• BMB Reports
• /
• 제50권4호
• /
• pp.208-213
• /
• 2017

Vascular endothelial growth factor (VEGF) is an essential cytokine that has functions in the formation of new blood vessels and regression of cardiac hypertrophy. VEGF/VEGF-receptor-1 (VEGFR1) signaling plays a key role in the regression of cardiac hypertrophy, whereas VEGF/VEGFR2 signaling leads to cardiac hypertrophy. In this study, we identified the prohypertrophic role of miR-374 using neonatal rat ventricular myocytes (NRVMs). Our results showed that overexpression of miR-374 activated G protein-coupled receptor-mediated prohypertrophic pathways by the inhibition of VEGFR1-dependent regression pathways. Luciferase assays revealed that miR-374 could directly target the 3'-untranslated regions of VEGFR1 and cGMP-dependent protein kinase-1. Collectively, these findings demonstrated that miR-374 was a novel pro-hypertrophic microRNA functioning to suppress the VEGFR1-mediated regression pathway.

• Natural Product Sciences
• /
• 제17권4호
• /
• pp.273-278
• /
• 2011

In this study, the effects of a methanol (MeOH) extract of Carduus crispus L. (Asteraceae) on adipogenesis was investigated in 3T3-L1 cells. To differentiate preadipocytes to adipocytes, confluent 3T3-L1 preadipocytes were treated with a hormone mixture, which included isobutylmethylxanthine, dexamethasone, and insulin (MDI). The methanol extract of C. crispus significantly decreased fat accumulation by inhibiting adipogenic signal transcriptional factors in MDI-induced 3T3-L1 cells in a dose-dependent manner. In MTT assays and on PI-staining, methanol extract of C. crispus inhibited the proliferation of 3T3-L1 cells during mitotic clonal expansion (MCE). The anti-adipogenic effect of the Carduus extract seemed to be associated with the upregulation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) pathways within the first 2 days after MDI treatment. These results suggest that methanol extract of C. crispus might be beneficial for the treatment of obesity.

• The Korean Journal of Physiology and Pharmacology
• /
• 제10권4호
• /
• pp.199-205
• /
• 2006

The functional expression of potassium $(K^+)$ channels has electrophysiologically been studied in bone cells from several species, however, their identity and regulation of gene expressions in bone cells are not well known. In the present study, to investigate how $K^+$ channel expressions are regulated by estrogen, we measured changes of transcript levels of various $Ca^{2+}$-activated ($K_{Ca}$) and ATP-sensitive $K^+$ channels in rat osteoblastic ROS 17/2.8 cells after treatment with estrogen. Application of 17${\beta}$-estradiol $(E_2)$ for 24 h and 48 h increased mRNA and protein expressions of inwardly rectifying $K^+$ channel (Kir) 6.2 and type 2 small conductance $K_{Ca}$ channel (SK2), respectively. Combined treatment of cells with 17${\beta}-E_2$ and ICI 182,780, a pure antiestrogen, suppressed 17${\beta}-E_2$-induced alterations of SK2 and Kir6.2 mRNA levels. In addition, treatment of cells with U0126, a specific inhibitor of extracellular receptor kinases (ERK)1/2, and SP600125, a specific inhibitor of c-jun N-terminal kinase (JNK) blocked the enhancing effects of 17${\beta}-E_2$ on SK2 and Kir6.2 protein expressions. On the other hand, blocking of p38 mitogen-activated protein kinase had no effect. Taken together, these results indicate that 17${\beta}-E_2$ modulates SK2 and Kir6.2 expressions through the estrogen receptor, involving ERK1/2 and JNK activations.

