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PubMed2002

PI3K is required for proliferation and migration of human pulmonary vascular smooth muscle cells.

Human vascular smooth muscle cell proliferation and migration contribute to vascular remodeling in pulmonary hypertension and atherosclerosis. The precise mechanisms that regulate structural remodeling of the vessel wall remain unknown. This study tests the hypothesis that phosphatidylinositol 3-kinase (PI3K) activation is both necessary and sufficient to mediate human pulmonary vascular smooth muscle (PVSM) cell proliferation and migration. Microinjection of human PVSM cells with a dominant-negative class IA PI3K inhibited platelet-derived growth factor (PDGF)-induced DNA synthesis by 65% (P < 0.001; chi(2) analysis) compared with cells microinjected with control plasmid, whereas microinjection of cells with a constitutively active class IA PI3K (p110*-CA) was sufficient to induce DNA synthesis (mitotic index of p110*-CA-microinjected cells was 15% vs. 3% in control cells; P < 0.01). Transfection of PVSM cells with p110*-CA was also sufficient to promote human PVSM cell migration. In parallel experiments, stimulation of human PVSM cells with PDGF induced PI3K-dependent activation of Akt, p70 S6 kinase, and ribosomal protein S6 but not mitogen-activated protein kinase. PDGF-induced proliferation and migration was inhibited by LY-294002. These results demonstrate that PI3K signaling is both necessary and sufficient to mediate human PVSM cell proliferation and migration and suggest that the activation of PI3K may play an important role in vascular remodeling.

Cell Division↗

PubMed2005

Involvement of tyrosine kinase and PI3K in the regulation of OAT3-mediated estrone sulfate transport in isolated rabbit renal proximal tubules.

It was shown previously that OAT3 activity was differentially regulated by protein kinases including MAPK, PKA, and PKC. The present study investigated the short-term effect of tyrosine kinase and phosphatidylinositol 3-kinase (PI3K) on OAT3-mediated organic anion transport in S2 segments of renal proximal tubules. Genistein, a tyrosine kinase inhibitor, and wortmannin, a PI3K inhibitor, inhibited transport of estrone sulfate, a prototypic substrate for OAT3, in a dose-dependent manner. Previously, we showed that epidermal growth factor (EGF) stimulated OAT3 activity via the MAPK pathway. In the present study, we investigated whether EGF-stimulated OAT3 activity was dependent on tyrosine kinase and PI3K. We showed that EGF stimulation of OAT3 was reduced by inhibition of tyrosine kinase or PI3K, suggesting that they play a role in the stimulatory process. Inhibitory effects also indicated that tyrosine kinase and PI3K are involved in the MAPK pathway for EGF stimulation of OAT3 in intact renal proximal tubules, with PI3K acting upstream and tyrosine kinase acting downstream of mitogen-activated/extracellular signal-regulated kinase kinase activation.

Androstadienes↗

PubMed2005

Sequential activation of class IB and class IA PI3K is important for the primed respiratory burst of human but not murine neutrophils.

It is well established that preexposure of human neutrophils to proinflammatory cytokines markedly augments the production of reactive oxygen species (ROS) to subsequent stimuli. This priming event is thought to be critical for localizing ROS to the vicinity of the inflammation, maximizing their role in the resolution of the inflammation, and minimizing the damage to surrounding tissue. We have used a new generation of isoform-selective phosphoinositide 3-kinase (PI3K) inhibitors to show that ROS production under these circumstances is regulated by temporal control of class I PI3K activity. Stimulation of tumor necrosis factor-alpha (TNF-alpha)-primed human neutrophils with N-formyl-methionyl-leucyl-phenylalanine (fMLP) results in biphasic activation of PI3K; the first phase is largely dependent on PI3Kgamma, and the second phase is largely dependent on PI3Kdelta. The second phase of PI3K activation requires the first phase; it is this second phase that is augmented by TNF-alpha priming and that regulates parallel activation of ROS production. Surprisingly, although TNF-alpha-primed mouse bone marrow-derived neutrophils exhibit superficially similar patterns of PI3K activation and ROS production in response to fMLP, these responses are substantially lower and largely dependent on PI3Kgamma alone. These results start to define which PI3K isoforms are responsible for modulating neutrophil responsiveness to infection and inflammation.

