Ligand-bound Thyroid Hormone Receptor Contributes to Reprogramming of Pancreatic Acinar Cells into Insulin-producing Cells

One goal of diabetic regenerative medicine is to instructively convert mature pancreatic exocrine cells into insulin-producing cells. We recently reported that ligand-bound thyroid hormone receptor α (TRα) plays a critical role in expansion of the β-cell mass during postnatal development. Here, we used an adenovirus vector that expresses TRα driven by the amylase 2 promoter (AdAmy2TRα) to induce the reprogramming of pancreatic acinar cells into insulin-producing cells. Treatment with l-3,5,3-triiodothyronine increases the association of TRα with the p85α subunit of phosphatidylinositol 3-kinase (PI3K), leading to the phosphorylation and activation of Akt and the expression of Pdx1, Ngn3, and MafA in purified acinar cells. Analyses performed with the lectin-associated cell lineage tracing system and the Cre/loxP-based direct cell lineage tracing system indicate that newly synthesized insulin-producing cells originate from elastase-expressing pancreatic acinar cells. Insulin-containing secretory granules were identified in these cells by electron microscopy. The inhibition of p85α expression by siRNA or the inhibition of PI3K by {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 prevents the expression of Pdx1, Ngn3, and MafA and the reprogramming to insulin-producing cells. In immunodeficient mice with streptozotocin-induced hyperglycemia, treatment with AdAmy2TRα leads to the reprogramming of pancreatic acinar cells to insulin-producing cells in vivo. Our findings suggest that ligand-bound TRα plays a critical role in β-cell regeneration during postnatal development via activation of PI3K signaling.     

Effects of Isoform-selective Phosphatidylinositol 3-Kinase Inhibitors on Osteoclasts: ACTIONS ON CYTOSKELETAL ORGANIZATION,SURVIVAL, AND RESORPTION*

Phosphatidylinositol 3-kinases (PI3K) participate in numerous signaling pathways, and control distinct biological functions. Studies using pan-PI3K inhibitors suggest roles for PI3K in osteoclasts, but little is known about specific PI3K isoforms in these cells. Our objective was to determine effects of isoform-selective PI3K inhibitors on osteoclasts. The following inhibitors were investigated (targets in parentheses): wortmannin and {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 (pan-p110), PIK75 (α), GDC0941 (α, δ), TGX221 (β), AS252424 (γ), and IC87114 (δ). In addition, we characterized a new potent and selective PI3Kδ inhibitor, GS-9820, and explored roles of PI3K isoforms in regulating osteoclast function. Osteoclasts were isolated from long bones of neonatal rats and rabbits. Wortmannin, {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, GDC0941, IC87114, and GS-9820 induced a dramatic retraction of osteoclasts within 15–20 min to 65–75% of the initial area. In contrast, there was no significant retraction in response to vehicle, PIK75, TGX221, or AS252424. Moreover, wortmannin and GS-9820, but not PIK75 or TGX221, disrupted actin belts. We examined effects of PI3K inhibitors on osteoclast survival. Whereas PIK75, TGX221, and GS-9820 had no significant effect on basal survival, all blocked RANKL-stimulated survival. When studied on resorbable substrates, osteoclastic resorption was suppressed by wortmannin and inhibitors of PI3Kβ and PI3Kδ, but not other isoforms. These data are consistent with a critical role for PI3Kδ in regulating osteoclast cytoskeleton and resorptive activity. In contrast, multiple PI3K isoforms contribute to the control of osteoclast survival. Thus, the PI3Kδ isoform, which is predominantly expressed in cells of hematopoietic origin, is an attractive target for anti-resorptive therapeutics.     

Phosphatidylinositol 3-Kinase and Rab5 GTPase Inversely Regulate the Smad Anchor for Receptor Activation (SARA) Protein Independently of Transforming Growth Factor-β1

