Insulin-like growth factor-I-stimulated Akt phosphorylation and oligodendrocyte progenitor cell survival require cholesterol-enriched membranes

Abstract

Previously we showed that insulin-like growth factor-I (IGF-I) promotes sustained phosphorylation of Akt in oligodendrocyte progenitor cells (OPCs) and that Akt phosphorylation is required for survival of these cells. The direct mechanisms, however, by which IGF-I promotes Akt phosphorylation are currently undefined. Recently, cholesterol-enriched membranes (CEMs) have been implicated in regulation of growth factor-mediated activation of the PI3K/Akt pathway and survival of mature oligodendrocytes; however, less is know about their role in OPC survival. In the present study, we investigate the role of CEMs in IGF-I-mediated Akt phosphorylation and OPC survival. We report that acute disruption of membrane cholesterol with methyl-beta-cyclodextrin results in altered OPC morphology and inhibition of IGF-I-mediated Akt phosphorylation. We also report that long-term inhibition of cholesterol biosynthesis with 25-hydroxycholesterol blocks IGF-I stimulated Akt phosphorylation and cell survival. Moreover, we show that the PI3K regulatory subunit, p85, Akt, and the IGF-IR are sequestered within cholesterol-enriched fractions in steady-state stimulation of the IGF-IR and that phosphorylated Akt and IGF-IR are present in cholesterol-enriched fractions with IGF-I stimulation. Together, the results of these studies support a role for CEMs or "lipid rafts" in IGF-I-mediated Akt phosphorylation and provide a better understanding of the mechanisms by which IGF-I promotes OPC survival.

Fig. 1

Cholesterol depletion alters OPC membrane integrity. OPCs were treated with 5 mM MβCD for 30 min at 37°C and were visualized by phase-contrast microscopy. A: Untreated cells exhibited normal, continuous processes extending from the cell body. B: Treatment with MβCD resulted in punctate, discontinuous processes (arrowheads). C: Posttreatment with 200 μM cholesterol–MβCD complexes reversed the effects of MβCD. Insets show magnified intact (A,C) or disrupted (B) processes.

Fig. 2

Acute cholesterol depletion blocks IGF-I-mediated Akt phosphorylation but not IGF-IR phosphorylation. OPCs were treated for 30 min with 10 ng/m IGF-I (C, control; I, IGF-I; with or without pretreatment with MβCD). Cell lysates were processed for SDS-PAGE and Western blot analysis. Data represent mean ± SEM (n = 3). A–D: Treatment with IGF-I induced phosphorylation of Akt (A,B; P < 0.0001). Pretreatment with MβCD blocked IGF-I-mediated Akt phosphorylation (A,B). Cholesterol repletion reversed the effects of MβCD (A,B; P < 0.0001). MβCD pretreatment had no effect on IGF-I-mediated IGF-IR phosphorylation compared with untreated control cells (C,D; P < 0.003). E,F: The recovery of Akt phosphorylation was dependent on the dose of cholesterol. Incubation with 200 μM cholesterol induced an approximately threefold increase in IGF-I-mediated Akt phosphorylation (⋆ ⋆P < 0.008) compared with incubation with 100 μM cholesterol (E,F; ⋆P < 0.04).

Fig. 3

Inhibition of cholesterol biosynthesis blocks IGF-I-mediated sustained Akt phosphorylation. A–C: OPCs were treated with 10 ng/ml IGF-I in the presence (+) or absence (−) of 25-hydroxycholesterol (25-HC) for 24 or 30 hr in serum-free media. A: Representative Western blots showing levels of P-Akt, total Akt, cleaved caspase-3, and β-actin. B,C: Quantification of levels of Akt phosphorylation (B) and cleaved caspase-3 (E). Data represent the mean ± SEM from two experiments (n = 3 for t0; n = 5 for all other treatment groups). B: IGF-I stimulated the phosphorylation of Akt over control at 24 hr (⋆P = 0.02, 24− vs. t0; P = 0.06 30− vs. t0). Treatment with 25-HC completely prevented Akt phosphorylation at 24 hr (⋆⋆P = 0.001 24+ vs. 24−). C: Treatment of OPCs with 25-HC resulted in a significant increase in cleaved caspase-3 at both 24 and 30 hr (⋆P = 0.03 24+ vs. 24−; P = 0.04 30+ vs. 30−).

Fig. 4

Isolation of detergent (Lubrol WX)-resistant membranes from OPCs. A: OPCs were lysed and solubilized in 0.5% Lubrol WX at 4°C. Lysates were mixed with 80% sucrose and subsequently overlaid with 35% and 5% sucrose. Sucrose gradients were centrifuged at 46,900 rpm for 18 hr at 4°C to separate buoyant raft membranes from bulk cellular material. Fractions 1–9 (equal volume) were taken from the top of each gradient and were used for SDS-PAGE and Western blot analysis. Flotillin-1, a marker for raft microdomains, was present in both buoyant “rafting” (2–3) fractions. GM-1 ganglioside, a raft marker, was detected in fractions 1 and 2 (GM-1 standard shown). Calnexin was localized only within nonbuoyant fractions. IGF-IR, p85, and Akt were detected strongly in nonbuoyant fractions but were also seen in buoyant fractions. B: Total cholesterol and protein distributions across fractions analyzed in A were determined as described in Materials and Methods. Squares (dashed line) represent total protein; lozenges (solid line) represent total cholesterol in each fraction. The majority of cholesterol is contained within the buoyant fractions. In contrast, the amount of protein per fractions is much greater in the nonbuoyant fractions. C: OPCs serum starved for 2 hr and treated or not with IGF-I (20 ng/ml) for 15 min were lysed and solubilized in 0.5% Lubrol WX at 4°C. Lysates were centrifuged at 13,000g for 20 min to separate detergent insoluble (caveolin-1- and cholesterol-enriched) fractions from the soluble supernatant. Equal protein fractions were analyzed by SDS-PAGE and Western blot analysis. Phosphorylated Akt and IGF-IR were increased in the insoluble fractions with IGF-I stimulation. Total Akt, IGF-1R, and p85 are also detected in insoluble fractions and were unchanged with IGF-I treatment. D: Total cholesterol (lozenges and solid line) and protein (squares and dashed line) distribution in detergent insoluble and soluble fractions that correlate with fractions analyzed in C. Most cholesterol is contained within the Lubrol WX-insoluble fractions.