Levels Of Phosphatidylinositol 3-kinase Research Articles

Amifostine attenuates bleomycin-induced pulmonary fibrosis in mice through inhibition of the PI3K/Akt/mTOR signaling pathway

Amifostine is a normal cell protection agent, not only used in the adjuvant therapy of lung cancer, ovarian cancer, breast cancer, nasopharyngeal cancer, bone tumor, digestive tract tumor, blood system tumor and other cancers in order to reduce the toxicity of chemotherapy drugs, and recent studies have reported that the drug can also reduce lung tissue damage in patients with pulmonary fibrosis, but its mechanism of action is not yet fully understood. In this study, we explored the potential therapeutic effects and molecular mechanisms of AMI on bleomycin (BLM)-induced pulmonary fibrosis in mice. A mouse model of pulmonary fibrosis was established using BLM. We then assessed histopathological changes, inflammatory factors, oxidative indicators, apoptosis, epithelial-mesenchymal transition, extracellular matrix changes, and levels of phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathway-related proteins in the BLM-treated mice to determine the effect of AMI treatment on these factors. BLM-treated mice had substantial lung inflammation and abnormal extracellular matrix deposition. Overall, treatment with AMI significantly improved BLM-induced lung injury and pulmonary fibrosis. More specifically, AMI alleviated BLM-induced oxidative stress, inflammation, alveolar cell apoptosis, epithelial-mesenchymal transition, and extracellular matrix deposition by regulating the PI3K/Akt/mTOR signaling pathway. This finding that AMI can alleviate pulmonary fibrosis in a mouse model by inhibiting activation of the PI3K/Akt/mTOR signaling pathway lays a foundation for potential future clinical application of this agent in patients with pulmonary fibrosis.

Ursolic Acid Suppresses Oncostatin M Expression through Blockade of PI3K/Akt/NF-κB Signaling Processes in Neutrophil-like Differentiated HL-60 Cells

Cytokine oncostatin M (OSM) plays an important role in a variety of inflammatory reactions and is mainly produced in neutrophils in inflammatory diseases. While natural pentacyclic triterpenoid ursolic acid (UA) possesses a wide range of beneficial effects, such as anti-oxidant, anti-tumor, and anti-inflammatory, the regulatory processes of OSM suppression by UA in neutrophils are still poorly understood. This study was aimed at examining how UA regulates OSM expression in neutrophil-like differentiated (d)HL-60 cells. Enzyme-linked immunosorbent assay, quantitative polymerase chain reaction, and immunoblotting were employed to analyze the effects of UA. Whereas stimulation with granulocyte-macrophage colony-stimulating factor (GM-CSF) led to elevations of OSM production and mRNA expression, these elevations were lowered by treatment with UA in neutrophil-like dHL-60 cells. When the cells were exposed to GM-CSF, phosphorylated levels of phosphatidylinositol 3-kinase, Akt, and nuclear factor-kB were upregulated. However, the upregulations were diminished by treatment with UA in neutrophil-like dHL-60 cells. The results of this study proposed that UA might relieve inflammatory diseases via inhibition of OSM.

Effect of Tongdu Tiaoshen acupuncture on PI3K/Akt/mTOR signaling pathway and autophagy-related proteins of hippocampus in rats with post-stroke depression

