Fibrosis, the result of excess deposition of extracellular matrix (ECM), in particular collagen, leads to scarring and loss of function in tissues that include the heart, lung, kidney and liver. The second messenger cAMP can inhibit the formation and extent of ECM during this late phase of inflammation, but the mechanisms for these actions of cAMP and of agents that elevate tissue cAMP levels are not well understood. In this article, we review the fibrotic process and focus on two recently recognized aspects of actions of cAMP and its effector Epac (Exchange protein activated by cAMP): (a) blunting of epithelial-mesenchymal transformation (EMT) and (b) down-regulation of Epac expression by profibrotic agents (e.g. TGF-b, angiotensin II), which may promote tissue fibrosis by decreasing Epac-mediated antifibrotic actions. Pharmacological approaches that raise cAMP or blunt the decrease in Epac expression by profibrotic agents may thus be strategies to block or perhaps reverse tissue fibrosis. LINKED ARTICLESThis article is part of a themed section on Novel cAMP Signalling Paradigms. To view the other articles in this section visit http://dx.doi. org/10.1111/bph.2012.166.issue-2 Abbreviations CF, cardiac fibroblast; ECM, extracellular matrix; Epac, Exchange protein activated by cyclic AMP; 8-Me-cAMP, 8-CPT-2′-O-Me-cAMP; N6-cAMP, N6-Benzoyl-cAMP; NSAIDs, non-steroidal anti-inflammatory drugs; a-SMA, a-smooth muscle actin Introduction to tissue fibrosis and cAMP signalling Tissue fibrosisTissue fibrosis (scarring) results from the excess deposition of extracellular matrix (ECM) and occurs as part of normal physiology (e.g. aging) and following injury, in particular, with recurrent or persistent stimulation of the inflammatory process. Tissue fibrosis often alters tissue function. For example, cardiac fibrosis contributes to diastolic dysfunction and a decrease in cardiac output that accompany advanced age (Lakatta, 2003;Chen and Frangogiannis, 2010). Inflammation that follows tissue injury produces a series of acute and late-phase responses. In its acute phase, inflammation is characterized by several cardinal features [calor, rubor, dolor and tumour (heat, redness, pain and swelling)], which can be treated by pharmacological agents, in particular nonsteroidal anti-inflammatory drugs (NSAIDs), whose major action is inhibition of COXs and thus the synthesis of prostaglandins.Much less is known regarding the mechanisms that mediate the late phase of inflammation during which resolution of the tissue infiltration of acute inflammatory cells occurs, but tissue fibrosis can be initiated. Recent efforts have helped identify mechanisms of the resolution of inflammation (Serhan, 2010;Maskrey et al., 2011;Wynn, 2011;Yates et al., 2011). Glucocorticoids, the principal class of drugs used to treat this phase of the inflammatory process, block protein synthesis, thereby decreasing the accumulation of collagens and other ECM proteins. However, the use of glucocorticoids is associated with numerous side effects. Pirfenidone is ...

The small GTP-binding protein ADP ribosylation factor 6 (ARF6) has recently been implicated in the internalization of G proteincoupled receptors (GPCRs), although its precise molecular mechanism in this process remains unclear. We have recently identified centaurin ␣ 1 as a GTPase activating protein (GAP) for ARF6. In the current study, we characterized the effects of centaurin ␣ 1 on the agonist-induced internalization of the ␤ 2 -adrenoceptor transiently expressed in human embryonic kidney (HEK) 293 cells. Using an enzyme-linked immunosorbent assay as well as confocal imaging of cells, we found that expression of centaurin ␣ 1 strongly inhibited the isoproterenolinduced internalization of ␤ 2 -adrenoceptor. On the other hand, expression of functionally inactive versions of centaurin ␣ 1 , including an R49C mutant, which has no catalytic activity, and a double pleckstrin homology (PH) mutant (DM; R148C/ R273C), which has mutations in both the PH domains of centaurin ␣ 1 , rendering it unable to translocate to the cell membrane, were unable to inhibit ␤ 2 -adrenoceptor internalization. In addition, a constitutively active version of ARF6, ARF6Q67L, reversed the ability of centaurin ␣ 1 to inhibit ␤ 2 -adrenoceptor internalization. Finally, expression of centaurin ␣ 1 also inhibited the agonist-induced internalization of ␤ 2 -adrenoceptor endogenously expressed in HEK 293 cells, whereas the R49C and DM mutant versions of centaurin ␣ 1 had no effect. Together, these data indicate that by acting as an ARF6 GAP, centaurin ␣ 1 is able to switch off ARF6 and so inhibit its ability to mediate ␤ 2 -adrenoceptor internalization. Thus, ARF6 GAPs, such as centaurin ␣ 1 , are likely to play a crucial role in GPCR trafficking by modulating the activity of ARF6.

