PDE4 inhibitors activate a mitochondrial apoptotic pathway in chronic lymphocytic leukemia cells that is regulated by protein phosphatase 2A

Chronic lymphocytic leukemia (CLL) cells, but not peripheral blood T cells, undergo apoptosis following treatment with inhibitors of type 4 cyclic nucleotide phosphodiesterase (PDE4), a process that correlates dose dependently with elevation of adenosine 3',5'-cyclic monophosphate (cAMP) in leukemic cells. We show that treatment of CLL cells with rolipram, a prototypic PDE4 inhibitor, and forskolin, an adenylate cyclase activator, induces mitochondrial depolarization, release of cytochrome c into the cytosol, caspase-9 and -3 activation, and cleavage of poly(adenosine diphosphate [ADP]-ribose)polymerase. Inhibitors of caspase-9, but not caspase-8, block rolipram/forskolin-induced CLL apoptosis. In a subset of CLL patients, B-cell lymphoma 2 (Bcl-2)-associated death promoter homolog (Bad), a proapoptotic Bcl-2 family member that when phosphorylated on specific serine residues is sequestered in the cytosol by 14-3-3, was dephosphorylated at Ser112 following rolipram/forskolin treatment of leukemic cells. Rolipram/forskolin treatment also induced Bad to accumulate in CLL heavy-membrane fractions, consistent with Bad translocation to mitochondria. To determine the mechanism for rolipram/forskolin-induced Bad dephosphorylation, we examined CLL phosphatase activity. Rolipram/forskolin treatment augmented protein phosphatase 2A (PP2A) activity, as well as levels of immunoreactive PP2A catalytic subunit. Treatment of CLL cells with a concentration of okadaic acid (5 nM) that selectively inhibits PP2A, reduced both rolipram/forskolin-induced mitochondrial cytochrome c release and mitochondrial depolarization. Okadaic acid restored Bad Ser112 phosphorylation and Bad association with 14-3-3 in rolipram/forskolin-treated CLL cells. These results suggest that PDE4 inhibitors may induce CLL apoptosis by activating PP2A-induced dephosphorylation of proapoptotic BH3-only Bcl-2 family members such as Bad.     

Insulin-mediated stimulation of protein kinase Akt: A potent survival signaling cascade for endothelial cells

Insulin exerts potent antiapoptotic effects in neuronal cells and has been suggested to promote angiogenesis. Therefore, we investigated whether insulin inhibits tumor necrosis factor-alpha (TNF-alpha)-induced apoptosis in human umbilical vein endothelial cells (HUVECs). Because insulin has been shown to stimulate the protein kinase Akt, we investigated whether activation of Akt contributes to the apoptosis-suppressive effect of insulin and characterized the downstream signaling pathway. Incubation with insulin dose-dependently prevented apoptosis induced by TNF-alpha (50 ng/mL). The extent of apoptosis suppression by insulin was similar to the effect of vascular endothelial growth factor. Pharmacological inhibition of Akt activation or overexpression of a dominant-negative Akt mutant prevented the antiapoptotic effect of insulin. Furthermore, we investigated the effect of TNF-alpha on Akt phosphorylation by Western blot analysis with the use of a phosphospecific Akt antibody. Incubation of HUVECs with TNF-alpha induced a marked dephosphorylation of Akt. Insulin counteracted this TNF-alpha-induced dephosphorylation of Akt. Furthermore, we investigated the downstream signaling events. Akt has been shown to mediate its apoptosis-suppressive effects via phosphorylation of Bad or caspase-9. However, incubation with insulin did not lead to enhanced phosphorylation of Bad at Ser 136 or Ser 112. In contrast, insulin inhibited caspase-9 activity and prevented caspase-9-induced apoptosis. Mutation of the Akt site within caspase-9 significantly reduced the apoptosis-suppressive effect of insulin. The present study demonstrates an important role for insulin-mediated Akt activation in the prevention of endothelial cell apoptosis, which may importantly contribute to cell homeostasis and the integrity of the endothelium. In endothelial cells, Akt seems to mediate its antiapoptotic effect, at least in part, via phosphorylation of caspase-9 rather than Bad.     

