, there is no ERbeta-mediated tamoxifen agonism when nonosteoblastic cells are tested. Taken together, these results suggest that the transcription of the c-fos gene is regulated by ER ligands possibly through non-ERE elements in ligand structure-, cell type-, and ER subtype-dependent manners.

Animals↗

PubMed2001

Brefeldin A inhibits osteoclastic bone resorption through induction of apoptosis.

Brefeldin A (BFA), a fungal metabolite with a macrocyclic lactone structure, has been developed for the treatment of cancer, and its major biological activity is the inhibition of intracellular protein transport from the endoplasmic reticulum to the cis-Golgi apparatus. In this study, we investigated the effect of BFA on osteoclastic pit formation in vitro. BFA reduced pit formation in a concentration-dependent manner, and the IC50 values on the pit number and the pit volume were 11.3 +/- 2.2 and 13.3 +/- 2.0 nM, respectively. In parallel with the inhibitory effect on pit formation, BFA also reduced the cell viability of osteoclasts-enriched bone cells with an IC50 value of 13.9 +/- 2.2 nM. These results suggest that the inhibition of bone resorption by BFA is caused by the induction of osteoclast cell death. BFA at a concentration of 100 nM induced DNA fragmentation in purified osteoclasts, assessed by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling and DNA ladder formation, demonstrating that BFA induces cell death of osteoclasts in an apoptotic manner. In addition, the accumulation of p53 proteins to the nuclei was observed in the osteoclasts treated with 100 nM BFA. These results, taken together, suggest that BFA inhibits osteoclastic bone resorption by inducing apoptosis in osteoclasts through a p53-dependent mechanism.

Animals↗

PubMed2001

Cloning and characterization of the functional promoter of mouse estrogen receptor beta gene.

The recently discovered estrogen receptor beta (ERbeta) exhibits some properties distinct from those of the classical estrogen receptor, ERalpha. To elucidate the mechanism underlying the regulation of ERbeta gene expression, we cloned and characterized the 5'-flanking (promoter) region of the mouse ERbeta (mERbeta) gene. A TATA-like motif was found in the 5'-flanking region, and transcription initiation sites were mapped 24 bp and 27 bp downstream from the motif by primer extension analysis and 5'-rapid amplification of cDNA ends. The mERbeta promoter contains several putative cis-acting elements, many of which are also found in the mouse ERalpha promoter. Therefore, the expression of both ERs may be partially regulated by a common mechanism. Luciferase assays revealed that the mERbeta promoter activity paralleled the endogenous expression levels of mERbeta in several cell lines. Successive 5'-deletion analyses showed that element(s) critical for its basal activity and negative regulatory element(s) are located in the sequences -90/+33 and -505/-372, respectively. The characterization of the mERbeta promoter will allow further studies to investigate the transcriptional regulation of mERbeta.

Amino Acid Sequence↗

PubMed2001

Quantification of the expression levels of lysosomal cysteine proteinases in purified human osteoclastic cells by competitive RT-PCR.

Cathepsin K is a lysosomal cysteine proteinase (LCP) predominantly expressed in osteoclasts. This study was conducted to evaluate the importance of human cathepsin K for osteoclastic bone resorption relative to that of other LCPs. To accomplish this, we quantitatively determined the expression levels of major LCPs (cathepsins B, K, L, and S) in human osteoclastic cells by using competitive RT-PCR. Giant cell tumor of bone (GCT) was used as a source of human osteoclastic cells, since the tissue was shown to contain a large number of cells satisfying the criteria for typical osteoclasts. The involvement of LCPs in the bone-resorption process by the GCT cells was confirmed by showing that trans-epoxysucciny-L-leucylamido-(4-guanidino) butane (E-64), a nonselective cysteine proteinase inhibitor, exerted an inhibitory effect on the pit formation. We isolated osteoclast-like cells (OLCs) positive for tartrate-resistant acid phosphatase (TRAP) and cathepsin K from the GCT tissue to a degree of almost 95% purity. In these cells, the expression of cathepsin K was shown to be approximately 20-, 130-, and 410-fold stronger than that of cathepsins B, L, and S, respectively. A similar result was obtained when human bone marrow cells in culture were used as another source of OLCs. Further, we found that cathepsin K was expressed in OLCs far more strongly than in several human nonosteoclastic cells including osteoblastic cell lines. The abundant and selective expression of cathepsin K in OLCs relative to that of other LCPs suggests that cathepsin K is mainly responsible for osteoclastic degradation of human bone matrix.

