Lipoxin B₄ Regulates Pro-Survival Kinase Signaling in the Mouse Retina and Human Astrocytes

Abstract

Purpose : To investigate the intracellular signaling kinases regulated by Lipoxin B₄ (LXB₄) in healthy mouse retinas and astrocytes.LXB₄ is neuroprotective and is produced under homeostatic conditions by astrocytes in the retina and directly acts on retinal ganglion cells (RGCs). We recently identified that LXB₄ reverses astrocyte reactivity in response to cytokines and the chronic mild ocular hypertension model.

Methods : LXB₄ regulation of kinase activity in vivo was investigated through intravitreal injections in the mouse retina. Primary cultures of human brain astrocytes were treated with IL-1α, TNF-α, and C1q cytokines for 24 hours to induce astrocyte reactivity. Astrocytes were treated with LXB₄ (1 mM) or vehicle for 20 mins before adding cytokines. Astrocytes and retinas were collected in a lysis buffer for kinase screening assays. Comprehensive kinase profiling was performed using a proteome profiler phospho-kinase array kit, enabling the detection of the relative phosphorylation of 37 human protein kinases.

Results : LXB₄ treatment elevated the phosphorylation of CREB (S133) by 80% and ERK (T202/Y204, T185/Y187) while reducing the phosphorylation of STAT3 (S727) compared with vehicle-treated mouse retinas (p<0.05, n=3). Control astrocytes treated with LXB₄ showed activation of beta-catenin and p70 S6 kinase (T421/S424), while reduced phosphorylation of p53 (S15), p53 (S392), RSK-1/2 (S221/S227), and STAT1 (Y701) confirming potent bioactivity in astrocytes (p<0.05, n=4). Among the 37 kinases analyzed, cytokine treatment induced elevated phosphorylation of p70 S6 kinase (S389) and STAT1 (Y701), suggesting potential signaling mechanisms activated in reactive astrocytes. Upon treatment with LXB₄, phosphorylation levels of STAT1 were significantly reduced by more than 70% in reactive astrocytes (n=3, p<0.05).

Conclusions : CREB, recognized as a downstream target of ERK and Akt, significantly contributes to promoting the survival of RGCs. Activating CREB (S133) by LXB₄ may represent a potential neuroprotective signaling mechanism in mouse retinas. The reduction in STAT1 (Y701) phosphorylation by LXB₄ in reactive astrocytes is intriguing, given that activation of STAT1 at Y701 residue is known to be pro-apoptotic and implicated in glaucomatous neurodegeneration.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.