• The Korean Journal of Physiology and Pharmacology
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• v.5 no.4
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• pp.287-297
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• 2001

Contraction of smooth muscle is initiated by an increase in cytosolic $Ca^{2+}$ leading to activation of $Ca^{2+}$/ calmodulin-dependnet myosin light chain (MLC) kinase and phosphorylation of MLC. The types of contraction and signaling mechanisms mediating contraction differ depending on the region. The involvement of these different mechanisms varies depending on the source of $Ca^{2+}$ and the kinetic of $Ca^{2+}$ mobilization. $Ca^{2+}$ mobilizing agonists stimulate different phospholipases $(PLC-{\beta},\;PLD\;and\;PLA_2)$ to generate one or more $Ca^{2+}$ mobilizing messengers $(IP_3\;and\;AA),$ and diacylglycerol (DAG), an activator of protein kinase C (PKC). The relative contributions of $PLC-{\beta},\;PLA_2$ and PLD to generate second messengers vary greatly between cells and types of contraction. In smooth muscle cell derived form the circular muscle layer of the intestine, preferential hydrolysis of $PIP_2$ and generation of $IP_3$ and $IP_3-dependent\;Ca^{2+}$ release initiate the contraction. In smooth muscle cells derived from longitudinal muscle layer of the intestine, preferential hydrolysis of PC by PLA2, generation of AA and AA-mediated $Ca^{2+}$ influx, cADP ribose formation and $Ca^{2+}-induced\;Ca^{2+}$ release initiate the contraction. Sustained contraction, however, in both cell types is mediated by $Ca^{2+}-independent$ mechanism involving activation of $PKC-{\varepsilon}$ by DAG derived form PLD. A functional linkage between $G_{13},$ RhoA, ROCK, $PKC-{\varepsilon},$ CPI-17 and MLC phosphorylation in sustained contraction has been implicated. Contraction of normal esophageal circular muscle (ESO) in response to acetylcholine (ACh) is linked to $M_2$ muscarinic receptors activating at least three intracellular phospholipases, i.e. phosphatidylcholine-specific phospholipase C (PC-PLC), phospholipase D (PLD) and the high molecular weight (85 kDa) cytosolic phospholipase $A_2\;(cPLA_2)$ to induce phosphatidylcholine (PC) metabolism, production of diacylglycerol (DAG) and arachidonic acid (AA), resulting in activation of a protein kinase C (PKC)-dependent pathway. In contrast, lower esophageal sphincter (LES) contraction induced by maximally effective doses of ACh is mediated by muscarinic $M_3$ receptors, linked to pertussis toxin-insensitive GTP-binding proteins of the $G_{q/11}$ type. They activate phospholipase C, which hydrolyzes phosphatidylinositol bisphosphate $(PIP_2),$ producing inositol 1, 4, 5-trisphosphate $(IP_3)$ and DAG. $IP_3$ causes release of intracellular $Ca^{2+}$ and formation of a $Ca^{2+}$-calmodulin complex, resulting in activation of myosin light chain kinase and contraction through a calmodulin-dependent pathway.

• Asian Pacific Journal of Cancer Prevention
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• v.16 no.4
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• pp.1375-1383
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• 2015

Background: Objective Myocardin-related transcription factor (MRTF)-A is a Rho signaling-responsive co-activator of serum response factor (SRF). The purpose of this study is to investigate the role of MRTF-A and AQP1 (aquaporin 1) in pathological vascular remodeling. Materials and Methods: MRTF-A, AQP1 and neointima expression was detected both in the wire injured femoral arteries of wild-type mice and the atherosclerotic aortic tissues of $ApoE^{-/-}$ mice. Expression of ICAM-1, matrix metallopeptidase 9 (MMP-9) and integrin ${\beta}1$ were also assayed. The intercourse relationship between the molecules were investigated by interfering RNA and inhibitor assay. Results: MRTF-A and AQP1 expression were significantly higher in the wire injured femoral arteries of wild-type mice and in the atherosclerotic aortic tissues of $ApoE^{-/-}$ mice than in healthy control tissues. Both in wire-injured femoral arteries in MRTF-A knockout ($Mkl1^{-/-}$) mice and atherosclerotic lesions in $Mkl1^{-/-}$; $ApoE^{-/-}$ mice, neointima formation were significantly attenuated and the expression of AQP1 were significantly decreased. Expression of ICAM-1, matrix metallopeptidase 9 (MMP-9) and integrin ${\beta}1$, three SRF targets and key regulators of cell migration, and AQP1 in injured arteries was significantly weaker in $Mkl1^{-/-}$ mice than in wild-type mice. In cultured vascular smooth muscle cells (VSMCs), knocking down MRTF-A reduced expression of these genes and significantly impaired cell migration. Underlying the increased MRTF-A expression in dedifferentiated VSMCs were the down-regulation of microRNA-300. Moreover, the MRTF-A inhibitor CCG1423 significantly reduced neointima formation following wire injury in mice. Conclusions: MRTF-A could be a novel therapeutic target for the treatment of vascular diseases.

