Yasunari Kanda

Reactive oxygen species mediate adipocyte differentiation in mesenchymal stem cells.

AIMSnMesenchymal stem cells (MSC) have the potential to differentiate into various cell lineages, including adipocytes and osteoblasts. The formation of adipose tissue involves the commitment of MSC to the preadipocyte lineage and the differentiation of preadipocytes into mature adipocytes. In the present study, we investigated the involvement of reactive oxygen species (ROS) in adipocyte differentiation from MSC.nnnMAIN METHODSnROS signaling was evaluated by the effects of antioxidant N-acetyl-l-cysteine (NAC) or shRNA against NAD(P)H oxidase in the multipotent mesenchymal stem cell line 10T1/2 cells. Intracellular ROS was measured using an H(2)DCF dye.nnnKEY FINDINGSnWe found that NAC blocked adipocyte differentiation in MSC. An H(2)DCF assay revealed that differentiation-inducing agents induced ROS generation. These data suggest that ROS is involved in adipocyte differentiation in MSC. Next, we examined the source of ROS. Knockdown of NAD(P)H oxidase 4 (Nox4) by RNA interference inhibited ROS production and adipocyte differentiation by differentiation-inducing agents. Furthermore, treatment with NAC blocked the transcriptional activation of CREB, and the expression of dominant-negative mutants of CREB inhibited adipocyte differentiation.nnnSIGNIFICANCEnThe findings suggest that the increase in the intracellular ROS level via Nox4 mediates adipocyte differentiation through CREB in MSC. This data will provide new insight into the drug development for obesity.

Putative membrane-bound estrogen receptors possibly stimulate mitogen-activated protein kinase in the rat hippocampus

We investigated whether 17beta-estradiol affects the activity of extracellular signal-regulated kinase (ERK) in the central nervous system in vivo. 17beta-Estradiol was administered intracerebroventricularly, and then ERK activity in the rat hippocampus was measured. We found that ERK activity in the rat hippocampus had increased to approximately threefold its basal level at 5 min. This rapid effect was mimicked by the membrane-impermeable estradiol, bovine serum albumin-conjugated 17beta-estradiol, and was not inhibited by tamoxifen and 7alpha,9-(4,4,5,5, 5-pentafluoropentylsulphinyl)nonylestra-1,3,5(10)-triene-3, 17beta-diol (ICI 182780), classical nuclear estrogen receptor antagonists. These data suggest that the rapid activation of ERK by estradiol in vivo is mediated through a putative membrane estrogen receptor in the rat hippocampus.

Neuroprotection by estrogen via extracellular signal-regulated kinase against quinolinic acid-induced cell death in the rat hippocampus

Extracellular signal‐regulated kinase (ERK) belongs to the family of mitogen‐activated protein kinases (MAPKs), which are serine‐threonine kinases activated by phosphorylation in response to a variety of mitogenic signals. We previously reported that 17β‐estradiol rapidly activates ERK in the rat hippocampus. However, the physiological role of this rapid activation of ERK by estrogen in vivo has not yet been elucidated. This study investigated whether ERK may participate in mediating the neuroprotective effects of estrogen against quinolinic acid (QA) toxicity in the rat hippocampus in vivo. Injection of QA into the hippocampi of male rats produced a loss of Nissl‐stained neurons in the CA1 after 24u2003h. Prior administration of 17β‐estradiol (50u2003pmol/animal) to the ventricles prevented the QA‐induced decrease in Nissl‐stained neurons. Pretreatment with U0126, an inhibitor of MAPK/ERK kinase, inhibited the rapid activation of ERK by 17β‐estradiol in the rat hippocampus. Moreover, the neuroprotective effects of 17β‐estradiol against QA toxicity were blocked by the pretreatment with U0126. U0126 alone did not produce a loss of neurons. These results indicate that ERK mediates estrogen neuroprotection after QA toxicity in the rat hippocampus.

Thrombin-induced p38 mitogen-activated protein kinase activation is mediated by epidermal growth factor receptor transactivation pathway.

