Akt Stimulation Research Articles

Emerging follicular activation strategies to treat women with poor ovarian response and primary ovarian insufficiency.

Female reproductive aging remains one of the key unsolved challenges in the field of reproductive medicine. This article reviews three of the most recent and cutting-edge strategies that are currently being investigated to address the issues of poor ovarian response (POR) and primary ovarian insufficiency (POI). Publications revealing the mechanism of mechanical disruption of the Hippo signaling pathway paved the way to studies on its potential application for fertility treatments. This, in combination with Akt stimulation, resulted in live births and ongoing pregnancies in women with POI. Building on previous reports on the effects of bone marrow transplants on fertility after chemotherapy, another approach involved autologous stem cell ovarian transplantation (ASCOT). The method proved effective in achieving live births in women previously diagnosed with POR. A third approach, intraovarian injection of autologous platelet-rich plasma, resulted in live births and ongoing pregnancies both spontaneously and via in vitro fertilization (IVF) in women with POI and POR. New paths are being charted to address the issues of POI and POR. Although these are preliminary studies that should be interpreted with caution, they represent great promise for the women affected by these conditions and the physicians treating them.

Ovarian Fragmentation and AKT Stimulation for Expansion of Fertile Lifespan.

Since the first baby was born after in vitro fertilization, the female infertility treatment has been well-developed, yielding successful outcomes. However, successful pregnancies for patients with premature ovarian insufficiency and diminished ovarian reserve are still difficult and diverse therapies have been suggested to improve the chances to have their genetically linked offspring. Recent studies demonstrated that the activation Akt pathway by using a phosphatase and tensin homolog enzyme inhibitor and a phosphatidylinositol-3 kinase stimulator can activate dormant primordial follicles in both mice and human ovaries. Subsequent researches suggested that the disruption of Hippo signaling pathway by ovarian fragmentation increased the expression of downstream growth factors and secondary follicle growth. Based on the combination of ovarian fragmentation and Akt stimulation, the in vitro activation (IVA) approach has resulted in successful follicle growth and live births in premature ovarian insufficiency patients. The approach with disruption of Hippo signaling only was also shown to be effective for treating poor ovarian responders with diminishing ovarian reserve, including advanced age women and cancer patients undergoing sterilizing treatments. This review aims to summarize the effectiveness of ovarian fragmentation and Akt stimulation on follicle growth and the potential of IVA in extending female fertile lifespan.

The PI3K/Akt pathway is not a main driver in HDL-mediated cell protection

High-density lipoproteins (HDLs) can protect cells against a variety of death-inducing stresses. This is often accompanied by activation of the anti-apoptotic Akt kinase but whether this activation mediates the protective functions of HDLs is still unclear. In this study, we evaluated the roles of PI3K/Akt signaling in endoplasmic reticulum (ER) stress- and starvation-induced cell death using pharmacological and genetic approaches to gain a better understanding of the relationship between Akt- and HDL-mediated protection. Three cell models were used for this purpose, a primary endothelial cell line, an insulinoma cell line and a colon adenocarcinoma cell line. Our results show that HDLs indeed elicited mild Akt activation in all the tested cellular models. PI3K is one of the main upstream proteins involved in Akt stimulation. In the three cellular models, LY294002, a PI3K inhibitor, only slightly blunted HDLs protection, indicating that HDLs induce PI3K-independent cell protection. Furthermore, genetic ablation or silencing of Akt did not abolish the protective effects of HDLs. This study demonstrates that the PI3K-Akt signaling pathway is not the main mediator of the cell protective functions of HDLs. Further investigation is therefore needed to identify the intrinsic mechanism of HDL-mediated cell protection.

Reducing the Levels of Akt Activation by PDK1 Knock-in Mutation Protects Neuronal Cultures against Synthetic Amyloid-Beta Peptides.