GTPase activating proteins (GAPs) of the centaurin family regulate the actin cytoskeleton and vesicle trafficking through inactivation of the ADP-ribosylation factor (ARF) family of small GTP-binding proteins. We report the functional characterisation of centaurin-␣ 2 , which is structurally related to the centaurin-␣ 1 ARF6 GAP. Centaurin-␣ 2 contains an N-terminal GAP domain followed by two pleckstrin homology (PH) domains (N-PH and C-PH). In vitro, GFP-centaurin-␣ 2 specifically binds the phosphatidylinositol (PI) 3-kinase lipid products, PI 3,4-P 2 and PI 3,4,5-P 3 (PIP 3 ), through its C-terminal PH domain. In agreement with this observation, GFPcentaurin-␣ 2 was recruited to the plasma membrane from the cytosol in EGF-stimulated cells in a PI-3-kinasedependent manner. Moreover, the C-PH domain is sufficient and necessary for membrane recruitment of centaurin-␣ 2 . Centaurin-␣ 2 shows sustained kinetics of PI-3-kinase-mediated membrane recruitment in EGFstimulated cells, owing to its binding to PI 3,4-P 2 . Centaurin-␣ 2 prevents ARF6 translocation to, and cortical actin formation at, the plasma membrane, which are phenotypic indications for ARF6 activation in EGFstimulated cells. Moreover, the constitutively active mutant of ARF6 reverses the effect of centaurin-␣ 2 on cortical actin formation. The membrane targeted centaurin-␣ 2 is constitutively active. Together, these studies indicate that centaurin-␣ 2 is recruited in a sustained manner to the plasma membrane through binding to PI 3,4-P 2 and thereby regulates actin reorganisation via ARF6.

The mechanism of cAMP-promoted apoptosis is not well defined. In wild-type (WT) murine S49 lymphoma cells, cAMP promotes apoptosis in a protein kinase A (PKA)-dependent manner. We find that treatment of WT S49 cells with 8-CPT-cAMP prominently increases the expression (as determined by DNA microarray analysis, real-time PCR and immunblotting) of cytotoxic T lymphocyte antigen-2α (CTLA-2α), a cathepsin L-like cysteine protease inhibitor. By contrast, CTLA-2α expression is only slightly increased by 8-CPT-cAMP treatment of D- S49 cells, which lack cAMP/PKA-promoted apoptosis. Raising endogenous cAMP (by use of forskolin or inhibition of phosphodiesterase [PDE] 4) or a PKA-selective, but not an Epac-selective, cAMP analogue, increases CTLA-2α mRNA expression; PKA, and not Epac, thus mediates the increase in CTLA-2α expression. An adenoviral CLTA-2α (Ad-CTLA-2α) construct induces apoptosis and enhances cAMP-promoted apoptosis in WT S49 cells but such cells do not have an increase in cathepsin L activity nor does a cathepsin L inhibitor alter cAMP-promoted apoptosis. 8-CPT-cAMP also increases CTLA-2α expression and induces apoptosis in murine cardiac fibroblasts; knockdown of CTLA-2α expression by siRNA blocks 8-CPT-cAMP-promoted apoptosis. Thus, cAMP increases CTLA-2α expression in murine lymphoma and cardiac fibroblasts and this increase in CTLA-2α contributes to cAMP/PKA-promoted apoptosis by mechanisms that are independent of the ability of CTLA-2α to inhibit cathepsin L.

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