Cooperation of Epac1/Rap1/Akt and PKA in prostaglandin E₂‐induced proliferation of human umbilical cord blood derived mesenchymal stem cells: Involvement of c‐Myc and VEGF expression

Abstract

Prostaglandin E(2) (PGE(2)) is well known to regulate cell functions through cAMP; however, the role of exchange protein directly activated by cAMP (Epac1) and protein kinase A (PKA) in modulating such functions is unknown in human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs). Therefore, we investigated the relationship between Epac1 and PKA during PGE(2)-induced hUCB-MSC proliferation and its related signaling pathways. PGE(2) increased cell proliferation, and E-type prostaglandin (EP) 2 receptor mRNA expression level and activated cAMP generation, which were blocked by EP2 receptor selective antagonist AH 6809. PGE(2) increased Epac1 expression, Ras-related protein 1 (Rap1) activation level, and Akt phosphorylation, which were inhibited by AH 6809, adenylyl cyclase inhibitor SQ 22536, and Epac1/Rap1-specific siRNA. Also, PGE(2) increased PKA activity, which was inhibited by AH 6809, SQ 22536, and PKA inhibitor PKI. HUCB-MSCs were incubated with the Epac agonist 8-pCPT-cAMP or the PKA agonist 6-phe-cAMP to examine whether Epac1/Rap1/Akt activation was independent of PKA activation. 8-pCPT-cAMP increased Akt phosphorylation but not PKA activity. 6-Phe-cAMP increased PKA activity, but not Akt phosphorylation. Additionally, an Akt inhibitor or PKA inhibitor (PKI) did not block the PGE(2) -induced increase in PKA activity or Akt phosphorylation, respectively. Moreover, PGE(2) increased glycogen synthase kinase (GSK)-3β phosphorylation and nuclear translocation of active-β-catenin, which were inhibited by Akt inhibitor or/and PKI. PGE(2) increased c-Myc and vascular endothelial growth factor (VEGF) expression levels, which were blocked by β-catenin siRNA. In conclusion, PGE(2) stimulated hUCB-MSC proliferation through β-catenin-mediated c-Myc and VEGF expression via Epac/Rap1/Akt and PKA cooperation.

Vascular Endothelial Cell Growth–Activated XBP1 Splicing in Endothelial Cells Is Crucial for Angiogenesis

“…Activated Akt phosphorylates GSK3β, resulting in suppression of GSK activity and the consequent β-catenin nuclear translocation. 31 As expected, overexpression of XBP1s did increase GSK3β phosphorylation (Figure 3A), suppress GSK activity ( Figure 3C), and increase β-catenin nuclear translocation ( Figure 3D and 3E) and target gene E2F2 expression analysis ( Figure 3F). It was further demonstrated that inhibition of Akt phosphorylation by LY294002 abolished the XBP1s-induced β-catenin nuclear translocation ( Figure 3G) and TCF-reporter gene expression ( Figure 3H).…”

JAK2V617F activates Lu/BCAM-mediated red cell adhesion in polycythemia vera through an EpoR-independent Rap1/Akt pathway

“…Rap1 is a small GTP-bound protein of the Ras superfamily that has been described as an upstream effector of Akt in several reports. [27][28][29] Ras-like proteins couple extracellular signals to various cellular responses and have the specificity of activating proteins at the inner surface of cell membranes. Rap1 is mainly involved in controlling cell adhesion, cell junction formation, cell secretion, and cell polarity.…”

The Prostanoid EP4 Receptor and Its Signaling Pathway

“…In vivo experiments have shown EP4-mediated angiogenesis as well (Kuwano et al, 2004;Rao et al, 2007;Zhang and Daaka, 2011). It has been suggested that Src, Epac/Rap1/Akt, and PKA signaling are involved in EP4-or PGE 2 -mediated VEGF expression in HeLa cells and mesenchymal stem cells (Liu et al, 2011;Jang et al, 2012). Taking this evidence together, it appears that EP4 receptor signaling contributes to vascular angiogenesis in both normal and cancer cells (Table 1).…”

“…These extensive studies have contributed significantly to our understanding that EP4 signaling plays a variety of roles not via the cAMP pathway alone but via others as well. It now appears that EP4 signaling is associated not only with G s a but also with G i a (Leduc et al, 2009), phosphatidylinositol 3-kinase (PI3K) (Regan, 2003), b-arrestin Kim et al, 2010), and b-catenin (Banu et al, 2009;Jang et al, 2012), as described in section II.C.…”