Thrombin is a potent mitogen for vascular smooth muscle cells (VSMC) and has been implicated its pathogenic role in vascular remodelling. However, the signalling pathways by which thrombin mediates its mitogenic response are not fully understood. We have previously reported that thrombin activates p38 mitogen‐activated protein kinase (p38 MAPK) by a tyrosine kinase‐dependent mechanism, and that p38 MAPK has a role in thrombin‐induced mitogenic response in rat VSMC. In the present study, we examine the involvement of epidermal growth factor (EGF) receptor in thrombin‐induced p38 MAPK activation. We found that thrombin induced EGF receptor tyrosine phosphorylation (transactivation) in A10 cells, a clonal VSMC cell line. A selective inhibitor of EGF receptor kinase (AG1478) inhibited the p38 MAPK activation in a dose‐dependent manner, whereas it had no effect on the response to platelet‐derived growth factor (PDGF). EGF receptor phosphorylation induced by thrombin was inhibited by BAPTA‐AM and GF109203X, which suggest a requirement for intracellular Ca2+ increase and protein kinase C. We next examined the effect of AG1478 on thrombin‐induced DNA synthesis. AG1478 inhibited thrombin‐induced DNA synthesis in a dose‐dependent manner. In contrast, PDGF‐induced DNA synthesis was not affected by AG1478. In conclusion, these data suggest that the EGF receptor transactivation and subsequent p38 MAPK activation is required for thrombin‐induced proliferation of VSMC.

Targeted DNA transfection into the mouse central nervous system using laser- induced stress waves

We investigated the feasibility of gene transfer into the mouse central nervous system (CNS) by applying nanosecond pulsed laser-induced stress waves (LISWs). Intraventricular or hippocampal injection of a reporter gene [enhanced green fluorescent protein (EGFP)] followed by application of LISWs showed this method to be efficient in the CNS of newborn and adult mice. Cells expressing EGFP reside at least 3.5 mm from the surface of the tissue, while no apparent damage was detected. Additionally, expression of EGFP was limited to the area that was exposed to LISWs. Using this method, the formulation of plasmid DNA by cationic transfer reagent polyethylenimine proved to be effective for improving transfer efficiency into the CNS.

The stimulation of β3-adrenoceptor causes phosphorylation of extracellular signal-regulated kinases 1 and 2 through a Gs- but not Gi-dependent pathway in 3T3-L1 adipocytes

Abstract The treatment of 3T3-L1 adipocytes with three β 3 -adrenoceptor agonists, (±)-( R *, R *)-(4-[2-([2-(3-chlorophenyl)-2-hydroxyethyl]amino)propyl]phenoxy)acetic acid (BRL37344), 4-[3-[(1,1-dimethylethyl)amino]-2-hydroxypropoxy]-1, 3-dihydro-2H-benzimidazol-2-one (CGP12177) and [(7S)7-{(2R)2-(3-chlorophenyl)-2-hydroxyethyl-amino}-5,6,7,8-tetrahydronapht-2-yl]ethyl oxyacetate, hydrochloride (SR58611) induces phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2). The phosphorylations were not affected by pretreatment of the adipocytes with pertussis toxin, whereas the same treatment completely abolished lisophosphatidic acid-induced phosphorylation of ERK1/2, suggesting the role of pertussis toxin-insensitive G protein in the ERK1/2 phosphorylation by stimulation with the β 3 -adrenoceptor agonists. The phosphorylation of ERK1/2 was mimicked by treating the adipocytes with cholera toxin, a direct activator of stimulatory G (G s ) protein. In addition, the ERK1/2 phosphorylations by the β 3 -adrenoceptor agonists were completely diminished by long-term treatment of the adipocytes with cholera toxin (100 ng/ml, 24 h), whereas that obtained with lisophosphatidic acid stimulation was not. Our findings strongly suggest that the three β 3 -adrenoceptor agonists induce ERK1/2 phosphorylation in 3T3-L1 adipocytes through a G s protein-dependent cascade.