Acid Phosphatase↗

PubMed2001

Potential role of Gab1 and phospholipase C-gamma in osmotic shock-induced glucose uptake in 3T3-L1 adipocytes.

Osmotic shock induces GLUT4 translocation and glucose uptake through a mechanism independent of PI 3-kinase, but dependent on tyrosine phosphorylation of cellular proteins. To identify the tyrosine phosphorylated proteins required for osmotic shock-stimulated glucose uptake, we examined tyrosine phosphorylation of candidate proteins, and found that the 60-80kDa species including paxillin and the 120-130kDa species including p130Cas, PYK2, FAK and Gab1 were tyrosine-phosphorylated in response to osmotic shock. Inhibition of actin polymerization by cytochalasin D significantly decreased the tyrosine phosphorylation of paxillin, p130Cas, PYK2 and FAK but not Gab1, but had no effect on 2-deoxyglucose (DOG) uptake, suggesting a role for Gab1 in osmotic shock-induced glucose transport. Also, we found that osmotic shock increases the association of phospholipase C-gamma (PLC-gamma) with Gab1 and stimulates tyrosine phosphorylation of PLC-gamma itself. The PLC inhibitor, U73122, inhibited osmotic shock-induced 2-DOG uptake. These results suggest that tyrosine phosphorylation of Gab1 and subsequent recruitment and activation of PLC-gamma may play a role in osmotic shock-induced glucose transport.

3T3 Cells↗

PubMed2001

Tensile stress induces bone morphogenetic protein 4 in preosteoblastic and fibroblastic cells, which later differentiate into osteoblasts leading to osteogenesis in the mouse calvariae in organ culture.

Mechanical stress is an important factor controlling bone remodeling, which maintains proper bone morphology and functions. However, the mechanism by which mechanical stress is transduced into biological stimuli remains unclear. Therefore, the purpose of this study is to examine how gene expression changes with osteoblast differentiation and which cells differentiate into osteoblasts. Tensile stress was applied to the cranial suture of neonatal mouse calvaria in a culture by means of helical springs. The suture was extended gradually, displaying a marked increase in cell number including osteoblasts. A histochemical study showed that this osteoblast differentiation began in the neighborhood of the existing osteoblasts, which can be seen by 3 h. The site of osteoblast differentiation moved with time toward the center of the suture, which resulted in an extension of osteoid. Scattered areas of the extended osteoid were calcified by 48 h. Reverse-transcription polymerase chain reaction (RT-PCR) revealed that tensile stress increased bone morphogenetic protein 4 (BMP-4) gene expression by 6 h and it remained elevated thereafter. This was caused by the induction of the gene in preosteoblastic cells in the neighborhood of osteoblasts and adjacent spindle-shaped fibroblastic cells. These changes were evident as early as 3 h and continued moving toward the center of the suture. The expression of Cbfa1/Osf-2, an osteoblast-specific transcription factor, followed that of BMP-4 and those cells positive with these genes appeared to differentiate into osteoblasts. These results suggest that BMP-4 may play a pivotal role by acting as an autocrine and a paracrine factor for recruiting osteoblasts in tensile stress-induced osteogenesis.

Alkaline Phosphatase↗

PubMed2001

Pioglitazone ameliorates tumor necrosis factor-alpha-induced insulin resistance by a mechanism independent of adipogenic activity of peroxisome proliferator--activated receptor-gamma.