Aging enhances the sensitivity of endothelial cells toward apoptotic stimuli: important role of nitric oxide

Advanced aging leads to impaired endothelial NO synthesis and enhanced endothelial cell apoptosis; therefore, we investigated the sensitivity of aged endothelial cells toward apoptotic stimuli and determined the role of NO. Human umbilical vein endothelial cells (HUVECs) were cultured until 14th passage. In aged cells, oxLDL and tumor necrosis factor-alpha-induced apoptosis and caspase-3-like activity were significantly enhanced more than 3-fold compared with young cells (passage 3). Because NO contributes to protection against endothelial cell death via S-nitrosylation of caspases, we determined endothelial NO synthase (eNOS) protein expression and the content of S-nitrosylated proteins. Aged HUVECs showed significantly reduced eNOS expression (35+/-10%) and a decrease in the overall S-NO content (33+/-3%), suggesting that eNOS downregulation may be involved in age-dependent increase of apoptosis sensitivity. Indeed, eNOS knockout endothelial cells showed a significantly enhanced apoptosis induction. Exogenous NO donors abolished increased apoptosis and caspase-3-like activity. In contrast, the application of shear stress, which exerts a profound apoptosis inhibitory effect via upregulation of NO synthesis in young cells, failed to inhibit apoptosis in aged cells. Moreover, no upregulation of eNOS protein expression and S-NO content in response to shear stress was detected in aged cells. Overexpression of wild-type eNOS completely restored the antiapoptotic effect of shear stress, whereas only a partial inhibitory effect was detected under steady conditions. Strikingly, transfection of constitutively active phosphomimetic eNOS (S1177D) further abrogated apoptosis in aged HUVECs. Thus, aging of endothelial cells is associated with decreased NO synthesis and concomitantly increased sensitivity of apoptosis, which may contribute to functional impairment of the endothelial monolayer.     

Vascular cell adhesion molecule-1 (CD106) is cleaved by neutrophil proteases in the bone marrow following hematopoietic progenitor cell mobilization by granulocyte colony-stimulating factor

BAD is a proapoptotic member of the BCL-2 family of proteins, which play a major role in regulating apoptosis in cytokine-dependent hematopoietic cells. The function of BAD is regulated by reversible phosphorylation. Deprivation of survival factors induces BAD dephosphorylation, resulting in apoptosis. Serine-threonine phosphatase activity dephosphorylated BAD in interleukin-3-dependent FL5.12 lymphoid cells. Inhibition of PP2A activity by treatment of cells with PP2A-selective inhibitors, okadaic acid and fostriecin, prevented BAD dephosphorylation in these cells. Conversely, BAD dephosphorylation was not inhibited by the PP1-selective inhibitor tautomycin. In cell-free extracts, BAD phosphatase activity was also inhibited by the PP2A-selective inhibitors okadaic acid and fostriecin, but not by the PP1-specific protein inhibitor I-2. Dissociation of 14-3-3 from BAD was a prerequisite for BAD dephosphorylation in vitro, suggesting a mechanism by which 14-3-3 can regulate the activation of the proapoptotic function of BAD in vivo. Significantly, the inhibition of BAD phosphatase activity rescued cell death induced by survival factor withdrawal in FL5.12 cells expressing wild-type BAD but not phosphorylation-defective mutant BAD. These data indicate that PP2A, or a PP2A-like enzyme, dephosphorylates BAD and, in conjunction with 14-3-3, modulates cytokine-mediated survival.     

Gatekeeper role of 14-3-3tau protein in HIV-1 gp120-mediated apoptosis of human endothelial cells by inactivation of Bad