The Akt kinase has been widely assumed for years as a key downstream effector of the PI3K signaling pathway in promoting neuronal survival. This notion was however challenged by the finding that neuronal survival responses were still preserved in mice with reduced Akt activity. Moreover, here we show that the Akt signaling is elevated in the aged brain of two different mice models of Alzheimer Disease. We manipulate the rate of Akt stimulation by employing knock-in mice expressing a mutant form of PDK1 (phosphoinositide-dependent protein kinase 1) with reduced, but not abolished, ability to activate Akt. We found increased membrane localization and activity of the TACE/ADAM17 α-secretase in the brain of the PDK1 mutant mice with concomitant TNFR1 processing, which provided neurons with resistance against TNFα-induced neurotoxicity. Opposite to the Alzheimer Disease transgenic mice, the PDK1 knock-in mice exhibited an age-dependent attenuation of the unfolding protein response, which protected the mutant neurons against endoplasmic reticulum stressors. Moreover, these two mechanisms cooperatively provide the mutant neurons with resistance against amyloid-beta oligomers, and might singularly also contribute to protect these mice against amyloid-beta pathology.

Aldosterone stimulates the cardiac sodium/bicarbonate cotransporter via activation of the g protein-coupled receptor gpr30

Some cardiac non-genomic effects of aldosterone (Ald) are reported to be mediated through activation of the classic mineralocorticoid receptor (MR). However, in the last years, it was proposed that activation of the novel G protein-coupled receptor GPR30 mediates certain non-genomic effects of Ald. The aim of this study was to elucidate if the sodium/bicarbonate cotransporter (NBC) is stimulated by Ald and if the activation of GPR30 mediates this effect. NBC activity was evaluated in rat cardiomyocytes perfused with HCO3−/CO2 solution in the continuous presence of HOE642 (sodium/hydrogen exchanger blocker) during recovery from acidosis using intracellular fluorescence measurements. Ald enhanced NBC activity (% of ΔJHCO3−; control: 100±5.82%, n=7 vs Ald: 151.88±11.02%, n=5; P<0.05), which was prevented by G15 (GPR30 blocker, 90.53±7.81%, n=7). Further evidence for the involvement of GPR30 was provided by G1 (GPR30 agonist), which stimulated NBC (185.13±18.28%, n=6; P<0.05) and this effect was abrogated by G15 (124.19±10.96%, n=5). Ald- and G1-induced NBC stimulation was abolished by the reactive oxygen species (ROS) scavenger MPG and by the NADPH oxidase inhibitor apocynin. In addition, G15 prevented Ald- and G1-induced ROS production. Pre-incubation of myocytes with wortmannin (PI3K-AKT pathway blocker) prevented Ald- or G1-induced NBC stimulation. In summary, Ald stimulates NBC by GPR30 activation, ROS production and AKT stimulation.

Differential Phosphatidylinositol-3-Kinase-Akt-mTOR Activation by Semliki Forest and Chikungunya Viruses Is Dependent on nsP3 and Connected to Replication Complex Internalization.

Many viruses affect or exploit the phosphatidylinositol-3-kinase (PI3K)-Akt-mammalian target of rapamycin (mTOR) pathway, a crucial prosurvival signaling cascade. We report that this pathway was strongly activated in cells upon infection with the Old World alphavirus Semliki Forest virus (SFV), even under conditions of complete nutrient starvation. We mapped this activation to the hyperphosphorylated/acidic domain in the C-terminal tail of SFV nonstructural protein nsP3. Viruses with a deletion of this domain (SFV-Δ50) but not of other regions in nsP3 displayed a clearly delayed and reduced capacity of Akt stimulation. Ectopic expression of the nsP3 of SFV wild type (nsP3-wt), but not nsP3-Δ50, equipped with a membrane anchor was sufficient to activate Akt. We linked PI3K-Akt-mTOR stimulation to the intracellular dynamics of viral replication complexes, which are formed at the plasma membrane and subsequently internalized in a process blocked by the PI3K inhibitor wortmannin. Replication complex internalization was observed upon infection of cells with SFV-wt and SFV mutants with deletions in nsP3 but not with SFV-Δ50, where replication complexes were typically accumulated at the cell periphery. In cells infected with the closely related chikungunya virus (CHIKV), the PI3K-Akt-mTOR pathway was only moderately activated. Replication complexes of CHIKV were predominantly located at the cell periphery. Exchanging the hypervariable C-terminal tail of nsP3 between SFV and CHIKV induced the phenotype of strong PI3K-Akt-mTOR activation and replication complex internalization in CHIKV. In conclusion, infection with SFV but not CHIKV boosts PI3K-Akt-mTOR through the hyperphosphorylated/acidic domain of nsP3 to drive replication complex internalization. SFV and CHIKV are very similar in terms of molecular and cell biology, e.g., regarding replication and molecular interactions, but are strikingly different regarding pathology: CHIKV is a relevant human pathogen, causing high fever and joint pain, while SFV is a low-pathogenic model virus, albeit neuropathogenic in mice. We show that both SFV and CHIKV activate the prosurvival PI3K-Akt-mTOR pathway in cells but greatly differ in their capacities to do so: Akt is strongly and persistently activated by SFV infection but only moderately activated by CHIKV. We mapped this activation capacity to a region in nonstructural protein 3 (nsP3) of SFV and could functionally transfer this region to CHIKV. Akt activation is linked to the subcellular dynamics of replication complexes, which are efficiently internalized from the cell periphery for SFV but not CHIKV. This difference in signal pathway stimulation and replication complex localization may have implications for pathology.

