Role of cAMP-Dependent Protein Kinase A Activity in Low-Dose Endothelial Monocyte-Activating Polypeptide-II-Induced Opening of Blood–Tumor Barrier

Abstract

Our previous studies demonstrated that low-dose endothelial monocyte-activating polypeptide-II (EMAP-II) can selectively increase the permeability of blood-tumor barrier (BTB). In addition, low-dose EMAP-II significantly decreases the cyclic adenosine monophosphate (cAMP) concentration and the protein kinase A (PKA) expression level in tumor tissues in the rat C6 glioma model. In this study, an in vitro BTB model was used to investigate the potential role of cAMP/PKA signaling cascade in EMAP-II-induced BTB hyperpermeability. Our data revealed that low-dose EMAP-II (0.05 nM) induced a significant decrease in total intracellular cAMP concentration and PKA activity in rat brain microvascular endothelial cells (RBMECs). Pretreatment with forskolin to increase intracellular cAMP nearly completely blocked the EMAP-II-induced decrease in transendothelial electric resistance and increase in horseradish peroxidase flux across the BTB. Similar pretreatment completely prevented the EMAP-II-induced changes in RhoA/Rho kinase activity, expression and distribution of tight junction-associated protein ZO-1, and myosin light chain phosphorylation, as well as actin cytoskeleton arrangement in RBMECs. Pretreatment with 6Bnz-cAMP to activate PKA significantly attenuated these EMAP-II-induced alterations in RBMECs. In summary, our present study demonstrates that the cAMP/PKA signaling cascade works as a crucial signaling pathway in EMAP-II-induced BTB hyperpermeability.

Functions for the cAMP/Epac/Rap1 Signaling Pathway in Low-Dose Endothelial Monocyte-Activating Polypeptide-II-Induced Opening of Blood–Tumor Barrier

“…In our previous and the present studies, the EMAP-II concentration used to induce opening of the BTB was 0.05 nM (Xie et al 2012;Li et al 2012Li et al , 2015. However, as a proinflammatory cytokine, EMAP-II might be toxic to the RBMECs.…”

“…Data from our recent study revealed that increased intracellular cAMP nearly completely blocked EMAP-II-induced BTB hyperpermeability in EMAP-IItreated BTB RBMECs. Additionally, elevated cAMP prevented EMAP-II-induced increase in RhoA/ROCK activity (Li et al 2015). PKA activation only partially attenuated the effects of EMAP-II, which suggests the involvement of a cAMP/PKA-independent signaling pathway.…”

“…To increase intracellular cAMP levels, forskolin (Sigma-Aldrich) was used for 1 h at 5 μM as previously described (Li et al 2015). A cAMP analogue, 8-(4-chlorophenylthio)-2′-O-methyladenosine-3′, 5′-cyclic monophosphate (8-pCPT-2′-O-Me-cAMP; BioLog, Germany) was used for 15 min at 0.5 mM to activate Epac.…”

“…Pretreatment with forskolin (5 μM, 1 h) has been shown to completely block EMAP-II-induced BTB hyperpermeability (Li et al 2015). To determine the role of cAMP elevation in EMAP-II-induced inactivation of Rap1, RBMECs were pretreated with forskolin followed by EMAP-II treatment.…”

“…Previous studies demonstrated that low-dose EMAP-II can increase BTB permeability via both paracellular and transcellular pathways (Xie et al 2010(Xie et al , 2012Li et al 2014). Our recent study showed that the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA)-dependent signaling pathway plays an important role in this process (Li et al 2015). Given that cAMP signaling has been reported through both PKA-dependent and PKA-independent mechanisms (Patterson et al 2000;Van Hinsbergh and Van Nieuw Amerongen 2002;Yuan 2002;Ishizaki et al 2003), the potential role of the cAMP/PKA-independent signaling cascade in EMAP-II-induced TJ disruption and BTB hyperpermeability warrants further investigation.…”