난포호르몬이 임신 자궁동맥 평활근세포에서 extracellular signal-regulated kinases 활성화에 미치는 영향

이용우 ( Yong Woo Lee ),김호연 ( Ho Yeon Kim ),박상민 ( Sang Min Park ),최수란 ( Soo Ran Choi ),정지윤 ( Ji Youn Chung ),문종수 ( Chong Soo Moon ) 대한산부인과학회 2011 Obstetrics & Gynecology Science Vol.54 No.9

목적: 내피세포를 제거한 자궁동맥 평활근세포에 난포호르몬을 투여하여 자궁동맥 평활근세포가 난포호르몬의 직접적인 표적조직인지와 난포 호르몬이 자궁동맥 평활근세포에서 직접적으로 extracellular signal-regulated kinases (ERK2/1) 활성화하는지 여부를 파악하여 난포호르몬의 자궁동맥 혈관확장 중에서 내피세포-비의존성 기전을 알아보기 위하여 본 연구를 시행하였다. 연구방법: 만삭임신의 양에서 절제된 자궁동맥을 대상으로 콜라겐 분해효소로 내피세포를 제거하였다. 내피세포가 제거된 자궁동맥 평활근 분절은 소화효소처리 후에 평활근세포를 수집하여 성장배지에서 계대배양하였다. 혈관 내피세포와 평활근 상태의 평가는 항평활근 알파-액틴 단 일클론 항체, 항내피세포 산화질소 합성효소 단일클론 항체, 항caveolin-1 다클론 항체를 처리하여 중복 면역형광 염색과 흐름세포측정으로 판정하였다. 자궁동맥 평활근세포에 난포호르몬을 농도를 달리하여(10-14 M에서 10-6 M까지) 투여하여 전체 세포 추출물에 대하여 항인산 특이 mitogen activated protein kinases (MAPKs) 다클론 항체로 ERK2/1 인산화의 western blot 분석을 하였다. 난포호르몬 수용체 길항제인 ICI 182,780으로 전처치한 후에 다시 난포호르몬을 낮은 농도(10-10 M)와 높은 농도(10-7 M)로 투여하여 인산화 ERK2/1의 western blot 분석을 하여 전처치하지 않은 경우와 비교하였다. 결과: 평활근세포의 전형적인 hill and valley 양상과 세포 내에 평활근 알파-액틴과 caveolin-1이 뚜렷하게 염색되었다. 흐름세포측정 분석에서 평활근 알파-액틴과 caveolin-1은 각각 99.96%와 99.98%가 발현되었지만 내피세포 산화질소 합성효소 단백질은 26.12%에서만 발현되었다. 난포호르몬의 농도의 증가에 따라 인산화 ERK2/1의 발현은 이상성 증가반응을 보였다. 난포호르몬이 저농도(10-14-10-10 M)에서는 ERK2/1의 인산화는 용량-의존적으로 증가하였지만, 고농도(10-9-10-8 M)에서는 ERK2/1의 인산화는 오히려 감소하였으며, 약물농도인 초고농도(10-7-10-5 M)에서는 ERK2/1의 인산화가 급격하게 증가하였다. 난포호르몬 수용체 길항제인 ICI 182,780으로 전처치하면 난포호르몬을 저농도(10-10 M) 혹은 고농도(10-7 M)로 투여하더라도 ERK2/1 인산화는 소멸되었다. Reverse transcription-polymerase chain reaction 분석에서 난포호르몬 알파-수용체와 베타-수용체는 자궁동맥 평활근과 자궁동맥 평활근세포에서 모두 발현되었다. 결론: 자궁동맥 평활근은 난포호르몬 수용체를 지니고 있는 난포호르몬의 표적조직이며, 난포호르몬의 혈관확장 작용에는 자궁동맥 평활근세포에서 직접적인 ERK2/1 활성화를 통한 혈관내피 비의존성 기전도 존재한다는 것을 확인할 수 있다. Objective: To explore the endothelium-independent mechanisms of estrogen induced uterine vasodilatation, this study was performed to determine whether uterine artery smooth muscle (UASM) cells are direct targets of estrogen, and estradiol (E2) stimulates extracellular signal-regulated kinases (ERK2/1) in endothelium denuded UASMs. Methods: The uterine arteries were obtained from late gestation pregnant sheep and the endothelium was denuded with collagenase digestion. The uterine artery smooth muscle segments were digested, collected and cultured. Endothelial integrity and smooth muscle status were assessed by Double immunofluorescence staining and flow cytometry. The UASM cells were treated with increasing concentrations of E2 (10-14 to 10-6 M), and pretreated with ICI 182,780 followed by different concentrations (10-10 and 10-7 M) of E2. Western blot analysis of ERK2/1 phosphorylation with a phospho-mitogen activated protein kinases (MAPKs) antibody were carried out to total cell extracts. Results: The loss of endothelial function and adequacy of smooth muscle integrity were confirmed. When challenged with increasing concentrations of E2, a bi-phase ERK2/1 phosphorylation was observed. Treatment with low doses (10-14 to 10-10 M) of E2, ERK2/1 phosphorylation was dose-dependently increased, whereas high doses (10-9 to 10-8 M) did not phosphorylate ERK2/1. However, treatment with pharmacological doses (10-7 to 10-6 M) drastically phosphorylated ERK2/1. In the presence of ICI 182,780, E2 induced ERK2/1 phosphorylation were abolished in both. Conclusion: It suggests that UASM is the target tissue of estrogen during uterine vasodilatation, and estrogen stimulation of ERK2/1 activation is mediated by an estrogen receptor-dependent mechanism. It also is presumed that endothelium independent mechanism exists in estrogen induced vasodilatation.

