Visfatin induces neurite outgrowth in PC12 cells via ERK1/2 signaling pathway

Kang, Young-Soon,Bae, Moon-Kyoung,Kim, Jee-Young,Jeong, Joo-Won,Yun, Il,Jang, Hye-Ock,Bae, Soo-Kyung Elsevier 2011 Neuroscience Letters Vol.504 No.2

<P><B>Abstract</B></P> <P>The angiogenic and inflammatory functions of visfatin and its effect on vascular cells, are fairly well known. However, its role within the nervous system remains largely unclear. To gain insight into this area, we studied the neuritogenic effect of visfatin on PC12 rat pheochromocytoma cells. We investigated whether visfatin gene expression, which is upregulated by hypoxia in cancer cells, is associated with neuritogenesis in PC12 cells. Using RT-PCR, Western blot analysis, ELISA, morphological observations, and immunostaining, we initially showed that CoCl<SUB>2</SUB>, a hypoxic mimetic agent, upregulated visfatin gene expression along with neurite outgrowth in PC12 cells. We also showed that visfatin stimulated neurite outgrowth in PC12 cells. Moreover, in PC12 cells, visfatin evoked the activation of the extracellular signal-regulated kinase 1/2 (ERK1/2), which is closely linked to neuritogenesis. Visfatin-induced outgrowth of neurites was prevented by inhibition of the ERK1/2 pathway. Taken together, our results demonstrate for the first time that visfatin induces neurite outgrowth in PC12 cells via the activation of an ERK-dependent pathway, and suggest that visfatin may exert various biological, physiological, and pathological functions in not only the vascular system but also the nervous system.</P> <P><B>Highlights</B></P> <P>▸ We investigated the neuritogenic effect of visfatin on PC12 cells. ▸ Visfatin expression was upregulated during cobalt chloride-induced neuritogenesis in PC12 cells. ▸ Visfatin induced neurite outgrowth in PC12 cells. ▸ Visfatin-induced outgrowth of neurites was mediated by ERK1/2 signaling pathway.</P>

PC12 세포에서 신경전달물질 방출을 저해하는 저분자 생리활성물질 FS390의 탐색

정연태(Yeun-Tai Chung),김희정(Hee-Jung Kim),이윤식(Yun-Sik Lee) 대한해부학회 2006 Anatomy & Cell Biology Vol.39 No.2

In vitro실험계에서 tritium-label된 norepinephrine ([3H]-NE)을 PC12세포에 incorporation시킨 후에 60mM의 고농도의 K+의 자극에 의해서 탈분극 후에 세포 외로 방출되는 [3H]-NE의 양을 scintillation countering하여 정량하는 독특한 실험계를 수립하였다. 이 조절성 분비 실험계에서, 미생물대사 산물로부터 신경전달물질의 방출에 영향을 미치는 생리활성물질을 탐색하기 위하여, 곰팡이, 방선균과 박테리아의 대사산물 1만 1,000여 샘플을 탐색한 결과, PC12세포에서 고농도의 K+의 자극에 의해서 탈분극 후에 유도되는 [3H]-NE의 방출을 효과적으로 저해하는 FS390을 방선균 유래의 대사산물로부터 얻었다. PC12세포와 rat cortical neurons에서 FS390은 고농도의 K+의 자극에 의한 탈분극 후에 유도되는 신경전달물질로서 ATP의 방출에도 유의한 저해 효과를 나타냈다. PC12세포에서 저해 효과는 ionopore로 알려진 ionomycin (1 μM)을 포함하는 저농도의 K+의 완충액을 처리하였을 때에도 보여졌다. 이들 결과로부터 FS390의 신경전달 물질의 방출에 대한 저해작용은 세포내 Ca2+ 유입 이후의 반응으로 추정되며 이 신경전달물질의 방출기구에 대한 해석을 위하여 FS390물질에 대한 정제 및 구조해석을 시도하였다. We established an in vitro experimental system using the following procedure. We first introduced tritium-labeled norepinephrine ([3H]-NE) into PC12 cells. The [3H]-NE incorporated-PC12 cells were stimulated by a high concentration (60 mM) of K+ buffer during 12 minutes. Then, we collected 100 μL supernatant and counted the amount of [3H]-NE release from PC12 cells with a scintillation counter. After screening fungal, Streptomyces spp. or bacterial product using this experimental sytem, we obtained FS390 from Streptomyces spp. which inhibited [3H]-NE release from PC12 cells. FS390 also inhibits the release of ATP as a neurotransmitter of PC12 cells and rat cortical neurons. The inhibitory effect was seen even when the PC12 cells were treated with low K+ buffer containing ionomycin (1 μM) as an ionopore. This result suggests that the inhibitory action of FS390 on neurotransmitter release appeared after the influx of Ca2+.