OBJECTIVE: HIV-1-associated dementia (HAD) is a major neurological complication often observed in the advanced stages of AIDS. We have reported that 14-3-3 proteins in cerebrospinal fluid, reflecting neuronal cell destruction, is a real-time marker of HAD progression. This study was designed to examine the role of 14-3-3 proteins in HAD. DESIGN: An in-vitro human umbilical vein endothelial cells (HUVEC) model of gp120 protein-induced apoptosis to study the protective role of 14-3-3 in HIV-1 gp120/CXCR4-mediated cell death. METHODS: The alpha-chemokine receptor-mediated cell death by HIV-1 envelope protein, gp120, the critical event that causes neuron loss and endothelial cell injury, was evaluated in HUVEC undergoing gp120-induced apoptosis through the CXCR4 receptor. We studied the effects of siRNA for each 14-3-3 isoform on the death of HUVEC treated with CXCR4-preferring gp120 (IIIB). RESULTS: Gp120 increased the expression of 14-3-3tau in HUVEC. The binding of Gp120 to CXCR4 induced apoptosis of HUVEC through decreased binding of 14-3-3tau to the pro-apoptotic molecule, Bad. Treatment of the cells with dsRNA against 14-3-3tau enhanced the gp120-mediated dephosphorylation of Bad and its association with Bcl-XL in mitochondria, accelerating the gp120-induced apoptosis, whereas suppression of Bad by RNAi rescued the cells from apoptosis triggered by gp120. CONCLUSIONS: The specific up-regulation of 14-3-3tau in HUVEC negatively regulated gp120/CXCR4-mediated cell death by protecting Bad dephosphorylation.     

Direct apoptotic effects of free fatty acids on human endothelial cells

Background and aimEndothelial cell injury is a key event in the pathogenesis of diabetes-associated atherosclerosis and vascular complications. Increased apoptosis may contribute to the loss of endothelial integrity and leads to cardiovascular disease. This study was designed to elucidate whether high levels of free fatty acids (FFA) cause apoptosis and if so what is the possible role of insulin signaling alteration(s) in determining this effect.Methods and resultsIn human umbilical vein endothelial cells (HUVECs) cultured for 72 h with high levels of FFA, apoptotic cells, detected by Annexin V-FITC and PI, were increased. Then we observed a decrease of Bcl-2/Bax ratio (pro-apoptotic condition), measured by RT–PCR and Western blot. As the Akt pathway is involved in insulin signaling and apoptosis, we investigated whether Akt mediated FFA apoptotic effects. HUVECs exposed to FFA showed an equal amount of total Akt protein content compared to controls. In HUVECs, FFA induced a significant decrease in phosphorylated active Akt. Furthermore, phosphorylated eNOS (active form) was decreased and cleaved caspase-9 (active form) was increased. These alterations were prevented when insulin at 10⁻⁸ M was added in culture medium containing FFA. The insulin anti-apoptotic effect was prevented by Ly29400, a PI3K/Akt inhibitor.ConclusionHigh levels of FFA cause HUVECs apoptosis through Akt inhibition; insulin can prevent these effects. Inappropriate FFA elevation may affect vascular endothelium by impairing cell survival via activation of apoptosis, thus contributing to the development of cardiovascular disease in Type 2 diabetic patients.     

Big mitogen-activated protein kinase (BMK1)/ERK5 protects endothelial cells from apoptosis

Blood flow that is steady and laminar is known to be atheroprotective. One likely mechanism is enhanced endothelial cell (EC) survival. Because the mitogen-activated protein kinases (MAPKs) are known regulators of cell survival, we investigated the role of Big MAPK-1 (BMK1 or ERK5), which is potently stimulated by fluid shear stress. To activate BMK1, we overexpressed constitutively active (CA)-MEK5 in bovine lung microvascular ECs (BLMECs). Cell apoptosis was induced by growth factor deprivation (0% serum for 24 hours). Analysis of cell viability with MTT assay showed that activation of BMK1 by CA-MEK5 significantly improved cell viability from 48% to 87% and decreased apoptotic cells from 49% to 10%. Growth factor deprivation induced caspase-3 activity 5.2-fold, which was inhibited (approximately 60%) by CA-MEK5 overexpression. In contrast, inhibiting BMK1 activity by overexpressing dominant-negative BMK1 (DN-BMK1) stimulated apoptosis in BLMECs. Steady laminar fluid shear stress inhibited BLMEC apoptosis, and this protective effect was also reduced significantly by overexpressing DN-BMK1. Analysis of antiapoptotic mechanisms showed that both shear stress and CA-MEK5 stimulated phosphorylation of Bad on Ser112 and Ser136, whereas DN-BMK1 inhibited phosphorylation. Phosphorylation of Bad induced by BMK1 activation was independent of Akt, PKA, or p90RSK kinase activity. These results suggest that BMK1 activation by steady laminar flow is atheroprotective by inhibiting EC apoptosis via phosphorylation of Bad.     