저칼륨혈증 흰쥐 신장에서 Akt, p-Akt, ERK 및 p-ERK 단백발현의 변화

배춘상(Choon Sang Bae),조혜정(Hye Jung Cho),안규윤(Kyu Yoon Ahn) 대한체질인류학회 2017 해부·생물인류학 (Anat Biol Anthropol) Vol.30 No.3

저칼륨혈증은 신장의 형태학적 변화와 대사성 알칼리증을 유발시키는 것으로 알려져 있다. 이전 결과들은 저칼륨혈증의 병태생리학적 변화에 K+평형 조절 이온채널, pump 유전자, NF-E2-related factor 2 (Nrf2) 전사유전자를 포함한 다양한 유전자가 관여할 것이라는 가능성을 제시하였다. 이에 본 연구는 칼륨제한 식이 기간에 따른 흰쥐 신장 내 Akt와 p-Akt 및 ERK와 p-ERK의 발현 및 분포의 변화를 Western 분석과 면역조직화학 방법으로 관찰함으로써, 저칼륨혈증이 신장에서 AKT/ERK 인산화에 영향을 미치는지를 확인하였다. Western 분석소견에서 Akt 및 p-Akt 단백질 발현은 칼륨제한 식이가 진행될수록 증가하는 양상을 보였으며, ERK 및 p-ERK 단백질발현은 칼륨제한 식이 2주군에서 정상 식이군에 비해 약간 증가하는 양상을 보였다. 면역조직화학 소견에서 Akt 단백의 면역반응성은 먼쪽곱슬세관, 겉질곧은부분 및 속질곧은부분에서 중등도의 발현을 보였다. 칼륨제한 식이군의 Akt의 면역반응성은 칼륨제한 식이가 진행될수록 바깥속질집합관에서 현저히 증가하였다. 칼륨제한 식이군의 p-Akt의 면역반응성은 칼륨제한 식이 2주군의 먼쪽곱슬세관, 치밀반점과 속질곧은부분에서 증가하였고 토리쪽곱슬세관에서는 중등도의 발현을 보였다. 칼륨제한 식이군의 ERK의 면역반응성은 칼륨제한 식이 2주군과 3주군의 바깥속질집합관에서 현저히 증가하였으며 먼쪽곱슬세관, 겉질집합관에서는 증등도의 증가를 보였다. 칼륨제한 식이군의 p-ERK의 발현부위는 정상식이군과 비교해 차이가 없었으나 면역반응성은 칼륨제한 식이 2주군의 바깥속 질집합관의 핵에서 현저히 증가하였다. 이상의 결과 저칼륨혈증 시 p-Akt의 발현은 칼륨제한 식이가 길어질수록 점진적으로 증가하였지만, p-ERK의 발현은 칼륨제한 식이 2주군에서 현저히 증가되었다. 따라서 저칼륨 상태에서의 Akt 및 ERK 인산화의 촉진은 이온채널 및 이온수송체 유전자 조절뿐만 아니라 세포 내 신호전달에 있어 중요한 역할에 관여할 것임을 시사해 주었다. Hypokalemia causes metabolic alkalosis and morphological changes of the kidney. K+ balance is regulated not only by ion channels or pump gene, but also by various genes including NF-E2-related factor 2 (Nrf2). Previous study suggested the possibility that Akt and ERK kinase may be involved in Nrf2 transcriptional gene activation. In present study, we investigate the alterations of Akt, p-Akt, ERK, p-ERK protein in both normal kidney and K+-deficient diet kidney using Western blot analysis, and immunohistochemisrty. Our western blot data showed that the expression of Akt and p-Akt was increased gradually in K+-depleted diet (from 1W-3W) compared to normal group. The expression of ERK and p-ERK was markedly increased in K+-depleted diet 2W in comparison with normal group. Based on our immunostaining results, Akt protein immunoreactivity was prominently increased in outer medullary collecting duct, especially in K+-depleted diet 2 weeks. The localization of p-Akt proteins in K+-depleted groups was not different from normal group, but the immunoreactivity was significantly increased in distal convoluted tubule, macula densa and outer medullary thick ascending limb in K+-depleted diet 1 and 2 weeks groups. ERK protein immunoreactivity was prominently increased in outer medullary collecting duct, especially in K+-depleted diet 2 and 3 weeks. The localization of p-ERK proteins in K+-depleted groups was not different from normal group, but the immunoreactivity was prominently increased in the nucleus of outer medullary collecting duct especially in K+-depleted diet 2 weeks. Taken together, we suggest that the expression of p-Akt was gradually increased in K+-depleted groups of kidney, but the expression of p-ERK was markedly increased in K+-depleted diet 2 week group. Hence, the promotion of AKT and ERK phosphorylation in hypokalemic condition may be involved in the regulation of ion channels, ion transporters and subsequent intracellular signal transduction.

