Biomedical subjects

Michinari Hamaguchi

Publications and source records attributed to Michinari Hamaguchi.

32 records · Page 2Linked to original sources

PubMed2003

The PLC-PKC cascade is required for IL-1beta-dependent Erk and Akt activation: their role in proliferation.

We investigated the signaling mechanisms that lead to IL-1beta-induced cell proliferation. Treatment of Balb 3T3 cells with IL-1beta activated two signaling pathways, Erk and Akt. IL-1beta also increased tyrosine phosphorylation of PLC-gamma in Src kinase-dependent manner. Pharmacological inhibition of the PLC-PKC cascade by using specific inhibitor for PLC-gamma (U73122) and PKC (GFX) strongly inhibited IL-1beta-induced Erk and Akt activation. Inhibition of MEK1 by its specific inhibitor, PD98059 substantially inhibited Erk activation. Similarly, inhibition of PI3K activation by its specific inhibitor LY294002 suppressed Akt phosphorylation. Moreover, IL-1beta-induced association of PLC-gamma with SHPS-1. SHPS-1 mutants lacking the tyrosine phosphorylation sites failed to associate with PLC-gamma. Finally, IL-1beta-induced proliferation of Balb 3T3 cells and inhibition of Erk and Akt signalings or their upstream signaling molecules, Src kinase and PKC by their inhibitors strongly inhibited IL-1beta-dependent cell proliferation. Taken together, our results suggest that a SHPS-1-PLC-gamma complex activate the PLC-PKC cascade, which is required for the activation of IL-1beta-dependent Erk and Akt signalings and cell proliferation.

Animals↗

PubMed2002

A role for SHPS-1/SIRPalpha1 in IL-1beta- and TNFalpha-dependent signaling.

We investigated the role of SHPS-1/SIRPalpha1 in IL-1beta- and TNFalpha-dependent signaling that leads to the activation of Erk 1/2 and Akt. Treatment of Balb3T3 cells with IL-1beta or TNFalpha activated tyrosine phosphorylation of SHPS-1, its association with SHP-2 and the phosphorylation of Erk 1/2 and Akt. PP1, a specific inhibitor for the Src family protein tyrosine kinases, strongly inhibited tyrosine phosphorylation of SHPS-1 and complex formation of SHPS-1 with SHP-2 by IL-1beta. In addition, PP1 substantially inhibited the IL-2beta- and TNFalpha-dependent activation of Erk 1/2 and Akt. Exogenous expression of either SHPS-1 mutants that lack SHP-2 binding function or a dominant negative mutant of SHP-2 markedly inhibited the activation of Erk 1/2 and Akt by IL-1beta, whereas wild type SHPS-1 did not. Moreover, IL-1beta-stimulation induced association of SHPS-1 with IL-1RAcP, a second subunit of IL-1 receptor, whereas expression of SHPS-1 mutant that lack SHP-2 binding function clearly blocked the association and tyrosine phosphorylation of endogenous SHPS-1. Taken together, our results strongly suggest that activation of Erk 1/2 and Akt by proinflammatory cytokines requires tyrosine phosphorylation of SHPS-1 and subsequent association of SHPS-1 with SHP-2.

3T3 Cells↗

PubMed2002

Stat3-dependent induction of BATF in M1 mouse myeloid leukemia cells.

Stat3 mediates cellular responses associated with proliferation, survival and differentiation, but the mechanisms underlying the diverse effects of this signaling molecule remain unknown. M1 mouse myeloid leukemia cells arrest growth and differentiate into macrophages following treatment with interleukin 6 (IL-6) or leukemia inhibitory factor (LIF), and recent studies have shown that Stat3 plays a central role in this process. Utilizing representational difference analysis, we demonstrate that expression of the mouse BATF gene is upregulated as an early response to IL-6/LIF stimulation and Stat3 activation in this cell system. Immunoblots using antibodies to BATF detected an increase in BATF protein in response to LIF/IL-6 stimulation. BATF is a member of the AP-1 family of basic leucine zipper transcription factors and functions to inhibit the transcriptional and biological functions of AP-1 activity in mammalian cells. BATF forms complexes with c-Jun in M1 cells and forced expression of BATF in the absence of Stat3 signaling results in a reduced rate of cellular growth. These results indicate that Stat3 mediates cellular growth by modulating AP-1 activity through the induction of BATF.

