Evidence indicates that cAMP, and thus cAMP-dependent protein kinase (PKA), is highly effective in prevention of increased vascular endothelial permeability induced by inflammatory mediators. Our preliminary findings indicate that regulation of permeability occurred at the level of cadherin/catenin linkage to actin and that cAMP inhibited RhoA activation, an important molecule in actin remodeling and barrier dysfunction. We showed that isoforms of PKI, the PKA inhibitor protein, are widely expressed by endothelial cells, suggesting an added level in permeability regulation and underscores the role of PKA. We propose to test the hypothesis that PKA prevents development of mediator-induced barrier dysfunction through stabilizing the linkage between the VE-cadherin/catenin protein complex with actin through signaling Rho GTPases. Since cAMP has PKA-independent actions, Aim 1 will first evaluate PKA-dependency of the cAMP signal in prevention of increases in permeability. PKA will be inhibited by use of adenoviruses containing PKI or pharmacologically and the permeability measured by transendothelial resistance and transvascular FITC-albumin leak in the cremaster muscle.
Aim 2 will investigate whether PKA stabilizes associations of VE-cadherin and catenins to actin using methods of detergent fractionation, co-precipitation, and immunofluorescent localization.
Aim 3 will investigate mechanisms by which PKA signal regulation of Rho, Rac, and Cdc42. The studies will determine whether PKA coordinates signaling intermediates (i.e., protein phosphatase 2B) to regulate Rho GTPase activation and GTP/GDP cycling. Rho GTPase activity will be assessed by affinity binding of activated proteins with GST-fusion target proteins and by subcellular translocation.
Aim 4 will investigate whether Rho GTPases regulate endothelial permeability by regulating the VE-cadherin/catenin linkage to actin. The effects of altered Rho GTPase activity by use of bacterial toxins such as C3 and CNF1 and expression plasmids containing dominant-negative and -active mutants of RhoA, Rac1, or Cdc42 will be determined.
Aim 5 will test the hypothesis that the level of PKL expression regulates the extent of barrier restrictiveness. PKI expression or function will be altered by use of adenoviruses containing PKIgamma or PKIbeta isoforms to investigate regulation of the cadherin/catenin proteins, Rho GTPase activity, and barrier function assessed by in vitro and in vivo assays of permeability.
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