ERK5 regulates invasiveness of osteosarcoma by inducing MMP‐9

Kim, Sang‐,Min,Lee, Hyewon,Park, Youn‐,Soo,Lee, Youbin,Seo, Sung Wook Wiley Subscription Services, Inc., A Wiley Company 2012 Journal of orthopaedic research Vol.30 No.7

<P><B>Abstract</B></P><P>The purpose of this study is to determine the role of ERK5 in cellular invasion of osteosarcoma (OS). The human OS cell line (MG63, SaOS, and U2OS) and primary OS cells were used for the study. The expression of ERK5 and MMP‐9 in each cell was examined by western blot or RT‐PCR. To evaluate the biological role of ERK5, proliferation assay (MTT) and invasion assay (BD Matrigel™) were performed after silencing ERK5 using siRNA. MMPs expressions were analyzed using RT‐PCR and zymography after silencing ERK5. ERK5 was distinctly overexpressed in U2OS and primary OS cell. Both of them also expressed MMP‐9, which was not shown in MG63 and SaOS in RT‐PCR. ERK5 silencing did not suppress the proliferation of OS cells. However, ERK5 silencing significantly reduced the number of invading cells in invasion assay. The expression of MMP‐9 was specifically reduced after silencing ERK5. The zymography showed that the enzyme activity of MMP‐9 was also reduced after ERK5 suppression. The expression of ERK5 regulates the invasion of OS cells by inducing MMP‐9 expression. Therefore, ERK5 may be a new therapeutic target in invasive OS expressing MMP‐9. © 2011 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 30:1040–1044, 2012</P>

Medicinal Chemistry : ERK1/2 antagonize AMPK-dependent regulation of FcεRI-mediated mast cell activation and anaphylaxis

( Seung Lark Hwang ),( Yue Lu ),( Xian Li Msc ),( Yong Deuk Kim ),( You Sook Cho ),( Yurn Dong Jahng ),( Jong Keun Son ),( Youn Ju Lee ),( Won Ku Kang ),( Yoshitaka Taketomi ),( Makoto Murakami ),( Ta 영남대학교 약품개발연구소 2015 영남대학교 약품개발연구소 연구업적집 Vol.25 No.-

