Expression Of Dominant Negative Akt Research Articles

Resveratrol induces autophagy impeding BAFF-stimulated B-cell proliferation and survival by inhibiting the Akt/mTOR pathway

Therapeutically targeting B cells has received great attention in the treatment of B-cell malignancies and autoimmune diseases. The B-cell activating factor (BAFF) is critical to the survival of normal and neoplastic B cells, and excess production of BAFF contributes to autoimmune diseases. Resveratrol, a natural polyphenolic compound, has a positive effect on the treatment of autoimmune diseases. However, how resveratrol affects BAFF-stimulated B-cell proliferation and survival is poorly understood. Here, we show that resveratrol increased autophagosome formation and ATG5/LC3-II levels and decreased p62 level, promoting autophagic flux/autophagy and thereby suppressing the basal or human soluble BAFF (hsBAFF)-stimulated proliferation and survival of normal and B-lymphoid (Raji) cells. This is supported by the findings that inhibition of autophagy with 3-methyladenine (3-MA, an inhibitor of Vps34) or ATG5 shRNA attenuates resveratrol-induced autophagy and -reduced proliferation/viability in B-cells. Inhibition of mTOR with rapamycin or knockdown of mTOR potentiated resveratrol-induced autophagy and inhibition of hsBAFF-stimulated B-cell proliferation/viability, while overexpression of wild-type mTOR conferred resistance to the actions of resveratrol. Similarly, inhibition of Akt with Akt inhibitor X or ectopic expression of dominant negative Akt reinforced resveratrol-induced autophagy and inhibition of hsBAFF-stimulated B-cell proliferation/viability, whereas expression of constitutively active Akt conferred resistance to the actions of resveratrol. Taken together, these results indicate that resveratrol induces autophagy impeding BAFF-stimulated proliferation and survival via blocking the Akt/mTOR signaling pathway in normal and neoplastic B cells. Our findings highlight that resveratrol has a great potential for prevention and treatment of excessive BAFF-elicited aggressive B-cell disorders and autoimmune diseases.

Metformin prevents BAFF activation of Erk1/2 from B-cell proliferation and survival by impeding mTOR-PTEN/Akt signaling pathway

B-cell activating factor (BAFF) is an essential cytokine for B-cell maturation, differentiation and survival, and excess BAFF induces aggressive or neoplastic B-cell disorders and contributes to development of autoimmune diseases. Metformin, an anti-diabetic drug, has recently garnered a great attention due to its anti-proliferative and immune-modulatory features. However, little is known regarding the effect of metformin on BAFF-stimulated B cells. Here, we show that metformin attenuated human soluble BAFF (hsBAFF)-induced cell proliferation and survival by blocking the Erk1/2 pathway in normal and B-lymphoid (Raji) cells. Pretreatment with U0126, knockdown of Erk1/2, or expression of dominant negative MKK1 strengthened metformin’s inhibition of hsBAFF-activated Erk1/2 and B-cell proliferation/viability, whereas expression of constitutively active MKK1 rendered high resistance to metformin. Further investigation found that overexpression of wild type PTEN or ectopic expression of dominant negative Akt potentiated metformin's suppression of hsBAFF-induced Erk1/2 activation and proliferation/viability in Raji cells, implying a PTEN/Akt-dependent mechanism involved. Furthermore, we noticed that metformin hindered hsBAFF-activated mTOR pathway in B cells. Inhibition of mTOR with rapamycin or knockdown of mTOR enhanced metformin’s suppression of hsBAFF-induced phosphorylation of S6K1, PTEN, Akt, and Erk1/2, as well as B-cell proliferation/viability. These results indicate that metformin prevents BAFF activation of Erk1/2 from cell proliferation and survival by impeding mTOR-PTEN/Akt signaling pathway in normal and neoplastic B-lymphoid cells. Our findings support that metformin has a great potential for prevention of excessive BAFF-induced aggressive B-cell malignancies and autoimmune diseases.

Rapamycin inhibits B-cell activating factor (BAFF)-stimulated cell proliferation and survival by suppressing Ca2+-CaMKII-dependent PTEN/Akt-Erk1/2 signaling pathway in normal and neoplastic B-lymphoid cells

