TRPC-mediated actin-myosin contraction is critical for BBB disruption following hypoxic stress

Abstract

Hypoxia-induced disruption of the blood-brain barrier (BBB) is the result of many different mechanisms, including alterations to the cytoskeleton. In this study, we identified actin-binding proteins involved in cytoskeletal dynamics with quantitative proteomics and assessed changes in subcellular localization of two proteins involved in actin polymerization [vasodilator-stimulated phosphoprotein (VASP)] and cytoskeleton-plasma membrane cross-linking (moesin). We found significant redistribution of both VASP and moesin to the cytoskeletal and membrane fractions of BBB endothelial cells after 1-h hypoxic stress. We also investigated activation of actin-myosin contraction through assessment of phosphorylated myosin light chain (pMLC) with confocal microscopy. Hypoxia caused a rapid and transient increase in pMLC. Blocking MLC phosphorylation through inhibition of myosin light chain kinase (MLCK) with ML-7 prevented hypoxia-induced BBB disruption and relocalization of the tight junction protein ZO-1. Finally, we implicate the transient receptor potential (TRP)C family of channels in mediating these events since blockade of TRPC channels and the associated calcium influx with SKF-96365 prevents hypoxia-induced permeability changes and the phosphorylation of MLC needed for actin-myosin contraction. These data suggest that hypoxic stress triggers alterations to cytoskeletal structure that contribute to BBB disruption and that calcium influx through TRPC channels contributes to these events.

Fig. 1.

iTRAQ proteomics confirmation of protein changes following hypoxic treatment. We found that our proteomic analysis, while identifying a similar set of proteins each time it was performed, was quite variable in the degree of change seen between data sets. Therefore we also used in-cell Western blot (ICWB) analysis to confirm changes seen in our proteomic data sets (data not shown). Actin levels as detected by iTRAQ were slightly elevated after 1 h of hypoxia, then fell at 3 and 6 h. Moesin, vasodilator-stimulated phosphoprotein (VASP), and myosin light chain (MLC) were unchanged in iTRAQ analysis. Data are presented as means ± SE for 3 iTRAQ experiments. A significant change was only found for actin levels after 1 h of hypoxia by one-way analysis of variance. *P < 0.05, ***P < 0.001 vs. control.

Fig. 2.

Subcellular localization of moesin and VASP is altered after exposure to hypoxic stress. We used differential detergent fractionation and conventional immunoblotting to assess the distribution of VASP and moesin after exposure to hypoxia (see materials and methods). VASP levels in the cytoplasm were not significantly altered from normoxic levels after either 1- or 6-h hypoxia, although there was a significant decrease in cytoplasmic VASP between the 1-h and 6-h hypoxia groups (^^P < 0.01 vs. 1-h hypoxia). VASP associated with the plasma membrane and the insoluble cytoskeletal fraction was significantly increased after 1-h hypoxia (***P < 0.001 and *P < 0.05, respectively) and dropped back to control levels at 6 h. Similarly, cytoplasmic levels of moesin were not altered, but levels of moesin associated with the membrane and cytoskeleton were significantly increased at 1 h (**P < 0.01 and ***P < 0.001, respectively). The 6-h levels were similar to control values. These data suggest an early and robust alteration in blood-brain barrier (BBB) endothelial cell cytoskeletal dynamics and structure that may contribute to barrier disruption. Data are presented as means ± SE for 3 fractionation studies.

Fig. 3.

Hypoxic stress causes rapid, transient phosphorylation of MLC. A: we used confocal microscopy (×63 objective) to assess MLC phosphorylation over the time course of hypoxic stress. WGA-TR, wheat germ agglutinin-tetramethylrhodamine. B: hypoxia caused a dramatic and significant increase in phosphorylated MLC (pMLC) within 15 min (***P < 0.001) that was quickly restored to basal levels. Levels of pMLC remained similar to control (normoxic) for the duration of the hypoxic stress (6 h). Data are presented as means ± SE for 3 confocal stacks.

Fig. 4.

Inhibition of myosin light chain kinase (MLCK) protects against hypoxia-induced BBB disruption. Cells were incubated with and without the MLCK inhibitor ML-7 (1 μM) and exposed to 6-h hypoxia, after which paracellular permeability was assessed as described in text. Hypoxia caused a significant increase in paracellular permeability (*P < 0.05 vs. normoxic control) that was blocked by incubation with ML-7. There was no significant effect of ML-7 on basal permeability. Data are presented as means ± SE for 10–14 measurements.

Fig. 5.

Inhibition of actin-myosin contraction protects tight junction structure. Zonula occludens 1 (ZO-1) is a tight junction accessory protein that links actin filaments to the plasma membrane at tight junctions (61). After 6-h hypoxic stress, ZO-1 immunofluorescence moves away from the cell-cell border into the cytoplasm, indicating disruption of the BBB. However, in cells treated with 1 μM ML-7, a MLCK inhibitor, ZO-1 localization at the plasma membrane is preserved after 6-h hypoxia. Representative fluorescent images are shown (×63 objective).

Fig. 6.

Inhibition of transient receptor (TRP)C channels prevents BBB disruption and MLC phosphorylation following hypoxic stress. We assessed barrier function (permeability assay) and MLC phosphorylation (confocal microscopy) after hypoxia. A: treatment with SKF-96365 (0.1, 1.0, and 10 μM), an inhibitor of TRPC channels, dose-dependently blocked BBB disruption following 6-h hypoxia (**P < 0.01 vs. control, ^P < 0.05 vs. hypoxia). B: treatment with 1 μM SKF-96365 also significantly reduced pMLC immunofluorescence following hypoxia (**P < 0.01 vs. hypoxia), indicating prevention of MLCK activation and/or RhoA/ROCK mediated inhibition of MLC phosphatase (MLCP). Data are presented as means ± SE for 8–10 measurements (A) or 3 confocal stacks (B).

Fig. 7.

Pathways implicated in cytoskeletal regulation are altered after hypoxia. Isobaric protein labeling followed by LC tandem mass spectrometry (MS/MS) identified a number of proteins that were up- or downregulated by hypoxia in BBB endothelial cells. We are particularly interested in proteins involved in actin filament polymerization (VASP, profiling, cofilin-1), actin-myosin contraction [MLC, MLCK, Ras homolog gene family A (RhoA)/Rho kinase (ROCK)], and anchoring of actin filaments to the plasma membrane (moesin, α-actinin). Arrowheads indicate activation/phosphorylation, while blocked arrows indicate inhibition. α-Act, α-actinin; α-cat, α-catenin; β-cat, β-catenin; Ca⁺⁺, calcium; CaM, calmodulin; Cof-1, cofilin-1; γ-cat, γ-catenin; MHC, myosin heavy chain; PFN, profilin.