Insulin-like growth factor-1 stimulates retinal cell proliferation via activation of multiple signaling pathways

Abstract

Insulin-like growth factor-1 (IGF-1) plays critical roles in the development of the central nervous system (CNS), including the retina, regulating cell proliferation, differentiation, and survival. Here, we investigated the role of IGF-1 on retinal cell proliferation using primary cultures from rat neural retina. Our data show that IGF-1 stimulates retinal cell proliferation and regulates the expression of neurotrophic factors, such as interleukin-4 and brain-derived neurotrophic factor. In addition, our results indicates that IGF-1-induced retinal cell proliferation requires activation of multiple signaling pathways, including phosphatidylinositol 3-kinase, protein kinase Src, phospholipase-C, protein kinase C delta, and mitogen-activated protein kinase pathways. We further show that activation of matrix metalloproteinases and epidermal growth factor receptor is also necessary for IGF-1 enhancing retinal cell proliferation. Overall, these results unveil potential mechanisms by which IGF-1 ensures retinal cell proliferation and support the notion that manipulation of IGF-1 signaling may be beneficial in CNS disorders associated with abnormal cell proliferation.

Keywords: Cell proliferation; Central nervous system; Epidermal growth factor; Insulin-like growth factor-1; Retina; Signaling pathway.

Graphical abstract

Fig. 1

IGF-1 stimulates transient cell proliferation in retinal cell cultures. (A,B) Primary cultures from rat neural retinas were treated with IGF-1 (0.1–100 ng/mL) for 48h, or (C,D) IGF-1 (10 ng/mL) for the indicated time intervals. Plots represent mean (±SEM) [³H]-thymidine uptake expressed as percentage of control group (set on 100%; white bars), with sample size provided within each bar. Data were analyzed by one-way ANOVA followed by Holm-Sidak's test (**p < 0.01, ***p < 0.001). EC₅₀ value was calculated in GraphPad Prism 8 using a nonlinear curve fit ([IGF-1] vs. normalized response - method of least squares with variable slope). (E) Representative images (bright-field) of retinal cells treated with IGF-1 (10 ng/mL) for 24h, 48h or 144h (scale bar = 20 μm).

Fig. 2

IGF-1 regulates expression of neurotrophic molecules in retinal cell cultures. (A) Primary retinal cultures were treated with an inhibitor of vesicular protein transport (brefeldin A; 30 ng/mL) or IGF-1 (10 ng/mL) for 24h, and cell proliferation was assessed by [³H]-thymidine incorporation assay. Plots represent mean (±SEM) [³H]-thymidine uptake expressed as percentage of control group (set on 100%; white bars), with sample size provided within each bar. Additionally, (B-D) cultured retinal cells were exposed to IGF-1 (10 ng/mL) for the indicated time periods for assessing IL-4 and BDNF levels by immunoblotting. Plots represent mean (±SEM) protein optical density (normalized to β-actin) expressed as fold change relative to control cultures (represented as a dashed line), with N = 3–5 experiments using independent retinal cultures. Data were analyzed by (A) one-way ANOVA followed by Holm-Sidak's test or (C,D) two-tailed paired Student's t-test (*p < 0.05, **p < 0.01, ***p < 0.001).

Fig. 3

IGF-1 requires receptor endocytosis and activity of multiple kinases to enhance retinal cell proliferation. Primary retinal cultures were treated with IGF-1 (10 ng/mL) or following drugs for 24h: (A) MDC (endocytosis inhibitor; 0.75 nM), (B) PD98059 (MAPK/ERK inhibitor; 37.5 μM), (C) JNKi (JNK inhibitor; 0.5 μM), (D) SB202190 (p38-MAPK inhibitor; 20 μM), (E) PP1 (Src inhibitor; 1 μM), (F) LY294002 (PI3K inhibitor; 25 μM), (G) chelerythrine chloride (pan-PKC inhibitor; 1.25 μM), (H) rottlerin (PKCδ inhibitor; 2 μM), or (I) U73122 (phospholipase-C inhibitor; 4 μM). Plots represent mean (±SEM) [³H]-thymidine uptake expressed as percentage of control group (set on 100%; white bars), with sample size provided within each bar. Data were analyzed by one-way ANOVA followed by Holm-Sidak's test (**p < 0.01, ***p < 0.001).

Fig. 4

Activation of EGFR mediates IGF-1-stimulated retinal cell proliferation. Primary retinal cultures were treated with IGF-1 (10 ng/mL), (A) AG-1478 (EGFR inhibitor; 2.5 μM), (B) MMP9i (MMP9 inhibitor; 20 μM), or (C) EGF (0.1 ng/mL) for 24h. Additionally, (D) cultured retinal cells were treated with IGF-1 for 24h, after which media were removed and novel medium containing EGF was applied for additional 24h, in parallel with primary retinal cultures exposed only to IGF-1 or EGF for 48h. Control cultures had their medium replaced by a novel medium after 24h (M199 → M199), which did not impact [³H]-thymidine uptake compared to control cultures without medium change (M199 48h). Plots represent mean (±SEM) [³H]-thymidine uptake expressed as percentage of control group (set on 100%; white bars), with sample size provided within each bar. Data were analyzed by one-way ANOVA followed by Holm-Sidak's test (**p < 0.01, ***p < 0.001).

Fig. 5

A simplified model of cellular signaling pathways underlying IGF-1-induced cell proliferation in the retina. IGF-1 activates PI3K/AKT pathway in retinal cells and increases protein levels and release of IL-4, which stimulates IGF-1R/PI3K/AKT pathway. Inhibition of protein vesicular secretion or PI3K activity prevents the mitogenic effect of IGF-1. IGF-1-induced retinal cell proliferation also requires activation of PKCδ, PLC, JNK, and MAPK/ERK (Ras-Raf-MEK-ERK) pathways. EGF is another neurotrophic factor that induces retinal cell proliferation by activating MAPK/ERK pathway. EGFR activation is also necessary for IGF-1 ensuring retinal cell proliferation. Furthermore, IGF-1-induced retinal cell proliferation requires activation of both MMP9 and Src. Although it has been reported that IGF-1 can transactivate EGFR in different cell types via MMP-mediated proteolytic release of EGF-like ligands or via Src activation, further studies are needed to investigate whether these mechanisms of EGFR transactivation also happen in retinal cells.