Kumiko Tanabe

Mechanisms of tumor necrosis factor-α-induced interleukin-6 synthesis in glioma cells

BackgroundInterleukin (IL)-6 plays a pivotal role in a variety of CNS functions such as the induction and modulation of reactive astrogliosis, pathological inflammatory responses and neuroprotection. Tumor necrosis factor (TNF)-α induces IL-6 release from rat C6 glioma cells through the inhibitory kappa B (IκB)-nuclear factor kappa B (NFκB) pathway, p38 mitogen-activated protein (MAP) kinase and stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK). The present study investigated the mechanism of TNF-α-induced IL-6 release in more detail than has previously been reported.MethodsCultured C6 cells were stimulated by TNF-α. IL-6 release from the cells was measured by an enzyme-linked immunosorbent assay, and the phosphorylation of IκB, NFκB, the MAP kinase superfamily, and signal transducer and activator of transcription (STAT)3 was analyzed by Western blotting. Levels of IL-6 mRNA in cells were evaluated by real-time reverse transcription-polymerase chain reaction.ResultsTNF-α significantly induced phosphorylation of NFκB at Ser 536 and Ser 468, but not at Ser 529 or Ser 276. Wedelolactone, an inhibitor of IκB kinase, suppressed both TNF-α-induced IκB phosphorylation and NFκB phosphorylation at Ser 536 and Ser 468. TNF-α-stimulated increases in IL-6 levels were suppressed by wedelolactone. TNF-α induced phosphorylation of STAT3. The Janus family of tyrosine kinase (JAK) inhibitor I, an inhibitor of JAK 1, 2 and 3, attenuated TNF-α-induced phosphorylation of STAT3 and significantly reduced TNF-α-stimulated IL-6 release. Apocynin, an inhibitor of NADPH oxidase that suppresses intracellular reactive oxygen species, significantly suppressed TNF-α-induced IL-6 release and mRNA expression. However, apocynin failed to affect the phosphorylation of IκB, NFκB, p38 MAP kinase, SAPK/JNK or STAT3.ConclusionThese results strongly suggest that TNF-α induces IL-6 synthesis through the JAK/STAT3 pathway in addition to p38 MAP kinase and SAPK/JNK in C6 glioma cells, and that phosphorylation of NFκB at Ser 536 and Ser 468, and NADPH oxidase are involved in TNF-α-stimulated IL-6 synthesis.

Involvement of p38 MAP kinase in TGF-beta-stimulated VEGF synthesis in aortic smooth muscle cells.

Although it is known that transforming growth factor (TGF)‐β induces vascular endothelial growth factor (VEGF) synthesis in vascular smooth muscle cells, the underlying mechanisms are still poorly understood. In the present study, we examined whether the mitogen‐activated protein (MAP) kinase superfamily is involved in TGF‐β‐stimulated VEGF synthesis in aortic smooth muscle A10 cells. TGF‐β stimulated the phosphorylation of p42/p44 MAP kinase and p38 MAP kinase, but not that of SAPK (stress‐activated protein kinase)/JNK (c‐Jun N‐terminal kinase). The VEGF synthesis induced by TGF‐β was not affected by PD98059 or U0126, specific inhibitors of the upstream kinase that activates p42/p44 MAP kinase. We confirmed that PD98059 or U0126 did actually suppress the phosphorylation of p42/p44 MAP kinase by TGF‐β in our preparations. PD169316 and SB203580, specific inhibitors of p38 MAP kinase, significantly reduced the TGF‐β‐stimulated synthesis of VEGF (each in a dose‐dependent manner). PD169316 or SB203580 attenuated the TGF‐β‐induced phosphorylation of p38 MAP kinase. These results strongly suggest that p38 MAP kinase plays a part in the pathway by which TGF‐β stimulates the synthesis of VEGF in aortic smooth muscle cells. J. Cell. Biochem. 82: 591–598, 2001.