SARA has been shown to be a regulator of epithelial cell phenotype, with reduced expression during TGF-β1-mediated epithelial-to-mesenchymal transition. Examination of the pathways that might play a role in regulating SARA expression identified phosphatidylinositol 3-kinase (PI3K) pathway inhibition as sufficient to reduce SARA expression. The mechanism of PI3K inhibition-mediated SARA down-regulation differs from that induced by TGF-β1 in that, unlike TGF-β1, PI3K-dependent depletion of SARA was apparent within 6 h and did not occur at the mRNA or promoter level but was blocked by inhibition of proteasome-mediated degradation. This effect was independent of Akt activity because neither reducing nor enhancing Akt activity modulated the expression of SARA. Therefore, this is likely a direct effect of p85α action, and co-immunoprecipitation of SARA and p85α confirmed that these proteins interact. Both SARA and PI3K have been shown to be associated with endosomes, and either {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 or p85α knockdown enlarged SARA-containing endocytic vesicles. Inhibition of clathrin-mediated endocytosis blocked SARA down-regulation, and a localization-deficient mutant SARA was protected against down-regulation. As inhibiting PI3K can activate the endosomal fusion-regulatory small GTPase Rab5, we expressed GTPase-deficient Rab5 and observed endosomal enlargement and reduced SARA protein expression, similar to that seen with PI3K inhibition. Importantly, either interference with PI3K via {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 or p85α knockdown, or constitutive activity of the Rab5 pathway, enhanced the expression of smooth muscle α-actin. Together, these data suggest that although TGF-β1 can induce epithelial-to-mesenchymal transition through reduction in SARA expression, SARA is also basally regulated by its interaction with PI3K.     

CpG-ODN Attenuates Pathological Cardiac Hypertrophy and Heart Failure by Activation of PI3Kα-Akt Signaling

Phosphoinositide-3-kinase α (PI3Kα) represents a potential novel drug target for pathological cardiac hypertrophy (PCH) and heart failure. Oligodeoxynucleotides containing CpG motifs (CpG-ODN) are classic agonists of Toll-like receptor 9 (TLR9), which typically activates PI3K-Akt signaling in immune cells; however, the role of the nucleotide TLR9 agonists in cardiac myocytes is largely unknown. Here we report that CpG-ODN C274 could both attenuate PCH and improve cardiac dysfunction by activating PI3Kα-Akt signaling cascade. In vitro studies indicated that C274 could blunt reactivation of fetal cardiac genes and cell enlargement induced by a hypertrophic agent, isoproterenol. The anti-hypertrophic effect of C274 was suppressed by a pan-PI3K inhibitor, {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, or a small interfering RNA targeting PI3Kα. In vivo studies demonstrated that PCH, as marked by increased heart weight (HW) and cardiac ANF mRNA, was normalized by pre-administration with C274. In addition, Doppler echocardiography detected cardiac ventricular dilation, and contractile dysfunction in isoproterenol-treated animals, consistent with massive replacement fibrosis, reflecting cardiac cell death. As expected, pre-treatment of mice with C274 could prevent cardiac dysfunction associated with diminished cardiac cell death and fibrosis. In conclusion, CpG-ODNs are novel cardioprotective agents possessing antihypertrophic and anti-cell death activity afforded by engagement of the PI3Kα-Akt signaling. CpG-ODNs may have clinical use curbing the progression of PCH and preventing heart failure.     

mTORC1 Regulates Flagellin-Induced Inflammatory Response in Macrophages

Bacterial flagellin triggers inflammatory responses. Phosphoinositide 3-kinase (PI3K) and mammalian target of rapamycin (mTOR) regulate the production of pro- and anti-inflammatory cytokines that are induced by extrinsic antigens, but the function of mTORC1 in flagellin-induced inflammatory response is unknown. The purpose of this study was to examine the role and the mechanism of PI3K/Akt/mTOR pathway in flagellin-induced cytokine expression in mouse macrophages. We observed that flagellin upregulated TNF-α time- and dose-dependently. Flagellin stimulated rapid (<15 min) PI3K/Akt/mTOR phosphorylation that was mediated by TLR5. Inhibition of PI3K with {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 and wortmannin, and of mTORC1 with rapamycin decreased flagellin-induced TNF-α and IL-6 expression and cell proliferation. The activation of NF-κB p65 and STAT3 was regulated by mTORC1 via degradation of IκBα and phosphorylation of STAT3 in response to flagellin, respectively. Thus, the PI3K/Akt/mTORC1 pathway regulates the innate immune response to bacterial flagellin. Rapamycin is potential therapy that can regulate host defense against pathogenic infections.     

Over-expression of microRNA-494 up-regulates hypoxia-inducible factor-1 alpha expression via PI3K/Akt pathway and protects against hypoxia-induced apoptosis

Background

Hypoxia-inducible factor-1 alpha (HIF-1α) is one of the key regulators of hypoxia/ischemia. MicroRNA-494 (miR-494) had cardioprotective effects against ischemia/reperfusion (I/R)-induced injury, but its functional relationship with HIF-1α was unknown. This study was undertaken to determine if miR-494 was involved in the induction of HIF-1α.