B-cell activating factor (BAFF) is a crucial survival factor for B cells, and excess BAFF contributes to development of autoimmune diseases. Recent studies have shown that rapamycin can prevent BAFF-induced B-cell proliferation and survival, but the underlying mechanism remains to be elucidated. Here we found that rapamycin inhibited human soluble BAFF (hsBAFF)-stimulated cell proliferation by inducing G1-cell cycle arrest, which was through downregulating the protein levels of CDK2, CDK4, CDK6, cyclin A, cyclin D1, and cyclin E. Rapamycin reduced hsBAFF-stimulated cell survival by downregulating the levels of anti-apoptotic proteins (Mcl-1, Bcl-2, Bcl-xL and survivin) and meanwhile upregulating the levels of pro-apoptotic proteins (BAK and BAX). The cytostatic and cytotoxic effects of rapamycin linked to its attenuation of hsBAFF-elevated intracellular free Ca2+ ([Ca2+]i). In addition, rapamycin blocked hsBAFF-stimulated B-cell proliferation and survival by preventing hsBAFF from inactivating PTEN and activating the Akt-Erk1/2 pathway. Overexpression of wild type PTEN or ectopic expression of dominant negative Akt potentiated rapamycin's suppression of hsBAFF-induced Erk1/2 activation and proliferation/viability in Raji cells. Interestingly, PP242 (mTORC1/2 inhibitor) or Akt inhibitor X, like rapamycin (mTORC1 inhibitor), reduced the basal or hsBAFF-induced [Ca2+]i elevations. Chelating [Ca2+]i with BAPTA/AM, preventing [Ca2+]i elevation using EGTA, 2-APB or verapamil, inhibiting CaMKII with KN93, or silencing CaMKII strengthened rapamycin's inhibitory effects. The results indicate that rapamycin inhibits BAFF-stimulated B-cell proliferation and survival by blunting mTORC1/2-mediated [Ca2+]i elevations and suppressing Ca2+-CaMKII-dependent PTEN/Akt-Erk1/2 signaling pathway. Our finding underscores that rapamycin may be exploited for prevention of excessive BAFF-induced aggressive B-cell malignancies and autoimmune diseases.

Cadmium results in accumulation of autophagosomes-dependent apoptosis through activating Akt-impaired autophagic flux in neuronal cells

Environmental exposure to cadmium (Cd) links to neurodegenerative disorders. Autophagy plays an important role in controlling cell survival/death. However, how autophagy contributes to Cd's neurotoxicity remains enigmatic. Here, we show that Cd induced significant increases in autophagosomes with a concomitant elevation of LC3-II and p62 in PC12 cells and primary neurons. Using autophagy inhibitor 3-MA, we demonstrated that Cd-increased autophagosomes contributed to neuronal apoptosis. Impairment of Cd on autophagic flux was evidenced by co-localization of mCherry and GFP tandem-tagged LC3 puncta in the cells. This is further supported by the findings that administration of chloroquine (CQ) potentiated the basic and Cd-elevated LC3-II and p62 levels, autophagosome accumulation and cell apoptosis, whereas rapamycin relieved the effects in the cells in response to Cd. Subsequently, we noticed that Cd evoked the phosphorylation of Akt and BECN1. Silencing BECN1 and especially expression of mutant BECN1 (Ser295A) attenuated Cd-increased autophagosomes and cell death. Of note, inhibition of Akt with Akt inhibitor X, or ectopic expression of dominant negative Akt (dn-Akt), in the presence or absence of 3-MA, significantly alleviated Cd-triggered phosphorylation of Akt and BECN1, autophagosomes, and apoptosis. Importantly, we found that Cd activation of Akt functioned in impairing autophagic flux. Collectively, these results indicate that Cd results in accumulation of autophagosomes-dependent apoptosis through activating Akt-impaired autophagic flux in neuronal cells. Our findings underscore that inhibition of Akt to improve autophagic flux is a promising strategy against Cd-induced neurotoxicity and neurodegeneration.

BAFF inhibits autophagy promoting cell proliferation and survival by activating Ca2+-CaMKII-dependent Akt/mTOR signaling pathway in normal and neoplastic B-lymphoid cells

B cell activating factor from the TNF family (BAFF) is implicated in not only the physiology of normal B cells, but also the pathophysiology of aggressive B cells related to malignant and autoimmune diseases. Autophagy plays a crucial role in balancing the beneficial and detrimental effects of immunity and inflammation. However, little is known about whether and how excessive BAFF mediates autophagy contributing to B-cell proliferation and survival. Here, we show that excessive human soluble BAFF (hsBAFF) inhibited autophagy with a concomitant reduction of LC3-II in normal and B-lymphoid (Raji) cells. Knockdown of LC3 not only potentiated hsBAFF inhibition of autophagy, but also attenuated hsBAFF activation of Akt/mTOR pathway, thereby diminishing hsBAFF-induced B-cell proliferation/viability. Further, we found that hsBAFF inhibition of autophagy was Akt/mTOR-dependent. This is supported by the findings that hsBAFF increased mTORC1-mediated phosphorylation of ULK1 (Ser757); Akt inhibitor X, mTORC1 inhibitor rapamycin, mTORC1/2 inhibitor PP242, expression of dominant negative Akt, or knockdown of mTOR attenuated hsBAFF-induced phosphorylation of ULK1, decrease of LC3-II level, and increase of cell proliferation/viability. Chelating intracellular free Ca2+ ([Ca2+]i) with BAPTA/AM or preventing [Ca2+]i elevation using EGTA or 2-APB profoundly blocked hsBAFF-induced activation of Akt/mTOR, phosphorylation of ULK1 and decrease of LC3-II, as well as increase of cell proliferation/viability. Similar effects were observed in the cells where CaMKII was inhibited by KN93 or knocked down by CaMKII shRNA. Collectively, these results indicate that hsBAFF inhibits autophagy promoting cell proliferation and survival through activating Ca2+-CaMKII-dependent Akt/mTOR signaling pathway in normal and neoplastic B-lymphoid cells. Our findings suggest that manipulation of intracellular Ca2+ level or CaMKII, Akt, or mTOR activity to promote autophagy may be exploited for prevention of excessive BAFF-induced aggressive B lymphocyte disorders and autoimmune diseases.

