HDAC9 nuclear/cytoplasmic shuttling in pulmonary vascular endothelial barrier regulation

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PROJECT SUMMARY Acute lung injury (ALI) is characterized by lung vascular endothelial (EC) barrier compromise resulting in pulmonary edema. Gram negative (G-) bacteria are the source of bacterial toxins such as lipopolysaccharides (LPS), which are potent triggers of ALI. Despite the use of potent antibiotics and aggressive intensive care support, the mortality of LPS-induced ALI remains high, primarily because the complex molecular mechanisms involved in EC barrier regulation are ill-defined. The Scientific Premise of this Project derives from our novel preliminary data indicating that the class IIa histone deacetylase, HDAC9, is upregulated in the lungs of patients with sepsis in concert with the inflammatory responses. Accordingly, the overexpression of HDAC9 in transgenic mice increases basal lung vascular permeability and exacerbates vascular barrier compromise induced by LPS. Conversely, deletion of HDAC9 gene improves lung function in LPS-induced murine ALI model. These data suggest pro-edemagenic role of HDAC9 expression in ALI. HDAC9 is nuclear-cytoplasmic protein and its cellular localization is regulated by phosphorylation. Phosphorylation leads to HDAC9 nuclear export thus inhibiting its activity in the nucleus and promoting interactions with extra-nuclear targets. We demonstrated that LPS increases HDAC9 phosphorylation accompanied by its nuclear export in EC. In contrary, de-phosphorylation of HDAC9 leads to nuclear import thus inhibiting its association with cytoplasmic proteins. We showed that HDAC9 interacts with protein phosphatase 2A (PP2A), which has a barrier-protective role in EC. Further, our novel data indicated that PP2A is activated by Gi-mediated EC barrier-protective agonists, HGF and the stable ATP analog, ATPγS, suggesting the link between Gi-mediated signaling and PP2A in EC barrier enhancement. While the mechanisms of Gi-mediated PP2A activation have not been described, our data suggest that they may include the activation of GAB1/Shp2-mediated signaling. Further, both HDAC9 and PP2A interact with AJ protein plakoglobin suggesting functional complex. However, whether the HDAC9-dependent EC barrier regulation involved HDAC9/PP2A/plakoglobin interaction and EC barrier-protective effect of PP2A is mediated through decreased HDAC9 phosphorylation is not yet established. Therefore, a primary goal of this Proposal will be to define a novel role for HDAC9/PP2A crosstalk in AJ-mediated EC barrier regulation. We hypothesize that the phosphorylation status of HDAC9 and its interaction with PP2A alter intracellular signaling to regulate the EC barrier via effect on AJ assembly. The SAs are: 1: To evaluate the role of HDAC9 phosphorylation/nuclear export in the LPS-induced adherens junctions-mediated endothelial barrier compromise in vitro and in vivo. 2: To determine the importance of PP2A-mediated HDAC9 dephosphorylation/nuclear import in the assembly of AJs and endothelial barrier strengthening in vitro and in vivo.