Aging is associated with decreased pancreatic acinar cell regeneration and phosphatidylinositol 3-kinase/Akt activation

BACKGROUND & AIMS: The effects of aging on pancreatic acinar cell proliferation have not been clearly defined. Phosphatidylinositol 3-kinase (PI3K)-mediated phosphorylation of Akt is a critical step for proliferation of various cell types and insulin secretion from pancreatic endocrine cells; however, its role in acinar cell proliferation is not known. The purpose of this study was to (1) delineate the effects of aging on pancreatic regeneration after partial pancreatectomy (Px) and (2) define the involvement of the PI3K/Akt pathway in pancreatic regeneration. METHODS: Following partial Px, pancreatic regeneration and activation of the PI3K pathway were compared in young and aged mice. Activation of the PI3K/Akt pathway was evaluated by Akt phosphorylation (pAkt). The role of the PI3K pathway in pancreatic regeneration after partial Px was assessed by effects of a pharmacologic PI3K inhibitor wortmannin or small interfering RNA (siRNA) to the p85alpha regulatory subunit. To confirm further the critical role of the PI3K/Akt pathway in pancreatic acinar cell proliferation, IGF-1-mediated cell proliferation was determined in cultured acinar cells pretreated with wortmannin or p85alpha siRNA. RESULTS: Pancreatic regeneration and pAkt expression after partial Px were significantly decreased with aging. Treatment with wortmannin or p85alpha siRNA reduced pancreatic regeneration after partial Px. The IGF-1-mediated cell proliferation in vitro was completely blocked by wortmannin or p85alpha siRNA but not by the MEK/ERK inhibitor PD98059. CONCLUSIONS: PI3K/Akt activation plays a critical role in the regeneration of pancreatic acini after resection. Furthermore, pancreatic regeneration is markedly attenuated in the aged pancreas most likely because of decreased PI3K/Akt activation.     

Glucose-potentiated chemotaxis in human vascular smooth muscle is dependent on cross-talk between the PI3K and MAPK signaling pathways

Atheroma formation involves the movement of vascular smooth muscle cells (VSMC) into the subendothelial space. The aim of this study was to determine the involvement of PI3K and MAPK pathways and the importance of cross-talk between these pathways, in glucose-potentiated VSMC chemotaxis to serum factors. VSMC chemotaxis occurred in a serum gradient in 25 mmol/L glucose (but not in 5 mmol/L glucose) in association with increased phosphorylation (activation) of Akt and ERK1/2 in PI3K and MAPK pathways, respectively. Inhibitors of these pathways blocked chemotaxis, as did an mTOR inhibitor. VSMC expressed all class IA PI3K isoforms, but microinjection experiments demonstrated that only the p110beta isoform was involved in chemotaxis. ERK1/2 phosphorylation was reduced not only by MAPK pathway inhibitors but also by PI3K and mTOR inhibitors; when PI3K was inhibited, ERK phosphorylation could be induced by microinjected activated Akt, indicating important cross-talk between the PI3K and ERK1/2 pathways. Glucose-potentiated phosphorylation of molecules in the p38 and JNK MAPK pathways inhibited these pathways but did not affect chemotaxis. The statin, mevinolin, blocked chemotaxis through its effects on the MAPK pathway. Mevinolin-inhibited chemotaxis was restored by farnesylpyrophosphate but not by geranylgeranylpyrophosphate; in the absence of mevinolin, inhibition of farnesyltransferase reduced ERK phosphorylation and blocked chemotaxis, indicating a role for the Ras family of GTPases (MAPK pathway) under these conditions. In conclusion, glucose sensitizes VSMC to serum, inducing chemotaxis via pathways involving p110beta-PI3K, Akt, mTOR, and ERK1/2 MAPK. Cross-talk between the PI3K and MAPK pathways is necessary for VSMC chemotaxis under these conditions.     

