p38信号通路参与LPS诱导的Raw264.7细胞骨架重构

应用免疫荧光标记、基因转染等方法，观察脂多糖（LPS）刺激对单核细胞系Raw264.7细胞骨架的影响，探讨p38家族不同亚型对LPS诱导的细胞骨架蛋白微管蛋白与肌动蛋白变化的调控作用结果显示，未受LPS刺激的细胞富含微管蛋白，微管蛋白交联形成辐射状的交联丝网，丝网在细胞中分布均匀；LPS刺激后，微管蛋白募集在细胞膜、核膜周围；p38α、p38β、p38γ亚型的特异性抑制剂FHPI对LPS诱导的微管蛋白募集无影响，而p38无活性突变体p38δ(AF)的基因转染，可抑制LPS诱导的细胞骨架微管蛋白的募集；肌动蛋白在静息的细胞内主要存在于细胞膜周围，LPS作用后，肌动蛋白在细胞中形成广泛分布的辐射状应激纤维；p38上游激酶活性诱变体MKK6b基因转染可诱导Raw细胞形成类似的应激纤维，而p38γ(AF)的基因转染，可抑制LPS诱导的细胞应激纤维的形成.上述结果表明,p38δ可能参与了LPS诱导的微管蛋白的重构；而LPS诱导Raw细胞应激纤维的形成，可能是通过p38γ蛋白激酶而发挥作用.

p38 Signaling Pathway Involved in LPS-induced Cytoskeleton Rearrangement in Raw264.7 Cells

Cytoskeletal proteins are major components of the cell backbone and regulate cell shape and function. The purpose of this study was to investigate the effect of p38 signaling pathway on lipopolysaccharide (LPS) induced cytoskeleton rearrangement in Raw264.7 cells. Indirect fluorescence microscopy and gene transfection techniques were used to detect the change of tubulin and actin in Raw264.7 after LPS stimulation. Ectopic expression of p38 MAP kinase was confirmed by immunostaining with an antibody to the Flag epitope tag. The fluorescence microscopy results showed that tubulin formed abundant networks in resting Raw264.7 cells, while tubulin recruited to the cell membrane and the nuclear membrane after LPS stimulation. FHPI, the specific inhibitor of p38α, p38β and p38γ, has no effect on the changes of tubulin after LPS stimulation, while overexpression of the dominant negative mutant of p38δ, p38δ (AF), could inhibit the tubulin recruiment in these cells. Meanwhile, LPS could induce the reorganization of actin from cortical microfilament into stress fibers. Overexpression of a constitutively active mutant of MKK6bE, an upstream kinase for p38 MAPK, could also induce the formation of stress fibers similar to that of LPS treatment. In contrast, transfection of the dominant negative mutant of p38γ, p38γ(AF), could inhibit the formation of actin stress fiber. Taken together, these findings suggest in Raw264.7 cells, LPS induced tubulin rearrangement is mediated by p38δ, while LPS stimulated actin reorganization is mediated by p38γ.