Background: Extracellular signal-regulated kinases 1/2 (ERK1/2) make important contributions to allergic responses via their regulation of degranulation, eicosanoid production, and cytokine expression by mast cells, yet the mechanisms underlying their positive effects on FcεRI-dependent signaling are not fully understood. Recently, we reported that mast cell activation and anaphylaxis are negatively regulated by AMP-activated protein kinase (AMPK). However, little is known about the relationship between ERK1/2-mediated positive and the AMPK-mediated negative regulation of FcεRI signaling in mast cells. Objective: We investigated possible interactions between ERK1/2 and AMPK in the modulation of mast cell signaling and anaphylaxis. Methods: Wild-type or AMPKa22/2 mice, or bone marrow. derived mast cells obtained from these mice, were treated with either chemical agents or small interfering RNAs that modulated the activity or expression of ERK1/2 or AMPK to evaluate the functional interplay between ERK1/2 and AMPK in FcεRI-dependent signaling. Results: The ERK1/2 pathway inhibitor U0126 and the AMPK activator 5-aminoimidazole-4-carboxamide-1-b-4-ribofuranoside similarly inhibited FcεRI-mediated mast cell signals in vitro and anaphylaxis in vivo. ERK1/2-specific small interfering RNA also mimicked this effect on FcεRI signals. Moreover, AMPKa2 knockdown or deficiency led to increased FcεRI-mediated mast cell activation and anaphylaxis that were insensitive to U0126 or activator 5-aminoimidazole-4-carboxamide-1-b-4-ribofuranoside, suggesting that the suppression of FcεRI signals by the inhibition of the ERK1/2 pathway relies largely on AMPK activation. ERK1/2 controlled AMPK activity by regulating its subcellular translocation. Conclusions: ERK1/2 ablated the AMPK-dependent negative regulatory axis, thereby activating FcεRI signals in mast cells. (J Allergy Clin Immunol 2014;134:714-21.)

Nrf2 전사유전자의 이온수송체 유전자 조절기전

조혜정,안규윤 대한체질인류학회 2019 해부·생물인류학 (Anat Biol Anthropol) Vol.32 No.4

저칼륨 상태에서 활성산소종의 양이 증가되고, 증가된 활성산소종은 Nrf2의 핵 내 이동을 촉진한다고 하고, Nrf2 활성화는 extracellular-regulated kinase (ERK), Jun N-terminal kinase (JNK), p38과 phosphatidyl-inositol 3-kinase (PI3K)라는 인산화효소가 작용하는 것으로 알려져 있다. 본 연구는 저칼륨 상태에서 어떤 인산화효소가Nrf2 활성화에 관여하는지, 그리고 활성화된 Nrf2가 이온수송체의 발현에 관련이 있는지 알아보고자 정상 및 칼륨제한 식이 흰쥐 신장과 CV-1, 293T 및 MEF (mouse embryonic fibroblast) 세포주를 정상 및 저칼륨 상태에서 배양하여각각 RT-PCR과 Western 분석으로 정량화하였다. 저칼륨 배양조건에서 증가된 Nrf2의 발현량이 LY294002와 SP600125로 처리하였을 때 감소하는 것을 관찰할 수 있었으나 PD98059와 SB203580로 처리하였을 때는 변화가 없었다. 이는 Nrf2 전사유전자 활성화에 Akt와 JNK 인산화효소가 관여할 가능성을 시사하였다. 칼륨제한 식이 신장조직에서는 ERK1/2 와 Akt의 활성화된 형태인 phospho- ERK1/2, phospho-Akt의 발현이 칼륨제한 식이가 길어질수록 증가하였고 JNK와 p38의 활성은 변화가 없었다. 저칼륨상태 CV-1 세포주에서는 phospho-ERK1/2, phospho-Akt의 발현이 증가함을 확인하였다. 또한 phospho- Nrf2가 칼륨제한 식이가 길어질수록 핵으로 많이 축적됨을 알 수 있었다. 저칼륨 배양조건 MEF-Nrf2 wild-type (+/+) 세포주에서는 Nrf2 발현이 증가하였지만 MEF- Nrf2 Hetero (+/- ) 세포주에서는 감소하였고, MEF-Nrf2 knock-out (- /- ) 세포주에서는 발현이 되지 않음을 확인하였다. kNBC1과 colonic H/K의 mRNA 발현 역시 저칼륨 배양조건 MEF-Nrf2 wild-type (+/+) 세포주에서는 증가하였으나 MEF-Nrf2 Hetero (+/- )와 MEF-Nrf2 knock-out (- /- ) 세포주에서는억제되었음을 관찰하였다. 이상의 결과로 저칼륨 상태에서 Nrf2는 ERK1/2와 Akt에 의해 활성화되고, 활성화된 Nrf2는 kNBC1과 colonic H/ K-ATPase 유전자의 전사를 조절할 것으로 추측하였다. The Nuclear factor-erythroid-2-related factor 2 (Nrf2) plays a key role in the cellular defense against oxidative stress. Low K+ increased the reactive oxygen species and it stimulate Nrf2 activation. Previous our study demonstrated that low potassium promoted expression of H/K-ATPase and kNBC1 by Nrf2 transcription factor in cultured models. In addition, phosphorylation of ERK, JNK, p38 and PI3K was involved in the activation of Nrf2 expression. This study aims to elucidate the mechanism which low potassium regulates Nrf2 expression through various in vitro and in vivo models. Using various kinase inhibitors, promotion of Nrf2 expression in low potassium condition was inhibited by LY294002 and SP600125 while PD98059 and SB203580 did not affect Nrf2, suggesting that phosphorylation of Akt and JNK is specifically involved in Nrf2 expression in low potassium condition. Kidney tissues from low potassium diet rats showed increased phospho-ERK1/2 and phospho-Akt in diet time dependent manner but no effect to JNK and p38 phosphorylation. Specifically, Phospho- Nrf2 was also increased in nuclear compartment by low potassium diet. In order to demonstrate direct evidence that low potassium regulates ionic transporters by Nrf2, Nrf2 knockout mice were employed. Mouse embryonic fibroblasts (MEF) were harvested for the study. As expected, low potassium promotes expression of Nrf2 and level of phospho-ERK1/2 and phospho-Akt in MEF-Nrf2 (+/+). Low potassium promoted expression of kNBC1 and H/K-ATPase in MEF-Nrf2 (+/+), but unchanged or even decreased in MEF-Nrf2 (+/- ) and MEFNrf2 (- /- ). Taken together, these results show that Nrf2 was activated by ERK1/2 and AKT in low potassium condition and further regulates expression of kNBC1 and colonic H/K-ATPase.