Animals↗

PubMed2005

Neuregulins rescue PC12-ErbB-4 cells from cell death induced by beta-amyloid peptide: involvement of PI3K and PKC.

Neuregulins (NRGs), which are highly expressed in the nervous system, bind and activate two receptor tyrosine kinases, ErbB-3 and ErbB-4. We previously showed that NRG mediates survival of PC12-ErbB-4 cells from apoptosis induced by serum deprivation, tumor necrosis factor-alpha treatment, or H2O2. These effects of NRGs are mediated by the phosphoinositide 3-kinase (PI3K) signaling pathway. In the present study, we show that NRG induces a significant protective effect from beta-amyloid 25-35 (Abeta[25-35]) peptide-induced cell death. The PI3K signaling pathway might be involved in this effect of NRG as the downstream effector of PI3K, protein kinase B (PKB/AkT), is activated by NRG in the presence of Abeta, and PKB/AkT activation is inhibited by the PI3K inhibitor, LY294002. In addition, our results demonstrate that Abeta-induced cell death is reduced by expression of activated PI3K. These results suggest that PI3K-dependent pathways might regulate the toxic effect of Abeta. In addition, Abeta induced alteration in the levels of the proapoptotic protein Bax. Neuregulin (NRG) treatment however, induced elevation in the levels of the antiapoptotic protein BclxL. The NRG-mediated BclxL elevation is regulated by protein kinase C (PKC), as NRG failed to elevate BclxL in the presence of the PKC inhibitor, GF109203X. Moreover, activation of PKC by phorbol 12-myristate 13-acetate markedly attenuated cell death induced by Abeta and induced elevation in BclxL levels. The results suggest that NRG might affect cell viability using two signaling pathways: activation of PI3K/PKB/AkT pathway and activation of PKC, which results in increasing levels of the antiapoptotic protein BclxL.

Amyloid beta-Protein Precursor↗

PubMed2001

Involvement of PI3K in PKCepsilon-mediated oncogenic signal in rat colonic epithelial cells.

We have recently demonstrated that overexpression of PKCepsilon is oncogenic in colonic epithelial cells. To test whether PI3K might be an upstream effector of PKCepsilon in cell transformation, we have overexpressed the p110alpha PI3K subunit in non-transformed D/WT colonic epithelial cells. Transfectants displayed the major in vitro features of transformed cells. Interestingly, no transformation occurred when p110alpha was co-transfected with a dead-kinase PKCepsilon mutant. The p85alpha subunit of PI3K, displaying a dominant-negative-like effect, was then transfected in PKCepsilon-transformed D/epsilon cells. The transformed profile of these cells was markedly reduced. To identify which by-products of PI3K might be involved in cell transformation we have transfected the D/WT cell line with cDNAs encoding the PI3 kinases hVps34 and C2beta. Overexpression of hVps34 did not cause cell transformation. Conversely, in vitro transformation was observed when C2beta was transfected into D/WT cells. These results indicate that phosphatidylinositol-3 monophosphate does not seem to be involved in cell transformation, and that phosphatidylinositol-3,4 bisphosphate and phosphatidylinositol-3,4,5 trisphosphate are more likely involved in this process. Thus, our data support the hypothesis of a linkage between PI3K and PKCepsilon, and indicate that PI3K may act as a source of second messengers responsible for oncogenic activation of PKCepsilon.

Animals↗

PubMed2004

[Activation of phosphatidylinositol 3-kinase-protein kinase B (PI3K-PKB) induced by 17beta-estradiol in endometrial carcinoma cell (Ishikawa)].

