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At least 1,171 records · Page 65Linked to original sources
PubMed2003

The insulin-like growth factor 1 receptor induces physiological heart growth via the phosphoinositide 3-kinase(p110alpha) pathway.

Insulin-like growth factor 1 (IGF1) was considered a potential candidate for the treatment of heart failure. However, some animal studies and clinical trials have questioned whether elevating IGF1 chronically is beneficial. Secondary effects of increased serum IGF1 levels on other tissues may explain these unfavorable results. The aim of the current study was to examine the role of IGF1 in cardiac myocytes in the absence of secondary effects, and to elucidate downstream signaling pathways and transcriptional regulatory effects of the IGF1 receptor (IGF1R). Transgenic mice overexpressing IGF1R in the heart displayed cardiac hypertrophy, which was the result of an increase in myocyte size, and there was no evidence of histopathology. IGF1R transgenics also displayed enhanced systolic function at 3 months of age, and this was maintained at 12-16 months of age. The phosphoinositide 3-kinase (PI3K)-Akt-p70S6K1 pathway was significantly activated in hearts from IGF1R transgenics. Cardiac hypertrophy induced by overexpression of IGF1R was completely blocked by a dominant negative PI3K(p110alpha) mutant, suggesting IGF1R promotes compensated cardiac hypertrophy in a PI3K(p110alpha)-dependent manner. This study suggests that targeting the cardiac IGF1R-PI3K(p110alpha) pathway could be a potential therapeutic strategy for the treatment of heart failure.

Animals↗
PubMed2003

Stable gene silencing in human monocytic cell lines using lentiviral-delivered small interference RNA. Silencing of the p110alpha isoform of phosphoinositide 3-kinase reveals differential regulation of adherence induced by 1alpha,25-dihydroxycholecalciferol and bacterial lipopolysaccharide.

Studying mononuclear phagocyte cell biology through genetic manipulation by non-viral transfection methods has been challenging due to the dual problems of low transfection efficiency and the difficulty in obtaining stable transfection. To overcome this problem, we developed a system for mediating RNA interference in monocytic cells. The p110alpha isoform of phosphoinositide 3-kinases (PI3Ks) was silenced using a lentiviral vector expressing short hairpin RNA (shRNA). This resulted in the generation of stable THP-1 and U-937 monocytic cell lines deficient in p110alpha. Notably, p110alpha was silenced without affecting levels of either the other class I(A) PI3K catalytic subunits p110beta and p110delta, or the p85alpha regulatory subunit. The role of p110alpha in mediating cell adherence was examined. Monocyte adherence induced in response to either lipopolysaccharide (LPS) or 1alpha,25-dihydroxycholecalciferol (D(3)) was blocked by the PI3K inhibitor LY294002. However, although adherence induced in response to D(3) was sensitive to silencing of p110alpha, LPS-induced adherence was not. Expression of the monocyte differentiation marker CD11b was also induced by D(3) in a PI3K-dependent manner and gene silencing using shRNA showed that p110alpha was also required for this effect. Taken together, these findings demonstrate that LPS and D(3) use distinct isoforms of class I(A) PI3K to induce functional responses and that lentiviral-mediated delivery of shRNA is a powerful approach to study monocyte biology.

Calcitriol↗
PubMed2004

The Hepatitis C virus NS5A protein activates a phosphoinositide 3-kinase-dependent survival signaling cascade.

Hepatitis C virus (HCV) establishes a persistent infection, with up to 80% of infected individuals proceeding to chronic hepatitis, which in many cases may result in liver cirrhosis and hepatocellular carcinoma (HCC); indeed HCV infection is increasingly associated with the development of HCC. The long time period (up to 30 years) between primary infection and the onset of HCC implies that HCV is not directly oncogenic but in some way predisposes patients to develop tumors, though the mechanism for this is unclear as yet. We report here that NS5A binds directly to the Src homology 3 domain of the p85 regulatory subunit of phosphoinositide 3-kinase (PI3K), and this interaction is mediated by a novel (non-proline-rich) motif within NS5A. Coimmunoprecipitation analysis revealed that NS5A bound native heterodimeric PI3K and enhanced the phosphotransferase activity of the catalytic (p110) subunit both in vitro and in human cell lines harboring a subgenomic HCV replicon or expressing NS5A alone. NS5A-mediated activation of PI3K resulted in increased phosphorylation and activity of Akt/protein kinase B and concomitantly provided protection against the induction of apoptosis in both replicon-harboring cells and cells stably expressing NS5A alone. These data suggest that stimulation of PI3K by NS5A may represent an indirect mechanism for development of HCC in HCV-infected patients and further suggests potential therapeutic strategies to counteract the occurrence of HCV-related HCC.