Celastrol Attenuates Cadmium-Induced Neuronal Apoptosis via Inhibiting Ca2+ -CaMKII-Dependent Akt/mTOR Pathway.

Cadmium (Cd), an environmental and industrial pollutant, affects the nervous system and consequential neurodegenerative disorders. Recently, we have shown that celastrol prevents Cd-induced neuronal cell death partially by suppressing Akt/mTOR pathway. However, the underlying mechanism remains to be elucidated. Here, we show that celastrol attenuated Cd-elevated intracellular-free calcium ([Ca2+ ]i ) level and apoptosis in neuronal cells. Celastrol prevented Cd-induced neuronal apoptosis by inhibiting Akt-mediated mTOR pathway, as inhibition of Akt with Akt inhibitor X or ectopic expression of dominant negative Akt reinforced celastrol's prevention of Cd-induced phosphorylation of S6K1/4E-BP1 and cell apoptosis. Furthermore, chelating intracellular Ca2+ with BAPTA/AM or preventing [Ca2+ ]i elevation using EGTA potentiated celastrol's repression of Cd-induced [Ca2+ ]i elevation and consequential activation of Akt/mTOR pathway and cell apoptosis. Moreover, celastrol blocked Cd-elicited phosphorylation of CaMKII, and pretreatment with BAPTA/AM or EGTA enhanced celastrol's suppression of Cd-increased phosphorylation of CaMKII in neuronal cells, implying that celastrol hinders [Ca2+ ]i -mediated CaMKII phosphorylation. Inhibiting CaMKII with KN93 or silencing CaMKII attenuated Cd activation of Akt/mTOR pathway and cell apoptosis, and this was strengthened by celastrol. Taken together, these data demonstrate that celastrol attenuates Cd-induced neuronal apoptosis via inhibiting Ca2+ -CaMKII-dependent Akt/mTOR pathway. Our findings underscore that celastrol may act as a neuroprotective agent for the prevention of Cd-induced neurodegenerative disorders. J. Cell. Physiol. 232: 2145-2157, 2017. © 2016 Wiley Periodicals, Inc.

Abstract 4616: A novel non-canonical Notch-AKT- NF-κB pathways in triple negative breast cancer cells

Abstract Triple negative breast cancer (TNBC) is a heterogeneous group of clinically aggressive diseases that account for approximately 15-20% of invasive breast cancers. TNBC patients have high risk of recurrence and metastasis, and current treatment options remain limited. There is strong evidence for the involvement of Notch signaling in TNBC. Notch1 is highly expressed in Basal-like 1 (BL1) and especially Mesenchymal-Stem-Like (MSL) TNBCs. Expression of Notch1 protein correlates with pAKT and nuclear NF-κB in TNBC specimens. Expression of Notch1 and its ligand Jagged1 correlate with poor prognosis. NF-κB also plays key roles in promoting cancer cell survival in TNBC. Notch-NF-κB cross-talk through several mechanisms has been demonstrated in T-cell acute leukemia, cervical cancer and Luminal A breast cancer cells, but not TNBC. Here, we demonstrate that Notch1 promotes cell survival in MDA-MB-231 cells, representative of MSL TNBC, in part by activating NF-κB. Notch activation by Jagged1-expressing stromal cells enhances transcription of the anti-apoptotic gene cIAP-2 (BIRC3), a known NF-κB target. This event is dependent on recruitment to the cIAP-2 promoter of NF-κB subunits, IKKα and Notch1. However, Notch1 is recruited to the chromatin later than NF-κB subunits and IKKα, suggesting that early, non-nuclear events contribute to this process. NF-κB activation correlates with AKT activation as revealed by S473 phosphorylation. Pharmacological inhibition of AKT or expression of dominant-negative AKT decreases NF-κB activity. Inhibition of Notch activity through GSIs (γ-secretase inhibitors) leads to decreased AKT phosphorylation. Our results indicate that AKT activation is independent of canonical nuclear Notch1 partner CBF-1. Short term exposure of MDA-MB-231 cells (MSL, PTEN wild-type), but not MDA-MB-468 cells (BL1, PTEN-null) to recombinant Jagged1 leads to AKT phosphorylation within 1 hour. This is suppressed by dual mTORC1/2 inhibitors, PI3K inhibitors and IKKα inhibitors but not Everolimus (mTORC1-selective inhibitor). Pharmacological inhibition of AKT, or mTORC1/2 or IKKα decreases AKT phosphorylation and cell proliferation. These observations support a model where canonical and non-canonical mechanisms downstream of Notch1 trigger AKT phosphorylation and NF-κB activation in PTEN wild type TNBC cells. Rapid AKT phosphorylation downstream of Notch1 requires mTORC2, PI3K and IKKα, and contributes to NF-κB activation. Notch1 has been shown to activate mTORC2 in T-cells, while IKKα has been reported to activate mTORC2 in other models. Our data indicate that this pathway may be active in some TNBC subtypes as well. Our data indicate that therapeutic regimens targeting this pathway deserve further investigation in PTEN-wild type but not PTEN-mutant TNBCs. Citation Format: Fokhrul Hossain, Yin Peng, Antonio Pannuti, Kandis Backus, Todd Eliot Golde, Barbara Osborne, Lucio Miele. A novel non-canonical Notch-AKT- NF-κB pathways in triple negative breast cancer cells. [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 4616.

