Aging is associated with decreased pancreatic acinar cell regeneration and phosphatidylinositol 3-kinase/Akt activation

BACKGROUND & AIMS: The effects of aging on pancreatic acinar cell proliferation have not been clearly defined. Phosphatidylinositol 3-kinase (PI3K)-mediated phosphorylation of Akt is a critical step for proliferation of various cell types and insulin secretion from pancreatic endocrine cells; however, its role in acinar cell proliferation is not known. The purpose of this study was to (1) delineate the effects of aging on pancreatic regeneration after partial pancreatectomy (Px) and (2) define the involvement of the PI3K/Akt pathway in pancreatic regeneration. METHODS: Following partial Px, pancreatic regeneration and activation of the PI3K pathway were compared in young and aged mice. Activation of the PI3K/Akt pathway was evaluated by Akt phosphorylation (pAkt). The role of the PI3K pathway in pancreatic regeneration after partial Px was assessed by effects of a pharmacologic PI3K inhibitor wortmannin or small interfering RNA (siRNA) to the p85alpha regulatory subunit. To confirm further the critical role of the PI3K/Akt pathway in pancreatic acinar cell proliferation, IGF-1-mediated cell proliferation was determined in cultured acinar cells pretreated with wortmannin or p85alpha siRNA. RESULTS: Pancreatic regeneration and pAkt expression after partial Px were significantly decreased with aging. Treatment with wortmannin or p85alpha siRNA reduced pancreatic regeneration after partial Px. The IGF-1-mediated cell proliferation in vitro was completely blocked by wortmannin or p85alpha siRNA but not by the MEK/ERK inhibitor PD98059. CONCLUSIONS: PI3K/Akt activation plays a critical role in the regeneration of pancreatic acini after resection. Furthermore, pancreatic regeneration is markedly attenuated in the aged pancreas most likely because of decreased PI3K/Akt activation.     

Regulation of phospholipid hydrolysis in streptolysin-O-permeabilized rat pancreatic acini.

To investigate the mechanism of phospholipid hydrolysis in pancreatic acinar cells, the effects of Ca2+, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) and cholecystokinin (CCK) on both polyphosphoinositide (PI) and phosphatidylcholine (PC) hydrolysis were studied in rat pancreatic acini permeabilized with the bacterial toxin, streptolysin-O. When acini were prelabeled with myo-[3H]inositol, permeabilized, and then incubated with various concentrations of free Ca2+ for 15 min, Ca2+ stimulated [3H]inositol phosphate release at a concentration of 100 nM and was maximally effective at 100 microM. Both GTP gamma S and CCK enhanced Ca(2+)-induced [3H]inositol phosphate release, although these agents had no effect in the absence of Ca2+. At a physiological concentration of Ca2+ (100 nM), CCK stimulated [3H]inositol phosphate release which was further enhanced by GTP gamma S. When acini were similarly prelabeled with [3H]choline before permeabilization, [3H]choline phosphate release was also stimulated by free Ca2+ over the concentration range from 100 nM to 10 microM. In contrast to PI hydrolysis, however, neither GTP gamma S, CCK, or GTP gamma S plus CCK had an additional effect on [3H]choline phosphate release stimulated by 100 nM-100 microM free Ca2+. Furthermore, Ca(2+)-induced [3H]choline phosphate release appeared to be due to the redistribution from cell to the medium rather than to an increase in choline phosphate production. Therefore, choline phosphate release following prelabeling with [3H]choline is not useful as an indicator of PC hydrolysis in permeabilized acini.     