BMB Reports : Biphasic activation of extracellular signal-regulated kinase (ERK) 1/2 in epidermal growth factor (EGF)-stimulated SW480 colorectal cancer cells

( Donghyun Joo ),( Jong Soo Woo ),( Kwang Hyun Cho ),( Seung Hyun Han ),( Tae Sun Min ),( Deok Chun Yang ),( Cheol Heui Yun ) 생화학분자생물학회(구 한국생화학분자생물학회) 2016 BMB Reports Vol.49 No.4

Cancer cells have different characteristics due to the genetic differences where these unique features may strongly influence the effectiveness of therapeutic interventions. Here, we show that the spontaneous reactivation of extracellular signalregulated kinase (ERK), distinct from conventional ERK activation, represents a potent mechanism for cancer cell survival. We studied ERK1/2 activation in vitro in SW480 colorectal cancer cells. Although ERK signaling tends to be transiently activated, we observed the delayed reactivation of ERK1/2 in epidermal growth factor (EGF)-stimulated SW480 cells. This effect was observed even after EGF withdrawal. While phosphorylated ERK1/2 translocated into the nucleus following its primary activation, it remained in the cytoplasm during late-phase activation. The inhibition of primary ERK1/2 activation or protein trafficking, blocked reactivation and concurrently increased caspase 3 activity. Our results suggest that the biphasic activation of ERK1/2 plays a role in cancer cell survival; thus, regulation of ERK1/2 activation may improve the efficacy of cancer therapies that target ERK signaling. [BMB Reports 2016; 49(4): 220-225]

좌골신경 재생 시 슈반세포의 이주와 재수초화 관련 요인들에 미치는 트레드밀 운동의 효과

서태범 ( Tae Beom Seo ),김경래 ( Kyung Rae Kim ),윤진환 ( Jin Hwan Yoon ) 한국스포츠정책과학원(구 한국스포츠개발원) 2010 체육과학연구 Vol.21 No.2

