Biomedical subjects

M Shibanuma

Publications and source records attributed to M Shibanuma.

28 records · Page 2Linked to original sources

PubMed1994

Production of hydrogen peroxide by transforming growth factor-beta 1 and its involvement in induction of egr-1 in mouse osteoblastic cells.

TGF-beta 1 controls the expression of numerous genes, including early response and cellular matrix genes. However, the signal-transducing mechanism underlying this regulation of gene expression is not fully understood. In this study, we investigated whether redox regulation plays a role in the TGF-beta 1 signal transduction in the mouse osteoblastic cell line (MC3T3-E1). The overall intracellular oxidized state of the cells, when measured using 2',7'-dichlorofluorescin diacetate by laser-scanning confocal microscopy, was increased transiently after the addition of TGF-beta 1. This increase was abolished by the addition of oxygen radical scavengers such as catalase and N-acetylcysteine. In a variant cell line lacking the TGF-beta 1 receptor, the intracellular oxidized state was not modulated by treatment with TGF-beta 1. We then examined the expression of early growth response-1 (egr-1) gene, which is inducible by TGF-beta 1 and H2O2. Radical scavengers inhibited the induction of egr-1 by TGF-beta 1, but not that by 12-O-tetradecanoylphorbol-13 acetate. A nuclear run-on assay indicated that this inhibition was at the transcriptional level. From transient expression experiments using chloramphenicol acetyltransferase gene linked to serially deleted egr-1 gene 5'-upstream region, the CArG element in the 5' flanking region of egr-1 was identified as an essential sequence in the transcriptional activation for both TGF-beta 1 and H2O2 stimulation. These findings suggest that H2O2 acts as a mediator for the TGF-beta 1-induced transcription of egr-1 gene.

3T3 Cells↗

PubMed1993

Cloning from a mouse osteoblastic cell line of a set of transforming-growth-factor-beta 1-regulated genes, one of which seems to encode a follistatin-related polypeptide.

Transforming growth factor(TGF)beta 1 is a potent inhibitor of growth in mouse osteoblastic MC3T3-E1 cells. To isolate genes that are induced by TGF beta 1, the differential screening method was adopted using a cDNA library constructed from cells treated with TGF beta 1 for 4 h. Six independent cDNA clones were isolated (TGF beta-stimulated clone, TSC-5, TSC-36, TSC-115, TSC-128, TSC-160 and TSC-161), the expression of which was increased by TGF beta 1-treatment with maximal expression at 6-10 h. The steady-state levels of TSC-36, TSC-128 and TSC-160 increased almost tenfold, whereas those of TSC-5, TSC-115 and TSC-161 were elevated at most threefold. From partial nucleotide sequences, TSC-160 was found to be identical to rrg (ras-recision gene, lysyl oxidase), and TSC-115 had 80% similarity with tropomyosin cDNA, whereas other genes seemed novel. Expression of TSC-36 and TSC-160 was dramatically decreased in v-Ki-ras-transformed MC3T3 cells or in transformed NIH 3T3 cells (DT), and was recovered to normal levels in a flat revertant (C11). A nearly full-length copy of TSC-36 cDNA was isolated, and an open reading frame indicated that it encodes a protein of 35 kDa. An antiserum was raised against the C-terminal peptide predicted from the nucleotide sequence, and a polypeptide with an approximate molecular mass of 38 kDa was detected in cultured medium of MC3T3-E1 cells. The amino acid sequence of TSC-36 protein was found to have some similarity with follistatin, an activin-binding protein, and a limited similarity with the secreted protein rich in cysteine (SPARC).

3T3 Cells↗

PubMed1992

Cell-cycle dependent phosphorylation of HSP28 by TGF beta 1 and H2O2 in normal mouse osteoblastic cells (MC3T3-E1), but not in their ras-transformants.

Transforming growth factor (TGF) beta 1 increased phosphorylation of a specific protein of approximately M(r) = 30,000 (p30) in mouse osteoblastic MC3T3-E1 cells. This protein, p30, was identified as one of the small heat shock proteins (HSP) 28 from the electrophoretic pattern on two-dimensional gels, and its peptide map compared with that of heat shock-inducible p28. The increase in phosphorylation of HSP 28 seemed to correlate with growth inhibition in this cell line, since it was increased by growth inhibitory agents, such as TGF beta 1, H2O2 and 12-O-tetradecanoylphorbol-13-acetate (TPA), but not by the growth stimulating agent, epidermal growth factor (EGF), and this phosphorylation was observed only when the cells were sensitive to growth inhibition by these agents, in the late G1 phase of the cell cycle. Furthermore, in ras-transformants, whose DNA synthesis was not inhibited by these agents, this phosphorylation was not increased by these stimuli. These results indicate that phosphorylation of HSP 28 may be coupled to inhibition of DNA synthesis in this cell line.

