• Proceedings of the PSK Conference
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• 2003.10b
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• pp.144.1-144.1
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• 2003

We previously reported that the activation of p$21^{WAFl/Cip1}$ transcription by histone deacetylase inhibitor apicidin was mediated through Spl sites and pointed to the possible participation of protein kinase C (PKC). In this study, we investigated the role and identity of the specific isoforms of PKC involved and identified phosphatidylinositol 3-kinase (PI 3-kinase) as an upstream effector in HeLa cells. Using an isoform-specific pharmacological inhibitor of PKC, a PKC$\varepsilon$ dominant-negative mutant, and antisense oligonucleotide to inhibit PKC$\varepsilon$ specifically, (omitted)

• Proceedings of the Korean Biophysical Society Conference
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• 2003.06a
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• pp.29-29
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• 2003

Compartmentation of intracellular signaling pathways serves as an important mechanism conferring the specificity of G protein-coupled receptor (GPCR) signaling. In the heart, stimulation of $\beta$$_2$-adrenoceptor ($\beta$$_2$-AR), a prototypical GPCR, activates a tightly localized protein kinase A (PKA) signaling, which regulates substrates at cell surface membranes, bypassing cytosolic target proteins (eg, phospholamban). Although a concurrent activation of $\beta$$_2$-AR-coupled $G_{i}$ proteins has been implicated in the functional compartmentation of PKA signaling, the exact mechanism underlying the restriction of the $\beta$$_2$-AR-PKA pathway remains unclear. In the present study, we demonstrate that phosphatidylinositol 3-kinase (PI3K) plays an essential role in confining the $\beta$$_2$-AR-PKA signaling. Inhibition of PI3K with LY294002 or wortmannin enables $\beta$$_2$-AR-PKA signaling to reach intracellular substrates, as manifested by a robust increase in phosphorylation of phospholamban, and markedly enhances the receptor-mediated positive contractile and relaxant responses in cardiac myocytes. These potentiating effects of PI3K inhibitors are not accompanied by an increase in $\beta$$_2$-AR-induced cAMP formation. Blocking $G_{i}$ or $G_{$\square$$\square$}$ signaling with pertussis toxin or $\beta$ARK-ct, a peptide inhibitor of $G_{$\square$$\square$}$, completely prevents the potentiating effects induced by PI3K inhibition, indicating that the pathway responsible for the functional compartmentation of $\beta$$_2$-AR-PKA siglaling sequentially involves $G_{i}$, $G_{$\square$$\square$}$, and PI3K. Thus, PI3K constitutes a key downstream event of $\beta$$_2$-AR- $G_{i}$ signaling, which confines and negates the concurrent $\beta$$_2$-AR/Gs-mediated PKA signaling.gnaling.

• The Korean Journal of Zoology
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• v.30 no.2
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• pp.193-209
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• 1987

Patterns of amylase secretion in mouse pancreatic fragments were studied over a period of time after the tissue was stimulated by acetyicholine and MNNG. MNNG is known to activate guanylate cyclase and thus increase the cGMP concentration in the pancreatic acinar cell. These amylase secretion patterns were studied to investigate the role of cGMP in reaction cascade during secretion response of the tissues stimulated by acetyicholine. Cellular response of amylase secretion in the pancreas by acetyicholine was divided into two phases. During the first phase, zymogen granules which had existed in the cells were secreted by the action of $Ca^2$+ and calmodulin immediately after secretagogue administration, this being known as the initial response. When the tissue was stimulated by acetylcholine in a $Ca^2$+-deficient medium or one containing trifluoperazine as a calmodulin antagonist, this initial response was reduced. In the second phase, newly formed zymogen granules were secreted as sustained response after protein synthesis was triggered by secretagogue. This response was provoked by an activation of protein kinase C. When either cycloheximide as a protein synthesis inhibitor or dibucaine as a protein kinase C inhibitor were added to the incubation medium, this sustained response was remarkablely depressed in the pancreatic fragments stimulated with acetylcholine. In the pancreatic acinar cell, phosphatidylinositol turnover plays an important role in the secretion response and hexachlorocyclohexane inhibits this phosphatidylinositol turnover. The pancreatic tissue treated with the hexachlorocyclohexane exhibited inhibition on both initial and sustained responses of amylase secretion by acetylcholine. MNNG also accelerated amylase secretion from the tissue gradually along incubation time. The 22 minutes fraction of the pancratic secretion after administration of both acetylcholine and MNNG showed higher amylase activity than the neighboring fractions. Guanylate cyclase potentiated the sustained response. Even if it is experimented with an indirect method, guanylate cyclase was found responsible for activation of the sustained response of a step prior to the action of protein kinase C. As conclusion, it was considered that amylase secretion in mouse pancreatic fragments stimulated by acetylcholine is a three phasic response.