MTOR Complexes Repress Hypertrophic Agonist-Stimulated Expression of Connective Tissue Growth Factor in Adult Cardiac Muscle Cells.

Connective tissue growth factor (CTGF) is a fibrogenic cytokine that promotes fibrosis in various organs. In the heart, both cardiomyocytes (CM) and cardiac fibroblasts have been reported as a source of CTGF expression, aiding cardiac fibrosis. Although the mammalian target of rapamycin (mTOR) forms 2 distinct complexes, mTORC1 and mTORC2, and plays a central role in integrating biochemical signals for protein synthesis and cellular homeostasis, we explored its role in CTGF expression in adult feline CM. CM were stimulated with 10 μM phenylephrine (PE), 200 nM angiotensin (Ang), or 100 nM insulin for 24 hours. PE and Ang, but not insulin, caused an increase in CTGF mRNA expression with the highest expression observed with PE. Inhibition of mTOR with torin1 but not rapamycin significantly enhanced PE-stimulated CTGF expression. Furthermore, silencing of raptor and rictor using shRNA adenoviral vectors to suppress mTORC1 and mTORC2, respectively, or blocking phosphatidylinositol 3-kinase (PI3K) signaling with LY294002 (LY) or Akt signaling by dominant-negative Akt expression caused a substantial increase in PE-stimulated CTGF expression as measured by both mRNA and secreted protein levels. However, studies with dominant-negative delta isoform of protein kinase C demonstrate that delta isoform of protein kinase C is required for both agonist-induced CTGF expression and mTORC2/Akt-mediated CTGF suppression. Finally, PE-stimulated CTGF expression was accompanied with a corresponding increase in Smad3 phosphorylation and pretreatment of cells with SIS3, a Smad3 specific inhibitor, partially blocked the PE-stimulated CTGF expression. Therefore, a PI3K/mTOR/Akt axis plays a suppressive role on agonist-stimulated CTGF expression where the loss of this mechanism could be a contributing factor for the onset of cardiac fibrosis in the hypertrophying myocardium.

Survivin expression promotes VEGF-induced tumor angiogenesis via PI3K/Akt enhanced β-catenin/Tcf-Lef dependent transcription.

Early in cancer development, tumour cells express vascular endothelial growth factor (VEGF), a secreted molecule that is important in all stages of angiogenesis, an essential process that provides nutrients and oxygen to the nascent tumor and thereby enhances tumor-cell survival and facilitates growth. Survivin, another protein involved in angiogenesis, is strongly expressed in most human cancers, where it promotes tumor survival by reducing apoptosis as well as favoring endothelial cell proliferation and migration. The mechanisms by which cancer cells induce VEGF expression and angiogenesis upon survivin up-regulation remain to be fully established. Since the PI3K/Akt signalling and β-catenin-Tcf/Lef dependent transcription have been implicated in the expression of many cancer-related genes, including survivin and VEGF, we evaluated whether survivin may favor VEGF expression, release from tumor cells and induction of angiogenesis in a PI3K/Akt-β-catenin-Tcf/Lef-dependent manner. Here, we provide evidence linking survivin expression in tumor cells to increased β-catenin protein levels, β-catenin-Tcf/Lef transcriptional activity and expression of several target genes of this pathway, including survivin and VEGF, which accumulates in the culture medium. Alternatively, survivin downregulation reduced β-catenin protein levels and β-catenin-Tcf/Lef transcriptional activity. Also, using inhibitors of PI3K and the expression of dominant negative Akt, we show that survivin acts upstream in an amplification loop to promote VEGF expression. Moreover, survivin knock-down in B16F10 murine melanoma cells diminished the number of blood vessels and reduced VEGF expression in tumors formed in C57BL/6 mice. Finally, in the chick chorioallantoid membrane assay, survivin expression in tumor cells enhanced VEGF liberation and blood vessel formation. Importantly, the presence of neutralizing anti-VEGF antibodies precluded survivin-enhanced angiogenesis in this assay. These findings provide evidence for the existance of a posititve feedback loop connecting survivin expression in tumor cells to PI3K/Akt enhanced β-catenin-Tcf/Lef-dependent transcription followed by secretion of VEGF and angiogenesis.Electronic supplementary materialThe online version of this article (doi:10.1186/1476-4598-13-209) contains supplementary material, which is available to authorized users.

