• BMB Reports
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• v.55 no.12
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• pp.627-632
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• 2022

Dickkopf-1 (DKK1) is a secreted protein that acts as an antagonist of the canonical WNT/β-catenin pathway, which regulates osteoblast differentiation. However, the role of DKK1 on osteoblast differentiation has not yet been fully clarified. Here, we investigate the functional role of DKK1 on osteoblast differentiation. Primary osteoprogenitor cells were isolated from human spinal bone tissues. To examine the role of DKK1 in osteoblast differentiation, we manipulated the expression of DKK1, and the cells were differentiated into mature osteoblasts. DKK1 overexpression in osteoprogenitor cells promoted matrix mineralization of osteoblast differentiation but did not promote matrix maturation. DKK1 increased Ca⁺ influx and activation of the Ca⁺/calmodulin-dependent protein kinase II Alpha (CAMK2A)-cAMP response element-binding protein 1 (CREB1) and increased translocation of p-CREB1 into the nucleus. In contrast, stable DKK1 knockdown in human osteosarcoma cell line SaOS2 exhibited reduced nuclear translocation of p-CREB1 and matrix mineralization. Overall, we suggest that manipulating DKK1 regulates the matrix mineralization of osteoblasts by Ca⁺-CAMK2A-CREB1, and DKK1 is a crucial gene for bone mineralization of osteoblasts.

• Biomolecules & Therapeutics
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• v.28 no.6
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• pp.549-560
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• 2020

Although DNA damage responses (DDRs) are reported to be involved in nitric oxide (NO) production in response to genotoxic stresses, the precise mechanism of DDR-mediated NO production has not been fully understood. Using a genotoxic agent aphidicolin, we investigated how DDRs regulate NO production in bovine aortic endothelial cells. Prolonged (over 24 h) treatment with aphidicolin increased NO production and endothelial NO synthase (eNOS) protein expression, which was accompanied by increased eNOS dimer/monomer ratio, tetrahydrobiopterin levels, and eNOS mRNA expression. A promoter assay using 5'-serially deleted eNOS promoters revealed that Tax-responsive element site, located at -962 to -873 of the eNOS promoter, was responsible for aphidicolin-stimulated eNOS gene expression. Aphidicolin increased CREB activity and ectopic expression of dominant-negative inhibitor of CREB, A-CREB, repressed the stimulatory effects of aphidicolin on eNOS gene expression and its promoter activity. Co-treatment with LY294002 decreased the aphidicolin-stimulated increase in p-CREB-Ser¹³³ level, eNOS expression, and NO production. Furthermore, ectopic expression of dominant-negative Akt construct attenuated aphidicolin-stimulated NO production. Aphidicolin increased p-ATM-Ser¹⁹⁸¹ and the knockdown of ATM using siRNA attenuated all stimulatory effects of aphidicolin on p-Akt-Ser⁴⁷³, p-CREB-Ser¹³³, eNOS expression, and NO production. Additionally, these stimulatory effects of aphidicolin were similarly observed in human umbilical vein endothelial cells. Lastly, aphidicolin increased acetylcholine-induced vessel relaxation in rat aortas, which was accompanied by increased p-ATM-Ser¹⁹⁸¹, p-Akt-Ser⁴⁷³, p-CREB-Ser¹³³, and eNOS expression. In conclusion, our results demonstrate that in response to aphidicolin, activation of ATM/Akt/CREB/eNOS signaling cascade mediates increase of NO production and vessel relaxation in endothelial cells and rat aortas.

