고포도당이 백서 사구체 메산지움 배양세포에서 p38 MAPK 활성화 및 Fibronectin 생성에 미치는 영향

유태현 ( Yu Tae Hyeon ),허종호 ( Heo Jong Ho ),류동열 ( Lyu Dong Yeol ),김현욱 ( Kim Hyeon Ug ),이수현 ( Lee Su Hyeon ),김진주 ( Kim Jin Ju ),정동섭 ( Jeong Dong Seob ),최규헌 ( Choe Gyu Heon ),이호영 ( Lee Ho Yeong ),한대석 ( Han 대한신장학회 2003 Kidney Research and Clinical Practice Vol.22 No.5

목 적 : 고포도당으로 자극한 백서 메산지움 배양세포에서 p38 mitogen activated protein kinase (MAPK)의 활성화와 p38 MAPK의 상부와 하부인자로 알려져 있는 MAPK kinase 3/6 (MKK3/6)와 c-AMP responsive element binding protein (CREB)의 활성화, 그리고 fibronectin 합성을 자극 시간에 따라 관찰하고, p38 MAPK 경로와 fibronectin 합성 사이의 연관성을 규명함으로서 당뇨병성 신증의 병태생리를 규명하고자 하였다. 방 법 : 백서 메산지움 배양세포를 정상 포도당 (5.6 mM), 고포도당 (30 mM), 또는 정상 포도당+만니톨 (24.4 mM)로 자극한 후 첫째 p38 MAPK; 둘째 p38 MAPK의 상부 인자인 MKK3/6; 셋째 p38 MAPK의 하부인자인 CREB; 넷째 MAPK phosphatase-1 (MKP-1)의 변화를 자극 시간 (3분-48시간)에 따라 Western blot을 이용하여 관찰하였으며, 다섯째 fibronectin의 변화는 RT-PCR과 ELISA를 이용하여 확인하였다. 결 과 : 고포도당으로 자극한 백서 사구체 메산지움 배양세포에서 p38 MAPK 경로가 활성화 되었다. Total p38 MAPK 단백은 변화가 없었으나, 활성화된 phospho-p38 MAPK 단백은 고포도당군에서 자극 10분 후부터 정상 포도당군에 비해 의의있게 증가되었으며 (평균 1.9배), phospho-CREB 단백 역시 고포도당군에서 자극 10분 후부터 의미있게 증가되었다 (평균 2.5배). 이에 비해 p38 MAPK의 상부인자인 phospho-MKK3/6는 고포도당군에서 자극 3분 후부터 의의있게 증가되었다 (평균 2.4배). 또한, MKP-1 단백 발현은 고포도당군에서 p38 MAPK의 활성이 증가하기 시작한 시기와 동일한 시기 (자극 10분 후)부터 증가하기 시작하였다 (평균 1.9배). Fibronectin mRNA와 세포 배양액내 fibronectin은 고포도당군에서 자극 48시간 후에 각각 1.7배와 1.5배 증가되었으며, 이러한 증가는 p38 MAPK 억제제인 SB203580 (1 μM)에 의해 각각 73%와 69% 억제되었다. 결 론 : 고포도당으로 자극한 백서 사구체 메산지움 배양세포에서 p38 MAPK 경로의 활성화를 확인하였으며, p38 MAPK 억제제가 고포도당에 의한 fibronectin의 합성 증가를 억제시키는 것으로 보아 당뇨병성 신증에서 관찰되어지는 fibronectin의 축적에 p38 MAPK 경로가 관여할 것으로 생각된다. Background : The p38 mitogen-activated protein kinase (MAPK) pathway is activated by several stress factors, potentially leading to cellular apoptosis and growth. Little is known about the pattern of p38 MAPK pathway activation in mesangial cells under high glucose conditions. We examined the activity and expression of p38 MAPK members, 1x78 MAPK, MAPK kinase 3/6 (MKK3/6), c-AMP responsive element binding protein (CREE), and MAPK phosphatase-l (MKP-1) in cultured rnesangial cells exposed to high glucose. Methods : Mesangial cells were subcultured from rat glomeruli isolated by sieving technique. After serum restriction for 48 hours rnesangial cells were exposed to 5.6 mM glucose (low glucose, LG), 5.6 mM glucose+24.4 mM mannitol (LG+M), or 30 mM glucose (high glucose, HG) for 3 minutes to 48 hours with or without SU203580. Western blot was performed to determine the activity and protein expression of p38 MAPK members. R`I-I`CR and ELISA were performed for fibronectin mRNA expression and fibronectin synthesis, respectively. Results : p38 MAPK and CREB activities were significantly increased in rnesangial cells exposed to FIG compared with LG or LG+M after 10 minutes and was sustained at higher levels to 48 hours (p<0.05), but total p38 MAPK and CREB protein expressions did not differ. MKP--1 showed a similar pattern as p 3 MAPK and CREE (p<O.O.i). MKK3/6 acitvity was significantly higher in HG cells after 3 minutes and remained at higher levels throught the study period (p<0.05). Fibronectin mRNA expression and fibronectin synthesis were significantly increased in mesangial cells exposed to HG after 48 hours (p<0.05). SB203580 (1 pM) pretreatment for 1 hour significantly reduced HG-induced fibronectin mRNA expression and fibronectin synthesis by 73% and 69%, respectively (p<0.05). Conclusion : p38 MAPK activity was increased in mesangial cells exposed to HG in parallel with increased MKK3/6 activity, resulting in CREB activation and increased fibronectin synthesis. This activated p38 MAPK may play a role in the pathogenesis of diabetic nephropathy.

