[INTRODUCTION]

Embryonic Stem (ES) cells are able to differentiate into a variety of cell types, which provide an attractive source of cells for disease modeling in vitro and the treatment of many degenerative diseases. An essential step for therapeutic and research applications of stem cells is the ability to differentiate them into specific cell types. Understanding the mechanisms of ES cell differentiation is crucial for developing more efficient and selective methods to direct the differentiation of ES cells to produce homogenous populations of particular cell types. However, molecular mechanisms that control the commitment of ES cells into a specific lineage are still poorly understood. In this study, we will demonstrate the physiological roles of p38 mitogen-activated protein kinase (p38 MAPK) in lineage commitment of mouse ES cells.

[METHODS AND RESULTS]

38 MAPK specific inhibitor SB203580 blocked cardiomyogenesis and committed ES cells into the neuronal lineage

Mouse ES cells are maintained in an undifferentiated state and retain the potential for unlimited proliferation in the presence of Leukemiainhibitory Factor (LIF). Removing LIP and cultured in serum containing medium, ES cells can be differentiated spontaneously into beating cardiomyocytes through embryonic body (EB) formation to mimic embryo development in vivo. Mitogen-activated protein (MAP) kinases are serine/threonine-specific protein kinases that respond to extracellular stimuli and regulate various cellular activities, such as gene expression, mitosis, differentiation, and cell survival/apoptosis. To investigate the role of three main MAP kinase and PI3- kinases in ES cell lineage commitment, EBs were treated with specific inhibitors between day 1 and day 6 during the differentiation process. The effect of the extracellular signal-regulated kinase (ERK)-specific inhibitor U0126, stress-activated protein kinase/c-Jun NH2-terminal kinase (JNK)-specific inhibitor SP6000125, p38 MAPK-specific inhibitor SB203580, and Phosphoinositide 3-kinase (PI3K)-specific inhibitor wortmannin, were evaluated. We found that ERK-specific inhibitor U0126 and p38 MAPK-specific inhibitor SB203580 significantly blocked spontaneous cardiac differentiation, with less than 5% of EBs containing beating foci at day 12. In contrast, more than 90% of untreated control EBs containing beating areas, which were confirmed with phenotypic observations (Fig. 1A). Interestingly, more than 90% of SB203580 treated EBs exhibited prominent growth when blocked cardiomyogenesis (Fig. 1B). These outgrowths showed positive immunofluorescence staining with anti-BIIItubulin antibody specific for neurons, whereas EBs treated with U0126 did not, which has the same effect on the inhibition of cardiac differentiation (Fig. 1C). These results demonstrate that the neural differentiation is induced specifically by SB203580, but does not due to the inhibition of cardiomyogenesis.

To further confirm the switch effect of SB203580 on cardiac versus neural differentiation, frozen sections from EBs treated with or without SB203580 between day 1 and day 6 were immunohistochemical staining at day 12. EBs with SB203580 treatment did not stain with a cardiac-specific marker a actinin, but did stain positively with anti-a TUJ antibody specific for neurons. And RT-PCR analysis showed that SB203580 treatment completely inhibited the expression of cardiac genes, including mef2c, mhc, and mlc2v, whereas induced significant increase in mRNA level of neural-specific genes, nestin, hes5, mash] , mash3, and map2.

p38 MAPK activity between day 3 and 4 determines cardiac or neural commitment of ES cells

To investigate the role of p38 MAPK in ES cell commitment, we first measured the p38 MAPK activity during early stages of differentiation by Western blot using antiphospho-p38 MAPK and antip38 MAPK antibodies. At day 0, when ES cells were cultured as monolayer, no detectable p38 MAPK activation was observed in whole cell lysates. Interestingly, after the EB formation, p38 MAPK was spontaneously activated and peaked from day 2 to day 6 without affecting the expression level of p38 MAPK protein. Next we determined at what time point p38 MAPK acts during the differentiation process by treating EBs with SB203580 at various time intervals. The treatment of SB203580 between day 3 and day 4 significantly promoted neuron differentiation, as did a treatment from day 1 to day 6, whereas SB203580 treated at other periods did not induce neurogenesis (Fig. 2A). RT-PCR analysis confirmed that the expression of neuronal markers hes5 and map2 were induced only when the inhibitor was applied between day 3 and day 4 or between day 1 and day 6, while the cardiomyocyte-specific genes mhc and mlc2v were strongly decreased (Fig. 2B). These results clearly demonstrated that the p38 MAPK activity between day 3 and 4 is critical for cardiac or neural commitment of ES cells.

p38 MAPK functions as a molecular switch in ES cell commitment into cardiomvocvtes versus neurons by controlling the expression of BMP-2

To further elucidate the molecular mechanism of p38 MAPK in governing the cell-fate choices in ES cells, we sought to identify downstream targets of p38 MAPK. Since p38 MAPK activity between day 3 and 4 is crucial for the lineage commitment, we investigated whether the mRNA expression of known genes involved in cell fate determination were affected by treating the EBs with or without SB203580 between day 3 and 4. Notably, RT-PCR analysis showed that the mRNA expression of bone morphogenetic protein 2 (bmp-2) was decreased with the SB203580 treatment. Further analysis revealed that bmp-2 was induced from day 4 and SB203580 treatment strongly repressed this induction.