Results

Quantitative RT-PCR showed that miR-494 was up-regulated to peak after 4 hours of hypoxia in human liver cell line L02. To investigate the role of miR-494, cells were transfected with miR-494 mimic or miR-negative control, followed by incubation under normoxia or hypoxia. Our results indicated that overexpression of miR-494 significantly induced the expression of p-Akt, HIF-1α and HO-1 determined by qRT-PCR and western blot under normoxia and hypoxia, compared to negative control (p < 0.05). While {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 treatment markedly abolished miR-494-inducing Akt activation, HIF-1α and HO-1 increase under both normoxic and hypoxic conditions (p < 0.05). Moreover, apoptosis detection using Annexin V indicated that overexpression of miR-494 significantly decreased hypoxia-induced apoptosis in L02 cells, compared to control (p < 0.05). MiR-494 overexpression also decreased caspase-3/7 activity by 1.27-fold under hypoxia in L02 cells.

Conclusions

Overexpression of miR-494 upregulated HIF-1α expression through activating PI3K/Akt pathway under both normoxia and hypoxia, and had protective effects against hypoxia-induced apoptosis in L02 cells. Thus, these findings suggested that miR-494 might be a target of therapy for hepatic hypoxia/ischemia injury.     

Inhibition of the phosphoinositide 3-kinase pathway decreases innate resistance to lipopolysaccharide toxicity in TLR4 deficient mice

Background

Upon lipopolysaccharide (LPS) stimulation, activation of both the Toll-like receptor 4 (TLR4) and phosphoinositide 3-kinase (PI3K) pathways serves to balance proinflammatory and anti-inflammatory responses. Although the antagonist to TLR4 represents an emerging promising target for the treatment of sepsis; however, the role of the PI3K pathway under TLR4-null conditions is not well understood. This goal of this study was to investigate the effect of inhibition of PI3K on innate resistance to LPS toxicity in a murine model.

Results

The overall survival of the cohorts receiving intraperitoneal injections of 100, 500, or 1000 μg LPS from Escherichia coli serotype 026:B6 after 7 d was 100%, 10%, and 10%, respectively. In contrast, no mortality was noted after 500-μg LPS injection in Tlr4^-/- mice. When the PI3K inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 was injected (1 mg/25 g body weight) 1 h prior to the administration of LPS, the overall survival of the Tlr4^-/- mice was 30%. In the Tlr4^-/- mice, the LPS injection induced no NF-κB activation but an increased Akt phosphorylation in the lung and liver, when compared to that of the C57BL/6 mice. Injection of 500 μg LPS led to a significant induction in O₂^- detected by electron paramagnetic resonance (EPR) spin trapping spectroscopy in the lung and liver at 3 and 6 h in C57BL/6 but not Tlr4^-/- mice. Addition of {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 only significantly increased the O₂^- level in the lung and liver of the Tlr4^-/- mice but not in the C57BL/6 mice following 500-μg LPS injection. In addition, the serum IL-1β and IL-2 levels were more elevated in C57BL/6 mice than in Tlr4^-/- mice. Notably, IL-1β and IL-2 were significantly increased in Tlr4^-/- mice but not in the C57BL/6 mice when the PI3K pathway was inhibited by {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 prior to LPS injection.

Conclusions

In this study, we demonstrate that innate resistance to LPS toxicity in Tlr4^-/- mice is impaired by inhibition of the PI3K pathway, with a corresponding increase in mortality and production of tissue O₂^- and inflammatory cytokines.     

The anti-apoptotic function of human ��A-crystallin is directly related to its chaperone activity

