Stulpinas Aurimas, Uzusienis Tomas, Imbrasaite Ausra, Krestnikova Natalija, Unguryte Ausra, Kalvelyte Audrone V
Dept. of Molecular Cell Biology, Institute of Biochemistry, Life Sciences Centre, Vilnius University, Saulėtekio al. 7, LT-10257, Lithuania.
Centre for Innovative Medicine, Santariškių g. 5, LT-08406, Lithuania.
Cell Signal. 2021 Aug;84:110034. doi: 10.1016/j.cellsig.2021.110034. Epub 2021 Apr 30.
The use of cultured cells as a tool for research, precision medicine, biopharmacy, and biomanufacturing is constantly increasing. In parallel, the role of cell-cell and cell-substratum contacts in cell functioning is increasingly validated. Adhesion signalling plays a key role here. The activity of cell fate-regulating signalling molecules is an important factor in determining cell behaviour, as well as their response to treatment, depending on cell phenotypic status and location in the body. Three cellular state models (adherent, single cells in suspension, and aggregated cells) were compared for cell signalling, including focal adhesion (FAK), mitogen-activated (MAPK), as well as Akt protein kinases, and transcription factor cJun, by using lung adenocarcinoma A549, muscle-derived stem Myo, as well as primary lung cancer cell lines. Survival of both A549 and Myo cells was dependent on kinases Akt and ERK in detached conditions. Intercellular contacts in aggregates promoted activation of Akt and ERK, and cell survival. Loss of contacts with the substrate increased phosphorylation of MAP kinases JNK and p38, while decreased Akt phosphorylation by processes involving FAK. Unexpectedly, detachment increased phosphorylation of antiapoptotic kinase ERK in A549, while in Myo stem cells ERK phosphorylation was downregulated. JNK target transcription factor cJun protein level was markedly diminished by contacts between cells possibly involving mechanism of proteasomal degradation. Furthermore, studies revealed the opposite dependence of molecules of the same signalling pathway - phospho-cJun and phospho-JNK - on cell culture density. Differences in ERK activation under detachment conditions indicate that targeting of prosurvival kinases during anoikis should be different in different cells. Moreover, the outcome of JNK activation in cells may depend on the amount of cJun, which is determined by cell-cell contacts.
将培养细胞用作研究、精准医学、生物制药和生物制造的工具的情况正在不断增加。与此同时，细胞间和细胞与基质接触在细胞功能中的作用也越来越得到证实。黏附信号传导在此起着关键作用。细胞命运调节信号分子的活性是决定细胞行为以及它们对治疗反应的重要因素，这取决于细胞的表型状态和在体内的位置。通过使用肺腺癌A549细胞、肌肉来源的干细胞Myo以及原发性肺癌细胞系，比较了三种细胞状态模型（贴壁细胞、悬浮单细胞和聚集细胞）的细胞信号传导，包括粘着斑激酶（FAK）、丝裂原活化蛋白激酶（MAPK）以及Akt蛋白激酶，还有转录因子cJun。在脱离条件下，A549细胞和Myo细胞的存活均依赖于激酶Akt和ERK。聚集体中的细胞间接触促进了Akt和ERK的激活以及细胞存活。与基质接触的丧失增加了丝裂原活化蛋白激酶JNK和p38的磷酸化，而通过涉及FAK的过程降低了Akt磷酸化。出乎意料的是，脱离增加了A549细胞中抗凋亡激酶ERK的磷酸化，而在Myo干细胞中ERK磷酸化被下调。细胞间接触可能通过蛋白酶体降解机制显著降低JNK靶转录因子cJun蛋白水平。此外，研究揭示了同一信号通路的分子——磷酸化cJun和磷酸化JNK——对细胞培养密度的相反依赖性。脱离条件下ERK激活的差异表明，在失巢凋亡过程中针对促存活激酶的靶向在不同细胞中应该是不同的。此外，细胞中JNK激活的结果可能取决于cJun的量，而cJun的量由细胞间接触决定。