OBJECTIVE: Cellular response to estradiol is mediated both by estrogen receptor (ER) binding to estrogen response element (ERE) and by non-nuclear actions like activation of signal transduction pathways such as mitogen activated protein kinase (MAPK) pathway. However, the signal transduction of estrogen involving phosphatidylinositol 3-kinase-protein kinase B (PI3K-PKB) is not clear in endometrial carcinoma. Our purpose was to study if PI3K-PKB signaling pathway could be activated rapidly by 17beta-E(2) through non-nuclear action and also, whether PI3K inhibitor, LY294002, could inhibit such non-nuclear action of 17beta-E(2) in endometrial carcinoma cell line Ishikawa. METHODS: Levels of phosphorylated PKB (Ser473 site, p-PKB) and total PKB were examined by western blotting in Ishikawa cells after stimulation with 17beta-E(2) at 1 x 10(-6) mol/L for different time periods and at varied doses for 30 min. Optimal time and appropriate dose for 17beta-E(2) to activate PKB in Ishikawa cells were observed. Inhibitory effect of LY294002 on activation of PKB induced by 17beta-E(2) was also studied. p-PKB/PKB ratio was used to indicate levels of activation of PKB. RESULTS: p-PKB/PKB at 15 min (0.533 +/- 0.029) was significantly higher than the control (0.361 +/- 0.029, P < 0.05). Maximal activation of PKB (maximal levels of p-PKB/PKB) took place at 30 min (0.666 +/- 0.021, P < 0.001) and the activation persisted at least 2 h after stimulation with 1 x 10(-6) mol/L 17beta-E(2) in Ishikawa cells. With increased doses of 17beta-E(2) (vehicle, 1 x 10(-10), 1 x 10(-8), 1 x 10(-6), 1 x 10(-4) mol/L), the activation of PKB (p-PKB/PKB was 0.300 +/- 0.098, 0.312 +/- 0.081, 0.625 +/- 0.100, 0.771 +/- 0.041, 0.902 +/- 0.043 accordingly) increased gradually (in comparison with vehicle, P > 0.05, < 0.05, < 0.05, < 0.001 accordingly). PI3K inhibitor, LY294002 could inhibit the activation of PKB induced by 17beta-E(2) in Ishikawa cells. Levels of PKB (p-PKB/PKB) decreased (0.443 +/- 0.032, 0.415 +/- 0.032, 0.111 +/- 0.035, 0, 0) gradually with increased concentrations (vehicle, 0.1, 10, 50, 100 micro mol/L) of LY294002 (compared with vehicle, P > 0.05, < 0.05, < 0.001, < 0.001) and 50 micro mol/L LY294002 completely blocked the activation of PKB induced by 17beta-E(2). CONCLUSIONS: 17beta-estradiol, by non-nuclear action, can activate promptly PI3K-PKB signaling pathway in endometrial carcinoma cell line, Ishikawa. This action of 17beta-E(2) is PI3K dependent and can be inhibited or blocked by LY294002.

Chromones↗

PubMed2026

Diosmetin Inhibits Bladder Cancer through Suppression of the PI3K-AKT Signaling Pathway and Activation of the p53 Signal Pathway Revealed by Network Pharmacology and In Vitro Experimental Verification.

