Lysophosphatidic acid induces GATA4 expression in the P19 stem cell line through activation of NF-κB.

A Awachat, S Thakur, A Baydoun. University of Hertfordshire, School of Life Sciences, AL10 9AB, UK

Lysophosphatidic acid (LPA) is an endogenous bioactive molecule with the potential of regulating stem cell differentiation through mechanisms which are as yet poorly defined. In particular, the transcriptional regulation of the process by LPA is unclear. Furthermore, there is currently no data to suggest whether ubiquitous transcription factors such as NF-κB play a role. To address this we have initiated a programme of research which has demonstrated that LPA induces the murine P19 stem cell line to differentiate into cardiomyocytes predominantly through activation of LPA receptor 4 and partially through LPA receptors 1 and 3. We now report that coupled to receptor activation is the induction of the cardiac specific transcription factor GATA4, through activation of NF-κB.

All experiments were carried out in P19 cells cultured in α-Minimal Essential Media (α-MEM) supplemented with 10% foetal bovine serum plus 1% penicillin and streptomycin. To initiate differentiation into cardiomyocytes, cells were trypsinised and allowed to aggregate into embryoid bodies (EBs) in sterile Petri dishes over 4 days in the presence of LPA at either 0.05 µM - 10 µM or at 5 µM. Controls were incubated in medium alone. The selective NF-κB inhibitor CAY10470 (0.001 - 0.1 nM) was added to cells 30 minutes prior to LPA. The EBs were subsequently transferred into 6-well plates and cultured in a monolayer for 6 days. Lysates were generated and subjected to western blotting for the expression of cardiac-specific myosin light chain-1v (MLC-1v). Total RNA was isolated from control and LPA treated cells and subjected to RT-PCR for GATA4 expression using the following primer sequence: forward - ACG GGC CAA CCC TGG AAG ACA; reverse - TGT CCC GTC CCA TCT CGC CT. Statistical analysis was carried out using a one-way ANOVA followed by the Bonferroni test in SPSS version 19.0. Statistical significance was set at p<0.01.

Incubation of cells with LPA often resulted in beating clusters within monolayers and in the expression of MLC-1v. The latter increased concentration-dependently in response to LPA, reaching a peak with 1 µM and plateauing thereafter. More importantly, the differentiation was significantly inhibited by CAY10470 which at the non-cytotoxic concentration of 0.01 nM inhibited MLC-1v expression by 81.3±4.7% (n=3; p<0.01). In parallel studies, LPA induced GATA4 in a time-dependent manner with levels of the transcript increasing by 1.2±0.2 fold above basal (n=3; p<0.001) half an hour after exposure to LPA and reached a peak at 2 hours, with the increase at the latter time point being 2.1±0.3 fold above basal (n=3; p<0.01). Pre-treating cells with 0.01 nM CAY10470 inhibited LPA induced GATA4 expression by 51.5±2.6% (n=3; p<0.01).

The current results confirm LPA to be an important endogenous biolipid activator of stem cell differentiation into cardiomyocytes. Furthermore, the data suggests that this action of LPA is critically dependent on the activation of NF-κB which precedes the induction of GATA4 expression. This, to our knowledge, is the first report to demonstrate the link between NF-κB, GATA4 and the differentiation of stem cells into cardiomyocytes.