• 대한약학회:학술대회논문집
• /
• 대한약학회 2001년도 Proceedings of International Convention of the Pharmaceutical Society of Korea
• /
• pp.109-113
• /
• 2001

Oxidized low-density lipoprotein (oxLDL) has been shown to modulate transactivations by the peroxisome proliferator activated receptor (PPAR)$\gamma$ and nuclear factor-kappa B (NF$\kappa$B). In this study, the oxLDL signaling pathways involved with the NF$\kappa$B transactivation were investigated by utilizing a reporter construct driven by three upstream NF$\kappa$B binding sites, and various pharmacological inhibitors. OxLDL and its constituent lysophophatidylcholine (lysoPC) induced a rapid and transient increase of intracellular calcium and stimulated the NF-KB transactivation in resting RAW264.7 macrophage cells in an oxidation-dependent manner. The NF$\kappa$B activation by oxLDL or lysoPC was inhibited by protein kinase C inhibitors or an intracellular calcium chelator. Tyrosine kinase or PI3 kinase inhibitors did not block the NF$\kappa$B transactivation. Furthermore, the oxLDL-induced NF$\kappa$B activity was abolished by the PPAR$\gamma$ ligands. When the endocytosis of oxLDL was blocked by cytochalasin B, the NF$\kappa$B transactivation by oxLDL was synergistically increased, while PPAR transactivation was blocked. These results suggest that oxLDL activates NF-$\kappa$B in resting macrophages via protein kinase C- and/or calcium-dependent pathways, which does not involve the endocytic processing of oxLDL. The endocytosis-dependent PPAR$\gamma$ activation by oxLDL may function as an inactivation route of the oxLDL induced NF$\kappa$B signal. Short heterodimer partner (SHP), specifically expressed in liver and a limited number of other tissues, is an unusual orphan nuclear receptor that lacks the conventional DNA-binding domain. In this work, we found that SHP expression is abundant in murine macrophage cell line RAW 264.7 but suppressed by oxLDL and its constituent I3-HODE, a ligand for peroxisome proliferator-activated receptor y. Furthermore, SHP acted as a transcription coactivator of nuclear factor-$\kappa$B (NF$\kappa$B) and was essential for the previously described NF$\kappa$B transactivation by lysoPC, one of the oxLDL constituents. Accordingly, NF$\kappa$B, transcriptionally active in the beginning, became progressively inert in oxLDL-treated RAW 264.7 cells, as oxLDL decreased the SHP expression. Thus, SHP appears to be an important modulatory component to regulate the transcriptional activities of NF$\kappa$B in oxLDL-treated, resting macrophage cells.

• 한방비만학회지
• /
• 제22권2호
• /
• pp.93-101
• /
• 2022

Objectives: This study aimed to observe the anti-diabetic effect and underlying mechanisms of Galgunhwanggumhwangryun-tang (GHH; Gegen-Qinlian-decoction) in the C2C12 myotubes. Methods: GHH (1.0 mg/ml) or metformin (0.75 mM) or insulin (100 nM) were treated in C2C12 myotubes after 4 days differentiation. The glucose uptake was assessed by 2-[N-(7-160 nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-d-glucose uptake by C2C12 cells. The expression of adenosine monophosphate-activated protein kinase (AMPK) and phosphorylation AMPK (pAMPK) were measured by western blot. We also evaluated gene expression of glucose transporter type 4 (Slc2a4, formerly known as GLUT4), glucokinase (Gk), carnitine palmitoyltransferase IA (Cpt1a), nuclear respiratory factors 1 (Nrf1), mitochondrial transcription factor A (Tfam), and peroxisome proliferator-activated receptor γ coactivator 1α (Ppargc1a) by quantitative real-time polymerase chain reaction. Results: GHH promoted glucose uptake in C2C12 myotubes. The expression of AMPK protein, which plays an essential role in glucose metabolism, was increased by treatment with GHH. GHH treatment tended to increase gene expression of Slc2a4, Gk, and Nrf1 but was not statistically significant. However, GHH significantly improved Tfam and Ppargc1a gene expression in C2C12 myotubes. Conclusions: In summary, GHH treatment promoted glucose uptake in C2C12 myotubes. We suggest that these effects are associated with increased gene expression involved in mitochondrial biosynthesis and oxidative phosphorylation, such as Tfam and Ppargc1a, and increased expression of AMPK protein.