• Reproductive and Developmental Biology
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• v.35 no.2
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• pp.159-165
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• 2011

To explore the role of histone deactylase (HDAC) activation in an in vivo model of hypertrophy, we studied the effects of Trichostatin A (TSA). TSA subjected to thoracic aortic banding (TAB)-induced pressure stress in mice. In histological observations, TAB in treated mice showed a significant hypertrophic response, whereas the sham operation remained nearly normal structure with partially blunted hypertrophy. TSA treatment had no effect (measured as HW/BW) on sham-operated animals. TAB animals treated with vehicle manifested a robust ~50% hypertrophic response (p<0.05 vs sham). TAB mice treated with 2 mg/kg/day TSA manifested a blunted growth responses, which was significantly diminished (p<0.05) compared with vehicle-treated TAB mice. TAB mice treated with a lower dose of TSA (0.5 mg/kg/day) manifested a similar blunting of hypertrophic growth (~25% increase in heart mass). Furthermore, to determine activity duration of TSA in vitro, 1 nM TSA was added to H9c2 cells. Histone acetylation was initiated at 4 hr after treatment, and it was peak up to 18 hr, then followed by significantly reduced to 30 hr. We also analyzed the expression of p53 following TSA treatment, wherein p53 expression was elevated at 4 hr, and it was maintained to 24 hr after treatment. ERK was activated at 8 hr, and maintained till 30 hr after treatment suggesting an intracellular signaling interaction between TSA and p53 expression Taken together, it is suggested that HDAC activation is required for pressure-overload growth of the heart. Eventually, these data suggest that histone acetylation may be a novel target for therapeutic intervention in pressure-overloaded cardiac hypertrophy.

• The Korean Journal of Physiology and Pharmacology
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• v.21 no.6
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• pp.687-694
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• 2017

Plumbagin, a hydroxy 1,4-naphthoquinone compound from plant metabolites, exhibits anticancer, antibacterial, and antifungal activities via modulating various signaling molecules. However, its effects on vascular functions are rarely studied except in pulmonary and coronary arteries where NADPH oxidase (NOX) inhibition was suggested as a mechanism. Here we investigate the effects of plumbagin on the contractility of skeletal artery (deep femoral artery, DFA), mesenteric artery (MA) and renal artery (RA) in rats. Although plumbagin alone had no effect on the isometric tone of DFA, $1{\mu}M$ phenylephrine (PhE)-induced partial contraction was largely augmented by plumbagin (${\Delta}T_{Plum}$, 125% of 80 mM KCl-induced contraction at $1{\mu}M$). With relatively higher concentrations (>$5{\mu}M$), plumbagin induced a transient contraction followed by tonic relaxation of DFA. Similar biphasic augmentation of the PhE-induced contraction was observed in MA and RA. VAS2870 and GKT137831, specific NOX4 inhibitors, neither mimicked nor inhibited ${\Delta}T_{Plum}$ in DFA. Also, pretreatment with tiron or catalase did not affect ${\Delta}T_{Plum}$ of DFA. Under the inhibition of PhE-contraction with L-type $Ca^{2+}$ channel blocker (nifedipine, $1{\mu}M$), plumbagin still induced tonic contraction, suggesting $Ca^{2+}$-sensitization mechanism of smooth muscle. Although ${\Delta}T_{Plum}$ was consistently observed under pretreatment with Rho A-kinase inhibitor (Y27632, $1{\mu}M$), a PKC inhibitor (GF 109203X, $10{\mu}M$) largely suppressed ${\Delta}T_{Plum}$. Taken together, it is suggested that plumbagin facilitates the PKC activation in the presence of vasoactive agonists in skeletal arteries. The biphasic contractile effects on the systemic arteries should be considered in the pharmacological studies of plumbagin and 1,4-naphthoquinones.

• Journal of Life Science
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• v.23 no.6
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• pp.812-824
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• 2013