Involvement of MAP kinases in TGF-β-stimulated vascular endothelial growth factor synthesis in osteoblasts

Transforming growth factor-beta (TGF-beta) reportedly induces vascular endothelial growth factor (VEGF) synthesis in osteoblast-like MC3T3-E1 cells. We have recently shown that TGF-beta activates p44/p42 mitogen-activated protein (MAP) kinase and p38 MAP kinase in these cells. In the present study, we investigated the exact mechanism of TGF-beta behind the synthesis of VEGF in MC3T3-E1 cells. PD98059 and U-0126, specific inhibitors of MEK, suppressed the VEGF synthesis induced by TGF-beta. U-0126 inhibited the TGF-beta-induced p44/p42 MAP kinase phosphorylation. SB203580 and PD169316, inhibitors of p38 MAP kinase, reduced the TGF-beta-stimulated VEGF synthesis. SB202474, a negative control for p38 MAP kinase inhibitor, did not affect the VEGF synthesis. A combination with PD98059 and SB203580 almost completely suppressed the TGF-beta-induced VEGF synthesis. Retinoic acid, which alone failed to affect VEGF synthesis, markedly enhanced the VEGF synthesis stimulated by TGF-beta. Retinoic acid enhanced the TGF-beta-increased levels of VEGF mRNA. The amplifications by retinoic acid of TGF-beta-increased VEGF synthesis and levels of VEGF mRNA were reduced by PD98059 or SB203580. The combination of PD98059 and SB203580 almost completely suppressed the enhancement by retinoic acid of VEGF synthesis induced by TGF-beta. Taken together, our results strongly suggest that both p44/p42 MAP kinase and p38 MAP kinase take part in TGF-beta-stimulated VEGF synthesis in osteoblasts, and that retinoic acid upregulates the VEGF synthesis.

Simvastatin stimulates VEGF release via p44/p42 MAP kinase in vascular smooth muscle cells

It has been shown that 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) modulate vascular smooth muscle cell functions. In the present study, we investigated the effect of simvastatin on vascular endothelial growth factor (VEGF) release, and the underlying mechanism, in a rat aortic smooth muscle cell line, A10 cells. Administration of simvastatin increased the VEGF level in rat plasma in vivo. In cultured cells, simvastatin significantly stimulated VEGF release in a dose-dependent manner. Simvastatin induced the phosphorylation of p44/p42 MAP kinase but not p38 MAP kinase or SAPK (stress-activated protein kinase)/JNK (c-Jun N-terminal kinase). PD98059 and U-0126, inhibitors of the upstream kinase that activates p44/p42 MAP kinase, significantly reduced the simvastatin-induced VEGF release in a dose-dependent manner. The phosphorylation of p44/p42 MAP kinase induced by simvastatin was reduced by PD98059 or U-0126. Moreover, a bolus injection of PD98059 truly suppressed the simvastatin-increased VEGF level in rat plasma in vivo. These results strongly suggest that p44/p42 MAP kinase plays a role at least partly in the simvastatin-stimulated VEGF release in vascular smooth muscle cells.

p38 MAP Kinase Is Required for Vasopressin-Stimulated HSP27 Induction in Aortic Smooth Muscle Cells

We previously showed that arginine vasopressin (AVP) stimulates heat shock protein 27 (HSP27) induction through protein kinase C activation in aortic smooth muscle A10 cells. In the present study, we examined whether the mitogen-activated protein (MAP) kinase superfamily is involved in the AVP-stimulated HSP27 induction in A10 cells. AVP stimulated the phosphorylation of p42/p44 MAP kinase and p38 MAP kinase. On the contrary, AVP had little effect on SAPK (stress-activated protein kinase)/JNK (c-Jun N-terminal kinase) phosphorylation. The HSP27 accumulation by AVP was not affected by PD98059, an inhibitor of the upstream kinase that activates p42/p44 MAP kinase. SB203580 and PD169316, specific inhibitors of p38 MAP kinase, suppressed the AVP-induced accumulation of HSP27. 12-O-tetradecanoylphorbol 13-acetate, an activator of protein kinase C, induced accumulation of HSP27 and was not inhibited by PD98059 but was inhibited by SB203580. Calphostin C and ET-18-OCH(3), inhibitors of protein kinase C, reduced the phosphorylation of p38 MAP kinase by AVP. SB203580 and PD169316 suppressed the AVP-increased levels in mRNA for HSP27. Dissociation of the aggregated HSP27 to the dissociated HSP27 was induced by AVP. These results strongly suggest that p38 MAP kinase takes part in the pathway of the AVP-stimulated induction of HSP27 in vascular smooth muscle cells.