흰쥐 해마에서 경련에 의해 발현 유도된 MKP-1에 의한 MAPK 활성 조절

유범희,강웅구,안용민,정선주,전송희,박주배,김용식 大韓神經精神醫學會 1999 신경정신의학 Vol.38 No.4

연구목적 : 전기경련 충격(Electroconvulsive shock, ECS) 및 카이닌산(kainic acid)에 의한 경련은 흰쥐 해마에서 mitogen activated protein kinase(MAPK)를 활성화시키며, 동시에 MAPK 불활성화 효소인 MAPK phosphatase-1(MKP-1)의 발현을 일으킨다. 이 연구의 목적은 경련에 의해 발현된 MKP-1이 역시 경련에 의해 활성화된 MAPK의 불활성화에 관여하는지를 알아보고자 하는 것이다. 방 법 : 흰쥐에 ECS를 가하여 해마에서 MKP-1 발현을 일으킨 뒤 다시 ECS를 가하고, 두 번째 ECS에 의한 MAPK의 일시적 활성화가 MKP-1의 발현상태에 의해 영향받는지를 알아보았다. 또한 흰쥐에 지속적인 MAPK 활성화 및 MKP-1 발현을 일으키는 카이닌산을 투여한 뒤 해마에서 MKP-1의 발현과 MAPK 활성과의 관계를 알아보았다. 결 과 : ECS후 해마에서 타이로신 인산화 면역블롯 및 효소활성으로 측정한 MAPK의 인산화 및 활성은 MKP-1의 발현이 일어나 있는 경우와 그렇지 않은 경우 사이에 차이를 보이지 않았다. 카이닌산의 투여에 의해 MAPK 활성화가 일어나는 경우, 뒤이어 MKP-1 발현이 일어나지만, 이렇게 발현된 MKP-1은 MAPK 활성을 충분히 감소시키지 못하였다. 결 론 : MKP-1은 흰쥐 해마에서 ECS 및 카이닌산에 의한 MAPK의 활성화를 차단하는데 큰 역할을 하지 않는다. Objectives : Both electroconvulsive shock(ECS)- and kainic acid-induced seizures activate mitogen-activated protein kinases(MAPKs) in rat hippocampus. They can also induce the expression of MAPK phosphatase-1(MKP-1) in rat hippocampus. MKP-1 is known as a specific MAPK deactivator. This study aimed to elucidate the role of MKP-1 in the deactivation of MAPKs in rat hippocampus. Methods : In order to induce MKP-1 in the hippocampus, ECS was given to the rats. At the time points when MKP-1 was sufficiently induced, the second ECS was given to them and the subsequent phosphorylation or activation of MAPKs were measured in the hippocampus. A second group of rats were injected with kainic acid and the relationship between MKP-1 expression and MAPK phosphorylation was examined in their hippocampi. Results : The expression of MKP-1 did not influence the phosphorylation or activation of MAPKs following ECS in rat hippocampus. Kainic acid-induced expression of MKP-1 did not significantly reduce the phosphorylation of MAPKs. Conclusion : MKP-1 did not play a significant role in the deactivation of MAPKs which were activated by ECS or kainic acid in rat hippocampus.