INTRODUCTION: Diosmetin, a naturally occurring flavonoid abundant in plants such as chrysanthemums, lemons, and oranges, has been reported to exhibit diverse antitumor properties. However, its potential efficacy against bladder cancer remains unexplored. This study aims to investigate the anti-bladder cancer effects of Diosmetin and elucidate the underlying mechanisms using network pharmacology combined with in vitro experiments. METHODS: Public databases were employed to identify shared targets between Diosmetin and bladder cancer. A Protein-Protein Interaction (PPI) network was constructed, followed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses to predict core targets and signaling pathways. The predicted mechanisms were subsequently validated through in vitro assays. RESULTS: A total of 48 common targets were identified. PPI network analysis revealed 22 hub genes, including AKT1 and MDM2. GO analysis indicated enrichment in 208 biological processes, 23 cellular components, and 38 molecular functions. KEGG analysis suggested that Diosmetin exerts anti-bladder cancer effects primarily through pathways such as Pathways in cancer, PI3K-AKT signaling, and Proteoglycans in cancer. Notably, the PI3K-AKT pathway showed the highest gene enrichment, indicating its potential prominence. In vitro experiments demonstrated that Diosmetin suppresses bladder cancer cell proliferation and induces apoptosis. Additionally, Diosmetin reduced the expression of p-PI3K, p-AKT, and MDM2, while upregulating p53 expression, suggesting involvement of both the PI3K-AKT and p53 pathways. DISCUSSION: These findings align with network pharmacology predictions and highlight the potential of Diosmetin as a multi-target agent against bladder cancer, warranting further in vivo investigation. CONCLUSION: Diosmetin inhibits bladder cancer cell proliferation and promotes apoptosis by suppressing the PI3K-AKT pathway and activating the p53 pathway.

Diosmetin↗

PubMed2002

Effects of pharmacologic antagonists of epidermal growth factor receptor, PI3K and MEK signal kinases on NF-kappaB and AP-1 activation and IL-8 and VEGF expression in human head and neck squamous cell carcinoma lines.

We previously reported that expression of angiogenesis factors interleukin-8 (IL-8) and vascular endothelial growth factor (VEGF) is promoted by coactivation of transcription factors nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1) by interleukin-1alpha in human head and neck squamous cell carcinomas (HNSCC). However, expression of IL-1 receptor antagonist incompletely blocked reporter gene activity and cytokine expression, suggesting that other upstream signals may contribute to activation. Overexpression and autocrine activation of epidermal growth factor receptor (EGFR) is detected in 90% of HNSCC, and EGFR inhibitors have been reported to inhibit IL-8 and VEGF expression, but the intermediary signal pathways and transcription factors by which EGFR modulates proangiogenic factors is unknown. EGFR can activate the phosphotidylinositol-3 kinase (PI3K) and mitogen-activated/extracellular signal-regulated kinase (MEK) pathways, which can potentially modulate activation of NF-kappaB and AP-1, respectively. In our study, we examined the effect of EGF and antagonists of EGFR, PI3K and MEK on NF-kappaB and AP-1 activation and IL-8 and VEGF expression in HNSCC cell lines UM-SCC-9 and 11B in which EGFR is overexpressed and activated. Recombinant EGF induced EGFR phosphorylation, activation of NF-kappaB and AP-1 reporter genes and IL-8 and VEGF expression, indicating that EGFR can mediate coactivation of both transcription factors and cytokine genes in HNSCC. EGFR antagonist PD153035 and anti-EGFR antibody C225 completely inhibited EGF-induced reporter activity and cytokine expression, but only partially inhibited constitutive activity. MEK inhibitor U0126 preferentially blocked AP-1 activity and expression of both IL-8 and VEGF, while PI3K inhibitor LY-294002 or a dominant negative inhibitor-kappaB preferentially blocked NF-kappaB activation and expression of IL-8 but not VEGF. EGFR, PI3K and MEK antagonists inhibited growth of HNSCC. We conclude that antagonists of EGFR, PI3K and MEK signal pathways have inhibitory activity against EGFR-induced NF-kappaB and AP-1 activation, IL-8 and VEGF expression and growth by HNSCC. Published 2002 Wiley-Liss, Inc.