Amino Acid Motifs↗
PubMed2003

Phosphoinositide 3-kinase-mediated activation of cofilin phosphatase Slingshot and its role for insulin-induced membrane protrusion.

Cofilin plays an essential role in actin filament dynamics and membrane protrusion in motile cells. Cofilin is inactivated by phosphorylation at Ser-3 by LIM kinase and reactivated by dephosphorylation by cofilin-phosphatase Slingshot (SSH). Although cofilin is dephosphorylated in response to various extracellular stimuli, signaling pathways regulating SSH activation and cofilin dephosphorylation have remained to be elucidated. Here we show that insulin stimulates the phosphatase activity of Slingshot-1L (SSH1L) and cofilin dephosphorylation in cultured cells, in a manner dependent on phosphoinositide 3-kinase (PI3K) activity. Consistent with this, the level of Ser-3-phosphorylated cofilin is increased in PTEN (phosphatase and tensin homolog deleted in chromosome 10)-overexpressing cells and decreased in PTEN-deficient cells. Insulin induced the accumulation of SSH1L and active Akt (a downstream effector of PI3K), together with a PI3K product phosphatidylinositol 3,4,5-trisphosphate, onto membrane protrusions. Cofilin, but not Ser-3-phosphorylated cofilin, accumulated in membrane protrusions in insulin-stimulated cells, indicating that cofilin is dephosphorylated in these areas. Finally, suppression of SSH1L expression by RNA interference abolished insulin-induced cofilin dephosphorylation and the membrane protrusion. These findings suggest that SSH1L is activated downstream of PI3K and plays a critical role in insulin-induced membrane protrusion by dephosphorylating and activating cofilin.

Actin Depolymerizing Factors↗
PubMed2004

Focal adhesion kinase is upstream of phosphatidylinositol 3-kinase/Akt in regulating fibroblast survival in response to contraction of type I collagen matrices via a beta 1 integrin viability signaling pathway.

The beta(1) integrin, functioning as a mechanoreceptor, senses a mechanical stimulus generated during collagen matrix contraction and down-regulates the phosphatidylinositol 3-kinase (PI3K)/Akt survival signal triggering apoptosis. The identities of integrin-associated signal molecules in the focal adhesion complex that are responsible for propagating beta(1) integrin viability signals in response to collagen matrix contraction are not known. Here we show that in response to collagen contraction focal adhesion kinase (FAK) is dephosphorylated. In contrast, enforced activation of beta(1) integrin by anti-beta(1) integrin antibody, which protects fibroblasts from apoptosis, preserves FAK phosphorylation. We demonstrate that ligation of beta(1) integrin by type I collagen or by enforced activation of beta(1) integrin by antibody promotes phosphorylation of FAK, p85 subunit of PI3K, and serine 473 of Akt. Wortmannin inhibited Akt but not FAK phosphorylation in response to enforced activation of beta(1) integrin by antibody. Blocking FAK by pharmacologic inhibition or by dominant negative FAK attenuated phosphorylation of p85 subunit of PI3K and Akt. Dominant negative FAK augmented fibroblast apoptosis during collagen contraction, and this was associated with diminished Akt activity. Constitutively active FAK augmented levels of p85 subunit of PI3K and Akt phosphorylation, and fibroblasts were protected from apoptosis. Our data identify a novel role for FAK, functioning upstream of PI3K/Akt, in transducing a beta(1) integrin viability signal in collagen matrices.