The involvement of phosphatidylinositol 3-kinase in gonadotropin-releasing hormone-induced gonadotropin alpha- and FSHbeta-subunit genes expression in clonal gonadotroph LbetaT2 cells

Akt/protein kinase B (Akt/PKB), which is activated by phosphatidylinositol-3 kinase (PI3-kinase), plays an important role in cell survival and cell proliferation. Using the well differentiated, clonal gonadotroph cell line, LbetaT2, we examined (1) whether Akt/PKB was activated by gonadotropin-releasing hormone (GnRH); (2) the contribution of PI3-kinase-Akt/PKB pathway in each of gonadotropin subunit gene expression; (3) crosstalk between extracellular signal-regulated kinase (ERK) and Akt/PKB pathways. Insulin-like growth factor-1 (IGF-1) was used as Akt/PKBs classic activator. Western blot analyses using antibodies specific for the phosphorylated forms of ERK and Akt/PKB demonstrated that both were rapidly phosphorylated following treatment with GnRH and IGF-1. Akt/PKB activation by GnRH and IGF-1 was completely eliminated in the presence of the PI3-kinase inhibitor, LY 294002, but not in the presence of an Akt/PKB inhibitor. Interestingly, the total amount of Akt/PKB protein was dramatically increased in the presence of LY 294002. Phosphorylation of ERK was significantly increased in the presence of LY 294002 alone, and was further increased when GnRH was used in combination with LY 294002. In experiments using a luciferase reporter construct containing the serum response element (SRE), a known target of the ERK pathway, LY 294002 but not the Akt/PKB inhibitor increased SRE-luciferase activity. GnRH-induced SRE-luciferase activity was significantly increased by LY 294002. GnRH stimulation resulted in gonadotropin LHbeta, FSHbeta, and alpha-subunit promoter activation, while IGF-1 failed to stimulate any of them. GnRH-induced gonadotropin promoter activities were not modulated in the presence of an Akt/PKB inhibitor, but treatment with LY 294002 or Wortmannin resulted in a significant increase in alpha- and FSHbeta-subunit promoter activation, both with and without GnRH. LY 294002, but not the Akt/PKB inhibitor, significantly inhibited cell proliferation. These results suggest that GnRH-induced gonadotropin gene expression is not regulated through the Akt/PKB pathway; however, PI3-kinase may be involved in the negative regulation of alpha- and FSHbeta-subunit gene expression as well as cell proliferation.     

The IGF-1 Signaling Pathway in Viral Infections

Insulin-like growth factor-1 (IGF-1) and the IGF-1 receptor (IGF-1R) belong to the insulin-like growth factor family, and IGF-1 activates intracellular signaling pathways by binding specifically to IGF-1R. The interaction between IGF-1 and IGF-1R transmits a signal through a number of intracellular substrates, including the insulin receptor substrate (IRS) and the Src homology collagen (Shc) proteins, which activate two major intracellular signaling pathways: the phosphatidylinositol 3-kinase (PI3K)/AKT and mitogen-activated protein kinase (MAPK) pathways, specifically the extracellular signal-regulated kinase (ERK) pathways. The PI3K/AKT kinase pathway regulates a variety of cellular processes, including cell proliferation and apoptosis. IGF1/IGF-1R signaling also promotes cell differentiation and proliferation via the Ras/MAPK pathway. Moreover, upon IGF-1R activation of the IRS and Shc adaptor proteins, Shc stimulates Raf through the GTPase Ras to activate the MAPKs ERK1 and ERK2, phosphorylate and several other proteins, and to stimulate cell proliferation. The IGF-1 signaling pathway is required for certain viral effects in oncogenic progression and may be induced as an effect of viral infection. The mechanisms of IGF signaling in animal viral infections need to be clarified, mainly because they are involved in multifactorial signaling pathways. The aim of this review is to summarize the current data obtained from virological studies and to increase our understanding of the complex role of the IGF-1 signaling axis in animal virus infections.     

