Optical Control of Phosphatidic Acid Signaling

Abstract

Phosphatidic acids (PAs) are glycerophospholipids that regulate key cell signaling pathways governing cell growth and proliferation, including the mTOR and Hippo pathways. Their acyl chains vary in tail length and degree of saturation, leading to marked differences in the signaling functions of different PA species. For example, in mTOR signaling, saturated forms of PA are inhibitory, whereas unsaturated forms are activating. To enable rapid control over PA signaling, we describe here the development of photoswitchable analogues of PA, termed AzoPA and dAzoPA, that contain azobenzene groups in one or both lipid tails, respectively. These photolipids enable optical control of their tail structure and can be reversibly switched between a straight trans form and a relatively bent cis form. We found that cis-dAzoPA selectively activates mTOR signaling, mimicking the bioactivity of unsaturated forms of PA. Further, in the context of Hippo signaling, whose growth-suppressing activity is blocked by PA, we found that the cis forms of both AzoPA and dAzoPA selectively inhibit this pathway. Collectively, these photoswitchable PA analogues enable optical control of mTOR and Hippo signaling, and we envision future applications of these probes to dissect the pleiotropic effects of physiological and pathological PA signaling.

Figure 1

Design and synthesis of the photoswitchable PA variants AzoPA and dAzoPA. (A) Chemical structures of PA and previously developed photoswitchable lipids, including PhoDAG, OptoDArG, AzoLPA, AzoPC, FAAzo4, and PhotoS1P. (B) Photoisomerization of an azobenzene-containing photolipid (shown: FAAzo4). (C) Synthesis of AzoPA from PhoDAG-1. (D) Synthesis of dAzoPA from a protected form of AzoLPA.

Figure 2

Photophysical characterization of AzoPA (A,B) and dAzoPA (C,D). (A,C) UV–vis spectra of AzoPA (A) and dAzoPA (C) in the dark-adapted (black, trans), 365-nm-adapted (gray, cis), and 460-nm-adapted (blue, trans) photostationary states (50 μM, DMSO). (B,D) Reversible cycling between photoisomers with alternating illumination at 365/460 nm for AzoPA (B) and dAzoPA (D). (E) Nuclear magnetic resonance (NMR) study (performed in DMSO-d₆) of the aromatic region of dAzoPA before and after irradiation with 365 or 460 nm and quantification of the trans form relative to the cis form.

Figure 3

Characterization of cellular uptake and metabolism of AzoPA and dAzoPA. (A) Schematic diagram of mammalian PA metabolism. CDP: Cytidine diphosphate, CPT: cholinephosphotransferase, DAGK: diacylglycerol kinase, LPAAT: lysophosphatidic acid acyl transferase, PAP: phosphatidic acid phosphatase, PLA: phospholipase A, PLD: phospholipase D. (B,C) HPLC trace of lipid extracts from NIH 3T3 cells treated with 20 μM AzoPA (B) or dAzoPA (C) in the light-induced cis form (red) or dark-adapted trans form (gray) for 1 h. Just prior to the analysis, all samples were irradiated with 460 nm light to switch azobenzene moieties into the trans forms and normalize the absorption. (D) Observed elution times of standard photolipid molecules on HPLC.

Figure 4

Optical control of mTOR signaling in NIH 3T3 cells. (A) Schematic depiction of the optical control of mTOR activation induced by PA. (B) Western blot analysis of NIH 3T3 cells treated with BSA (4 mg/mL) or indicated lipids (100 μM) for 1 h, probing for p-S6K, S6K, or actin as a loading control. (C,D) Western blot analysis of cells treated with AzoPA (C) or dAzoPA (D) in their light-induced cis form or dark-adapted trans form at different concentrations (0–20 μM) for 30 min. (E,F) Quantification of p-S6K levels in (C) and (D). Horizontal bars indicate mean (n = 6), and vertical error bars indicate standard deviation. Statistical significance was calculated using an unpaired two-tailed Student’s t test. *, p < 0.05; **, p < 0.01; ***, p < 0.001. (G) Confocal microscopy images of NIH 3T3 cells treated with the indicated lipids (5 μM) for 30 min and immunostained for p-S6. Green, p-S6; blue, DAPI (nuclei). Scale bars: 50 μm. (H) Quantification of immunofluorescence analysis. The plots show average p-S6 intensity per cell in each image. Black horizontal bars indicate mean (n = 10–14), and vertical error bars indicate standard deviation. Statistical significance was calculated using one-way ANOVA, followed by Tukey’s HSD test.

Figure 5

Optical control of Hippo signaling in NIH 3T3 cells. (A) Schematic depiction of the optical control of Hippo deactivation induced by PA. (B) Confocal microscopy images of NIH 3T3 cells treated with BSA (4 mg/mL) or the indicated lipids (50 μM) for 1 h and immunostained for YAP. (C) Quantification of nuclear YAP levels in (B). The plots show mean nuclear YAP intensity in each cell. Black horizontal bars indicate mean (n = 645), and vertical error bars indicate standard deviation. (D) Confocal microscopy images of NIH 3T3 cells treated with BSA (4 mg/mL) or indicated lipids (10 μM) for 1 h with or without 365 nm light (5 ms every 15 s) and immunostained for YAP. (E,F) Quantification of nuclear YAP levels in (C) and (D). The plots show mean nuclear YAP intensity in each cell. Black horizontal bars indicate mean (n = 342 (C) and 465 (D)), and vertical error bars indicate standard deviation. Statistical significance was calculated using one-way ANOVA, followed by Tukey’s HSD test. Green, YAP; blue, DAPI (nuclei). Scale bars: 50 μm.

Figure 6

Optical control of Hippo signaling in NIH 3T3 cells with the inhibition of LPA receptor signaling. (A,B) Confocal microscopy images of NIH 3T3 cells treated with AzoLPA (100 nM) or dAzoPA (10 μM) for 1 h with or without 365 nm light and with or without LPA receptor inhibitor (LPAi: Ki16425, 10 μM) and immunostained for YAP. Green, YAP; blue, DAPI (nuclei). Scale bars: 50 μm. (C,D) Quantification of nuclear YAP levels in (A). The plots show mean nuclear YAP intensity in each cell. Black horizontal bars indicate mean (n = 540 (C) and 811 (D)), and vertical error bars indicate standard deviation. Statistical significance was calculated using one-way ANOVA, followed by Tukey’s HSD test.