말초조직염증 혹은 신경손상 후 흰쥐 등쪽뿌리신경절에서 mitogen-activated protein kinase의 활성화

배재천(Jae-Chun Bae),이혜림(Hye-Lim Lee),이경민(Kyung-Min Lee),황성헌(Sung-Hun Hwang),손선주(Sun-Ju Sohn),김동선(Dong-Sun Kim),조희중(Hee-Jung Cho) 대한해부학회 2003 Anatomy & Cell Biology Vol.36 No.3

Mitogen-activated protein kinase (MAPK)계열에는 extracellular signal-regulated kinase (ERK), p38 MAPK와 c-Jun Nterminal kinase (JNK) 등이 이에 속한다. 그리고 이러한 MAPK전달계는 세포 외부로부터의 자극을 전달하여 세포내 반응을 일으키는데 있어서 매우 중요한 역할을 하는 것으로 보고되어져 있다. ERK는 성장인자, 산화 스트레스, 세포내 칼슘 증가 혹은 glutamate 수용체의 자극 등에 의하여, p38 MAPK 및 JNK는 cytokine, 열쇼크, 자외선 및 허혈 등에 의하여 활성화되는 것으로 알려져 있다. 한편 말초조직에 염증을 유발시키거나 말초신경의 손상 후 축삭이 퇴행을 일으키면 이곳으로부터 cytokine, 성장인자 및 기타 물질들이 생성되어 등쪽뿌리신경절(DRG)로 역행성이동을 하여 동통과민화를 야기하게 된다. 본 연구에서는 말초조직염증이나 신경손상 후 DRG에서 MAPK이 어떻게 활성화되는지를 알아보기 위하여 면역조직염색 법으로 관찰하여 다음의 결과를 얻을 수 있었다. 1. 말초조직염증을 유발시키면 동측의 L5 DRG에서 phosphorylated (P)-ERK, P-p38 MAPK 및 P-JNK 면역반응신경세포수 의 유의한 증가가 관찰되었다. 2. 좌골신경절단의 경우 동측의 DRG에서 P-ERK 혹은 P-p38 MAPK 면역반응성 신경세포수의 유의한 감소와 P-JNK 면 역반응성 신경세포수의 유의한 증가를 볼 수 있었다. 3. 좌골신경의 만성협착손상(chronic constriction injury, CCI)시 DRG에서 MAPK의 활성화는 말초조직염증유발시의 그것 과 유사하였다. 4. 말초조직염증이나 CCI 후의 ERK, p38 MAPK 및 JNK의 활성화는 작은직경의 신경세포에서, 좌골신경절단 후의 JNK 의 활성화는 모든 직경 신경세포에서 관찰되었다. 이러한 연구결과들로 미루어 보아 말초조직염증이나 좌골신경의 CCI 후 생성되는 cytokine이나 성장인자들이 DRG에서 의 P-ERK, P-p38 MAPK 및 P-JNK의 발현에 관여할 것이며 이러한 MAPK계열들은 동통과민화에 중요한 역할을 할 것으 로 생각된다. The mitogen-activated protein kinase (MAPK) family has three members: the extracellular signal-regulated kinase (ERK), p38 MAPK and c-Jun N-terminal kinase (JNK). There is substantial evidence indicating that the MAPK cascade plays a pivotal role in transducing extracellular stimuli into intracellular responses in all cells. The ERK is activated in response to growth factors, oxidative stress, increases in intracellular calcium levels, or glutamate receptor stimulation. The p38 MAPK and JNK are activated by stress signals such as inflammtory cytokines, heat shock, ultraviolet light, and ischemia. It has been shown that cytokines, growth factors, or other agents released and are retrograde-transported to the dorsal root ganglia (DRG) as a result of peripheral tissue inflammation or the degeneration of axons following peripheral nerve injuries which cause hyperalgesia. In the present study, we investigated the activation of MAPKs in rat DRG by means of immunohistochemistry following peripheral inflammation or nerve injuries. The results obtained were as follows; 1. Peripheral tissue inflammation induced significant increase in the percentage of phosphorylated (P)-ERK, P-p38 or P-JNK immunoreactive neurons in the ipsilateral L5 DRG. 2. Following axotomy, the percentage of P-ERK or P-p38 MAPK immunoreactive neurons decreased significantly and that of P-JNK showed significant increase in the ipsilateral side. 3. Chronic constriction injury of the sciatic nerve (CCI) induced similar changes with those following peripheral inflammation in the activation of MAPKs in the DRG neurons. 4. The activation of ERK, p38 MAPK and JNK following inflammation and CCI was observed primarily in small neurons, while that of JNK following axotomy was found in neurons of all sizes. These results suggest that cytokines or growth factors released as a result of peripheral inflammation or CCI of the sciatic nerve may modulate expression of P-ERK, P-p38 MAPK and P-JNK in the DRG and that MAPKs may play an important roles in pain hypersensitivity.