To observe the improvement of Tongdu Tiaoshen acupuncture on depression-like behavior in rats with post-stroke depression (PSD), and to explore its possible mechanism. A total of 48 SD rats were randomized into a sham-operation group, a model group, a Tongdu Tiaoshen group and a non-meridian-non-acupoint group, 12 rats in each group. In the the model group, the Tongdu Tiaoshen group and the non-meridian-non-acupoint group, PSD model was established by the compound method of cerebral medial arterial occlusion, chronic unpredictable mild stress and isolation. Acupuncture was applied at "Dazhui"(GV 14), "Shuigou"(GV 26), "Baihui"(GV 20) and "Shenting" (GV 24) in the Tongdu Tiaoshen group, and at the bilateral dorsum hollows between 3rd and 4th metatarsal bones of the forelegs in the non-meridian-non-acupoint group, needles were retained for 40 min in the both groups, once a day, 6 times a week for 4 weeks. Before model establishment and after intervention, the ethology indice of Zea Longa neurological behavioral score, open-field test and sucrose preference test were evaluated in the 4 groups. The levels of phosphatidylinositol 3-kinase (PI3K), phosphorylated protein kinase B (p-Akt), phosphorylated mammalian target of rapamycin (p-mTOR), Beclin1 and LC3B-Ⅱ/Ⅰin the CA1 region of hippocampus were detected by western blot. The expressions of Beclin1, LC3B-ⅡmRNA in the CA1 region of hippocampus were detected by real-time PCR. After model established, compared with the sham-operation group, the Zea Longa neurological behavioral scores were increased (P<0.05), the scores of horizontal and vertical motion of the open-field test and the consumption of sucrose solution were decreased in the model group, the Tongdu Tiaoshen group and the non-meridian-non-acupoint group (P<0.05). After intervention, compared with the sham-operation group, the Zea Longa neurological behavioral score, the expression of Beclin1, LC3B-Ⅱ/Ⅰ and LC3B-Ⅱin the CA1 region of hippocampus were increased (P<0.05), the scores of horizontal and vertical motion of the open-field test, the consumption of sucrose solution and the expression of PI3K, p-Akt and p-mTOR were decreased in the model group (P<0.05). After intervention, compared with the model group and the non-meridian-non-acupoint group, the Zea Longa neurological behavioral score, the expression of Beclin1, LC3B-Ⅱ/Ⅰ and LC3B-Ⅱin the CA1 region of hippocampus were decreased (P<0.05), the scores of horizontal and vertical motion of the open-field test, the consumption of sucrose solution and the expression of PI3K, p-Akt and p-mTOR were increased in the Tongdu Tiaoshen group (P<0.05). Tongdu Tiaoshen acupuncture can effectively improve the depression-like behavior in PSD rats, its mechanism may relate to the activation of PI3K/Akt/mTOR signaling pathway and the inhibition of hippocampal neuron autophagy.

Investigating the effects of Pirfenidone on TGF-\u03b21 stimulated non-SMAD signaling pathways in Dupuytren\u2019s disease -derived fibroblasts

BackgroundDupuytren’s disease (DD) is a progressive, debilitating condition of the hand that can eventually cause contractures of the affected fingers. Transforming growth factor- β1 (TGF-β1) has been reported to play a key role in DD pathology. Increased expression of TGF-β1 has shown to be the main stimulator of myofibroblast activity and in DD contractures. Pirfenidone (PFD), a small active molecule possess the ability to inhibit TGF-β1-mediated action in various fibrotic disorders. Our recent published findings show that PFD reduced TGF-β1-mediated cellular functions implicated in DD through SMAD signaling pathways. In the present study, the effect of PFD on TGF-β1-mediated non-SMAD signaling pathways were investigated in both carpal tunnel (CT) - and DD-derived fibroblasts.MethodsFibroblasts harvested from Dupuytren’s disease (DD) and carpal tunnel (CT) tissues were cultured in the presence or absence of TGF-β1 (10 ng/ml) and/or PFD (800 μg/ml). Cell lysates were analyzed using Western blots. Equal amounts of proteins were loaded to determine the phosphorylation levels of phosphatidylinositol-3 kinase (PI3K/AKT), extracellular regulated kinases (ERK1/2), p38 mitogen-activated protein kinase and Rho family related myosin light chain (MLC).ResultsWe show that the TGF-β1-induced phosphorylation of AKT was significantly decreased by the addition of PFD (800 μg/mL) in both CT- and DD-derived fibroblasts. Interestingly, there was no significant difference in the phosphorylation levels of both ERK and p38 on TGF-β1- induced cells in both CT-and DD-derived fibroblasts. But, PFD significantly decreased the TGF- β1-induced phosphorylation levels of ERK1/2 in both CT- and DD- cells. In contrast, PFD significantly decreased the basal and TGF- β1-induced phosphorylation levels of p38 in DD-derived fibroblasts. TGF- β1-induced phosphorylation levels of MLC was decreased by PFD in DD-derived fibroblasts.ConclusionsThese in-vitro results indicate for the first time that PFD has the potential to inhibit TGF-β1-induced non-SMAD signaling pathways in both CT- and DD-derived fibroblasts but pronounced statistically significant inhibition on all molecules was observed only in DD-derived fibroblasts. Our previous studies show that PFD can inhibit TGF-β1- induced SMAD signaling pathway proteins, namely p- SMAD2/SMAD3. These broad and complementary actions suggest PFD as a promising candidate to inhibit the TGF-β1- mediated molecular mechanisms leading to DD fibrosis.

Therapeutic Effect of External Application of Ligustrazine Combined with Holistic Nursing on Pressure Sores.

