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11 recordsLinked to original sources
PubMed2026

PIP3 antagonist as a molecular regulator in MSC-derived cardiomyocytes: Potential in vitro therapeutic implications for conotruncal heart defects.

Conotruncal heart defects (CTDs) account for approximately one-third of all congenital heart defects. Elevated levels of phosphatidylinositol (3,4,5)-trisphosphate (PIP3) may contribute to CTD pathogenesis. PIP3 plays a pivotal role in mechanotransduction-based biological processes and remodeling of cardiac cytoskeletal proteins. Here, we aimed to evaluate the efficacy of the 322PESB derivative compound as a molecular regulator that antagonizes PIP3 binding pleckstrin homology (PH) domain of the Akt protein using mesenchymal stem cell-derived cardiomyocyte. Human adipose-derived MSCs (Ad-MSCs) were isolated. Immunophenotypic features of the hAd-MSCs were characterized according to minimal criteria of the international society for cellular therapy (ISCT) including immunophenotyping and trilineage differentiation potential. Subsequently, the differentiated hAd-MSCs were cultured in cardiomyogenesis-inducing medium. Successfully differentiated cardiomyocytes were assessed by measuring the expression levels of cardiomyocyte-specific genes using RT-qPCR. PIP3-primed cardiomyocytes were treated with 10 and 30 μmol/L of a 322PESB derivative molecule. The results showed a typical MSCs with high expression levels of CD73 (77.55%), CD90 (87.59%) and CD105 (91.88%) and that was accompanied by low expression levels of CD34 (0.59%) and CD45 (1.78%). After 21 days of MSC culture, cardiomyocyte-like cells with prominent striations were observed. Subsequent confirmation by RT-qPCR quantification of ADRB1 and MLC2a expression levels showed an average increase of 2.9-fold and 2.1-fold, respectively, in induced cardiomyocytes. Compared with the untreated control, PIP3 ELISA assay showed a significant increase in PIP3 levels in PIP3(10 nmol/L)-primed cardiomyocytes treated with 10 and 30 μmol/L of the 322PESB molecule derivative by 485.804 and 3564.164 ng/mL, respectively. In this study, we conducted the first promising molecular regulator with potential therapeutic implications for CTD patients. Further functional animal model and clinical phase studies are recommended.

Cardiomyocyte↗
PubMed1989

The identification of a novel inositol lipid, phosphatidylinositol trisphosphate (PIP3), in rat cerebrum using in vivo techniques.

Rats received intraventricular injections of 20 uCi of [3H]-myo-inositol, and were sacrificed 24 hrs later by high-power head-focused microwave fixation. Two inositol lipid extraction methods were compared: The Hauser and Eichberg method yielded higher recovery of inositol lipids, but a lower inositol phosphate content. The Schacht method yielded reduced radiolabel in the lipid fractions, but increased water soluble phosphates. Both methods extracted a novel inositol lipid (PIP3) which contained inositol tetrakisphosphate (IP4) as its polar head group. This was determined by alkaline hydrolysis and analyzed by high performance liquid chromatography with authentic IP4 standard. Furthermore, preliminary studies of the fatty acid composition indicated a similarity with other inositol lipids. The radiolabel ratio of PIP2:PIP3 was 5:1. In summary, we have isolated a novel inositol phospholipid in rat brain, PIP3, the parent compound for inositol tetrakisphosphate (IP4).

Animals↗
PubMed1989

Transient increase in phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol trisphosphate during activation of human neutrophils.

We recently showed that phosphatidylinositol trisphosphate (PIP3) was present in a unique lipid fraction generated in neutrophils during activation. Here, we demonstrate that the band containing this fraction isolated from thin layer chromatography consists primarily of PIP3 and that only small amounts of radiolabeled PIP3 exist prior to activation. In addition, high performance liquid chromatography of deacylated phospholipids from stimulated cells reveals an increase in a fraction eluting ahead of glycerophosphoinositol 4,5-P2. After removal of the glycerol we found that it coeluted with inositol 1,3,4-P3 when resubjected to high performance liquid chromatography. Thus, we have detected a second, novel form of phosphatidylinositol bisphosphate in activated neutrophils, PI-(3,4)P2. The elevation of PIP3 through the formyl peptide receptor is blocked by pretreatment with pertussis toxin, implicating mediation of the increase in PIP3 by a guanosine triphosphate-binding (G) protein. The rise in PIP3 is not secondary to calcium elevation. Buffering the rise in intracellular calcium did not diminish the increase in PIP3. The elevation of PIP3 appears to occur during activation with physiological agonists, its level varying with the degree of activation. Leukotriene B4, which elicits many of the same responses as stimulation of the formyl peptide receptor but with minimal oxidant production, stimulates a much attenuated rise in PIP3. Isoproterenol, which inhibits oxidant production also reduces the rise in PIP3. Hence formation of PI(3,4)P2 and PIP3 (presumed to be PI(3,4,5)P3) correlates closely with the early events of neutrophil activation.