Tumor necrosis factor (TNF)-alpha is one of the candidate mediators of insulin resistance associated with obesity, a major risk factor for the development of type 2 diabetes. The insulin resistance induced by TNF-alpha is antagonized by thiazolidinediones (TZDs), a new class of insulin-sensitizing drugs. The aim of the current study was to dissect the mechanism whereby pioglitazone, one of the TZDs, ameliorates TNF-alpha-induced insulin resistance in 3T3-L1 adipocytes. Pioglitazone restored insulin-stimulated 2-deoxyglucose (DOG) uptake, which was reduced by TNF-alpha, with concomitant restorations in tyrosine phosphorylation and protein levels of insulin receptor (IR) and insulin receptor substrate (IRS)-1, as well as association of the p85 regulatory subunit of phosphatidylinositol (PI) 3-kinase with IRS-1 and PI 3-kinase activity. Adenovirus-mediated gene transfer of either wild-type human peroxisome proliferator-activated receptor (PPAR)-gamma2 or a mutant carrying a replacement at the consensus mitogen-activated protein kinase phosphorylation site (hPPAR-gamma2-S112A) promoted adipogenesis of 3T3-L1 fibroblasts and restored TNF-alpha-induced decrease of triglyceride in adipocytes as effectively as pioglitazone. Overexpression of the PPAR-gamma proteins in TNF-alpha-treated adipocytes restored protein levels of IR/IRS-1, but did not improve insulin-stimulated tyrosine phosphorylation of IR/IRS-1 or insulin-stimulated 2-DOG uptake. These results indicate that the ability of pioglitazone to restore insulin-stimulated tyrosine phosphorylation of IR/IRS-1, which is necessary for amelioration of TNF-alpha-induced insulin resistance, may be independent of the adipogenic activity of PPAR-gamma that regulates protein levels of IR/IRS-1.

3T3 Cells↗

PubMed2000

Tensile stress induced osteoblast differentiation and osteogenesis in mouse calvarial suture in culture: possible involvement of BMP-4 and other genes.

Mechanical stress is one of the most potent inducer of bone formation. The mechanism by which cells receive and transduce the signal into osteogenesis, however, remains unknown. Previous studies have demonstrated that mechanical stress causes changes in expression levels of many genes in osteoblasts and osteocytes both in vivo and in vitro. However, none of these changes are specific to bone cells. Moreover it is not clear which types of cells contributed to the increased osteoblasts induced by mechanical stress. The purpose of this study, therefore, was to identify which cells differentiate into osteoblasts and to examine how the expression of genes that are specific to osteogenic cells changes.

Animals↗

PubMed1999

Matrix metalloproteinases and lysosomal cysteine proteases in osteoclasts contribute to bone resorption through distinct modes of action.

The effects of inhibitors of matrix metalloproteinases (MMPs) and lysosomal cysteine proteases on osteoclastic pit formation in dentine slices were investigated. A nonspecific cysteine protease inhibitor, E-64, inhibited pit formation on naked slices in a concentration-dependent manner, and at 10 microM E-64 reduced the pit volume by 70%. However, up to 10 microM of the MMP inhibitor, BB-94, did not show any inhibition of pit formation. On the other hand, on slices coated with reconstituted basement membrane, both BB-94 and E-64 at 10 microM showed a marked decrease in pit volume by 73% and 68%, respectively. By a combination of treatment with both BB-94 and E-64, pit formation could be completely suppressed. These results suggest that MMPs are necessary for the migration of precursor and/or immature osteoclasts to bone surface through basement membranes, while cysteine proteases are essential for the osteoclastic degradation of bone collagen.

Animals↗

PubMed1999

Differential effects of palmitate on glucose uptake in rat-1 fibroblasts and 3T3-L1 adipocytes.