Impaired glucose-stimulated insulin secretion, enhanced intraperitoneal insulin tolerance, and increased beta-cell mass in mice lacking the p110gamma isoform of phosphoinositide 3-kinase

Phosphoinositide 3-kinase (PI3 kinase) has been implicated in G protein-coupled receptor regulation of pancreatic beta-cell growth and glucose-stimulated insulin secretion. The G protein-activated p110gamma isoform of PI3 kinase was detected in insulinoma cells, mouse islets, and human islets. In 7- to 10-wk-old mice, knockout of p110gamma reduced the plasma insulin response to ip glucose injection and impaired first and second phase glucose-stimulated insulin secretion from pancreata perfused ex vivo. The p110gamma -/- mice responded to preinjection with the glucagon-like peptide-1 receptor agonist exendin 4, such that plasma glucose and insulin responses to ip glucose injection were not different from wild types. Mice lacking p110gamma were not diabetic and were only slightly glucose intolerant (ip glucose injection) compared with wild types, in part due to enhanced responsiveness to insulin as determined by an ip insulin tolerance test. Despite severely reduced insulin secretion in these animals, the p110gamma -/- mice had greater pancreatic insulin content, and an increased beta-cell mass due to beta-cell hypertrophy. These surprising results suggest that the G protein-coupled p110gamma isoform of PI3 kinase is not central to the development or maintenance of sufficient beta-cell mass but positively regulates glucose-stimulated insulin secretion.     

Deletion of the phosphoinositide 3-kinase p110gamma gene attenuates murine atherosclerosis

Inflammatory cell activation by chemokines requires intracellular signaling through phosphoinositide 3-kinase (PI3-kinase) and the PI3-kinase-dependent protein serine/threonine kinase Akt. Atherosclerosis is a chronic inflammatory process driven by oxidatively modified (atherogenic) lipoproteins, chemokines, and other agonists that activate PI3-kinase. Here we show that macrophage PI3-kinase/Akt is activated by oxidized low-density lipoprotein, inflammatory chemokines, and angiotensin II. This activation is markedly reduced or absent in macrophages lacking p110gamma, the catalytic subunit of class Ib PI3-kinase. We further demonstrate activation of macrophage/foam cell PI3-kinase/Akt in atherosclerotic plaques from apolipoprotein E (apoE)-null mice, which manifest an aggressive form of atherosclerosis, whereas activation of PI3-kinase/Akt was undetectable in lesions from apoE-null mice lacking p110gamma despite the presence of class Ia PI3-kinase. Moreover, plaques were significantly smaller in apoE-/-p110gamma-/- mice than in apoE-/-p110gamma+/+ or apoE-/-p110gamma+/-mice at all ages studied. In marked contrast to the embryonic lethality seen in mice lacking class Ia PI3-kinase, germ-line deletion of p110gamma results in mice that exhibit normal viability, longevity, and fertility, with relatively well tolerated defects in innate immune and inflammatory responses that may play a role in diseases such as atherosclerosis and multiple sclerosis. Our results not only shed mechanistic light on inflammatory signaling during atherogenesis, but further identify p110gamma as a possible target for pharmacological intervention in the primary and secondary prevention of human atherosclerotic cardiovascular disease.     

Phosphoinositide 3-kinase mediates enhanced spontaneous and agonist-induced contraction in aorta of deoxycorticosterone acetate-salt hypertensive rats

Arteries from deoxycorticosterone acetate (DOCA)-salt and N(omega)-nitro-L-arginine (L-NNA) hypertensive but not normotensive rats develop spontaneous tone. LY294002 and wortmannin, phosphoinositide 3-kinase (PI3-kinase) inhibitors, eliminate spontaneous tone. We hypothesized that PI3-kinase protein and/or activity was increased in hypertension and contributed to the observed enhanced contractility. PI3-kinase activity assays revealed 2-fold higher activity in thoracic aorta from DOCA-salt [systolic blood pressure (SBP)=184+/-5 mm Hg] compared with sham rats (SBP=111+/-2 mm Hg). Western analyses of aortic homogenates revealed the presence of p85alpha, p110alpha, p110beta, and p110delta but not p110gamma PI3-kinase subunits; p110delta protein was elevated in aorta of hypertensive rats as compared with sham. Aortic homogenates from L-NNA rats also had elevated p110beta protein density, but neither L-NNA nor DOCA-salt had differences in p85alpha and p110alpha. Total Akt density was unaltered, but pAkt was significantly lower in homogenates from DOCA-salt rats. LY294002 (20 micromol/L) and nifedipine (50 nmol/L) abolished Ca2+-induced spontaneous tone in aorta from DOCA-salt rats. However, LY294002 did not alter BayK8644-induced contraction, indicating that LY294002 does not inhibit L-type Ca2+ channels directly. PTEN (phosphatase and tensin homolog) and pPTEN were expressed but not different in aorta from DOCA-salt and sham rats. LY294002 corrected the enhanced contraction to KCl and norepinephrine in aorta from DOCA-salt rats. These data support an increase in PI3-kinase activity and p110delta density in aorta from L-NNA and DOCA-salt rats. Importantly, this increase contributes to the enhanced contractility observed in two models of hypertension.     