대장 점막의 전암 병변과 암종에서 Nuclear Factor, p38 Mitogen-Activated Protein Kinase 및 Cyclin D1 단백 발현의 면역조직화학 분석

이상대 ( Sang Dae Lee ),이태진 ( Tae Jin Lee ),박언섭 ( Eon Sub Park ) 대한소화기학회 2008 대한소화기학회지 Vol.52 No.6

목적: Nuclear Factor-κB p65 (NF-κB p65)와 Nuclear Factor-κB1 p50 (NF-κB p50)은 세포증식과 세포자멸사, 시토카인 생성 및 종양화에 역할을 하는 것으로 알려져 있고, 최근 p38 Mitogen-Activated Protein Kinase (MAPK)/NF-κB/cyclin D1 신호전달 경로가 인간 종양 발생에 중요한 부분을 담당한다는 연구 결과가 있다. 이번 연구에서는 대장의 전암 병변과 선암종에서 NF-κB p65, NF-κB p50, p38 MAPKα, cyclin D1의 단백 발현을 알아보고자 하였다. 대상 및 방법: 정상 점막 20예, 저등급 선종 20예, 고등급 선종 20예, 선암종 64예의 파라핀 포매 조직을 이용하여 NF-κB p65, NF-κB p50, p38 MAPKα, cyclin D1 단백에 대한 면역조직화학 염색을 실시하였다. 결과: NF-κB p65과 NF-κB p50 및 p38 MAPKα 단백의 발현은 정상 점막에서 선암종으로 진행하면서 유의하게 증가하였다. 선암종에서 NF-κB p50 단백은 저등급 분화도인 경우, 림프절 전이가 있는 경우, 병기가 높은 경우에 발현 빈도가 높았고, p38 MAPKα 단백은 종양 병기가 높은 경우, 림프절 전이가 있는 경우, 병기가 높은 경우에 발현 빈도가 높았다. NF-κB p65, NF-κB p50, p38 MAPKα, cyclin D1 단백은 서로 연관성 있게 발현하였고, 선암종에서 NF-κB p65, NF-κB p50, p38 MAPKα, cyclin D1 단백이 모두 발현되는 예가 많았다. 결론: NF-κB p65, NF-κB p50, p38 MAPKα 단백 발현은 각각 대장암의 발생 단계에서 역할을 하고 NF-κB p50과 p38 MAPKα 단백 발현은 대장암의 임상-병리 인자들과 연관성이 있어서 암의 진행과 연관성이 있으며, NF-κB p65, NF-κB p50, p38 MAPKα, cyclin D1 단백은 서로 연관성있게 발현하여 NF-κB/p38 MAPKα/cyclin D1 신호전달 경로가 대장암의 발생과 진행에 역할을 하는 것으로 생각한다. Background/Aims: Nuclear factor-κB p65 (NF-κB p65), nuclear factor-κB1 p50 (NF-κB p50) have been shown to play a role in cell proliferation, apoptosis, cytokine production, and oncogenesis. Recently, p38 mitogen-activated protein kinase (MAPK)/ NF-κB/ cyclin D1 signaling pathway has been shown to play an important part in the pathogenesis of human cancers. This study was designed to investigate the expression of NF-κB p65, NF-κB p50, p38 MAPKα, and cyclin D1 proteins in premalignant lesions of colon and colorectal adenocarcinoma. Methods: Paraffin sections of 20 normal mucosa, 20 low-grade tubular adenoma, 20 high-grade tubular adenoma and 64 adenocarcinoma tissues were analysed immunohistochemically for the expression of NF-κB p65, NF-κB p50, p38 MAPKα, and cyclin D1 proteins. Results: The expression of NF-κB p65, NF-κB p50, and p38 MAPKα proteins were significantly higher in adenocarcinoma tissue in comparison with that in normal mucosa, low-grade tubular adenoma, and high-grade tubular adenoma tissues. Expression of NF-κB p50 was more frequent in poorly differentiated histologic grade, presence of nodal metastasis, and advanced stage. Expression of p38 MAPKα protein was higher in advanced tumor stage, presence of nodal metastasis and advanced stage. Synchronous expression of NF-κB p65, NF-κB p50, p38 MAPKα, and cyclin D1 proteins were significantly higher in adenocarcinoma tissue. Conclusions: With the increased expression of NF-κB p65, NF-κB p50, and p38 MAPKα proteins, p38 MAPK/ NF-κB/ cyclin D1 signaling pathway may play a role in the pathogenesis of colorectal carcinoma.

