Among circulating hematopoietic cells, B-CLL uniquely expresses functional EPAC1, but EPAC1-mediated Rap1 activation does not account for PDE4 inhibitor-induced apoptosis

Abstract

IntroductionCyclic nucleotide signaling in lymphoid cells is regulated by a diverse set of phosphodiesterase (PDE) families, and selective inhibitors of these PDEs have proven to be both of therapeutic interest and of use in the analysis of lymphoid signal transduction pathways. At a minimum, lymphoid cells express PDE1B, PDE3B, PDE4A, B, and D, and PDE7A; all enzymes that can catabolize 3Ј:5Ј cyclic adenosine monophosphate (cAMP). [1][2][3][4][5] In studies of the mature B-cell malignancy B-cell chronic lymphocytic leukemia (B-CLL), we and others have found that both nonspecific PDE inhibitors such as theophylline and the PDE4-specific inhibitor rolipram induce apoptosis in leukemic cells over a 48-to 72-hour period. [6][7][8] While rolipram also induces apoptosis in peripheral B cells, albeit superimposed on a high normal basal apoptotic rate, the same agent has little or no apoptotic effect on peripheral T cells. 8 PDE4 inhibitors induce a mitochondrial apoptotic pathway in B-CLL cells with release of cytochrome c, caspase 9 and 3 activation, and PARP (poly-adenosine diphosphate [ADP] ribose polymerase) cleavage. This pathway may be triggered by PDE4 inhibitor-induced up-regulation of serine/threonine phosphatase protein phosphatase 2A (PP2A) activity with resultant dephosphorylation of the proapoptotic BH3-only Bcl2 family member BAD, release of BAD from the cytosolic adapter protein 14-3-3, and translocation of BAD to mitochondria. 9 In contrast, although PDE3B is expressed in B-CLL, when used alone PDE3 inhibitors do not induce apoptosis in B-CLL. 10 PDE7A is expressed and regulated in B-CLL cells, but reagents that allow selective inhibition of this PDE over the 48 to 72 hours required for apoptosis studies have not yet become available. 11 While most studies of cAMP signaling in lymphoid cells have focused on protein kinase A (PKA), recently a novel family of cAMP effector proteins, exchange protein directly activated by cAMP 1 (EPAC1) and EPAC2 (also known as cAMP-guanine nucleotide exchange factor I/II [GEFI/II]) has been identified in other cell lineages. 12-14 Upon binding cAMP, EPAC catalyzes release of guanosine 5Ј-diphosphate (GDP) from the Ras family guanosine 5Ј-triphosphatases (GTPases) Rap1 and Rap2, allowing the more abundant intracellular GTP to bind and convert the GTPase to an active, signaling conformation. 12 In an effort to develop a reagent that would allow selective analysis of EPACinduced signaling, Enserink et al 15 subsequently identified a cAMP analog, 8CPT-2Me-cAMP (8-(4-chloro-phenylthio)-2Ј-O-methyladenosine-3Ј,5Ј-cAMP), that activates EPAC while having little or no activity on PKA. While prior studies implicated Rap1 in a lineage-dependent manner in either cAMP-induced activation or inhibition of extracellular signal-regulated kinase (ERK), experiments using 8CPT-2Me-cAMP failed to support a role for EPAC or The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked ''advertisem...

Genetic Dissection of the Murine Lupus Susceptibility LocusSle2: Contributions to Increased Peritoneal B-1a Cells and Lupus Nephritis Map to Different Loci

“…We have recently shown that B6.Sle2 mice produce lower levels of type I IFN, and that the number of B-1a cells could be increased by anti-IFN-␣-blocking Ab, or decreased with IFN-␣ injections. 4 Finally, PDE4 (encoded by Pde4b, which is potentially in the Sle2b interval) is a phosphodiesterase that has been implicated in B cell chronic lymphocytic leukemia proliferation (40). Further refinement of the genetic maps through the generation of additional recombinants will be necessary to reduce significantly the list of candidate genes, and specifically address the contribution of these three genes.…”

Cutting Edge: Macrophage Inhibition by Cyclic AMP (cAMP): Differential Roles of Protein Kinase A and Exchange Protein Directly Activated by cAMP-1

“…A functional role for Epac-1 in integrin-mediated adhesion has been shown in nonmyeloid cell lines (6,7), whereas Epac-2 appears to be important for pancreatic ␤ cell insulin granule exocytosis (8). In leukocytes, Epac-1 mRNA transcripts were detected in circulating human B cells, but not in peripheral blood T cells, monocytes, or neutrophils (9). Apart from this, no information about Epac-1 expression in primary myeloid cells, or its role in such cells, is available.…”

Exchange Protein Directly Activated by Cyclic AMP Increases Melanoma Cell Migration by a Ca2+-Dependent Mechanism

“…Two isoforms of Epac, Epac1 and Epac2, were shown to mediate cAMP signaling through activation of a small-molecular weight G protein, Rap1 (4). In cancer cells, reports show that Epac mediates cell adhesion in Ovcar3 cells (5), apoptosis (6), and growth arrest (7) in B lymphoma cells, formation of embryonic vasculogenic networks in melanoma cells (8), and proliferation of prostate carcinoma cells (7). We have previously reported that Epac increases melanoma cell migration by a heparan sulfate-related mechanism (9).…”