Animals↗

PubMed1992

Isolation of a gene encoding a putative leucine zipper structure that is induced by transforming growth factor beta 1 and other growth factors.

A gene sequence (TSC-22) that is induced by transforming growth factor (TGF) beta 1 was isolated by differential screening of a lambda gt10 cDNA library constructed from poly(A)+ RNA of mouse osteoblastic cells treated with TGF-beta 1 for 2 h. TSC-22 gene expression was transcriptionally activated by TGF-beta 1. It was also induced by phorbol 12-myristate 13-acetate, serum, cholera toxin, or dexamethasone, but not appreciably by epidermal growth factor. Its induction was rapid and transient, reaching a peak 2 h after TGF-beta 1 treatment, and was resistant to cycloheximide like that of c-jun. The nucleotide sequences of TSC-22 cDNA showed no homology with any known gene sequence. The open reading frame and in vitro translation product indicated that the gene encodes a polypeptide of 143 amino acids with a molecular mass of 18 kDa that contains a putative leucine zipper structure. Polyclonal antibody was raised against TSC-22 protein expressed in Escherichia coli cells, and the antibody detected a 18-kDa protein in both the cytoplasmic and nuclear fractions of [35S]Met-labeled cells. These results indicate that the TSC-22 gene is a new member of the family of early response genes, and encodes a small polypeptide that is a putative transcriptional regulator.

Amino Acid Sequence↗

PubMed1991

Transcriptional activation of early-response genes by hydrogen peroxide in a mouse osteoblastic cell line.

H2O2, like other oxidants, is known to act as a mitogen at low concentrations in resting Balb/3T3 or mouse epidermal JB6 cells. We described previously that H2O2 induces some early response genes in Balb/3T3 cells. We extended these observations using another cell line, MC3T3 (mouse osteoblastic) cells by examination of transcriptional activity of these genes and by using inhibitors of protein kinases. H2O2 increased the expressions of c-fos, c-jun, egr-1 and JE genes which are known to be early response genes and are induced by mitogenic stimuli in many types of cells. Exogenous addition of H2O2 increased the mRNA levels of these genes, the kinetics of increase being similar to those of their inductions by a phorbol ester or serum. Nuclear run-on transcription showed that this induction occurred at the transcriptional level. H2O2 at 0.1-0.2 mM induced maximal expressions of c-fos and c-jun, whereas 0.3 mM H2O2 was required for induction of stress-induced heme oxygenase mRNA. The inductions of c-fos and c-jun were inhibited by 50 microM H7, a protein kinase inhibitor that is relatively specific for protein kinase C, but were not affected by H9, relatively specific for cAMP-dependent protein kinase. In cells pretreated with 12-O-tetradecanoylphorbol 13-acetate, however, in which protein kinase was supposed to be downregulated, H2O2 induced c-fos and heme oxygenase as efficiently as in untreated cells. H2O2 did not increase the phosphorylation of p80 protein, which is known to be a substrate for protein kinase C. Thus, H2O2 seemed to induce c-fos and c-jun by activating protein kinases distinct from protein kinase C. Activity of the chloramphenicol acetyltransferase gene under control of the serum-response element of human c-fos genes was increased by H2O2 treatment, whereas that under control of cAMP-response element was not affected. These results indicate that the inductions by H2O2 of c-fos and possibly other early response genes are mediated through activation of the serum-response element in their enhancer.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1991

Release of H2O2 and phosphorylation of 30 kilodalton proteins as early responses of cell cycle-dependent inhibition of DNA synthesis by transforming growth factor beta 1.

Transforming growth factor beta 1 (TGF-beta 1) and H2O2 both inhibited DNA synthesis of mouse osteoblastic (MC3T3) cells in the late G1 phase of the cell cycle. TGF-beta 1 stimulated cells to release H2O2 in the late G1 phase, but not in the G0 phase, even though TGF-beta 1 receptors were present in both phases. The inhibition of DNA synthesis caused by TGF-beta 1 was partly decreased by the addition of catalase. TGF-beta 1 and H2O2 increased the phosphorylation of the same proteins with a molecular weight of 30,000 in cells in the late G1 phase, and the increase by TGF-beta 1 was abolished at least partly by catalase. The results suggest that H2O2 is one of the mediators of inhibition of DNA synthesis by TGF-beta 1.