BackgroundThis study aimed to explore the therapeutic effect of external application of ligustrazine combined with holistic nursing on pressure sores, as well as the underlying mechanism.Material/MethodsFrom February 2014 to March 2015, a total of 32 patients with Phase II and Phase III pressure sores were enrolled and randomly assigned to an experimental group or a control group. The clinical data were comparable between the 2 groups. In addition to holistic nursing, the patients in the experimental group received 4 weeks of continuous external application of ligustrazine, whereas patients in the control group received compound clotrimazole cream. Therapeutic effect and healing time were recorded. HaCaT cells were used as an in vitro model for mechanism analysis of the effect of ligustrazine in treating pressure sores. After culturing with different concentrations of ligustrazine or the inhibitor of AKT (LY294002) for 72 h, cell viability, clone formation numbers, and levels of phosphatidyl inositol 3-kinase (PI3K), p-AKT, and p-mammalian target of rapamycin (mTOR) were determined.ResultsCompared to the control group, the total effective rate in the experimental group was significantly higher, and the healing time was significantly reduced. Cell viability and clone formation numbers were significantly upregulated by ligustrazine in a dose-dependent manner. Both the cell viability and clone formation numbers were significantly inhibited by application of LY294002.ConclusionsOur results suggest that ligustrazine combined with holistic nursing is an effective treatment of pressure sores. The protective effect may be associated with the promotion of cell growth by activation of the PI3K/AKT pathway.

Abstract 4671: Inhibition of phosphatidylinositol 3-kinase suppresses propagation of drug-tolerant cancer cell subpopulations enriched by 5-fluorouracil

Abstract The majority of advanced gastric cancer has been treated with 5-fluorouracil (5-FU)-based chemotherapy after complete resection, but a considerable number of patients experience relapse within 5 years after the resection. It has been considered that extremely small drug-tolerant cancer cell subpopulations are responsible for the relapse after chemotherapy. Previously, we established a 5-FU-tolerant gastric cancer cell line, MKN45T, which requires a higher drug concentration to suppress its growth relative to its parental, MKN45. Using the pair of cell lines, we developed an orthotopic xenograft (OX) model that mimics advanced human gastric cancer by transplantation of the cells into the gastric submucosal layer of nude mice. The OX of the 5-FU-tolerant cell line, MKN45T, showed an aggressive tumorigenicity, metastasis, and peritoneal dissemination, in contrast to the parental cell line, MKN45. Proteomic screening using a colony lysate array, a special format of reverse-phase protein array that allows us to quantify the protein levels in individual drug-tolerant subpopulations (i.e., colonies), revealed the level of phosphatidylinositol 3-kinase (PI3K) phosphorylation was increased according to the 5-FU dose-escalation. The prevalence of cells with active PI3K also increased with 5-FU treatment in OX. In contrast, phosphorylation of Akt and mTOR proteins were slightly decreased by the 5-FU treatment. Interestingly, PTEN protein expression was almost completely suppressed in OX after the 5-FU treatment. To see whether PI3K inhibitors have potency in the reduction of tumors with persistent growth after 5-FU treatment, PI3K inhibitors, including LY294002, Queracetin, Wortmannin, GNE493, and GDC0941, were tested by means of colony formation assay. Among them, simultaneous administration of 5-FU and GDC0941 significantly suppressed colony formation, which led us to examine the inhibitory effect of GDC0941 for tumor growth in the OX model. Sequential administration of 5-FU and GDC0941 prohibited the propagation of 83% (5/6) of OX tumors in the stomach as well as other organs. Western blot was then performed to clarify the growth suppression mechanisms by GDC0941 for PI3K-activated tumors triggered by 5-FU. The Akt/PI3K/mTOR and PTEN pathway analysis in response to single/combinational drug administration revealed that the majority (75%, 6/8) of gastric cancer cell lines tested exhibited an increased level of PI3K phosphorylation with 5-FU administration. The increased level of PI3K phosphorylation with 5-FU, which subsequently induces Akt and mTOR activation, was effectively suppressed by GDC0941 administration, particularly with a considerably decreased level of p70 S6 kinase phosphorylation. These results suggest that administration of 5-FU followed by GDC0941 may suppress relapse after complete resection with 5-FU-based chemotherapy for gastric cancer. Citation Format: Satoshi S. Nishizuka, Kaoru Ishida, Yukimi Ohmori, Kohei Kume, Chie Ito, Akari Konta, Yuka Koizumi, Mamoru Nukatsuka, Takashi Kobunai, Teiji Takechi, Takeshi Iwaya. Inhibition of phosphatidylinositol 3-kinase suppresses propagation of drug-tolerant cancer cell subpopulations enriched by 5-fluorouracil. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4671.

