Biased signalling properties of LPA molecular species in human lung fibroblasts

A Sattikar, EM Rosethorne, TC Kent. Novartis Institutes for Biomedical Research, Horsham, UK

Lysophosphatidic acid (LPA) is a potent endogenous signalling molecule involved in a number of cellular processes including cell proliferation, apoptosis and migration. Several molecular species of LPA exist1,2 but little is known about their signalling properties. The aim of this project was to investigate the signalling profiles of different LPA molecular species in two assay formats using primary human lung fibroblasts (HLFs) which express a high level of endogenous LPA1 receptors (in house data). We compared the responses of eleven LPA molecular species in the HLFs in a calcium flux assay and an ERK1/2 phosphorylation (pERK1/2) imaging assay. HLFs (8000 cells.well-1) were plated in 384-well, black clear bottom plates and incubated overnight at 37ºC/5% CO2. Following a 16h serum-starvation step, Fluo-4 calcium dye was added to cells and incubated for 1h. Agonist was added and calcium flux measured using the fluorescence reader on the FDSS7000. Cells were then fixed with 4% formaldehyde and standard IFC protocol was followed under permeabilised conditions using a pERK1/2 specific antibody and Hoechst to stain nuclei. Cells were imaged using an InCell2000 using DAPI (nuclei) and FITC (pERK) filters. Images were subsequently analysed for cell intensity. Four of the molecular species (16:0, 17:0, 18:2 & C18:1 LPA) had significant differences in their EC50 & efficacy values between the two assays (p<0.05, un-paired t-test). These four molecular species & 18:1 LPA (control) were completely inhibited by the LPA1 antagonist AM966 in both assays, demonstrating the responses are solely due to LPA1 receptor activation in HLFs. As the LPA1 receptor is known to signal via Gi/o, Gq/11, G12/13 pathways1, we investigated the effects of Pertussis toxin (PTx) treatment on the response of the selected LPA molecular species. HLFs were treated with 150ng/ml PTx during the serum starvation period. As shown in Table 1, the concentration response of the four selected LPA molecular species is only partially inhibited in the calcium assay, but it is completely inhibited in the pERK1/2 assay.

Table 1 Potency of LPA molecular species in calcium flux & pERK1/2 assay, ± PTx treatment (mean ± SEM; n=3)

Interestingly, of the four selected ligands, for all the molecular species except 17:0, the pERK response was less potent than the calcium response. This suggests there may be some bias in the way the different molecular species signal between the two pathways. In summary, we have characterized the calcium and pERK responses downstream of the LPA1 receptor for a number of different LPA molecular species, and there appears to be a potential signalling bias between these pathways in the HLFs. The calcium flux response appears to be both Gi/o & Gq/11 dependent, while the pERK1/2 response appears to be completely Gi/o dependent for the LPA molecular species tested in the HLFs.

1. Tigyi G, Br J Pharmacol 161:241, 2010

2. Yanagida K & Ishii S, Journal of Biochemistry 150:223, 2011