백서 해마에서 카이닌산에 의한 조기유전자의 발현과 p42, p44 MAPK 및 EIK-1 인산화의 발달 단계에 따른 변화

정희연,김수진,김종흔,정선주,박주배,김용식,조수철 大韓神經精神醫學會 1999 신경정신의학 Vol.38 No.4

연구목적 : 어린 백서에게 카이닌산(kainic acid, KA)을 주사하여 발작을 일으킨 후, 해마에서 조기유전자 -c-fos, junB, 및 TIS1의 발현 유도 양상을 발달 단계별로 조사하여 전기경련충격(electroconvulsive shock, ECS)에서의 결과와 비교함으로써 백서 뇌 신호전달계의 성숙과정을 파악하고자 하였다. 그리고 KA 주사후 p42, p44 mitogen activated protein kinase(MAPK)의 인산화 및 그에 의해 활성화되는 것으로 알려진 전사인자 Elk-1의 인산화를 관찰하여, KA 주사후 MAPK 신호절달계를 통한 c-fos의 발현로 알려진 전사인자 Elk-1의 인산화를 관찰하여, KA 주사후 MAPK 신호전달계를 통한 c-fos의 발현 경로를 발달 단계별로 밝히고자 하였다. 방 법 : 생후 7, 14 21일 된 수컷 백서에서 KA를 복강내 주사한 후, 백서 해마에서 조기유전자의 발현 양상은 northern blot analysis로, p42, p44 MAPK와 Elk-1의 인산화는 immunoblotting으로 관찰하였다. 결 론 : 생후 7일된 백서의 해마에서는 ECS와는 달리, KA에 의한 세 가지 조기유전자의 발현이 관찰되지 않았다. 그러나 생후 14일부터 이들 조기유전자의 뚜렷한 발현을 관찰할 수 있었고 생후 21일에는 성숙한 백서와 같은 수준의 발현 양상을 볼 수 있었다. 세 가지 유전자 모두 백서의 연령과 KA 주사 후 시간에 따른 발현 유도 양상은 비슷하였다. p42, p44 MAPK는 생후 7일부터 상당한 수준의 기저치 인산화가 관찰되었으나, KA에 의한 인산화 증가는 생후 14일부터 관찰되었다. Elk-1의 인상화 역시 생후 7일부터 높은 수준으로 관찰되었으나 KA 주사 후 시간에 따른 Elk-1 인산화의 변화는 관찰할 수 없었다. 결 론 : ECS와 KA가 조기유전자 발현 양상에 차이를 보이는 것은 이들 유전자의 발현과 관련되어 활성화되는 신호전달경로의 차이 때문으로 생각된다. 백서 해마에서 KA에 의한 MAPK 활성화에 관여하는 신호 전달 기구는 연령이 높아짐에 따라 점차 성숙하고, MAPK의 활성화로 전달된 신호는 Elk-1 이외의 다른 경로를 통해 c-fos 발현을 조절하는 것으로 추측된다. Objectives : In order to investigate the maturational process of intracellular signal transduction system in rat brain, we studied the induction of the immediate early genes(IEGs)-c-fos, iunB, and TIS1 in each developmental stage after kainic acid(KA)-induced seizure in young rat hippocampus and then compared these with the results after electroconvulsive shock(ECS) And to elucidate the induction mechanism of c-fos via mitogen-activated protein kinase(MAPK) by KA in each developmental stage, we investigated the phosphorylation of p42, p44 MAPK and Elk-1 after KA treatment in young rat hippocampus. Methods : We examined the induction patterns of IEGs by northern blot analysis, and the phosphorylation of p42, p44 MAPK and Elk-1 by immunoblotting in rat hippocampus at post-natal day 7, 14, and 21(P7, P14 & P21), respectively after intraperitoneal injection of KA. Results : Unlike ECS, KA did not induce c-fos, junB, and TIS1 in P7 hippocampus. But these genes were apparently induced at P14 and to an adult level at P21. These three IEGs showed similar temporal patterns of induction in each developmental stage. Although the basal level of phosphorylated 42p, 44p MAPK was considerable in P7 rat hippocampus, the increase or phosphorylation after KA treatment was observed at P14. While the phosphorylation of Elk-1 was detected with high basal level in P7 rat, the amount of phosphorylated Elk-1 was not changed after KA treatment. Conclusion : Our results suggest that the differences in IEGs induction patterns between KA and ECS may be due to the differences in the activated signal transduction pathways. And our results also implicate that the signal transduction system involved in MAPK phosphorylation after KA treatment mature with aging and c-fos induction via MAPK activation may be regulated through some pathways other than Elk-1 in rat hippocampus.