Dopamine d1-like receptors suppress proliferation of vascular smooth muscle cell induced by insulin-like growth factor-1

Objective: Proliferation of vascular smooth muscle cells (VSMCs) participates in the pathogenesis and development of cardiovascular diseases, including essential hypertension and atherosclerosis. Our previous study found that stimulation of D₁-like dopamine receptors inhibited insulin-induced proliferation of VSMCs. Insulin-like growth factor-1 (IGF-1) and insulin share similar structure and biological effect. However, whether or not there is any effect of D₁-like receptors on IGF-1-induced proliferation of VSMCs is not known. Therefore, we investigated the inhibitory effect of D₁-like dopamine receptors on the IGF-1-induced VSMCs proliferation in this study.

Method: VSMC proliferation was determined by [³H]-thymidine incorporation, the uptake of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and cell number. Phosphorylated/non-phosphorylated IGF-1 receptor, Akt, mTOR and p70S6K expressions were determined by immunoblotting. The oligodeoxynucleotides were transfected to A10 cells to identify the effect of D₁ and D₅ receptors, respectively.

Results: IGF-1 increased the proliferation of VSMCs, while in the presence of fenoldopam, IGF-1-mediated stimulatory effect was reduced. Use of either antisense for D₁ or D₅ receptor partially inhibited the fenoldopam-induced antiproliferation effect of VSMCs. Use of both D₁ and D₅ receptor antisenses completely blocked the inhibitory effect of fenoldopam. In the presence of PI3k and mTOR inhibitors, the IGF-1-mediated proliferation of VSMCs was blocked. Moreover, IGF-1 increased the phosphorylation of PI3k and mTOR. The inhibitory effect of fenoldopam on VSMC proliferation might be due to the inhibition of IGF-1 receptor expression and IGF-1 phosphorylation, because in the presence of fenoldopam, the stimulatory effect of IGF-1 on phosphorylation of IGF-1 receptor, PI3k and mTOR is reduced, the IGF-1 receptor expression was reduced in A10 cells.

Conclusion: Activation of the D₁-like receptors suppressed the proliferative effect of IGF-1 in A10 cells via the inhibition of the IGF-1R/Akt/mTOR/p70S6K pathway and downregulated the expression of IGF-1 receptor.     

Dueling for dual inhibition: Means to enhance effectiveness of PI3K/Akt/mTOR inhibitors in AML

The phosphatidylinositol 3-kinase/protein kinase B (Akt)/mechanistic target of rapamycin (PI3K/Akt/mTOR) pathway is amplified in 60–80% of patients with acute myelogenous leukemia (AML). Since this complex pathway is crucial to cell functions such as growth, proliferation, and survival, inhibition of this pathway would be postulated to inhibit leukemia initiation and propagation. Inhibition of the mTORC1 pathway has met with limited success in AML due to multiple resistance mechanisms including direct insensitivity of the mTORC1 complex, feedback activation of the PI3k/Akt signaling network, insulin growth factor-1 (IGF-1) activation of PI3K, and others. This review explores the role of mTOR inhibition in AML, mechanisms of resistance, and means to improve outcomes through use of dual mTORC1/2 inhibitors or dual TORC/PI3K inhibitors. How these inhibitors interface with currently available therapies in AML will require additional preclinical experiments and conduct of well-designed clinical trials.     

Platelet factor 4 inhibits FGF2-induced endothelial cell proliferation via the extracellular signal-regulated kinase pathway but not by the phosphatidylinositol 3-kinase pathway