p190RhoGAP and Rap-dependent RhoGAP (ARAP3) inactivate RhoA in response to nerve growth factor leading to neurite outgrowth from PC12 cells

Chan-Young Jeon,김희준,Jae-Yong Lee,김재봉,김성찬,Jae-Bong Park 생화학분자생물학회 2010 Experimental and molecular medicine Vol.42 No.5

Rat pheochromocytoma (PC12) cells have been used to investigate neurite outgrowth. Nerve growth factor (NGF) has been well known to induce neurite outgrowth from PC12 cells. RhoA belongs to Ras-related small GTP-binding proteins, which regulate a variety of cellular processes, including cell morphology alteration,actin dynamics, and cell migration. NGF suppressed GTP-RhoA levels after 12 h in PC12 cells and was consistently required for a long time to induce neurite outgrowth. Constitutively active (CA)-RhoA suppressed neurite outgrowth from PC12 cells in response to NGF, whereas dominant-negative (DN)-RhoA stimulated it, suggesting that RhoA inactivation is essential for neurite outgrowth. Here, we investigated the mechanism of RhoA inactivation. DN-p190RhoGAP abrogated neurite outgrowth, whereas wild-type (WT)-p190RhoGAP and WT-Src synergistically stimulated it along with accelerating RhoA inactivation, suggesting that p190RhoGAP, which can be activated by Src, is a major component in inhibiting RhoA in response to NGF in PC12 cells. Contrary to RhoA, Rap1was activated by NGF, and DN-Rap1 suppressed neurite outgrowth, suggesting that Rap1 is also essential for neurite outgrowth. RhoA was co-immunoprecipitated with Rap1, suggesting that Rap1 interacts with RhoA. Furthermore, a DN-Rap-dependent RhoGAP (ARAP3) prevented RhoA inactivation, abolishing neurite formation from PC12 cells in response to NGF. These results suggest that NGF activates Rap1, which, in turn, up-regulates ARAP3 leading to RhoA inactivation and neurite outgrowth from PC12cells. Taken together, p190RhoGAP and ARAP3 seem to be two main factors inhibiting RhoA activity during neurite outgrowth in PC12 cells in response to NGF.

Nerve Growth Factor로 분화된 PC12 세포에서 GABA 및 NMDA 수용체의 전기생리학적 특성

김종욱,송대규,배재훈,박원균 啓明大學校 醫科大學 2002 계명의대학술지 Vol.21 No.1

NGF로 분화시킨 PC12 세포에서 NMDA 수용체 및 GABA 수용체의 전기생리학적 특성을 관찰하고자 배양 7∼14일 사이의 PC12 세포를 이용하여 whole-cell patch clamp 방법으로 안정막전압 및 막전압의 변동에 따른 이들 수용체 전류의 특성을 기록하였다. 관류액에 20 μM APV를 투여한 후 안정막전압은 유의한 변동이 없었다. 막전압을 -80 mV에서 -10 mV까지 단계적으로 고정하였을 때 발생하는 NMDA 수용체 전류는 막전압이 과분극될 때는 내향성, 탈분극될 때는 외향성의 전류가 측정되며 그 크기는 과분극에 비하여 탈분극 시 유발전류의 증가폭이 점점 더 크게 나타나는 막전압에 의존적이었다. 활동전압 및 glutamate 수용체를 차단한 후 20 μM GABA를 투여한 후 안정막전압이 약간 과분극되는 세포에서 막전압을 -80 mV에서 -10 mV까지 단계적으로 고정하였을 때 발생하는 GABA 수용체 유발전류는 막전압이 과분극될 때는 외향성, 탈분극될 때는 내향성의 전류가 측정되며 30 mV까지는 막전압의 변동과 유발전류사이에 직선적인 관계가 관찰되었다. 이상으로 NGF로 분화시킨 PC12 세포주에서 NMDA 수용체 및 GABA 수용체의 전기생리학적 특성은 생체의 신경계에서 발견되는 수용체 특성과 유사한 것으로 생각되며, PC12세포는 이들 수용체에 대한 다양한 전기생리학적 실험의 연구재료로 충분히 이용될 수 있을 것으로 생각된다. Nerve growth factor (NGF), which has been used for the differentiation of PC12 cells in culture, not only promotes the survival and differentiation of neurons but also affects the suructural and functional properties. The aim of this study was to investigate the current properties of NMDA and GABA receptors by using whole-cell patch clamp technique in NGF differentiated PC12 cells cultured for 7∼14 days. Membrane potential did not change from the resting potential of -48 mV by the infusion of a NMDA receptor blocker, APV, (50 μM) in the perfusion solution. NMDA components of the evoked currents at the membrane potential, changing from -80 mV to -10 mV. showed a voltage dependency in the current-potential relationship. When action potential and glutamate receptors were blocked, membrane potential was hyperpolarized by the infusion of GABA (20 μM) in some PC12 cells, but not in other cells. In the hyperpolarized cells, GABA components of the evoked currents at the membrane potential, changing from -80 mV to -10 mV, showed a linear correlation between the currents and the membrnae potential. In conclusion, the electrophysiological properties of NMDA and GABA receptors in NGF differentiated PC12 cells may be similar to those in the biological neurons. Therefore, it seems that PC12 cells appear to be suited for the studies on function and signal transmission of these receptors.