Mechanisms of interleukin-1β-induced GDNF release from rat glioma cells

Glial cell line-derived neurotrophic factor (GDNF) is highly expressed both in neurons and astrocytes in injured tissues. Astrocytes support neurons by releasing neurotrophic factors including GDNF. It has been reported that various agents including cytokines such as interleukin (IL)-1beta induce GDNF mRNA expression and the release in astrocytes. However, the mechanism behind the GDNF synthesis and release remains unclear. Herein, we investigated the mechanisms of the IL-1beta-induced GDNF release from rat C6 glioma cells. IL-1beta time dependently stimulated GDNF release from C6 cells. IL-1beta induced the phosphorylation of inhibitor kappa B (IkappaB), p38 mitogen-activated protein (MAP) kinase, p44/p42 MAP kinase, stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) and signal transducer and activator of transcription (STAT) 3. The IL-1beta-stimulated levels of GDNF were suppressed by wedelolactone, an inhibitor of IkappaB kinase, SB203580, an inhibitor of p38 MAP kinase, PD98059, an inhibitor of MAP kinase kinase 1/2 or Janus family of tyrosine kinase (JAK) inhibitor I, an inhibitor of upstream kinase of STAT3. On the contrary, SP600125, an inhibitor of SAPK/JNK, failed to reduce the IL-1beta-effect. These results strongly suggest that IL-1beta stimulates GDNF release through the pathways of IkappaB-nuclear factor kappa B, p38 MAP kinase, p44/p42 MAP kinase and JAK-STAT3, but not through the SAPK/JNK pathway in glioma cells.

Midazolam suppresses interleukin-1β-induced interleukin-6 release from rat glial cells.

BackgroundPeripheral-type benzodiazepine receptor (PBR) expression levels are low in normal human brain, but their levels increase in inflammation, brain injury, neurodegenerative states and gliomas. It has been reported that PBR functions as an immunomodulator. The mechanisms of action of midazolam, a benzodiazepine, in the immune system in the CNS remain to be fully elucidated. We previously reported that interleukin (IL)-1β stimulates IL-6 synthesis from rat C6 glioma cells and that IL-1β induces phosphorylation of inhibitory kappa B (IκB), p38 mitogen-activated protein (MAP) kinase, stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase 1/2, and signal transducer and activator of transcription (STAT)3. It has been shown that p38 MAP kinase is involved in IL-1β-induced IL-6 release from these cells. In the present study, we investigated the effect of midazolam on IL-1β-induced IL-6 release from C6 cells, and the mechanisms of this effect.MethodsCultured C6 cells were stimulated by IL-1β. IL-6 release from C6 cells was measured using an enzyme-linked immunosorbent assay, and phosphorylation of IκB, the MAP kinase superfamily, and STAT3 was analyzed by Western blotting.ResultsMidazolam, but not propofol, inhibited IL-1β-stimulated IL-6 release from C6 cells. The IL-1β-stimulated levels of IL-6 were suppressed by wedelolactone (an inhibitor of IκB kinase), SP600125 (an inhibitor of SAPK/JNK), and JAK inhibitor I (an inhibitor of JAK 1, 2 and 3). However, IL-6 levels were not affected by PD98059 (an inhibitor of MEK1/2). Midazolam markedly suppressed IL-1β-stimulated STAT3 phosphorylation without affecting the phosphorylation of p38 MAP kinase, SAPK/JNK or IκB.ConclusionThese results strongly suggest that midazolam inhibits IL-1β-induced IL-6 release in rat C6 glioma cells via suppression of STAT3 activation. Midazolam may affect immune system function in the CNS.

