Makio Hayakawa

Tyrosine Phosphorylation of β-Catenin and Plakoglobin Enhanced by Hepatocyte Growth Factor and Epidermal Growth Factor in Human Carcinoma Cells

The effect of hepatocyte growth factor /scatter factor (HGF/SF) and epidermal growth factor (EGF) on cadherin-mediated adhesion of human carcinoma cells was studied. HGF/SF induced scattering of colonic adenocarcinoma HT29 and gastric adenocarcinomas MKN7 and MKN74 cells. Likewise, EGF induced scattering of HT29 and MKN7 cells. These cells expressed E-cadherin, which was concentrated at cell-cell contact sites. When the scattering of these cells was induced by HGF/SF or EGF, the E-cadherin concentration at cell-cell boundaries tended to decrease. Irnmunoblotting analyses, however, demonstrated that these growth factor treatments did not alter the expression of E-cadherin and E-cadherin-associated proteins, α- and β-catenin and plakoglobin. β-Catenin, plakoglobin and an unidentified 115-kDa molecule associated with E-cadherin were found to be phosphorylated at tyrosine residues, and these phosphorylations were enhanced by the growth factor treatments. These results suggest that HGF/SF and EGF may modulate ...

Evidence that reactive oxygen species do not mediate NF‐κB activation

It has been postulated that reactive oxygen species (ROS) may act as second messengers leading to nuclear factor (NF)‐κB activation. This hypothesis is mainly based on the findings that N‐acetyl‐L‐cysteine (NAC) and pyrrolidine dithiocarbamate (PDTC), compounds recognized as potential antioxidants, can inhibit NF‐κB activation in a wide variety of cell types. Here we reveal that both NAC and PDTC inhibit NF‐κB activation independently of antioxidative function. NAC selectively blocks tumor necrosis factor (TNF)‐induced signaling by lowering the affinity of receptor to TNF. PDTC inhibits the IκB–ubiquitin ligase activity in the cell‐free system where extracellular stimuli‐regulated ROS production does not occur. Furthermore, we present evidence that endogenous ROS produced through Rac/NADPH oxidase do not mediate NF‐κB signaling, but instead lower the magnitude of its activation.

Novel insights into FGD3, a putative GEF for Cdc42, that undergoes SCFFWD1/β‐TrCP‐mediated proteasomal degradation analogous to that of its homologue FGD1 but regulates cell morphology and motility differently from FGD1

We previously demonstrated that FGD1, the Cdc42 guanine nucleotide exchange factor (GEF) responsible for faciogenital dysplasia, is targeted by the ubiquitin ligase SCFFWD1/β‐TrCP upon phosphorylation of two serine residues in its DSGIDS motif and subsequently degraded by the proteasome. Here we show that FGD3, which was identified as a homologue of FGD1 but has been poorly characterized, has conserved the same motif and is down‐regulated similarly by SCFFWD1/β‐TrCP. Although FGD3 and FGD1 share strikingly similar Dbl homology (DH) domains and adjacent pleckstrin homology (PH) domains, both of which are responsible for guanine nucleotide exchange, there also exist remarkable differences in their structures. Indeed, FGD1 and FGD3 induced significantly different morphological changes in HeLa Tet‐Off cells: whereas FGD1 induced long finger‐like protrusions, FGD3 induced broad sheet‐like protrusions when the level of GTP‐bound Cdc42 was significantly increased by the inducible expression of FGD3. Furthermore, FGD1 and FGD3 reciprocally regulated cell motility: when inducibly expressed in HeLa Tet‐Off cells, FGD1 stimulated cell migration whereas FGD3 inhibited it. Thus we demonstrate that the highly homologous GEFs, FGD1 and FGD3 play different roles to regulate cellular functions but that their intracellular levels are tightly controlled by the same destruction pathway through SCFFWD1/β‐TrCP.