Carcinoma, Squamous Cell↗

PubMed2004

TGF-beta1 enhances betaig-h3-mediated keratinocyte cell migration through the alpha3beta1 integrin and PI3K.

betaig-h3 is an extracellular matrix (ECM) protein whose expression is highly induced by transforming growth factor beta1 (TGF-beta1). We previously demonstrated that betaig-h3 has two alpha3beta1 integrin-interacting motifs, which promote adhesion, migration, and proliferation of human keratinocytes. Both betaig-h3 and TGF-beta1 have been suggested to play important roles in the healing of skin wounds. In this study, we demonstrate that TGF-beta1 enhances keratinocyte adhesion and migration toward betaig-h3 through the alpha3beta1 integrin. TGF-beta1 did not increase the amount of the alpha3beta1 integrin on the cell surface, but rather increased its affinity for betaig-h3. LY294002, an inhibitor of PI3K, blocked the basal and TGF-beta1-enhanced cell migration but not adhesion to betaig-h3. A constitutively active mutant of PI3K stimulated cell migration but not adhesion to betaig-h3. The PI3K pathway is also not associated with the affinity of the alpha3beta1 integrin to betaig-h3. TGF-beta1 induced phosphorylation of AKT and FAK. Taken together, these data suggest that TGF-beta1 increases affinity of the alpha3beta1 integrin to betaig-h3, resulting in enhanced adhesion and migration of keratinocytes toward betaig-h3. TGF-beta1 also enhances migration through PI3K, but PI3K is not associated with either the binding affinity of the alpha3beta1 integrin or its adhesion to betaig-h3.

Cell Movement↗

PubMed2004

Regulation of interleukin-6-mediated PI3K activation and neuroendocrine differentiation by androgen signaling in prostate cancer LNCaP cells.

BACKGROUND: Neuroendocrine (NE) differentiation in prostate cancer has been suggested to be one of the early events in the development of androgen independence. In the human prostate cancer LNCaP cell line, treatment with interleukin-6 (IL-6) induces NE-like differentiation, which is similar to the phenomena observed in advanced stages of prostate cancer progression. In this study, we investigate how androgen plays a role in IL-6-mediated NE differentiation in LNCaP cell line. METHODS: Western blot, co-immunoprecipitation (co-IP), and GST pull-down assays were performed to detect the protein expression and protein-protein interaction. PI3K kinase assay was used to measure PI3K activity. RESULTS: Addition of androgen blocks IL-6-mediated PI3K activation and NE differentiation in LNCaP cells. In vivo and in vitro protein interaction assays suggested that androgen receptor (AR) can directly interact with IL-6 transducer gp130. In addition, androgen treatment enhances the interaction between AR and gp130, interrupts the IL-6-induced gp130-mediated PI3K activation, which may lead to inhibition of IL-6-mediated NE differentiation in LNCaP cells. CONCLUSIONS: Our results suggest androgen and AR can regulate IL-6-mediated LNCaP cell NE differentiation via directly modulating the IL-6-PI3K pathway.

Blotting, Western↗

PubMed1997

Identification and cDNA cloning of a novel mammalian C2 domain-containing phosphoinositide 3-kinase, HsC2-PI3K.

Phosphoinositide (PI) 3-kinases have been shown to have critical roles in signal transduction, cell transformation and intracellular protein trafficking. Reverse-transcription polymerase chain reaction methods, using degenerate primers derived from the lipid kinase consensus region, were utilised to identify PI 3-kinases in the normal human breast. Here we report the cDNA cloning of a novel human PI 3-kinase isoform, HsC2-PI3K. This PI 3-kinase is most closely related to the recently described C2 domain-containing family of PI 3-kinases which includes Drosophila PI3K_68D/cpk and murine cpk-m/p170. Sequence analysis suggests that HsC2-PI3K is a second distinct mammalian member of the C2 domain-containing PI 3-kinase family. Northern blot analysis of human tissues indicates that HsC2-PI3K is widely expressed. Fluorescence in situ hybridisation has mapped HsC2-PI3K to chromosome 1q32.

Amino Acid Sequence↗

PubMed2000

Cardiotrophin-1 phosphorylates akt and BAD, and prolongs cell survival via a PI3K-dependent pathway in cardiac myocytes.

