PI3K/AKT/mTOR訊息傳遞路徑在脂多醣誘導巨噬細胞表現G-CSF中所扮演的角色

Abstract

Oct-2是因為能結合到許多基因上的octamer motif（ATTTGCAT）而命名的，Oct-2是屬於POU family的一員，POU family的成員除了Oct-2之外，還有Oct-1、Pit-1以及Unc-86，在之前的研究指出，Oct-2在神經細胞內以及B淋巴球中是非常重要的轉錄因子，然而Oct-2在巨噬細胞內的表現和功能卻還尚未明瞭，在我們之前的研究指出，脂多醣LPS會誘導巨噬細胞表現Oct-2，並且進一步的發現在LPS作用下，巨噬細胞表現Oct-2是藉由PI3K/AKT/mTOR pathway來調節其蛋白質的合成，因此在巨噬細胞中，Oct-2或許是個調節細胞內發炎基因表現的重要轉錄因子。 Granulocyte colony stimulating factor (G-CSF)是一個血球生長因子，它能維持嗜中性球先驅細胞的生長並幫助其分化成嗜中性球，在體內一些發炎性刺激，像是IL-1, LPS, and TNF-α可以誘導巨噬細胞、內皮細胞和纖維母細胞表現G-CSF，雖然已知道G-CSF會受到刺激而表現，但是對於調控G-CSF表現的分子機制卻還不清楚。在本篇論文中，我們探討了在LPS作用下，巨噬細胞表現G-CSF是否會經由PI3K/AKT/mTOR pathway來調節，並且探討了Oct-2在LPS誘導G-CSF表現的過程中所扮演的角色，我們將RAW264.7細胞先給予不同濃度的PI3K、AKT或是mTOR的抑制劑來阻斷PI3K/AKT/mTOR pathway，以RT-PCR來偵測LPS誘導六小時後G-CSF mRNA的表現，在培養液中的G-CSF蛋白是利用ELISA assay偵測，結果發現，隨著抑制劑的濃度的增加，LPS所誘導的G-CSF mRNA和蛋白質表現會逐漸降低，並且這些抑制劑也能降低LPS誘導Oct-2表現。除此之外，NF-κB transactivation activity和 DNA binding affinity都會受到這些抑制劑而降低。 在chromatin immunoprecipitation (ChIP) assay中顯示在LPS作用下，Oct-2會結合到G-CSF啟動子上，而另一個octamer binding protein-Oct-1則是不會結合到啟動子上；在PI3K、AKT或是mTOR的抑制劑加入之後，便會使得Oct-2減少結合到G-CSF啟動子上。進一步地，我們將細胞轉染表現shRNA的質體，利用RNA interference的方式來knockdown Oct-2的表現，結果顯示，由LPS誘導的G-CSF的表現會因為Oct-2被knockdown之後而明顯的減少，因此由實驗結果得知，在LPS作用下，巨噬細胞表現G-CSF是需要活化PI3K/AKT/mTOR pathway，並且除了NF-κB之外，Oct-2調節LPS所誘導的G-CSF基因轉錄中是相當重要的轉錄因子。

The Oct-2 factor was originally identified on the basis of its ability to bind to the octamer motif ATGCAAAT which is found in the promoters of several genes. Oct-2 belongs to the POU family composed of Oct-1, Oct-2, Pit-1, and Unc-86. In the previous studies, Oct-2 has been known as an important transcription factor for B cells and neuron cells. However, expression and function of Oct-2 in the macrophages is mostly unknown. Our recent results showed that expression of Oct-2 in the macrophages was induced by LPS. Moreover, our data suggest that LPS-induced increase of Oct-2 protein is through PI3K/AKT/mTOR signaling pathway. Therefore, Oct-2 may act as a mediator in response to inflammatory stimuli. Granulocyte colony stimulating factor (G-CSF) is a hematopoietic growth factor. It supports the survival and stimulates the proliferation of neutrophil progenitors and promotes their differentiation into mature neutrophils. Inflammatory stimuli such as IL-1, LPS, and TNF-α can induce G-CSF production in macrophages, endothelial cells, and fibroblast, but the molecular mechanism was not clear. In our studies, we tested if LPS induces G-CSF expression through PI3K/AKT/mTOR pathway, and if Oct-2 plays a role in LPS-induced G-CSF expression. RAW264.7 macrophages were pretreated with inhibitors of PI3K, AKT, or mTOR before LPS was added for 6 hours and then G-CSF mRNA was determined by RT-PCR and protein in medium was determined by ELISA assay, and its RNA was determined by RT-PCR. The results showed that different concentration of PI3K, AKT, and mTOR inhibitors gradually prevented LPS-induced increase of G-CSF and inhibitors also downregulated LPS-induced Oct-2 expression. Additionally, NF-κB transactivation activity and DNA binding affinity was reduced by these inhibitors. Chromatin immunoprecipitation (ChIP) assay showed that in LPS-treated cells, Oct-2, but not Oct-1 was recruited to the octamer motif of the G-CSF promoter. Furthermore, pretreated with inhibitors of PI3K, AKT, or mTOR before LPS was added resulted in less Oct-2 binding to the promoter of G-CSF. When shRNA was transfected into cells to knockdown Oct-2, LPS-induced G-CSF expression was significantly reduced. Taken together, our data suggest that LPS-induced G-CSF expression depends on the activation of PI3K/AKT/mTOR pathway and besides NF-κB, Oct-2 plays an important role in the transcription regulation of G-CSF expression induced by LPS.