Thrombin activates p38 mitogen-activated protein kinase in vascular smooth muscle cells

Thrombin is a potent mitogen for vascular smooth muscle cells. However, the signaling pathways by which thrombin mediates its mitogenic response are not fully understood. The ERK (extracellular signal-regulated protein kinase) and JNK (c-Jun N-terminal kinase) members of the mitogen-activated protein kinase (MAPK) family are reported to be activated by thrombin. We have investigated the response to thrombin of another member of the MAPK family, p38 MAPK, which has been suggested to be activated by both stress and inflammatory stimuli in vascular smooth muscle cells. We found that thrombin induced time- and dose-dependent activation of p38 MAPK. Maximal stimulation of p38 MAPK was observed after a 10-min incubation with 1 unit ml(-1) thrombin. GF109203X, a protein kinase C inhibitor, and prolonged treatment with phorbol 12-myristate 13-acetate partially inhibited p38 MAPK activation. A tyrosine kinase inhibitor, genistein, also inhibited p38 MAPK activation in a dose-dependent manner. p38 MAPK activation was inhibited by overexpression of betaARK1ct (beta-adrenergic receptor kinase I C-terminal peptide). p38 MAPK activation was also inhibited by expression of dominant-negative Ras, not by dominant-negative Rac. We next examined the effect of a p38 MAPK inhibitor, SB203580, on thrombin-induced proliferation. SB203580 inhibited thrombin-induced DNA synthesis in a dose-dependent manner. These results suggest that thrombin activates p38 MAPK in a manner dependent on Gbetagamma, protein kinase C, a tyrosine kinase, and Ras, that p38 MAPK has a role in thrombin-induced mitogenic response in the cells.

Adrenaline increases glucose transport via a Rap1-p38MAPK pathway in rat vascular smooth muscle cells.

Adrenaline has been implicated in the pathogenesis of atherosclerosis. However, little is known regarding the role of adrenaline in glucose transport in VSMC.

Dopamine D2 receptor stimulation promotes the proliferation of neural progenitor cells in adult mouse hippocampus.

We initially examined the effects of apomorphine in vitro using mouse embryonic and adult neural progenitor cells. The effects of apomorphine treatment led to dose-dependent increases in the number of embryonic and adult neural progenitor cells, and dopamine D2 receptor antagonist treatment significantly reduced the increases induced by apomorphine. Next, we investigated the effects of apomorphine in vivo in the adult mouse hippocampus. The effects of single-dose apomorphine administration led to an increase of approximately 30% in the number of bromodeoxyuridine-positive cells in the dentate gyrus. Moreover, the chronic apomorphine administration induced an increase in the number of bromodeoxyuridine-positive cells by about 30%. Thus, we suggest that the stimulation of dopamine D2 receptors increases the proliferation of neural progenitor cells both in vivo and in vitro.

Stimulation of β3‐adrenoceptors causes phosphorylation of p38 mitogen‐activated protein kinase via a stimulatory G protein‐dependent pathway in 3T3‐L1 adipocytes

This study deals with phosphorylation and activation of p38 mitogen‐activated protein kinase (MAPK) via β3‐adrenoceptor (AR) and the signal transduction pathway in 3T3‐L1 adipocytes. β3‐AR agonist BRL37344A (10u2003nM) caused phosphorylation and activation of p38 MAPK in 3T3‐L1 adipocytes but not in fibroblasts. BRL37344A and also the other β3‐AR agonists, CGP12177A and SR58611A, caused p38 MAPK phosphorylation in dose‐dependent manners. The p38 MAPK phosphorylations by BRL37344A (10u2003nM), CGP12177A (100u2003nM), and SR58611A (10u2003nM) were not antagonized by β1‐ and β2‐ARs antagonist 1‐propranolol (100u2003nM) but blocked by β3‐AR antagonist SR59230A (10u2003μM), suggesting the phosphorylation was caused via β3‐AR. The phosphorylations of p38 MAPK were completely abolished by treatment with cholera toxin (CTX) but not pertussis toxin (100u2003ngu2003ml−1, 24u2003h). Activation of Gs by CTX (100u2003ngu2003ml−1) and adenylyl cyclase by forskolin mimicked p38 MAPK phosphorylation. p38 MAPK phosphorylation by BRL37344A was reduced to almost 50% by cyclic AMP‐dependent protein kinase (PKA) inhibitors such as H89 (10u2003μM) and PKI (10u2003μM). A src‐family tyrosine kinases inhibitor PP2 (1u2003μM) also halved the p38 MAPK phosphorylation. Combined use of H89 (10u2003μM) and PP2 (10u2003μM) did not bring about further inhibition. These results suggest that β3‐AR caused phosphorylation of p38 MAPK via Gs protein and partly through a pathway involving PKA and src‐family kinase(s), although the contribution of the unidentified pathway remains to be clarified.