JNK and p38 mitogen-activated protein kinase pathways contribute to porcine epidemic diarrhea virus infection

Lee, Changhee,Kim, Youngnam,Jeon, Ji Hyun Elsevier 2016 VIRUS RESEARCH Vol.222 No.-

<P><B>Abstract</B></P> <P>The mitogen-activated protein kinase (MAPK) pathways, which are central building blocks in the intracellular signaling network, are often manipulated by viruses of diverse families to favor their replication. Among the MAPK family, the extracellular signal-regulated kinase (ERK) pathway is known to be modulated during the infection with porcine epidemic diarrhea virus (PEDV); however, involvement of stress-activated protein kinases (SAPKs) comprising p38 MAPK and c-Jun NH<SUB>2</SUB>-terminal kinase (JNK) remains to be determined. Therefore, in the present study, we investigated whether activation of p38 MAPK and JNK cascades is required for PEDV replication. Our results showed that PEDV activates p38 MAPK and JNK1/2 up to 24h post-infection, whereas, thereafter their phosphorylation levels recede to baseline levels or even fall below them. Notably, UV-irradiated inactivated PEDV, which can enter cells but cannot replicate inside them, failed to induce phosphorylation of p38 MAPK and JNK1/2 suggesting that viral biosynthesis is essential for activation of these kinases. Treatment of cells with selective p38 or JNK inhibitors markedly impaired PEDV replication in a dose-dependent manner and these antiviral effects were found to be maximal during the early times of the infection. Furthermore, direct pharmacological inhibition of p38 MAPK or JNK1/2 activation resulted in a significant reduction of viral RNA synthesis, viral protein expression, and progeny release. However, independent treatments with either SAPK inhibitor did not inhibit PEDV-induced apoptotic cell death mediated by activation of mitochondrial apoptosis-inducing factor (AIF) suggesting that SAPKs are irrelevant to the apoptosis pathway during PEDV infection. In summary, our data demonstrated critical roles of the p38 and JNK1/2 signaling pathways in facilitating successful viral infection during the post-entry steps of the PEDV life cycle.</P> <P><B>Highlights</B></P> <P> <UL> <LI> PEDV infection activated p38 MAPK and JNK1/2 <I>in vitro</I>. </LI> <LI> UV-inactivated virus failed to induce p38 MAPK and JNK1/2 activation. </LI> <LI> Pharmacological inhibition of p38 MAPK or JNK activation impaired PEDV replication. </LI> <LI> SAPK cascades do not affect the apoptosis pathway during PEDV infection. </LI> <LI> PEDV exploits the p38 MAPK and JNK signaling pathways for optimal replication. </LI> </UL> </P>

Expression of the Drosophila p38b Gene Promoter during Development and in the Immune Response

Park, Joung-Sun,Kim, Young-Shin,Park, So-Young,Yoo, Mi-Ae 한국유전학회 2003 Genes & Genomics Vol.25 No.3

p38 MAPK have been extensively studied as a stress-responsive kinase and recently its role during development was reported. However, the mechanisms by which p38 gene expression is regulated remains unknown. In this study, to investigate expression of the Drosophila p38b (D-p38b) gene, we established transgenic flies bearing the D-p38b-lacZ fusion genes containing the D-p38b promoter region (-901 to +208 with respect to the transcription initiation site). Levels of the D-p38b mRNA examined by RT-PCR and analyses of expression patterns of the D-p38b-lacZ in transgenic flies indicated that the D-p38b gene is expressed throughout development. Expression of the D-p38b-lacZ gene during embryogenesis was similar to that of the D-p38b gene in situ reported by other group. The results indicated that the promoter region is sufficient for endogenous expression of the D-p38b gene. Strong expression of the D-p38b-lacZ gene was detected in the brain and ganglion, imaginal disc, salivary gland, gut and fat body among larval tissues and in the gut, fat body and reproductive systems of adult female and male. Interestingly, upregulated expression of the D-p38b-lacZ gene in the fat body and gut by injury was detected. Transgenic flies bearing the D-p38b-lacZ fusion gene promise to be useful for further studies on the mechanisms of regulation of the D-p38b gene expression during development and in the immune response.

Keratins regulate Hsp70-mediated nuclear localization of p38 mitogen-activated protein kinase

Lee, So-Young,Kim, Sujin,Lim, Younglan,Yoon, Han-Na,Ku, Nam-On The Company of Biologists Ltd. 2019 Journal of cell science Vol.132 No.18