Non-esterified fatty acids are thought to be one of the causes for insulin resistance. However, the molecular mechanism of fatty acid-induced insulin resistance is not clearly known. In this study, we first examined the effect of palmitate on insulin signaling in 3T3-L1 adipocytes. We found that 1h treatment with 1 mmol/l palmitate had no effect on insulin binding, tyrosine phosphorylation of insulin receptors, 185 kDa proteins and Shc, and PI3 kinase activity in 3T3-L1 adipocytes. Then, the effects of palmitate on MAP kinase activity and glucose uptake in fully differentiated 3T3-L1 adipocytes were compared with those in poorly differentiated 3T3-L1 cells and in HIRc-B cells. Palmitate treatment had no effect on MAP kinase activity in fully differentiated 3T3-L1 adipocytes, while it inhibited MAP kinase in poorly differentiated 3T3-L1 cells and HIRc-B cells. Glucose transport in 3T3-L1 adipocytes treated with palmitate for 1 h, 4 h and 16 h was higher than that in control cells, but palmitate treatment caused a rightward shift of the insulin-dose responsive curve for glucose uptake in HIRc-B cells. Palmitate treatment did not significantly affect basal and insulin-stimulated GLUT4 translocation. When the cells were treated with PD98059, a specific MEK inhibitor, insulin-stimulated glucose uptake was not affected in 3T3-L1 adipocytes, while it was almost completely inhibited in HIRc-B cells. These results suggest the primary effect of palmitate on adipocytes may not involve insulin resistance of adipocytes themselves.

3T3 Cells↗

PubMed1999

Breast cancer cells express cathepsins B and L but not cathepsins K or H.

Lysosomal cysteine proteinases (cathepsins) are considered to play a role in bone degradation mediated by metastatic breast cancers. To evaluate which cathepsin contributes to the osteolysis, we quantitatively determined the expression levels of four cathepsins in two breast cancer cell lines, MCF-7 and MDA-MB-231, by competitive RT-PCR. Cathepsin K, which is the most abundant cathepsin in osteoclasts, was not detected in either cell lines. We also failed to detect cathepsin H mRNA. By contrast, we found significant expression of cathepsins B and L in both cell lines. By Northern blot analysis cathepsin B mRNA was detected in a single form in these cells, whereas osteoclasts contained multiple forms of the mRNA. Cathepsin B protein was also detected by Western blotting as a single immunoreactive band corresponding to its mature enzyme. These findings suggest that osteolysis associated with metastatic breast cancers takes place in a different way from osteoclast-mediated bone resorption.

Adenocarcinoma↗

PubMed1998

IRS pleckstrin homology domains bind to acidic motifs in proteins.

Using a yeast two-hybrid system, we identified several proteins that interact with the PH domains in IRS-1 and IRS-2, including Lon protease, myeloblast protein, and nucleolin. Although the roles of these molecules in insulin action are not yet known, each protein contained an acidic motif that interacted with the PH domain of IRS-2. However, only the acidic motif in nucleolin bound to IRS-1, suggesting that the PH domain in IRS-1 and IRS-2 are not identical. Moreover, synthetic peptides based on the acidic motif in Lon protease and myeloblast protein inhibited the binding of nucleolin to the PH domain of IRS-2 but not to the PH domain of IRS-1, confirming the selectivity of these PH domains. The ability to bind acidic motifs may be a specific function of the PH domain in IRS proteins, because the PH domains in betaARK, phospholipase Cgamma, or spectrin did not bind nucleolin. In 32D cells, nucleolin bound to both IRS-1 and IRS-2, and expression of the acidic motif of nucleolin inhibited insulin-stimulated tyrosine phosphorylation of IRS-1 and IRS-2. These results suggest that the binding of acidic motifs to the PH domain of IRS-1 and IRS-2 disrupts coupling to the activated insulin receptor. Our results are consistent with the hypothesis that the PH domain in the IRS proteins may ordinarily bind acidic peptide motifs in membrane proteins or other acidic membrane elements that couple IRS proteins to activated membrane receptors.

ATP-Dependent Proteases↗

PubMed1998

Involvement of heat shock protein 90 in the degradation of mutant insulin receptors by the proteasome.