αA-crystallin is a molecular chaperone and an antiapoptotic protein. This study investigated the mechanism of inhibition of apoptosis by human αA-crystallin and determined if the chaperone activity of αA-crystallin is required for the antiapoptotic function. αA-crystallin inhibited chemical-induced apoptosis in Chinese hamster ovary (CHO) cells and HeLa cells by inhibiting activation of caspase-3 and -9. In CHO cells, it inhibited apoptosis induced by the overexpression of human proapoptotic proteins, Bim and Bax. αA-crystallin inhibited doxorubicin-mediated activation of human procaspase-3 in CHO cells and it activated the PI3K/Akt cell survival pathway by promoting the phosphorylation of PDK1, Akt and phosphatase tensin homologue in HeLa cells. The phosphoinositide 3 kinase (PI3K) activity was increased by αA-crystallin overexpression but the protein content was unaltered. Downregulation of PI3K by the expression of a dominant-negative mutant or inhibition by {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 abrogated the ability of αA-crystallin to phosphorylate Akt. These antiapoptotic functions of αA-crystallin were enhanced in a mutant protein (R21A) that shows increased chaperone activity than the wild-type (Wt) protein. Interestingly, a mutant protein (R49A) that shows decreased chaperone activity was far weaker than the Wt protein in its antiapoptotic functions. Together, our study results show that αA-crystallin inhibits apoptosis by enhancing PI3K activity and inactivating phosphatase tensin homologue and that the antiapoptotic function is directly related to its chaperone activity.     

Heat shock protein 90β inhibits apoptosis of intestinal epithelial cells induced by hypoxia through stabilizing phosphorylated Akt

Intestinal epithelial cell (IEC) apoptosis induced by hypoxia compromise intestinal epithelium barrier function. Both Akt and Hsp90 have cytoprotective function. However, the specific role of Akt and Hsp90β in IEC apoptosis induced by hypoxia has not been explored. We confirmed that hypoxia-induced apoptosis was reduced by Hsp90β overexpression but enhanced by decreasing Hsp90β expression. Hsp90β overexpression enhanced BAD phosphorylation and thus reduced mitochondrial release of cytochrome C. Reducing Hsp90β expression had opposite effects. The protective effect of Hsp90β against apoptosis was negated by {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, an Akt inhibitor. Further study showed that Akt phosphorylation was enhanced by Hsp90β, which was not due to the activation of upstream PI3K and PDK1 but because of stabilization of pAkt via direct interaction between Hsp90β and pAkt. These results demonstrate that Hsp90β may play a significant role in protecting IECs from hypoxia-induced apoptosis via stabilizing pAkt to phosphorylate BAD and reduce cytochrome C release. [BMB Reports 2013;46(1): 47-52]     

Phosphoinositide-3-kinase/akt - dependent signaling is required for maintenance of [Ca2+]i,ICa, and Ca2+ transients in HL-1 cardiomyocytes

The phosphoinositide 3-kinases (PI3K/Akt) dependent signaling pathway plays an important role in cardiac function, specifically cardiac contractility. We have reported that sepsis decreases myocardial Akt activation, which correlates with cardiac dysfunction in sepsis. We also reported that preventing sepsis induced changes in myocardial Akt activation ameliorates cardiovascular dysfunction. In this study we investigated the role of PI3K/Akt on cardiomyocyte function by examining the role of PI3K/Akt-dependent signaling on [Ca²⁺]_i, Ca²⁺ transients and membrane Ca²⁺ current, I_Ca, in cultured murine HL-1 cardiomyocytes. {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 (1–20 μM), a specific PI3K inhibitor, dramatically decreased HL-1 [Ca²⁺]_i, Ca²⁺ transients and I_Ca. We also examined the effect of PI3K isoform specific inhibitors, i.e. α (PI3-kinase α inhibitor 2; 2–8 nM); β (TGX-221; 100 nM) and γ (AS-252424; 100 nM), to determine the contribution of specific isoforms to HL-1 [Ca²⁺]_i regulation. Pharmacologic inhibition of each of the individual PI3K isoforms significantly decreased [Ca²⁺]_i, and inhibited Ca²⁺ transients. Triciribine (1–20 μM), which inhibits AKT downstream of the PI3K pathway, also inhibited [Ca²⁺]_i, and Ca²⁺ transients and I_Ca. We conclude that the PI3K/Akt pathway is required for normal maintenance of [Ca²⁺]_i in HL-1 cardiomyocytes. Thus, myocardial PI3K/Akt-PKB signaling sustains [Ca²⁺]_i required for excitation-contraction coupling in cardiomyoctyes.     

