Genome-wide analysis of gene expression in jnk1-/-jnk2-/- immortalized mouse embryonic fibroblasts compared to wildtype counterparts co-cultivated with differentiating keratinocytes

Analysis of JNK-dependent fibroblast-derived soluble factors at gene expression level. The hypothesis tested in the present study was that loss of c-Jun N-terminal kinases 1 and 2, JNK1 and JNK2, in MEFs causes a strong alteration of the gene expression program coding for soluble factors, which promote an efficient keratinocyte differentiation. Results provide important information of the repertoire of fibroblast transcripts encoding secreted proteins, which is severely disarranged upon loss of JNK activity under the in vitro co-culture conditions applied. In vitro transwell co-culture experiments were performed using jnk1-/-jnk2-/- or wildtype immortalized mouse embryonic fibroblasts (MEFs) and differentiating primary normal human epidermal keratinocytes (NHEK) over a time course of 6 days. Every second day, fibroblast-loaded inserts were changed resulting in 3 triads (triads 1, 2, and 3). Total RNA was obtained from jnk1-/-jnk2-/- and wildtype immortalized mouse embryonic fibroblasts (MEFs) prior to co-cultivation (day 0) and of each triad 1, 2, or 3.

本研究针对基因表达层面上依赖于c-Jun氨基末端激酶（c-Jun N-terminal kinases, JNK）的成纤维细胞源性可溶性因子开展分析。本研究验证的核心假说为：在小鼠胚胎成纤维细胞（mouse embryonic fibroblasts, MEFs）中敲除c-Jun氨基末端激酶1与2（即JNK1与JNK2），会显著改变编码可溶性因子的基因表达程序，此类可溶性因子可有效促进角质形成细胞分化。本研究结果为解析编码分泌蛋白的成纤维细胞转录本谱系提供了重要依据；在本研究采用的体外共培养条件下，JNK活性缺失会导致该转录本谱系发生严重紊乱。本研究采用Transwell体外共培养体系，以JNK1与JNK2双敲除（jnk1-/-jnk2-/-）或野生型永生化小鼠胚胎成纤维细胞（MEFs），与处于分化阶段的原代正常人表皮角质形成细胞（normal human epidermal keratinocytes, NHEK）进行为期6天的共培养并设置完整时间梯度。每间隔2日更换一次搭载成纤维细胞的小室，最终得到3组独立生物学重复（分别编号为重复1、2与3）。分别于共培养起始前（第0天），以及3组生物学重复的各对应时间节点，从JNK1与JNK2双敲除及野生型永生化小鼠胚胎成纤维细胞中提取总RNA。