The Coordinate Cellular Response to Insulin-like Growth Factor-I (IGF-I) and Insulin-like Growth Factor-binding Protein-2 (IGFBP-2) Is Regulated through Vimentin Binding to Receptor Tyrosine Phosphatase β (RPTPβ)

Insulin-like growth factor-binding protein-2 (IGFBP-2) functions coordinately with IGF-I to stimulate cellular proliferation and differentiation. IGFBP-2 binds to receptor tyrosine phosphatase β (RPTPβ), and this binding in conjunction with IGF-I receptor stimulation induces RPTPβ polymerization leading to phosphatase and tensin homolog inactivation, AKT stimulation, and enhanced cell proliferation. To determine the mechanism by which RPTPβ polymerization is regulated, we analyzed the protein(s) that associated with RPTPβ in response to IGF-I and IGFBP-2 in vascular smooth muscle cells. Proteomic experiments revealed that IGF-I stimulated the intermediate filament protein vimentin to bind to RPTPβ, and knockdown of vimentin resulted in failure of IGFBP-2 and IGF-I to stimulate RPTPβ polymerization. Knockdown of IGFBP-2 or inhibition of IGF-IR tyrosine kinase disrupted vimentin/RPTPβ association. Vimentin binding to RPTPβ was mediated through vimentin serine phosphorylation. The serine threonine kinase PKCζ was recruited to vimentin in response to IGF-I and inhibition of PKCζ activation blocked these signaling events. A cell-permeable peptide that contained the vimentin phosphorylation site disrupted vimentin/RPTPβ association, and IGF-I stimulated RPTPβ polymerization and AKT activation. Integrin-linked kinase recruited PKCζ to SHPS-1-associated vimentin in response to IGF-I and inhibition of integrin-linked kinase/PKCζ association reduced vimentin serine phosphorylation. PKCζ stimulation of vimentin phosphorylation required high glucose and vimentin/RPTPβ-association occurred only during hyperglycemia. Disruption of vimetin/RPTPβ in diabetic mice inhibited RPTPβ polymerization, vimentin serine phosphorylation, and AKT activation in response to IGF-I, whereas nondiabetic mice showed no difference. The induction of vimentin phosphorylation is important for IGFBP-2-mediated enhancement of IGF-I-stimulated proliferation during hyperglycemia, and it coordinates signaling between these two receptor-linked signaling systems.

Akt signaling dynamics in individual cells.

The protein kinase Akt (for which there are three isoforms) is a key intracellular mediator of many biological processes, yet knowledge of Akt signaling dynamics is limited. Here, we have constructed a fluorescent reporter molecule in a lentiviral delivery system to assess Akt kinase activity at the single cell level. The reporter, a fusion between a modified FoxO1 transcription factor and clover, a green fluorescent protein, rapidly translocates from the nucleus to the cytoplasm in response to Akt stimulation. Because of its long half-life and the intensity of clover fluorescence, the sensor provides a robust readout that can be tracked for days under a range of biological conditions. Using this reporter, we find that stimulation of Akt activity by IGF-I is encoded into stable and reproducible analog responses at the population level, but that single cell signaling outcomes are variable. This reporter, which provides a simple and dynamic measure of Akt activity, should be compatible with many cell types and experimental platforms, and thus opens the door to new insights into how Akt regulates its biological responses.