Autoradiography↗
PubMed1990

Is there a relationship between phosphatidylinositol trisphosphate and F-actin polymerization in human neutrophils?

Stimulation of human neutrophils with the chemoattractant N-formyl peptide caused rapid polymerization of F-actin as detected by right angle light scatter and 7-nitrobenz-2-oxa-1,3-diazol (NBD)-phallacidin staining of F-actin. After labeling neutrophils with 32P, exposure to N-formyl peptide induced a fast decrease of phosphatidylinositol 4-bisphosphate (PIP)2, a slow increase of phosphatidic acid, and a rapid rise of phosphatidylinositol 4-trisphosphate (PIP3). Formation of PIP3 as well as actin polymerization was near maximal at 10 s after stimulation. Half-maximal response and PIP3 formation at early time points resulted from stimulation of neutrophils with 0.01 nM N-formyl peptide or occupation of about 200 receptors. Sustained elevation of PIP3, prolonged right angle light scatter response, and F-actin formation required higher concentrations of N-formyl peptide, occupation of thousands of receptors, and high binding rates. When ligand binding was interrupted with an antagonist, F-actin rapidly depolymerized, transient light scatter response recovered immediately, and elevated [32P]PIP3 levels decayed toward initial values. However, recovery of [32P]PIP2 was not influenced by the antagonist. Based on the parallel time courses and dose response of [32P] PIP3, the right angle light scatter response, and F-actin polymerization, PIP3 is more likely than PIP2 to be involved in modulation of actin polymerization and depolymerization in vivo.

Actins↗
PubMed1992

C5a reduces formyl peptide-induced actin polymerization and phosphatidylinositol(3,4,5)trisphosphate formation, but not phosphatidylinositol (4,5) bisphosphate hydrolysis and superoxide production, in human neutrophils.

We investigated phospholipid signal transduction, calcium flux, O2- anion production and actin polymerization after stimulation with the C fragment and chemoattractant, C5a, and then determined how C5a pretreatment affected subsequent responses to formyl peptide in human neutrophils. We have previously demonstrated that the novel lipids, phosphatidylinositol trisphosphate (PIP3) and phosphatidylinositol(3,4)P2 (PI(3,4)P2), rise transiently in neutrophils after activation with formyl peptide. Furthermore, the rise in PIP3 parallels actin polymerization. In this study, neutrophils activated with C5a exhibited two distinct G protein-dependent signal pathways involving different phosphoinositides: 1) [32P]PI(4,5)P2 hydrolysis and [32P]PA production, and 2) the transient formation of D-3-phosphorylated phosphoinositides, [32P]PIP3 and [32P]PI(3,4)P2. When neutrophils were preincubated with C5a for 5 min before stimulation with formyl peptide, [32P]PI(4,5)P2 hydrolysis was unchanged, and [32P]PA production and O2- formation were slightly enhanced compared with controls stimulated with formyl peptide in the absence of C5a. In contrast, [32P]PIP3 production, right angle light scatter, and actin polymerization were all reduced 35 to 40%. Therefore, these data support the hypothesis that PIP3 plays a role in chemotaxis but not superoxide formation.

Actins↗
PubMed1989

Polyphosphoinositides produced by phosphatidylinositol 3-kinase are poor substrates for phospholipases C from rat liver and bovine brain.

