cAMP-specific PDE (phosphodiesterase) 4 isoforms underpin compartmentalized cAMP signalling in mammalian cells through targeting to specific signalling complexes. Their importance is apparent as PDE4 selective inhibitors exert profound anti-inflammatory effects and act as cognitive enhancers. The p38 MAPK (mitogen-activated protein kinase) signalling cascade is a key signal transduction pathway involved in the control of cellular immune, inflammatory and stress responses. In the present study, we show that PDE4A5 is phosphorylated at Ser147, within the regulatory UCR1 (ultraconserved region 1) domain conserved among PDE4 long isoforms, by MK2 (MAPK-activated protein kinase 2, also called MAPKAPK2). Phosphorylation by MK2, although not altering PDE4A5 activity, markedly attenuates PDE4A5 activation through phosphorylation by protein kinase A. This modification confers the amplification of intracellular cAMP accumulation in response to adenylate cyclase activation by attenuating a major desensitization system to cAMP. Such reprogramming of cAMP accumulation is recapitulated in wild-type primary macrophages, but not MK2/3-null macrophages. Phosphorylation by MK2 also triggers a conformational change in PDE4A5 that attenuates PDE4A5 interaction with proteins whose binding involves UCR2, such as DISC1 (disrupted in schizophrenia 1) and AIP (aryl hydrocarbon receptor-interacting protein), but not the UCR2-independent interacting scaffold protein β-arrestin. Long PDE4 isoforms thus provide a novel node for cross-talk between the cAMP and p38 MAPK signalling systems at the level of MK2.

Stem cells have been studied for many years for their potential to repair damaged organs in the human body. Although many different mechanisms have been suggested as to how stem cells may initiate and facilitate repair processes, much remains unknown. Recently, there has been considerable interest in the idea that stem cells may exert their effects in vivo via paracrine actions. This could involve the release of cytokines, growth factors or secreted extracellular vesicles. This article reviews the role that paracrine actions may play in tissue regeneration. In particular, it considers how microvesicles, as a mediator or modulator of paracrine action, can be exploited as a tool for non-cell-based therapies in regenerative medicine.

No abstract

A survey of PDE4 inhibitors reveals that some compounds trigger intracellular aggregation of PDE4A4 into accretion foci through association with the ubiquitin-binding scaffold protein p62 (SQSTM1). We show that this effect is driven by inhibitor occupancy of the catalytic pocket and stabilization of a "capped state" in which a sequence within the enzyme's upstream conserved region 2 (UCR2) module folds across the catalytic pocket. Only certain inhibitors cause PDE4A4 foci formation, and the structural features responsible for driving the process are defined. Switching to the UCR2-capped state induces conformational transition in the enzyme's regulatory N-terminal portion, facilitating protein association events responsible for reversible aggregate assembly. PDE4-selective inhibitors able to trigger relocalization of PDE4A4 into foci can therefore be expected to exert actions on cells that extend beyond simple inhibition of PDE4 catalytic activity and that may arise from reconfiguring the enzyme's protein association partnerships.

No abstract

No abstract

Pathfinder cells (PCs), a novel cell type derived from the pancreas of adult rats, have been demonstrated to stimulate recovery of tissue structure and function in two animal models of acute tissue damage to date-streptozotocin (STZ)-induced diabetes and ischemia-reperfusion damage to the kidney. In repaired tissue, PCs and their progeny typically represent only 0.02% of the repaired tissue, suggesting that they act via a paracrine mechanism on native cells in the damaged area. Extracellular vesicles are strong candidates for mediating such a paracrine effect. Therefore, we studied the effects of two PC-derived extracellular vesicle fractions on tissue repair in the STZ diabetes model, one containing primarily microvesicles and the second containing predominantly exosomes. Treatment of STZ-induced diabetic mice with the microvesicles preparation led to blood glucose, insulin, glucagon, and C-peptide levels similar to those found with PC treatment. Furthermore, analysis of the histopathology of the pancreas indicated islet regeneration. In contrast, the exosome fraction demonstrated no repair activity, and STZ diabetic mice treated with exosome preparations had blood glucose values that were indistinguishable from those of vehicle-only treated controls. Therefore, we conclude that exosomes play no part in PC action as detected by this assay, whereas microvesicles provide all or a large component of the paracrine activity of PCs. Because they act to stimulate repair of multiple tissues, PC-derived microvesicles may similarly have the potential to stimulate repair of many damaged tissues, identifying a very significant cell-free therapeutic opportunity in regenerative medicine.

linescanning after 488nm excitation and recording emission at 505-530nm in intact Fluo-3-loaded cardiomyocytes (2uM) at 37 C and at [Ca2þ] 1.2mM and 5.0mM. These studies showed that spontaneous wave frequency was higher at 5.0mM than 1.2mM Ca2þ. Post-MI HF cardiomyocytes had ~twice the wave frequency compared to sham-operated controls. Regular ExTr post-MI improved exercise capacity and induced reverse remodeling. ExTr also reduced the frequency of spontaneous waves at both Ca2þ 1.2mM and 5.0mM, although it did not completely normalize spontaneous Ca2þ waves. ExTr also increased the ratio between aborted and complete waves at Ca2þ 1.2mM, but not Ca2þ 5.0mM. No effects were found on spontaneous wave velocity. This suggests that ExTr partly improved the control of diastolic Ca2þ by reducing the frequency of spontaneous Ca2þ waves and by improving the ability of the cardiomyocyte to eliminate a spontaneous wave after its generation, but before its propagation. Finally, we repeated these studies in the presence of the nitric oxide synthase inhibitor L-NAME, to study the contribution of nitric oxide. This did not have any effects.