Hippo signaling disruption and Akt stimulation of ovarian follicles for infertility treatment

Primary ovarian insufficiency (POI) and polycystic ovarian syndrome are ovarian diseases causing infertility. Although there is no effective treatment for POI, therapies for polycystic ovarian syndrome include ovarian wedge resection or laser drilling to induce follicle growth. Underlying mechanisms for these disruptive procedures are unclear. Here, we explored the role of the conserved Hippo signaling pathway that serves to maintain optimal size across organs and species. We found that fragmentation of murine ovaries promoted actin polymerization and disrupted ovarian Hippo signaling, leading to increased expression of downstream growth factors, promotion of follicle growth, and the generation of mature oocytes. In addition to elucidating mechanisms underlying follicle growth elicited by ovarian damage, we further demonstrated additive follicle growth when ovarian fragmentation was combined with Akt stimulator treatments. We then extended results to treatment of infertility in POI patients via disruption of Hippo signaling by fragmenting ovaries followed by Akt stimulator treatment and autografting. We successfully promoted follicle growth, retrieved mature oocytes, and performed in vitro fertilization. Following embryo transfer, a healthy baby was delivered. The ovarian fragmentation-in vitro activation approach is not only valuable for treating infertility of POI patients but could also be useful for middle-aged infertile women, cancer patients undergoing sterilizing treatments, and other conditions of diminished ovarian reserve.

Inhibition of mTOR Signaling by Quercetin in Cancer Treatment and Prevention

Quercetin is an abundant micronutrient in our daily diet. Several beneficial health effects are associated with the dietary uptake of this bioflavonoid, including alleviating effects on chronic inflammation and atherosclerosis. A variety of in vitro data indicate a possible use of quercetin for cancer treatment purposes through its interaction with multiple cancer-related pathways. Among these, recent data reveal that quercetin can inhibit mTOR activity in cancer cells. Inhibition of the mTOR signaling pathway by quercetin has directly been described and can further be deduced from its interference with PI3K-dependent Akt stimulation, AMP-dependent protein kinase activation and hamartin upregulation. The ability of quercetin to interfere with both mTOR activity and activation of the PI3K/Akt signaling pathway gives quercetin the advantage to function as a dual-specific mTOR/PI3K inhibitor. The mTOR complex, often hyperactivated in cancer, is a crucial regulator of homeostasis controlling essential pathways leading to cell growth, protein biosynthesis and autophagy. The ability of quercetin to inhibit mTOR activity by multiple pathways makes this otherwise safe bioflavonoid an interesting tool for the treatment of cancers and other diseases associated with mTOR deregulation.

Systematic Analysis Reveals Elongation Factor 2 and α-Enolase as Novel Interaction Partners of AKT2

AKT2 is one of the three isoforms of the protein kinase AKT being involved in the modulation of cellular metabolism. Since protein-protein interactions are one possibility to convey specificity in signal transduction, we performed AKT2-protein interaction analysis to elucidate their relevance for AKT2-dependent cellular functions. We identified heat shock protein 90 kDa (HSP90), Cdc37, heat shock protein 70 kDa (HSP70), 78 kDa glucose regulated protein (GRP78), tubulin, GAPDH, α-enolase and elongation factor 2 (EF2) as AKT2-interacting proteins by a combination of tandem affinity purification and mass spectrometry in HEK293T cells. Quantitative MS-analysis using stable isotope labeling by amino acids in cell culture (SILAC) revealed that only HSP90 and Cdc37 interact stably with AKT2, whereas the other proteins interact with low affinity with AKT2. The interactions of AKT2 with α-enolase and EF2 were further analyzed in order to uncover the functional relevance of these newly discovered binding partners. Despite the interaction of AKT2 and α-enolase, which was additionally validated by proximity ligation assay (PLA), no significant impact of AKT on α-enolase activity was detected in activity measurements. AKT stimulation via insulin and/or inhibition with the ATP-competitive inhibitor CCT128930 did not alter enzymatic activity of α-enolase. Interestingly, the direct interaction of AKT2 and EF2 was found to be dynamically regulated in embryonic rat cardiomyocytes. Treatment with the PI3-kinase inhibitor LY294002 before stimulation with several hormones stabilized the complex, whereas stimulation alone led to complex dissociation which was analyzed in situ with PLA. Taken together, these findings point to new aspects of AKT2-mediated signal transduction in protein synthesis and glucose metabolism.