Animals↗

PubMed2002

LSSIG is a novel murine leukocyte-specific GPCR that is induced by the activation of STAT3.

G-protein-coupled receptors (GPCRs) transduce the signal of a wide variety of chemokines, cytokines, neurotransmitters, hormones, odorants, and others to regulate the biologic homeostasis, including hematopoiesis and immunity. Here we report the molecular cloning of leukocyte-specific STAT-induced GPCR (LSSIG), which is a novel murine orphan GPCR with the highest homology to human GPR43. The mRNA expression of LSSIG was clearly induced in M1 leukemia cells during the leukemia inhibitory factor (LIF)-induced differentiation to macrophages, and the induction was evidently signal transducers and activators of transcription 3 (STAT3)-dependent. GPR43 expression was also strongly induced in HL-60 and U937 leukemia cells during the differentiation to monocytes. Further analysis showed that the expression of both LSSIG and GPR43 is highly restricted in hematopoietic tissues. Cytokine-stimulation induced LSSIG and GPR43 in bone marrow cells, and monocytes and neutrophils, respectively. These results suggest that LSSIG and GPR43 might play pivotal roles in differentiation and immune response of monocytes and granulocytes.

Animals↗

PubMed2002

SHPS-1: a budding molecule against cancer dissemination.

The expression of SHPS-1 is down-regulated by several oncogene products such as v-Src in fibroblasts. In addition, the down-regulation of SHPS-1 is also found inhuman breast cancer tissues compared with the matched normal tissues. On the other hand, forced expression of SHPS-1 suppresses anchorage-independent cell growth of v-Src-transformed cells in vitro as well as peritoneal dissemination of the cells in nude mice. Surprisingly, the extracellular region (EC) of SHPS-1 seems to take part on the inhibitory effect. Because the EC domain interacts with fibronectin and DTT abrogates the inhibitory effects of the EC, we speculate that super-fibronectin may function with the EC as a suppressor of cancer cells. Here we show that SHPS-1 expression provides a unique potential that links suppression of anchorage-independent cell growth and cancer dissemination.

3T3 Cells↗

PubMed2002

Hyaluronan-CD44s signaling regulates matrix metalloproteinase-2 secretion in a human lung carcinoma cell line QG90.

We investigated the production of matrix metalloproteinase (MMP) by hyaluronan(HA) stimulation in a human cancer cell line, QG90, that expresses a large amount of CD44s, a HA receptor. Treatment of QG90 with HA strongly activated MMP-2 secretion in a time- and dose-dependent manner. We found that expression of antisense CD44s in QG90 cells substantially inhibited the HA-dependent secretion of MMP-2, whereas overexpression of full-length CD44s augmented the HA-dependent secretion of MMP-2. In addition, pretreatment of cells with the neutralizing anti-CD44 antibody significantly inhibited both the HA-dependent MMP-2 secretion and the HA-dependent activation of mitogen-activated protein kinase in a dose-dependent manner. Similarly, treatment of cells with a Ras farnesyltransferase inhibitor, manumycin A, strongly inhibited the HA-dependent MMP-2 secretion. Moreover, in vitro invasiveness of QG90 and its activation by HA were clearly suppressed by the expression of antisense CD44s. In addition, treatment of cells with anti-CD44, a mitogen-activated protein/extracellular signal-regulated kinase kinase 1 inhibitor, PD98059, or phosphatidylinositol 3'-kinase inhibitors, wortmannin and LY294002, effectively blocked the HA-dependent activation of the invasiveness. In contrast, overexpression of full-length CD44 substantially activated the invasiveness of QG90. Taken together, HA-CD44s signaling plays a key role in the HA-dependent secretion of MMP-2 and, hence, in the invasiveness of QG90 cells.

Carcinoma, Small Cell↗

PubMed2002

SHPS-1, a multifunctional transmembrane glycoprotein.

Src homology 2 (SH2) domain-containing protein tyrosine phosphatase substrate 1 (SHPS-1) is a member of the signal regulatory protein (SIRP) family. The amino-terminal immunoglobulin-like domain of SHPS-1 is necessary for interaction with CD47, a ligand for SHPS-1, which plays an important role in cell-cell interaction. The intracellular region of SHPS-1, on the other hand, may act as a scaffold protein, binding to various adapter proteins. Interestingly, increasing evidence has shown that SHPS-1 is involved in various biological phenomena, including suppression of anchorage-independent cell growth, negative regulation of immune cells, self-recognition of red blood cells, mediation of macrophage multinucleation, skeletal muscle differentiation, entrainment of circadian clock, neuronal survival and synaptogenesis. Recent progress has been made in attributing these novel exciting functions. Here we discuss how this interesting molecule works and consider its true role in biology.