Cyclic stretch increases the expression of fibronectin through cPLA2, STAT3, and Akt phosphorylation in renal tubular epithelial cells (690.18)

Extracellular matrix accumulation contributes to the progression of chronic renal disease. Recent in vivo data indicate that unilateral ureteral obstruction (UUO), a model for chronic obstructive nephropathy, induces profibrotic renal tubular cell injury via STAT3 activation. A characterization of obstructive nephropathy and UUO is an increase in intrarenal pressure resulting in tubular mechanical stretch. However, in the kidney, the influence of mechanical strain on renal tubular epithelial (RTE) cells remains unclear. It is possible that tubular mechanical strain may attribute to increased STAT3 activation leading to accumulation of the extracellular matrix protein, fibronectin, in RTE cells. Human proximal tubular HK‐2 cells were either or not subjected to cyclic mechanical stretch on a uniform biaxial strain device. Fibronectin expression increased significantly in response to cyclic stretch, which was attenuated by inhibition of cPLA2, but not sPLA2 or iPLA2. In addition, treatment of cells with arachidonic acid resulted in an increased expression of fibronectin. Cyclic stretch also stimulated phosphorylation of STAT3 and Akt, and inhibition of STAT3, Akt, or its upstream activator phosphatidyl inositol 3‐kinase (PI3‐K), attenuated stretch‐induced fibronectin accumulation. Expression of dominant negative Akt or transfection of small interfering RNA targeting STAT3 significantly blocked stretch‐induced increase in fibronectin, indicating a role for these kinases in fibronectin accumulation. Taken together, these results provide the first evidence for a role of cPLA2, STAT3, and Akt in stretch‐induced fibronectin expression in RTE cells.

Abstract 1173: AKT-mTOR pathway mediates mutant p53 gain-of-function by inhibiting autophagy

Abstract Nearly half of human cancers harbor p53 mutations, which are predominantly through missense mutations that result in accumulation of mutant p53 proteins in cancer cells. Persistent evidence supports that the mutant p53 proteins not only lose tumor suppressor activity, but also acquire gain-of-function abilities to promote carcinogenesis, metastasis, tumor recurrence and chemoresistance. Indeed, knock-in mouse models bearing the mutant p53R172H (equivalent to human R175H mutation) at physiological level in vivo display gain-of-function phenotypes, such as enhanced cell transformation potentials and tumor metastasis. However, the molecular mechanism of mutated p53R172H conferring gain-of-function remains poorly understood. In this study, utilizing mouse embryonic fibroblasts (MEFs) and lung adenocarcinoma cells (93-1) derived from p53R172H knock-in mice, we observed that AKT was preferentially activated in p53R172H mutant cells over p53-deficient cells. This activation of AKT allows p53R172H mutant cells to bypass contact inhibition, grow in an anchorage-independent manner and form tumors after subcutaneous injection into SCID mice. In contrast, expression of dominant negative AKT or shRNA-mediated AKT knockdown in p53R172H mutant cells inhibited colony formation in soft-agar and suppressed tumor growth in mouse xenograft tumor model. To directly dissect the essential role of AKT in p53R172H mutant-initiated tumorigenesis in vivo, we bred AKT1 knockout mice to p53R172H mutant knock-in mice. Loss of AKT1 significantly reduced the incidence of tumor development and extended the life span from around 3 months in AKT1-proficient and p53R172H mutant mice (AKT1+/+; p53R172H/R172H) to about 12 months in AKT1-deficient and p53R172H mutant mice (AKT1−/−; p53R172H/R172H). To understand how the activated AKT conferred mutant p53 gain-of-function, we demonstrated that p53R172H mutant activated AKT-mTOR signaling pathway that led to an inhibition of autophagy both in vitro and in vivo. Furthermore, induction of autophagy in p53R172H mutant lung cancer cells through suppressing AKT-mTOR pathway pharmacologically by the dual kinase inhibitor NVP-BEZ235 treatment or genetically by RNAi-mediated knockdown of mTOR dramatically inhibited p53R172H mutant-induced colony formation in soft agar and tumor growth in SCID mice. Our results support that AKT plays a crucial role in mediating mutant p53 gain-of-function phenotypes in vivo through the inhibition of autophagy by the activated AKT-mTOR signaling pathway. These findings provide the proof-of-principle that targeting the AKT-mTOR-autophagy pathway might have promising therapeutic benefits for cancer patients carrying such p53 mutations. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1173. doi:1538-7445.AM2012-1173

