Figures and data
Metabolomic screening identifies LPC-16:0 as a pannexin agonist.
a Schematic of the metabolite screen. Organic extracts of mouse liver tissues were fractionated via reverse-phase chromatography and assessed for their ability to stimulate Panx1+GS or Panx2 using whole-cell patch-clamp. Active fractions were analyzed by HPLC-HRMS. b Extracted ion chromatograms for fractions #5-8 from the second round of Panx1 activity-guided fractionation. c Chemical structure of LPC-16:0. d Representative whole-cell patch-clamp traces. e Quantification of peak current densities triggered by LPC-16:0. Wildtype pannexins were expressed in indicated cells. f ATP release induced by application of LPC-16:0 to Panx1-expressing GnTI- (left; 10 μM at 3 min) and Panx2-expressing HEK293 (right; 30 μM at 3 min) cells. Data are expressed as percent of total ATP released upon membrane solubilization. N=6-8. P values were calculated using unpaired t-test with unequal variances. An asterisk denotes P< 0.01. Error bars represent s.e.m. g Representative whole cell currents of Panx1 and Panx2 stimulated by LPC-16:0 in buffers containing different anions and cations. h Quantification of peak current densities in different buffers. Voltage-clamp recordings were performed at -60 mV. Blue bars indicate application of LPC-16:0 (7 μM), and orange bars indicate application of carbenoxolone (50 μM). N=5-20. One-way ANOVA followed by Dunnett’s test was used to assess statistical significance. V indicates the vector control.
Cellular mVenus-quench assays reveal a series of lysophospholipids as pannexin agonists.
a Cartoon illustrating the principle of the mVenus quench assay. b-e Representative traces (b for Panx1 and d for Panx2) and quantification of initial mVenus quenching rates (c for Panx1 and e for Panx2). LPC-16:0 (30 μM) was applied with or without CBX (50 μM), and the maximum mVenus quenching was measured after cell solubilization with 1% Triton-X100. N=8-14. P values were calculated using unpaired t-test with unequal variances. f and g Initial mVenus quenching rates of Panx1 expressed in GnTI- cells (f) and Panx2 expressed in HEK293 cells (g). Pannexin activation was measured following addition of 60 μM sn-1 LPCs (LPC-12:0-20:0), sn-2 LPC (LPC2-16:0), a monounsaturated sn-1 (LPC-18:1), or other sn-1 lysophospholipids with different headgroups (LPA-16:0, LPI-16:0, LPE-16:0, and SPC-18:1). N=4-14. P values were calculated using one-way ANOVA followed by Dunnett’s t-test. Asterisks denote P<0.01. Error bars represent s.e.m.
Select released signaling metabolites following LPC-16:0 stimulation of Panx1.
a Metabolites enriched in the conditioned media (CM) of cells expressing Panx1 treated with LPC-16:0 that were previously identified as Panx1 permeant using apoptotic T cells38. b Comparative analysis by HPLC-HRMS of CM from cells expressing Panx1+LPC-16:0 versus CM from vector-expressing cells treated with LPC-16:0. Volcano plot depicts subset of features detected in negative ion mode. Unadjusted P-values calculated by unpaired, two-sided t-test (see Methods for details). c Additional metabolite discovered in this study with known roles in immunomodulation. Panx1 was expressed in GnTI- cells and the released metabolites were analyzed 45 min after the stimulation with LPC-16:0 (10 μM). N=4. P values were calculated using one-way ANOVA. Asterisks denote P<0.05. Error bars represent s.e.m.
Functional reconstitution of Panx1 confirms direct activation by LPC-16:0.
a Schematic representation of YO-PRO-1 uptake assay. b Relative YO-PRO-1 fluorescence triggered by LPC-16:0 (100 μM) with or without CBX (50 μM). Asterisks indicate P< 0.01 using unpaired t-test. N=6-15. c Dose-response profile of Panx1 treated with LPC-16:0. Dose responses were fitted with the Hill equation, and the EC50 values are indicated. N=10-13. Error bars represent s.e.m.
Pannexins mediate lysophospholipid signaling.
a Schematic illustrating lysophospholipid signaling. b-e Pannexin activities triggered by extracellularly applied stimuli. Normalized initial mVenus quenching rates are shown for PLA1 (b), sPLA2 (c), and major metabolic products of PLA2 (d), mastoparan with or without PLA2 inhibitors (QCT and CPZ), a Src kinase inhibitor (PP2), or a caspase inhibitor (ZVAD)(e). V indicates the vector control. Panx1 was expressed in GnTI- cells and Panx2 was expressed in HEK293 cells. N=4-14. f Panx1-dependent mVenus quenching induced by synovial fluids obtained from canine patients with mild (-) or moderate/severe (+/++) pain. The activity of each fraction was normalized to the effect of LPC-16:0 (30 μM). Each point represents a different patient. N=10-12. P values were calculated using unpaired Student’s t-test with unequal variances (b, c, and f) or using one-way ANOVA, followed by Dunnett’s t-test (d and e). Asterisks indicate P<0.01. Error bars represent s.e.m.
Knockdown of endogenous Panx1 reduces LPC-16:0-triggered release of cleaved IL-1β.
a Panx1 protein expression levels in PMA-differentiated/LPS-primed THP-1 control (shRNA-empty vector) cells or two different shPanx1 knockdown (KD) lines. b Cleaved IL-1β released into culture supernatant following stimulation with 50 µM LPC-16:0 for 1.5 h. A representative blot for control and Panx1 knockdown cells is shown. c Densitometry (ImageJ) was used to quantify the LPC-16:0-induced release of cleaved IL-1β from control and Panx1 KD-b cells relative to the release from control cells only primed with LPS. d Relative amount of the retained pro-IL-1β normalized to total protein in the cells. P values were calculated using unpaired Student’s t-test. * indicates P<0.0032 and ** indicates P<0.0021. Error bars represent s.d.
Schematic summary of Panx1-mediated signaling.
a Panx1 activation in dying cells. In dying cells, the C-terminal tails of Panx1 are cleaved by caspases, initiating an irreversible mode of Panx1 activation. This leads to the release of "find-me" signals, such as ATP, which play a role in attracting phagocytic cells to the site of cell death. b Panx1 activation in living cells. In living cells, activation of membrane receptors such as NMDA, P2X7, TNF-α, and α1-adrenergic receptors stimulates the production of lysophospholipids via cytoplasmic phospholipase A (PLA) enzymes. Lysophospholipids are abundant in extracellular microvesicles and oxidized low-density lipoproteins (oxLDLs). They are also produced by secreted PLAs during pathological conditions, including atherosclerosis and joint or metabolic diseases. These lysophospholipids reversibly activate Panx1, leading to the release of signaling molecules crucial for inflammation and pain.