Animals↗

PubMed2002

Forced expression of NESH suppresses motility and metastatic dissemination of malignant cells.

To characterize the function of a novel Src homology 3 (SH3) adapter protein termed NESH, we have established transfectants stably expressing NESH. We observed that every clone of NESH transfectants caused a marked reduction in motility, although the clones exhibited no significant differences in intrinsic cell growth compared with the control cells in vitro. The NESH transfectants also exhibited significant reduction in tumor metastatic potential in vivo. We found that NESH expression is frequently lost in invasive cancer cell lines despite its ubiquitous expression in normal tissues. The SH3 domain of NESH seems to interact with p21-activated kinase (PAK), which is involved in regulation of cell motility. Furthermore, a significant decrease in PAK phosphorylation at (402)Thr was found in NESH transfectants. Taken together, these results suggest that NESH inhibits ectopic metastasis of tumor cells as well as cell migration through interaction with intracellular molecules such as PAK that are essential for cell motility.

3T3 Cells↗

PubMed2002

Repair by Src kinase of function-impaired RET with multiple endocrine neoplasia type 2A mutation with substitutions of tyrosines in the COOH-terminal kinase domain for phenylalanine.

An oncogenic mutant of c-RET as a receptor-type tyrosine kinase, termed RET-MEN2A, displays both cell-transforming activity in vivo and strong catalytic activity in vitro. In this study, we compared the activities of mutant RET-MEN2A with substitutions of each of nine tyrosines for phenylalanine (Y1062F, Y1015F, Y981F, Y952F, Y928F, Y905F, Y900F, Y864F, and Y826F), which had been transfected into NIH 3T3 cells. In RET-MEN2A with the Y905F mutation, the cell-transforming activity was drastically reduced with a great reduction in the in vitro catalytic activity. Unexpectedly, we found that in vitro kinase activity was severely impaired in RET-MEN2A with Y981F, Y952F, or Y928F mutation, which displayed near-normal cell-transforming activity and only a partially impaired tyrosine phosphorylation level in vivo. Phosphoamino acid analysis actually demonstrated some increase in phosphotyrosine in the Y905F mutant but no or barely detectable increase in the Y981F, Y952F, or Y928F mutant after incubation for in vitro kinase assay. This suggested a crucial role of the Y981/Y952/Y928-linked structural integrity of the COOH end of the catalytic domain of RET in starting Y905 autophosphorylation. Interestingly, the apparent defect in intrinsic kinase activity in vitro in the Y981F, Y952F, or Y928F mutant, but not the reduction in activity in the Y905F mutant, could be partially repaired or restored by c-Src or, more extensively, by v-Src, which promoted Y905 phosphorylation in trans. A complex was shown to be formed between v-Src and RET-MEN2A through association of both with a cholesterol-rich membrane microdomain known as "a raft," possibly for efficient contact of submembranous domains of Src and RET to promote phosphorylation of Y905 of the latter. Finally, endogenous c-Src was shown to promote Y905 phosphorylation of the Y981F mutant in vivo. These results reveal a novel Src kinase-mediated repair mechanism of otherwise function-impaired mutant RET kinases.

3T3 Cells↗

PubMed2002

Abnormal accumulation of hyaluronan matrix diminishes contact inhibition of cell growth and promotes cell migration.

Elevated hyaluronan biosynthesis and matrix deposition correlates with cell proliferation and migration. We ectopically expressed three isoforms of hyaluronan synthase (HAS1, HAS2, or HAS3) in nontransformed rat 3Y1 cells and observed a de novo, massive formation of a hyaluronan matrix that resulted in a partial loss of contact-mediated inhibition of cell growth and migration. All three HAS transfectants showed an enhanced motility in scratch wound assays, and a significant increase in their confluent cell densities. In high-density cultures, the HAS transfectants had a fibroblastic cell shape and markedly formed overlapping cell layers. This phenotype was more pronounced in the HAS2 transfectants than HAS1 or HAS3 transfectants, and occurred with significant alterations in the microfilament organization and N-cadherin distribution at the cell-cell border. Inhibition of a phosphatidylinositol 3-kinase (PI3-kinase) pathway resulted in reacquisition of the normal phenotype of HAS2 transfectants, suggesting that the intracellular PI3-kinase signaling regulates diminution of contact inhibition induced by formation of the massive hyaluronan matrix. Our observations suggest that hyaluronan and its matrix can modulate contact inhibition of cell growth and migration, and provide evidence for functional differences between hyaluronan synthesized by the different HAS proteins.