IGF-I increases the expression of fibronectin by Nox4-dependent Akt phosphorylation in renal tubular epithelial cells

Extracellular matrix accumulation contributes to the progression of chronic kidney disease. Many growth factors including insulin-like growth factor-I (IGF-I) enhance matrix protein accumulation. Proximal tubular epithelial cells (PTCs) synthesize matrix proteins. NADPH oxidases are major sources of reactive oxygen species (ROS), important signaling molecules that mediate biological responses in a variety of cells and tissue. We investigated the mechanism by which IGF-I regulates fibronectin accumulation in PTCs and the role of a potential redox-dependent signaling pathway. IGF-I induces an increase in NADPH-dependent superoxide generation, enhances the release of hydrogen peroxide, and increases the expression of NADPH oxidase 4 (Nox4) in PTCs. IGF-I also stimulates phosphorylation of Akt, and inhibition of Akt or its upstream activator phosphatidylinositol 3-kinase attenuates IGF-I-induced fibronectin accumulation. Expression of dominant negative Akt also inhibits IGF-I-induced expression of fibronectin, indicating a role for this kinase in fibronectin accumulation. Expression of dominant negative adenovirus Nox4 inhibits IGF-I-induced NADPH oxidase activity, Akt phosphorylation, and fibronectin protein expression. Moreover, transfection of small interfering RNA targeting Nox4 decreases Nox4 protein expression and blocks IGF-I-induced Akt phosphorylation and the increase in fibronectin, placing Nox4 and ROS upstream of Akt signaling pathway. To confirm the role of Nox4, PTCs were infected with adenovirus construct expressing wild-type Nox4. Ad-Nox4, but not control Ad-green fluorescent protein, upregulated Nox4 expression and increased NADPH oxidase activity as well as fibronectin expression. Taken together, these results provide the first evidence for a role of Nox4 in IGF-I-induced Akt phosphorylation and fibronectin expression in tubular epithelial cells.

An APPL1/Akt signaling complex regulates dendritic spine and synapse formation in hippocampal neurons

The formation and plasticity of dendritic spines and synapses, which are poorly understood on a molecular level, are critical for cognitive functions, such as learning and memory. The adaptor protein containing a PH domain, PTB domain, and leucine zipper motif (APPL1) is emerging as a critical regulator of various cellular processes in non-neuronal cells, but its function in the nervous system is not well understood. Here, we show that APPL1 localizes to dendritic spines and synapses and regulates the development of these structures in hippocampal neurons. Knockdown of endogenous APPL1 using siRNA led to a significant decrease in the number of spines as well as synapses and this defect could be rescued by expression of siRNA-resistant APPL1. Expression of exogenous APPL1 increased the spine and synaptic density and the amount of surface GluR1-containing α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs). Deletion of the C-terminal phosphotyrosine binding domain of APPL1, which binds the serine/threonine kinase Akt, resulted in a significant decrease in the spine and synaptic density, suggesting a role for Akt in regulating the development of these structures. Consistent with this, knockdown of Akt with siRNA or expression of dominant negative Akt led to a dramatic decrease in spine and synapse formation. In addition, APPL1 increased the amount of active Akt in spines and synapses and the effects of APPL1 on these structures were dependent on Akt, indicating that Akt is an effector of APPL1 in the regulation of these processes. Moreover, APPL1 signaling modulates spine and synapse formation through p21-activated kinase (PAK). Thus, our results indicate that APPL1 signaling through Akt and PAK is critical for spine and synaptic development and point to a role for APPL1 and its effectors in regulating cognitive function.

Akt promotes chemoresistance in human ovarian cancer cells by modulating cisplatin-induced, p53-dependent ubiquitination of FLICE-like inhibitory protein

Although Akt is a determinant of cisplatin (cis-diaminedichloroplatinum (CDDP)) resistance in ovarian cancer cells, which is related in part to its inhibitory action on p53 activation, precisely how Akt confers CDDP resistance is unclear. In this study, we show that CDDP induced p53-dependent Fas-associated death domain-like interleukin-1beta-converting enzyme (FLICE)-like inhibitory protein (FLIP) degradation in chemosensitive ovarian cancer cells but not their resistant counterparts. CDDP induced FLIP-p53-Itch interaction, colocalization and FLIP ubiquitination in chemosensitive but not chemoresistant ovarian cancer cells. Moreover, although activated Akt inhibited CDDP-induced FLIP degradation and apoptosis in sensitive cells, these responses were facilitated by dominant-negative Akt expression in chemoresistant cells. Inhibition of Akt function also facilitated p53-FLIP interaction and FLIP ubiquitination, which were attenuated by p53 silencing. These results suggest that Akt confers resistance, in part, by modulating CDDP-induced, p53-dependent FLIP ubiquitination. Understanding the precise etiology of chemoresistance may improve treatment for ovarian cancer.