• Journal of Ginseng Research
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• v.46 no.3
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• pp.426-434
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• 2022

Aim: This study investigates the effects of ginsenoside Rb1 (GsRb1) on methamphetamine (METH)-induced toxicity in SH-SY5Y neuroblastoma cells and METH-induced conditioned place preference (CPP) in adult Sprague-Dawley rats. It also examines whether GsRb1 can regulate these effects through the NR2B/ERK/CREB/BDNF signaling pathways. Methods: SH-SY5Y cells were pretreated with GsRb1 (20 mM and 40 mM) for 1 h, followed by METH treatment (2 mM) for 24 h. Rats were treated with METH (2 mg/kg) or saline on alternating days for 10 days to allow CPP to be examined. GsRb1 (5, 10, and 20 mg/kg) was injected intraperitoneally 1 h before METH or saline. Western blot was used to examine the protein expression of NR2B, ERK, P-ERK, CREB, P-CREB, and BDNF in the SH-SY5Y cells and the rats' hippocampus, nucleus accumbens (NAc), and prefrontal cortex (PFC). Results: METH dose-dependently reduced the viability of SH-SY5Y cells. Pretreatment of cells with 40 µM of GsRb1 increased cell viability and reduced the expression of METH-induced NR2B, p-ERK, p-CREB and BDNF. GsRb1 also attenuated the expression of METH CPP in a dose-dependent manner in rats. Further, GsRb1 dose-dependently reduced the expression of METH-induced NR2B, p-ERK, p-CREB, and BDNF in the PFC, hippocampus, and NAc of rats. Conclusion: GsRb1 regulated METH-induced neurotoxicity in vitro and METH-induced CPP through the NR2B/ERK/CREB/BDNF regulatory pathway. GsRb1 could be a therapeutic target for treating METH-induced neurotoxicity or METH addiction.

• The Korean Journal of Physiology and Pharmacology
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• v.21 no.6
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• pp.579-589
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• 2017

Anesthetics are used extensively in surgeries and related procedures to prevent pain. However, there is some concern regarding neuronal degeneration and cognitive deficits arising from regular anesthetic exposure. Recent studies have indicated that brain-derived neurotrophic factor (BDNF) and cyclic AMP response element-binding protein (CREB) are involved in learning and memory processes. Genistein, a plant-derived isoflavone, has been shown to exhibit neuroprotective effects. The present study was performed to examine the protective effect of genistein against isoflurane-induced neurotoxicity in rats. Neonatal rats were exposed to isoflurane (0.75%, 6 hours) on postnatal day 7 (P7). Separate groups of rat pups were orally administered genistein at doses of 20, 40, or 80 mg/kg body weight from P3 to P15 and then exposed to isoflurane anesthesia on P7. Neuronal apoptosis was detected by TUNEL assay and FluoroJade B staining following isoflurane exposure. Genistein significantly reduced apoptosis in the hippocampus, reduced the expression of proapoptotic factors (Bad, Bax, and cleaved caspase-3), and increased the expression of Bcl-2 and Bcl-xL. RT-PCR analysis revealed enhanced BDNF and TrkB mRNA levels. Genistein effectively upregulated cAMP levels and phosphorylation of CREB and TrkB, leading to activation of cAMP/CREB-BDNF-TrkB signaling. PI3K/Akt signaling was also significantly activated. Genistein administration improved general behavior and enhanced learning and memory in the rats. These observations suggest that genistein exerts neuroprotective effects by suppressing isoflurane-induced neuronal apoptosis and by activating cAMP/CREB-BDNF-TrkB-PI3/Akt signaling.

• Journal of Life Science
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• v.28 no.1
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• pp.37-42
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• 2018

Non-steroidal anti-inflammatory drug-activated gene-1 (NAG-1) is a transforming growth factor beta (TGF-${\beta}$) superfamily gene associated with pro-apoptotic and anti-tumorigenic activities. In the present study, we investigated if caffeic acid phenethyl ester (CAPE) derived from propolis could induce the expression of anti-tumorigenic gene NAG-1. Our results indicate that CAPE significantly induced NAG-1 expression in a time- and concentration-dependent manner in HCT116 cells. We also found that CAPE induced NAG-1 expression in a concentration-dependent manner in another human colorectal cancer cell line, LOVO. In addition, CAPE triggered apoptosis, which was detected with Western blot analysis using poly-(ADP-ribose) polymerase antibody. NAG-1 induction by CAPE was not dependent on transcription factor p53, which was confirmed with Western blot analysis using p53 null HCT116 cells. The luciferase assay results indicated that the new cis-elements candidates were located between -474 and -1,086 of the NAG-1 gene promoter. CAPE dramatically induced activating transcription factor 3 (ATF3) expression, but not cAMP response element-binding protein (CREB), which shares the same binding sites with ATF3. The co-transfection experiment with pCG-ATF3 and pCREB showed that only ATF3 was associated with NAG-1 up-regulation by CAPE, whereas CREB had no effect. In conclusion, the results suggest that CAPE could induce the expression of anti-tumorigenic gene NAG-1 mainly through ATF3.