Agar↗

PubMed2002

A role for focal adhesion kinase in hyluronan-dependent MMP-2 secretion in a human small-cell lung carcinoma cell line, QG90.

Hyluronan (HA), a nonsulfated high-molecular mass glycoaminoglycan, has been assigned as a clinical marker for the progression of various tumors. We found that HA stimulation of QG90, a cell line derived from human small-cell lung carcinoma, activates the secretion of matrix metalloproteinase-2 (MMP-2) in a focal adhesion kinase (FAK)-dependent manner. HA stimulation of QG90 cells activated MMP-2 secretion in a time-dependent manner. Larger sizes of HA seemed to have higher activities than smaller size one in MMP-2 secretion. Under HA stimulation, tyrosine phosphorylation of cellular proteins including FAK was activated. By use of antisense oligonucleotide to FAK, we found that FAK signaling was required for the activation of MMP-2 secretion and for the sustained activation of MAP kinase by HA treatment. These results strongly suggest that FAK-MAPK signaling is involved, at least in part, in HA-dependent activation of MMP-2 secretion in QG90 cells.

Carcinoma, Small Cell↗

PubMed2002

Establishment of immortal normal and ataxia telangiectasia fibroblast cell lines by introduction of the hTERT gene.

To establish immortal human cells, we introduced the human catalytic subunit of telomerase (hTERT) gene into skin fibroblast cells obtained from normal and ataxia telangiectasia (AT) individuals of Japanese origin. After hTERT introduction, these cells continue to grow beyond a population doubling number of 200 while maintaining their original radiosensitivity. Inductions of p53, phosphorylation of Ser15 in p53, and induction of p21 by X-ray irradiation in immortal cells derived from normal individual were not affected by the hTERT introduction. Both normal and AT immortal cells exhibited an apparent inhibition of growth as original primary cells when they reached confluence. Karyotype analysis has revealed that they are in a diploid range. These results suggest that cells immortalized by hTERT introduction retain their original characteristics except for immortalization, and that they may be useful for analyzing various effects of radiation on human cells.

Ataxia Telangiectasia↗

PubMed2002

In search of a function for the E3B1/Abi2/Argbp1/NESH family (Review).

An E3B1/Abi2/Argbp1/NESH protein family has recently emerged from analysis of the sequence and structural similarities. These are adaptor proteins possessing homeobox homology domain, proline rich region and Src-homology 3 (SH3) domain that interact with Abl-family tyrosine kinases. Some of the members of this family are involved in cytoskeletal reorganization that plays important roles in membrane-ruffling, lamellipodia formation and cell migration. Furthermore, it seems likely that this family may also regulate cancer development through modulation of the oncogenic Abl kinase. The knowledge of this status is helpful in thinking of the regulatory mechanisms to cell metastasis and to malignant transformation of cancer. We discuss how these interesting molecules work and consider their involvement in cancer.

Adaptor Proteins, Signal Transducing↗

PubMedDate not supplied

Expression of p73 gene, cell proliferation and apoptosis in breast cancer: Immunohistochemical and clinicopathological study.

p73 is a member of p53 tumor suppressor protein family. In spite of similarities of three meaningful domains between p53 and p73, the exact function of p73 protein is not established. To approach the relevance of p73 expression to a pathological parameter in human breast cancer, the apoptotic index (AI) and the mitotic index (MI) were examined for 75 patients with the breast cancer after mastectomies. We found that both of the AI and MI in p73-positive cases were significantly higher compared to the p73-negative cases, suggesting that the p73-positive cases in the breast cancer were in advanced grade. In six cases of breast cancer with distant metastasis, however, the p73 expression was slight and showing low AI but high MI. Advanced breast cancer with distant metastasis seemed to lose the p73 expression and gain the function of evading apoptosis. Altogether, these data demonstrate that p73 expression is well correlated with AI in breast cancer.

Adenocarcinoma↗