Adenoviridae↗
PubMed2004

Protein phosphatase-2C alpha as a positive regulator of insulin sensitivity through direct activation of phosphatidylinositol 3-kinase in 3T3-L1 adipocytes.

During differentiation, expression of protein phosphatase-2Calpha (PP2Calpha) is increased in 3T3-L1 adipocytes. To elucidate the role of PP2Calpha in insulin signaling, we overexpressed wild-type (WT) PP2Calpha by adenovirus-mediated gene transfer in 3T3-L1 adipocytes. Overexpression of PP2Calpha-WT enhanced the insulin sensitivity of glucose uptake without any changes in the early steps of insulin signaling. Infection with adenovirus 5 expressing PP2Calpha-WT increased phosphatidylinositol 3-kinase (PI3K) activities in the immunoprecipitate using antibody against the p85 or p110 subunit under both basal and insulin-stimulated conditions, followed by activation of downstream steps in the PI3K pathway, such as phosphorylation of Akt, glycogen synthase kinase-3, and atypical protein kinase C. In contrast, overexpression of the phosphatase-defective mutant PP2Calpha(R174G) did not produce such effects. Furthermore, overexpression of PP2Calpha-WT (but not PP2Calpha(R174G)) decreased the (32)P-labeled phosphorylation state as well as the gel mobility shift of the p85 subunit, suggesting that dephosphorylation of the p85 subunit by PP2Calpha activation might stimulate PI3K catalytic activity. Moreover, knockdown of PP2Calpha by transfection of small interfering RNA led to a significant decrease in Akt phosphorylation. In addition, microinjection of anti-PP2Calpha antibody or PP2Calpha small interfering RNA led to decreased insulin-stimulated GLUT4 translocation. In conclusion, PP2Calpha is a new positive regulator of insulin sensitivity that acts through a direct activation of PI3K in 3T3-L1 adipocytes.

3T3-L1 Cells↗
PubMed2004

Group IB secretory phospholipase A2 promotes matrix metalloproteinase-2-mediated cell migration via the phosphatidylinositol 3-kinase and Akt pathway.

Secretory phospholipase A(2) (sPLA(2)), abundantly expressed in various cells including fibroblasts, is able to promote proliferation and migration. Degradation of collagenous extracellular matrix by matrix metalloproteinase (MMP) plays a role in the pathogenesis of various destructive disorders, such as rheumatoid arthritis, tumor invasion, and metastasis. Here we show that group IB PLA(2) increased pro-MMP-2 activation in NIH3T3 fibroblasts. MMP-2 activity was stimulated by group IB PLA(2) in a dose- and time-dependent manner. Consistent with MMP-2 activation, sPLA(2) decreased expression of type IV collagen. These effects are due to the reduction of tissue inhibitor of metalloproteinase-2 (TIMP-2) and the activation of the membrane type1-MMP (MT1-MMP). The decrease of TIMP-2 levels in conditioned media and the increase of MT1-MMP levels in plasma membrane were observed. In addition, treatment of cells with decanoyl Arg-Val-Lys-Arg-chloromethyl ketone, an inhibitor of pro-MT1-MMP, suppressed sPLA(2)-mediated MMP-2 activation, whereas treatment with bafilomycin A1, an inhibitor of H(+)-ATPase, sustained MMP-2 activation by sPLA(2). The involvement of phosphatidylinositol 3-kinase (PI3K) and Akt in the regulation of MMP-2 activity was further suggested by the findings that PI3K and Akt were phosphorylated by sPLA(2). Expression of p85alpha and Akt mutants, or pretreatment of cells with LY294002, a PI3K inhibitor, attenuated sPLA(2)-induced MMP-2 activation and migration. Taken together, these results suggest that sPLA(2) increases the pro-MMP-2 activation and migration of fibroblasts via the PI3K and Akt-dependent pathway. Because MMP-2 is an important factor directly involved in the control of cell migration and the turnover of extracellular matrix, our study may provide a mechanism for sPLA(2)-promoted fibroblasts migration.

Amino Acid Chloromethyl Ketones↗
PubMed2004

Transformation potential of Ras isoforms correlates with activation of phosphatidylinositol 3-kinase but not ERK.