Growth factors and cytokines trigger survival signaling in a wide variety of cell systems, including cardiac myocytes. Participation of the phosphatidylinositol 3-OH kinase (PI3K)/Akt pathway in survival signaling has already been described in some cell types, but its involvement in the survival of cardiac myocytes is as yet unknown. Recently, CT-1, an interleukin 6-related cytokine, was shown to have survival-promoting, anti-apoptotic effects on cultured cardiac myocytes. However, roles of PI3K-dependent pathways in this signaling have not been elucidated. In the present study, therefore, we examined the participation of the PI3K/Akt pathway in CT-1-induced, survival-promoting signaling in cultured ventricular myocytes. It was found that CT-1 phosphorylated and activated Akt, and the effect was blocked by the PI3K inhibitors LY294002 and wortmannin. CT-1 also phosphorylated the pro-apoptotic factor, BAD, and the BAD phosphorylation was inhibited by LY294002, suggesting that phosphorylation of BAD is one of the key events by which the PI3K/Akt pathway mediates CT-1-induced survival signaling. Further, CT-1 PI3K-dependently prolonged the survival of serum-starved ventricular myocytes by preventing apoptosis. In summary, our findings show that PI3K-dependent survival signals contribute to CT-1-mediated ventricular myocyte survival. In vivo, the death of ventricular myocytes leads to heart failure, and downregulation of survival signals and/or augmentation of pro-apoptotic signals are likely to be important components of disease processes. Thus, the extent to which CT-1 and the PI3K/Akt pathway mitigate such pathological processes, in vivo, is an important question for the future.

Animals↗

PubMed1997

PI3K signal and DNA repair: a short commentary.

PI3K was originally discovered as a lipid kinase involved in the phosphorylation of the inositol ring in position -3, leading to the synthesis of phosphatidyl-inositol-3-4 bisphosphate. The enzyme purified from rat liver is an heterodimer of two subunits of 85 and 110 KD respectively: it phosphorylates the D3 hydroxyl of phosphoinositides to produce phosphatidyl-inositol-3-phosphate. So far the function of the 3-phospho-inositide is unclear. It is likely that the entire phospholipid serves as a second messenger, since no phospholipase C has yet been found that can cleave the inositol group with a 3 phosphate residue. However the activation targets of this second messenger are still poorly known. Recently a novel/serine/theronine kinase was insolated by three groups and called differently RAC, PKB and AKT. It exhibits sequence homology with protein kinase A and C at the carboxyl terminal, whereas the aminoterminal domain has a plectrin homology. Activation of ATK is inhibited by wortmannin, a specific inhibitor of PI3K at very low concentrations. Furthermore inositol-3-phosphate can activate ATK in vitro. In addition very recently, a linkage of G-protein coupled receptors to the MAP kinase signalled pattern through PI3K has been discovered. But what is downstream of this pathway? 70S6 kinase is an attractive candidate since this kinase, involved in protein synthesis, is activated by AKT in vivo. Interestingly AKT is the cellular protooncogene of v-ATK and this implies that ATK induces a pathway of oncogenic transformation. AKT is inhibited by dominant negative mutants of ras and thus involved in the ras-raf-MAP kinase pathway. The role of PI3K is still indefinite but it must have a paramount importance in cell signalling since nearly all growth factor receptors recruit this enzyme and that the activity of fundamental growth factor receptors like PDGF, EGF and insulin are blocked by the specific inhibitor wortmannin, leading to the conclusion that the PI3K signal is much important in mitogenesis, protein synthesis, membrane ruffling, cell transformation and cell cycle progression.

Animals↗

PubMed2005

Activation of PI3K-Akt-GSK3beta pathway mediates hepatocyte growth factor inhibition of RANTES expression in renal tubular epithelial cells.