좌골신경 손상 후 슈반세포의 기능적 활동성 증가는 축삭 재생을 촉진시킨다. Cell division cycle 2(Cdc2)는 세포 주기(cell cycle)의 G2와 M 기에서 작용하는 원형적 cyclic-dependent kinase이고 Extracellular signal-regulated kinase 1/2(ERK1/2)는 손상 신호에 대한 세포의 반응을 조절한다. 그러나 재생하는 좌골신경의 슈반세포에서 이러한 단백질들의 역할과 운동에 의한 조절 기전은 연구가 부족한 실정이다. 그러므로 이 연구에서는 좌골신경 손상 후 트레드밀 운동이 슈반세포의 증식에 관여하는 Cdc2와 ERK1/2의 인산화(p-ERK1/2)를 증가시키고, 이러한 단백질의 증가가 슈반세포의 이주를 촉진시키는지를 확인하고자 하였다. 좌골신경 손상 1, 3, 7, 14, 21 그리고 28일 후 저강도 트레드밀 운동에 의한 Cdc2와 p-ERK1/2의 발현 양상을 확인하기 위해, 적출된 좌골신경은 western blot 분석을 사용한 결과 손상 7일 후, Cdc2와 p-ERK1/2의 발현은 비운동 그룹에 비해 트레드밀 운동 그룹에서 통계적으로 유의하게 증가하였다. 또한 트레드밀 운동은 생체외의 이주 실험에서 슈반세포의 이주를 촉진시켰다. 손상된 좌골신경의 원위부에서 재수초화의 진행 양상을 확인하기 위해 MBP(myelin basic protein) 항체를 이용한 면역형광 염색법을 수행한 결과 5mm 원위부에서, 재수초화 형성은 비운동 그룹보다 트레드밀 운동군에서 향상되었다. 이러한 결과들을 종합해 보면, 운동에 의한 원위부 슈반세포의 Cdc2와 p-ERK1/2 발현 증가는 재생의 마지막 단계인 재수초화와 밀접한 관련이 있다는 새로운 증거를 제시하고 있다. Increased functional activity of Schwann cells in the injured peripheral nerves is closely related with axonal regeneration, and physical activity in animal models with sciatic nerve injury has been shown to enhance nerve regeneration. Cell division cycle 2 (Cdc2) is a prototypical cyclin-dependent kinase that promotes G2-M phase transition in the cell cycle. Extracellular signal-regulated kinase 1/2 (ERK1/2) regulates neuronal responses to injury signal. In this research, we report that treadmill training (TMT) after sciatic nerve injury facilitates migration and remyelination of proliferating Schwann cells via the increases of Cdc2 and p-Erk1/2 (phosphorylated Erk1/2) protein levels. To investigate exercise-regulated Cdc2 and p-ERK1/2, sciatic nerves in all groups were analyzed using the western blotting method. Cdc2 and p-ERK1/2 were increased more in the TMT group than the sedentary group in the 7th day after injury. TMT also elevated migration of Schwann cells in vitro. We performed immunofluorescence staining with anti-MBP (myelin basic protein) antibody to identify remyelination. TMT increased the number of remyelinated axons in the region 5 mm distal to the injury site. Thus, the present data provide a new evidence that increased Cdc2 and p-ERK1/2 activity in proliferating Schwann cells may play an important role in exercise-mediated enhancement of axonal regeneration in the injured peripheral nerve.

Reactive Oxygen Species Mediate ET-1-Induced Activation of ERK1/2 Signaling in Cultured Feline Esophageal Smooth Muscle Cells

Hyun Ju Song,Ji Soo Kim,Myong Jae Lee,Yoon Sung Nam,손의동 대한약학회 2007 Archives of Pharmacal Research Vol.30 No.9

Reactive oxygen species (ROS) have been shown to play a critical role in propagating the signals of several growth factors, peptide hormones, and cytokines, such as epidermal growth factor, insulin, and interleukin-1. We investigated a possible role for ROS generation in mediating the action of ET-1 on activation of ERK1/2 in cultured feline esophageal smooth muscle cells (ESMC). Confluent layers of ESMC were stimulated by 10nM ET-1; activation of ERK was examined by western blot analysis with phospho-specific antibodies of ERKs. ET-1 induced ERK1/2 phosphorylation in a dose- and time- dependent manner. ERK1/2 activation by ET-1 reached the maximal levels at 5min showing slight activation up to 20min, and then slowly declined. It was confirmed that the activation of ERK1/2 was reduced by MEK inhibitor PD98059. We observed the dose-dependent inhibitory effect of diphenyleneiodonium (DPI), an inhibitor of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase on the ET- 1-enhanced ERK1/2 phosphorylation in ESMC. Pretreatment of ESMC with N-acetylcysteine, a ROS scavenger, also attenuated the ET-1-induced ERK1/2 activation. In addition, DPI significantly inhibited the ET-1- induced ROS production when ROS was measured as a function of DCF fluorescence. The results suggest that ROS might be critical mediators of the ET-1- induced ERK1/2 signaling events in ESMC.