The Ras oncoproteins activate the Raf-MEK-ERK kinase pathway, which plays an important role in cellular transformation. We observed that H-RasV12 exhibited a higher transforming potential than either K-RasV12 or N-RasV12 in both NIH3T3 fibroblasts and RIE-1 rat epithelial cell cultures. Surprisingly N-Ras and K-Ras were more potent than H-Ras in activation of mitogen-activated protein (MAP) kinase activity and ternary complex factor-dependent transcription. In contrast, H-Ras was more effective in activation of phosphatidylinositol 3-kinase (PI3K) and AKT. Co-expression of constitutively active AKT, a downstream target of PI3K, cooperated with H-RasV12, K-RasV12, or N-RasV12 in transformation. Furthermore co-expression of the constitutively active MEK and AKT resulted in focus formation, while neither active MEK1 nor active AKT alone transformed NIH3T3 cells. Our data demonstrated that the transforming potential of Ras was not directly correlated with the ability of Ras to activate the MAP kinase cascade. In contrast, the ability to activate PI3K and AKT correlated with the ability of Ras to induce cellular transformation, suggesting an important role of PI3K-AKT in cellular transformation. Our data also demonstrated that, under these assay conditions, activation of the MAP kinase cascade was not sufficient to induce NIH3T3 cell transformation.

Animals↗
PubMed2004

Regulation of embryonic stem cell self-renewal by phosphoinositide 3-kinase-dependent signaling.

The maintenance of murine embryonic stem (ES) cell self-renewal is regulated by leukemia inhibitory factor (LIF)-dependent activation of signal transducer and activator of transcription 3 (STAT3) and LIF-independent mechanisms including Nanog, BMP2/4, and Wnt signaling. Here we demonstrate a previously undescribed role for phosphoinositide 3-kinases (PI3Ks) in regulation of murine ES cell self-renewal. Treatment with the reversible PI3K inhibitor, LY294002, or more specific inhibition of class I(A) PI3K via regulated expression of dominant negative Deltap85, led to a reduction in the ability of LIF to maintain self-renewal, with cells concomitantly adopting a differentiated morphology. Inhibition of PI3Ks reduced basal and LIF-stimulated phosphorylation of PKB/Akt, GSK3alpha/beta, and S6 proteins. Importantly, LY294002 and Deltap85 expression had no effect on LIF-induced phosphorylation of STAT3 at Tyr(705), but did augment LIF-induced phosphorylation of ERKs in both short and long term incubations. Subsequently, we demonstrate that inhibition of MAP-Erk kinases (MEKs) reverses the effects of PI3K inhibition on self-renewal in a time- and dose-dependent manner, suggesting that the elevated ERK activity observed upon PI3K inhibition contributes to the functional response we observe. Surprisingly, upon long term inhibition of PI3Ks we observed a reduction in phosphorylation of beta-catenin, the target of GSK-3 action in the canonical Wnt pathway, although no consistent alterations in cytosolic levels of beta-catenin were observed, indicating this pathway is not playing a major role downstream of PI3Ks. Our studies support a role for PI3Ks in regulation of self-renewal and increase our understanding of the molecular signaling components involved in regulation of stem cell fate.

Animals↗
PubMed2004

Extracellular ATP-induced proliferation of adventitial fibroblasts requires phosphoinositide 3-kinase, Akt, mammalian target of rapamycin, and p70 S6 kinase signaling pathways.