<P>Intermediate filament protein keratin 8 (K8) binds to heat shock protein 70 (Hsp70) and p38 MAPK, and is phosphorylated at Ser74 by p38α (MAPK14, hereafter p38). However, a p38 binding site on K8 and the molecular mechanism of K8-p38 interaction related to Hsp70 are unknown. Here, we identify a p38 docking site on K8 (Arg148/149 and Leu159/161) that is highly conserved in other intermediate filaments. A docking-deficient K8 mutation caused increased p38-Hsp70 interaction and enhanced p38 nuclear localization, indicating that the p38 dissociated from mutant K8 makes a complex with Hsp70, which is known as a potential chaperone for p38 nuclear translocation. Comparison of p38 MAPK binding with keratin variants associated with liver disease showed that the K18 I150V variant dramatically reduced binding with p38, which is similar to the effect of the p38 docking-deficient mutation on K8. Because the p38 docking site on K8 (Arg148/149 and Leu159/161) and the K18 Ile150 residue are closely localized in the parallel K8/K18 heterodimer, the K18 I150V mutation might interfere with K8-p38 interaction. These findings show that keratins, functioning as cytoplasmic anchors for p38, modulate p38 nuclear localization and thereby might affect a number of p38-mediated signal transduction pathways.</P><P><B>Summary:</B> Keratin 8 interacts with p38 MAPK via a specific docking site and modulates formation of the p38-Hsp70 complex, which regulates the subcellular localization of p38 MAPK.</P>

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.

Diagnostic Significance of p38 Isoforms (p38α, p38β, p38γ, p38δ) in Head and Neck Squamous Cell Carcinoma: Comparative Serum Level Evaluation and Design of Novel Peptide Inhibitor Targeting the Same

Vishal Sahu,Lokesh Nigam,Vertica Agnihotri,Abhishek Gupta,Shashank Shekhar,Naidu Subbarao,Suman Bhaskar,Sharmistha Dey 대한암학회 2019 Cancer Research and Treatment Vol.51 No.1

Purpose The p38 mitogen-activated protein kinase (MAPKs) play a crucial role in the production of pro-inflammatory cytokines and over-expression of it increase cytokines which promote cancer. Among four isoforms, p38α has been well studied in head and neck squamous cell carcinoma (HNSCC) and other cancers as a therapeutic target. p38δ has recently emerged as a potential disease-specific drug target. Elevated serum p38α level in HNSCC was reported earlier from our lab. This study aims to estimate the levels of p38 MAPK-isoforms in the serum of HNSCC and design peptide inhibitor targeting the same. Materials and Methods Levels of p38 MAPK isoforms in the serum of HNSCC and healthy controls were quantified by surface plasmon resonance technology. The peptide inhibitor for p38 MAPK was designed by molecular modeling using Grid-based Ligand Docking with Energetics tools and compared with known specific inhibitors. Results We have observed highly elevated levels of all four isoforms of p38 MAPK in serum of HNSCC patients compared to the control group. Further, serum p38α, p38β, and p38δ levels were down regulated after therapy in follow-up patients, while p38γ showed no response to the therapy. Present study screened designed peptide WFYH as a specific inhibitor against p38δ. The specific inhibitor of p38δ was found to have no effect on p38α due to great structural difference at ATP binding pocket. Conclusion In this study, first time estimated the levels of p38 MAPK isoforms in the serum of HNSCC. It can be concluded that p38 MAPK isoforms can be a diagnostic and prognostic marker for HNSCC and p38δ as a therapeutic target.

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.

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.

신생 백서신장에서 안지오텐신 전환효소 억제가 MAPK Family 발현에 미치는 영향에 관한 연구

최병민,오미혜,유기환 대한소아과학회 2004 小兒科 Vol.47 No.3

Sigma-1 receptor-mediated increase in spinal p38 MAPK phosphorylation leads to the induction of mechanical allodynia in mice and neuropathic rats

Moon, J.Y.,Roh, D.H.,Yoon, S.Y.,Kang, S.Y.,Choi, S.R.,Kwon, S.G.,Choi, H.S.,Han, H.J.,Beitz, A.J.,Lee, J.H. Academic Press 2013 Experimental neurology Vol.247 No.-

The direct activation of the spinal sigma-1 receptor (Sig-1R) produces mechanical allodynia (MA) and thermal hyperalgesia (TH) in mice. In addition, the blockade of the spinal Sig-1R prevents the induction of MA, but not TH in chronic constriction injury (CCI)-induced neuropathic rats. The present study was designed to investigate whether the increase in spinal p38 MAPK phosphorylation (p-p38 MAPK) mediates Sig-1R-induced MA or TH in mice and the induction of MA in neuropathic rats. MA and TH were evaluated using von Frey filaments and a hot-plate apparatus, respectively. Neuropathic pain was produced by CCI of the right sciatic nerve in rats. Western blot assay and immunohistochemistry were performed to determine the changes of p-p38 MAPK expression in the spinal cord. Intrathecal (i.t.) injection of PRE084, a selective Sig-1R agonist, into naive mice time-dependently increased the expression of p-p38 MAPK, which was blocked by pretreatment with BD1047, a Sig-1R antagonist. I.t. pretreatment with SB203580, a p38 MAPK inhibitor also dose-dependently inhibited PRE084-induced MA, whereas TH induction was not affected. In CCI rats, i.t. injection of BD1047 during the induction phase (postoperative days 0 to 5) reduced the CCI-induced increase in p-p38 MAPK. In addition, i.t. SB203580 treatment during the induction phase also suppressed the development of CCI-induced MA, but not TH. Conversely, i.t. SB203580 treatment during the maintenance phase (postoperative days 15 to 20) had no effect on CCI-induced MA or TH. These results demonstrate that the increase in spinal p-p38 MAPK is closely associated with the induction of Sig-1R mediated MA, but not TH. Sigma-1 receptor modulation of p-p38 MAPK also plays an important role in the induction, but not the maintenance, of MA in neuropathic pain.