• The Korean Journal of Physiology and Pharmacology
• /
• 제16권6호
• /
• pp.447-453
• /
• 2012

TLR6 forms a heterodimer with TLR2 and TLR4. While proinflammatory roles of TLR2 and TLR4 are well documented, the role of TLR6 in inflammation is poorly understood. In order to understand mechanisms of action of TLR6 in inflammatory responses, we investigated the effects of FSL-1, the TLR6 ligand, on expression of chemokine CCL2 and cytokine IL-$1{\beta}$ and determined cellular factors involved in FSL-1-mediated expression of CCL2 and IL-$1{\beta}$ in mononuclear cells. Exposure of human monocytic leukemia THP-1 cells to FSL-1 resulted not only in enhanced secretion of CCL2 and IL-$1{\beta}$, but also profound induction of their gene transcripts. Expression of CCL2 was abrogated by treatment with OxPAPC, a TLR-2/4 inhibitor, while treatment with OxPAPC resulted in partially inhibited expression of IL-$1{\beta}$. Treatment with FSL-1 resulted in enhanced phosphorylation of Akt and mitogen-activated protein kinases and activation of protein kinase C. Treatment with pharmacological inhibitors, including SB202190, SP6001250, U0126, Akt inhibitor IV, LY294002, GF109203X, and RO318220 resulted in significantly attenuated FSL-1-mediated upregulation of CCL2 and IL-$1{\beta}$. Our results indicate that activation of TLR6 will trigger inflammatory responses by upregulating expression of CCL2 and IL-$1{\beta}$ via TLR-2/4, protein kinase C, PI3K-Akt, and mitogen-activated protein kinases.

• International Journal of Oral Biology
• /
• 제30권1호
• /
• pp.9-15
• /
• 2005

Recently, we reported that high extracellular calcium increased receptor activator of nuclear factor-${\kappa}B$ ligand (RANKL) expression via p44/42 mitogen-activated protein kinase (p44/42 MAPK) activation in mouse osteoblasts. However, the mechanism for p44/42 MAPK activation by high extracellular calcium is unclear. In this study, we examined the role of intracellular calcium increase in high extracellular calcium-induced RANKL induction and p44/42 MAPK activation. Primary cultured mouse calvarial osteoblasts were used. RANKL expression was highly induced by 10 mM calcium treatment. Ionomycin, a calcium ionophore, also increased RANKL expression and activated p44/42 MAPK. U0126, an inhibitor of MEK1/2, an upstream activator of p44/42 MAPK, blocked the RANKL induction by both high extracellular calcium and ionomycin. High extracellular calcium increased the phosphorylation of proline-rich tyrosine kinase 2 (Pyk2), one of the known upstream regulators of p44/42 MAPK activation. Bisindolylmaleimide, an inhibitor of protein kinase C, did not block RANKL induction and p44/42 MAPK activation induced by high extracellular calcium. 2-Aminoethoxydiphenyl borate, an inhibitor of inositol 1,4,5-trisphosphate (IP3) receptor, blocked the RANKL induction by high extracellular calcium. It also partially suppressed the activation of Pyk2 and p44/42 MAPK. Cyclosporin A, an inhibitor of calcineurin, also inhibited high calcium-induced RANKL expression in dose dependent manner. However, cyclosporin A did not affect the activation of Pyk2 and p44/42 MAPK by high extracellular calcium treatment. These results suggest that 1) the increase in intracellular calcium via IP3-mediated calcium release is necessary for RANKL induction by high extracellular calcium treatment, 2) Pyk2 activation, but not protein kinase C, following the increase in intracellular calcium might be involved in p44/42 MAPK activation, and 3) calcineurin-NFAT activation by the increase in intracellular calcium is involved in RANKL induction by high extracellular calcium treatment.

• 대한의생명과학회지
• /
• 제20권2호
• /
• pp.62-69
• /
• 2014

Previous our study demonstrated that ailanthoidol (3-deformylated 2-arylbenzo[b]furan), a neolignan from Zanthoxylum ailanthoides or Salvia miltiorrhiza Bunge, is a novel anti-inflammatory agent. In this investigation, we examined the anti-adipogenic effect of ailanthoidol. Our data showed that ailanthoidol suppressed lipid droplet formation and adipocyte differentiation in 3T3-L1 cells. Treatment of the 3T3-L1 adipocytes with ailanthoidol resulted in an attenuation of the releases of leptin and interleukin-6. The expression of peroxisome proliferator-activated receptor $(PPAR){\gamma}$ and CCAAT/enhancer-binding protein $(C/EBP){\alpha}$, the central transcriptional regulators of adipogenesis, was decreased by treatment with ailanthoidol. Additionally, ailanthoidol treatment increased the phosphorylation levels of 5' adenosine monophosphate-activated protein kinase. These results suggest that ailanthoidol effectively suppresses adipogenesis and that it exerts its role mainly through the significant down-regulation of $PPAR{\gamma}$ and $C/EBP{\alpha}$ expression. Our findings provide important insights into the mechanisms underlying the anti-adipogenic activity of ailanthoidol.