Hepatocyte growth factor (HGF) was recently reported to ameliorate renal inflammation in a rat model of chronic renal failure. HGF exerted its action through suppression of RANTES expression in renal tubules. In the present study, we utilized an in vitro model of human kidney proximal tubule epithelial cells (HKC) to elucidate the mechanisms of RANTES suppression by HGF. HGF significantly suppressed basal and TNF-alpha-induced mRNA and protein expression of RANTES in a time and dose dependent fashion. HGF elicited PI3K-Akt activation and inhibited GSK3, a downstream transducer of PI3K-Akt, by inhibitory phosphorylation at Ser-9. When the PI3K-Akt pathway was blocked by wortmannin, HGF inhibition of RANTES was abrogated, demonstrating that the PI3K-Akt pathway is necessary for HGF action. In addition, specific inhibition of GSK3 activity by lithium ion suppressed basal and TNF-alpha-induced RANTES expression, reminiscent of the action of HGF. To further investigate the role of GSK3 in modulating RANTES expression, we examined the effect of forced expression of wild type GSK3beta or an uninhibitable mutant GSK3beta, in which the regulatory Ser-9 residue is changed to alanine (S9A-GSK3beta) in HKC. Overexpression of wild type GSK3beta did not alter the inhibitory action of HGF on RANTES. In contrast, expression of S9A-GSK3beta abolished HGF inhibition of basal and TNF-alpha stimulated RANTES expression. These findings suggest that PI3K-Akt activation and subsequent inhibitory phosphorylation of GSK3beta are required for HGF-induced suppression of RANTES in HKC.

Cell Line↗

PubMed2005

Thrombopoietin (TPO) induces c-myc expression through a PI3K- and MAPK-dependent pathway that is not mediated by Akt, PKCzeta or mTOR in TPO-dependent cell lines and primary megakaryocytes.

Thrombopoietin (TPO) and its receptor (c-Mpl) are the major regulators of megakaryocyte and platelet production and serve a critical and non-redundant role in hematopoietic stem cell (HSC) biology. TPO signals through the Jak-STAT, Ras-Raf-MAPK, and PI3K pathways, and promotes survival, proliferation, and polyploidization in megakaryocytes. The proto-oncogene c-myc also plays an important role in many of these same processes. In this work we studied the regulated expression of c-myc in megakaryocytic cell lines and primary cells by quantitative real-time RT-PCR. We found that TPO induced expression of c-myc in 1 h in both hematopoietic cell lines (UT-7 and BaF3/Mpl) and mature murine megakaryocytes. The TPO-induced expression of c-myc was blocked by a phosphatidylinositol 3-kinase (PI3K) inhibitor, suggesting that TPO stimulated c-myc expression through a PI3K-dependent pathway. Of interest, our study showed that overexpression of active Akt did not rescue the effect of PI3K blockade on c-myc expression, rather, enhanced it. In addition, inhibitors of protein kinase C (PKC)zeta and the target of rapamycin (mTOR) also failed to affect c-myc mRNA expression, while c-myc mRNA expression was reduced by inhibition of the mitogen activated protein kinase (MAPK) pathway. Therefore, we conclude that TPO stimulates c-myc expression in primary megakaryocytes through a PI3K- and MAPK-dependent pathway that is not mediated by Akt, PKCzeta or mTOR.

Animals↗

PubMed2006

Role of PI3K and AKT specific isoforms in ovarian cancer cell migration, invasion and proliferation through the p70S6K1 pathway.

