J Mol Neurosci (2015) 57:1–10 DOI 10.1007/s12031-015-0594-6
Functions for the cAMP/Epac/Rap1 Signaling Pathway in Low-Dose Endothelial Monocyte-Activating Polypeptide-II-Induced Opening of Blood–Tumor Barrier Zhen Li 1 & Xiao-bai Liu 2 & Yun-hui Liu 1 & Yi-xue Xue 3,4 & Ping Wang 3,4 & Li-bo Liu 3,4 & Yi-long Yao 1 & Jun Ma 3,4
Received: 15 April 2015 / Accepted: 28 May 2015 / Published online: 5 June 2015 # Springer Science+Business Media New York 2015
Abstract Previous studies have demonstrated that low-dose endothelial monocyte-activating polypeptide-II (EMAP-II) induces blood–tumor barrier (BTB) hyperpermeability via both paracellular and transcellular pathways. In a recent study, we revealed that cAMP/PKA-dependent and cAMP/PKAindependent signaling pathways are both involved in EMAP-II-induced BTB hyperpermeability. The present study further investigated the exact mechanisms through which the cAMP/PKA-independent signaling pathway affects EMAPII-induced BTB hyperpermeability. In an in vitro BTB model, low-dose EMAP-II (0.05 nM) induced a significant decrease in Rap1 activity in RBMECs. Pretreatment with forskolin to elevate intracellular cAMP concentration completely blocked EMAP-II-induced Rap1 inactivation. Epac/Rap1 activation by 8-pCPT-2′-O-Me-cAMP significantly prevented EMAPII-induced activation of RhoA/ROCK. Furthermore, 8-
Electronic supplementary material The online version of this article (doi:10.1007/s12031-015-0594-6) contains supplementary material, which is available to authorized users. * Yun-hui Liu [email protected] 1
Department of Neurosurgery, Shengjing Hospital, China Medical University, Shenyang, Liaoning Province 110004, People’s Republic of China
2
The 96th Class, 7-Year Program, China Medical University, Shenyang, Liaoning Province 110001, People’s Republic of China
3
Department of Neurobiology, College of Basic Medicine, China Medical University, Shenyang, Liaoning Province 110001, People’s Republic of China
4
Institute of Pathology and Pathophysiology, China Medical University, Shenyang, Liaoning Province 110001, People’s Republic of China
pCPT-2′-O-Me-cAMP pretreatment significantly inhibited EMAP-II-induced decreases in TEER and increases in HRP flux. Pretreatment also significantly prevented EMAP-IIinduced changes in MLC phosphorylation, actin cytoskeleton arrangement, and expression and distribution of ZO-1 in RBMECs. This study demonstrates that the cAMP/Epac/ Rap1 signaling cascade is a crucial pathway in EMAP-IIinduced BTB hyperpermeability. Keywords Endothelial monocyte-activating polypeptide-II . Blood-tumor barrier . Permeability . cAMP . Epac . Rap1
Introduction The blood–brain barrier (BBB) is formed by endothelial cells in cerebral microvessels and is a critical interface regulating molecular flux between blood and the brain. It contributes to homoeostasis of the central nervous system microenvironment and provides protection from pathogens and toxins. Two recent review articles described methods that have been used in pharmaceutical research to regulate BBB function and to assess the ability of drug-like molecules to permeate the BBB (Shityakov et al. 2013; Salvador et al. 2014). Brain tumor capillaries constitute the blood–tumor barrier (BTB), which has different structural and functional characteristics compared to normal brain capillaries that form the BBB. Although the BTB is more permeable than the BBB, it still impedes entry of antitumor drugs into brain tumor tissues (Black and Ningaraj 2004; Idbaih et al. 2008). Therefore, methods to selectively open the BTB are crucial for effective glioma chemotherapy. Endothelial monocyte-activating polypeptide-II (EMAPII) is a proinflammatory cytokine that has been used in antitumor therapy research (Reznikov et al. 2011; Awasthi et al.
2
2012). Previous studies demonstrated that low-dose EMAP-II can increase BTB permeability via both paracellular and transcellular pathways (Xie et al. 2010, 2012; Li 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. Elevation of cAMP may trigger cAMP-dependent, PKAindependent regulation of the EC barrier by small GTPases (Bos 2003; Birukova et al. 2007). In previous studies, significant increases in RhoA/Rho kinase (ROCK) activity in EMAP-II-treated rat brain microvascular endothelial cells (RBMECs) from a BTB model were observed (Xie et al. 2012; Li et al. 2012). 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. Rap1, a member of the Ras family of GTPases, is enriched at endothelial cell–cell contacts and activated by cAMP through a PKA-independent pathway. Epac, a cAMP-activated guanine nucleotide exchange factor for Rap GTPases, has been shown to regulate the integrity of endothelial junctions, state of the actin cytoskeleton, and stability of microtubules in ECs (Kooistra et al. 2005; Sehrawat et al. 2008). Based on these findings, we hypothesized that the mechanisms through which cAMP influences EMAP-II-induced BTB hyperpermeability likely includes activation of both PKA and Epac/Rap1 signaling pathways. The purpose of this study was to determine whether the cAMP/Epac/Rap1 signaling pathway is involved in EMAPII-induced BTB hyperpermeability and to explore potential signaling mechanisms.