Essential role for the p110delta isoform in phosphoinositide 3-kinase activation and cell proliferation in acute myeloid leukemia

The phosphoinositide 3-kinase (PI3K)/Akt signaling pathway has been shown to be frequently activated in blast cells from patients with acute myeloid leukemia (AML) and to contribute to survival and proliferation of these cells. Of the 8 distinct mammalian isoforms of PI3K, it is the class I PI3Ks (p110alpha, p110beta, p110gamma, and p110delta) that are responsible for Akt activation. It is not known which PI3K isoform is critical in AML. Here we show that the p110delta isoform of PI3K is consistently expressed at a high level in blast cells from AML, in contrast to the other class I isoforms, the expression of which was very variable among patients. IC87114, a p110delta-selective inhibitor, suppressed both constitutive and Flt-3-stimulated Akt activation in blasts to the same extent as Ly294002, an inhibitor of all PI3K isoforms. Moreover, IC87114 inhibited AML cell proliferation without affecting the proliferation of normal hematopoietic progenitor cells. These observations identify p110delta as a potential therapeutic target in AML.     

Tripartite motif containing protein 27 negatively regulates CD4 T cells by ubiquitinating and inhibiting the class II PI3K-C2β

The K(+) channel KCa3.1 is required for Ca(2+) influx and the subsequent activation of CD4 T cells. The class II phosphatidylinositol 3 kinase C2β (PI3KC2β) is activated by the T-cell receptor (TCR) and is critical for KCa3.1 channel activation. Tripartite motif containing protein 27 (TRIM27) is a member of a large family of proteins that function as Really Interesting New Gene (RING) E3 ubiquitin ligases. We now show that TRIM27 functions as an E3 ligase and mediates lysine 48 polyubiquitination of PI3KC2β, leading to a decrease in PI3K enzyme activity. By inhibiting PI3KC2β, TRIM27 also functions to negatively regulate CD4 T cells by inhibiting KCa3.1 channel activity and TCR-stimulated Ca(2+) influx and cytokine production in Jurkat, primary human CD4 T cells, and Th0, Th1, and Th2 CD4 T cells generated from TRIM27(-/-) mice. These findings provide a unique mechanism for regulating class II PI3Ks, and identify TRIM27 as a previously undescribed negative regulator of CD4 T cells.     

CCK-stimulated changes in pancreatic acinar morphology are mediated by rho

BACKGROUND/AIMS: Treatment of isolated pancreatic acini with high concentrations of cholecystokinin (CCK) is known to induce rapid changes in the cellular morphology. The signalling pathways remain to be characterized. METHODS: Pancreatic acini were permeabilized by digitonin and incubated with various agents. The acinar morphology was investigated by microscopy. The activation of p125 focal adhesion kinase was determined by Western blot analysis. Amylase was measured photometrically. RESULTS: The functionality of the permeabilized acini was tested by measuring stimulated amylase release. 300 microM GTP gamma S was almost as efficient as CCK to stimulate amylase release, while 300 microM GDP beta S inhibited the CCK-stimulated amylase release. Stimulation of permeabilized acini with 0.1 microM CCK induced similar morphological changes as in unpermeabilized acini. Incubation of permeabilized acini with GTP gamma S mimicked the CCK-induced changes, whereas a preincubation with GDP beta S prevented the CCK effects on the acinar morphology. Inhibition of the small G protein rho, which activates p125 focal adhesion kinase, by Clostridium botulinum C3 transferase also prevented the CCK-stimulated morphological changes. Preincubation of intact acini with cell-permeable inhibitors of protein kinase C, MEK or p38MAPK, or with the intracellular calcium chelator BAPTA/AM was without significant effect on the CCK-stimulated changes. CONCLUSION: The CCK-induced morphological changes seem to be mediated by G protein signalling via the small G protein rho and the associated activation of p125 focal adhesion kinase.     