The ability of three pure types of bovine brain phospholipase C (PLC) and one pure rat liver PLC to utilize as substrates the recently discovered phosphatidylinositol 3-phosphate (PI-3-P), a putative phosphatidylinositol 3,4-bisphosphate (PI-3,4-P2), and phosphatidylinositol trisphosphate (PIP3) was investigated. PI-3-P, PI-3,4-P2, and PIP3 are the products of phosphorylation of PI, PI-4-P, and PI-4,5-P2, respectively, by phosphoinositide 3-kinase activities that are associated with certain protein-tyrosine kinases. Although these new phospholipids have been found in intact cells, PI-3,4-P2 and PIP3 appear only after stimulation of quiescent cells with growth factors such as platelet-derived growth factor (Auger, K. R., Serunian, L. A., Soltoff, S. P., Libby, P., and Cantley, L. C. (1989) Cell 57, 167-175) and after transformation by certain oncoproteins (L. A. Serunian, K. R. Auger, T. M. Roberts, and L. C. Cantley, manuscript in preparation). Mixtures of [3H]PI-4-P plus [32P]PI-3-P or [3H]PI-4,5-P2 plus [32P]PI-3,4-P2 or PIP3 alone were used as substrates for PLCs in vitro. After incubation with enzyme followed by extraction with chloroform/methanol/HCl, the ratio of 3H/32P in the aqueous layer revealed the selective hydrolysis of PI-4-P and PI-4,5-P2 over PI-3-P and PI-3,4-P2. High performance liquid chromatography analysis of the aqueous layer containing reaction products confirmed that only PI-4-P and PI-4,5-P2, were hydrolyzed to inositol 1,4-P2 and inositol 1,4,5-P3, respectively. These findings suggest that the turnover of PI-3-P, PI-3,4-P2, and PIP3 occurs independently of the turnover of PI-4-P and PI-4,5-P2.

Animals↗
PubMed1990

Production of novel polyphosphoinositides in vivo is linked to cell transformation by polyomavirus middle T antigen.

Phosphatidylinositol 3-kinase associates with the polyomavirus middle T antigen (PyMTAg)-pp60c-src complex in polyomavirus-transformed cells. Here we show that anti-PyMTAg immunoprecipitates from PyMTAg-transformed NIH 3T3 cells have lipid kinase activities that phosphorylate phosphatidylinositol, phosphatidylinositol-4-bisphosphate, and phosphatidylinositol-4,5-bisphosphate at the D-3 position of the inositol ring to produce three new polyphosphoinositides: phosphatidylinositol-3-phosphate (PI-3-P), phosphatidylinositol-3,4-bisphosphate (PI-3,4-P2), and phosphatidylinositol trisphosphate (PIP3), respectively. PI-3-P was detected in intact parental and PyMTAg-transformed NIH 3T3 fibroblasts at both low and high cell densities. However, parental NIH 3T3 fibroblasts produced no detectable PI-3,4-P2 or PIP3 at high density. In contrast, growing, subconfluent cells and wild-type PyMTAg-transformed cells at high density had greatly enhanced incorporation of [3H]-inositol into these highly phosphorylated lipids. Cells transfected with a transformation-defective mutant of PyMTAg had undetectable levels of PI-3,4-P2 and PIP3 at high density. Thus, the synthesis of novel polyphosphoinositides by lipid kinase activity associated with PyMTAg correlates with cell growth and transformation.

Animals↗
PubMed1989

PDGF-dependent tyrosine phosphorylation stimulates production of novel polyphosphoinositides in intact cells.

A phosphatidylinositol (PI) kinase activity associated with certain protein tyrosine kinases important in cell proliferation phosphorylates the 3' hydroxyl position of PI to produce phosphatidylinositol-3-phosphate (PI-3-P). Here we report that, in addition to PI-3' kinase activity, anti-phosphotyrosine (alpha-P-tyr) immunoprecipitates from platelet-derived growth factor (PDGF)-stimulated smooth muscle cells (SMC) contain lipid kinase activities that utilize the substrates phosphatidylinositol-4-phosphate (PI-4-P) and phosphatidylinositol-4,5-bisphosphate (PI-4,5-P2). These activities are absent in alpha-P-tyr immunoprecipitates from quiescent SMC. The product of PI-4-P phosphorylation appears to be phosphatidylinositol-3,4-bisphosphate (PI-3,4-P2), a lipid not previously reported. The product of PI-4,5-P2 phosphorylation is phosphatidylinositol-trisphosphate (PIP3). PI-3-P was detected in quiescent SMC and increased only slightly in response to PDGF. PIP3 and the putative PI-3,4-P2 appeared only after the addition of mitogen. Both the temporal production of these novel phospholipids after PDGF stimulation and the observation of the enzymatic activities that produce them in alpha-P-tyr immunoprecipitates suggest that these phospholipids are excellent candidates for mediators of the PDGF mitogenic response.

