Lipid Products Of Phosphatidylinositol 3-kinase Research Articles

Elevated PI3K signaling drives multiple Breast Cancer subtypes

Most human breast tumors have mutations that elevate signaling through a key metabolic pathway that is induced by insulin and a number of growth factors. This pathway serves to activate an enzyme known as phosphatidylinositol 3' kinase (PI3K) as well as to regulate proteins that signal in response to lipid products of PI3K. The specific mutations that activate this pathway in breast cancer can occur in genes coding for tyrosine kinase receptors, adaptor proteins linked to PI3K, catalytic and regulatory subunits of PI3K, serine/threonine kinases that function downstream of PI3K, and also phosphatidylinositol 3' phosphatase tumor suppressors that function to antagonize this pathway. While each genetic change results in net elevation of PI3K pathway signaling, and all major breast cancer subtypes show pathway activation, the specific mutation(s) involved in any one tumor may play an important role in defining tumor subtype, prognosis and even sensitivity to therapy. Here, we describe mouse models of breast cancer with elevated PI3K signaling, and how they may be used to guide development of novel therapeutics.

Akt‐mediated regulation of NFκB and the essentialness of NFκB for the oncogenicity of PI3K and Akt

The serine/threonine kinase Akt (cellular homolog of murine thymoma virus akt8 oncogene), also known as PKB (protein kinase B), is activated by lipid products of phosphatidylinositol 3-kinase (PI3K). Akt phosphorylates numerous protein targets that control cell survival, proliferation and motility. Previous studies suggest that Akt regulates transcriptional activity of the nuclear factor-kappaB (NFkappaB) by inducing phosphorylation and subsequent degradation of inhibitor of kappaB (IkappaB). We show here that NFkappaB-driven transcription increases in chicken embryonic fibroblasts (CEF) transformed by myristylated Akt (myrAkt). Accordingly, both a dominant negative mutant of Akt and Akt inhibitors repress NFkappaB-dependent transcription. The degradation of the IkappaB protein is strongly enhanced in Akt-transformed cells, and the loss of NFkappaB activity by introduction of a super-repressor of NFkappaB, IkappaBSR, interferes with PI3K- and Akt-induced oncogenic transformation of CEF. The phosphorylation of the p65 subunit of NFkappaB at serine 534 is also upregulated in Akt-transformed cells. Our data suggest that the stimulation of NFkappaB by Akt is dependent on the phosphorylation of p65 at S534, mediated by IKK (IkappaB kinase) alpha and beta. Akt phosphorylates IKKalpha on T23, and this phosphorylation event is a prerequisite for the phosphorylation of p65 at S534 by IKKalpha and beta. Our results demonstrate two separate functions of the IKK complex in NFkappaB activation in cells with constitutive Akt activity: the phosphorylation and consequent degradation of IkappaB and the phosphorylation of p65. The data further support the conclusion that NFkappaB activity is essential for PI3K- and Akt-induced oncogenic transformation.

Non-Opsonic Phagocytosis of Legionella pneumophila by Macrophages Is Mediated by Phosphatidylinositol 3-Kinase

Background Legionella pneumophila, is an intracellular pathogen that causes Legionnaires' disease in humans, a potentially lethal pneumonia. L. pneumophila has the ability to enter and replicate in the host and is essential for pathogenesis.Methodology/Principal FindingsPhagocytosis was measured by cell invasion assays. Construction of PI3K mutant by PCR cloning and expression of dominant negative mutant was detected by Western blot. PI3K activity was measured by 32P labeling and detection of phospholipids products by thin layer chromatography. Infection of macrophages with virulent L. pneumophila stimulated the formation of phosphatidylinositol 3-phosphate (PIP3), a phosphorylated lipid product of PI3K whereas two structurally distinct phosphatidylinositol 3 kinase (PI3K) inhibitors, wortmannin and LY294002, reduced L. pneumophila entry into macrophages in a dose-dependent fashion. Furthermore, PI3K activation led to Akt stimulation, a serine/threonine kinase, which was also inhibited by wortmannin and LY294002. In contrast, PI3K and protein kinase B (PKB/Akt) activities were lower in macrophages infected with an avirulent bacterial strain. Only virulent L. pneumophila increased lipid kinase activity present in immunoprecipitates of the p85α subunit of class I PI3K and tyrosine phosphorylated proteins. In addition, macrophages expressing a specific dominant negative mutant of PI3K reduced L. pneumophila entry into these cells.Conclusion/SignificanceEntry of L. pneumophila is mediated by PI3K/Akt signaling pathway. These results suggest an important role for PI3K and Akt in the L. pneumophila infection process. They point to possible novel strategies for undermining L. pneumophila host uptake and reducing pathogenesis of Legionnaires' disease.