Insulin-like growth factor-1 and PTEN deletion enhance cardiac L-type Ca2+ currents via increased PI3Kalpha/PKB signaling

Ca2+ influx through the L-type Ca2+ channel (I(Ca,L)) is a key determinant of cardiac contractility and is modulated by multiple signaling pathways. Because the regulation of I(Ca,L) by phosphoinositide-3-kinases (PI3Ks) and phosphoinositide-3-phosphatase (PTEN) is unknown, despite their involvement in the regulation of myocardial growth and contractility, I(Ca,L) was recorded in myocytes isolated from mice overexpressing a dominant-negative p110alpha mutant (DN-p110alpha) in the heart, lacking the PI3Kgamma gene (PI3Kgamma(-/-)) or with muscle-specific ablation of PTEN (PTEN(-/-)). Combinations of these genetically altered mice were also examined. Although there were no differences in the expression level of CaV1.2 proteins, basal I(Ca,L) densities were larger (P<0.01) in PTEN(-/-) myocytes compared with littermate controls, PI3Kgamma(-/-), or DN-p110alpha myocytes and showed negative shifts in voltage dependence of current activation. The I(Ca,L) differences seen in PTEN(-/-) mice were eliminated by pharmacological inhibition of either PI3Ks or protein kinase B (PKB) as well as in PTEN(-/-)/DN-p110alpha double mutant mice but not in PTEN(-/-)/PI3Kgamma(-/-) mice. On the other hand, application of insulin-like growth factor-1 (IGF-1), an activator of PKB, increased I(Ca,L) in control and PI3Kgamma(-/-), while having no effects on I(Ca,L) in DN-p110alpha or PTEN(-/-) mice. The I(Ca,L) increases induced by IGF-1 were abolished by PKB inhibition. Our results demonstrate that IGF-1 treatment or inactivation of PTEN enhances I(Ca,L) via PI3Kalpha-dependent increase in PKB activation.     

Altered heart rate and sinoatrial node function in mice lacking the cAMP regulator phosphoinositide 3-kinase-gamma

Ablation of the enzyme phosphoinositide 3-kinase (PI3K)gamma (PI3Kgamma(-/-)) in mice increases cardiac contractility by elevating intracellular cAMP and enhancing sarcoplasmic reticulum Ca(2+) handling. Because cAMP is a critical determinant of heart rate, we investigated whether heart rate is altered in mice lacking PI3Kgamma. Heart rate was similar in anesthetized PI3Kgamma(-/-) and wild-type (PI3Kgamma(+/+)) mice. However, IP injection of atropine (1 mg/kg), propranolol (1 mg/kg), or both drugs in combination unmasked elevated heart rates in PI3Kgamma(-/-) mice, suggesting altered sinoatrial node (SAN) function. Indeed, spontaneous action potential frequency was approximately 35% greater in SAN myocytes isolated from PI3Kgamma(-/-) mice compared with PI3Kgamma(+/+) mice. These differences in action potential frequency were abolished by intracellular dialysis with the cAMP/protein kinase A antagonist Rp-cAMP but were unaffected by treatment with ryanodine to inhibit sarcoplasmic reticulum Ca(2+) release. Voltage-clamp experiments demonstrated that elevated action potential frequencies in PI3Kgamma(-/-) SAN myocytes were more strongly associated with cAMP-dependent increases in L-type Ca(2+) current (I(Ca,L)) than elevated hyperpolarization-activated current (I(f)). In contrast, I(Ca,L) was not increased in working atrial myocytes, suggesting distinct subcellular regulation of L-type Ca(2+) channels by PI3Kgamma in the SAN compared with the working myocardium. In summary, PI3Kgamma regulates heart rate by the cAMP-dependent modulation of SAN function. The effects of PI3Kgamma ablation in the SAN are unique from those in the working myocardium.     