Extracellular nucleotides are increasingly recognized as important regulators of growth in a variety of cell types. Recent studies have demonstrated that extracellular ATP is a potent inducer of fibroblast growth acting, at least in part, through an ERK1/2-dependent signaling pathway. However, the contributions of additional signaling pathways to extracellular ATP-mediated cell proliferation have not been defined. By using both pharmacologic and genetic approaches, we found that in addition to ERK1/2, phosphatidylinositol 3-kinase (PI3K), Akt, mammalian target of rapamycin (mTOR), and p70 S6K-dependent signaling pathways are required for ATP-induced proliferation of adventitial fibroblasts. We found that extracellular ATP acting in part through G(i) proteins increased PI3K activity in a time-dependent manner and transient phosphorylation of Akt. This PI3K pathway is not involved in ATP-induced activation of ERK1/2, implying activation of independent parallel signaling pathways by ATP. Extracellular ATP induced dramatic increases in mTOR and p70 S6K phosphorylation. This activation of the mTOR/p70 S6 kinase (p70 S6K) pathway in response to ATP is because of independent contributions of PI3K/Akt and ERK1/2 pathways, which converge on the level of p70 S6K. ATP-dependent activation of mTOR and p70 S6K also requires additional signaling inputs perhaps from pathways operating through Galpha or Gbetagamma subunits. Collectively, our data demonstrate that ATP-induced adventitial fibroblast proliferation requires activation and interaction of multiple signaling pathways such as PI3K, Akt, mTOR, p70 S6K, and ERK1/2 and provide evidence for purinergic regulation of the protein translational pathways related to cell proliferation.

Adenosine Triphosphate↗
PubMed2004

Interleukin-18 is a pro-hypertrophic cytokine that acts through a phosphatidylinositol 3-kinase-phosphoinositide-dependent kinase-1-Akt-GATA4 signaling pathway in cardiomyocytes.

In patients with congestive heart failure, high serum levels of the proinflammatory cytokine interleukin (IL)-18 were reported. A positive correlation was described between serum IL-18 levels and the disease severity. IL-18 has also been shown to induce atrial natriuretic factor (ANF) gene expression in adult cardiomyocytes. Because re-expression of the fetal gene ANF is mostly associated with hypertrophy, a hallmark of heart failure, we hypothesized that IL-18 induces cardiomyocyte hypertrophy. Treatment of the cardiomyocyte cell line HL-1 with IL-18 induced hypertrophy as characterized by increases in protein synthesis, phosphorylated p70 S6 kinase, and ribosomal S6 protein levels as well as cell surface area. Furthermore, IL-18 induced ANF gene transcription in a time-dependent manner as evidenced by increased ANF secretion and ANF promoter-driven reporter gene activity. Investigation into possible signal transduction pathways mediating IL-18 effects revealed that IL-18 activates phosphoinositide 3-kinase (PI3K), an effect that was blocked by wortmannin and LY-294002. IL-18 induced Akt phosphorylation and stimulated its activity, effects that were abolished by Akt inhibitor or knockdown. IL-18 stimulated GATA4 DNA binding activity and increased transcription of a reporter gene driven by multimerized GATA4-binding DNA elements. Pharmacological inhibition or knockdown studies revealed that IL-18 induced cardiomyocyte hypertrophy and ANF gene transcription via PI3K, PDK1, Akt, and GATA4. Most importantly, IL-18 induced ANF gene transcription and hypertrophy of neonatal rat ventricular myocytes via PI3K-, Akt-, and GATA4-dependent signaling. Together these data provide the first evidence that IL-18 induces cardiomyocyte hypertrophy via PI3K-dependent signaling, defines a mechanism of IL-18-mediated ANF gene transcription, and further supports a role for IL-18 in inflammatory heart diseases including heart failure.

Androstadienes↗
PubMed2005

WAVE3-mediated cell migration and lamellipodia formation are regulated downstream of phosphatidylinositol 3-kinase.

WAVE3 is a member of the WASP/WAVE family of protein effectors of actin reorganization and cell movement. The precise role of WAVE3 in cell migration and its regulation, however, have not been elucidated. Here we show that endogenous WAVE3 was found to be concentrated in the lamellipodia at the leading edge of migrating MDA-MB-231 cells. Platelet-derived growth factor (PDGF) treatment induced lamellipodia formation as well as two-dimensional migration of cells in the wound-closure assay and chemotactic migration toward PDGF in three-dimensional migration chambers. Knockdown of WAVE3 expression by RNA interference prevented the PDGF-induced lamellipodia formation and cell migration. Treatment of cells with LY294002, an inhibitor of phosphatidylinositol 3-kinase (PI3K), also abrogated the PDGF-induced lamellipodia formation and cell migration, suggesting that PI3K may be required for WAVE3 activity. WAVE3 and the PI3K regulatory subunit, p85, were found to interact in a yeast two-hybrid screen, which was confirmed through co-immunoprecipitation. The WAVE3-p85 interaction was mediated by the N-terminal region of WAVE3 and the C-terminal SH2 domain of p85. These results imply that the WAVE3-mediated migration in MDA-MB-231 cells via lamellipodia formation is activated downstream of PI3K and induced by PDGF. The findings of the WAVE3-p85 partnership also suggest a potential regulatory role for p85 in WAVE3-dependent actin-cytoskeleton reorganization and cell migration.