Pleckstrin-2 selectively interacts with phosphatidylinositol 3-kinase lipid products and regulates actin organization and cell spreading

Pleckstrin-2 (PLEK2) has been implicated to be regulated by phosphatidylinositol (PI) 3-kinase, while pleckstrin1 (PLEK1) has been suggested to be a major PKC substrate in platelets. In this paper, we confirmed that PLEK2 specifically bound to the PI 3-kinase products in vitro and explored its behavior. PLEK2 was found to be expressed in various adherent cell lines, while PLEK1 expression was restricted to non-adherent cells in the protein level. Expression of PLEK2 in COS1 cells induced formation of protrusive F-actin structure and enhanced the actin rearrangements induced on collagen- or fibronectin-coated plates. A PLEK2 mutant incapable of binding to the PI 3-kinase products did not show any effect on actin rearrangement. Knockdown of PLEK2 by shRNA inhibited spreading of HCC2998 adenocarcinoma cells. PLEK2 colocalized with Rac and was suggested to be oligomerized. These results suggest that PLEK2 is involved in actin rearrangement in a PI 3-kinase dependent manner.

Metformin improves atypical protein kinase C activation by insulin and phosphatidylinositol-3,4,5-(PO4)3 in muscle of diabetic subjects

Metformin is widely used for treating type 2 diabetes mellitus, but its actions are poorly understood. In addition to diminishing hepatic glucose output, metformin, in muscle, activates 5'-AMP-activated protein kinase (AMPK), which alone increases glucose uptake and glycolysis, diminishes lipid synthesis, and increases oxidation of fatty acids. Moreover, such lipid effects may improve insulin sensitivity and insulin-stimulated glucose uptake. Nevertheless, the effects of metformin on insulin-sensitive signalling factors in human muscle have only been partly characterised to date. Interestingly, other substances that activate AMPK, e.g., aminoimidazole-4-carboxamide-1-beta-D: -riboside (AICAR), simultaneously activate atypical protein kinase C (aPKC), which appears to be required for the glucose transport effects of AICAR and insulin. Since aPKC activation is defective in type 2 diabetes, we evaluated effects of metformin therapy on aPKC activity in muscles of diabetic subjects during hyperinsulinaemic-euglycaemic clamp studies. After metformin therapy for 1 month, basal aPKC activity increased in muscle, with little or no change in insulin-stimulated aPKC activity. Metformin therapy for 8 to 12 months improved insulin-stimulated, as well as basal aPKC activity in muscle. In contrast, IRS-1-dependent phosphatidylinositol (PI) 3-kinase activity and Ser473 phosphorylation of protein kinase B were not altered by metformin therapy, whereas the responsiveness of muscle aPKC to PI-3,4,5-(PO(4))(3), the lipid product of PI 3-kinase, was improved. These findings suggest that the activation of AMPK by metformin is accompanied by increases in aPKC activity and responsiveness in skeletal muscle, which may contribute to the therapeutic effects of metformin.