CTRP9 Ameliorates Pulmonary Arterial Hypertension Through Attenuating Inflammation and Improving Endothelial Cell Survival and Function.

Endothelial dysfunction and inflammation are believed to be 2 primary instigators of pulmonary arterial hypertension (PH). C1q/TNF-related protein 9 (CTRP9) plays important roles in anti-inflammation and improvement of epithelial function. However, the role of CTRP9 in the progression of PH remains still unclear. In this study, the role and mechanism of CTRP9 in the PH progression were explored. First, serum CTRP9 contents and CTRP9 mRNA expression in the pulmonary artery epithelial cells from patients with PH were detected. Our data on enzyme-linked immunosorbent assay and real-time quantitative Polymerase Chain Reaction showed that CTRP9 mRNA and protein content were markedly downregulated in the patients with PH. Then the pcDNA-CTRP9 expression vector or CTRP9 siRNA was transfected into the primary pulmonary artery epithelial cells from the patients with PH in vitro. CTRP9 overexpression significantly improved endothelial NOS protein expression and reduced the secretion of endothelin-1 (ET-1) and matrix metalloproteinase-2 (MMP-2), whereas knockdown of CTRP9 sharply reduced eNOS protein expression and promoted the secretion of ET-1 and MMP-2 in the cultured human epithelial cells. Moreover, the levels of phosphatidylinositol 3-kinase (PI3K) and pAkt were reduced in the epithelial cells and CTRP9 overexpression activated the PI3K/Akt pathway. CTRP9 could inhibit cell apoptosis and eNOS expression reduction in the cells pretreated with the PI3K/Akt inhibitor LY294002 and resist LY294002-induced ET-1 and MMP-2 secretion. Finally, to verify the role of CTRP9 in the progression of PH in vivo, the pcDNA-CTRP9 expression vector or CTRP9 siRNA was intravenously injected into rats with PH. Pulmonary arterial pressures of the rats were notably reduced by the pcDNA-CTRP9 injection and elevated by the CTRP9 siRNA injection. In conclusion, CTRP9 ameliorated PH by attenuating inflammation and improving endothelial cell survival and function.

Prognostic significance of constitutive phosphatidylinositol 3-kinase/Akt and mitogen-activated protein kinase phosphorylation in acute myeloid leukemia

Acute myeloid leukemia (AML) is a malignant hematopoietic disease with poor clinical course and outcome. There is a constant need for new prognostic factors that could facilitate patient risk stratification. The aim of our research was to determine the phosphorylation levels of phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen-activated protein kinase (MAPK) pathways in leukemic cells, their relation to P-glycoprotein (P-gp) expression/activity and their prognostic significance in adult de novo AML. A total of 118 patients with AML were enrolled in the study. In a multivariate Cox regression analysis we found that P-gp activity and Akt phosphorylation were independent poor prognostic factors of overall survival (OS). In contrast, phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2) represented a favorable prognostic factor of OS and relapse-free survival (RFS). A negative correlation between P-gp activity and p38 phosphorylation level was found, implying a possible role of this MAPK pathway in P-gp regulation. In addition, we found correlation between Akt and p38 phosphorylation levels, indicative of co-activation of two signaling cascades in AML.

Topical N-acetylcysteine accelerates wound healing in vitro and in vivo via the PKC/Stat3 pathway.

N-Acetylcysteine (Nac) is an antioxidant administered in both oral and injectable forms. In this study, we used Nac topically to treat burn wounds in vitro and in vivo to investigate mechanisms of action. In vitro, we monitored glutathione levels, cell proliferation, migration, scratch-wound healing activities and the epithelialization-related proteins, matrixmetalloproteinase-1 (MMP-1) and proteins involved in regulating the expression of MMP-1 in CCD-966SK cells treated with Nac. Various Nac concentrations (0.1, 0.5, and 1.0 mM) increased glutathione levels, cell viability, scratch-wound healing activities and migration abilities of CCD-966SK cells in a dose-dependent manner. The MMP-1 expression of CCD-966SK cells treated with 1.0 mM Nac for 24 h was significantly increased. Levels of phosphatidylinositol 3-kinase (PI3K), protein kinase C (PKC), janus kinase 1 (Jak1), signal transducer and activator of transcription 3 (Stat3), c-Fos and Jun, but not extracellular signal-regulated protein kinases 1 and 2 (Erk1/2), were also significantly increased in a dose-dependent manner compared to the controls. In addition, Nac induced collagenous expression of MMP-1 via the PKC/Stat3 signaling pathway. In vivo, a burn wound healing rat model was applied to assess the stimulation activity and histopathological effects of Nac, with 3.0% Nac-treated wounds being found to show better characteristics on re-epithelialization. Our results demonstrated that Nac can potentially promote wound healing activity, and may be a promising drug to accelerate burn wound healing.