Platelet factor 4 (PF-4) is a member of the chemokine family with powerful antiangiogenic properties. The mechanism by which PF-4 inhibits endothelial cell proliferation is unclear. We investigated the effects of PF-4 on the intracellular signal transduction induced by basic fibroblast growth factor (FGF2). We found that PF-4 (10 microg/mL) inhibited the FGF2-induced proliferation of adrenal cortex capillary endothelial (ACE) cells. The inhibition of MEK1/2 (mitogen-activated protein kinase kinase) by PD98059 or of PI3K (phosphatidylinositol 3-kinase) by Ly294002 abolished the proliferation induced by FGF2, suggesting that ACE cell proliferation required dual signaling through both the extracellular signal-regulated kinase (ERK) and PI3K pathways. Ly294002 had no significant effect on ERK phosphorylation, whereas PD98059 had a weak effect on the phosphorylation of Akt, suggesting that 2 separate cascades are required for ACE cell proliferation. The addition of PF-4 (10 microg/mL) significantly inhibited ERK phosphorylation (95%), showing that PF-4 acted directly on or upstream from this kinase. Surprisingly, PF-4 did not affect FGF2-induced Akt phosphorylation. This suggests that PF-4 disrupts FGF2 signaling via an intracellular mechanism of inhibition. To exclude the possibility that PF-4 inhibited the binding of FGF2 to only one FGF receptor, preferentially activating the ERK pathway, we investigated the effect of PF-4 on FGF2-induced ERK and Akt phosphorylation, using mutant heparan sulfate-deficient Chinese hamster ovary cells transfected with the FGF-R1 cDNA. The addition of PF-4 (1 microg/mL) significantly inhibited ERK phosphorylation (90%), with no effect on Akt phosphorylation, suggesting that PF-4 acts downstream from the FGF-R1 receptor. In conclusion, this is the first report showing that PF-4 inhibits FGF2 activity downstream from its receptor.     

β-Arrestins在胰岛素/胰岛素样生长因子-1信号转导中的作用

β-Arrestins是G蛋白耦联受体信号转导通路的负调节因子,越来越多的证据表明,β-arrestins也能作用于细胞内的多种信号分子,调节胰岛素/胰岛素样生长因子-1(IGF-1)信号转导通路.在胰岛素的刺激下,β-arrestin 2能够募集蛋白激酶B(Akt)和酪氨酸激酶Src到胰岛素受体,从而调节胰岛素介导的糖代谢效应;而β-arrestin 1则与胰岛素受体底物-1(IRS-1)竞争性结合泛素连接酶Mdm2,从而减少IRS-1的泛素化和降解,促进磷脂酰肌醇3激酶(PI3K)通路的信号转导.在IGF-1介导的信号转导通路中,β-arrestin 1结合并介导了IGF-1受体(IGF-1R)的内吞,促进胞外信号调节激酶活化,正性调节丝裂原活化蛋白激酶通路.此外,β-arrestin 1与IGF-1R相耦联后,能越过信号分子IRS-1而激活PI3K,进而活化Akt,表现出对P13K途径的正性调控作用.     

Tyrosine phosphorylation-dependent and -independent role of Shc in the regulation of IGF-1-induced mitogenesis and glycogen synthesis.

To examine the functional role of Shc tyrosine phosphorylation in IGF-1 signaling, wild-type (WT)-Shc and Y239,240,317F (3F)-Shc were transiently transfected into L6 myoblasts. IGF-1 signaling was compared among the transfected cells. IGF-1-induced tyrosine phosphorylation of Shc and its subsequent association with Grb2 were increased in WT-Shc cells, whereas they were decreased in 3F-Shc cells compared with those in parental L6 cells. Consistent with their changes, IGF-1-induced MAPK activation and thymidine incorporation were enhanced in WT-Shc cells, whereas they were again decreased in 3F-Shc cells. It is possible that Shc and insulin receptor substrate (IRS)-1 can interact competitively, via their phosphotyrosine binding (PTB) domains, with the activated IGF-1 receptor. In this regard, IGF-1-induced tyrosine phosphorylation of IRS-1 was decreased by overexpressing both WT-Shc and 3F-Shc cells. Consistent with the decrease, IGF-1-induced IRS-1 association with the p85 subunit of PI3K and activation of PI3K and Akt were reduced in both WT-Shc and 3F-Shc cells. As a result, IGF-1-induced glycogen synthesis was also decreased in both cells. Furthermore, expression of Shc PTB domain alone inhibited IGF-1 stimulation of Akt and glycogen synthesis. These results indicate that tyrosine phosphorylation of Shc is important for IGF-1 stimulation of MAPK leading to mitogenesis and that Shc, via its PTB domain, negatively regulates IGF-1-induced glycogen synthesis by competing with IRS-1, which is not relevant to Shc tyrosine phosphorylation.     