Gαq Inhibits Cardiac L-type Ca2+ Channels through Phosphatidylinositol 3-Kinase

Cardiac myocyte contractility is initiated by Ca2+ entry through the voltage-dependent L-type Ca2+ channel (LTCC). To study the effect of Galpha q on the cardiac LTCC, we utilized two transgenic mouse lines that selectively express inducible Galpha q-estrogen receptor hormone-binding domain fusion proteins (Galpha qQ209L-hbER or Galpha qQ209L-AA-hbER) in cardiac myocytes. Both of these proteins inhibit phosphatidylinositol (PI) 3-kinase (PI3K) signaling, but Galpha qQ209L-AA-hbER cannot activate the canonical Galpha q effector phospholipase Cbeta (PLCbeta). L-type Ca2+ current (I(Ca,L)) density measured by whole-cell patch clamping was reduced by more than 50% in myocytes from both Galpha q animals as compared with wild-type cells, suggesting that inhibition of the LTCC by Galpha q does not require PLCbeta. To investigate the role of PI3K in this inhibitory effect, I(Ca,L) was measured in the presence of various phosphoinositides infused through the patch pipette. Infusion of PI 3,4,5-trisphosphate (PI(3,4,5)P3) into wild-type myocytes did not affect I(Ca,L), but it fully restored I(Ca,L) density in both Galpha q transgenic myocytes to wild-type levels. By contrast, PI 4,5-bisphosphate (PI(4,5)P2) or PI 3,5-bisphosphate had no effect. Infusion with p110beta/p85alpha or p110gamma PI3K in the presence of PI(4,5)P2 also restored I(Ca,L) density to wild-type levels. Last, infusion of either PTEN, a PI(3,4,5)P3 phosphatase, or the pleckstrin homology domain of Grp1, which sequesters PI(3,4,5)P3, reduced the peak I(Ca,L) density in wild-type myocytes by approximately 30%. Taken together, these results strongly suggest that the inhibitory effect of Galpha q on the cardiac LTCC is mediated by inhibition of PI3K.

Insulin-Sensitive Protein Kinases (Atypical Protein Kinase C and Protein Kinase B/Akt): Actions and Defects in Obesity and Type II Diabetes

Glucose transport into muscle is the initial process in glucose clearance and is uniformly defective in insulin-resistant conditions of obesity, metabolic syndrome, and Type II diabetes mellitus. Insulin regulates glucose transport by activating insulin receptor substrate-1 (IRS-1)-dependent phosphatidylinositol 3-kinase (PI3K) which, via increases in PI-3,4,5-triphosphate (PIP(3)), activates atypical protein kinase C (aPKC) and protein kinase B (PKB/Akt). Here, we review (i) the evidence that both aPKC and PKB are required for insulin-stimulated glucose transport, (ii) abnormalities in muscle aPKC/PKB activation seen in obesity and diabetes, and (iii) mechanisms for impaired aPKC activation in insulin-resistant conditions. In most cases, defective muscle aPKC/PKB activation reflects both impaired activation of IRS-1/PI3K, the upstream activator of aPKC and PKB in muscle and, in the case of aPKC, poor responsiveness to PIP(3), the lipid product of PI3K. Interestingly, insulin-sensitizing agents (e.g., thiazolidinediones, metformin) improve aPKC activation by insulin in vivo and PIP3 in vitro, most likely by activating 5'-adenosine monophosphate-activated protein kinase, which favorably alters intracellular lipid metabolism. Differently from muscle, aPKC activation in the liver is dependent on IRS-2/PI3K rather than IRS-1/PI3K and, surprisingly, the activation of IRS-2/PI3K and aPKC is conserved in high-fat feeding, obesity, and diabetes. This conservation has important implications, as continued activation of hepatic aPKC in hyperinsulinemic states may increase the expression of sterol regulatory element binding protein-1c, which controls genes that increase hepatic lipid synthesis. On the other hand, the defective activation of IRS-1/PI3K and PKB, as seen in diabetic liver, undoubtedly and importantly contributes to increases in hepatic glucose output. Thus, the divergent activation of aPKC and PKB in the liver may explain why some hepatic actions of insulin (e.g., aPKC-dependent lipid synthesis) are increased while other actions (e.g., PKB-dependent glucose metabolism) are diminished. This may explain the paradox that the liver secretes excessive amounts of both very low density lipoprotein triglycerides and glucose in Type II diabetes. Previous reviews from our laboratory that have appeared in the Proceedings have provided essentials on phospholipid-signaling mechanisms used by insulin to activate several protein kinases that seem to be important in mediating the metabolic effects of insulin. During recent years, there have been many new advances in our understanding of how these lipid-dependent protein kinases function during insulin action and why they fail to function in states of insulin resistance. The present review will attempt to summarize what we believe are some of the more important advances.