Ovarian cancer is the leading cause of death from gynecological malignancy for women. The amplification of the PI3K catalytic subunit (p110alpha) and the lost function of PTEN are frequently detected in ovarian cancer cells. PI3K plays an important role in tumorigenesis. To specifically inhibit PI3K activity in ovarian cancer cells, we constructed small interfering RNA (siRNA) against p110alpha. The expression of p110alpha siRNA significantly decreased cell migration, invasion, and proliferation compared to the siSCR control cells. The expression of p110alpha siRNA induced CDK inhibitor p27(KIP1) levels, and decreased levels of cyclin D1, CDK4, and phosphorylated retinoblastoma protein. PI3K transmits the mytogenic signal through AKT. AKT has three isoforms in the cells: AKT1, AKT2 and AKT3. We found that inhibition of AKT1 is sufficient to affect cell migration, invasion, and proliferation. Expression of AKT1 siRNA had a similar effect as p110alpha siRNA in the cells. We showed the roles of specific PI3K and AKT isoforms in the cells, which are important to understanding the mechanism of PI3K/AKT signaling in ovarian cancer cells. Both p110alpha and AKT1 siRNA-expressing cells decreased the activation of p70S6K1. Inhibition of p70S6K1 activity by its siRNA also decreased cell migration, invasion, and proliferation associated with the induction of p27(KIP1) levels, and with the inhibition of cell cycle-associated proteins including cyclin D1, CDK2, and phosphorylated retinoblastoma protein. This study demonstrates the important role of the PI3K/AKT/mTOR/p70S6K1 pathway in cell proliferation, migration, and invasion in ovarian cancer cells by using siRNA-mediated gene silencing as a reverse genetic method.

Catalytic Domain↗

PubMed2006

Loss of class IA PI3K signaling in muscle leads to impaired muscle growth, insulin response, and hyperlipidemia.

The evolutionarily conserved phosphoinositide 3-kinase (PI3K) signaling pathway mediates both the metabolic effects of insulin and the growth-promoting effects of insulin-like growth factor-1 (IGF-1). We have generated mice deficient in both the p85alpha/p55alpha/p50alpha and the p85beta regulatory subunits of class I(A) PI3K in skeletal muscles. PI3K signaling in the muscle of these animals is severely impaired, leading to a significant reduction in muscle weight and fiber size. These mice also exhibit muscle insulin resistance and whole-body glucose intolerance. Despite their ability to maintain normal fasting and fed blood glucose levels, these mice show increased body fat content and elevated serum free fatty acid and triglyceride levels. These results demonstrate that in vivo p85 is a critical mediator of class I(A) PI3K signaling in the regulation of muscle growth and metabolism. Our finding also indicates that compromised muscle PI3K signaling could contribute to symptoms of hyperlipidemia associated with human type 2 diabetes.

Animals↗

PubMed2005

Mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3 kinase (PI3K) pathways differently regulate retinal pigment epithelial cell-mediated collagen gel contraction.

Retinal pigment epithelial (RPE) cell-mediated extracellular matrix contraction is believed to contribute to developing proliferative vitreoretinopathy. It has been shown that platelet-derived growth factor (PDGF) and its intracellular signaling pathway, including mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3 kinase (PI3K), are mainly involved in this process. The aim of this study is to investigate how these downstream signaling pathways are related to RPE-mediated collagen gel contraction. We performed the gel contraction assay to evaluate the effect of PDGF in cultured ARPE-19 cells under the presence or absence of PD98059, MAPK inhibitor or wortmannin, PI3K inhibitor. Experiments treated with neutralizing antibody for various subtypes of integrin were also performed and the effect on PDGF-induced gel contraction was investigated. Expression changes of integrin alpha1, alpha2 and beta1 after PDGF stimulation was evaluated using quantitative real-time PCR and flow cytometry. The results showed that PDGF up-regulated ARPE-19 cell-mediated gel contractile activity. PDGF-induced collagen gel contraction was attenuated under presence of PD98059, wortmannin, or neutralizing antibody for integrin alpha1, alpha2, or beta1, all of which are critical subset for binding with type I collagen. The expression of integrin alpha1 and alpha2 was increased after PDGF stimulation in both real-time PCR and flow cytometry, however beta1 expression was not increased. PD98059 significantly attenuated integrin alpha1 and alpha2 expressions. However, wortmannin did not have the same effect. In conclusion, PDGF promotes ARPE-19 cell-mediated gel contraction via both MAPK and PI3K. This was probably due to an increased expression of integrin alpha1 and alpha2, which is mediated by MAPK, but not by PI3K. PI3K may regulate collagen gel contraction by another mechanism other than the up-regulation of integrin expression.

Androstadienes↗