Alterations of PI3K and Akt signaling pathways in the hippocampus and hypothalamus of Wistar rats treated with highly palatable food

Background/objectivesHighly palatable food (HPF), which is enriched in simple sugars and saturated fat, contributes to obesity and insulin resistance in humans. These metabolic changes are associated with serious complications of the central nervous system, including an elevated risk of cognitive dysfunction. We, herein, treated rats with HPF and then examined the insulin-signaling pathway, in particular, the levels of phosphatidylinositol-3 kinase (PI3K), Akt, and insulin receptor substrate-1 (IRS-1) in the hippocampus and hypothalamus.MethodsAdult Wistar rats fed with HPF (heated or not during preparation) for 4 months and then measured the levels of PI3K, Akt, and IRS-1 in the hippocampus and hypothalamus, by western blotting and quantitative real-time polymerase chain reaction.ResultsWe observed changes in body weight, glucose intolerance, and lipidemia, confirming that peripheral metabolic alterations were induced using this model. Hippocampal PI3K and hypothalamic Akt were affected in rats that are submitted to chronic exposure to an HPF diet. Moreover, heated HPF caused differentiated alterations in the regulatory subunit of PI3K in the hippocampus.DiscussionOur data suggest that this diet alters insulin signaling differentially in each brain region, and that hippocampal changes induced by this diet could contribute to the understanding of cognitive impairments that are dependent on the hippocampus.

G-CSF downregulates natural killer cell-mediated cytotoxicity in donors for hematopoietic SCT

In G-CSF-mobilized hematopoietic SCT (HSCT), natural killer (NK) cells have a critical role in GVHD and GVL effects. However, regulation of NK cell response to G-CSF remains unclear. This study assayed G-CSF effects in both HSCT donors and NK-92MI cells. The donors who received G-CSF had significantly decreased NK cell cytotoxicity. Levels of phosphatidylinositol 3-kinase (PI3K) and phosphorylated (p)-Akt, but not mammalian target of rapamycin (mTOR), were downregulated in NK cells from G-CSF-injected donors. G-CSF also decreased cytotoxicity without affecting viability and NF-κB of NK-92MI cells. PI3K and p-ERK expression were also decreased in G-CSF-treated NK-92MI cells, and their inhibitors, wortmannin and PD98059, respectively, both enhanced the downregulation of cytotoxicity. These effects were accompanied by decreased expression of a cytotoxicity-related gene, triosephosphate isomerase (TPI). Wortmannin, but not PD98059, enhanced the downregulation of TPI in G-CSF-treated NK-92MI cells, indicating a correlation between PI3K and TPI. We conclude that G-CSF-impaired NK cell cytotoxicity may accompany PI3K/Akt signaling. The effect is transient and NK cells may recover after G-CSF clearance, suggesting that G-CSF-mobilized HSCT may benefit both acute GVHD prevention and late-phase GVL promotion in HSCT recipients.

Proanthocyanidins inhibit mitogenic and survival-signaling in vitro and tumor growth in vivo

We have previously shown that treatment of human epidermoid carcinoma A431 cells with grape seed proanthocyanidins (GSPs) induces apoptosis of A431 cells. Here, we report that treatment of A431 cells with GSPs inhibits constitutive as well as EGF-induced higher levels of phosphorylated proteins of MAPK family in a dose-dependent manner. This effect is associated with the reactivation of MAP kinase phosphatases. Western blot analysis reveals that GSPs decrease: (i) the levels of phosphatidylinositol 3-kinase (PI3K) and the phosphorylation of Akt at ser473, and (ii) the constitutive activation of NF-kappaB/p65. As NF-kappaB-targeted genes play crucial roles in tumor cell proliferation and differentiation, we assessed the effect of GSPs on proteins encoded by these genes. Treatment with GSPs results in inhibition of the expression of COX-2, iNOS, PCNA, cyclin D1 and MMP-9 in A431 cells compared with non-GSPs-treated controls. Treatment of athymic nude mice with GSPs by oral gavage (50 or 100 mg/kg body weight/mouse) reduces the growth of A431-xenografts in mice, which is associated with the inhibition of tumor cell proliferation in xenografts as indicated by the inhibition of mRNA expression of PCNA and cyclin D1, and of NF-kappaB activity. Together, the data suggest that GSPs might be effective in the treatment of skin cancers.