Na,K-ATPase, Scaffolding Actin Reorganization

The Na,K-ATPase is best known for its role in ion homeostasis; however, it is emerging as a regulator of signaling processes as well. The pump is composed of two subunits, a catalytic, ion-transporting α subunit and a β subunit, which is required for proper targeting to the plasma membrane. Decreases in Na,K-ATPase have been associated with cancer and increased cell motility. Barwe et al. studied how the abundance of the noncatalytic β subunit affected cell motility of Maloney sarcoma virus-transformed Madin-Darby canine kidney (MSV-MDCK) cells. They compared cells expressing very low levels of the β subunit, which had previously been shown to have a high motility phenotype, with cells overexpressing the β subunit and found that motility was decreased and the formation of lamellipodia was stimulated in the overexpressing cells. The abundance of active Rac1 (a guanosine triphosphatase that regulates actin reorganization) was increased, and the activity of phosphatidylinositol 3-kinase (PI3K) was also increased in the cells overexpressing the β subunit. Each of these effects required the C-terminal domain of the β subunit, suggesting a protein-protein interaction. Using a glutathione pull-down assay, the authors found that annexin II interacted with the β subunit C-terminal domain, and this interaction was confirmed by coimmunoprecipitation assays. The interaction between the β subunit and annexin II was inhibited if cells were treated with a pharmacological inhibitor of PI3K. The catalytic α subunit was important as well. Although overexpression of the α subunit alone had no effect on motility, the α subunit interacted with the p85 subunit of PI3K, and the abundance of phosphorylated p85 correlated with the abundance of the α subunit. Inhibition of the ion transport activity of Na,K-ATPase with ouabain did not alter the suppression of motility caused by overexpression of the β subunit. The authors propose that the α subunit, when delivered to the plasma membrane by proper assembly with the β subunit, interacts with the p85 subunit, allowing PI3K to be active. Annexin II interacts with the lipid products of PI3K and interacts with the β subunit of the Na,K-ATPase and sequesters active Rac1, leading to actin reorganization, the formation of lamellipodia, and decreased motility. S. P. Barwe, G. Anilkumar, S. Y. Moon, Y. Zheng, J. P. Whitelegge, S. A. Rajasekaran, A. K. Rajasekaran, Novel role for Na,K-ATPase in phosphatidylinositol 3-kinase signaling and suppression of cell motility. Mol. Biol. Cell 16 , 1082-1094 (2005). [Abstract] [Full Text]

Protein Kinase C βII Regulates Akt Phosphorylation on Ser-473 in a Cell Type- and Stimulus-specific Fashion

Akt (= protein kinase B), a subfamily of the AGC serine/threonine kinases, plays critical roles in survival, proliferation, glucose metabolism, and other cellular functions. Akt activation requires the recruitment of the enzyme to the plasma membrane by interacting with membrane-bound lipid products of phosphatidylinositol 3-kinase. Membrane-bound Akt is then phosphorylated at two sites for its full activation; Thr-308 in the activation loop of the kinase domain is phosphorylated by 3-phosphoinositide-dependent kinase-1 (PDK1) and Ser-473 in the C-terminal hydrophobic motif by a putative kinase PDK2. The identity of PDK2 has been elusive. Here we present evidence that conventional isoforms of protein kinase C (PKC), particularly PKCbetaII, can regulate Akt activity by directly phosphorylating Ser-473 in vitro and in IgE/antigen-stimulated mast cells. By contrast, PKCbeta is not required for Ser-473 phosphorylation in mast cells stimulated with stem cell factor or interleukin-3, in serum-stimulated fibroblasts, or in antigen receptor-stimulated T or B lymphocytes. Therefore, PKCbetaII appears to work as a cell type- and stimulus-specific PDK2.

Defective Activation of Protein Kinase C-z in Muscle by Insulin and Phosphatidylinositol-3,4,5,-(PO4)3in Obesity and Polycystic Ovary Syndrome

