Modulation of FAK, Akt, and p53 by stress release of the fibroblast-populated collagen matrix.
Modulation of FAK, Akt, and p53 by stress release of the fibroblast-populated collagen matrix.
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DOI：
10.1016/j.jss.2003.12.002
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发表时间：
2004-08
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影响因子：
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通讯作者：
M. A. Carlson;M. Longaker;Jon S Thompson
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文献类型：
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作者：
M. A. Carlson;M. Longaker;Jon S Thompson
成纤维细胞在三维胶原基质中的存活部分取决于基质与组织培养塑料的刚性锚定。我们假设，粘着斑激酶（FAK）和蛋白激酶B（Akt）将被激活，p53的水平将是低的刚性锚定（附）胶原基质;锚定（脱离）的损失被假设有相反的effects.MATERIALS AND METHODS人类包皮成纤维细胞培养在附着牛胶原基质分离前48小时作为自由浮动的矩阵。在释放后的不同时间点，对基质裂解物进行目标蛋白印迹，并对整个基质和细胞离心涂片制备物进行末端脱氧核苷酸转移酶介导的dUTP缺口末端标记测定。照射后的单层成纤维细胞作为p53蛋白量的阳性对照，在贴壁基质和脱离基质中（脱离后24小时），MTT法脱氧核苷酸转移酶介导的dUTP缺口末端标记阳性率分别为0.7 ± 0.3%和5.3 ± 1.7%（P < 0.05，非配对t检验）。FAK和Akt在附着的基质中被磷酸化（活化）;在基质脱离的4小时内，两种活化形式几乎完全丧失。辐射单层成纤维细胞的p53，mdm 2和p21的水平增加。与此相反，p53，mdm 2，和p21的水平只是在所附的矩阵中的检测水平，但在2-4小时内诱导5- 10倍后matrixdetachment.CONCLUSIONSFAK和Akt被激活在所附的成纤维细胞填充的胶原蛋白基质，而p53的水平相对较低，基质脱离下调FAK和Akt的活性，并诱导p53。胶原基质的机械锚定状态通过可能涉及FAK的机制调节包埋的成纤维细胞的存活。
BACKGROUNDFibroblast survival in a three-dimensional collagen matrix is dependent in part upon the rigid anchorage of the matrix to tissue culture plastic. We hypothesized that focal adhesion kinase (FAK) and protein kinase B (Akt) would be activated and that the p53 level would be low in the rigidly anchored (attached) collagen matrix; loss of anchorage (detachment) was hypothesized to have the opposite effects.MATERIALS AND METHODSHuman foreskin fibroblasts were cultured in attached bovine collagen matrices for 48 h before detachment as free-floating matrices. At various time points postrelease, matrix lysates were blotted for the proteins of interest, and the terminal deoxynucleotidyltransferase-mediated dUTP nick-end label assay was performed on both whole matrices and cytospin preparations. Irradiated monolayer fibroblasts were used as positive controls for the amount of p53 protein.RESULTSTerminal deoxynucleotidyltransferase-mediated dUTP nick-end label positivity in attached versus detached matrices (at 24 h post detachment) was 0.7 ± 03 versus 5.3 ± 1.7% (P < 0.05, unpaired t test). FAK and Akt were phosphorylated (activated) in the attached matrix; there was a near complete of loss of both activated forms within 4 h of matrix detachment. Irradiated monolayer fibroblasts had increased levels of p53, mdm2, and p21. In contrast, the p53, mdm2, and p21 levels were just at the level of detection in the attached matrix, but were induced 5- to 10-fold within 2–4 h after matrix detachment.CONCLUSIONSFAK and Akt are activated in the attached fibroblast-populated collagen matrix whereas the p53 level is relatively low; matrix detachment downregulates FAK and Akt activity and induces p53. The state of mechanical anchorage of the collagen matrix regulates the survival of embedded fibroblasts through a mechanism which may involve FAK.