We previously reported three families with type A insulin-resistant syndrome who had mutations, either Asp1179 or Leu1193, in the kinase domain of the insulin receptor. The extreme insulin resistance of these patients was found to be caused by the decreased number of insulin receptors on the cell surface, due to the intracellular rapid degradation (Imamura, T., Takata, Y., Sasaoka, T., Takada, Y., Morioka, H., Haruta, T., Sawa, T., Iwanishi, M., Yang, G. H., Suzuki, Y., Hamada, J., and Kobayashi, M. (1994) J. Biol. Chem. 269, 31019-31027). In the present study, we first examined whether these mutations caused rapid degradation of unprocessed proreceptors, using the exon 13 deleted mutant insulin receptors (DeltaEx13-IR), which were accumulated in the endoplasmic reticulum as unprocessed proreceptors. The addition of Asp1179 or Leu1193 mutation to DeltaEx13-IR caused accelerated degradation of the unprocessed DeltaEx13-IR in the transfected COS-7 cells. Next, we tested whether these mutant receptors were degraded by the proteasome. Treatment with proteasome inhibitors Z-Leu-Leu-Nva-H (MG-115) or Z-Leu-Leu-Leu-H (MG-132) prevented the accelerated degradation of these mutant receptors, resulting in increased amounts of the mutant receptors in the COS-7 cells. Essentially the same results were obtained in the patient's transformed lymphocytes. Finally, we found that these mutant receptors bound to heat shock protein 90 (Hsp90). To determine whether Hsp90 played an important role in the accelerated receptor degradation, we examined the effect of anti-Hsp90 antibody on the mutant receptor degradation. The microinjection of anti-Hsp90 antibody into cells prevented the accelerated degradation of both Asp1179 and Leu1193 mutant insulin receptors. Taken together, these results suggest that Hsp90 is involved in dislocation of the mutant insulin receptors out of the endoplasmic reticulum into the cytosol, where the mutant receptors are degraded by the proteasome.

Animals↗

PubMed1998

Production and characterization of two variants of human cystatin SA encoded by two alleles at the CST2 locus of the type 2 cystatin gene family.

Two variants of cystatin SA encoded by two alleles at the CST2 locus of the type 2 human cystatin gene family were expressed in Escherichia coli. One, termed cystatin SA1, is identical to cystatin SA [S. Isemura, E. Saitoh, and K. Sanada J. Biochem. 102, 693-704, 1987]. Another, termed cystatin SA2, carries two amino acid substitutions (59Gly-->Asp; 120Glu-->Asp), one of which is in the so-called QXVXG region (the first hairpin loop) and another in the C-terminal portion of the molecule. Four recombinant cystatins [full-sized cystatin SA1, two N-terminally truncated cystatin SA1 lacking four residues (WSPQ) and six residues (WSPQEE), and full-sized cystatin SA2] were purified from the periplasmic fractions of E. coli cells. Two N-terminally truncated recombinant cystatin SA1 inhibited bovine cathepsin C with 2- to 20-fold lower Ki values than that of the full-sized one. In the inhibition of papain and ficin, however, both of the N-terminally truncated cystatin SA1 displayed a 10-fold higher Ki value than that of full-sized one. In the inhibition of papain, ficin, and recombinant human cathepsin K, recombinant cystatin SA2 showed, respectively, 3826-, 1090-, and 30-fold higher Ki values compared with those of SA1. Recombinant cystatin SA2 inhibited bovine cathepsin C with a 50-fold lower Ki value compared with that of SA1. Recombinant cystatin SA1 did not inhibit human cathepsin H but SA2 inhibited it slightly (Ki = 528 nM). Neither of the recombinant variants inhibited bovine cathepsin B. Our data supply evidence indicating that the amino acid sequence of the first hairpin loop of the cystatin superfamily is important in the inhibition of papain, ficin, cathepsin C, cathepsin H, and cathepsin K.

Amino Acid Sequence↗

PubMed1998

Suppression of prostanoid formation and regulation of peripheral circulation after surgery using thrombin inhibitor (MD805).