Differential Effect of Harmalol and Deprenyl on Dopamine-Induced Mitochondrial Membrane Permeability Change in PC12 Cells

( Chung Soo Lee ) 한국응용약물학회 2004 Biomolecules & Therapeutics(구 응용약물학회지) Vol.12 No.1

N/A Opening of the mitochondrial permeability transition pore has been recognized to be involved in cell death. The present study investigated the effect of β-carbolines(harmaline and harmalol) and deprenyl on the dopamine-induced change in the mitochondrial membrane permeability and cell death in differentiated PC12 cells. Cell death due to 250μM dopamine was inhibited by caspase inhibitors (z-IETD.fmk, z-LEHD.fmk and z-DQMD,fmk) and antioxidants (N-acetylcysteine. ascorbate, superoxide dismutase, catalase and carboxy-PTIO). β-Carbolines prevented the dopamine-induced cell death in PC12 cells, while deprenyl did not inhibit cell death. β-Carbolines decreased the condensation and fragmentation of nuclei caused by dopamine in PC12 cells. β-Carbolines inhibited the decrease in mitochondrial transmembrane potential, cytochrome c release, formation of reactive oxygen specier and depletion of GSH caused by dopamine in PC12 cells, whereas deprenyl did not decrease dopamine-induced mitochondrial damage. β-Carboline, deprenyl and antioxidants depressed the formations of nitric oxide and melanin in dopamine-treated PC12 cells. The results suggest that cell death due to dopamine in PC12 cells is mediated by caspase-8, -9 and -3. Unlike deprenyl, β-carbolines may attenuate the dopamine- induced cell death in PC12 cells by suppressing change in the mitochondrial membrane permeability through inhibition of the toxic action of reactive oxygen and nitrogen species.

Molecular Mechanism of NO-induced Cell Death of PC12 Cells by $IFN{\gamma}\;and\;TNF{\alpha}$

Yi, Seh-Yoon,Han, Seon-Kyu,Lee, Jee-Yeon,Yoo, Young-Sook The Korean Society of Toxicogenomics and Toxicopro 2005 Molecular & cellular toxicology Vol.1 No.3

Nitric oxide (NO) is a small, diffusible, and highly reactive molecule, which plays dichotomous regulatory roles under physiological and pathological conditions. NO promotes apoptosis in some cells, and inhibits apoptosis in other cells. In the present study, we attempted to characterize the NO signaling pathway and cellular response in PC12 cells treated with cytokines. $IFN{\gamma}\;and\;TNF{\alpha}$ treatment resulted in a synergistic increase of nitrite accumulation, with the induction of inducible nitric oxide synthase (iNOS) in the PC12 cells. Moreover, as nitrite concentration increased, cell viability decreased. In order to explore MAP kinase involvement in nitric oxide production resultant from $IFN{\gamma}\;and\;TNF{\alpha}$ stimulation, we measured the activation of MAP kinase using specific MAP kinase inhibitors. PC12 cells pretreated with SB203580, a p38 MAP kinase-specific inhibitor, resulted in the inhibition of iNOS expression and NO production. However, PD98059, an ERK/MAP kinase-specific inhibitor, was not observed to exert such an effect. In addition, Stat1 activated by $IFN{\gamma}\;and\;TNF{\alpha}$ was interacted with p38 MAPK. These data suggest that p38 MAP kinase mediates cytokine-mediated iNOS expression in the PC12 cells, and Jak/Stat pathway interferes with p38 MAPK signaling pathway.

