Mark Harbeck

Cell physiology of cAMP sensor Epac.

Epac is an acronym for the exchange proteins activated directly by cyclic AMP, a family of cAMP‐regulated guanine nucleotide exchange factors (cAMPGEFs) that mediate protein kinase A (PKA)‐independent signal transduction properties of the second messenger cAMP. Two variants of Epac exist (Epac1 and Epac2), both of which couple cAMP production to the activation of Rap, a small molecular weight GTPase of the Ras family. By activating Rap in an Epac‐mediated manner, cAMP influences diverse cellular processes that include integrin‐mediated cell adhesion, vascular endothelial cell barrier formation, and cardiac myocyte gap junction formation. Recently, the identification of previously unrecognized physiological processes regulated by Epac has been made possible by the development of Epac‐selective cyclic AMP analogues (ESCAs). These cell‐permeant analogues of cAMP activate both Epac1 and Epac2, whereas they fail to activate PKA when used at low concentrations. ESCAs such as 8‐pCPT‐2′‐O‐Me‐cAMP and 8‐pMeOPT‐2′‐O‐Me‐cAMP are reported to alter Na+, K+, Ca2+ and Cl− channel function, intracellular [Ca2+], and Na+–H+ transporter activity in multiple cell types. Moreover, new studies examining the actions of ESCAs on neurons, pancreatic beta cells, pituitary cells and sperm demonstrate a major role for Epac in the stimulation of exocytosis by cAMP. This topical review provides an update concerning novel PKA‐independent features of cAMP signal transduction that are likely to be Epac‐mediated. Emphasized is the emerging role of Epac in the cAMP‐dependent regulation of ion channel function, intracellular Ca2+ signalling, ion transporter activity and exocytosis.

A cAMP and Ca2+ coincidence detector in support of Ca2+-induced Ca2+ release in mouse pancreatic β cells

The blood glucose‐lowering hormone glucagon‐like peptide‐1 (GLP‐1) stimulates cAMP production, promotes Ca2+ influx, and mobilizes an intracellular source of Ca2+ in pancreatic β cells. Here we provide evidence that these actions of GLP‐1 are functionally related: they reflect a process of Ca2+‐induced Ca2+ release (CICR) that requires activation of protein kinase A (PKA) and the Epac family of cAMP‐regulated guanine nucleotide exchange factors (cAMPGEFs). In rat insulin‐secreting INS‐1 cells or mouse β cells loaded with caged Ca2+ (NP‐EGTA), a GLP‐1 receptor agonist (exendin‐4) is demonstrated to sensitize intracellular Ca2+ release channels to stimulatory effects of cytosolic Ca2+, thereby allowing CICR to be generated by the uncaging of Ca2+ (UV flash photolysis). This sensitizing action of exendin‐4 is diminished by an inhibitor of PKA (H‐89) or by overexpression of dominant negative Epac. It is reproduced by cell‐permeant cAMP analogues that activate PKA (6‐Bnz‐cAMP) or Epac (8‐pCPT‐2′‐O‐Me‐cAMP) selectively. Depletion of Ca2+ stores with thapsigargin abolishes CICR, while inhibitors of Ca2+ release channels (ryanodine and heparin) attenuate CICR in an additive manner. Because the uncaging of Ca2+ fails to stimulate CICR in the absence of cAMP‐elevating agents, it is concluded that there exists in β cells a process of second messenger coincidence detection, whereby intracellular Ca2+ release channels (ryanodine receptors, inositol 1,4,5‐trisphosphate (IP3) receptors) monitor a simultaneous increase of cAMP and Ca2+ concentrations. We propose that second messenger coincidence detection of this type may explain how GLP‐1 interacts with β cell glucose metabolism to stimulate insulin secretion.

Synthetic Analogs of FTY720 [2-Amino-2-(2-[4-octylphenyl]ethyl)-1,3-propanediol] Differentially Regulate Pulmonary Vascular Permeability in Vivo and in Vitro

Novel therapies are needed to address the vascular endothelial cell (EC) barrier disruption that occurs in inflammatory diseases such as acute lung injury (ALI). We previously demonstrated the potent barrier-enhancing effects of both sphingosine 1-phosphate (S1P) and the structurally similar compound FTY720 [2-amino-2-(2-[4-octylphenyl]ethyl)-1,3-propanediol] in inflammatory lung injury. In this study, we examined the therapeutic potential of several novel FTY720 analogs to reduce vascular leak. Similar to S1P and FTY720, the (R)- and (S)-enantiomers of FTY720 phosphonate and enephosphonate analogs produce sustained EC barrier enhancement in vitro, as seen by increases in transendothelial electrical resistance (TER). In contrast, the (R)- and (S)-enantiomers of FTY720-regioisomeric analogs disrupt EC barrier integrity in a dose-dependent manner. Barrier-enhancing FTY720 analogs demonstrate a wider protective concentration range in vitro (1–50 μM) and greater potency than either S1P or FTY720. In contrast to FTY720-induced EC barrier enhancement, S1P and the FTY720 analogs dramatically increase TER within minutes in association with cortical actin ring formation. Unlike S1P, these FTY720 analogs exhibit differential phosphorylation effects without altering the intracellular calcium level. Inhibitor studies indicate that barrier enhancement by these analogs involves signaling via Gi-coupled receptors, tyrosine kinases, and lipid rafts. Consistent with these in vitro responses, the (S)-phosphonate analog of FTY720 significantly reduces multiple indices of alveolar and vascular permeability in a lipopolysaccharide-mediated murine model of ALI (without significant alterations in leukocyte counts). These results demonstrate the capacity for FTY720 analogs to significantly decrease pulmonary vascular leakage and inflammation in vitro and in vivo.

