Lysophosphatidic Acid and IL-6 Trans-signaling Interact via YAP/TAZ and STAT3 Signaling Pathways in Human Trabecular Meshwork Cells

Abstract

PURPOSE. Lysophosphatidic acid (LPA) and soluble interleukin-6 receptor (sIL6R) are elevated in primary open angle glaucoma (POAG). LPA and IL6 modulate in response to biomechanical stimuli and converge on similar fibrotic phenotypes. Thus, we determined whether LPA and IL6 trans-signaling (IL6/sIL6R) interact via Yes-associated protein (YAP)/Transcriptional coactivator with a PDZ-binding motif (TAZ) or Signal transducer and activator of transcription 3 (STAT3) pathways in human trabecular meshwork (hTM) cells. METHODS. Confluent primary hTM cells were serum starved for 24 hours, and treated with vehicle, LPA (20 μM), IL6 (100 ng/mL)/sIL6R (200 ng/mL), or both (LPA + IL6/sIL6R) for 24 hours, with or without a YAP inhibitor (verteporfin; 2 μM) or STAT3 inhibitor (2 μM). Expression of key receptors and ligands, signaling mediators, actomyosin machinery, cell contractility, and extracellular matrix (ECM) targets of both signaling pathways was determined by immunocytochemistry, RT-qPCR, and Western blotting. RESULTS. LPA and IL6 trans-signaling coupling overexpressed/activated receptors and ligands, glycoprotein-130, IL6, and autotaxin; signaling mediators, YAP, TAZ, Pan-TEAD, and phosphorylated STAT3 (pSTAT3); actomyosin and contractile machinery components, myosin light chain 2 (MLC2), phosphorylated MLC2, rho-associated protein kinase 1, filamentous actin, and α-smooth muscle actin; and fibrotic ECM proteins, collagen I and IV, fibronectin, laminin, cysteine-rich angiogenic inducer 61, and connective tissue growth factor in hTM cells; mostly beyond LPA or IL6 trans-signaling alone. Verteporfin inhibited YAP, TAZ, and pSTAT3, with concomitant abrogation of aforementioned fibrotic targets; the STAT3 inhibitor was only partially effective. CONCLUSIONS. These data suggest synergistic crosstalk between LPA and IL6 transsignaling, mediated by YAP, TAZ, and pSTAT3. By completely inhibiting these mediators, verteporfin may be more efficacious in ameliorating LPA and/or IL6 trans-signalinginduced ocular hypertensive phenotypes in hTM cells.

The lysophosphatidic acid axis in fibrosis: Implications for glaucoma

“…140 Pan-TEAD was markedly increased by a combination of LPA and IL-6, neither of which had an effect alone. 140 This suggests that there is crosstalk between the respective signalling pathways of LPA and IL-6. Furthermore, LPA treatment upregulated signal transducer and activator of transcription 3 which is involved in IL-6 signaling.…”

“…148 LPA treatment of TM cells activated YAP and TAZ via the LPA 1 and LPA 3 receptors, which in turn increased levels of CCN2, CYR61 and ECM proteins fibronectin, laminin, collagen I and α-SMA. 139,140 LPA, IL-6 or a combination of LPA and IL-6 all elevated YAP and TAZ levels in TM cells. 140 Pan-TEAD was markedly increased by a combination of LPA and IL-6, neither of which had an effect alone.…”

“…139,140 LPA, IL-6 or a combination of LPA and IL-6 all elevated YAP and TAZ levels in TM cells. 140 Pan-TEAD was markedly increased by a combination of LPA and IL-6, neither of which had an effect alone. 140 This suggests that there is crosstalk between the respective signalling pathways of LPA and IL-6.…”

“…139 LPA treatment of TM cells caused significant upregulation of LPA 1 and LPA 4 and downregulation of LPA 2 and LPA 6 . 140 Moreover, treatment of TM cells with IL-6 significantly increased LPA 6 . 140 Elevated IOP in glaucoma occurs as a result of increased resistance to AH outflow through the conventional pathway.…”

“…140 Moreover, treatment of TM cells with IL-6 significantly increased LPA 6 . 140 Elevated IOP in glaucoma occurs as a result of increased resistance to AH outflow through the conventional pathway. The regulation of AH outflow is complex, but involves mechanical stretch, cytoskeletal changes, the ECM, and locally acting mediators 141 including LPA (Figure 5).…”