Schizandra arisanensis extract attenuates cytokine-mediated cytotoxicity in insulin-secreting cells

AIM: To explore the bioactivity of an ethanolic extract of Schizandra arisanensis (SA-Et) and isolated constituents against interleukin-1β and interferon-γ-mediated β cell death and abolition of insulin secretion.METHODS: By employing BRIN-BD11 cells, the effects of SA-Et administration on cytokine-mediated cell death and abolition of insulin secretion were evaluated by a viability assay, cell cycle analysis, and insulin assay. The associated gene and protein expressions were also measured. In addition, the bioactivities of several peak compounds collected from the SA-Et were tested against cytokine-mediated β cell death.RESULTS: Our results revealed that SA-Et dose-dependently ameliorated cytokine-mediated β cell death and apoptosis. Instead of suppressing inducible nitric oxide synthase/nitric oxide cascade or p38MAPK activity, suppression of stress-activated protein kinase/c-Jun NH2-terminal kinase activity appeared to be the target for SA-Et against the cytokine mix. In addition, SA-Et provided some insulinotropic effects which re-activated the abolished insulin exocytosis in cytokine-treated BRIN-BD11 cells. Finally, schiarisanrin A and B isolated from the SA-Et showed a dose-dependent protective effect against cytokine-mediated β cell death.CONCLUSION: This is the first report on SA-Et ameliorating cytokine-mediated β cell death and dysfunction via anti-apoptotic and insulinotropic actions.     

Butyrate-Induced Differentiation of Colon Cancer Cells Is PKC and JNK Dependent

Butyric acid, a short–chain fatty acid physiologically present in human large gut, is derived from bacterial fermentation of complex carbohydrates. It has been shown to reduce the growth and motility of colon cancer cell lines and to induce cell differentiation and apoptosis. Apoptosis is considered a result of normal colonocyte terminal differentiation in vivo. The aim of this study was to characterize the cellular mechanisms regulating differentiation of colon cancer cells stimulated with sodium butyrate (NaB). The two human colon cancer cell lines Caco-2 and HT-29 were treated with NaB at physiologically relevant concentrations. Alkaline phosphatase (ALP) activity, a marker of colonocyte differentiation, was increased 48 hr after treatment with 1 mM NaB. Higher doses of NaB (5 and 10 mM) induced apoptosis of the cells and failed to stimulate the colonocyte differentiation. Therefore, we assumed that butyrate augments cell differentiation and induces apoptosis, acting via various intracellular mechanisms, and butyrate-mediated programmed cell death cannot be considered a consequence of colonocyte terminal differentiation. The effect of NaB on ALP activity was significantly attenuated in the presence of inhibitors of protein kinase C and JNK. Inhibition of MEK–ERK signal transduction pathways augmented the impact of butyrate on colonocyte differentiation. These results suggest that butyrate could influence the colonocyte differentiation via modulation of the activity of cellular protein kinases and signal transduction.     

α-硫辛酸抑制糖基化终末产物诱导血管内皮细胞凋亡的研究

目的研究α-硫辛(ALA)对晚期糖基化终末产物(AGEs)诱导血管内皮细胞凋亡的抑制作用及机制。方法体外培养人脐静脉内皮细胞,加不同培养液孵育60 min,以人血清白蛋白(HSA)培养液作为对照组,以AGEs-HSA 200 mg/L培养液体外培养60 min为AGEs-HSA组,以ALA 200 μg/ml加入200 mg/L AGEs为ALA组,采用Hoechst 33258染色检测细胞凋亡,Western blot法检测NF-κB p65核蛋白表达,ELISA法测定天冬氨酸蛋白酶3(caspase-3)活性。结果 AGEs以浓度依赖方式促进内皮细胞凋亡。与对照组比较,AGEs-HSA组细胞NF-κB p65蛋白表达量明显减低,caspase-3活性明显增高(P0.05);与AGEs HSA组比较,ALA组内皮细胞凋亡明显减少,NF-κB p65蛋白表达量明显增加,caspase-3活性明显降低(P0.05)。结论 ALA可能通过增加NF-κB表达,抑制caspase-3激活的AGEs诱导内皮细胞凋亡。     