PC12 세포에서 신경전달물질 방출을 저해하는 저분자 생리활성물질 S8877의 탐색

강주섭,지옥화,이윤식 대한암예방학회 2006 Journal of cancer prevention Vol.11 No.1

We established an in vitro experimental system using the following procedure. We first introduced tritium-labeled norepinephrine ([3H]-NE) into PC12 cells. The [3H]-NE incorporated into PC12 cells were then stimulated by a high concentration (60 mM) of K+ buffer during 12 minutes. Then, we collected 100μl supernatant and counted the amount of [3H]-NE release from PC12 cells with a scintillation counter. After screening fungal, Streptomyces spp. or bacterial product using this experimental system, we obtained S8877 from Streptomyces spp. which inhibited [3H]-NE release from PC12 cells. S8877 also inhibits the release of ATP as a neurotransmitter of PC12 cells and rat cortical neurons. The inhibitory effect was seen even when the PC12 cells were treated with low K+ buffer containing ionomycin (1μM) as an ionophore. This result suggests that the inhibitory action of S8877 on neurotransmitter release appeared after the influx of Ca2+. (Cancer Prev Res 11, 22-29, 2006)

Butyrate-induced differentiation of PC12 cells to chromaffin cells involves cell adhesion and induction of extracellular proteins and cell adhesion proteins

허지인,오수진,고윤정,김정현,강홍준,박성훈,김현석,신종연,이성영,김민주,민본홍,김성찬,박재봉,김재봉,이재용 한국통합생물학회 2010 Animal cells and systems Vol.14 No.4

PC12 cells were differentiated into the cells of chromaffin phenotype by butyrate treatment. Cells were aggregated and formed tight cell adhesion. To investigate the molecular change in this differentiation, we examined expression levels of cell adhesion proteins and extracellular proteins during butyrate induced-differentiation of PC12 cells. Integrin β1, integrin α7, E cadherin, VCAM, collagen-I, fibronectin, desmoglein and connexin were increased during differentiation. The levels of clusterin and secreted clusterin were also increased. These increased levels of cell adhesion proteins and extracellular proteins appear to induce cell aggregation and tight cell adhesion. The levels of p21, p27 and p16 were increased probably because of differentiation-related growth arrest during differentiation. Prolonged incubation of butyrate up to 1 day was required for differentiation. Signal transduction pathways for this differentiatiom could not be identified since various inhibitors had no effect. The results showed that butyrateinduced differentiation of PC12 cells

PC-12 세포에서 Cisplatin에 의한 Apoptosis의 기전

이영우,오세옥,조원호,이상호 대한신경외과학회 1998 Journal of Korean neurosurgical society Vol.27 No.11

PC12 세포에서 신경전달물질 방출을 저해하는 생리활성물질 FS11052의 탐색

이윤식,이존화,Lee, Yun-Sik,Lee, John Hwa 대한수의학회 2006 大韓獸醫學會誌 Vol.46 No.2

We established an in vitro experimental system using the following procedure. We first introduced tritium-labeled norepinephrine ([$^3$H]-NE) into PC12 cells, The [$^3$H]-NE incorporated into PC12 cells were then stimulated by a high concentration (60 mM) of $K^+$ buffer during 12 minutes. Then, we collected $100{\mu}l$ supernatant and counted the amount of [$^3$H]-NE release from PC12 cells with a scintillation counter. After screening fungal, Streptomyces spp. or bacterial product using this experimental sytem, we obtained FS11052 from Streptomyces spp. which inhibited [$^3$H]-NE release from PC12 cells. FS11052 also inhibits the release of ATP as a neurotransmitter of PC12 cells and rat cortical neurons, The inhibitory effect was seen even when the PC12 cells were treated with low $K^-$ buffer containing ionomycin ($1{\mu}M$) as an ionopore. This result suggests that the inhibitory action of FS11052 on neurotransmitter release appeared after the influx of $Ca^{2+}$.