Reserpine, an anti-hypertensive drug, is able to positively modulate several phenotypes associated with $A{\beta}$ toxicity in a Caenorhabditis elegans model of Alzheimer's disease (AD). We investigated into the therapeutic effects of reserpine on mammalian neurodegenerative disorders, and found that significant alteration of the key factors influencing AD was detected in Tg2576 mice after reserpine treatment for 30 days. The aggressive behavior of Tg2576 mice was significantly improved upon reserpine treatment, whereas their social contact was consistently maintained. Furthermore, the levels of $A{\beta}$-42 peptide in the hippocampus of the brain and blood serum were lower in the reserpine-treated group than in the vehicle-treated group. Among g-secretase components, the expression levels of PS-2, Pen-2, and APH-1 were slightly lower in reserpine-treated Tg2576 mice, although a significant change in nicastrin (NCT) expression was not detected. Furthermore, the serum level of nerve growth factor (NGF) increased in reserpine-treated Tg2576 mice compared with vehicle-treated mice. Among down-stream effectors of the NGF receptor TrkA signaling pathway, reserpine treatment induced elevation of TrkA phosphorylation and reduction of ERK phosphorylation. In addition, in the NGF receptor $p75^{NTR}$ signaling pathway, the expression levels of $p75^{NTR}$ and Bcl-2 were enhanced in reserpine-treated Tg2576 mice compared with vehicle-treated mice, whereas the expression level of RhoA declined. Overall, these results suggest that reserpine can help relieve AD pathogenesis in Tg2576 mice through downregulation of $A{\beta}$-42 deposition, alteration of ${\gamma}$-secretase components, and regulation of NGF metabolism.

• Tuberculosis and Respiratory Diseases
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• v.71 no.4
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• pp.259-265
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• 2011

Background: Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors, gefitinib and erlotinib, are effective therapies for non-small cell lung cancer (NSCLC) patients whose tumors harbor somatic mutations in EGFR. The mutations are, however, only found in about 30% of Asian NSCLC patients and all patients ultimately develop resistance to these agents. Ionizing radiation has been shown to induce autophosphorylation of EGFR and activate its downstream signaling pathways. In the present study, we have tested whether the effect of gefitinib treatment can be enhanced after ionizing radiation. Methods: We compared the PC-9 and A549 cell line with its radiation-resistant derivatives after gefitinib treatment with cell proliferation and apoptosis assay. We also analyzed the effect of gefitinib after ionizing radiation in PC-9, A549, and NCI-H460 cells. Cell proliferation was determined by MTT assay and induction of apoptosis was evaluated by flow cytometry. Caspase 3 activation and PARP cleavage were evaluated by western blot analysis. Results: PC-9 cells having mutated EGFR and their radiation-resistant cells showed no significant difference in cell viability. However, radiation-resistant A549 cells were more sensitive to gefitinib than were their parental cells. This was attributable to an increased induction of apoptosis. Gefitinib-induced apoptosis increased significantly after radiation in cells with wild type EGFR including A549 and NCI-H460, but not in PC-9 cells with mutated EGFR. Caspase 3 activation and PARP cleavage accompanied these findings. Conclusion: The data suggest that gefitinib-induced apoptosis could increase after radiation in cells with wild type EGFR, but not in cells with mutated EGFR.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.34 no.1
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• pp.28-35
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• 2008

Purpose: The nitric oxide (NO) release by inducible nitric oxide synthase (iNOS) is the key events in macrophage response to lipopolysaccharide (LPS) which is suggested to be a crucial mediator for inflammatory and innate immune responses. NO is an important mediator involved in many host defense action and may also lead to a harmful host response to bacterial infection. However, given the importance of iNOS in a variety of pathophysiological conditions, control of its expression and signaling events in response to LPS has been the subject of considerable investigation. Materials and Methods: The Raw264.7 macrophage cell line was used to observe LPS-stimulated iNOS expression. The expression of iNOS is observed by Western blot analysis and real-time RT-PCR. Protein kinase C $(PKC)-{\alpha}$ overexpressing Raw264.7 cells are established to determine the involvement of $PKC-{\alpha}$ in LPS-mediated iNOS expression. $NF-{\kappa}B$ activity is measured by $I{\kappa}B{\alpha}$ degradation and $NF-{\kappa}B$ luciferase activity assay. Results: We found that various PKC isozymes regulate LPS-induced iNOS expression at the transcriptional and translational levels. The involvement of $PKC-{\alpha}$ in LPS-mediated iNOS induction was further confirmed by increased iNOS expression in $PKC-{\alpha}$ overexpressing cells. $NF-{\kappa}B$ dependent transactivation by LPS was observed and $PKC-{\alpha}$ specific inhibitory peptide abolished this activation, indicating that $NF-{\kappa}B$ activation is dependent on $PKC-{\alpha}$. Conclusion: Our data suggests that $PKC-{\alpha}$ is involved in LPS-mediated iNOS expression and that its downstream target is $NF-{\kappa}B$. Although $PKC-{\alpha}$ is a crucial mediator in the iNOS regulation, other PKC isozymes may contribute LPS-stimulated iNOS expression. This finding is needed to be elucidated in further study.