The p110beta isoform of phosphoinositide 3-kinase signals downstream of G protein-coupled receptors and is functionally redundant with p110gamma

The p110 isoforms of phosphoinositide 3-kinase (PI3K) are acutely regulated by extracellular stimuli. The class IA PI3K catalytic subunits (p110alpha, p110beta, and p110delta) occur in complex with a Src homology 2 (SH2) domain-containing p85 regulatory subunit, which has been shown to link p110alpha and p110delta to Tyr kinase signaling pathways. The p84/p101 regulatory subunits of the p110gamma class IB PI3K lack SH2 domains and instead couple p110gamma to G protein-coupled receptors (GPCRs). Here, we show, using small-molecule inhibitors with selectivity for p110beta and cells derived from a p110beta-deficient mouse line, that p110beta is not a major effector of Tyr kinase signaling but couples to GPCRs. In macrophages, both p110beta and p110gamma contributed to Akt activation induced by the GPCR agonist complement 5a, but not by the Tyr kinase ligand colony-stimulating factor-1. In fibroblasts, which express p110beta but not p110gamma, p110beta mediated Akt activation by the GPCR ligands stromal cell-derived factor, sphingosine-1-phosphate, and lysophosphatidic acid but not by the Tyr kinase ligands PDGF, insulin, and insulin-like growth factor 1. Introduction of p110gamma in these cells reduced the contribution of p110beta to GPCR signaling. Taken together, these data show that p110beta and p110gamma can couple redundantly to the same GPCR agonists. p110beta, which shows a much broader tissue distribution than the leukocyte-restricted p110gamma, could thus provide a conduit for GPCR-linked PI3K signaling in the many cell types where p110gamma expression is low or absent.     

磷脂酰肌醇3-激酶γ对急性胰腺炎小鼠胰腺腺泡细胞自噬作用的影响

目的 探讨磷脂酰肌醇3-激酶γ(PI3Kγ)对小鼠实验性急性胰腺炎胰腺腺泡细胞自噬作用的影响,并探讨其意义.方法 野生型C57 BL/6小鼠和PI3Kγ基因敲除小鼠各18只,按数字表法随机分为对照组(6只)和急性胰腺炎(AP)组(12只),采用蛙皮素50μg/kg体重腹腔内注射7次、每次间隔1h的方法制备AP模型.首次注射后7h处死小鼠,光镜下观察胰腺病理学变化,免疫荧光检测自噬泡主要组成蛋白LC3颗粒,荧光分光光度计测定胰蛋白酶活性,蛋白质印迹法检测自噬相关蛋白beclin1、LC3-Ⅱ和p62的表达.结果 AP组野生型小鼠和PI3Kγ基因敲除小鼠的胰腺自噬空泡数量分别为(5.14±0.85)、(2.25±0.54)个/每高倍视野(HPF),LC3荧光免疫颗粒数量分别为(78.6±9.38)、(26.4±4.21)个/HPF,胰蛋白酶活性分别为(0.827±0.126)、(0.358±0.098) pmol/mg蛋白,各组间差异均具有统计学意义(P值均＜0.05).野生型小鼠的p62蛋白表达较PI3Kγ基因敲除小鼠显著减弱(0.11比0.92,P＜0.05),面LC3 -Ⅱ、beclin1蛋白表达较PI3Kγ基因敲除小鼠显著增强(1.82比0.93,1.43比1.05,P值均＜0.05).结论 AP时PI3Kγ可能通过增强小鼠胰腺腺泡细胞的自噬作用,促进胰蛋白酶原的活化及诱导腺泡细胞坏死.     

Effect of somatostatin on cholecystokinin-induced amylase release in rat pancreatic acini.

