Endogenous leukemia inhibitory factor protects photoreceptor cells against light-induced degeneration

Abstract

Purpose: Expression of leukemia inhibitory factor (LIF) by a subset of Müller glia cells has recently been implicated in an endogenous survival response to photoreceptor injury in a model of inherited retinal degeneration. To investigate whether such a LIF-controlled survival pathway might be commonly induced upon photoreceptor injury independently of the nature of the toxic stimulus, we analyzed the role of LIF during light-induced retinal degeneration.

Methods: Lif(+/-) and Lif(-/-) mice were exposed to 15,000 lx of white light for 2 h. Retinal morphology and rhodopsin content were analyzed nine days after light exposure. Gene expression studies were done using real-time PCR. Protein levels were determined by western blotting using specific antibodies.

Results: A lack of LIF reduced survival of photoreceptor cells after light exposure. In the absence of LIF several genes encoding molecules involved in the Janus kinase/signal transducer and activator of transcription (Jak/STAT) signaling pathway were not activated after light exposure. Presence or absence of LIF did not affect AKT (also known as protein kinase B, PKB) signaling and had only a mild effect on extracellular regulated kinase (ERK) phosphorylation. Stress-induced glial fibrillary acidic protein (GFAP) induction was minimal in the absence of LIF.

Conclusions: Our results suggest that increased retinal expression of LIF is a general response to photoreceptor injury. Independent of the nature of the toxic insult (gene mutation, light), LIF may activate an endogenous rescue pathway that protects viable photoreceptor cells, leading to an increased photoreceptor survival in the stressed retina. This defense system may depend on the Jak/STAT pathway and may involve endothelin 2 (EDN2) but not (or only minimally) AKT and ERK1,2 signaling.

Figure 1

Lack of LIF increases light-induced photoreceptor degeneration. A: Retinal morphology of Lif^+/– and Lif^–/– mice was analyzed before (upper panels) or at 9 days after exposure to 15,000 lx of white light for 2 h (lower panels). Fewer photoreceptors survived light exposure in the lower central retina (the most affected region) in the absence of LIF in Lif^–/– mice. Shown are representative sections of at least three animals. B: Rhodopsin levels were determined spectrophotometrically at 9 days after light exposure as a quantitative assessment of surviving rod photoreceptors in the whole retina. Rhodopsin levels after light exposure were expressed relatively to the respective dark controls, which were set to 100%. Note that values represent the average rhodopsin content of the whole retina, whereas the morphological pictures (A) show only the most affected areas. Abbreviations: retinal pigment epithelium (RPE); rod outer segments (ROS); rod inner segments (RIS); outer nuclear layer (ONL); inner nuclear layer (INL). Number of animals (N) is indicated for each group. The asterisk (*) indicates a p value of 0.0164 as calculated by a two-tailed t-test.

Figure 2

Lack of LIF prevents activation of STAT3 signaling. Relative levels of Lif, Clc, Stat3, and Socs3 mRNAs (as indicated) were analyzed by real-time PCR in retinas of Lif^+/– and Lif^–/– mice before (controls) or at 12 h after exposure to 15,000 lx of white light for 2 h. RNA levels were expressed relative to levels in Lif^+/– controls, which were set to 1. β-actin served as reference. Shown are means±SD of n=3.

Figure 3

Lack of LIF alters the protein response pattern after light exposure. Levels of proteins (as indicated) were analyzed by western blotting in retinas of Lif^+/– and Lif^–/– mice before (controls, C) or at 12 h after exposure to 15,000 lx of white light for 2 h (L). Shown are representative blots of n=3.

Figure 4

Lack of LIF alters gene expression after light exposure. Relative levels of mRNAs (as indicated) were analyzed by real-time PCR in retinas of Lif^+/– and Lif^–/– mice before (controls) or at 12 h after exposure to 15,000 lx of white light for 2 h. RNA levels were expressed relative to levels in Lif^+/– controls, which were set to 1. β-actin served as reference gene for the relative quantification. Bars show mean values±SD (n=3).