Insulin resistance occurs frequently in metabolic syndrome components, obesity, and the polycystic ovary syndrome, and is partly due to impaired glucose transport into skeletal muscle, but underlying mechanisms are uncertain. Atypical protein kinase C and protein kinase B, operating downstream of phosphatidylinositol 3-kinase, mediate insulin effects on glucose transport, but their importance in these syndromes is poorly understood. Presently, we examined these signaling factors in muscle biopsies obtained during euglycemic/hyperinsulinemic clamp studies. In lean subjects, insulin provoked approximately twofold increases in muscle atypical protein kinase C activity. In obese subjects and obese subjects who had evidence of the polycystic ovary syndrome, insulin-stimulated glucose disposal and atypical protein kinase C activation were diminished, whereas activation of insulin receptor substrate-1-dependent phosphatidylinositol 3-kinase and protein kinase B trended lower, but not significantly. Interestingly, direct activation of atypical protein kinase C by phosphatidylinositol-3,4,5-(PO(4))(3), the lipid product of phosphatidylinositol 3-kinase, was readily apparent in immunoprecipitates prepared from muscles of lean subjects, but to a lesser degree or poorly if at all in subjects who were obese or had the obesity/polycystic ovary syndrome. Our findings suggest that activation of muscle atypical protein kinase C by insulin and phosphatidylinositol-3,4,5-(PO(4))(3) is defective and may contribute to skeletal muscle insulin resistance in women who are obese, or have obesity associated with the polycystic ovary syndrome.

Mevastatin suppresses lipopolysaccharide-induced Rac activation in the human monocyte cell line THP-1

Background. Rac is a member of the Rho family of small guanosine triphosphatases that regulate the actin cytoskeleton. Activation of Rac requires lipid modification that can be blocked by statins. Lipopolysaccharide-induced rearrangements in the actin cytoskeleton lead to changes in cell adhesion and motility that play a role in the cellular response to infection. The lipid products of phosphatidylinositol-3 kinase (PI3K) modulate Rac effector pathways and have been linked to the activation of the Rac-guanosine triphosphatase. We hypothesize that lipopolysaccharide stimulation leads to Rac activation and that this may be inhibited by statin pretreatment. Furthermore, signaling downstream of Rac is linked to PI3K; therefore, we hypothesize that a signaling complex between PI3K and Rac may be involved. Methods. THP-1 cells were maintained in 1% fetal calf serum with or without 20 μmol/L mevastatin for 24 hours, followed by lipopolysaccharide stimulation. Active Rac was precipitated from THP-1 total cell lysate and then detected by immunoblotting. The PI3K-Rac complex was immunoprecipitated from total cell lysate, and the p85 regulatory subunit of PI3K was detected by immunoblotting. Results. Lipopolysaccharide stimulation activated Rac. Rac activation was suppressed by pretreatment with mevastatin. The p85 subunit of PI3K was associated with Rac. Conclusion. Lipopolysaccharide stimulation leads to Rac activation in THP-1 cells, which may be suppressed with mevastatin pretreatment. There is an association between Rac and PI3K that demonstrates a role for PI3K in the activation of downstream Rac effector pathways. (Surgery 2003;134:306-11.)

Mechanisms by which cAMP increases bile acid secretion in rat liver and canalicular membrane vesicles.

Bile acid secretion induced by cAMP and taurocholate is associated with recruitment of several ATP binding cassette (ABC) transporters to the canalicular membrane. Taurocholate-mediated bile acid secretion and recruitment of ABC transporters are phosphatidylinositol 3-kinase (PI3K) dependent and require an intact microtubular apparatus. We examined mechanisms involved in cAMP-mediated bile acid secretion. Bile acid secretion induced by perfusion of rat liver with dibutyryl cAMP was blocked by colchicine and wortmannin, a PI3K inhibitor. Canalicular membrane vesicles isolated from cAMP-treated rats manifested increased ATP-dependent transport of taurocholate and PI3K activity that were reduced by prior in vivo administration of colchicine or wortmannin. Addition of a PI3K lipid product, phosphoinositide 3,4-bisphosphate, but not its isomer, phosphoinositide 4,5-bisphosphate, restored ATP-dependent taurocholate in these vesicles. Addition of a decapeptide that activates PI3K to canalicular membrane vesicles increased ATP-dependent transport above baseline activity. In contrast to effects induced by taurocholate, cAMP-stimulated intracellular trafficking of the canalicular ABC transporters was unaffected by wortmannin, and recruitment of multidrug resistance protein 2, but not bile salt excretory protein (bsep), was partially decreased by colchicine. These studies indicate that trafficking of bsep and other canalicular ABC transporters to the canalicular membrane in response to cAMP is independent of PI3K activity. In addition, PI3K lipid products are required for activation of bsep in the canalicular membrane. These observations prompt revision of current concepts regarding the role of cAMP and PI3K in intracellular trafficking, regulation of canalicular bsep, and bile acid secretion.