Bile acids enhance the activity of the insulin receptor and glycogen synthase in primary rodent hepatocytes

Previously, we demonstrated that deoxycholic acid (DCA)-induced ERK1/2 and AKT signaling in primary hepatocytes is a protective response. In the present study, we examined the regulation of the phosphatidylinositol 3 (PI3) kinase/AKT/glycogen synthase (kinase) 3 (GSK3)/glycogen synthase (GS) pathway by bile acids. In primary hepatocytes, DCA activated ERBB1 (the epidermal growth factor receptor), ERBB2, and the insulin receptor, but not the insulin-like growth factor 1 (IGF-1) receptor. DCA-induced activation of the insulin receptor correlated with enhanced phosphorylation of insulin receptor substrate 1, effects that were both blocked by the insulin receptor inhibitor AG1024 and by expression of the dominant negative IGF-1 receptor (K1003R), which inhibited in trans. Expression of the dominant negative IGF-1 receptor (K1003R) also abolished DCA-induced AKT activation. Bile acid-induced activation of AKT and phosphorylation of GSK3 were blunted by the ERBB1 inhibitor AG1478 and abolished by AG1024. Bile acids caused activation of GS to a similar level induced by insulin (50 nM); both were blocked by inhibition of insulin receptor function and the PI3 kinase/AKT/GSK3 pathway. In conclusion, these findings suggest that bile acids and insulin may cooperate to regulate glucose storage in hepatocytes.     

Role of phosphoinositide 3-kinase and extracellular signal-regulated kinase pathways in granulocyte macrophage-colony-stimulating factor failure to delay fas-induced neutrophil apoptosis in elderly humans

Fas-stimulated neutrophils from elderly individuals show impaired granulocyte macrophage-colony-stimulating factor (GM-CSF)-induced apoptosis cell rescue. Herein, this defect was found to be associated with a significant reduction in GM-CSF-mediated Akt and extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation. Using Akt and ERK1/2 inhibitors, we demonstrated that both kinases were critical for GM-CSF antiapoptotic effects. Whereas Akt inhibition also affected GM-CSF-dependent ERK1/2 phosphorylation, ERK1/2 inhibition did not affect GM-CSF-induced Akt phosphorylation, suggesting that phosphoinositide 3-kinase (PI3-K)/Akt and ERK1/2 are activated in series and that PI3-K is located upstream of ERK1/2 along the GM-CSF-dependent signaling pathway. No age-associated changes in GM-CSF receptor expression were observed. Interestingly, both suppressors of cytokine signaling (SOCS)1 and SOCS3 proteins were significantly higher in unstimulated neutrophils from elderly individuals and, unlike in young individuals, did not further increase following GM-CSF cell triggering. These results indicate that defective PI3-K/Akt/ERK1/2 activation, likely dependent on elevated SOCS1 and SOCS3 levels, may affect the GM-CSF capacity to delay neutrophil apoptosis in elderly persons.     

Signal transduction pathways in androgen-dependent and -independent prostate cancer cell proliferation

In a previous report, we showed that increased activation of Akt, a downstream effector of phosphoinositide 3-kinase (PI3K) together with decreased activation of extracellular-signal-regulated kinase (ERK), a member of the mitogen-activated protein kinase (MAPK) family, predicted poor clinical outcome in prostate cancer (Kreisberg et al. 2004 Cancer Research 64 5232-5236). We now show that Akt activation, but not ERK activation, is correlated with proliferation in human prostate tumors as estimated by the expression of the cell proliferation antigen Ki67. We verified these results in vitro, using the androgen-dependent prostate cancer cell line LNCaP and its androgen-independent clone C4-2 as models of prostate cancer of good and poor clinical outcome, respectively. C4-2 cells expressed higher Akt activation, lower ERK activation and increased proliferation compared with LNCaP cells, similar to cases of poor clinical outcome. The PI3K inhibitor LY294002, but not the MAPK/ERK kinase inhibitor PD98059, induced growth arrest in both cell lines. Transient transfection with constitutively active Akt increased proliferation while dominant negative Akt decreased it, thus showing that Akt plays an important role in prostate cancer proliferation. Akt regulates the expression and activation of the androgen receptor. Androgen receptor inhibition with Casodex induced growth arrest in LNCaP cells, but not in C4-2 cells. Another PI3K downstream effector, p70 S6 kinase, requires prior phosphorylation by mammalian target of rapamycin (mTOR) for complete activation. Activation of p70 S6 kinase was higher in C4-2 compared with LNCaP cells. Rapamycin, an mTOR inhibitor, had a growth-inhibitory effect in C4-2 cells, but not in LNCaP cells. Our data suggest a shift from a Casodex-sensitive proliferation pathway in LNCaP cells to a rapamycin-sensitive pathway in C4-2 cells.     