Involvement of Rho-kinase in tumor necrosis factor-α-induced interleukin-6 release from C6 glioma cells

Tumor necrosis factor (TNF)-alpha stimulated interleukin (IL)-6 release and induced the phosphorylation of myosin phosphatase targeting subunit (MYPT)-1, a Rho-kinase substrate. The IL-6 release was significantly suppressed by Y-27632 and fasudil, Rho-kinase inhibitors. Although IkappaB inhibitor suppressed the TNF-alpha-induced IL-6 release, the Rho-kinase inhibitors did not affect the TNF-alpha-induced IkappaB phosphorylation. TNF-alpha induced the phosphorylation of p38 mitogen-activated protein (MAP) kinase, stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK), and p44/p42 MAP kinase. The TNF-alpha-induced IL-6 release was suppressed by SB203580, a p38 MAPK inhibitor, or SP600125, a SAPK/JNK inhibitor, but not by PD98059, a MAP kinase/extracellular signal-regulated kinase kinase inhibitor. The Rho-kinase inhibitors attenuated the TNF-alpha-induced phosphorylation of both p38 MAP kinase and SAPK/JNK. Rho-kinase, which has been used for the clinical treatment of cerebral vasospasms, may be involved in other central nervous system (CNS) disorders such as traumatic injury, stroke, neurodegenerative disease and neuropathic pain. TNF-alpha, a proinflammatory cytokine that affects the CNS through cytokines, such as IL-6, release from neurons, astrocytes and microglia. Therefore, we investigated the involvement of Rho-kinase in the TNF-alpha-induced IL-6 release from rat C6 glioma cells. These results strongly suggest that Rho-kinase regulates the TNF-alpha-induced IL-6 release at a point upstream from p38 MAPK and SAPK/JNK in C6 glioma cells. Therefore, Rho-kinase inhibitor may be considered to be a new clinical candidate for the treatment of CNS disorders in addition to cerebral vasospasms.

Clonidine premedication modifies responses to adrenoceptor agonists and baroreflex sensitivity