The effect of somatostatin on cholecystokinin-induced amylase release was investigated in isolated rat pancreatic acini. Acini were isolated by enzymatic digestion and incubated in a HEPES buffered Ringer's solution with testing reagents for 30 minutes at 37 degrees C. The activity of released amylase, cAMP, and inositol phosphate formation were measured. Intracellular calcium concentration ([Ca2+]i) was also checked. Somatostatin 14 and octreotide, a somatostatin analog, inhibited CCK-stimulated amylase release in a concentration-dependent manner. The inhibitory effect of octreotide on CCK-induced amylase release was not shown when the acini were treated with 8-Br-cAMP, irrespective of the presence of IBMX. Forskolin potentiated CCK-induced amylase release and this effect was blocked by octreotide treatment; although CCK-8 (3 x 10(-11) M) failed to stimulate cAMP formation, octreotide significantly inhibited basal cAMP formation in the acini. The increase of [Ca2+]i in response to CCK was inhibited by octreotide. However, CCK-induced inositol phosphate formation was not changed by 10(-9) M octreotide. Octreotide had no effect on CCK-stimulated tyrosine phosphorylation, and tyrosine phosphatase inhibitors (NaF and Na2WO4) did not influence the effect of octreotide on CCK-induced amylase release. From these results, we conclude that octreotide inhibits CCK-induced amylase release by inhibiting basal cAMP formation and decreasing the [Ca2+]i stimulated by CCK.     

The phosphoinositide 3-kinase inhibitor LY294002 enhances cardiac myocyte contractility via a direct inhibition of Ik,slow currents

OBJECTIVE: Phosphoinositide 3-kinase (PI3K) is a key component in regulating myocardial growth, survival and contractility. LY294002 and wortmannin are two PI3K inhibitors used widely to establish the role of PI3K. The goal of this study was to examine the effects of acute application of LY294002 and wortmannin on cardiac myocyte contractility and underlying mechanisms. METHODS: Patch-clamp, indo-1 epifluorescence and video-edge detection techniques were used to measure outward K(+) currents, action potentials (AP), Ca(2+) transients and shortening of myocytes isolated from mouse left ventricular free wall. RESULTS: In field-stimulated myocytes, LY294002 (10 micromol/l) increased Ca(2+) transient amplitude by 23%, and cell shortening amplitude by 60% in the absence or presence of wortmannin, while wortmannin alone had no effect. LY294002 (but not wortmannin) prolonged AP duration by specifically inhibiting slowly inactivating K(+) currents (i.e., the 4-aminopyrydine-sensitive I(k,slow1) and the tetraethylammonium-sensitive I(k,slow2)), leading to an increase in sarcoplasmic reticular Ca(2+) levels. It appeared that the AP prolongation was responsible for elevated contractility since AP-clamp of myocytes with prolonged APs (recorded in LY294002-treated myocytes) induced a 29% increase in cell shortening compared with control APs, while LY294002 application did not increase contractility in voltage-clamp studies using either step or AP depolarizations. CONCLUSIONS: The putative PI3K inhibitor LY294002 increases Ca(2+) release and myocyte contractility via direct inhibition of cardiac I(k,slow) and AP prolongation, thus limiting the usefulness of this agent in the analyses of the role of PI3K in heart function.     

Role of the p110delta PI 3-kinase in integrin and ITAM receptor signalling in platelets

We have investigated the function of the p110delta catalytic subunit of phosphoinositide 3-kinase (PI 3-kinase) in platelets using p110delta knock-out (p110delta(-/-)) mice and p110delta knock-in (p110delta(D910A/D910A)) mice, which express a catalytically inactive form of the enzyme. Aggregation to threshold concentrations of the GPVI-specific agonist, CRP, was partially reduced in p110delta(-/-) and p110delta(D910A/D910A) platelets. This inhibition was overcome by higher concentrations of CRP. The degree of inhibition was considerably weaker than that induced by LY294002 and wortmannin, which inhibit all isoforms of PI 3-kinase. p110delta(-/-) platelets showed decreased spreading on fibrinogen- or von Willebrand factor (VWF)-coated surfaces under static conditions, whereas they spread normally on collagen. LY294002 had a more pronounced inhibitory effect on spreading on all three surfaces. Adhesion and aggregate formation of p110delta(-/-) platelets to collagen or fibrinogen/VWF at intermediate/high rates of shear were normal. This study demonstrates a minor role for the p110delta catalytic subunit in mediating platelet activation by the collagen receptor GPVI and integrin alphaIIbeta3. The more pronounced inhibitory effect of LY294002 and wortmannin indicates that other isoforms of PI 3-kinase play a more significant role in signalling by the two platelet glycoprotein receptors.     