• YAKHAK HOEJI
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• v.47 no.1
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• pp.31-36
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• 2003

To investigate the effect of protein kinase on melanin production via cAMP-dependent pathway, we measured the melanin amount and tyrosinase activity in B16 melanoma cells stimulated by alpha-melanocyte stimulating hormone (MSH), forskolin and 8-Br-cAMP. MSH, forskolin and 8-Br-cAMP significantly increased both melanin production and tyrosinase activity in B16 cells. Melanin production and tyrosinase activity by MSH are significantly inhibited by cyclic AMP-dependent protein kinase inhibitor (KT5720) and protein kinase C down-regulation treated with PMA. Bisindolmaleimide (1$\mu$M), protein kinase C inhibitor, significantly inhibited melanin production and tyrosinase activity stimulated by MSH, forskolin and 8-Br-cAMP with the following order of potency: MSH＞forskolin＞8-Br-cAMP. Tyrosine kinase inhibitor, genistein and DHC, significantly inhibited both, but the inhibitory effect was more potent in 8-Br-cAMP-stimulated B16 cells than MSH-stimulated cells. NFkB inhibitor (parthenolide) significantly inhibited melanin production and tyrosinase activity. Neither melanin production nor tyrosinase activity induced by MSH, forskolin and 8-Br-cAMP were affected by KN-62 (calmodulin-dependent protein kinase II inhibitor), PD098059 (mitogen-activated protein kinase inhibitor, MAPKK) and worthmannin (phosphatidylinositol 3-kinase inhibitor). These results suggest that both protein kinase C and tyrosine kinase are involved in melanin production by cyclic AMP-dependent pathway and NFkB pathway may play an important role in cyclic AMP-dependent melanin production in B16 melanoma cells.

• BMB Reports
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• v.34 no.5
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• pp.421-427
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• 2001

Osteoclasts, cells primarily involved in bone resorption, originate from the hematopoietic precursor cells of the monocyte/macrophage lineage and differentiate into multinucleated mature forms. We developed an in vitro osteoclast culture system using embryonic chicken bone marrow cells. This culture system can be utilized in studies on the differentiation and function of osteoclasts. Phosphatidylinositol 3-kinase (PI3-kinase) and mitogen-activated protein kinases (MAPKs) have been implicated in diverse cellular functions including proliferation, migration, and survival. Using the developed avian osteoclast culture system, we examined the involvement of these kinases in osteoclast differentiation by employing specific inhibitors of the kinases. We Found that the inhibition of the PI 3-kinase, p38, or ERK interfered with osteoclast formation, suggesting that the signaling pathways that involve these molecules participate in the process of chicken osteoclast differentiation.

• Biomedical Science Letters
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• v.15 no.1
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• pp.61-66
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• 2009

Leflunomide is an immunomodulatory agent used for the treatment of rheumatoid arthritis (RA). Leflunomide known as a regulator of iNOS synthesis which largely decreases NO production in diverse cell type. However, the effect of leflunomide on chondrocyte is still poorly understood. In our previous studies, we have shown that direct production of Nitric oxide (NO) by treating chondrocytes with NO donor, sodium nitroprusside (SNP), causes apoptosis via p38 mitogen-activated protein kinase in association with elevation of p53 protein level, caspase-3 activation. In this study, we characterized the molecular mechanism by which A77 1726 inhibit apoptosis. We found that A77 1726 inhibit NO-induced apoptosis as determined by MTT (Thiazolyl Blue Tetrazolium Bromide) assay and DNA fragmentation. The inhibition of apoptosis by A77 1726 was accompanied by increased PI-3 kinase and AKT activities. So, inhibition of phosphatidylinositol (PI)-3kinase with LY294002 rescued apoptosis. Triciribine, the specific inhibitor of AKT, also abolished anti-apoptotic effect. Our results indicate that A77 1726, the active metabolite of leflunomide, mediates NO-induced apoptosis in chondrocytes by modulating up-regulation of PI-3 kinase and AKT.

• Toxicological Research
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• v.17
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• pp.251-256
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• 2001

The expression of phase II detoxifying enzymes is affected by a variety of compounds and the induction of the enzymes plays an essential role in chemoprevention. A variety of phytochemicals such as sulfur-containing chemoprotective agents (SCC) may trigger cellular signals and activate phase II gene expression through ARE activation. see induces glutathione S-transferases. Studies were conducted to investigate the role of mitogen-activated protein (MAP) kinase and phosphatidylinositol 3-kinase (PI3-kinase) in the induction of GST (e.g. rGSTA2) by sec. We also studied the MAP kinase pathway responsible for the GST expression by see and compared that with the pathway activated by oxidative stress as a result of sulfur amino acids deprivation (SAAD). see inhibited phosphorylation of ERK1/2 although the effect of see on JNK and p38 MAP kinase was minimal. Wortmannin and LY294002. PI3-kinase inhibitors. abolished the increases in rGSTA2 mRNA and protein levels by SCC. Deprivation of cystine and methionine caused oxidative stress in H4IIE cells. as evidenced by a decrease in the reduced glutathione and an increase in prooxidant production. Electrophoretic mobility shift assay revealed that the ARE complex consisting of Nrf-1/2 and Maf proteins was activated 12~48 h. The rGSTA2 mRNA and protein levels were increased by SAAD. Activation of ARE and induction of rGSTA2 were both completely inhibited by PI3-kinase inhibitors. Inhibition of p38 MAP kinase by SB203580 prevented the ARE-mediated rGSTA2 induction. The results of this study showed that PI3-kinase might play an essential role in the ARE-mediated rGSTA2 induction by see or SAAD and that the dual MAP kinase pathways were responsible for the enzyme induction.