Phorbol 12-Myristate 13-Acetate Induces MUC16 Expression via PKCδ and p38 in Human Airway Epithelial Cells

배창훈,김학수,송시연,김용대 대한이비인후과학회 2012 Clinical and Experimental Otorhinolaryngology Vol.5 No.3

Objectives. Phorbol 12-myristate 13-acetate (PMA) is widely used as a protein kinase C (PKC) activator, PKC is involved in the secretion of mucins. MUC16, one of the membrane-bound mucins, is produced in human airway epithelial cells. However, the effect and signaling pathway of PMA on MUC16 expression in human airway epithelial cells has not been reported. Therefore, the effect and brief signaling pathway of PMA on MUC16 expression were investigated in human airway epithelial cells in this study. Methods. In the mucin-producing human NCI-H292 airway epithelial cells and the primary cultures of normal nasal epithelial cells, the effect and signaling pathway of PMA on MUC16 expression were investigated using reverse transcriptase-polymerase chain reaction (RT-PCR), real-time PCR, enzyme immunoassay, and immunoblot analysis with several specific inhibitors and small interfering RNA (siRNA) for p38 mitogen-activated protein kinase (MAPK). Results. PMA increased MUC16 expression, and activated phosphorylation of p38 MAPK. However, it did not activate phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2). SB203580 (p38 MAPK inhibitor) inhibited PMA-induced MUC16 expression, while U0126 (ERK1/2 inhibitor) did not. In addition, the knockdown of p38 MAPK by p38MAPK siRNA significantly blocked PMA-induced MUC16 mRNA expression. Rottlerin (PKCδ inhibitor) inhibited PMA-induced MUC16 expression, and also inhibited the phosphorylation of activated p38 MAPK by PMA. Conclusion. These results show for the first time that PMA-induced MUC16 expression is regulated by activation of the PKCδ and p38 MAPK signaling pathway in human airway epithelial cells. Objectives. Phorbol 12-myristate 13-acetate (PMA) is widely used as a protein kinase C (PKC) activator, PKC is involved in the secretion of mucins. MUC16, one of the membrane-bound mucins, is produced in human airway epithelial cells. However, the effect and signaling pathway of PMA on MUC16 expression in human airway epithelial cells has not been reported. Therefore, the effect and brief signaling pathway of PMA on MUC16 expression were investigated in human airway epithelial cells in this study. Methods. In the mucin-producing human NCI-H292 airway epithelial cells and the primary cultures of normal nasal epithelial cells, the effect and signaling pathway of PMA on MUC16 expression were investigated using reverse transcriptase-polymerase chain reaction (RT-PCR), real-time PCR, enzyme immunoassay, and immunoblot analysis with several specific inhibitors and small interfering RNA (siRNA) for p38 mitogen-activated protein kinase (MAPK). Results. PMA increased MUC16 expression, and activated phosphorylation of p38 MAPK. However, it did not activate phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2). SB203580 (p38 MAPK inhibitor) inhibited PMA-induced MUC16 expression, while U0126 (ERK1/2 inhibitor) did not. In addition, the knockdown of p38 MAPK by p38MAPK siRNA significantly blocked PMA-induced MUC16 mRNA expression. Rottlerin (PKCδ inhibitor) inhibited PMA-induced MUC16 expression, and also inhibited the phosphorylation of activated p38 MAPK by PMA. Conclusion. These results show for the first time that PMA-induced MUC16 expression is regulated by activation of the PKCδ and p38 MAPK signaling pathway in human airway epithelial cells.