Myostatin inhibits IGF-I-induced myotube hypertrophy through Akt

Myostatin is a highly conserved negative regulator of skeletal muscle growth. Loss of functional myostatin in cattle, mice, sheep, dogs, and humans results in increased muscle mass. The molecular mechanisms responsible for this increase in muscle growth are not fully understood. Previously, we have reported that phenylephrine-induced cardiac muscle growth and Akt activation are enhanced in myostatin knockout mice compared with controls. Here we report that skeletal muscle from myostatin knockout mice show increased Akt protein expression and overall activity at baseline secondary to an increase in Akt mRNA. We examined the functional role of myostatin modulation of Akt in C2C12 myotubes, a well-established in vitro model of skeletal muscle hypertrophy. Adenoviral overexpression of myostatin attenuated the insulin-like growth factor-I (IGF-I)-mediated increase in myotube diameter, as well as IGF-I-stimulated Akt phosphorylation. Inhibition of myostatin by overexpression of the NH(2)-terminal portion of myostatin was sufficient to increase myotube diameter and Akt phosphorylation. Coexpression of myostatin and constitutively active Akt (myr-Akt) restored the increase in myotube diameter. Conversely, expression of dominant negative Akt (dn-Akt) with the inhibitory myostatin propeptide blocked the increase in myotube diameter. Of note, ribosomal protein S6 phosphorylation and atrogin-1/muscle atrophy F box mRNA were increased in skeletal muscle from myostain knockout mice. Together, these data suggest myostatin regulates muscle growth at least in part through regulation of Akt.

Identification of 4-(2-(4-Amino-1,2,5-oxadiazol-3-yl)-1-ethyl-7-{[(3S)-3-piperidinylmethyl]oxy}-1H-imidazo[4,5-c]pyridin-4-yl)-2-methyl-3-butyn-2-ol (GSK690693), a Novel Inhibitor of AKT Kinase

Overexpression of AKT has an antiapoptotic effect in many cell types, and expression of dominant negative AKT blocks the ability of a variety of growth factors to promote survival. Therefore, inhibitors of AKT kinase activity might be useful as monotherapy for the treatment of tumors with activated AKT. Herein, we describe our lead optimization studies culminating in the discovery of compound 3g (GSK690693). Compound 3g is a novel ATP competitive, pan-AKT kinase inhibitor with IC 50 values of 2, 13, and 9 nM against AKT1, 2, and 3, respectively. An X-ray cocrystal structure was solved with 3g and the kinase domain of AKT2, confirming that 3g bound in the ATP binding pocket. Compound 3g potently inhibits intracellular AKT activity as measured by the inhibition of the phosphorylation levels of GSK3beta. Intraperitoneal administration of 3g in immunocompromised mice results in the inhibition of GSK3beta phosphorylation and tumor growth in human breast carcinoma (BT474) xenografts.

Myosin 5a Is an Insulin-Stimulated Akt2 (Protein Kinase Bβ) Substrate Modulating GLUT4 Vesicle Translocation

Phosphatidylinositol 3-kinase activation of Akt signaling is critical to insulin-stimulated glucose transport and GLUT4 translocation. However, the downstream signaling events following Akt activation which mediate glucose transport stimulation remain relatively unknown. Here we identify an Akt consensus phosphorylation motif in the actin-based motor protein myosin 5a and show that insulin stimulation leads to phosphorylation of myosin 5a at serine 1650. This Akt-mediated phosphorylation event enhances the ability of myosin 5a to interact with the actin cytoskeleton. Small interfering RNA-induced inhibition of myosin 5a and expression of dominant-negative myosin 5a attenuate insulin-stimulated glucose transport and GLUT4 translocation. Furthermore, knockdown of Akt2 or expression of dominant-negative Akt (DN-Akt) abolished insulin-stimulated phosphorylation of myosin 5a, inhibited myosin 5a binding to actin, and blocked insulin-stimulated glucose transport. Taken together, these data indicate that myosin 5a is a newly identified direct substrate of Akt2 and, upon insulin stimulation, phosphorylated myosin 5a facilitates anterograde movement of GLUT4 vesicles along actin to the cell surface.