ERK½ antagonize AMPK-dependent regulation of FcεRI-mediated mast cell activation and anaphylaxis

Hwang, S.L.,Lu, Y.,Li, X.,Kim, Y.D.,Cho, Y.S.,Jahng, Y.,Son, J.K.,Lee, Y.J.,Kang, W.,Taketomi, Y.,Murakami, M.,Moon, T.C.,Chang, H.W. Mosby 2014 The journal of allergy and clinical immunology Vol.134 No.3

Background: Extracellular signal-regulated kinases ½ (ERK½) make important contributions to allergic responses via their regulation of degranulation, eicosanoid production, and cytokine expression by mast cells, yet the mechanisms underlying their positive effects on FcεRI-dependent signaling are not fully understood. Recently, we reported that mast cell activation and anaphylaxis are negatively regulated by AMP-activated protein kinase (AMPK). However, little is known about the relationship between ERK½-mediated positive and the AMPK-mediated negative regulation of FcεRI signaling in mast cells. Objective: We investigated possible interactions between ERK½ and AMPK in the modulation of mast cell signaling and anaphylaxis. Methods: Wild-type or AMPKα2<SUP>-/-</SUP> mice, or bone marrow-derived mast cells obtained from these mice, were treated with either chemical agents or small interfering RNAs that modulated the activity or expression of ERK½ or AMPK to evaluate the functional interplay between ERK½ and AMPK in FcεRI-dependent signaling. Results: The ERK½ pathway inhibitor U0126 and the AMPK activator 5-aminoimidazole-4-carboxamide-1-β-4-ribofuranoside similarly inhibited FcεRI-mediated mast cell signals in vitro and anaphylaxis in vivo. ERK½-specific small interfering RNA also mimicked this effect on FcεRI signals. Moreover, AMPKα2 knockdown or deficiency led to increased FcεRI-mediated mast cell activation and anaphylaxis that were insensitive to U0126 or activator 5-aminoimidazole-4-carboxamide-1-β-4-ribofuranoside, suggesting that the suppression of FcεRI signals by the inhibition of the ERK½ pathway relies largely on AMPK activation. ERK½ controlled AMPK activity by regulating its subcellular translocation. Conclusions: ERK½ ablated the AMPK-dependent negative regulatory axis, thereby activating FcεRI signals in mast cells.

Clinicopathologic Significance of BRAF Mutation and Extracellular Signal Regulated Kinase 1/2 Expression in Patients With a Colorectal Adenocarcinoma

김형욱,김범규,차성재,박용검,이태진 대한대장항문학회 2015 Annals of Coloproctolgy Vol.31 No.1

Purpose: BRAF mutation and expression of extracellular signal regulated kinase (ERK) are linked with colorectal carcinogenesis through the serrated pathway. BRAF and ERK1/2 play important roles in the activation of mitogen-activated protein (MAP) kinase signaling pathways. The present study investigated the clinicopathologic outcomes of BRAF mutation and ERK1/2 expression in patients with colorectal cancer (CRC) and the possibility of using them as prognostic indicators. Methods: Dual-priming oligonucleotide-based multiplex polymerase chain reaction for BRAFV600E mutation and immunohistochemical analysis of ERK1/2 were performed using 65 formalin-fixed, paraffin-embedded samples from patients with CRC. We analyzed the dependences of the clinicopathologic features on BRAF mutation and ERK1/2 expression. Results: Out of 65 samples from CRC patients, BRAF mutation was detected in 3 (4.6%). The 3 patients with BRAF mutation presented with T3 CRC with lymph node metastasis (stage III) showing moderately or poorly differentiated histology. ERK1 and ERK2 were positively detected in 73.8% and 15.4% of the patients with CRC, respectively. ERK1 expression was significantly correlated with lymph node metastasis (P = 0.049). ERK2 expression was significantly correlated with tumor emboli (P < 0.05), tumor invasion (P = 0.035), lymph node metastasis (P = 0.017), and stage (P = 0.02). Conclusion: BRAF mutation and ERK1/2 expression may be associated with advanced or more aggressive CRC. These molecular markers might play prognostic roles in CRC developed through the serrated pathway.