CD36 mediates H2O2-induced calcium influx in lung microvascular endothelial cells

Elevated levels of reactive oxygen species and intracellular Ca²⁺ play a key role in endothelial barrier dysfunction in acute lung injury. We previously showed that H₂O₂-induced increases in intracellular calcium concentrations ([Ca²⁺]_i) in lung microvascular endothelial cells (LMVECs) involve the membrane Ca²⁺ channel, transient receptor potential vanilloid-4 (TRPV4) and that inhibiting this channel attenuated H₂O₂-induced barrier disruption in vitro. We also showed that phosphorylation of TRPV4 by the Src family kinase, Fyn, contributes to H₂O₂-induced Ca²⁺ influx in LMVEC. In endothelial cells, Fyn is tethered to the cell membrane by CD36, a fatty acid transporter. In this study, we assessed the effect of genetic loss or pharmacological inhibition of CD36 on Ca²⁺ responses to H₂O₂ H₂O₂-induced Ca²⁺ influx was attenuated in LMVEC isolated from mice lacking CD36 (CD36^-/-). TRPV4 expression and function was unchanged in LMVEC isolated from wild-type (WT) and CD36^-/- mice, as well as mice with deficiency for Fyn (Fyn^-/-). TRPV4 immunoprecipitated with Fyn, but this interaction was decreased in CD36^-/- LMVEC. The amount of phosphorylated TRPV4 was decreased in LMVEC from CD36^-/- mice compared with WT controls. Loss of CD36 altered subcellular localization of Fyn, while inhibition of CD36 fatty acid transport with succinimidyl oleate did not attenuate H₂O₂-induced Ca²⁺ influx. Lastly, we found that CD36^-/- mice were protected from ischemia-reperfusion injury in vivo. In conclusion, our data suggest that CD36 plays an important role in H₂O₂-mediated lung injury and that the mechanism may involve CD36-dependent scaffolding of Fyn to the cell membrane to facilitate TRPV4 phosphorylation.