백서 해마에서 카이닌산에 의한 조기유전자의 발현과 p42, p44 MAPK 및 Elk-1 인산화의 발달 단계에 따른 변화

정희연,김수진,김종흔,정선주,박주배,김용식,조수철 대한신경정신의학회 1999 신경정신의학 Vol.38 No.4

연구목적: 어린 백서에게 카이닌산(kainic acid, KA)을 주사하여 발작을 일으킨 후, 해마에서 조기유전자 -c-fos, junB, 및 TIS1의 발현 유도 양상을 발달 단계별로 조사하여 전기경련충격(electroconvulsive shock, ECS) 에서의 결과와 비교함으로써 백서 뇌 신호전달계의 성숙과정을 파악하고자 하였다. 그리고 KA 주사후 p42, p44 mitogen activated protein kinase(MAPK)의 인산화 및 그에 의해 활성화되는 것으로 알려진 전사인 자 Elk-1의 인산화를 관찰하여, KA 주사후 MAPK 신호전달계를 통한 c-fos의 발현 경로를 발달 단계별 로 밝히고자 하였다. 방 법: 생후 7, 14 21일 된 수컷 백서에서 KA를 복강내 주사한 후, 백서 해마에서 조기유전자의 발현 양상은 northern blot analysis로, p42, p44 MAPK와 Elk-1의 인산화는 immunoblotting으로 관찰하였다. 결 론: 생후 7일된 백서의 해마에서는 ECS와는 달리, KA에 의한 세 가지 조기유전자의 발현이 관찰되지 않았다. 그 러나 생후 14일부터 이들 조기유전자의 뚜렷한 발현을 관찰할 수 있었고 생후 21일에는 성숙한 백서와 같은 수준의 발현 양상을 볼 수 있었다. 세 가지 유전자 모두 백서의 연령과 KA 주사 후 시간에 따른 발현 유도 양상은 비슷하였다. p42, p44 MAPK는 생후 7일부터 상당한 수준의 기저치 인산화가 관찰되었으나, KA에 의 한 인산화 증가는 생후 14일부터 관찰되었다. Elk-1의 인산화 역시 생후 7일부터 높은 수준으로 관찰되었 으나 KA 주사 후 시간에 따른 Elk-1 인산화의 변화는 관찰할 수 없었다. 결 론: ECS와 KA가 조기유전자 발현 양상에 차이를 보이는 것은 이들 유전자의 발현과 관련되어 활성화되는 신 호전달경로의 차이 때문으로 생각된다. 백서 해마에서 KA에 의한 MAPK 활성화에 관여하는 신호전달 기구 는 연령이 높아짐에 따라 점차 성숙하고, MAPK의 활성화로 전달된 신호는 Elk-1 이외의 다른 경로를 통 해 c-fos 발현을 조절하는 것으로 추측된다 Objectives:In order to investigate the maturational process of intracellular signal transduction system in rat brain, we studied the induction of the immediate early genes(IEGs)-c-fos, junB, and TIS1 in each developmental stage after kainic acid(KA)-induced seizure in young rat hippocampus and then compared these with the results after electroconvulsive shock(ECS). And to elucidate the induction mechanism of c-fos via mitogen-activated protein kinase(MAPK) by KA in each developmental stage, we investigated the phosphorylation of p42, p44 MAPK and Elk-1 after KA treatment in young rat hippocampus. Methods:We examined the induction patterns of IEGs by northern blot analysis, and the phosphorylation of p42, p44 MAPK and Elk-1 by immunoblotting in rat hippocampus at post-natal day 7, 14, and 21(P7, P14 & P21), respectively after intraperitoneal injection of KA. Results:Unlike ECS, KA did not induce c-fos, junB, and TIS1 in P7 hippocampus. But these genes were apparently induced at P14 and to an adult level at P21. These three IEGs showed similar temporal patterns of induction in each developmental stage. Although the basal level of phosphorylated 42p, 44p MAPK was considerable in P7 rat hippocampus, the increase of phosphorylation after KA treatment was observed at P14 . While the phosphorylation of Elk-1 was detected with high basal level in P7 rat, the amount of phosphorylated Elk-1 was not changed after KA treatment. Conclusion:Our results suggest that the differences in IEGs induction patterns between KA and ECS may be due to the differences in the activated signal transduction pathways. And our results also implicate that the signal transduction system involved in MAPK phosphorylation after KA treatment mature with aging and c-fos induction via MAPK activation may be regulated through some pathways other than Elk-1 in rat hippocampus

Comparative expression analysis of defense-related genes in both transgenic and nontransgenic Brassica juncea (var.) Varuna harbouring overexpressed MAPK3 gene in response to infection by Albugo candida

Modak Annayasa,Singh Brij Raj,Dubey Ashutosh,Tewari A. K.,Taj Gohar 한국작물학회 2022 Journal of crop science and biotechnology Vol.25 No.1

White rust, a devastating disease of Brassicaceae family, causes nearly 60% reduction in yield of Brassica juncea (Indian mustard). This dreadful disease is caused by oomycetes fungi Albugo candida. A comparative expression analysis of MAPKs cascade and some of defense-related genes in nontransgenic and transgenic (harbouring overexpressed MAPK3) cultivar of Brassica juncea were done to reveal the defense signalling pathway against biotrophic fungi Albugo candida. Disease indexing was also done to compare the disease severity in both transgenic and nontransgenic Brassica juncea cultivars. Lower disease index in transgenic Brassica suggests its tolerance to Albugo candida. Higher expression level of MAPK6 in comparison to MAPK3 in the transgenic Brassica indicates that MAPK6 mimics a portion of MAPK3 signalling pathway and work in a direct cascade for production of defense-related proteins. Overexpression of MAPK3 gene was observed in downregulating the expression of MAPK4 in transgenic Brassica juncea. The expression of two important transcription factors, such as WRKY33 and WRKY29 were also analyzed in the present study and were found to be higher in transgenic plant overexpressing MAPK3. The expression of three defense-related genes (OASTL-B, ACD2 and CSD2) was found to be increased in transgenic plants harbouring overexpressed MAPK3 gene; however, expression level of CYP20-3 was found higher in nontransgenic Brassica plant in response to pathogen infection. These four defense-related genes were also subjected to in silico studies, such as secondary structure, tertiary structure and putative phosphorylation sites prediction in addition to protein–protein interaction determination with potential MAPKs cascade and WRKY29, WRKY33.