Folic Acid Represses Hypoxia-Induced Inflammation in THP-1 Cells through Inhibition of the PI3K/Akt/HIF-1α Pathway

Though hypoxia has been implicated as a cause of inflammation, the underlying mechanism is not well understood. Folic acid has been shown to provide protection against oxidative stress and inflammation in patients with cardiovascular disease and various models approximating insult to tissue via inflammation. It has been reported that hypoxia-induced inflammation is associated with oxidative stress, upregulation of hypoxia-inducible factor 1-alpha (HIF-1α), and production of pro-inflammatory molecules. Whether folic acid protects human monocytic cells (THP-1 cells) against hypoxia-induced damage, however, remains unknown. We used THP-1 cells to establish a hypoxia-induced cellular injury model. Pretreating THP-1 cells with folic acid attenuated hypoxia-induced inflammatory responses, including a decrease in protein and mRNA levels of interleukin (IL)-1β and tumor necrosis factor-alpha (TNF-α), coupled with increased levels of IL-10. Folic acid also reduced hypoxia-induced Akt phosphorylation and decreased nuclear accumulation of HIF-1α protein. Both {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 (a selective inhibitor of phosphatidyl inositol-3 kinase, PI3K) and KC7F2 (a HIF-1α inhibitor) reduced levels of hypoxia-induced inflammatory cytokines. We also found that insulin (an Akt activator) and dimethyloxallyl glycine (DMOG, a HIF-1α activator) induced over-expression of inflammatory cytokines, which could be blocked by folic acid. Taken together, these findings demonstrate how folic acid attenuates the hypoxia-induced inflammatory responses of THP-1 cells through inhibition of the PI3K/Akt/HIF-1α pathway.     

IGF-1-Induced Enhancement of PRNP Expression Depends on the Negative Regulation of Transcription Factor FOXO3a

The conformational conversion of the cellular prion protein (PrP^C) into its β-sheet-rich scrapie isoform (PrP^Sc) causes fatal prion diseases, which are also called transmissible spongiform encephalopathies (TSEs). Recent studies suggest that the expression of PrP^C by the PRNP gene is crucial for the development of TSEs. Therefore, the identification of the exogenous and endogenous stimulating factors that regulate PRNP expression would help to understand the pathogenesis of TSEs. Here, we demonstrate that forkhead box O3a (FOXO3a) negatively regulates PRNP expression by binding to the PRNP promoter, which is negatively regulated by insulin-like growth factor 1 (IGF-1). Our results show that the IGF-1-induced enhancement of PRNP mRNA and protein levels is due to the activation of the PI3K-Akt signaling pathway. The activation of Akt then induces the phosphorylation of FOXO3a, leading to its translocation from the nucleus to the cytoplasm and preventing its binding to the PRNP promoter. Treatment with the PI3K-Akt inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 induces the nuclear retention of FOXO3a, which leads to a decrease in PRNP expression. We present a new IGF-1-PI3K-Akt-FOXO3a pathway, which influences PRNP expression. The results of this work are vital for understanding the function of PrP^C and for future therapeutic approaches to human TSEs.     

Roles of PI3K/Akt and c-Jun Signaling Pathways in Human Papillomavirus Type 16 Oncoprotein-Induced HIF-1α, VEGF,and IL-8 Expression and In Vitro Angiogenesis in Non-Small Cell Lung Cancer Cells

Background and Objectives

Human papillomavirus (HPV)-16 infection may be related to non-smoking associated lung cancer. Our previous studies have found that HPV-16 oncoproteins promoted angiogenesis via enhancing hypoxia-inducible factor-1α (HIF-1α), vascular endothelial growth factor (VEGF), and interleukin-8 (IL-8) expression in non-small cell lung cancer (NSCLC) cells. In this study, we further investigated the roles of PI3K/Akt and c-Jun signaling pathways in it.

Methods

Human NSCLC cell lines, A549 and NCI-H460, were stably transfected with pEGFP-16 E6 or E7 plasmids. Western blotting was performed to analyze the expression of HIF-1α, p-Akt, p-P70S6K, p-P85S6K, p-mTOR, p-JNK, and p-c-Jun proteins. VEGF and IL-8 protein secretion and mRNA levels were determined by ELISA and Real-time PCR, respectively. The in vitro angiogenesis was observed by human umbilical vein endothelial cells (HUVECs) tube formation assay. Co-immunoprecipitation was performed to analyze the interaction between c-Jun and HIF-1α.

Results

HPV-16 E6 and E7 oncoproteins promoted the activation of Akt, P70S6K, P85S6K, mTOR, JNK, and c-Jun. {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, a PI3K inhibitor, inhibited HPV-16 oncoprotein-induced activation of Akt, P70S6K, and P85S6K, expression of HIF-1α, VEGF, and IL-8, and in vitro angiogenesis. c-Jun knockdown by specific siRNA abolished HPV-16 oncoprotein-induced HIF-1α, VEGF, and IL-8 expression and in vitro angiogenesis. Additionally, HPV-16 oncoproteins promoted HIF-1α protein stability via blocking proteasome degradation pathway, but c-Jun knockdown abrogated this effect. Furthermore, HPV-16 oncoproteins increased the quantity of c-Jun binding to HIF-1α.

