EPAC1 promotes adaptive responses in human arterial endothelial cells subjected to low levels of laminar fluid shear stress: Implications in flow-related endothelial dysfunction.

Blood flow-associated fluid shear stress (FSS) dynamically regulates the endothelium's ability to control arterial structure and function. While arterial endothelial cells (AEC) subjected to high levels of laminar FSS express a phenotype resistant to vascular insults, those exposed to low levels of laminar FSS, or to the FSS associated with oscillatory blood flow, are less resilient. Despite numerous reports highlighting how the cAMP-signaling system controls proliferation, migration and permeability of human AECs (HAECs), its role in coordinating HAEC responses to FSS has received scant attention. Herein we show that the cAMP effector EPAC1 is required for HAECs to align and elongate in the direction of flow, and for the induction of several anti-atherogenic and anti-thrombotic genes associated with these events. Of potential therapeutic importance, EPAC1 is shown to play a dominant role the in response of HAECs to low levels of laminar FSS, such as would be found within atherosclerosis-prone areas of the vasculature. Moreover, we show that EPAC1 promotes these HAEC responses to flow by regulating Vascular Endothelial Growth Factor Receptor-2 and Akt activation, within a VE-cadherin (VECAD)/PECAM1-based mechanosensor. We submit that these findings are consistent with the novel proposition that promoting EPAC1-signaling represents a novel means through which to promote expression of an adaptive phenotype in HAECs exposed to non-adaptive FSS-encoded signals as a consequence of vascular disease.

Cyclic nucleotide phosphodiesterases (PDEs): coincidence detectors acting to spatially and temporally integrate cyclic nucleotide and non-cyclic nucleotide signals.

The cyclic nucleotide second messengers cAMP and cGMP each affect virtually all cellular processes. Although these hydrophilic small molecules readily diffuse throughout cells, it is remarkable that their ability to activate their multiple intracellular effectors is spatially and temporally selective. Studies have identified a critical role for compartmentation of the enzymes which hydrolyse and metabolically inactivate these second messengers, the PDEs (cyclic nucleotide phosphodiesterases), in this specificity. In the present article, we describe several examples from our work in which compartmentation of selected cAMP- or cGMP-hydrolysing PDEs co-ordinate selective activation of cyclic nucleotide effectors, and, as a result, selectively affect cellular functions. It is our belief that therapeutic strategies aimed at targeting PDEs within these compartments will allow greater selectivity than those directed at inhibiting these enzymes throughout the cells.

Genetic and physical interaction of Ssp1 CaMKK and Rad24 14-3-3 during low pH and osmotic stress in fission yeast.

The Ssp1 calmodulin kinase kinase (CaMKK) is necessary for stress-induced re-organization of the actin cytoskeleton and initiation of growth at the new cell end following division in Schizosaccharomyces pombe. In addition, it regulates AMP-activated kinase and functions in low glucose tolerance. ssp1(-) cells undergo mitotic delay at elevated temperatures and G2 arrest in the presence of additional stressors. Following hyperosmotic stress, Ssp1-GFP forms transient foci which accumulate at the cell membrane and form a band around the cell circumference, but not co-localizing with actin patches. Hyperosmolarity-induced localization to the cell membrane occurs concomitantly with a reduction of its interaction with the 14-3-3 protein Rad24, but not Rad25 which remains bound to Ssp1. The loss of rad24 in ssp1(-) cells reduces the severity of hyperosmotic stress response and relieves mitotic delay. Conversely, overexpression of rad24 exacerbates stress response and concomitant cell elongation. rad24(-) does not impair stress-induced localization of Ssp1 to the cell membrane, however this response is almost completely absent in cells overexpressing rad24.

The karyopherin Sal3 is required for nuclear import of the core RNA interference pathway protein Rdp1.

RNA-dependent RNA polymerase activity is required for RNA interference (RNAi) in many lower eukaryotes including the fission yeast Schizosacchromyces pombe. Together with Ago1 and Dcr1, the RNA-dependent RNA polymerase Rdp1 is critical for RNA-dependent transcriptional- and post-transcriptional gene silencing. Although the bulk of Rdp1 is localized to the nucleus, Ago1 and Dcr1 are primarily cytoplasmic. This may reflect the fact that Rdp1 is required early in the RNAi pathway to generate double strand RNA from transcripts that originate from centromeric loci. The relatively large size of Rdp1 (139.4 kD) precludes passive diffusion of the enzyme into the nucleus suggesting that karyopherin-dependent transport is involved in nuclear targeting of this enzyme. In this study, we report that the karyopherin/importin ß3 homolog Sal3 is required for nuclear import of Rdp1 in S. pombe. Loss of nuclear Rdp1 was associated with substantially reduced transcriptional gene silencing, and surprisingly, post-transcriptional gene silencing which occurs in the cytoplasm of other eukaryotes, was also significantly affected. Together, these results identify Sal3 as a modulator of RNAi-dependent transcriptional gene silencing as well as a potential link between nuclear import and post-transcriptional gene silencing.