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Lipid rafts mediate H₂O₂ prosurvival effects in cultured endothelial cells

Baohua Yang^*, Tin N. Oo and Victor Rizzo^*,1

^* Cardiovascular Research Center, Department of Anatomy and Cell Biology, Temple University School of Medicine, Philadelphia, Pennsylvania, USA; and

Center for Cardiovascular Research, Albany Medical College, Albany, New York, USA

¹ Correspondence: Cardiovascular Research Center and Department of Anatomy and Cell Biology, Temple University School of Medicine, 3420 North Broad St., MRB826, Philadelphia, PA 19140. E-mail: rizzov{at}temple.edu

SPECIFIC AIMS

The aim of this study was to directly evaluate the functional significance of lipid rafts in reactive oxygen species (ROS) signaling transduction in endothelial cells. Here, we provide evidence that lipid rafts are important regions for propagation of compensatory survival pathways activated in response to cytotoxic levels of H₂O₂.

PRINCIPAL FINDINGS

1. Enhancement of H₂O₂-induced apoptosis in cholesterol-depleted endothelial cell monolayers
Consistent with previous studies, 500 µM H₂O₂ induced apoptosis in endothelial cell cultures. We detected Annexin V binding and DNA fragmentation events via TUNEL analysis following H₂O₂ treatment (data not shown). After 4 h of incubation, the proportion of TUNEL-positive cells was statistically greater in peroxide-treated cultures compared to untreated monolayers (Fig. 1 ). Concomitant with these observations, caspase 3 activation occurred within 1 h of H₂O₂ exposure and progressively increased during the 4 h experimental period (data not shown). To address whether rafts play a role in H₂O₂-induced apoptosis, monolayers were pretreated with methyl-ßbeta;-cyclodextrin (CD) to deplete plasma membrane cholesterol resulting in disruption of raft structural integrity. Here, CD-pretreated cells showed heightened sensitivity to the cytotoxic effects of peroxide (Fig. 1) . Additionally, CD-pretreated cells showed heightened activation of caspase 3 after H₂O₂ treatment (data not shown).

View larger version (9K): [in this window] [in a new window]   Figure 1. Hydrogen peroxide-induced apoptosis is exacerbated following cholesterol depletion. Bovine aortic endothelial cells (BAEC) were pretreated with 10 mM methyl-ßbeta;-cyclodextrin (CD) for 30 min followed by bolus administration of H2O2 (500 µM). Representative fluorescence micrographs showing H2O2 induced apoptosis, as detected by TUNEL assay, after 4 h of exposure. Data are reported as mean ± SD; *P < 0.05. Comparisons between H2O2-treated cells with and without CD were significant at P < 0.05 (#).

2. Akt is activated within lipid rafts and contributes to prosurvival signaling
Akt is a crucial prosurvival signaling factor in several cell types exposed to a variety of proapoptotic stimuli. In endothelial cells exposed to apoptotic stressors, Akt is rapidly and transiently activated, an event that occurs predominantly in a PI3 kinase-dependent manner. Consistent with these reports, we found that wortmannin heightened peroxide-induced apoptotic events (Fig. 2 A and B), thus verifying Akt’s role as an antiapoptotic mediator.

View larger version (29K): [in this window] [in a new window]   Figure 2. Lipid rafts participation in H2O2-induced Akt prosurvival signaling. A) BAEC were pretreated with Wortmannin (2 µM) for 1 h followed by bolus administration of H2O2 for times indicated. Inhibition of PI3 kinase (Wort) showed enhanced H2O2-induced caspase 3 cleavage. B) The number of cells containing fragmented DNA (TUNEL assay) was significantly greater in wortmannin-treated monolayers subjected to H2O2 (4 h) compared to H2O2 alone. C) Control and CD-pretreated BAEC were exposed to H2O2 for 15 min and then processed to purify plasma membranes. The plasma membranes were sonicated and subfractionated by sucrose gradient (5% to 30%) centrifugation to isolate light buoyant density lipid rafts domains from bulk membranes. Plasma membrane fractions were Western-blotted with indicated primary antibodies. (D) H2O2 enhanced phosphorylation of Akt over nontreated control cells. Disassembling rafts with CD significantly attenuated H2O2-induced Akt phosphorylation. Data are reported as mean ± SD; *P < 0.05. Comparisons between H2O2-treated cells with and without CD were significant at P < 0.05 (#).

Next, we tested whether lipid raft domains play a role in propagation of H₂O₂ induced signaling to Akt. We found that Akt was distributed in both light buoyant density membrane fractions, as well as in heavier membrane fractions. Interestingly, H₂O₂ appeared to induce phosphorylation of the pool of Akt specifically associated with raft membranes (Fig. 2C ). In addition, CD pretreatment significantly attenuated H₂O₂-induced Akt phosphorylation (Fig. 2D ). To further explore the significance to Akt compartmentation to rafts, BAEC were pretreated with CD before H₂O₂ treatment. Figure 2C (right) shows that depletion of plasma membrane cholesterol effectively caused lipid raft disruption, as evidenced by a significant shift in the raft marker, caveolin, out of light buoyant density membrane fractions into heavier membrane fractions. In addition, raft ablation resulted in a similar redistribution of Akt. More importantly, the phosphorylation of Akt observed after H₂O₂ treatment remained attenuated, suggesting the raft localization plays an important role in Akt’s responsiveness to oxidative stress.