The FWD1/β‐TrCP‐mediated degradation pathway establishes a ‘turning off switch’ of a Cdc42 guanine nucleotide exchange factor, FGD1

FWD1/β‐TrCP is the F‐box protein that functions as the receptor subunit of the SCFFWD1/β‐TrCP ubiquitin ligase and has been shown to be responsible for the degradation of important signaling molecules such as IκBs and β‐catenin. Protein substrates of FWD1/β‐TrCP contain a consensus DSGΨXS motif (where Ψ represents a hydrophobic residue and X represents any amino acid). Recognition by FWD1/β‐TrCP requires phosphorylation of the conserved serines in that motif. Here we show that FGD1, a Cdc42 guanine nucleotide exchange factor (GEF), is a novel target of the SCFFWD1/β‐TrCP ubiquitin ligase. A mutant FGD1 protein, FGD1(SA), in which both of the critical serine residues in the DSGΨXS motif have been replaced by alanines, does not interact with FWD1/β‐TrCP and exhibits increased stability. Morphological changes induced by wild‐type FGD1 (FGD1(WT)) are reduced by the co‐expression of SCFFWD1/β‐TrCP whereas those induced by FGD1(SA) are not affected. FGD1(SA)‐expressing cells show a higher level of cell motility than FGD1(WT)‐expressing cells. We present a novel ‘turning off’ mechanism for the inactivation of FGD1, an upstream regulator for Cdc42.

Prostaglandins antagonize fibroblast proliferation stimulated by tumor necrosis factor

Tumor necrosis factor (TNF) is known to be a mitogen for human diploid FS-4 fibroblasts. We have shown in an earlier study (Hori et al. (1989) J. Cell. Physiol. 141, 275-280) that indomethacin further enhances the cell proliferation stimulated by TNF. Since indomethacin inhibits the activity of cyclooxygenase, the role of prostaglandins in TNF-stimulated cell growth was examined. Cell growth stimulated by TNF and indomethacin was inhibited by exogenously added prostaglandins (PGE2, PGF2 alpha, and PGD2), among which PGE2 caused the greatest inhibition of cell growth. Treatment of FS-4 cells with 10 ng/ml TNF resulted in the release of prostaglandins (PGE2, 6-keto-PGF1 alpha, PGA2, PGD2, and PGF2 alpha) 2 to 4 fold over that of untreated cells. The amount of all these prostaglandins increased in a time-dependent manner over 6 h after treatment. In both TNF-treated and control cells, PGE2 was released as the predominant prostaglandin. Furthermore, when PGE2 production and DNA synthesis were determined in FS-4 cells treated with increasing doses of indomethacin, these two cellular responses were inversely affected by indomethacin. These data show that prostaglandins induced by TNF antagonize growth stimulatory action of TNF.

Critical role of farnesoid X receptor for hepatocellular carcinoma cell proliferation

Farnesoid X receptor (FXR), a pivotal factor maintaining bile acid homeostasis, has been recently shown to be a critical factor required for liver regeneration. The elucidation of the mechanism how FXR controls the proliferation of hepatocellular carcinoma cells is useful to establish the therapy for liver cancer. Here, we show that FXR plays a crucial role in the proliferation of human hepatocellular carcinoma cell line, HepG2, Huh7 and HLE. The treatment of HepG2 with FXR siRNA elevates the level of p16/INK4a expression resulting in the inhibition of cell proliferation. By contrast, FXR activation reduces p16/INK4a expression and stimulates the cell proliferation. The ectopic expression of the active form of Ras that causes strong activation of extracellular signal-regulated kinase (ERK) leads to the decrease in FXR expression, suggesting that FXR expression is negatively regulated via Ras/ERK pathway. The elevation of p16/INK4a expression and the inhibition of cell proliferation by FXR knockdown are also observed in Huh7 and HLE. In this study, we have suggested a novel mechanism by which hepatocellular carcinoma cell proliferation is regulated: FXR stimulates cell proliferation by suppressing the p16/INK4a expression, whereas Ras/ERK pathway down-regulates the FXR expression, leading to the suppressed cell proliferation in hepatocellular carcinoma cell lines.

Overexpression of oxidized protein hydrolase protects COS-7 cells from oxidative stress-induced inhibition of cell growth and survival

Oxidized protein hydrolase (OPH) preferentially degrades oxidatively damaged proteins in vitro and is widely distributed in various cells and tissues. The role of OPH in intact cells exposed to oxidative stress was examined. For this purpose, using COS-7, a cell line derived from African green monkey kidney, COS-7-OPH cells that stably overexpressed OPH were established. When COS-7-OPH cells were exposed to oxidative stress induced by H(2)O(2) and paraquat, accumulation of protein carbonyls in the cells was apparently lower than that of parental COS-7 cells, and COS-7-OPH cells were significantly resistant to the oxidative stress compared with parental COS-7 cells. The majority of overexpressed OPH in the cells was found to be located uniformly in cytosol, and its location was not altered by H(2)O(2)-induced oxidative stress. Above results indicate that OPH in intact cells plays a preventive role against oxidative stress and suggest that OPH relieves cells from accumulation of oxidatively damaged proteins.