ANTI-APOPTOTIC EFFECTS OF SUBSTANCE P IN ANTI-FAS INDUCED APOPTOSIS OF HUMAN TENOCYTES

IntroductionSubstance P (SP) and its preferred receptor, the neurokinin-1 receptor (NK-1 R), are expressed in human tenocytes and this is particularly seen in the tenocytes in cases of tendinosis. Hypercellularity,...

Donepezil rescues the medial septum cholinergic neurons via nicotinic ACh receptor stimulation in olfactory bulbectomized mice

Olfactory bulbectomy (OBX) causes cognitive dysfunction by degeneration of cholinergic neurons in the medial septum. Here, we define an involvement of nicotinic acetylcholine receptor (nAChR) in neuroprotective effect of donepezil in the septum neurons of OBX mice. Neuroprotective effects on the medial septal cholinergic neurons were assessed after chronic donepezil administration in OBX mice. We also measured Akt and ERK phosphorylation to define the neuroprotective mechanism of donepezil. We found that treatment with donepezil (1 - 3 mg/kg) for 15 consecutive days completely rescued cholinergic neurons in the OBX mice with concomitant improved memory. Reduction of both protein kinase B (Akt) and extracellular signal-regulated kinase (ERK) phosphorylation were restored by chronic donepezil administration (1 - 3 mg/kg) in OBX mouse medial septum. Both phosphorylated Akt and ERK immunoreactivities were localized in cell bodies of choline acetyltransferase (ChAT)-positive cholinergic cells in the medial septum. Enhancement of Akt and ERK phosphorylation seen following donepezil administration was totally blocked by pre-administration of mecamylamine (10 μM), a nicotinic acetylcholine receptor antagonist. Donepezil increases phosphorylation of Akt and ERK via nAChR stimulation in the medial septum cholinergic neurons. The Akt and ERK stimulation by donepezil is associated with its ability of neuroprotection in the medial septum and memory improvement.

Suppression of EGFR endocytosis by dynamin depletion reveals that EGFR signaling occurs primarily at the plasma membrane

The role of endocytosis in the control of EGF receptor (EGFR) activation and cell signaling was explored by using mouse fibroblasts in which dynamin was conditionally depleted. Dynamin is a GTPase shown to play an important role in the control clathrin mediated endocytosis of EGFR and other cell surface receptors. In this report, we demonstrate that EGF binding activity and the display of high and low affinity EGFRs on the cell surface are not affected by dynamin depletion. By contrast, dynamin depletion leads to a strong inhibition of EGFR endocytosis, robust enhancement of EGFR autophosphorylation and ubiquitination, and slower kinetics of EGFR degradation. Surprisingly, MAPK stimulation induced by either low or high EGF concentrations is not affected by dynamin depletion. While a similar initial Akt response is detected in control or dynamin depleted fibroblasts, a somewhat more sustained Akt stimulation is detected in the dynamin depleted cells. These experiments demonstrate that dynamin-mediated endocytosis leads to attenuation of EGFR activation and degradation and that stimulation of the MAPK response and Akt activation are primarily mediated by activated EGFR located in the plasma membrane.

Akt2 interacts with Snail1 in the E-cadherin promoter

Snail1 is a transcriptional factor essential for triggering epithelial-to-mesenchymal transition. Moreover, Snail1 promotes resistance to apoptosis, an effect associated to PTEN gene repression and Akt stimulation. In this article we demonstrate that Snail1 activates Akt at an additional level, as it directly binds to and activates this protein kinase. The interaction is observed in the nucleus and increases the intrinsic Akt activity. We determined that Akt2 is the isoform interacting with Snail1, an association that requires the pleckstrin homology domain in Akt2 and the C-terminal half in Snail1. Snail1 enhances the binding of Akt2 to the E-cadherin (CDH1) promoter and Akt2 interference prevents Snail1 repression of CDH1 gene. We also show that Snail1 binding increases Akt2 intrinsic activity on histone H3 and have identified Thr45 as a residue modified on this protein. Phosphorylation of Thr45 in histone H3 is sensitive to Snail1 and Akt2 cellular levels; moreover, Snail1 upregulates the binding of phosphoThr45 histone H3 to the CDH1 promoter. These results uncover an unexpected role of Akt2 in transcriptional control and point out to phosphorylation of Thr45 in histone H3 as a new epigenetic mark related to Snail1 and Akt2 action.