3. Erk1/2 prosurvival effects in response to H₂O₂ are dependent on plasma membrane cholesterol content
Previous studies have shown that activation of ERK1/2 can confer protection from detrimental effects of ROS. Here, we observed that depleting cholesterol from plasma membrane raft compartments inhibited H₂O₂ activation of ERK1/2 (data not shown). Furthermore, H₂O₂-induced ERK1/2 phosphorylation was fully restored on repletion of plasma membrane cholesterol (data not shown). Pretreatment with PD 98059 effectively blocked ERK1/2 phosphorylation (data not shown) and served to enhance both H₂O₂ activation of caspase 3 activity and the number of TUNEL-positive cells (data not shown), substantiating the prosurvival role of ERK1/2 in H₂O₂-induced cytotoxicity reported previously. In addition, we found that wortmannin and PD98059 did not alter H₂O₂-induced ERK1/2 or Akt phosphorylation levels, respectively (data not shown). These data suggest that both Akt and ERK1/2 pathways function in parallel to mediated prosurvival signaling pathways in endothelial cells subjected to oxidative stress. More importantly, our data collectively suggest that plasma membrane rafts constitute a common, proximal point of signaling transduction that relays prosurvival messages through Akt and ERK1/2.

CONCLUSIONS AND SIGNIFICANCE

The results from this study demonstrate a functional relationship between plasma membrane cholesterol content and prosurvival signaling pathways stimulated in response to oxidative stress. Although it is possible that alteration in plasma membrane cholesterol can directly have lipid effects on signaling independent of cholesterol-rich rafts, the localization of both PI3 kinase and Akt, and in some cases enrichment, in rafts and caveolae suggest that raft domains may serve as a site for H₂O₂-induced signaling. In further support of this concept, upstream mediators of PI3 kinase, such as Src-like kinases, are also localized in raft and caveolae membranes. H₂O₂ can rapidly inactivate phosphatases resulting in loss of opposition to actions of Src-like kinases. Hence, H₂O₂ inactivation of phosphatases would result in Src induction of PI3 kinase activity and Akt phosphorylation. Given these observations, it could be predicted then that disruption of raft structures would alter effective propagation of these pathways. Indeed, our data showing that raft ablation through depletion of plasma membrane cholesterol caused the loss of Akt from the lipid rafts and attenuated activation of Akt by H₂O₂ supports this mechanism of H₂O₂-induced signaling.

Our data also show that lipid rafts mediate H₂O₂-induced activation of ERK1/2. A potential mechanism whereby rafts can effect ERK1/2 activation involves transactivation of epidermal growth factor receptor (EGFr) by H₂O₂. Because EGFr can localize to raft domains, it would be expected that alteration of raft integrity would induce loss of receptor compartmentation or disorganization of the signaling machinery necessary to propagate epidermal growth factor (EGF) receptor responses to ERK1/2 after an oxidant chal-lenge. To substantiate a mechanistic relationship between rafts and EGF receptors, experiments that directly examine localization and/or transactivation of EGFr in cholesterol-depleted endothelial cell cultures subjected to H₂O₂ would be required.

In summary, endothelial cell cultures respond to oxidative stress by activating several signaling pathways (Fig. 3 ). Our observations extend the general concept that compartmentalization of lipid and protein signaling mediators within the plasma membrane provides an efficient means to respond to external stimuli, including oxidants. Moreover, regulation of lipid raft or caveolae expression may be an important mechanism utilized by endothelial cells to adjust their sensitivity to ROS.

View larger version (20K): [in this window] [in a new window]   Figure 3. Summary diagram: Rafts and caveolae as a proximal relay point for prosurvival signaling in response to ROS. On exposure to exogenous H2O2, Akt localized within cholesterol- and sphingolipid lipid-rich plasma membrane microdomains (rafts/caveolae) becomes activated and functions as a crucial prosurvival signaling factor that inactivates apoptotic caspase molecules. The activation of ERK1/2 also confers protection from detrimental effects of ROS through a mechanism that relies on rafts/caveolae and potentially through transactivation of lipid raft localized epidermal growth factor receptor (EGFr).

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5359fje

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This Article Abstract Full Text Full Text (PDF) All Versions of this Article: fj.05-5359fjev1 20/9/1501    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Yang, B. Articles by Rizzo, V. Search for Related Content PubMed PubMed Citation Articles by Yang, B. Articles by Rizzo, V.