Assessment of PI3 kinase as a druggable target in melanoma

8521 Background: PI3 (phosphatidylinositol-3) kinase is central to a major intracellular signal transduction pathway that influences numerous cellular functions, including growth, differentiation and survival. Drugs that target PI3 kinase have demonstrated preclinical activity, and are undergoing further development. The PI3 kinase inhibitor, SF1126 is expected to enter clinical trials in 2007. The hallmarks for a valuable drug target include differential expression between normal and malignant cells and an association with disease progression. PI3 kinase expression has not been thoroughly evaluated in melanoma. We sought to determine the expression and prognostic value of PI3 kinase in a large cohort of primary and metastatic melanomas and to compare the expression with that of benign nevi. Methods: Tissue microarrays containing 548 melanomas and 540 benign nevi were employed to assess PI3 kinase expression. We used a novel method of in situ automated quantitative analysis (AQUA) of protein levels. We applied S100 conjugated to Cy3 to identify a melanoma tumor mask within a histospot, and measured PI3 kinase expression levels using Cy5 conjugated antibodies within the mask. Continuous AQUA scores were correlated with clinical and pathological variables. Results: PI3 kinase expression was higher in melanomas than in nevi by unpaired t-tests (p &lt; 0.0001), and was significantly higher in metastatic than in primary specimens (p &lt; 0.0001). High PI3 kinase scores were associated with Clark levels of IV-V (p = 0.0126), Breslow thickness &gt; 2mm (p = 0.044) and absence of tumor infiltrating lymphocytes (p = 0.014). High PI3 kinase expression strongly correlated with decreased survival by Cox univariate analysis (p = 0.0024), but was not independent of disease stage. Conclusions: PI3 kinase expression is remarkably higher in malignant melanocytes than in benign nevi. Moreover, high PI3 kinase expression is associated with disease aggression, making PI3 kinase an attractive drug target for melanoma. This is the first large study assessing PI3 kinase levels in melanoma in a quantitative fashion. Assessment of the association between PI3 kinase levels and response to therapy is warranted. No significant financial relationships to disclose.

Transactivation of the IGF-1R by aldosterone

Activation of epithelial sodium channels (ENaC) by aldosterone, insulin, or insulin-like growth factor-1 (IGF-1) in renal epithelial cells (including the Xenopus laevis renal cell line A6) appears to share some common signaling elements subsequent to the initial insulin or IGF-1 receptor activation. Previously, the convergence point for insulin or IGF-1 and aldosterone signaling was assumed to be downstream of the receptor at the level of phosphatidylinositol 3-kinase (PI3-K); however, this study shows aldosterone directly transactivates the IGF-1 receptor (IGF-1R). In A6 cells, 10-min exposure to aldosterone increased the phosphorylation of the IGF-1 receptor, insulin receptor substrate-1 (IRS-1), and Akt (PKB). Furthermore, aldosterone activated PI3-K and phosphorylation of the most downstream element, Akt, was blocked by the specific PI3-K inhibitor LY-294002. Transactivation requires aldosterone binding to the mineralocorticoid/glucocorticoid receptor and does not require transcription.

Mimp, a Mitochondrial Carrier Homologue, Inhibits Met-HGF/SF-Induced Scattering and Tumorigenicity by Altering Met-HGF/SF Signaling Pathways

We have recently shown that Mimp, a mitochondrial carrier protein homologue, is induced by Met-hepatocyte growth factor/scatter factor (HGF/SF) signaling and decreases the mitochondrial membrane potential in DA3 mammary adenocarcinoma cells. We show here that induction of Mimp leads to growth arrest in response to HGF/SF by arresting cells at the S phase of the cell cycle. Induction of Mimp or its transient expression does not lead to apoptosis. Mimp also attenuates HGF/SF-induced cellular scattering in vitro and tumor growth in vivo. The exogenous induction of Mimp at levels similar to its endogenous induction by HGF/SF increases the level of the Met protein and its phosphorylation by HGF/SF but reduces the levels of Shc and prevents the HGF/SF-induced tyrosine phosphorylation of Grb2 and Shc. In contrast, the level of phosphatidylinositol 3-kinase (PI3K) increases following Mimp induction and the level of phosphorylated PI3K in response to HGF/SF is unaffected by the exogenous induction of Mimp. Moreover, exogenous Mimp prevents the HGF/SF-induced transcription of the serum response element-luciferase reporter gene. Our results show that Mimp expression reduces Met-HGF/SF-induced proliferation and scattering by attenuating and altering the downstream signaling of Met. These data show a new link between a tyrosine kinase growth factor receptor and a mitochondrial carrier homologue that regulates cellular growth, motility, and tumorigenicity.