Stimulation of prostaglandin production by hepatocyte growth factor in human gastric carcinoma cells

Hepatocyte growth factor (HGF), a protein with pleiotropic biological activity affecting cell growth and motility, was found to markedly activate prostaglandin production in human gastric carcinoma TMK‐1 cells. HPLC analysis revealed that HGF stimulated the production of prostaglandin E2 (PGE2), which is the major prostaglandin produced in these cells. HGF maximally stimulated PGE2 production at a concentration of 10 , and it was a more potent stimulator of PGE2 production than epidermal growth factor (EGF), which is known to stimulate prostaglandin production in various cell lines. The simultaneous addition of HGF and EGF caused no further stimulation of the PGE2 production observed in HGF‐treated cells. We showed also that HGF increased the arachidonate release from TMK‐1 cells, which release was completely suppressed by the addition of phospholipase A2 (PLA2) inhibitors. Further studies in vitro showed that HGF enhanced cellular activities of cytosolic PLA2 and cyclooxygenase 1.5‐fold each. These results indicate that HGF stimulates prostaglandin production through increases in both cytosolic PLA2 and cyclooxygenase activities.

Hypoxia downregulates farnesoid X receptor via a hypoxia‐inducible factor‐independent but p38 mitogen‐activated protein kinase‐dependent pathway

Farnesoid X receptor (FXR), a member of the nuclear receptor superfamily, has been shown to play pivotal roles in bile acid homeostasis by regulating the biosynthesis, conjugation, secretion and absorption of bile acids. Accumulating data suggest that FXR signaling is involved in the pathogenesis of liver and metabolic disorders. Here we show that FXR expression is significantly suppressed in HepG2 cells exposed to hypoxia. Concomitantly, the expression of the bile salt export pump, known as an FXR target gene product and responsible for the excretion of bile acids from the liver, is also decreased under hypoxia. Overexpression of hypoxia‐inducible factor (HIF)‐1α does not mimic the suppressive effect of hypoxia on FXR expression. Furthermore, simultaneous knockdown of HIF‐1α, HIF‐2α and HIF‐3α fails to restore the FXR expression level under hypoxia, indicating that HIF is not involved in hypoxia‐evoked FXR downregulation. Instead, we demonstrate that p38 mitogen‐activated protein kinase is an indispensable factor for FXR downregulation under hypoxia. Thus, we propose a novel liver disorder model in which two signaling molecules, p38 mitogen‐activated protein kinase and FXR, may contribute to the linkage of two pathogenic conditions, i.e. ischemia, a condition accompanying hypoxia, and cholestasis, a condition with intrahepatic accumulation of cytotoxic bile acids.

Solubilization of human placental tumor necrosis factor receptors as a complex with a guanine nucleotide-binding protein.

Human placental membranes exhibited high-affinity receptors for tumor necrosis factor (TNF) (Kd = 5.6 x 10(-10) M) with a density of 1.2-1.7 x 10(10) sites/mg protein. The receptors were solubilized from these membranes with 1% Nonidet P-40, and the solubilized receptor was adsorbed to Con A-Sepharose and wheat germ agglutinin agarose columns, indicating that the TNF receptor derived from human placenta contains carbohydrate chains recognized by these lectins. TNF binding activity was eluted from a column of Sephacryl S-300 as a single peak of Mr 300 kDa. The solubilized receptor was further purified by TNF-Sepharose prepared by coupling of TNF to tresyl-activated Sepharose 4B. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the purified sample resolved five major bands of Mr 90, 78, 41, 35, and 11 kDa, suggesting that these polypeptides constitute a multimeric complex with a molecular mass of 300 kDa, as observed in gel filtration study. Furthermore, the TNF-Sepharose-bound fraction demonstrated GTP gamma S binding and GTPase activity. Immunoblot analysis showed that the 41- and 35-kDa polypeptides were recognized by antisera against alpha subunits and beta subunit of GTP-binding proteins, respectively. These results suggest that the native TNF receptor couples to a guanine nucleotide-binding protein to form a large complex structure in human placental membranes.