To determine whether downregulation of BMP-2 expression was a key factor for SB203580 to induce neurogenesis, recombinant human bone morphogenetic protein-2 (rhBMP-2) was applied to the EBs from day 4 to 6 in the presence of SB203580 from day 3 to 6. Predictably, rhBMP-2 remarkably repressed the neuron differentiation induced by SB203580 treatment (Fig. 3A). Quantification analysis indicated that SB203580 treatment from day 3 to 6 resulted in nearly 80% of neural induction, whereas addition of rhBMP-2 decreased this rate to less than 5% (Fig. 3B). Consistent with the microscopic analysis, RTPCR confirmed that rhBMP-2 strongly inhibited the expression of neuron-specific gene map2 induced by SB203580 Meanwhile, the expression of cardiomyocyte-specific genes mhc and mlc2v were slightly recovered by the addition of rhBMP-2. Furthermore, treated the EBs with BMP-2 antagonist Noggin blocked the cardiomyogenesis, conversely, promoted neuronal differentiation, similar to the SB203580 treatment. These results indicate that p38 MAPK controls ES cell lineage commitment by regulating the expression of BMP-2.

BMP-2 is a direct transcriptional target of MEF2C, a well-known substrate of n38 MAPK

SB203580 treatment also resulted in a dramatic decrease in mef2c expression, which had a similar expression pattern as bmp-2 during the differentiation process. MEF2C is a crucial transcription factor controlling the activation of many cardiac-specific genes. Interestingly, p38 MAPK is a well-known regulator of MEF2C, which can phosphorylate MEF2C directly on residues located within the activation domain, suggesting that p38 MAPK may induce cardiomyogenesis via direct regulation of MEF2C. Moreover, highly conserved consensusbinding site for MEF2 was detected in both mouse and human proximal bmp-2 promoters. Therefore, we speculate that MEF2C may directly regulate the transcription of bmp-2. To test this hypothesis, we first carried out luciferase reporter assays with HeLa cells using a proximal promoter region (-1703/-1 bp) of mouse bmp-2 containing the MEF2 site. Overexpression of MEF2C increased luciferase activity 3-fold, whereas SB203580 treatment repressed its transactivation (Fig. 4A). To determine whether MEF2C can bind to the bmp-2 promoter region, chromatin immunoprecipitation (ChIP) analysis was performed at day 6 under spontaneous differentiation conditions. The mouse bmp-2 promoter region encompassing the -656/-635 bp MEF2 site was precipitated in the presence of an anti-MEF2C specific antibody, indicating that MEF2C can bind to the bmp-2 promoter(Fig. 4B, up-left). but does not bind to the promoter region of control bandl gene present on the same chromosome (Fig. 4B, up-right). Furthermore. SB203580 treatment inhibited this binding of MEF2C to the MEF2 site (Fig. 4B, down-left). As a positive control, the region promoter mouse mef2c, which itself is a target of MEF2 transcription factor, was also precipitated similar to bmp-2 (Fig. 4B, middle). These data suggest that bmp-2 is a potential transcriptional target of MEF2C.

[CONCLUSIONS]

In this study, our results demonstrate that p38 MAPK functions as a molecular switch in ES cell commitment into cardiomyocytes versus neurons. During the differentiation process, the high p38 MAPK activity between day 3 and 4 is crucial for cardiomyogenesis of ES cells, and specific inhibition of this activity can direct the differentiation of ES cells from cardiomyocytes toward neurons. Furthermore, our data show that BMP-2 is a critical downstream target of p38 MAPK and appears to be regulated directly by transcription factor MEF2C, a well-known substrate of p38 MAPK.

Fig. 1 . Effects of MAP kinase specific inhibitors on ES cell differentiation.

Fig. 2 SB203580 treatment between day 3 and 4 specifically blocks cardiomyogenesis and induces neurogenesis.

Fig. 3 BMP-2 inhibits SB203580-induced neurogenesis.

Fig. 4 MEF2C controls BMP-2 promoter activity.