• Journal of Physiology & Pathology in Korean Medicine
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• v.26 no.3
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• pp.281-286
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• 2012

The present study was designed to investigate the effect of Rehmannia glutinosa on phosphorylation of extracellular signal-regulated kinase(ERK) and cAMP response element-binding protein(CREB) in the acute cocaine-treated rats. Rats orally received vehicle or extract of Rehmannia glutinosa 1 h prior to saline (1 ml/kg, i.p.) or cocaine hydrochloride (20 mg/kg, i.p.) treatment. Rats were sacrificed 15 min after a single intraperitoneal injection of saline or cocaine. Rehmannia glutinosa at dose of 50 mg/kg significantly decreased phosphorylation of ERK, CREB and Elk-1 in the nucleus accumbens and striatum of the cocaine-treated rat brain by immunocytochemistry. These results suggest that Rehmannia glutinosa may contribute to the effects of cocaine on gene expression and on behaviors.

• The Korean Journal of Physiology and Pharmacology
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• v.19 no.2
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• pp.89-97
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• 2015

The aim of this study was to investigate the involvement of dopaminergic receptors (DR) in behavioral sensitization, as measured by locomotor activity, and the over-expression of cocaine- and amphetamine-regulated transcript (CART) peptides after repeated administration of cocaine in mice. Repeated administrations of cocaine induced behavioral sensitization and CART over-expression in mice. The levels of striatal CART mRNA were significantly increased on the $3^{rd}$ day. CART peptides were over-expressed on the $5^{th}$ day in the striata of behaviorally sensitized mice. A higher proportion of $CART^+$ cells in the cocaine-treated mice were present in the nucleus accumbens (NAc) shell than in the dorsolateral (DL) part of caudate putamen (CP). The concomitant administration of both $D_1R$ and $D_2R$ antagonists, SCH 23390 ($D_1R$ selective) and raclopride ($D_2R$ selective), blocked cocaine induced-behavioral sensitization, CART over-expression, and cyclic adenosine 5'-monophosphate (cAMP)/ protein kinase A (PKA)/phospho-cAMP response element-binding protein (pCREB) signal pathways. SCH 23390 more predominantly inhibited the locomotor activity, CART over-expression, pCREB and PKA activity than raclopride. Cocaine induced-behavioral sensitization was also attenuated in the both $D_1R$ and $D_2R$ knockout (KO) mice, respectively. CART over-expression and activated cAMP/PKA/pCREB signal pathways were inhibited in the $D_1R$-KO mice, but not in the $D_2R$-KO mice. It is suggested that behavioral sensitization, CART over-expression and activated cAMP/PKA/pCREB signal pathways induced by repeated administration of cocaine could be more predominantly mediated by $D_1R$.

• Korean Journal of Biological Psychiatry
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• v.16 no.1
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• pp.5-14
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• 2009