Simultaneous Optical Measurements of Cytosolic Ca2+ and cAMP in Single Cells

Understanding the temporal and spatial integration of the Ca2+ and adenosine 3′,5′-monophosphate (cAMP) signaling pathways requires concurrent measurements of both second messengers. Here, we describe an optical technique to simultaneously image cAMP and Ca2+ concentration gradients in MIN6 mouse insulinoma cells using Epac1-camps, a Förster (or fluorescence) resonance energy transfer (FRET)-based cAMP biosensor, and Fura-2, a fluorescent indicator of Ca2+. This real-time imaging method allows investigation of the dynamic organization and integration of multiple levels of signal processing in single living cells.

MicroRNA regulation of integrins.

MicroRNAs (miRNAs) are a family of small RNAs that are ∼20 nucleotides in length and are nontranslated. To date, more than 700 miRNAs have been identified, and their involvement in many essential cellular processes is now apparent. By binding with target messenger RNAs (mRNA), miRNAs are able to regulate both mRNA stability and mRNA translational efficiency. Integrins are a family of transmembrane proteins that both regulate cell matrix interactions and serve as receptors that mediate intracellular signaling and a variety of cellular processes, including inflammatory responses, immunoresponses, and tumorigenesis. Integrin expression may also be regulated by miRNAs, which can also modulate integrin signaling and function. Integrins are heterodimer adhesion proteins comprised of an α and a β subunit. Cumulatively, there are 18 α subunits and 8 β subunits that can combine to form 24 distinct αβ receptor complexes. In addition, each integrin can be classified into 1 of 4 groups based on its extracellular binding ligand: collagen, laminin, RGD (Arg-Gly-Asp) or leukocyte-specific receptors. Collagen ligand integrins include integrins α1 and α2 subunits, known to be regulated by specific miRNAs. Among the laminin ligand integrins, there are no integrin α subunits known to be regulated by miRNA. As for the RGD ligand integrins, integrin α5 is the only α subunit found to be regulated by miRNAs (miR-31, miR-17-92 cluster, and miR-148 b). Finally, among the α subunits that comprise the leukocyte-specific receptor ligand integrins, integrins αD, αL, αM, and αX have shown regulation by different miRNAs. As for the integrin β subunits, regulation by miRNAs has been reported for all but β5 and β6 to date. However, computational predictions suggest that numerous miRNAs potentially regulate a variety of target integrins. These predictions will undoubtedly guide future investigations of mechanisms underlying integrin expression mechanism and may ultimately yield new therapeutic tools.

Serum- and Glucocorticoid-induced Protein Kinase 1 (SGK1) is Regulated by Store-Operated Ca2+ Entry and Mediates Cytoprotection Against Necrotic Cell Death

Background: Mechanisms underlying SGK1 activation are incompletely understood in epithelial cells. Results: Store-operated Ca2+ entry up-regulates SGK1, thereby modulating the lethal effects of Ca2+ overloading on mitochondrial membrane potential. Conclusion: Ca2+-induced SGK1 activates cytoprotective signaling and modifies mitochondrial function in epithelial cells. Significance: This work reveals a cytoprotective role for SGK1 in necrosis and has potential relevance for epithelial cell protection and cancer treatment. Serum and glucocorticoid-regulated kinase 1 (SGK1) encodes a phosphatidylinositol 3-kinase-dependent serine/threonine kinase that is rapidly induced in response to cellular stressors and is an important cell survival signal. Previous studies have suggested that an increase in cytoplasmic Ca2+ concentration ([Ca2+]c) is required for increased SGK1 expression, but the subcellular source of Ca2+ regulating SGK1 transcription remains uncertain. Activation of endoplasmic reticulum stress (ERS) with thapsigargin (TG) increased SGK1 mRNA and protein expression in MDA-MB-231 cells. Intracellular Ca2+ imaging revealed that store-operated Ca2+ entry played a prominent role in SGK1 induction by TG. Neither ERS nor release of Ca2+ from the ER was sufficient to activate SGK1. Prolonged elevation of intracellular Ca2+ levels, however, triggered cell death with a much greater proportion of the cells undergoing necrosis rather than apoptosis. A relative increase in the percentage of cells undergoing necrosis was observed in cells expressing a short hairpin RNA targeted to the SGK1 gene. Necrotic cell death evoked by cytoplasmic Ca2+ overloading was associated with persistent hyperpolarization of the inner mitochondrial membrane and a modest increase in calpain activation, but did not involve detectable caspase 3 or caspase 7 activation. The effects of cytoplasmic Ca2+ overloading on mitochondrial membrane potential were significantly reduced in cells expressing SGK1 compared with SGK1-depleted cells. Our findings indicate that store-operated Ca2+ entry regulates SGK1 expression in epithelial cells and suggest that SGK1-dependent cytoprotective signaling involves effects on maintaining mitochondrial function.