Actins↗
PubMed2005

Fibroblast growth factor-2 is a downstream mediator of phosphatidylinositol 3-kinase-Akt signaling in 14,15-epoxyeicosatrienoic acid-induced angiogenesis.

To determine the efficacy of cytochrome P450 2C9 metabolites of arachidonic acid, viz. 5,6-, 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acids (EETs), in inducing angiogenesis, we have studied their effects on human dermal microvascular endothelial cell (HDMVEC) tube formation and migration. All four EETs stimulated HDMVEC tube formation and migration in a dose-dependent manner. Because 14,15-EET was found to be slightly more efficacious than 5,6-, 8,9-, and 11,12-EETs in stimulating HDMVEC tube formation and migration, we next focused on elucidation of the signaling mechanisms underlying its angiogenic activity. 14,15-EET stimulated Akt and S6K1 phosphorylation in Src- and phosphatidylinositol 3-kinase (PI3K)-dependent manner in HDMVECs. Inhibition of Src and PI3K-Akt-mTOR signaling by both pharmacological and dominant-negative mutant approaches suppressed 14,15-EET-induced HDMVEC tube formation and migration in vitro and Matrigel plug angiogenesis in vivo. In addition, 14,15-EET induced the expression of fibroblast growth factor-2 (FGF-2) in Src- and PI3K-Akt-dependent and mTOR-independent manner in HDMVECs. Neutralizing anti-FGF-2 antibodies completely suppressed 14,15-EET-induced HDMVEC tube formation and migration in vitro and Matrigel plug angiogenesis in vivo. Together, these results show for the first time that Src and PI3K-Akt signaling via targeting in parallel with FGF-2 expression and mTOR-S6K1 activation plays an indispensable role in 14,15-EET-induced angiogenesis.

8,11,14-Eicosatrienoic Acid↗
PubMed2005

Phosphoinositide 3-kinase signaling to Akt promotes keratinocyte differentiation versus death.

Signaling pathways regulating the differentiation program of epidermal cells overlap widely with those activated during apoptosis. How differentiating cells remain protected from premature death, however, is still poorly defined. We show here that the phosphoinositide 3-kinase (PI3K)/Akt pathway is activated at early stages of mouse keratinocyte differentiation both in culture and in the intact epidermis in vivo. Expression of active Akt in keratinocytes promotes growth arrest and differentiation, whereas pharmacological blockade of PI3K inhibits the expression of "late" differentiation markers and leads to death of cells that would otherwise differentiate. Mechanistically, the activation of the PI3K/Akt pathway in keratinocyte differentiation depends on the activity of the epidermal growth factor receptor and Src families of tyrosine kinases and the engagement of E-cadherin-mediated adhesion. During this process, PI3K associates increasingly with cadherin-catenin protein complexes bearing tyrosine phosphorylated YXXM motifs. Thus, the PI3K signaling pathway regulates the choice between epidermal cell differentiation and death at the cross-talk between tyrosine kinases and cadherin-associated catenins.

Amino Acid Motifs↗
PubMed2005

Galpha q 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.

Animals↗
PubMed2005

Estrogen prevents cardiomyocyte apoptosis through inhibition of reactive oxygen species and differential regulation of p38 kinase isoforms.