Critical roles of c-Kit tyrosine residues 567 and 719 in stem cell factor-induced chemotaxis: contribution of src family kinase and PI3-kinase on calcium mobilization and cell migration

Stem cell factor (SCF) has crucial roles in proliferation, survival, and differentiation of hematopoietic stem cells and mast cells through binding to c-Kit receptor (KIT). Chemotaxis is another unique function of SCF. However, little is known about the intracellular signaling pathway of SCF/KIT-mediated cell migration. To investigate the signaling cascade, we made a series of 22 KIT mutants, in which tyrosine (Y) residue was substituted for phenylalanine (F) in the cytoplasmic domain, and introduced into BAF3 cells or 293T cells. On stimulation with SCF, BAF3 expressing KIT(WT)(WT) showed cell migration and Ca(2+) mobilization. Among 22 YF mutants, Y567F, Y569F, and Y719F showed significantly reduced cell migration and Ca(2+) mobilization compared to WT. In Y567F, Lyn activation on SCF stimulation decreased and C-terminal Src kinase (Csk) suppressed KIT-mediated Ca(2+) influx and cell migration, suggesting that Y567-mediated Src family kinase (SFK) activation leads to Ca(2+) influx and migration. Furthermore, we found that p38 mitogen-activated protein kinase (p38 MAPK) and Erk1/2 were also regulated by Y567/SFK and involved in cell migration, and that p38 MAPK induced Ca(2+) influx, thereby leading to Erk1/2 activation. In Y719F, the binding of phosphatidylinositol 3'-kinase (PI3K) to KIT was lost and KIT-mediated cell migration and Ca(2+) mobilization were suppressed by PI3K chemical inhibitors or dominant-negative PI3K, suggesting the involvement of Y719-mediated PI3K pathway in cell migration. Combination of Csk and the PI3K inhibitor synergistically reduced cell migration, suggesting the cooperation of SFK and PI3K. Taken together, these results indicate that 2 major KIT signaling pathways lead to cell migration, one is Y567-SFK-p38 MAPK-Ca(2+) influx-Erk and the other is Y719-PI3K-Ca(2+) influx.     

Calcium-sensing receptor induces proliferation through p38 mitogen-activated protein kinase and phosphatidylinositol 3-kinase but not extracellularly regulated kinase in a model of humoral hypercalcemia of malignancy

Using H-500 rat Leydig cancer cells as a model of humoral hypercalcemia of malignancy (HHM), we previously showed that high Ca(2+) induces PTH-related peptide (PTHrP) secretion via the calcium-sensing receptor (CaR) and mitogen- and stress-activated kinases, e.g. MAPK kinase 1 (MEK1), p38 MAPK, and stress-activated protein kinase 1/c-Jun N-terminal kinase. Because cellular proliferation is a hallmark of malignancy, we studied the role of the CaR in regulating the proliferation of H-500 cells. Elevated Ca(2+) has a mitogenic effect on these cells that is mediated by the CaR, because the calcimimetic NPS R-467 also induced proliferation. Inhibition of phosphatidylinositol 3-kinase (PI3K) and p38 MAPK but not MEK1 abolished the mitogenic effect. Activation of PI3K by elevated Ca(2+) was documented by phosphorylation of its downstream kinase, protein kinase B. Because protein kinase B activation promotes cell survival, we speculated that elevated Ca(2+) might protect H-500 cells against apoptosis. Using terminal uridine deoxynucleotidyl nick end labeling staining, we demonstrated that high Ca(2+) (7.5 mM) and NPS R-467 indeed protect cells against apoptosis induced by serum withdrawal compared with low Ca(2+) (0.5 mM). Because the CaR induces PTHrP secretion, it is possible that the mitogenic and antiapoptotic effects of elevated Ca(2+) could be indirect and mediated via PTHrP. However, blocking the type 1 PTH receptor with PTH (7-34) peptide did not alter either high Ca(2+)-induced proliferation or protection against apoptosis. Taken together, our data show that activation of PI3K and p38 MAPK but not of MEK1/ERK by the CaR promotes proliferation of H-500 cells as well as affords protection against apoptosis. These effects are likely direct without the involvement of PTHrP in an autocrine mode.     