Objectives : Most of the mechanisms reported for antidepressant drugs are the enhancement of neurite outgrowth and neuronal survival in the rat hippocampus. Neural cell adhesion molecule 140(NCAM140) has been implicated as having a role in cell-cell adhesion, neurite outgrowth, and synaptic plasticity. In this report, we have performed to elucidate a correlation among chronic antidepressant treatments, NCAM140 expression, and activation of phosphorylated cyclicAMP responsive element binding protein(pCREB) which is a downstream molecule of NCAM140-mediated intracellular signaling pathway in the rat hippocampus. Methods : Fluoxetine(10mg/kg) was injected acutely(daily injection for 5days) or chronically(daily injection for 14days) in adult rats. RNA and protein were extracted from the rat hippocampus, respectively. Real-time RT-PCR was performed to analyze the expression pattern of NCAM140 gene and western blot analyses for the activation of the phosphorylation ratio of CREB. Results : Chronic fluoxetine treatments increased NCAM140 expression 1.3 times higher than control in rat hippocampus. pCREB immunoreactivity in the rat hippocampus with chronic fluoxetine treatment was increased 4.0 times higher than that of control. Conclusion : Chronic fluoxetine treatment increased NCAM140 expression and pCREB activity in the rat hippocampus. Our data suggest that NCAM140 and pCREB may play a role in the clinical efficacy of antidepressants promoting the neurite outgrowth and neuronal survival.

• Herbal Formula Science
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• v.27 no.2
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• pp.121-136
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• 2019

Objective : To investigate the effect of sihogayonggolmoryeotang (SY) on Single Prolonged Stress(SPS)-induced Post Traumatic Stress Disorder(PTSD). Method : To confirm the effects of SY on SPS-induced PTSD, Changes in body weight, sucrose intake open field test(OFT) and forced swimming test(FST)were observed. After behavioral tests, the plasma corticosterone(CORT) from the abdominal aorta, serotonin(5-HT) from prefrontal cortex, hippocampus, amygdala and striatum, norepinephrine(NE) and dopamine(DA) from hippocampus was measured by ELISA. mRNA expression of brain-derived neurotrophic factor(BDNF) and cAMP response element-binding protein(CREB) in hippocampus was measured by RT-PCR. Result : Weight change and sucrose intakes of rats in 14th day after the administration of SY were significantly increased in the SPS + SY450 group compared to the SPS group (p<0.05). Numbers of crossing in the central zone in the OFT were significantly increased in the SPS + SY450 group (p<0.05) compared with the SPS group. The immobility time of FST was significantly decreased in SPS + SY450 group compared with SPS group (p<0.05). The change of plasma CORT concentration was significantly decreased in SPS + SY450 group compared with that in SPS group (p<0.05). The change of 5-HT concentration was significantly increased in the SPS + SY450 group at hippocampus and amygdala compared with the SPS group (p<0.05). The concentration of DA was significantly increased in the SPS + SY450 group compared with the SPS group (p<0.05). The expression of BDNF and CREB were significantly increased in SPS + SY450 group compared with the SPS group (p<0.05). Conclusion : SY administration lowered the increase of CORT caused by PTSD and increases the 5-HT concentration and reversed the decreased expression of NE and DA and BDNF and CREB by PTSD. It is postulated that SY is effective in treating PTSD by restoring cognitive function, memory impairment, unstable emotional disturbances.

• Biomolecules & Therapeutics
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• v.19 no.1
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• pp.45-51
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• 2011

These experiments were designed to use typical makers from behaviors and molecular basis in addicted animals of morphine and cocaine. Morphine has been widely abused with a high physical dependence liability. Morphine withdrawal activates the intracellular cAMP signaling pathway and further leads to changes in the expression of the cAMP response element binding protein (CREB), which may be important to the development and expression of morphine dependence. From these experiments, repeated morphine (10 mg/kg, twice per day for 7 days) developed physical dependence. Withdrawal signs were precipitated by naloxone and also increased the expression of the CREB. In addition, repeated exposure of cocaine (15 mg/kg) to mice develops locomotor sensitization and produced lasting behavioral sensitivity. Cocaine- and amphetamine-regulated transcript peptide (CART) peptide was up-regulated by repeated administration of cocaine in the striatum. Therefore, repeated morphine induced the development of physical dependence and increased pCREB. In addition, repeated cocaine induced locomotor sensitization and over-expressed CART peptide. In conclusion, the development of physical dependence and pCREB for morphine, and locomotor sensitization and CART peptide over-expression for cocaine would be useful markers to predict the abuse potential of opioid analgesics and pychostimulant drugs in animals, respectively.