Conclusions

PI3K/Akt signaling pathway and c-Jun are involved in HPV-16 oncoprotein-induced HIF-1α, VEGF, and IL-8 expression and in vitro angiogenesis. Moreover, HPV-16 oncoproteins promoted HIF-1α protein stability possibly through enhancing the interaction between c-Jun and HIF-1α, thus making a contribution to angiogenesis in NSCLC cells.     

Evidence That Atypical Protein Kinase C-λ and Atypical Protein Kinase C-ζ Participate in Ras-mediated Reorganization of the F-actin Cytoskeleton

Expression of transforming Ha-Ras L61 in NIH3T3 cells causes profound morphological alterations which include a disassembly of actin stress fibers. The Ras-induced dissolution of actin stress fibers is blocked by the specific PKC inhibitor GF109203X at concentrations which inhibit the activity of the atypical aPKC isotypes λ and ζ, whereas lower concentrations of the inhibitor which block conventional and novel PKC isotypes are ineffective. Coexpression of transforming Ha-Ras L61 with kinase-defective, dominant-negative (DN) mutants of aPKC-λ and aPKC-ζ, as well as antisense constructs encoding RNA-directed against isotype-specific 5′ sequences of the corresponding mRNA, abrogates the Ha-Ras–induced reorganization of the actin cytoskeleton. Expression of a kinase-defective, DN mutant of cPKC-α was unable to counteract Ras with regard to the dissolution of actin stress fibers. Transfection of cells with constructs encoding constitutively active (CA) mutants of atypical aPKC-λ and aPKC-ζ lead to a disassembly of stress fibers independent of oncogenic Ha-Ras. Coexpression of (DN) Rac-1 N17 and addition of the phosphatidylinositol 3′-kinase (PI3K) inhibitors wortmannin and {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 are in agreement with a tentative model suggesting that, in the signaling pathway from Ha-Ras to the cytoskeleton aPKC-λ acts upstream of PI3K and Rac-1, whereas aPKC-ζ functions downstream of PI3K and Rac-1.This model is supported by studies demonstrating that cotransfection with plasmids encoding L61Ras and either aPKC-λ or aPKC-ζ results in a stimulation of the kinase activity of both enzymes. Furthermore, the Ras-mediated activation of PKC-ζ was abrogated by coexpression of DN Rac-1 N17.     

Effects of Bone Marrow-Derived Mesenchymal Stem Cells on the Axonal Outgrowth through Activation of PI3K/AKT Signaling in Primary Cortical Neurons Followed Oxygen-Glucose Deprivation Injury

Background

Transplantation with bone marrow-derived mesenchymal stem cells (BMSCs) improves the survival of neurons and axonal outgrowth after stroke remains undetermined. Here, we investigated whether PI3K/AKT signaling pathway is involved in these therapeutic effects of BMSCs.

Methodology/Principal Findings

(1) BMSCs and cortical neurons were derived from Sprague-Dawley rats. The injured neurons were induced by Oxygen–Glucose Deprivation (OGD), and then were respectively co-cultured for 48 hours with BMSCs at different densities (5×10³, 5×10⁵/ml) in transwell co-culture system. The average length of axon and expression of GAP-43 were examined to assess the effect of BMSCs on axonal outgrowth after the damage of neurons induced by OGD. (2) The injured neurons were cultured with a conditioned medium (CM) of BMSCs cultured for 24 hours in neurobasal medium. During the process, we further identified whether PI3K/AKT signaling pathway is involved through the adjunction of {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 (a specific phosphatidylinositide-3-kinase (PI3K) inhibitor). Two hours later, the expression of pAKT (phosphorylated AKT) and AKT were analyzed by Western blotting. The length of axons, the expression of GAP-43 and the survival of neurons were measured at 48 hours.

Results

Both BMSCs and CM from BMSCs inreased the axonal length and GAP-43 expression in OGD-injured cortical neurons. There was no difference between the effects of BMSCs of 5×10⁵/ml and of 5×10³/ml on axonal outgrowth. Expression of pAKT enhanced significantly at 2 hours and the neuron survival increased at 48 hours after the injured neurons cultured with the CM, respectively. These effects of CM were prevented by inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002.