From human and animal studies, estrogen is known to protect the myocardium from an ischemic insult. However, there is limited knowledge regarding mechanisms by which estrogen directly protects cardiomyocytes. In this report, we employed an in vitro model, in which cultured rat cardiomyocytes underwent prolonged hypoxia followed by reoxygenation (H/R), to study the cardioprotective mechanism of estrogen. 17-beta-estradiol (E2) acting via estrogen receptors inhibited H/R-induced apoptosis of cardiomyocytes. Mitochondrial reactive oxygen species (ROS) generated from H/R activated p38alpha MAPK, and inhibition of p38alpha with SB203580 significantly prevented H/R-induced cell death. E2 suppressed ROS formation and p38alpha activation by H/R and concomitantly augmented the activity of p38beta. Unlike p38alpha, p38beta was little affected by H/R. Dominant negative p38beta protein expression decreased E2-mediated cardiomyocyte survival and ROS suppression during H/R stress. The prosurvival signaling molecule, phosphoinositol-3 kinase (PI3K), has previously been linked to cell survival following ischemia-reperfusion injury. Here, E2-activated PI3K was found to inhibit ROS generated from H/R injury, leading to inhibition of downstream p38alpha. We further linked these signaling pathways in that p38beta was activated by E2 stimulation of PI3K. Thus, E2 differentially modulated two major isoforms of p38, leading to cardiomyocyte survival. This was achieved by signaling through PI3K, integrating cell survival mediators.

Animals↗
PubMed2006

Up-regulation of the KLF2 transcription factor by fluid shear stress requires nucleolin.

We have previously characterized the regulation of the KLF2 transcription factor gene by describing an induction complex that binds to and regulates its promoter. In the present study, by using DNA affinity chromatography and mass spectrometry, we have identified nucleolin as an additional protein that binds to a palindromic response region in the KLF2 promoter. The presence of nucleolin on the KLF2 promoter in macrophages was verified by electrophoretic mobility shift assays. Interestingly, in mouse and human endothelial cell lines, electrophoretic mobility shift assays and chromatin immunoprecipitation analyses indicated that nucleolin binds the KLF2 promoter only upon application of fluid shear stress. Pretreatment of the endothelial cells with LY294002, a specific inhibitor of phosphatidylinositol 3-kinase (PI3K), blocked the shear stress-induced binding of nucleolin to the promoter, demonstrating its PI3K-dependent regulation. Additionally, nucleolin exhibited dynamic flow-specific, PI3K-dependent alterations in size. Anti-nucleolin antibodies interacted with a 110-kDa form in static endothelial cells and with several catalytic forms that changed in abundance after the application of shear stress. Immunoprecipitation experiments demonstrated that fluid flow induced the interaction of nucleolin with the p85 regulatory subunit of PI3K. Finally, introduction of small interfering RNAs targeting the nucleolin genetic sequence selectively reduced nucleolin expression and was sufficient to block the induction of KLF2 by shear stress. These data support a general role for nucleolin in gene regulation and identify it as a novel factor involved in regulation of KLF2 expression.

Amino Acid Sequence↗
PubMed2006

Induction of Krox-24 by endogenous cannabinoid type 1 receptors in Neuro2A cells is mediated by the MEK-ERK MAPK pathway and is suppressed by the phosphatidylinositol 3-kinase pathway.

Neuro2a cells endogenously express cannabinoid type 1 (CB1) receptors. CB1 stimulation with HU210 activated ERK and induced the transcription factor Krox-24. A functional MEK-ERK pathway is an important requirement for CB1-mediated Krox-24 induction as blockade of MEK signaling by UO126 reduces both basal and CB1-mediated activation of Krox-24. CB1 receptor stimulation did not activate either JNK or p38 MAPK pathways or the pro-proliferation phosphatidylinositol 3-kinase (PI3K)-Akt pathway. However, serum removal or blockade of PI3K signaling by LY294002 transiently stimulated basal Krox-24 expression and increased CB1-mediated induction of Krox-24. This was consistent with a transient increase in pMEK, pERK, and pCREB levels following PI3K blockade. These data demonstrate that CB1-mediated activation of the Krox-24 transcription factor is negatively regulated through the PI3K-Akt pathway and reveals several points of signaling cross-talk between these two important kinase pathways.

Animals↗