Role of phosphatidylinositol 3-kinase in the development of hepatocyte preconditioning

BACKGROUND & AIMS: Ischemic preconditioning has been proved effective in reducing ischemia/reperfusion injury during liver surgery. However, the mechanisms involved are still poorly understood. Here, we have investigated the role of phosphatidylinositol 3-kinase (PI3K) in the signal pathway leading to hepatic preconditioning. METHODS: PI3K activation was evaluated in isolated rat hepatocytes preconditioned by 10-minute hypoxia followed by 10-minute reoxygenation. RESULTS: Hypoxic preconditioning stimulated phosphatidylinositol-3,4,5-triphosphate production and the phosphorylation of PKB/Akt, a downstream target of PI3K. Conversely, PI3K inhibition by wortmannin or LY294002 abolished hepatocyte tolerance against hypoxic damage induced by preconditioning. PI3K activation in preconditioned hepatocytes required the stimulation of adenosine A 2A receptors and was mimicked by adenosine A 2A receptors agonist CGS21680. In the cells treated with CGS21680, PI3K activation was prevented either by inhibiting adenylate cyclase and PKA with, respectively, 2,5-dideoxyadenosine and H89 or by blocking Galphai-protein and Src tyrosine kinase with, respectively, pertussis toxin and PP2. H89 also abolished the phosphorylation of adenosine A 2A receptors. However, the direct PKA activation by forskolin failed to stimulate PI3K. This suggested that PKA-phosphorylated adenosine A 2A receptors may activate PI3K by coupling it with Galphai-protein through Src. We also observed that, by impairing PI3K-mediated activation of phospholypase Cgamma (PLCgamma), wortmannin and LY294002 blocked the downstream transduction of preconditioning signals via protein kinase C (PKC) delta/ isozymes. CONCLUSIONS: PI3K is activated following hepatocyte hypoxic preconditioning by the combined stimulation of adenosine A 2A receptors, PKA, Galphai protein, and Src. By regulating PKC-/delta-dependent signals, PI3K can play a key role in the development of hepatic tolerance to hypoxia/reperfusion.     

Role of the p110δ PI 3-kinase in integrin and ITAM receptor signalling in platelets

We have investigated the function of the p110δ catalytic subunit of phosphoinositide 3-kinase (PI 3-kinase) in platelets using p110δ knock-out (p110δ^–/–) mice and p110δ knock-in (p110δ^D910A/D910A) mice, which express a catalytically inactive form of the enzyme. Aggregation to threshold concentrations of the GPVI-specific agonist, CRP, was partially reduced in p110δ^–/– and p110δ^D910A/D910A platelets. This inhibition was overcome by higher concentrations of CRP. The degree of inhibition was considerably weaker than that induced by LY294002 and wortmannin, which inhibit all isoforms of PI 3-kinase. p110δ^–/– platelets showed decreased spreading on fibrinogen- or von Willebrand factor (VWF)-coated surfaces under static conditions, whereas they spread normally on collagen. LY294002 had a more pronounced inhibitory effect on spreading on all three surfaces. Adhesion and aggregate formation of p110δ^–/– platelets to collagen or fibrinogen/VWF at intermediate/high rates of shear were normal. This study demonstrates a minor role for the p110δ catalytic subunit in mediating platelet activation by the collagen receptor GPVI and integrin αIIbβ3. The more pronounced inhibitory effect of LY294002 and wortmannin indicates that other isoforms of PI 3-kinase play a more significant role in signalling by the two platelet glycoprotein receptors.