Nitric oxide modulates chromatin folding in human endothelial cells via protein phosphatase 2A activation and class II histone deacetylases nuclear shuttling

Nitric oxide (NO) modulates important endothelial cell (EC) functions and gene expression by a molecular mechanism which is still poorly characterized. Here we show that in human umbilical vein ECs (HUVECs) NO inhibited serum-induced histone acetylation and enhanced histone deacetylase (HDAC) activity. By immunofluorescence and Western blot analyses it was found that NO induced class II HDAC4 and 5 nuclear shuttling and that class II HDACs selective inhibitor MC1568 rescued serum-dependent histone acetylation above control level in NO-treated HUVECs. In contrast, class I HDACs inhibitor MS27-275 had no effect, indicating a specific role for class II HDACs in NO-dependent histone deacetylation. In addition, it was found that NO ability to induce HDAC4 and HDAC5 nuclear shuttling involved the activation of the protein phosphatase 2A (PP2A). In fact, HDAC4 nuclear translocation was impaired in ECs expressing small-t antigen and exposed to NO. Finally, in cells engineered to express a HDAC4-Flag fusion protein, NO induced the formation of a macromolecular complex including HDAC4, HDAC3, HDAC5, and an active PP2A. The present results show that NO-dependent PP2A activation plays a key role in class II HDACs nuclear translocation.     

Ingestion of an exogenous ketone monoester improves the glycemic response during oral glucose tolerance test in individuals with impaired glucose tolerance: A cross-over randomized trial

Aims/IntroductionAs a low‐carbohydrate diet and the use of sodium–glucose transporter‐2 inhibitors are both known to increase D‐beta‐hydroxybutyrate levels, the effect of these levels on glucose metabolism has attracted attention. We investigated the acute effects of ketone monoester (KM) ingestion on blood glucose levels during the 75‐g oral glucose tolerance test (OGTT) in participants with impaired glucose tolerance.Materials and MethodsNine Japanese adults aged 48–62 years (4 men, 5 women) with impaired glucose tolerance participated in this study. After participants fasted overnight, we carried out OGTT for 180 min with and without KM ingestion on two separate days in a randomized cross‐over design. We compared the area under the curve (AUC) of D‐beta‐hydroxybutyrate, glucose, insulin, C‐peptide, glucagon and free fatty acids during OGTT.ResultsThe AUC of D‐beta‐hydroxybutyrate during OGTT was significantly higher with KM than without KM (KM 5995.3 ± 1257.1 mmol/L·h; without KM 116.1 ± 33.9 mmol/L·h, P < 0.0001), and the AUC of glucose with KM was significantly lower than that without KM (KM 406.6 ± 70.6 mg/dL·h; without KM 483.2 ± 74.3 mg/dL·h, P < 0.0001). This improved glucose excursion was associated with enhanced AUC of insulin during the first half (0–90 min) of OGTT, even though the AUC of C‐peptide during this period was unchanged. In contrast, the AUC of insulin, C‐peptide, glucagon and free fatty acids during 180 min of OGTT were similar in both conditions.ConclusionThe ingestion of KM decreased the AUC of glucose during 75‐g OGTT in Japanese individuals with impaired glucose tolerance, and the mechanism might involve elevated levels of circulating early phase insulin.     

The effect of carotenoids on the expression of cell surface adhesion molecules and binding of monocytes to human aortic endothelial cells