• Biomolecules & Therapeutics
• /
• 제29권1호
• /
• pp.41-51
• /
• 2021

Src family kinases (SFKs), an important group of non-receptor tyrosine kinases, are suggested to be excessively activated during various types of tissue fibrosis. The present study investigated the effect of KF-1607, an orally active and a newly synthesized Src kinase inhibitor (SKI) with proposed low toxicity, in preventing the progression of renal interstitial fibrosis. Unilateral ureteral obstruction (UUO) surgery was performed in 6-week-old male C57BL/6 mice to induce renal interstitial fibrosis. Either KF-1607 (30 mg/kg, oral gavage) or PP2 (2 mg/kg, intraperitoneal injection), a common experimental SKI, was administered to mice for seven days, started one day prior to surgery. UUO injury-induced SFK expression, including Src, Fyn, and Lyn kinase. SFK inhibition by KF-1607 prevented the progression of tubular injury in UUO mice, as indicated by decreases in albuminuria, urinary KIM-1 excretion, and kidney NGAL protein expression. Renal tubulointerstitial fibrosis was attenuated in response to KF-1607, as shown by decreases in α-SMA, collagen I and IV protein expression, along with reduced Masson's trichrome and collagen-I staining in kidneys. KF-1607 also inhibited inflammation in the UUO kidney, as exhibited by reductions in F4/80 positive-staining and protein expression of p-NFκB and ICAM. Importantly, the observed effects of KF-1607 were similar to those of PP2. A new pan Src kinase inhibitor, KF-1607, is a potential pharmaceutical agent to prevent the progression of renal interstitial fibrosis.

• Animal Bioscience
• /
• 제36권6호
• /
• pp.851-860
• /
• 2023

Objective: This study aims to evaluate the target genes of gga-miR-20a-5p and the regulated immune responses in the chicken macrophage cell line, HD11, by the exosome-mediated delivery of miR-20a-5p. Methods: Exosomes were purified from the chicken macrophage cell line HD11. Then, mimic gga-miR-20p or negative control miRNA were internalized into HD11 exosomes. HD11 cells were transfected with gga-miR-20a-5p or negative control miRNA containing exosomes. After 44 h of transfection, cells were incubated with or without 5 ㎍/mL poly(I:C) for 4 h. Then, expression of target genes and cytokines was evaluated by quantitative realtime polymerase chain reaction. Results: Using a luciferase reporter assay, we identified that gga-miR-20a-5p directly targeted interferon gamma receptor 2 (IFNGR2), mitogen-activated protein kinase 1 (MAPK1), mitogen-activated protein kinase kinase kinase 5 (MAP3K5), and mitogen-activated protein kinase kinase kinase 14 (MAP3K14). Moreover, the exosome-mediated delivery of gga-miR-20a-5p successfully repressed the expression of IFNGR2, MAPK1, MAP3K5, and MAP3K14 in HD11 cells. The expressions of interferon-stimulated genes (MX dynamin like GTPase 1 [MX1], eukaryotic translation initiation factor 2A [EIF2A], and oligoadenylate synthase-like [OASL]) and proinflammatory cytokines (interferon-gamma [IFNG], interleukin-1 beta [IL1B], and tumor necrosis factor-alpha [TNFA]) were also downregulated by exosomal miR-20a-5p. In addition, the proliferation of HD11 cells was increased by exosomal miR-20a-5p. Conclusion: The exosome-mediated delivery of gga-miR-20a-5p regulated immune responses by controlling the MAPK and apoptotic signaling pathways. Furthermore, we expected that exosomal miR-20a-5p could maintain immune homeostasis against highly pathogenic avian influenza virus H5N1 infection by regulating the expression of proinflammatory cytokines and cell death.