Inhibition of phosphatidylinositol 3-kinase signaling negates the growth advantage imparted by a mutant epidermal growth factor receptor on human glioblastoma cells.

In de novo glioblastoma multiforme, loss of the tumour suppressor protein PTEN can coincide with the expression of a naturally occurring mutant epidermal growth factor receptor known as deltaEGFR. DeltaEGFR signals constitutively via the phosphatidylinositol 3-kinase (PI3K)/protein kinase Akt and mitogen-activated protein kinase pathways. In human U87MG glioblastoma cells that lack PTEN, deltaEGFR expression enhances tumourigenicity by increasing cellular proliferation. Inhibition of PI3K signaling with the pharmacologic inhibitor wortmannin, or by the reconstitution of physiological levels of PTEN to dephosphorylate the lipid products of PI3K, negated the growth advantage imparted by deltaEGFR on U87MG cells. PTEN reconstitution suppressed the elevated PI3K signaling, without affecting mitogen-activated protein kinase signaling and caused a delay in G1 cell cycle progression that was concomitant with increased cyclin-dependent protein kinase inhibitor p21CIP1/WAF1 protein levels. Our study provides insight into the mechanism by which deltaEGFR may contribute to glioblastoma development.

Defective activation of atypical protein kinase C zeta and lambda by insulin and phosphatidylinositol-3,4,5-(PO4)(3) in skeletal muscle of rats following high-fat feeding and streptozotocin-induced diabetes.

Insulin-stimulated glucose transport in skeletal muscle is thought to be effected at least partly through atypical protein kinase C isoforms (aPKCs) operating downstream of phosphatidylinositol (PI) 3-kinase and 3-phosphoinositide-dependent protein kinase-1 (PDK-1). However, relatively little is known about the activation of aPKCs in physiological conditions or insulin-resistant states. Presently, we studied aPKC activation in vastus lateralis muscles of normal chow-fed and high-fat-fed rats and after streptozotocin (STZ)-induced diabetes. In normal chow-fed rats, dose-dependent increases in aPKC activity approached maximal levels after 15-30 min of stimulation by relatively high and lower, presumably more physiological, insulin concentrations, achieved by im insulin or ip glucose administration. Insulin-induced activation of aPKCs was impaired in both high-fat-fed and STZ-diabetic rats, but, surprisingly, IRS-1-dependent and IRS-2-dependent PI 3-kinase activation was not appreciably compromised. Most interestingly, direct in vitro activation of aPKCs by PI-3,4,5-(PO(4))(3), the lipid product of PI 3-kinase, was impaired in both high-fat-fed and STZ-diabetic rats. Defects in activation of aPKCs by insulin and PI-3,4,5-(PO(4))(3) could not be explained by diminished PDK-1-dependent phosphorylation of threonine-410 in the PKC-zeta activation loop, as this phosphorylation was increased even in the absence of insulin treatment in high-fat-fed rats. 1) muscle aPKCs are activated at relatively low, presumably physiological, as well as higher supraphysiological, insulin concentrations; 2) aPKC activation is defective in muscles of high-fat-fed and STZ-diabetic rats; and 3) defective aPKC activation in these states is at least partly due to impaired responsiveness to PI-3,4,5-(PO(4))(3), apparently at activation steps distal to PDK-1-dependent loop phosphorylation.

Bile acid depletion and repletion regulate cholangiocyte growth and secretion by a phosphatidylinositol 3-kinase–dependent pathway in rats