Several large epidemiological studies have shown a correlation between elevated plasma carotenoid levels and decreased risk of cardiovascular disease (CVD). One proposed mechanism for the beneficial effect of carotenoids is through functional modulation of potentially atherogenic processes associated with the vascular endothelium. To test this, we incubated confluent human aortic endothelial cell (HAEC) cultures (passages 4–8) for 24 h with each of the five most prevalent carotenoids in human plasma, which are -carotene, β-carotene, β-cryptoxanthin, lutein, and lycopene, at an approximate concentration of 1 μmol/l. Carotenoids were solubilized in 0.7% (v/v) tetrahydrofuran and incorporated into FBS before adding to cell culture medium. Due to disparate solubilities in aqueous medium, final concentrations of -carotene, β-carotene, β-cryptoxanthin, lutein, and lycopene were 1.7, 1.1, 0.7, 0.9, and 0.3 μmol/l and monolayers accumulated 647, 158, 7, 113, and 9 pmol/mg protein, respectively. Monolayers were then stimulated with IL-1β (5 ng/ml) for 6 h with subsequent determination of cell surface expression of adhesion molecules as measured by an enzyme-linked immunosorbent assay (ELISA). To assess endothelial cell adhesion to monocytes, IL-1β-stimulated monolayers were incubated for 10 min with ⁵¹Cr-labeled U937 monocytic cells and adhesion determined by isotope counting. Pre-incubation of HAEC with β-carotene, lutein and lycopene significantly reduced VCAM-1 expression by 29, 28, and 13%, respectively. Pre-incubation with β-carotene and lutein significantly reduced E-selectin expression by 38 and 34%, respectively. Pre-treatment with β-carotene, lutein and lycopene significantly reduced the expression of ICAM-1 by 11, 14, and 18%, respectively. While other carotenoids were ineffective, lycopene attenuated both IL-1β-stimulated and spontaneous HAEC adhesion to U937 monocytic cells by 20 and 25%, respectively. Thus, among the carotenoids, lycopene appears to be most effective in reducing both HAEC adhesion to monocytes and expression of adhesion molecules on the cell surface.     

Protein phosphatase 2A regulates life and death decisions via Akt in a context-dependent manner

Here, we show how targeting protein phosphatase 2A (PP2A), a key regulator of cellular protein phosphorylation, can either induce or prevent apoptosis depending on what other signals the cell is receiving. The oncoprotein polyoma small T interacts with PP2A to regulate survival. In the presence of growth factors, small T induces apoptosis. Akt activity, which usually promotes survival, is required for this death response, because inhibitors of Akt or PI3 kinase protect cells from death. The activation of Akt under these conditions is partial, characterized by T308 phosphorylation but not S473 phosphorylation. In the absence of growth factors, small T protects from cell death. Here, small T uses PP2A to promote phosphorylation of Akt on both T308 and S473. This effect results in a different pattern of phosphorylation of Akt substrates and shifts Akt from a proapoptotic (presence of growth factors) to an antiapoptotic mode (absence of growth factors). An intriguing possibility is that Akt phosphorylation could be therapeutically disregulated to decrease the survival of cancer cells.     

Ethanol stimulates endothelial cell angiogenic activity via a Notch- and angiopoietin-1-dependent pathway

AIMS: Our aims were to determine the effect of alcohol (EtOH) on endothelial angiogenic activity and to delineate the cell signalling mechanisms involved. METHODS AND RESULTS: Treatment of human umbilical vein endothelial cells (HUVECs) with EtOH (1-100 mM, 24 h) dose-dependently increased their network formation on Matrigel (an index of angiogenesis) with a maximum response (2.5- to 3-fold increase) at 25 mM. Ethanol also stimulated the proliferation (by cell count and proliferating cell nuclear antigen expression) and migration (by scratch wound assay) of HUVECs. In parallel cultures, EtOH stimulated Notch receptor (1 and 4) and Notch target gene (hrt-1, -2, and -3) mRNA and protein expression and enhanced CBF-1/RBP-Jk promoter activity. EtOH also stimulated, at the mRNA and protein level, the expression of angiopoietin-1 (Ang1) and its Tie2 receptor in these cells. Knockdown of Notch 1 or 4 by siRNA or inhibition of Notch-mediated, CBF-1/RBP-Jk-regulated gene expression by the Epstein-Barr virus-encoded protein RPMS-1 inhibited both ethanol-induced Ang1/Tie2 expression in HUVECs and their network formation on Matrigel. Moreover, knockdown of Ang1 or Tie2 by siRNA inhibited ethanol-induced endothelial network formation. CONCLUSION: These data demonstrate that ethanol, at levels consistent with moderate consumption, enhances endothelial angiogenic activity in vitro by stimulating a novel Notch/CBF-1/RBP-JK-Ang1/Tie2-dependent pathway. These actions of ethanol may be relevant to the cardiovascular effects of alcohol consumption purported by epidemiological studies.     