胚性幹細胞(ES細胞)は生体外で自己複製能を保ちながら、すべての細胞に分化誘導することが可能なため、疾患モデル研究や再生医療への応用に注目が集まっている。マウスES細胞は3.5日胚から樹立された多能性幹細胞であり、適切な分化誘導法によって心筋細胞や神経細胞などの様々な細胞系へと分化誘導することが可能である。目的とする細胞へと効率よく高純度に分化誘導するために、ES細胞の分化を制御する分子メカニズムの解明は非常に重要であるが、細胞分化決定の分子機構は依然として不明な点が多い,「Role of p38 Mitogen-Activated Protein Kinase in lineage commitment of mouse ES cells(p38 MAPキナーゼのマウスES細胞分化決定における役割)」と題した本論文においては、ES細胞の分化過程においてp38MAPキナーゼが活性化され、転写因子MEF2Cを介して骨形成因子BMP-2の発現を制御し、心筋分化を促進すると同時に神経分化を抑制することを見出している。

1.p38MAPキナーゼ阻害薬(SB203580)は心筋分化を抑制して神経分化を促進する

ES細胞の分化過程において、MAPキナーゼを中心とした各種阻害薬(ERK系阻害薬U0126、P38阻害薬SB203580、JNK阻害薬SP600125)とPI3キナーゼ阻害薬wortmaninを用いてその分化に及ぼす影響を検討した。その結果、ERK系阻害薬U0126とp38阻害薬SB203580を添加すると、拍動するEBの割合は、阻害薬非存在下の90%以上から5%以下にまで顕著に抑制された。一方、JNK阻害薬SP600125とPI3キナーゼ阻害薬wortmaninを添加した場合には、顕著な影響が認められなかった。興味深いことに、p38阻害薬であるSB203580は、心筋分化を抑制する同時に、胚葉体(EB)の周りに突起伸長を誘導した。神経マーカーである抗BIII tubulin抗体を用いて免疫染色した結果、この突起伸長が陽性像を示した。一方、SB203580と同様に心筋分化を抑制するERK系阻害薬U0126を添加した場合には、陽性像が観察されなかった。これらの結果より、心筋分化の抑制が単に神経分化を促進したのではなく、p38の阻害薬SB203580は特異的に神経分化を促進することが示唆された。

2.p38は時期特異的に心筋と神経への分化決定を制御する

分化過程におけるp38の活性状態を、活性化型であるリン酸化p38を特異的に認識する抗体を用いてWestern Blottingにより検討した。その結果、分化開始直前のday0の単層培養時にはp38の活性が認められなかったが、EBの形成とともに、p38がday2からday6の期間で持続的に活性化された。次に、day1からday6までの分化過程に、一定の期間のみSB203580を添加して、p38の作用時期を検討した。day3-4とday1-6にSB203580を添加すると、顕著に心筋分化を抑制し、神経分化を促進した。さらに、Day12に細胞を回収し、RT-PCRで心筋と神経分化のマーカー分子を解析した結果、day3-4とday1-6にSB203580を添加した細胞では、無添加の対照と比較して、神経マーカー分子の発現が強く誘導され、逆に、心筋マーカー分子の発現が抑制された。これらの結果から、SB203580がday3-4の時期に特異的に心筋分化を抑制し、神経分化を促進することが示された。

3.p38シグナル経路の下流に骨形成因子BMP-2が存在する

p38の下流標的分子の同定に向けて、ES細胞分化決定に重要な時期であるday3-4にSB203580を添加してRT-PCRにより心筋や神経の分化決定に関わる因子を探索した。その結果、SB203580の添加によりBMP-2遺伝子の発現に変化が見られた。SB203580の存在下でBMP-2を添加すると、SB203580依存的な神突起伸長が顕著に抑制された。さらに、BMP-2阻害因子であるNogginを用いてBMPシグナルを遮断すると、SB203580と同様に心筋分化を顕著に抑制し、神経分化を促進することが示された。これらの結果より、p38はその下流の因子であるBMP-2を介して、心筋分化を制御すると同時に、神経分化を抑制することが明らかにされた。

4.p38の基質であるMEF2CがBMP-2の転写発現を制御する

p38の基質であるMEF2Cは、心臓の発生に必須な転写因子である。BMP-2のプロモーター領域にはMEF2転写因子の応答配列がマウスのみならずヒトにも存在し、ES細胞の分化過程において、MEF2CはBMP-2と類似する発現パターンを示した。そこで、マウスBMP-2遺伝子のMEF2応答配列を含むプロモーター領域をPCRにより増幅し、ルシフェラーゼ遺伝子と融合させたベクターを作成してプロモーター解析を行った。BMP-2プロモーターの転写活性は、MEF2Cを過剰発現すると顕著に上昇し、SB203580を添加すると抑制された。さらに、クロマチン免疫沈降実験により、MEF2CがBMP-2プロモーターのMEF2応答配列に結合することが示された。これらの結果から、転写因子MEF2Cが直接にBMP-2の発現を制御していることが示された。

本論文から、p38はES細胞の分化において分子スイッチとして機能し、心筋と神経分化の運命決定を制御していることが明らかにされた。また、p38シグナル経路の下流に骨形成因子BMP-2が存在すること、さらに、p38の基質である転写因子MEF2CがBMP-2の発現を制御していることが示された。以上を要するに、本論文は、ES細胞の分化決定におけるMAPキナーゼの役割とそのシグナル伝達系について、新たに重要な知見を提示しており、博士(薬学)の学位として十分な価値があるものと認められる。