Animals↗

PubMed1990

Stimulation by hydrogen peroxide of DNA synthesis, competence family gene expression and phosphorylation of a specific protein in quiescent Balb/3T3 cells.

Treatment of quiescent Balb/3T3 clone A31-1-1 cells with 0.1-0.2 mM H2O2 in the presence of 1 microM insulin induced DNA synthesis 20-24 h later at almost the same level as that in cells treated with 10% serum. Treatment with 0.1-0.2 mM H2O2 alone did not induce DNA synthesis and was not toxic to the cells. Cell cycle analysis indicated that treatment with H2O2 plus insulin induced progression of the cell cycle from the quiescent state. The amounts of mRNA for competence family genes such as c-fos, KC and JE were increased by the addition of H2O2. Under these conditions H2O2 caused rapid phosphorylation of a protein of 78 kDa with a pI of 6.3 (p78). Phosphorylation of p78 increased on treatment with TPA and serum as well. Catalase reduced the increase in phosphorylation of p78 induced by TPA and serum. Endogenous production of H2O2 was observed within 10 min after treatment of quiescent cells with platelet derived growth factor (PDGF) or 12-O-tetradecanoylphorbol-13-acetate (TPA). These results indicate that H2O2 at certain concentrations mimics the action of competence factors on resting Balb/3T3 cells.

Animals↗

PubMed1988

Superoxide as a signal for increase in intracellular pH.

We examined the role of superoxide in the increase in intracellular pH (pHi) of human histiocytic leukemia U937 cells treated with 4 beta-phorbol-12,13-didecanoate (4 beta-PDD) or serum. 4 beta-PDD or serum induced a rapid increase in pHi, and antioxidants such as superoxide dismutase (SOD), vitamin E, and butylated hydroxyanisole (BHA) were found to inhibit the amiloride-sensitive increase in pHi induced by 4 beta-PDD. SOD inhibited the increase in pHi caused by serum, and essentially the same was found in concanavalin A-stimulated mouse thymocytes. Also, a superoxide-generating system, xanthine-xanthine oxidase (X-XOD), increased pHi of U937 cells as much as 4 beta-PDD or serum. From these findings, it appears that superoxide is the basis for the modulation of pHi.

Amiloride↗

PubMed1987

Effects of the protein kinase C inhibitor H-7 and calmodulin antagonist W-7 on superoxide production in growing and resting human histiocytic leukemia cells (U937).

Serum, phorbol 12,13-didecanoate (PDD) and 1-oleoyl-2-acetoy-sn-glycerol (OAG) stimulated O2- release in human histiocytic leukemia U937 cells. The kinetics of O2- release caused by PDD but not by serum or OAG in growing cells differed from those in resting cells. Both the protein kinase C inhibitor 1-(5-isoquinolinylsulfonyl) 2-methylpiperidine (H-7) and calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) reduced the superoxide generation induced by these stimuli. H-7 inhibited the O2- release either from growing or resting cells but the effect of W-7 varied according to the growth phase. From these results, it is suggested that activation of protein kinase C and calmodulin-dependent process has an important role in O2(-)-release induced by serum, OAG and PDD, and that the mechanism for PDD-induced O2(-)-release is different in growing and resting cells.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1987

Inhibition of proto-oncogene c-fos transcription by inhibitors of protein kinase C and ion transport.

Both 4 beta-phorbol 12,13-didecanoate (PDD) and calcium ionophore A23187 induced c-fos mRNA accumulation with similar kinetics in human monocyte-like cells (U937). Their effects were additive. Cells pretreated with PDD were not induced to accumulate c-fos mRNA by PDD but remained responsive to the induction by A23187. Similarly cells pretreated with A23187 responded to PDD but not to A23187. Nuclear run-off transcription assay indicated that increase in the c-fos mRNA level after the second inducer treatment corresponded to transcriptional activation of the c-fos gene. The accumulation of c-fos mRNA and the transcriptional activation of c-fos induced by PDD or A23187 were inhibited by the protein kinase inhibitor H-7 and by quinidine and amiloride. The induction by A23187, but not that by PDD, was also inhibited by 4-aminopyridine, or tetraethylammonium. From these results, it is proposed that activation of protein kinase C and Na+ or K+ transport are required for c-fos induction caused by PDD and A23187.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