조골세포에서 p-38 MAP kinase의 nitric oxide 및 interieukin-6 생성조절에 관한 연구

이경원(Kyung-Won Lee),이도훈(Doe-Hoon Lee),강경화(Kyung-Hwa Kang),김상철(Sang-Cheol Kim) 대한치과교정학회 2003 대한치과교정학회지 Vol.33 No.3

치아이동 시 발생한는 골흡수에서 이미 여러 cytokine의 중요성이 강조된 바 있으며 이 가운데 intreleukin-6는 구강 및 연골조직 등에서 많은 연구의 초점이 되어 왔으나 확실한 기전은 아직까지 정확히 확립되어 있지 못하다. 골흡수 시조골세포에서 유리되는 Interleukin-6 (IL-6)와 nitric oxide (NO) 등이 골흡수의 조절자로 최근 대구되고 있으며 Mitogen-activated protein kinase (MAPK)의 활성화로 인해 염증성 cytokine등이 유리될 수 있음이 최근 macropage등이 증명된 바 있다. 그러므로 치아이동을 비롯한 구강 내 여러 염증의 조건에서 골흡수의 대표인자 IL-6및 NO 유리가 MAPK등의 활성 등을 통해 조절된 수 있는 가능성을 시사하고 있다. 본 연구에서 조골세포 특징을 대부분 가지고 있는 조골세포주, MC3T3E1에서 p-38 MAP kinase을 매개로 NO 와 IL-6가 유리됨을 확인하고자 하였다. 10% Fetal Bovine Serum이 첨가된 -MEM 배양약으로 배양한 조골세포주인 MC3T3E1 세포에 tumor necrosis factor-α (TNF-α), interferon-r (IFN-r) 및 lipopolysacchalide(LPS)등의 단독처리 시 NO와 IL-6의 증가는 확인되지 않았으나 TNF-α/IFN-r 혹은 LPS/IFN-r 등의 처치시 NO 와 IL-6이 유의한 증가를 보였으며, NO발현에 직접 관여하는 inducible nitric oxide synthase (iNOS)와 IL-6 단백질 및 mRNA의 발현을 관찰하였다. 또한 specific p-38 MAP kinase inhibitor인 SB203580의 NO와 IL-6의 생성 억제를 관찰하고 단백질과 mRAN 발현억제를 통해서도 확인함으로써 SB203580은 transcription 단계에서 NO와 IL-6의 생성을 조절하고 있음을 시사하여 주고 있다. TNF-α/IFN-r 혹은 LPS/IFN-r 처치 시 p-38 MAP Kinase의 활성을 관찰하였으나 단독 처치 시 역시 p-38 MAP Kinase의 활성율 확인함으로써 NO 와 IL-6유리를 확인하였으며, 또한 이들의 생성기전중의 하나로 p-38 MAP Kinase 가 transcription 단계에서 관여하고 있음을 확인하였다. Tooth movement is the result of bone metabolism in the periodontium, where various cytokines take important roles. Interleukin-6(116) and nitrous oxide (NO) were reported to be secreted from osteoblasts in the process of bone resorption. The mechanism of the process has not been clearly understood, but the activation of mitogen-activated protein kinase (MAPK) was known to be an important process in the release of the inflammatory cytokines in macrophages. In this regard, to prove the role of MAPK in the release of IL-6 and NO in MC3T3E-1 osteoblasts, Northern blot analysis, Western blot analysis and immune complex kinase assay were used. As a result, the treatment of MC3T3E-l osteoblast cultures with combined interferon-y(IFN-r), lipopolysaccharide,(LPS) and tumor necrosis factor-a(TNF-a) induces expressions of inducible nitric oxide synthase (iNOS) and IL-6, resulting in sustained releases of large amounts of NO and IL-6. However, IFN-r, LPS, and TNF-a individually induce a non-detectable or small amount of NO and IL-6 in AMC3T3E-1 osteoblasts. The role of MAPK activation in the early intracellular signal transduction involved in iI1OS and IL-6 transcription in the combined agents-stimulated osteoblasts has been investigated. The p38 MAPK pathway is specifically involved in the combined agents-induced NO and IL-6 release, since NO and IL-6 release in the presence of a specific inhibitor of p38 MAPK, 4-(4fuorophenyi)-2-(4-metylsulfinylphenyl)-5-(4pyridyl)imidazole) (SB203580), were significantly diminished, In contrast, PD98059, a specific inhibitor of MEK1, had no effect on NO and IL-6 release. Northern blot analysis showed that the p38 MAPK pathway controlled the iNOS and IL-6 transcription level. These data suggest that p38 MAPK play an important role in the secretion of NO and IL-6 in LPS/IFNr- or TNF-a/IFN-r-treated MC3T3E-1 osteoblasts.