Conjugated bile acids regulate hepatocyte glycogen synthase activity in vitro and in vivo via Galphai signaling.

The regulation of glycogen synthase activity by bile acids in primary hepatocytes and in the intact liver was investigated. Bile acids (deoxycholic acid, DCA; taurocholic acid, TCA) activated AKT and glycogen synthase (GS) in primary rat hepatocytes. Incubation with a phosphatidyl inositol-3 kinase inhibitor or expression of dominant-negative AKT in primary rat hepatocytes abolished activation of AKT and GS by DCA and TCA. TCA, but not DCA, activated Galpha(i) proteins in primary rat hepatocytes. Treatment of cells with pertussis toxin or expression of dominant-negative Galpha(i) blocked TCA-induced activation of AKT and of GS but did not alter AKT or GS activation caused by DCA. TCA caused activation of AKT and GS in intact rat liver. Expression of dominant-negative Galpha(i) reduced TCA-induced activation of AKT and of GS in intact rat liver. Together, our findings demonstrate that bile acids are physiological regulators of glycogen synthase in rat liver and that conjugated bile acids use a Galpha(i)-coupled G protein-coupled receptor to regulate GS activity in vitro and in vivo.

Downregulation of catalase by reactive oxygen species via PI 3 kinase/Akt signaling in mesangial cells

Reactive oxygen species (ROS) contribute to many glomerular diseases by targeting mesangial cells. ROS have been shown to regulate expression of many antioxidant enzymes including catalase. The mechanism by which the expression of catalase protein is regulated by ROS is not precisely known. Here we report that increased intracellular ROS level by hydrogen peroxide (H(2)O(2)) reduced the expression of catalase. H(2)O(2) increased phosphorylation of Akt kinase in a dose-dependent and sustained manner with a concomitant increase in the phosphorylation of FoxO1 transcription factor. Further analysis revealed that H(2)O(2) promoted rapid activation of phosphatidylinositol (PI) 3 kinase. The PI 3 kinase inhibitor Ly294002 and expression of tumor suppressor protein PTEN inhibited Akt kinase activity, resulting in the attenuation of FoxO1 phosphorylation and preventing the downregulating effect of H(2)O(2) on catalase protein level. Dominant negative Akt attenuated the inhibitory effect of H(2)O(2) on expression of catalase. Constitutively active FoxO1 increased the expression of catalase. However, dominant negative FoxO1 inhibited catalase protein level. Catalase transcription was reduced by H(2)O(2) treatment. Furthermore, expression of dominant negative Akt and constitutively active FoxO1 increased catalase transcription, respectively. These results demonstrate that ROS downregulate the expression of catalase in mesangial cells by PI 3 kinase/Akt signaling via FoxO1 as a target.

Human Chorionic Gonadotropin Modulates Prostate Cancer Cell Survival after Irradiation or HMG CoA Reductase Inhibitor Treatment

The impact of human chorionic gonadotropin (hCG) on prostate carcinoma viability was investigated. Treatment of LNCaP and PC-3 cells with hCG modestly reduced cell viability within 96 h. Treatment of cells with hCG followed by exposure to ionizing radiation enhanced radiosensitivity. Exposure of LNCaP cells to hCG promoted activation of epidermal growth factor receptor (ERBB1) via a Galpha(i)-, mitogen-activated protein kinase kinase (MEK)1/2-, and metalloprotease-dependent paracrine mechanism, effects that were further enhanced after radiation exposure, and that were causal in prolonged intense activation of poly(ADP-ribose) polymerase (PARP). Inhibition of ERBB1, MEK1, or PARP1 function suppressed the radiosensitizing properties of hCG. Radiosensitization was also, in part, dependent upon c-Jun NH2-terminal kinase 1/2 signaling. PARP1-dependent radiosensitization was suppressed by a pan-caspase inhibitor and by knockdown of apoptosis-inducing factor expression. Inhibition of phosphatidylinositol 3-kinase, expression of dominant-negative AKT, or treatment with the HMG CoA reductase inhibitor lovastatin suppressed AKT phosphorylation and enhanced the cytotoxic effects of hCG. The enhancing effect of lovastatin was reproduced by incubation with a geranylgeranyl transferase inhibitor and blocked by coexposure to geranylgeranyl pyrophosphate. Treatment with hCG and lovastatin decreased expression of BCL-(XL) and XIAP, and increased expression of IkappaB. The cytotoxic effects of hCG were enhanced by expression of dominant-negative IkappaB, and they were abolished by coexpression of activated AKT. Expression of activated AKT maintained BCL-(XL) levels in cells expressing dominant-negative IkappaB. The promotion of hCG lethality by lovastatin was abolished by overexpression of BCL-(XL), and was dependent upon activation of caspase-9. Thus, hCG, in combination with radiation and lovastatin, may represent a novel approach to kill prostate cancer cells.