1-Phosphatidylinositol 4-Kinase↗
PubMed1992

Activation of human neutrophils by mastoparan. Reorganization of the cytoskeleton, formation of phosphatidylinositol 3,4,5-trisphosphate, secretion up-regulation of complement receptor type 3 and superoxide anion production are stimulated by mastoparan.

In human neutrophils, mastoparan induced rapid F-actin polymerization which was followed by a slow and sustained depolymerization to below the initial F-actin content. Incubation of neutrophils with pertussis toxin inhibited mastoparan-stimulated actin polymerization; however it did not prevent sustained depolymerization of F-actin. Analyses of phospholipids performed in parallel revealed that mastoparan stimulated rapid formation of phosphatidylinositol 3,4,5-trisphosphate (PIP3) and consumption of phosphatidylinositol 4,5-bisphosphate (PIP2). Pertussis toxin treatment blocked mastoparan-induced formation of PIP3. Furthermore, mastoparan stimulated the release of N-acetylglucosaminidase from primary granules. Cytochalasin B enhanced mastoparan-stimulated secretion. Mastoparan triggered superoxide radical production in a cytochalasin B-sensitive manner and induced complement type 3 receptor (CR3) up-regulation.

Acetylglucosaminidase↗
PubMed1991

Phosphatidylinositol-3 kinase is activated in v-src, v-yes, and v-fps transformed chicken embryo fibroblasts.

PI-3 kinase activity has been shown to associate with p60v-src. We found that immunoprecipitates of p60v-src exhibit an activity that catalyzes the formation of PI-3-P, PI-3,4-P2 and PIP3 from PI, PI-4-P, and PI-4,5-P2, respectively. Transformation of chicken embryo fibroblasts (CEF) by p60v-src of Rous sarcoma virus (RSV) caused elevation of PI-3-P, PI-3,4-P2, and PIP3, suggesting that the PI-3 kinase may be activated in these cells. Similar elevations were seen in cells transformed with the v-yes or v-fps oncogenes, but not with v-ros or v-ras. We have established also a system that allows the binding of PI-3 kinase to purified p60v-src in vitro, reproducing the binding seen in vivo. This assay indicated that more PI-3 kinase activity binds to purified p60v-src in cell lysates from CEF transformed with v-yes or v-fps, suggesting that some modification or over-expression of PI-3 kinase takes place in these cells.

1-Phosphatidylinositol 4-Kinase↗
PubMed1990

Regulation of phosphoinositide-specific phospholipase C.

The receptors involved in the regulation of phospholipase C by hormones, neurotransmitters and other ligands have seven transmembrane-spanning hydrophobic regions (seven-helix motif) and no known enzymatic activity. Furthermore these receptors can be isolated as complexes with guanine nucleotide binding (G) proteins. Guanine nucleotides affect the binding of hormones that stimulate phospholipase C and it has been possible to see activation of GTPase activity in membranes upon addition of these ligands. Further indirect evidence for a Gp (p stands for phospholipase C activation) protein is the finding that in membranes agonist activation of phospholipase C requires the presence of GTP gamma S a non-hydrolyzable analog of GTP. Furthermore, fluoride is able to activate phospholipase C but its inhibition of phosphatidylinositol-4' kinase (PI-4' kinase) can interfere with efforts to demonstrate this in intact cells. There are four major isozymes of phospholipase C that have been cloned and sequenced. Recently it was found that phospholipase C-gamma as well as PI-3'-kinase are substrates for phosphorylation on tyrosine residues by the EGF and PDGF receptors. The PI-3' kinase is able to convert phosphatidylinositol 4,5-bisphosphate (PIP2) to phosphatidylinositol 3,4,5-trisphosphate (PIP3) but the function of this lipid is unknown since it is not a substrate for any known phospholipase C. While much has been learned about the structure and regulation of the phosphoinositide specific kinases and phosphodiesterase enzymes this is a relatively new field in which we can expect many advances during the next few years.

Animals↗