Conclusions/Significance

BMSCs promote axonal outgrowth and the survival of neurons against the damage from OGD in vitro by the paracrine effects through PI3K/AKT signaling pathway.     

TGF-β1 Protects against Mesangial Cell Apoptosis via Induction of Autophagy

Autophagy can lead to cell death in response to stress, but it can also act as a protective mechanism for cell survival. We show that TGF-β1 induces autophagy and protects glomerular mesangial cells from undergoing apoptosis during serum deprivation. Serum withdrawal rapidly induced autophagy within 1 h in mouse mesangial cells (MMC) as determined by increased microtubule-associated protein 1 light chain 3 (LC3) levels and punctate distribution of the autophagic vesicle-associated-form LC3-II. We demonstrate that after 1 h there was a time-dependent decrease in LC3 levels that was accompanied by induction of apoptosis, evidenced by increases in cleaved caspase 3. However, treatment with TGF-β1 resulted in induction of the autophagy protein LC3 while suppressing caspase 3 activation. TGF-β1 failed to rescue MMC from serum deprivation-induced apoptosis upon knockdown of LC3 by siRNA and in MMC from LC3 null (LC3^−/−) mice. We show that TGF-β1 induced autophagy through TAK1 and Akt activation, and inhibition of PI3K-Akt pathway by {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 or dominant-negative Akt suppressed LC3 levels and enhanced caspase 3 activation. TGF-β1 also up-regulated cyclin D1 and E protein levels while down-regulating p27, thus stimulating cell cycle progression. Bafilomycin A1, but not MG132, blocked TGF-β1 down-regulation of p27, suggesting that p27 levels were regulated through autophagy. Taken together, our data indicate that TGF-β1 rescues MMC from serum deprivation-induced apoptosis via induction of autophagy through activation of the Akt pathway. The autophagic process may constitute an adaptive mechanism to glomerular injury by inhibiting apoptosis and promoting mesangial cell survival.     

Regulation of PP2AC Carboxylmethylation and Cellular Localisation by Inhibitory Class G-Protein Coupled Receptors in Cardiomyocytes

The enzymatic activity of the type 2A protein phosphatase (PP2A) holoenzyme, a major serine/threonine phosphatase in the heart, is conferred by its catalytic subunit (PP2A_C). PP2A_C activity and subcellular localisation can be regulated by reversible carboxylmethylation of its C-terminal leucine309 (leu309) residue. Previous studies have shown that the stimulation of adenosine type 1 receptors (A1.Rs) induces PP2A_C carboxylmethylation and altered subcellular distribution in adult rat ventricular myocytes (ARVM). In the current study, we show that the enzymatic components that regulate the carboxylmethylation status of PP2A_C, leucine carboxylmethyltransferase-1 (LCMT-1) and phosphatase methylesterase-1 (PME-1) are abundantly expressed in, and almost entirely localised in the cytoplasm of ARVM. The stimulation of G_i-coupled A1.Rs with N⁶-cyclopentyladenosine (CPA), and of other G_i-coupled receptors such as muscarinic M₂ receptors (stimulated with carbachol) and angiotensin II AT₂ receptors (stimulated with {"type":"entrez-protein","attrs":{"text":"CGP42112","term_id":"874777115"}}CGP42112) in ARVM, induced PP2A_C carboxylmethylation at leu309 in a concentration-dependent manner. Exposure of ARVM to 10 µM CPA increased the cellular association between PP2A_C and its methyltransferase LCMT-1, but not its esterase PME-1. Stimulation of A1.Rs with 10 µM CPA increased the phosphorylation of protein kinase B at ser473, which was abolished by the PI3K inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 (20 µM), thereby confirming that PI3K activity is upregulated in response to A1.R stimulation by CPA in ARVM. A1.R-induced PP2A_C translocation to the particulate fraction was abrogated by adenoviral expression of the alpha subunit (Gα_t1) coupled to the transducin G-protein coupled receptor. A similar inhibitory effect on A1.R-induced PP2A_C translocation was also seen with {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 (20 µM). These data suggest that in ARVM, A1.R-induced PP2A_C translocation to the particulate fraction occurs through a G_iPCR-Gβγ-PI3K mediated intracellular signalling pathway, which may involve elevated PP2A_C carboxylmethylation at leu309.