Serine 1179 Phosphorylation of Endothelial Nitric Oxide Synthase Increases Superoxide Generation and Alters Cofactor Regulation.

Endothelial nitric oxide synthase (eNOS) is responsible for maintaining systemic blood pressure, vascular remodeling and angiogenesis. In addition to producing NO, eNOS can also generate superoxide (O2 -.) in the absence of the cofactor tetrahydrobiopterin (BH4). Previous studies have shown that bovine eNOS serine 1179 (Serine 1177/human) phosphorylation critically modulates NO synthesis. However, the effect of serine 1179 phosphorylation on eNOS superoxide generation is unknown. Here, we used the phosphomimetic form of eNOS (S1179D) to determine the effect of S1179 phosphorylation on superoxide generating activity, and its sensitivity to regulation by BH4, Ca2+, and calmodulin (CAM). S1179D eNOS exhibited significantly increased superoxide generating activity and NADPH consumption compared to wild-type eNOS (WT eNOS). The superoxide generating activities of S1179D eNOS and WT eNOS did not differ significantly in their sensitivity to regulation by either Ca2+ or CaM. The sensitivity of the superoxide generating activity of S1179D eNOS to inhibition by BH4 was significantly reduced compared to WT eNOS. In eNOS-overexpressing 293 cells, BH4 depletion with 10mM DAHP for 48 hours followed by 50ng/ml VEGF for 30 min to phosphorylate eNOS S1179 increased ROS accumulation compared to DAHP-only treated cells. Meanwhile, MTT assay indicated that overexpression of eNOS in HEK293 cells decreased cellular viability compared to control cells at BH4 depletion condition (P<0.01). VEGF-mediated Serine 1179 phosphorylation further decreased the cellular viability in eNOS-overexpressing 293 cells (P<0.01). Our data demonstrate that eNOS serine 1179 phosphorylation, in addition to enhancing NO production, also profoundly affects superoxide generation: S1179 phosphorylation increases superoxide production while decreasing sensitivity to the inhibitory effect of BH4 on this activity.

Novel Mechanism for Nicotinamide Phosphoribosyltransferase Inhibition of TNF-α–mediated Apoptosis in Human Lung Endothelial Cells

&NA; Nicotinamide phosphoribosyltransferase (NAMPT) exists as both intracellular NAMPT and extracellular NAMPT (eNAMPT) proteins. eNAMPT is secreted into the blood and functions as a cytokine/enzyme (cytozyme) that activates NF‐&kgr;B signaling via ligation of Toll‐like receptor 4 (TLR4), further serving as a biomarker for inflammatory lung disorders such as acute respiratory distress syndrome. In contrast, intracellular NAMPT is involved in nicotinamide mononucleotide synthesis and has been implicated in the regulation of cellular apoptosis, although the exact mechanisms for this regulation are poorly understood. We examined the role of NAMPT in TNF‐&agr;‐induced human lung endothelial cell (EC) apoptosis and demonstrated that reduced NAMPT expression (siRNA) increases EC susceptibility to TNF‐&agr;‐induced apoptosis as reflected by PARP‐1 cleavage and caspase‐3 activation. In contrast, overexpression of NAMPT served to reduce degrees of TNF‐&agr;‐induced EC apoptosis. Inhibition of nicotinamide mononucleotide synthesis by FK866 (a selective NAMPT enzymatic inhibitor) failed to alter TNF‐&agr;‐induced human lung EC apoptosis, suggesting that NAMPT‐dependent NAD+ generation is unlikely to be involved in regulation of TNF‐&agr;‐induced EC apoptosis. We next confirmed that TNF‐&agr;‐induced EC apoptosis is attributable to NAMPT secretion into the EC culture media and subsequent eNAMPT ligation of TLR4 on the EC membrane surface. Silencing of NAMPT expression, direct neutralization of secreted eNAMPT by an NAMPT‐specific polyclonal antibody (preventing TLR4 ligation), or direct TLR4 antagonism all served to significantly increase EC susceptibility to TNF‐&agr;‐induced EC apoptosis. Together, these studies provide novel insights into NAMPT contributions to lung inflammatory events and to novel mechanisms of EC apoptosis regulation.