Materials and Methods RBMEC Culture and Cell Viability Assay Neonatal Wistar rats age 0.05). EMAP-II Decreases Rap1 Activity in RBMECs of the BTB To investigate the role of Rap1 in EMAP-II-induced BTB hyperpermeability, we first detected alterations in Rap1 activity in EMAP-II-treated RBMECs. In an in vitro BTB model, Rap1 activity in RBMECs decreased significantly as early as 0.25 h after EMAP-II treatment (P
Functions for the cAMP/Epac/Rap1 Signaling Pathway in Low-Dose Endothelial Monocyte-Activating Polypeptide-II-Induced Opening of Blood–Tumor Barrier Zhen Li 1 & Xiao-bai Liu 2 & Yun-hui Liu 1 & Yi-xue Xue 3,4 & Ping Wang 3,4 & Li-bo Liu 3,4 & Yi-long Yao 1 & Jun Ma 3,4
Received: 15 April 2015 / Accepted: 28 May 2015 / Published online: 5 June 2015 # Springer Science+Business Media New York 2015
Abstract Previous studies have demonstrated that low-dose endothelial monocyte-activating polypeptide-II (EMAP-II) induces blood–tumor barrier (BTB) hyperpermeability via both paracellular and transcellular pathways. In a recent study, we revealed that cAMP/PKA-dependent and cAMP/PKAindependent signaling pathways are both involved in EMAP-II-induced BTB hyperpermeability. The present study further investigated the exact mechanisms through which the cAMP/PKA-independent signaling pathway affects EMAPII-induced BTB hyperpermeability. In an in vitro BTB model, low-dose EMAP-II (0.05 nM) induced a significant decrease in Rap1 activity in RBMECs. Pretreatment with forskolin to elevate intracellular cAMP concentration completely blocked EMAP-II-induced Rap1 inactivation. Epac/Rap1 activation by 8-pCPT-2′-O-Me-cAMP significantly prevented EMAPII-induced activation of RhoA/ROCK. Furthermore, 8-
Electronic supplementary material The online version of this article (doi:10.1007/s12031-015-0594-6) contains supplementary material, which is available to authorized users. * Yun-hui Liu [email protected] 1
Department of Neurosurgery, Shengjing Hospital, China Medical University, Shenyang, Liaoning Province 110004, People’s Republic of China
2
The 96th Class, 7-Year Program, China Medical University, Shenyang, Liaoning Province 110001, People’s Republic of China
3
Department of Neurobiology, College of Basic Medicine, China Medical University, Shenyang, Liaoning Province 110001, People’s Republic of China
4
Institute of Pathology and Pathophysiology, China Medical University, Shenyang, Liaoning Province 110001, People’s Republic of China
pCPT-2′-O-Me-cAMP pretreatment significantly inhibited EMAP-II-induced decreases in TEER and increases in HRP flux. Pretreatment also significantly prevented EMAP-IIinduced changes in MLC phosphorylation, actin cytoskeleton arrangement, and expression and distribution of ZO-1 in RBMECs. This study demonstrates that the cAMP/Epac/ Rap1 signaling cascade is a crucial pathway in EMAP-IIinduced BTB hyperpermeability. Keywords Endothelial monocyte-activating polypeptide-II . Blood-tumor barrier . Permeability . cAMP . Epac . Rap1
Introduction The blood–brain barrier (BBB) is formed by endothelial cells in cerebral microvessels and is a critical interface regulating molecular flux between blood and the brain. It contributes to homoeostasis of the central nervous system microenvironment and provides protection from pathogens and toxins. Two recent review articles described methods that have been used in pharmaceutical research to regulate BBB function and to assess the ability of drug-like molecules to permeate the BBB (Shityakov et al. 2013; Salvador et al. 2014). Brain tumor capillaries constitute the blood–tumor barrier (BTB), which has different structural and functional characteristics compared to normal brain capillaries that form the BBB. Although the BTB is more permeable than the BBB, it still impedes entry of antitumor drugs into brain tumor tissues (Black and Ningaraj 2004; Idbaih et al. 2008). Therefore, methods to selectively open the BTB are crucial for effective glioma chemotherapy. Endothelial monocyte-activating polypeptide-II (EMAPII) is a proinflammatory cytokine that has been used in antitumor therapy research (Reznikov et al. 2011; Awasthi et al.
2
2012). Previous studies demonstrated that low-dose EMAP-II can increase BTB permeability via both paracellular and transcellular pathways (Xie et al. 2010, 2012; Li 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. Elevation of cAMP may trigger cAMP-dependent, PKAindependent regulation of the EC barrier by small GTPases (Bos 2003; Birukova et al. 2007). In previous studies, significant increases in RhoA/Rho kinase (ROCK) activity in EMAP-II-treated rat brain microvascular endothelial cells (RBMECs) from a BTB model were observed (Xie et al. 2012; Li et al. 2012). 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. Rap1, a member of the Ras family of GTPases, is enriched at endothelial cell–cell contacts and activated by cAMP through a PKA-independent pathway. Epac, a cAMP-activated guanine nucleotide exchange factor for Rap GTPases, has been shown to regulate the integrity of endothelial junctions, state of the actin cytoskeleton, and stability of microtubules in ECs (Kooistra et al. 2005; Sehrawat et al. 2008). Based on these findings, we hypothesized that the mechanisms through which cAMP influences EMAP-II-induced BTB hyperpermeability likely includes activation of both PKA and Epac/Rap1 signaling pathways. The purpose of this study was to determine whether the cAMP/Epac/Rap1 signaling pathway is involved in EMAPII-induced BTB hyperpermeability and to explore potential signaling mechanisms.
Materials and Methods RBMEC Culture and Cell Viability Assay Neonatal Wistar rats age 0.05). EMAP-II Decreases Rap1 Activity in RBMECs of the BTB To investigate the role of Rap1 in EMAP-II-induced BTB hyperpermeability, we first detected alterations in Rap1 activity in EMAP-II-treated RBMECs. In an in vitro BTB model, Rap1 activity in RBMECs decreased significantly as early as 0.25 h after EMAP-II treatment (P