PurposeTo evaluate the effects of clonidine on responses to adrenoceptor agonists and baroreflex sensitivity, we examined arterial blood pressure (AP) responses to phenylephrine and heart rate (HR) responses to isoproterenol and baroreflex sensitivity (HR response to AP changes due to phenylephrine or nitroglycerin).MethodsWe studied 60 anaesthetized patients who either did or did not receive 5 μg·kg−1 clonidinepo before they were anaesthetized. After induction of general anaesthesia, the patients received 3 gmg·kg−1 phenylephrine, 0.02 μg·kg−1 isoproterenol, or 2–3 μg·kg−1 nitroglycerin, and haemodynamic measurements were taken. Baroreflex sensitivity was expressed as the slope of the linear regression line (msec·mmHg−1; in msec of R-R interval change vs mmHg change in systolic arterial pressure) following the administration of phenylephrine and nitroglycerin.ResultsPatients who received clonidine had greater augmented responses in AP to phenylephrine and in HR to isoproterenol (47.2 ± 15.6% vs 23.7 ± 11.9% for increase in systolic AP and 59.8 ± 22.6% vs 26.2 ± 11.0% for increase in HR,P < 0.05 respectively). There were no differences between the baroreflex sensitivities in the pressor (phenylephrine) test groups (3.77 ± 1.08 vs 4.41 ± 1.66 msec·mmHg−1). In contrast, the slopes of depressor (nitroglycerin) test groups were decreased in patients receiving clonidine (1.98 ± 0.73 vs 3.68 ± 1.72 msec mmHg−1,P < 0.05).ConclusionThe results suggest that premedication with clonidine might enhance critical hypotension during anaesthesia and surgery, but restoration both of AP and HR decrease can be achieved effectively by phenylephrine and isoproterenoliv, respectively.RésuméObjectifAfin d’évaluer les effets de la clonidine sur les réponses des récepteurs adrénergiques agonistes et sur la sensibilité baroréflexe, nous avons enregistré les changements de tension artérielle (TA) liés à la phényléphrine, les modifications de fréquence cardiaque (FC) liées à l’isoprotérénol et la sensibilité baroréflexe (les réactions de la FC aux changements de TA liées à la phényléphrine ou à la nitroglycérine).MéthodeNous avons étudié 60 patients qui avaient reçu ou non 5 μg·kg−1 de clonidinepo avant l’anesthésie. Après l’induction de l’anesthésie générale, les patients ont reçu 3 μg·kg−1 de phényléphrine, 0,02 μg·kg−1 d’isoprotérénol ou 2–3 μg·kg−1 de nitroglycérine, et on a procédé aux mesures hémodynamiques. La sensibilité baroréflexe était exprimée par la pente du tracé de régression linéaire (msec·mmHg−1; en msec de changement dans l’intervalle R-R vs en mmHg de changement dans la tension artérielle systolique) à la suite de l’administration de phényléphrine et de nitroglycérine.RésultatsLes patients qui ont reçu de la clonidine ont présenté des augmentations plus importantes de TA, en réaction à la phényléphrine, et de FC, en réaction à l’isoprotérénol (47,2 ± 15,6 % vs 23,7 ± 11,9 % concernant l’augmentation de la TA systolique et 59,8 ± 22,6 % vs 26,2 ± 11,0% concernant l’augmentation de FC,P < 0,05 respectivement). Il n’y a pas eu de différence de sensibilité baroréflexe entre les groupes testés pour les réactions au médicament stimulant, phényléphrine, (3,77 ± 1,08 vs 4,41 ± 1,66 msec·mmHg−1). En comparaison, les pentes des groupes du test hypotenseur (nitroglycérine) s’abaissaient chez les patients ayant reçu de la clonidine (1,98 ± 0,73 vs 3,68 ± 1,72 msec·mmHg−1,P < 0,05).ConclusionLes résultats laissent voir que la prémédication avec de la clonidine a pu favoriser une hypotension critique pendant l’anesthésie et la chirurgie, mais que le rétablissement de TA et de FC plus basses pouvait être réalisé efficacement avec de la phényléphrine et de l’isoprotérénoliv, respectivement.

Inhibitory effects of propofol on intracellular signaling by endothelin-1 in aortic smooth muscle cells.

Background Blood pressure decreases when propofol is administered. However, the exact mechanism underlying the vascular effects of propofol has not yet been elucidated. Endothelin produced by vascular endothelial cells is a potent vasoactive peptide that elicits prolonged contraction of vascular smooth muscle cells. The effects of propofol on endothelin‐1‐induced intracellular signaling in an aortic smooth muscle cell line, A10 cells, were examined. Methods Cultured A10 cells were pretreated with propofol for 20 min and then stimulated with endothelin‐1. The effect of propofol on the endothelin‐1‐induced Ca2+ influx into A10 cells was evaluated by measuring intracellular45 Ca2+. The effects of propofol on the endothelin‐1‐induced activation of phosphatidylinositol‐hydrolyzing phospholipase C and phosphatidylcholine‐hydrolyzing phospholipase D were evaluated by measuring the formation of inositol phosphates and choline, respectively. The effect of propofol on endothelin‐1 binding to its receptor was determined by an [sup 125 I] endothelin‐1‐binding assay. Results Propofol inhibited the the endothelin‐1‐induced Ca2+ influx, but this was significant only at supuraclinical concentrations. The endothelin‐1‐stimulated formation of inositol phosphates was significantly suppressed by propofol. However, propofol had no effect on the formation of inositol phosphates induced by NaF, an activator of heterotrimeric guanosine triphosphate (GTP)‐binding proteins. Propofol inhibited the endothelin‐1‐induced formation of choline. Propofol had no effect on the binding of endothelin‐1 to its receptor. Conclusions These results suggest that propofol inhibits endothelin‐1‐induced intracellular signaling in vascular smooth muscle cells. The inhibitory effect of propofol might be exerted at a point between the endothelin‐1 receptor and its GTP‐binding protein. However, because all significant effects are observed at high concentrations, clinical relevance is unclear.