Thrombin activation of PI3K/PDK1/Akt signaling promotes cyclin D1 upregulation and RPE cell proliferation

The retinal pigment epithelium (RPE) plays an essential role in the survival and function of the neural retina. RPE uncontrolled proliferation leads to the development of proliferative ocular pathologies, among which proliferative vitreoretinopathy (PVR) is the main cause of retinal surgery failure. Upon the breakdown of the BRB due to trauma or metabolic imbalance the contact of RPE with serum-contained thrombin has been shown to stimulate the proliferation of otherwise quiescent RPE cells. Although the molecular mechanisms involved in this effect are still undetermined, thrombin proteolytic activation of protease-activated G protein coupled receptor-1 (PAR-1) activates PI3K and Akt, known to play an essential role in proliferation. The present study demonstrates that: 1) thrombin stimulates Ser 473 Akt phosphorylation without affecting Thr 308 basal phosphorylation in RPE cells; 2) thrombin-induced Akt stimulation promotes cyclin D1 accumulation through the phosphorylation/ inhibition of GSK-3β, thus preventing Thr 286 cyclin D1 phosphorylation, nuclear export and degradation; 3) Akt signaling requires the upstream activation of PI3K and PLC. Since the pharmacological inhibition of these pathways or the silencing of cyclin expression prevent thrombin-induced RPE cell proliferation, these results contribute relevant evidence for establishing the mechanism involved in the development of proliferative eye diseases.

Arrestin-2 Differentially Regulates PAR4 and ADP Receptor Signaling in Platelets

Arrestins can facilitate desensitization or signaling by G protein-coupled receptors (GPCR) in many cells, but their roles in platelets remain uncharacterized. Because of recent reports that arrestins can serve as scaffolds to recruit phosphatidylinositol-3 kinases (PI3K)s to GPCRs, we sought to determine whether arrestins regulate PI3K-dependent Akt signaling in platelets, with consequences for thrombosis. Co-immunoprecipitation experiments demonstrate that arrestin-2 associates with p85 PI3Kα/β subunits in thrombin-stimulated platelets, but not resting cells. The association is inhibited by inhibitors of P2Y12 and Src family kinases (SFKs). The function of arrestin-2 in platelets is agonist-specific, as PAR4-dependent Akt phosphorylation and fibrinogen binding were reduced in arrestin-2 knock-out platelets compared with WT controls, but ADP-stimulated signaling to Akt and fibrinogen binding were unaffected. ADP receptors regulate arrestin recruitment to PAR4, because co-immunoprecipitates of arrestin-2 with PAR4 are disrupted by inhibitors of P2Y1 or P2Y12. P2Y1 may regulate arrestin-2 recruitment to PAR4 through protein kinase C (PKC) activation, whereas P2Y12 directly interacts with PAR4 and therefore, may help to recruit arrestin-2 to PAR4. Finally, arrestin2(-/-) mice are less sensitive to ferric chloride-induced thrombosis than WT mice, suggesting that arrestin-2 can regulate thrombus formation in vivo. In conclusion, arrestin-2 regulates PAR4-dependent signaling pathways, but not responses to ADP alone, and contributes to thrombus formation in vivo.

Essential role of HDL on endothelial progenitor cell proliferation with PI3K/Akt/cyclin D1 as the signal pathway

High-density lipoprotein (HDL) is known as an important factor in vascular wall remodeling that also affects gene expression in cell proliferation and differentiation. In this article, the role of HDL on endothelial progenitor cell (EPC) proliferation, angiogenesis and the signal pathway involved was studied, particularly the influence of HDL in strengthening the promoting effect of EPCs on wound healing of the arterial wall in hypercholesterolemic rats. Mononuclear cells isolated from rat bone marrow displayed characteristics of EPCs after cultivation. The role of HDL on EPC function and the signal pathway involved were studied by Western blotting, in vitro migration and 'tube' formation. Re-endothelialization and the number of circulating EPCs were compared between normal rats, hypercholesterolemic rats and hypercholesterolemic rats with HDL treatment. Results showed that HDL participated in the healing process by promoting EPC proliferation, migration and 'tube' formation. HDL activates cyclin D1 via phosphatidylinositol 3-kinase (PI3K)/Akt stimulation. Inhibition of PI3K/Akt via pharmacological or small interfering RNA approaches significantly attenuated HDL-induced EPC migration, proliferation and 'tube' formation. Results of experiments in vivo showed that HDL increased the number of circulating EPCs and promoted re-endothelialization in wound healing. These findings demonstrate for the first time that PI3K/Akt-dependent cyclin D1 activation plays an essential role in HDL-induced EPC proliferation, migration and angiogenesis.