Gene structure, molecular characterization and transcriptional expression of two p38 isoforms (MAPK11 and MAPK14) from rock bream (Oplegnathus fasciatus)

Umasuthan, N.,Bathige, S.D.N.K.,Noh, J.K.,Lee, J. Academic Press 2015 FISH AND SHELLFISH IMMUNOLOGY Vol.47 No.1

The p38 kinases are one of the four subgroups of mitogen-activated protein kinase (MAPK) superfamily which are involved in the innate immunity. The p38 subfamily that includes four members namely p38α (MAPK14), p38β (MAPK11), p38γ (MAPK12) and p38δ (MAPK13), regulates the activation of several transcription factors. In this study, a p38β (OfMAPK11) homolog and a p38α (OfMAPK14) homolog of Oplegnathus fasciatus were identified at genomic level. Results clearly showed that both MAPK11 and MAPK14 are well-conserved at both genomic structural- and amino acid (aa)-levels. Genomic sequences of OfMAPK11 (~15.6 kb) and OfMAPK14 (~13.4 kb) had 12 exons. A comparison of exon-intron structural arrangement of these genes from different vertebrate lineages indicated that all the exon lengths are highly conserved, except their terminal exons. Full-length cDNAs of OfMAPK11 (3957 bp) and OfMAPK14 (2504 bp) encoded corresponding proteins of 361 aa and 360 aa, respectively. Both OfMAPK proteins harbored a Ser/Thr protein kinases catalytic domain (S_TKc domain) which includes an activation loop with a dual phosphorylation site (TGY motif) and several specific-binding sites for ATP and substrates. Molecular modeling of the activation loop and substrate binding sites of rock bream MAPKs revealed the conservation of crucial residues and their orientation in 3D space. Transcripts of OfMAPKs were ubiquitously detected in eleven tissues examined, however at different levels. The modulation of OfMAPKs' transcription upon pathogen-associated molecular patterns (PAMPs: flagellin, lipopolysaccharide and poly I:C) and pathogens (Edwardsiella tarda, Streptococcus iniae and rock bream iridovirus) was investigated. Among the seven examined tissues, the flagellin-challenge upregulated the mRNA level of both OfMAPKs in the head kidney. Meanwhile, modulation of OfMAPK mRNA expression in the liver upon other immune-challenges varied in a time-dependent manner. Collectively, these results suggest that OfMAPKs are true members of p38 subfamily, which might be induced by different immune stimuli.

기초 : 톡소포자충 감염 신경교종세포의 Mitogen-Activated Protein Kinase Pathway 활성화

박병현 ( Byung Hyun Park ),이현성 ( Hyun Sung Lee ),이종수 ( Jong Soo Lee ),양승환 ( Seung Hwan Yang ),신대환 ( Dae Whan Shin ),이영하 ( Young Ha Lee ) 대한뇌종양학회 2006 대한뇌종양학회지 Vol.5 No.2

Objective:Gliomas are the most common primary brain tumors in adults. Toxoplasma gondii is an obligate intracellular parasite with a high affinity for brain cells. Mitogen-activated protein kinases(MAPKs) are known to regulate the host cell invasion, although little is known regarding MAPK signaling in Toxoplasma-infected glioma cells. Methods:U87MG and U373MG cells were infected with tachyzoites of the RH strain of T. gondii, and the kinetics of MAPK activation were determined by immunoblotting. We also determined the effects of MAPK inhibitors on glioma cell growth and T. gondii replication using the 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetraterazolium bromide(MTT; Sigma) method and [3H]-uracil incorporation assays. Results:There were no significant differences in the levels of unphosphorylated MAPK at different incubation times in the Toxoplasma-infected U373MG cells. T. gondii induced the activation of extracellular signal-regulated kinase(ERK) 1/2, p30MAPK, and c-Jun N-terminal kinase(JNK)1/2 within 30 minutes of infection, and showed differential kinetics of activation for each MAPK. The activation of ERK1/2, p38MAPK, and JNK1/2 in Toxoplasma-infected glioma cells was blocked by PD98059, SB202190, and SB203580, respectively. T. gondii replication was inhibited in a dose-dependent manner by the addition of MAPK inhibitors to Toxoplasma-infected glioma cells. Conclusion:Infection with T. gondii induces the activation of MAPKs in glioma cells, and this activation can be blocked by the addition of kinase-specific inhibitors. These results suggest that the MAPK pathway plays a role in the invasion of glioma cells with T. gondii.