Multifunction Steroid Receptor Coactivator, E6-Associated Protein, Is Involved in Development of the Prostate Gland

In this study we report that deletion of E6-associated protein (E6-AP) in mice results in a smaller prostate gland compared with that in normal wild-type animals. To investigate the mechanism(s) by which E6-AP affects prostate gland growth and development, we carried out both in vitro and in vivo experiments. In this study we show that E6-AP interacts with androgen receptor (AR) in a hormone-dependent manner and enhances the transactivation function of AR. Our in vivo data from E6-AP-null prostate glands show that the level of AR protein is elevated while the level of the AR target protein, probasin, is decreased. In contrast, the level of AR protein is decreased, and its target protein is increased in an E6-AP-overexpressing stable cell line, suggesting that E6-AP modulates both the protein level and the activity of AR. In addition, we show that the levels of phosphatidylinositol 3-kinase, total Akt, and phosphorylated Akt are decreased in E6-AP-null prostate, suggesting that E6-AP deletion down-regulates the signaling of the phosphatidylinositol 3-kinase-Akt pathway. We also show that RhoA negatively regulates AR function, and RhoA levels are increased in E6-AP-null prostate. Furthermore, expression levels of p53, Bax, active caspases, and apoptotic index are increased in E6-AP-null prostate. Collectively, our data suggest that E6-AP deletion attenuates the growth and development of the prostate gland by interfering with AR function as well as by stimulating p53-mediated apoptosis.

Intracellular Delivery of Phosphatidylinositol (3,4,5)-Trisphosphate Causes Incorporation of Glucose Transporter 4 into the Plasma Membrane of Muscle and Fat Cells without Increasing Glucose Uptake

Insulin stimulates glucose uptake into muscle and fat cells by translocating glucose transporter 4 (GLUT4) to the cell surface, with input from phosphatidylinositol (PI) 3-kinase and its downstream effector Akt/protein kinase B. Whether PI 3,4,5-trisphosphate (PI(3,4,5)P(3)) suffices to produce GLUT4 translocation is unknown. We used two strategies to deliver PI(3,4,5)P(3) intracellularly and two insulin-sensitive cell lines to examine Akt activation and GLUT4 translocation. In 3T3-L1 adipocytes, the acetoxymethyl ester of PI(3,4,5)P(3) caused GLUT4 migration to the cell periphery and increased the amount of plasma membrane-associated phospho-Akt and GLUT4. Intracellular delivery of PI(3,4,5)P(3) using polyamine carriers also induced translocation of myc-tagged GLUT4 to the surface of intact L6 myoblasts, demonstrating membrane insertion of the transporter. GLUT4 translocation caused by carrier-delivered PI(3,4,5)P(3) was not reproduced by carrier-PI 4,5-bisphosphate or carrier alone. Like insulin, carrier-mediated delivery of PI(3,4,5)P(3) elicited redistribution of perinuclear GLUT4 and Akt phosphorylation at the cell periphery. In contrast to its effect on GLUT4 mobilization, delivered PI(3,4,5)P(3) did not increase 2-deoxyglucose uptake in either L6GLUT4myc myoblasts or 3T3-L1 adipocytes. The ability of exogenously delivered PI(3,4,5)P(3) to augment plasma membrane GLUT4 content without increasing glucose uptake suggests that input at the level of PI 3-kinase suffices for GLUT4 translocation but is insufficient to stimulate glucose transport.

Insulin improves postischemic recovery of function through PI3K in isolated working rat heart.