Regulation of G-Protein Signaling by RKTG via Sequestration of the Gβγ Subunit to the Golgi Apparatus

Upon ligand binding, G-protein-coupled receptors (GPCRs) impart the signal to heterotrimeric G proteins composed of alpha, beta, and gamma subunits, leading to dissociation of the G alpha subunit from the G betagamma subunit. While the G alpha subunit is imperative for downstream signaling, the G betagamma subunit, in its own right, mediates a variety of cellular responses such as GPCR desensitization via recruiting GRK to the plasma membrane and AKT stimulation. Here we report a mode of spatial regulation of the G betagamma subunit through alteration in subcellular compartmentation. RKTG (Raf kinase trapping to Golgi apparatus) is a newly characterized membrane protein specifically localized at the Golgi apparatus. The N terminus of RKTG interacts with G beta and tethers G betagamma to the Golgi apparatus. Overexpression of RKTG impedes the interaction of G betagamma with GRK2, abrogates the ligand-induced change of subcellular distribution of GRK2, reduces isoproterenol-stimulated phosphorylation of the beta2-adrenergic receptor (beta 2AR), and alters beta 2AR desensitization. In addition, RKTG inhibits G betagamma- and ligand-mediated AKT phosphorylation that is enhanced in cells with downregulation of RKTG. Silencing of RKTG also alters GRK2 internalization and compromises ligand-induced G beta translocation to the Golgi apparatus. Taken together, our results reveal that RKTG can modulate GPCR signaling through sequestering G betagamma to the Golgi apparatus and thereby attenuating the functions of G betagamma.

α2-Adrenergic Receptors Attenuate Secretagogue-Induced Endocytosis and Promote Exocytosis of Intestinal NHE2 and NHE3

Adrenergic agonists, through activation of intestinal epithelial alpha2-adrenergic receptors (alpha2AR), inhibit electrolyte secretion and promote absorption. The mechanisms of action to promote basal Na(+) absorption and inhibit stimulated secretion are not understood completely. The effects of alpha2-agonists on Na(+) transport were studied in a cell line, Caco2-3B, derived from the Caco2 cell line engineered to permanently express human alpha2A-adrenergic receptors. Serosal, but not mucosal, addition of the alpha2AR agonist N-(2,6-dichlorophenyl)-4,5-dihydro-1H-imidazol-2-amine (clonidine) increased Caco2-3B apical (22)Na(+) uptake, an effect not seen in the Caco2 parent line that lacks alpha2AR expression. This effect was blocked by the alpha2AR antagonist 17alpha-yohmban-16alpha-carboxylic acid methyl ester (yohimbine). Increased Na(+) uptake was paralleled by increased apical surface abundance of the sodium/hydrogen exchangers NHE2 and NHE3. No changes in total cell NHE2 and NHE3 expression were observed. Clonidine also inhibited both cAMP and Ca(2+)-induced decreases in apical Na(+) uptake and apical membrane NHE2 and NHE3 endocytosis stimulated by these agents. alpha2AR actions were mediated via stimulation of phospholipase C, and metabolism of arachidonic acid by an epoxygenase activity followed epidermal growth factor release and activation of the epidermal growth factor receptor, resulting in phosphatidylinositol-3-kinase and Akt stimulation. In summary, activation of intestinal epithelial alpha2AR significantly blocks the inhibition of apical Na(+) transporters by cAMP- and Ca(2+)-mediated pathways and also directly increases apical sodium/hydrogen exchange activities. By both blocking electrolyte secretion and promoting absorption, alpha2-agonists could be potent antidiarrheal agents that could directly counteract the actions of toxigenic pathogens and other secretagogues causing secretory diarrhea.