• Biomolecules & Therapeutics
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• v.29 no.6
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• pp.650-657
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• 2021

Metformin is an anti-diabetic drug and has anticancer effects on various cancers. Several studies have suggested that metformin reduces cell proliferation and stimulates cell-cycle arrest and apoptosis. However, the definitive molecular mechanism of metformin in the pathophysiological signaling in endometrial tumorigenesis and metastasis is not clearly understood. In this study, we examined the effects of metformin on the cell viability and apoptosis of human cervical HeLa and endometrial HEC-1-A and KLE cancer cells. Metformin suppressed cell growth in a dose-dependent manner and dramatically evoked apoptosis in HeLa cervical cancer cells, while apoptotic cell death and growth inhibition were not observed in endometrial (HEC-1-A, KLE) cell lines. Accordingly, the p27 and p21 promoter activities were enhanced while Bcl-2 and IL-6 activities were significantly reduced by metformin treatment. Metformin diminished the phosphorylation of mTOR, p70S6K and 4E-BP1 by accelerating adenosine monophosphate-activated kinase (AMPK) in HeLa cancer cells, but it did not affect other cell lines. To determine why the anti-proliferative effects are observed only in HeLa cells, we examined the expression level of liver kinase B1 (LKB1) since metformin and LKB1 share the same signalling system, and we found that the LKB1 gene is not expressed only in HeLa cancer cells. Consistently, the overexpression of LKB1 in HeLa cancer cells prevented metformin-triggered apoptosis while LKB1 knockdown significantly increased apoptosis in HEC-1-A and KLE cancer cells. Taken together, these findings indicate an underlying biological/physiological molecular function specifically for metformin-triggered apoptosis dependent on the presence of the LKB1 gene in tumorigenesis.

• Tuberculosis and Respiratory Diseases
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• v.70 no.5
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• pp.390-396
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• 2011

Background: Hyaluronan (HA) is an unbranched glycosaminoglycan. It has been proposed that HA acts as a vehicle for cytokines due to the strong negative charge on its surface. We hypothesized that HA would function like a cytokine scavenger and reduce the inflammatory signaling cascade and this would lead to improved survival in rats suffering with endotoxemia. Methods: Endotoxin (Salmonella, 10 mg/kg) or an equal amount of 0.9% NaCl (NS) was injected into the jugular vein of rats. HA (1,600 kDa, 0.35%) or NS was given at 0.1 mL/kg/h for 3 hours. HA or NS infusion was started at 4 hour after endotoxin injection. The rats were divided into the control and HA groups (n=16 for each group). The mean arterial pressure (MAP) was monitored during HA or normal saline infusion. Survival was assessed every 12 hours for 3 days throughout the experiment. Results: The survival rate (%) of the rats treated with HA was higher (60%) than that of the controls (20%) when HA was infused 4 hours after lipopolysaccharide (LPS) injection. The bronchoalveolar lavage (BAL) fluid of the animals surviving HA or NS infusion 4 hours after LPS showed that the total cell counts and number of neutrophils were significantly (p < 0.01) reduced in the HA treated groups compared with that of the controls (total cell count, $9.2{\times}10^4$/mL vs. $61{\times}10^4$/mL; neutrophils, $21{\times}10^4$/mL vs. $0.2{\times}10^4$/mL, respectively). There was no significant MAP difference between the HA or control groups either with or without endotoxin. Conclusion: Infusion of hyaluronan (1,600 kDa) reduced the BAL total cell count and the number of neutrophils and it improved the survival rate of the endotoxemic rats.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.33 no.6
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• pp.636-642
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• 2007

Background: CpG DNA plays an important role in immune cell function. This study examined whether the temporal control of toll-like receptor (TLR)9 by CpG DNA can regulate the expression of matrix metalloproteinase-9(MMP-9). Methods and materials: Macrophages were cultured in the presence of 10% FBS. For the various MMP genes analysis, RT-PCR and real-time PCR were performed. In addition, zymography assay performed for the MMP activity. The phosphorylation assay did for the ERK1/2 and NF${\kappa}B$ activation, and luciferase promoter assay was for the NF${\kappa}B$ activity. Results: CpG DNA induced the mRNA expression of MMP-2, MMP-9, and MMP-13, but not of MMP-7, MMP-8, and MMP-12, in a time-dependent manner. Especially, the mRNA expression of MMP-9 was strongly induced by CpG DNA using real-time RT-PCR. The TLR9 inhibitor, chloroquine, suppressed CpG DNA-induced MMP-9 expression and its activity. Moreover, CpG DNA induced the phosphorylation of ERK and the inhibition of ERK by U0126 suppressed CpG DNA-induced MMP-9 expression and its activity. CpG DNA stimulated $I{\kappa}B-{\alpha}$ degradation and luciferase activity. In addition, pretreatment of SN-50, the inhibitor of NF${\kappa}B$, strongly blocked the CpG DNA-induced MMP-9 expression and activity. Conclusion: These observations suggest that CpG DNA may play important roles in the activation of macrophages by regulating the production of MMP-9 via the sequential TLR9-ERK-NF${\kappa}B$ signaling pathway.