Actions of PP2A on the MAP kinase pathway and apoptosis are mediated by distinct regulatory subunits

Individual subunits of protein phosphatase 2A (PP2A), protein phosphatase 4, and protein phosphatase 5 were knocked out in Drosophila Schneider 2 cells by using RNA interference. Ablation of either the scaffold (A) or catalytic (C) subunits of PP2A caused the disappearance of all PP2A subunits. Treating cells with double-stranded RNA targeting all four of the Drosophila PP2A regulatory subunits caused the disappearance of both the A and C subunits. The loss of PP2A subunits was associated with decreased protein stability indicating that only the heterotrimeric forms of PP2A are stable in intact cells. Ablation of total PP2A by using double-stranded RNA against either the A or C subunit, or specific ablation of the R2/B regulatory subunit, enhanced insulin-induced ERK activation. These results indicated that the R2/B subunit targets PP2A to the mitogen-activated protein (MAP) kinase cascade in Schneider 2 cells, where it acts as a negative regulator. A severe loss of viability occurred in cells in which total PP2A or both isoforms of the Drosophila R5/B56 subunit had been ablated. The reduced viability of these cells correlated with the induction of markers of apoptosis including membrane blebbing and stimulation of caspase-3-like activity. These observations indicated that PP2A has a powerful antiapoptotic activity that is specifically mediated by the R5/B56 regulatory subunits. In contrast to PP2A, ablation of protein phosphatase 4 caused only a slight reduction in cell growth but had no effect on MAP kinase signaling or apoptosis. Depletion of protein phosphatase 5 had no effects on MAP kinase, cell growth, or apoptosis.     

Extracellular ATP induces assembly and activation of the myosin light chain phosphatase complex in endothelial cells

OBJECTIVES: Extracellular ATP stabilizes the endothelial barrier and inactivates the contractile machinery of endothelial cells. This inactivation relies on dephosphorylation of the regulatory myosin light chain (MLC) due to an activation of the MLC phosphatase (MLCP). To date, activation and function of MLCP in endothelial cells are only partially understood. METHODS: Here, the mechanism of extracellular ATP-mediated activation of MLCP was analyzed in human endothelial cells from umbilical veins. Cells were transfected with the endogenous protein phosphatase 1 (PP1)-specific inhibitor-2 (I-2). RESULTS: Overexpression of I-2 led to inhibition of PP1 activity and abrogation of the ATP-induced dephosphorylation of MLC. This indicates that the PP1 catalytic subunit is the principal phosphatase catalyzing the MLC dephosphorylation induced by extracellular ATP. As demonstrated by immunoprecipitation analysis, extracellular ATP recruits the PP1delta catalytic subunit and the myosin phosphatase targeting subunit (MYPT1) to form a complex. ATP stimulated dephosphorylation of MYPT1 at the inhibitory phosphorylation sites threonine 850 and 696. However, extracellular ATP failed to stimulate MYPT1 dephosphorylation in I-2-overexpressing cells. CONCLUSIONS: The present study shows for the first time that, in endothelial cells, extracellular ATP causes activation of MLCP through recruitment of PP1delta and MYPT1 into a MLCP holoenzyme complex and PP1-mediated reduction of the inhibitory phosphorylation of MYPT1.     

尼古丁通过抑制JAK/STAT通路对HUVECs增殖和凋亡的影响研究

目的探究尼古丁通过抑制JAK/STAT通路促进人脐静脉血管内皮细胞(HUVECs)增殖和凋亡的作用机制。方法将HUVECs分为4组,即对照组、尼古丁1组、尼古丁2组、尼古丁3组,分别在培养基中加入终浓度为0、10-8、10-7、10-6 mol/L的尼古丁培养24 h。显微镜观察细胞形态。CCK-8和流式细胞术分别用于检测细胞活力和凋亡。Western blot和PCR分别用于检测蛋白和mRNA的表达水平。结果显微镜观察尼古丁会使HUVECs呈现萎缩状态。尼古丁可剂量依赖性的抑制HUVECs活力,并且剂量依赖性的促进HUVECs凋亡。尼古丁可显著的抑制p-JAK/t-JAK和p-STAT/t-STAT的水平。尼古丁可显著的提高Caspase-3和Bax mRNA的水平,抑制Bcl-2 mRNA的水平。结论尼古丁可能通过下调JAK/STAT通路调节凋亡相关蛋白的表达,抑制HUVECs活力并促进其凋亡。