位於G-CSF 3'端UTR之SLDE序列在SB203580誘導下增加G-CSF mTNA穩定度的角色

Abstract

顆粒性白血球群落刺激因子(G-CSF)為造血性醣蛋白家族中的一員，也是一種細胞激素，主要由巨噬細胞及單核球細胞所分泌，具有促進噬中性白血球分化、增生及移動的功能。細胞受到LPS刺激活化p38 MAPK會使G-CSF mRNA穩定度增加，增加G-CSF表現。G-CSF mRNA的穩定度主要由 3'UTR的ARE及SLDE所調控。許多研究顯示給予SB203580抑制p38 MAPK活性後，會使在3’UTR帶有ARE的mRNA半衰期變短，進而降低其表現量。但是本實驗室先前的研究結果發現，以小鼠巨噬細胞Raw264.7經LPS刺激之下預先給予SB203580，會增加G-CSF mRNA的穩定度，更增加LPS誘導的G-CSF mRNA及蛋白質的表現量，顯示SB203580對G-CSF表現的影響與對其他帶有ARE的細胞激素的mRNA作用是不同的。本研究的目的是探討SLDE在SB203580增 加LPS誘導的G-CSF mRNA穩定度所扮演的角色。 實驗結果發現當細胞不受刺激時G-CSF 3’UTR會使mRNA不穩定；而給予LPS及SB203580後會提高mRNA穩定度。將3’UTR接到luciferase後，以site direct mutagenesis分析發現SB203580提高LPS誘導的G-CSF mRNA穩定度是透過3’UTR SLDE內TTTAATATTTA這段高保留性序列。此外，不同的p38 MAPKs抑制劑對G-CSF mRNA及蛋白質的量有不同的影響，其中預先給予SB203580、SB202190及PD169316都會提高G-CSF mRNA穩定度進而提高LPS誘導的mRNA及蛋白質表現，而SKF86002及SB239063則不會影響G-CSF蛋白質表現，但是SKF86002也會些微增加約1.8倍的mRNA表現。最後我們也發現在沒有LPS刺激的情況下，SB203580、SB202190及PD169316也會透G-CSF 3’UTR來增加G-CSF mRNA穩定度，顯示這些抑制劑增加G-CSF mRNA穩定度可能不是藉由抑制p38 MAPKs磷酸酶活性所導致。綜合以上結果，SB203580會透過SLDE中高保留性的序列提高G-CSF mRNA穩定度，而且SB203580增加G-CSF mRNA的作用可能不是藉由抑制p38 MAPKs磷酸酶活性所導致。

Granulocyte colony stimulating factor (G-CSF) is not only a member of hematopoietic growth factor but also a cytokine. G-CSF is known to control the production, differentiation and migration of neutrophils. LPS increasing G-CSF mRNA stability by activating p38 MAPK kinase activity, thus increases G-CSF production. There are two destabilizing elements in G-CSF 3’UTR-adenosine uridine-rich element (ARE) and stem-loop destabilizing element (SLDE) that regulate G-CSF mRNA stability. Numerous studies show that SB203580, a pyridinyl imidazole p38 MAPK inhibitors, reduces the half-life of the ARE-containing mRNA by inhibiting p38 MAPK kinase activity. However, our previous studies showed that SB203580 enhances LPS-induced G-CSF production by increasing mRNA stability. The aim of this study is to investigate the possible role of SLDE in SB203580 mediated increase in G-CSF mRNA stability in LPS treated macrophage. Our results show that G-CSF 3’UTR decreases mRNA stability in unstimulated cells, but stabilized mRNA when p38 MAPKs activity was activated by LPS. We find that the consensus sequence “TTTAATATTTA” rather than the stem-loop structure in G-CSF 3’UTR SLDE is essential for the SB230580 enhanced LPS-increased G-CSF mRNA stability. Moreover, various p38 MAPK inhibitors had different effects on the expression levels of G-CSF mRNA and protein. The levels of LPS-induced G-CSF mRNA and protein in Raw 264.7 were increased by SB203580, SB202190 and PD169316, but not by SB239063. Furthermore, we discovered that SB203580, SB202190 and PD169316 increased G-CSF mRNA stability when p38 MAPK kinase activity was inactivated. Taken together, these results suggest that SB203580 increases G-CSF mRNA stability through the highly conserved sequence and this effect is independent p38 MAPKs kinase inhibition.