Background & Aims: We tested the hypothesis that during bile duct obstruction, increased biliary bile acids trigger cholangiocyte proliferation and secretion by a phosphatidylinositol 3-kinase (PI3-K)–dependent pathway. Methods: In bile duct–incannulated (BDI) rats, bile duct obstruction present for 7 days was relieved for 24 hours by external bile drainage. During the 24-hour drainage period, animals received either Krebs Ringer Henseleit (the bile-depleted group), or sodium taurocholate (the bile-depleted, taurocholate-infused group). We evaluated cholangiocyte proliferation and secretin-stimulated ductal secretion. Apical bile acid transporter (ABAT) expression and bile acid transport activity was determined. In pure preparations of cholangiocytes, we examined the effect of taurocholate (in the absence or presence of wortmannin or PI 3,4-bisphosphate the lipid product of PI3-K) on cholangiocyte proliferation and secretin-stimulated cyclic adenosine 3',5'-monophosphate (cAMP) levels. Results: Bile depletion reduced cholangiocyte proliferation and secretin-stimulated ductal secretion and ABAT expression and bile acid transport activity compared with 1-week BDI control rats. In bile-depleted, taurocholate-infused rats, cholangiocyte proliferation and secretion and ABAT expression and bile acid transport activity were maintained at levels similar to those seen in BDI control rats. In vitro, taurocholate stimulation of DNA replication and secretin-stimulated cAMP levels was blocked by wortmannin. The inhibitory effect of wortmannin on taurocholate stimulation of cholangiocyte proliferation and secretion was prevented by PI 3,4-bisphosphate. Conclusions: Bile acid uptake by ABAT and the PI3-K pathway are important for bile acids to signal cholangiocyte proliferation. In bile duct obstruction, increased biliary bile acid concentration and ABAT expression initiate increased cholangiocyte proliferation and secretion.GASTROENTEROLOGY 2002;123:1226-1237

PTEN Modulates Vascular Endothelial Growth Factor-Mediated Signaling and Angiogenic Effects

Phosphatidylinositol 3-kinase is activated by vascular endothelial growth factor (VEGF), and many of the angiogenic cellular responses of VEGF are regulated by the lipid products of phosphatidylinositol 3-kinase. The tumor suppressor PTEN has been shown to down-regulate phosphatidylinositol 3-kinase signaling, yet the effects of PTEN on VEGF-mediated signaling and angiogenesis are unknown. Inhibition of endogenous PTEN in cultured endothelial cells by adenovirus-mediated overexpression of a dominant negative PTEN mutant (PTEN-C/S) enhanced VEGF-mediated Akt phosphorylation, and this effect correlated with decreases in caspase-3 cleavage, caspase-3 activity, and DNA degradation after induction of apoptosis with tumor necrosis factor-alpha. Overexpression of PTEN-C/S also enhanced VEGF-mediated endothelial cell proliferation and migration. In contrast, overexpression of wild-type PTEN inhibited the anti-apoptotic, proliferative, and chemotactic effects of VEGF. Moreover, PTEN-C/S increased the length of vascular sprouts in the rat aortic ring assay and modulated VEGF-mediated tube formation in an in vitro angiogenesis assay, whereas PTEN-wild type inhibited these effects. Taken together, these findings demonstrate that PTEN potently modulates VEGF-mediated signaling and function and that PTEN is a viable target in therapeutic approaches to promote or inhibit angiogenesis.

Direct Interaction between the Cytoplasmic Tail of ADAM 12 and the Src Homology 3 Domain of p85α Activates Phosphatidylinositol 3-Kinase in C2C12 Cells

ADAM 12, a member of the ADAM family of transmembrane metalloprotease-disintegrins, has been implicated previously in the differentiation of skeletal myoblasts. In the present study, we show that the cytoplasmic tail of mouse ADAM 12 interacts in vitro and in vivo with the Src homology 3 domain of the p85alpha regulatory subunit of phosphatidylinositol (PI) 3-kinase. By site-directed mutagenesis, we have identified three p85alpha-binding sites in ADAM 12 involving PXXP motifs located at amino acids 825-828, 833-836, and 884-887. Using green fluorescent protein (GFP)-pleckstrin homology (PH) domain fusion protein as a probe for PI 3-kinase lipid products, we have further demonstrated that expression of ADAM 12 in C2C12 cells resulted in translocation of GFP-PH to the plasma membrane. This suggests that transmembrane ADAM 12, by providing docking sites for the Src homology 3 domain of p85alpha, activates PI 3-kinase by mediating its recruitment to the membrane. Because PI 3-kinase is critical for terminal differentiation of myoblasts, and because expression of ADAM 12 is up-regulated at the onset of the differentiation process, ADAM 12-mediated activation may constitute one of the regulatory mechanisms for PI 3-kinase during myoblast differentiation.

FMet-Leu-Phe stimulates proinflammatory cytokine gene expression in human peripheral blood monocytes: the role of phosphatidylinositol 3-kinase.