Rho-GDP dissociation inhibitor alpha downregulated by low shear stress promotes vascular smooth muscle cell migration and apoptosis: a proteomic analysis

AIMS: Low shear stress (LSS) plays a significant role in vascular remodelling during atherogenesis, which involves migration, proliferation, and apoptosis of vascular smooth muscle cells (VSMCs). The aim of the present study is to elucidate the molecular mechanisms by which LSS induces vascular remodelling. METHODS AND RESULTS: Using proteomic techniques, two-dimensional electrophoresis, and mass spectrometry, the protein profiles of Sprague-Dawley rat aorta cultured under two levels of shear stress, 5 and 15 dyn/cm(2), were determined. The results showed a significantly lower expression of protein-Rho-GDP dissociation inhibitor alpha (Rho-GDIalpha) in the LSS vessels. Rho-GDIalpha signalling mechanisms and effects on VSMC migration and apoptosis were then studied to understand the role of Rho-GDIalpha in the LSS-induced vascular remodelling. A decrease in Rho-GDIalpha expression by using target small interfering RNA (siRNA) transfection caused increases in the phosphorylation of Rac1 and Akt and enhancements of VSMC migration and apoptosis. Treatment with the PI3K/Akt-specific inhibitor wortmannin significantly decreased Akt phosphorylation, but had no effect on Rho-GDIalpha expression and Rac1 phosphorylation. Wortmannin was able to reverse the Rho-GDIalpha siRNA-induced enhancement of VSMC migration, but not VSMC apoptosis. CONCLUSION: The results indicate that the LSS-induced VSMC migration and apoptosis are mediated by a downregulation of Rho-GDIalpha. The effect of Rho-GDIalpha on VSMC migration is mediated by the PI3K/Akt pathway, but its effect on VSMC apoptosis is not.     

Glimepiride induces proliferation and differentiation of rat osteoblasts via the PI3-kinase/Akt pathway

Glimepiride is a third-generation sulfonylurea agent and is widely used in the treatment of type 2 diabetes mellitus. In addition to the stimulatory effects on pancreatic insulin secretion, glimepiride has also been reported to have extrapancreatic functions including activation of PI3 kinase (PI3K) and Akt in rat adipocytes and skeletal muscle. PI3-kinase and Akt are important signaling molecules in the regulation of proliferation and differentiation in various cells. This study investigated the actions of glimepiride in rat osteoblasts and the role of PI3K/Akt pathway. Cell proliferation was determined by measuring absorbance at 550 nm. Supernatant assay was used for measuring alkaline phosphatase activity. Western blot analysis was used for determining collagen I, insulin receptor substrate-1/2, PI3K/Akt, and endothelial nitric oxide synthase expression. We found that glimepiride significantly enhanced proliferation and differentiation of osteoblasts and led to activation of several key signaling molecules including insulin receptor substrate-1/2, PI3K/Akt, and endothelial nitric oxide synthase. Furthermore, a specific inhibitor of PI3K abolished the stimulatory effects of glimepiride on proliferation and differentiation. Taken together, these observations provide concrete evidence that glimepiride activates the PI3K/Akt pathway; and this activation is likely required for glimepiride to stimulate proliferation and differentiation of rat osteoblasts.     

High dose human insulin and insulin glargine promote T24 bladder cancer cell proliferation via PI3K-independent activation of Akt

Background

This study was to investigate the effects of human insulin and insulin glargine on proliferation of T24 human bladder cancer cells and the implication of the PI3K/Akt and MEK/ERK1/2 pathways.