• Toxicological Research
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• v.22 no.3
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• pp.283-291
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• 2006

The survival and growth of epithelial cells depends on adhesion to the extracellular matrix. An adhesion signal may regulate the initiation of differentiation, since epidermal keratinocytes differentiate as they leave the basement membrane. A metabolically dead cornified cell envelope is the end point of epidermal differentiation so that this process may be viewed as a specialized form of programmed cell death. In order to investigate the precise cellular signaling events loading to terminal differentiation of keratinocytes, we have utilized HaCaT cells to monitor the biological consequences of $Ca^{2+}$ stimulation and numerous downstream signaling pathways, including activation of the extracellular signal-regulated protein kinase(ERK) pathway and activation of phosphatidylinositol 3-kinase(PI3K). The results presented in this study show that $Ca^{2+}$ function as potent agents for the differentiation of HaCaT keratinocytes, and this differentiation depends or the activation of ERK, Protein kinase B(PKB) and p70 ribosomal protein S6 kinase(p70S6K). Finally, the results show that the expression of Activator protein 1(AP-1; c-Jun and c-Fos) increased following $Ca^{2+}$-mediated differentiation of HaCaT cells, suggesting that ERK-mediated AP-1 expression is critical for initiating the terminal differentiation of keratinocytes.

• Journal of Applied Biological Chemistry
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• v.66
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• pp.236-243
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• 2023

Platelets act a fundamental role in primary- and secondary-hemostasis, however, platelet activation may cause thrombosis simultaneously. Therefore, control of platelet aggregation is crucial in preventing thrombosis-mediated diseases. Recently, the development of insect materials is attracting attention. Among the highly nutritious functional food sources, insects such as two-spotted cricket (Gryllus bimaculatus). Gryllus bimaculatus (G. bimaculatus) contains high protein and unsaturated fatty acids and has been registered as a food material September 2015 by the Ministry of Food and Drug Safety of Korea. In this study, we examined whether G. bimaculatus extract (GBE) inhibits platelet aggregation, intracellular calcium mobilization, thromboxane A₂ production and glycoprotein IIb/IIIa (integrin αIIb/β3) activation. We investigated whether GBE can regulate signaling molecules, such as 1, 4, 5-triphosphate receptor type I, extracellular signal-regulated kinase, cytosolic phospholipase A₂, mitogen-activated protein kinases p38, vasodilator-stimulated phosphoprotein, phosphatidylinositol-3 kinase, Akt, glycogen synthase kinase-3α/β, and SYK. Taken together, GBE is a potential therapeutic drug candidate to prevent platelet-related thrombosis and cardiovascular disease.

• Archives of Pharmacal Research
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• v.27 no.7
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• pp.757-762
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• 2004

The protective adaptive response to electrophiles and reactive oxygen species is mediated by the induction of phase II detoxifying genes including glutathione S-transferases (GSTs). NF-E2-related factor-2 (Nrf2) phosphorylation by protein kinase C (PKC) is a critical event for its nuclear translocation in response to oxidative stress. Previously, we have shown that peroxynitrite plays a role in activation of Nrf2 and Nrf2 binding to the antioxidant response element (ARE) via the pathway of phosphatidylinositol 3-kinase (PI3-kinase) and that nitric oxide synthase in hepatocytes is required for GSTA2 induction. In view of the importance of PKC and Pl3-kinase in Nrf2-mediated GST induction, we investigated the role of these kinases in peroxynitrite formation for GSTA2 induction by oxidative stress and determined the relationship between PKC and PI3-kinase. Although PKC activation by phorbol 12-myristate-13-acetate (PMA) did not increase the extents of constitutive and inducible GSTA2 expression, either PKC depletion by PMA or PKC inhibition by staurosporine significantly inhibited GSTA2 induction by tert-butylhydroquinone (t-SHa) a prooxidant chemical. Therefore, the basal PKC activity is req- uisite for GSTA2 induction. 3-Morpholinosydnonimine (SIN-1), which decomposes and yields peroxynitrite, induced GSTA2, which was not inhibited by PKC depletion, but slightly enhanced by PKC activation, suggesting that PKC promotes peroxynitrite formation for Nrf2-mediated GSTA2 induction. Treatment of cells with S-nitroso-N-acetyl-penicillamine (SNAP), an exogenous NO donor, in combination with t-BHQ may produce peroxynitrite. GSTA2 induction by SNAP ＋ t-BHQ was not decreased by PKC depletion, but rather enhanced by PKC activation, showing that the activity of PKC might be required for peroxynitrite formation. LY294002 a P13-kinase inhibitor blocked GSTA2 induction by t-BHQ, which was reversed by PMA-induced PKC activation. These results provide evidence that PKC may playa role in formation of peroxynitrite that activates Nrf2 for GSTA2 induction and that PKC may serve an activator for GSTA2 induction downstream of PI3-kinase.