We studied the effects of thrombin generation due to surgical stress on prostanoid formation and peripheral circulation in anesthetized dogs. Three experimental groups were used, consisting of a control group (group 1), a thoracotomized group (group 2), and a thoracotomized group treated with thrombin inhibitor (MD805: a synthetic arginine derivative) (group 3). The plasma concentrations of thrombin-antithrombin III complex (TAT) and prostanoids were measured along with the hemodynamic parameters. The plasma concentrations of TAT and thromboxane B2 significantly increased 1h after a thoracotomy in group 2. However, neither concentration increased after a thoracotomy in group 3. The flow ratio of the brachial and femoral arteries to cardiac output significantly decreased 1h after a thoracotomy in group 2. This study indicates that thromboxane A2 was thus synthesized by the stimulation of endogenous thrombin, while it also reduced the peripheral blood flow after surgery.

Animals↗

PubMed1997

A new method to synthesize competitor RNAs for accurate analyses by competitive RT-PCR.

A method to synthesize competitor RNAs as internal standards for competitive RT-PCR is improved by using the long accurate PCR (LA-PCR) technique. Competitor templates synthesized by the new method are almost the same in length, and possibly in secondary structure, as target mRNAs to be quantified except that they include the short deletion within the segments to be amplified. This allows the reverse transcription to be achieved with almost the same efficiency from both target mRNAs and competitor RNAs. Therefore, more accurate quantification can be accomplished by using such competitor RNAs.

Polymerase Chain Reaction↗

PubMed1997

Cathepsin K antisense oligodeoxynucleotide inhibits osteoclastic bone resorption.

Cathepsin K is a recently identified cysteine protease which is abundantly and selectively expressed in osteoclasts. To evaluate the contribution of cathepsin K to bone resorption processes, we investigated the effect of cathepsin K antisense phosphothiorate oligodeoxynucleotide (S-ODN) on the bone-resorbing activity of osteoclasts. Rabbit osteoclasts were cultured on dentine slices for 24 h in the presence or absence of antisense S-ODN in a medium containing 100 nM TfxTM-50, polycationic liposome, as a carrier of the S-ODN. Uptake of the S-ODN by osteoclasts was confirmed microscopically using fluorescein-labeled S-ODN. The treatment with antisense significantly decreased the amount of cathepsin K protein in osteoclasts. The antisense inhibited the osteoclastic pit formation in a concentration-dependent fashion. At 10 microM the antisense reduced the total pit number and area and average pit depth by 46, 52, and 30%, respectively. The sense and mismatch S-ODNs, which were used as negative controls, had no effect on either the cathepsin K protein level or the pit formation. A nonspecific cysteine protease inhibitor, E-64, also reduced pit formation in a concentration-dependent manner with maximum reductions at 1 microM of 46, 48, and 35% in the above pit parameters. The inhibitory effect of the antisense almost equal to that of E-64 demonstrates that cathepsin K is a cysteine protease playing a crucial role in osteoclastic bone resorption.

Animals↗

PubMed1997

Localization of cathepsin K in human osteoclasts by in situ hybridization and immunohistochemistry.

We have recently cloned cathepsin K from a human bone cDNA library. Since cathepsins are proposed to be involved in the degradation of mineralized bone matrix, we have investigated, by in situ hybridization and immunocytochemistry, the expression of the cathepsin K mRNA transcripts and protein in sections of bone and giant cell tumor to determine which cells express this enzyme. Within all tissues studied, cathepsin K was highly expressed in osteoclasts. Furthermore, the expression of cathepsin K mRNA in giant cell tumor tissue appeared to be confined to the periphery of the osteoclast indicating a compartmentalization of the mRNA. Immunohistochemistry confirmed the specific localization of cathepsin K to the osteoclast. In actively resorbing osteoclasts, the immunostaining was localized at the ruffled border, whereas in osteoclasts in sections of giant cell tumor, staining was observed in lysosomal vacuoles, which in some cases were seen to fuse with the cell membrane. Other cells within the bone, such as osteoblasts and osteocytes, did not express either the cathepsin K transcript or protein. However, there were very low levels of cathepsin K detected in a population of mononuclear cells, possibly representing osteoclast progenitor cells, within the marrow/stromal layer. The specific localization of cathepsin K within osteoclasts would therefore indicate the potential role of this enzyme in the bone resorptive process.

Blotting, Northern↗