Regulation of Oct4 expression by Jun-N terminal kinase/cJun signaling in murine embryonic stem cells

Oct4 is a POU class V transcription factor that functions to maintain the pluripotent state of embryonic stem cells by activating or repressing the transcription of hundreds of target genes (Boyer et al. 2005). Aberrant expression of Oct4 has been reported in a number of cancers, suggesting that it may also play a role in cellular transformation (Tai et al. 2005). This study examines the role of the proto-oncogene cJun and its upstream kinase Jun N terminal kinase (JNK) in the regulation of Oct4 expression and the pluripotent state. Our laboratory identified a putative AP-1 binding site approximately 2500 bp upstream of the Oct4A transcription start site. We modulated the cJun/JNK pathway in two murine embryonic stem cell lines through chemical treatments and transient transfection of wild type and mutant GFP cJun constructs. Our results show mESCs overexpressing cJun forms or treated with 10 ng/ml, 50 ng/ml anisomycin and 50 μM JNK inhibitor SP600125 have an increase in the Oct4A isoform by immunocytochemistry and Western blot analysis, but not the total amount of Oct4. The potency of these cells is also affected. Increased JNK/cJun activity increased in alkaline phosphatase activity but resulted in a significant reduction of pancreatic islet like cluster size, number, insulin expression and the neuronal network between clusters. Loss of JNK/cJun activity had the opposite result: transfection of the transcriptionally inactive cJun mutant L40/42A or treatment with SP600125 resulted in increased number, size and neuronal network formation among the pancreatic islet like clusters. JNK modulation by chemical treatment also affected the formation of cardiomyocyte beating clusters. These results indicate the importance of the regulation of JNK/cJun pathway in stem cell potency, potentially through changes in the expression level of Oct4A. Future studies exploring the role of Oct4A in pluripotency and JNK/cJun activity in early embryonic development could provide insight into the role of this isoform in cellular transformation.