Methods

After exposure to insulin or glargine at the indicated concentrations for certain time courses, in the absence or presence of inhibitor for MEK (PD98059) or PI3K (LY294002), T24 cell proliferation was evaluated by CCK-8 assay. Phosphorylation of Akt and ERK1/2 was analyzed by Western blot.

Results

Insulin and glargine similarly induced phosphorylation of Akt and slight increases in T24 cell proliferation at 10-100 IU/L. LY294002 remarkably reduced T24 cell proliferation in all groups. However, in the presence of LY294002, cell growth was still promoted by insulin and glargine relative to LY294002-treated group. Accordingly, LY294002 profoundly reduced protein levels of pAkt, while insulin and glargine increased pAkt in T24 cells pretreated with LY294002 as compared with cells treated with LY294002 alone. PD98059 reduced pERK while enhanced T24 cell proliferation. Insulin and glargine increased pERK at 15, 30, 60 min, not at 24 h.

Conclusions

High dose human insulin and insulin glargine similarly promoted T24 bladder cancer cell proliferation via PI3K-independent activation of Akt.     

Autocrine IGF-1/IGF-1R signaling is responsible for constitutive PI3K/Akt activation in acute myeloid leukemia: therapeutic value of neutralizing anti-IGF-1R antibody

Background

Alterations in the PI3K/Akt pathway are found in a wide range of cancers and the development of PI3K inhibitors represents a promising approach to cancer therapy. Constitutive PI3K activation, reflecting an intrinsic oncogenic deregulation of primary blast cells, is detected in 50% of patients with acute myeloid leukemia. However, the mechanisms leading to this activation are currently unknown. As we previously reported IGF-1 autocriny in acute myeloid leukemia cells, we investigated whether IGF-1 signaling was involved in the constitutive activation of PI3K.

Design and Methods

We analyzed the IGF-1/IGF-1R signaling pathway and PI3K activity in 40 acute myeloid leukemia bone marrow samples. Specific inhibition of IGF-1/IGF-1R signaling was investigated using neutralizing anti-IGF-1R, anti-IGF-1 antibodies or IGF-1 short interfering RNA. The anti-leukemic activity of the neutralizing anti-IGF-1R was tested by analyzing its effects on leukemic progenitor clonogenicity, blast cell proliferation and survival.

Results

In all samples tested, we found that functional IGF-1R was constantly expressed in leukemic cells. In the acute myeloid leukemia samples with PI3K activation, we found that the IGF-1R was constitutively phosphorylated, although no IGF-1R activating mutation was detected. Specific inhibition of IGF-1R signaling with neutralizing anti-IGF-1R strongly inhibited the constitutive phosphorylation of both IGF-1R and Akt in 70% of the PI3K activated samples. Moreover, both incubation with anti-IGF-1 antibody and IGF-1 short interfering RNA inhibited Akt phosphorylation in leukemic cells. Finally, neutralizing anti-IGF-1R treatment decreased the clonogenicity of leukemic progenitors and the proliferation of PI3K activated acute myeloid leukemia cells.

Conclusions

Our current data indicate a critical role for IGF-1 autocriny in constitutive PI3K/Akt activation in primary acute myeloid leukemia cells and provide a strong rationale for targeting IGF-1R as a potential new therapy for this disease.     

Akt is a major downstream target of PI3-kinase involved in angiotensin II-induced proliferation

Different signal transduction cascades have been implicated in angiotensin II (Ang II)-mediated cell growth, such as the extracellular signal-regulated kinase 1/2 (ERK1/2) and the phosphatidylinositol 3-kinase (PI3K) pathways. To identify the downstream targets of PI3K involved in Ang II-induced proliferation, we used both rat aortic smooth muscle (RASM) cells and a CHO cell line stably expressing the rat AT1A receptor. The ERK1/2 and PI3K pathways are independently activated and implicated in Ang II-mediated DNA synthesis and cell number increase in these 2 cell lines. In addition, a specific inhibitor of Akt inhibited Ang II-induced Akt phosphorylation, DNA synthesis and proliferation in CHO-AT1A or RASM cells. A dominant-negative mutant of Akt was also found to selectively block Ang II-induced proliferation of CHO-AT1A cells. To further elucidate the signaling events leading to Akt activation, we used an AT1 receptor mutant (AT1AD74E), deficient for Gq protein coupling, and the intracellular calcium chelator BAPTA-AM. Although altered Akt and ERK1/2 activation was observed in the CHO-AT1AD74E cell line, blockade of intracellular calcium elevation did not affect phosphorylation of these kinases. These results provide the first evidence of a specific and necessary role of Akt in Ang II-induced proliferation through a Gq protein-dependent calcium-independent pathway.     