The fMLP-stimulated release of proinflammatory cytokines such as IL-1 by human peripheral blood monocytes is an important component of the inflammatory process. The signaling mechanisms used by fMLP to stimulate the release of cytokines are still incompletely understood. We previously demonstrated that fMLP-stimulated NF-kappaB activation in PBMC and now we present evidence that the lipid products of phosphatidylinositol 3-kinase (PI 3-kinase) are required for fMLP-stimulated activation of NF-kappaB. Pretreatment with the PI 3-kinase inhibitors, wortmannin and LY294002, effectively blocked fMLP-induced IL-1beta gene expression as well as NF-kappaB activation. Transient transfection of THP1 cells with a dominant-negative mutant of the PI 3-kinase p85 subunit also abrogated fMLP-induced kappaB activity. These results suggest a potential role of fMLP in the transcription of proinflammatory cytokines and provide the first evidence that such regulation may occur through PI 3-kinase activity.

Phosphoinositide 3-kinase lipid products regulate ATP-dependent transport by sister of P-glycoprotein and multidrug resistance associated protein 2 in bile canalicular membrane vesicles.

Bile acid transport and secretion in hepatocytes require phosphatidylinositol (PI) 3-kinase-dependent recruitment of ATP-dependent transporters to the bile canalicular membrane and are accompanied by increased canalicular PI 3-kinase activity. We report here that the lipid products of PI 3-kinase also regulate ATP-dependent transport of taurocholate and dinitrophenyl-glutathione directly in canalicular membranes. ATP-dependent transport of taurocholate and dinitrophenyl-glutathione in isolated canalicular vesicles from rat liver was reduced 50-70% by PI 3-kinase inhibitors, wortmannin, and LY294002, at concentrations that are specific for Type I PI 3-kinase. Inhibition was reversed by addition of lipid products of PI 3-kinase (PI 3,4-bisphosphate and, to a lesser extent, PI 3-phosphate and PI 3,4,5-trisphosphate) but not by PI 4, 5-bisphosphate. A membrane-permeant synthetic 10-mer peptide that binds polyphosphoinositides and leads to activation of PI 3-kinase in macrophages doubled PI 3-kinase activity in canalicular membrane vesicles and enhanced taurocholate and dinitrophenyl-glutathione transport in canalicular membrane vesicles above maximal ATP-dependent transport. The effect of the peptide was blocked by wortmannin and LY294002. PI 3-kinase activity was also necessary for function of the transporters in vivo. ATP-dependent transport of taurocholate and PI 3-kinase activity were reduced in canalicular membrane vesicles isolated from rat liver that had been perfused with taurocholate and wortmannin. PI 3,4-bisphosphate enhanced ATP-dependent transport of taurocholate in these vesicles above control levels. Our results indicate that PI 3-kinase lipid products are necessary in vivo and in vitro for maximal ATP-dependent transport of bile acid and nonbile acid organic anions across the canalicular membrane. Our results demonstrate regulation of membrane ATP binding cassette transporters by PI 3-kinase lipid products.

Phosphatidylinositol 3-Kinase Translocates onto Liver Endoplasmic Reticulum and May Account for the Inhibition of Glucose-6-phosphatase during Refeeding

By using a rapid procedure of isolation of microsomes, we have shown that the liver glucose-6-phosphatase activity was lowered by about 30% (p < 0.001) after refeeding for 360 min rats previously unfed for 48 h, whereas the amount of glucose-6-phosphatase protein was not lowered during the same time. The amount of the regulatory subunit (p85) and the catalytic activity of phosphatidylinositol 3-kinase (PI3K) were higher by a factor of 2.6 and 2.4, respectively (p < 0.01), in microsomes from refed as compared with fasted rats. This resulted from a translocation process because the total amount of p85 was the same in the whole liver homogenates from fasted and refed rats. The amount of insulin receptor substrate 1 (IRS1) was also higher by a factor of 2.6 in microsomes from refed rats (p < 0. 01). Microsome-bound IRS1 was only detected in p85 immunoprecipitates. These results strongly suggest that an insulin-triggered mechanism of translocation of PI3K onto microsomes occurs in the liver of rats during refeeding. This process, via the lipid products of PI3K, which are potent inhibitors of glucose-6-phosphatase (Mithieux, G., Danièle, N., Payrastre, B., and Zitoun, C. (1998) J. Biol. Chem. 273, 17-19), may account for the inhibition of the enzyme and participate to the inhibition of hepatic glucose production occurring in this situation.