Insulin improves contractile function after ischemia, but does not increase glucose uptake in the isolated working rat heart. We tested the hypothesis that the positive inotropic effect of insulin is independent of the signaling pathway responsible for insulin-stimulated glucose uptake. We inhibited this pathway at the level of phosphatidyl inositol 3-kinase (PI3K) with wortmannin. Hearts were perfused for 70 min at physiological workload with Krebs-Henseleit buffer containing [2-(3)H] glucose (5 mM, 0.05 microCi/ml) and oleate (0.4 mM, 1% BSA) in the presence (WM, n = 5) or absence (control, n = 7) of wortmannin (WM, 3 micromol/L). After 20 min, hearts were subjected to 15 min of total global ischemia followed by 35 min of reperfusion. Insulin (1 mU/ml) was added at the beginning of reperfusion (WM + insulin n = 8, insulin n = 8). Cardiac power before ischemia was 8.1 +/- 0.7 mW. Recovery of contractile function after ischemia was significantly increased in the presence of insulin (73.5 +/- 8.9% vs. 38.5 +/- 6.7%, p < 0.01). The addition of wortmannin completely abolished the effect of insulin on recovery (32.6 +/- 6.4%). Glucose uptake was 1.84 +/- 0.32 micromol/min/g dry before ischemia and was slightly elevated during reperfusion (2.68 +/- 0.35 micromol/min/g dry, n.s.). Insulin did not affect postischemic glucose uptake. In the presence of wortmannin, glucose uptake was lowest during reperfusion (n.s.). The results suggest that PI3K is involved in the insulin-induced improvement in postischemic recovery of contractile function. This effect of insulin is independent of its effect on glucose uptake.

Association of SH2-containing inositol phosphatase 2 with the insulin resistance of diabetic db/db mice.

SH-2-containing inositol 5'-phosphatase 2 (SHIP-2) is a physiologically important lipid phosphatase that functions to hydrolyze phosphatidylinositol (PI) 3-kinase product PI(3,4,5)P3 to PI(3,4)P2 in the negative regulation of insulin signaling. We investigated whether SHIP-2 is associated with the insulin resistance of diabetic db/db mice. The amount of SHIP-2 protein was elevated in quadriceps muscle and epididymal fat tissue, but not in the liver, of db/db mice relative to that in control db/+m mice. In accordance with the enhanced expression of SHIP-2, its localization at the membrane preparation was increased in the skeletal muscle and fat tissue of db/db mice. Insulin stimulation of PI 3-kinase activity was modestly decreased in skeletal muscle, fat tissue, and liver of db/db mice compared with that of db/+m mice. In addition to the modest decrease at the level of PI 3-kinase, the activity of Akt and protein kinase C (PKC)-zeta/lambda, which are downstream molecules of PI 3-kinase, was more severely reduced in the skeletal muscle and fat tissue, but not in liver of db/db mice. Treatment with the insulin-sensitizing agent rosiglitazone decreased the elevated expression of SHIP-2 in the skeletal muscle and fat tissue of db/db mice. Insulin-induced Akt activation and PKC-zeta/lambda phosphorylation were restored to the control level, although insulin-stimulated PI 3-kinase activation was minimally affected in the skeletal muscle and fat tissue of db/db mice. These results indicate that SHIP-2 is a novel molecule associated with insulin resistance in the skeletal muscle and fat tissue, and that insulin-induced activity of the downstream molecules of PI 3-kinase is decreased, at least in part, by the elevated expression of SHIP-2 in diabetic db/db mice.

Motility Enhancement by Tumor-Derived Mutant E-Cadherin Is Sensitive to Treatment with Epidermal Growth Factor Receptor and Phosphatidylinositol 3-Kinase Inhibitors

Diffuse-type gastric and lobular breast cancers are characterized by frequent mutations in the cell adhesion molecule E-cadherin. Here we report that tumor-associated mutations of E-cadherin enhanced random cell movement of transfected MDA-MB-435S mammary carcinoma cells as compared to wild-type (wt) E-cadherin-expressing cells. The mutations included in frame deletions of exons 8 or 9 and a point mutation in exon 8 which all affect putative calcium-binding sites within the linker region of the second and third extracellular domain. Motility enhancement by mutant E-cadherin was investigated by time-lapse laser scanning microscopy. Increased cell motility stimulated by mutant E-cadherin was influenced by cell–matrix interactions. The motility-increasing activity of mutant E-cadherin was blocked by application of pharmacological inhibitors of epidermal growth factor receptor and phosphatidylinositol (PI) 3-kinase. Investigation of the activation status of PI 3-kinase and the downstream signaling molecules Akt/protein kinase B and MAP kinase p44/42 showed that these kinases are not more strongly activated in mutant E-cadherin-expressing cells than in wt E-cadherin-expressing cells. Instead, the basal level of PI 3-kinase is necessary for mutant E-cadherin-enhanced cell motility. Our data suggest a critical role of E-cadherin mutations for the fine tuning of tumor cell motility.