IL-2- and IL-15-induced activation of the rapamycin-sensitive mTORC1 pathway in malignant CD4+ T lymphocytes

We examined functional status, activation mechanisms, and biologic role of the mTORC1 signaling pathway in malignant CD4(+) T cells derived from the cutaneous T-cell lymphoma (CTCL). Whereas the spontaneously growing CTCL-derived cell lines displayed persistent activation of the TORC1 as well as the PI3K/Akt and MEK/ERK pathways, the IL-2-dependent cell lines activated the pathways in response to IL-2 and IL-15 but not IL-21. Activation of mTORC1 and MEK/ERK was nutrient dependent. The mTORC1, PI3K/Akt, and MEK/ERK pathways could also be activated by IL-2 in the primary leukemic, mitogen-preactivated CTCL cells. mTORC1 activation was also detected in the CTCL tissues in the lymphoma stage-dependent manner with the highest percentage of positive cells present in the cases with a large cell transformation. Rapamycin inhibited mTORC1 signaling and suppressed CTCL cell proliferation but showed little effect on their apoptotic rate when used as a single agent. Activation of the mTORC1, PI3K/Akt, and MEK/ERK pathways was strictly dependent on the Jak3 and Jak1 kinases. Finally, mTORC1 activation was transduced preferentially through the PI3K/Akt pathway. These findings document the selective gammac-signaling cytokine-mediated activation of the mTORC1 pathway in the CTCL cells and suggest that the pathway represents a therapeutic target in CTCL and, possibly, other T-cell lymphomas.     

Insulin and insulin-like growth factor-1 stimulate proliferation and type I collagen accumulation by human hepatic stellate cells: differential effects on signal transduction pathways.

Insulin and insulin-like growth factor (IGF-1) are mitogenic for fibroblasts and smooth muscle cells. IGF-1 increases in inflamed and fibrotic tissues and induces proliferation of rat hepatic stellate cells (HSC). This study evaluates the potential roles of these hormones in the development of liver fibrosis. Insulin and IGF-1 receptor expression was evaluated by immunohistochemistry in both cultured human HSC and human liver tissue. Phosphorylation of both 70-kd S6 kinase and extracellular-regulated kinase (ERK), cell proliferation, type I collagen gene expression, and accumulation in HSC culture media were evaluated by Western blot, immunohistochemistry for bromodeoxyuridine (BrdU), Northern blot, and enzyme-linked immunosorbent assay, respectively. Insulin and IGF-1 receptors were detected in HSC in vitro and in liver sections from patients with chronic active hepatitis. Insulin and IGF-1 induced 70-kd S6 kinase phosphorylation in HSC, whereas IGF-1 only induced ERK phosphorylation. Insulin and IGF-1 stimulated HSC proliferation in a dose-dependent fashion, with IGF-1 being four to five times more potent than insulin. Cell exposure to specific inhibitors showed that both phosphatidylinositol 3-kinase (PI3-K) and ERK are involved in IGF-1-induced mitogenesis, whereas insulin stimulated mitogenesis through a PI3-K-dependent ERK-independent pathway. IGF-1 increased type I collagen gene expression and accumulation in HSC culture media through a PI3-K- and ERK-dependent mechanism. In conclusion, insulin and IGF-1, which stimulate HSC mitogenesis and collagen synthesis, may act in concert to promote liver fibrosis in vivo by a differential activation of PI3-K- and ERK1-dependent pathways.