Lysophosphatidic acid induced lineage commitment of stem cells to cardiomyocytes is critically dependent on the sequential activation of JNK2 and JNK1 in the P19 teratocarcinoma stem cell line

G Maan, S Thakur, A Baydoun. University of Hertfordshire, Hatfield, UK

Introduction: The signalling mechanisms that regulate stem cell (SC) differentiation are poorly understood and often the generation of cardiomyocytes (CMs) from SCs has been achieved using chemical agents with limited clinical applications. We have recently demonstrated that the endogenous biolipid, lysophosphatidic acid (LPA), has the potential to effectively induce differentiation of the P19 teratocarcinoma SC line into CMs. As part of our programme of studies, we have therefore examined whether LPA utilizes the c-Jun N-terminal kinases (JNK) to mediate its effects. The JNKs are a subgroup of the mitogen activated protein kinases that regulate survival, apoptosis, differentiation, and inflammatory responses in various cell types and may therefore be a key signalling pathway that lead to lineage commitment of SCs when exposed to LPA.

Methods: All experiments were carried out in the murine P19 teratocarcinoma stem cell line cultured in α- minimal essential medium (MEM) supplemented with 10% foetal bovine serum and 1% penicillin-streptomycin. Cells were allowed to aggregate into embryoid bodies in the presence of 5µM LPA in sterile petri dishes over the course of 4 days to commence the differentiation process. When used, the JNK inhibitor SP600125 (10µM) was added 30 minutes prior to the LPA, while control cells were cultured without an inducing agent or inhibitor. Embryoid bodies were transferred into 6-well plates and cultured in a monolayer for 12 days. Lysates were collected on day 6 and 12 followed by western blotting probing for cardiac specific myosin light chain-1v (MLC-1v) and downstream targets of JNK. In parallel experiments, the widely used differentiating agent dimethyl sulfoxide (DMSO) was used at 1% as a positive control.

Results: Consistent with our previous findings, treatment of P19 cells with 5µM LPA resulted in their differentiation into CMs as confirmed by the detection of MLC-1v. The efficiency of differentiation achieved at this concentration of LPA was comparable to that seen with 1% DMSO. LPA induced the rapid phosphorylation of JNK2 which occurred within 1 minute of exposure of cells to LPA but was no longer detectable 24 hours after treatment. In contrast, the JNK1 isoform appeared to be phosphorylated 18 hours after exposure to LPA and was detectable in lysates from SC derived CMs at day 6. High levels of phosphorylated AP-1 subunits c-Jun and JunD were also detected in the LPA induced differentiated cells when compared to undifferentiated cells and the expression of both was abolished upon treatment with 10µM of SP600125 which also significantly reduced the MLC-1v expression.

Conclusion: Our current data confirm that LPA induces P19 SC differentiation into CMs as efficiently as 1% DMSO. More importantly, LPA appears to mediate its effects by utilising the JNK pathway which further regulates the AP-1 subunits c-Jun and JunD. The process may be sequential with JNK2 being transiently activated followed by the sustained phosphorylation of JNK1. The significance of this pattern of activation of the JNKs and the precise role of each isoform as well as those of the AP-1 subunits are currently being investigated.