Lipopolysaccharide induces lung fibroblast proliferation through Toll-like receptor 4 signaling and the phosphoinositide3-kinase-Akt pathway

Pulmonary fibrosis is characterized by lung fibroblast proliferation and collagen secretion. In lipopolysaccharide (LPS)-induced acute lung injury (ALI), aberrant proliferation of lung fibroblasts is initiated in early disease stages, but the underlying mechanism remains unknown. In this study, we knocked down Toll-like receptor 4 (TLR4) expression in cultured mouse lung fibroblasts using TLR4-siRNA-lentivirus in order to investigate the effects of LPS challenge on lung fibroblast proliferation, phosphoinositide3-kinase (PI3K)-Akt pathway activation, and phosphatase and tensin homolog (PTEN) expression. Lung fibroblast proliferation, detected by BrdU assay, was unaffected by 1 mug/mL LPS challenge up to 24 hours, but at 72 hours, cell proliferation increased significantly. This proliferation was inhibited by siRNA-mediated TLR4 knockdown or treatment with the PI3K inhibitor, Ly294002. In addition, siRNA-mediated knockdown of TLR4 inhibited the LPS-induced up-regulation of TLR4, down-regulation of PTEN, and activation of the PI3K-Akt pathway (overexpression of phospho-Akt) at 72 hours, as detected by real-time PCR and Western blot analysis. Treatment with the PTEN inhibitor, bpV(phen), led to activation of the PI3K-Akt pathway. Neither the baseline expression nor LPS-induced down-regulation of PTEN in lung fibroblasts was influenced by PI3K activation state. PTEN inhibition was sufficient to exert the LPS effect on lung fibroblast proliferation, and PI3K-Akt pathway inhibition could reverse this process. Collectively, these results indicate that LPS can promote lung fibroblast proliferation via a TLR4 signaling mechanism that involves PTEN expression down-regulation and PI3K-Akt pathway activation. Moreover, PI3K-Akt pathway activation is a downstream effect of PTEN inhibition and plays a critical role in lung fibroblast proliferation. This mechanism could contribute to, and possibly accelerate, pulmonary fibrosis in the early stages of ALI/ARDS.     

Stat3 in resident macrophages as a repressor protein of inflammatory response

Inflammation is counterbalanced by anti-inflammatory cytokines such as IL-10, in which Stat3 mediates the signaling pathway. In this study, we demonstrate that resident macrophages, but not other cell types, are important targets of IL-10 in a murine model of acute peritonitis. Injection of thioglycollate i.p. induced a considerable number of neutrophils and macrophages in the peritoneum, which was significantly augmented in mice with a cell-type specific disruption of the Stat3 gene in macrophages and neutrophils (LysMcre/Stat3flox/- mice). The augmented leukocyte infiltration was accompanied by increased peritoneal levels of TNF-alpha, MIP-2, KC chemokine (KC), and MCP-1/CCL2. Stat3 was tyrosine phosphorylated in peritoneal resident macrophages as well as infiltrating leukocytes in the littermate controls, suggesting that Stat3 in either or both of these cells might play a regulatory role in inflammation. The peritoneal levels of TNF-alpha, MIP-2, KC, and MCP-1 were similarly elevated in LysMcre/Stat3flox/- mice rendered leukopenic by cyclophosphamide treatment as compared with the controls. Adoptive transfer of resident macrophages from LysMcre/Stat3flox/- mice into the control littermates resulted in increases in the peritoneal level of TNF-alpha, MIP-2, KC, and MCP-1 after i.p. injection of thioglycollate. Under these conditions, control littermates harboring LysMcre/Stat3flox/- macrophages exhibited an augmented leukocyte infiltration relative to those received control macrophages. Taken together, these data provide evidence that resident macrophages, but not other cell types, play a regulatory role in inflammation through a Stat3 signaling pathway. Stat3 in resident macrophages appears to function as a repressor protein in this model of acute inflammation.     

Expression of PTEN and Akt phosphorylation in lipopolysaccharide-treated NIH3T3 cells

PTEN is a tumor suppressor gene encoding a phosphatase, and it negatively regulates cell survival mediated by the phosphoinositol 3-kinase (PI3-Kinase)-Akt pathway. To elucidate PTEN expression and its effect on the PI3-kinase-Akt pathway in fibroblasts and macrophages, we investigated the expression of PTEN and the phosphorylation status of Akt in NIH3T3 and RAW264.7 cells treated with LPS. Phosphorylation of Akt was induced by LPS treatment in a dose-dependent manner in RAW264.7 cells, but not in NIH3T3 cells. LPS induced the expression of PTEN in a dose and time-dependent manner in NIH3T3 cells (0-1 microg/ml, 0-6h). However, LPS did not stimulate PTEN expression in RAW264.7 cells. These data indicate the existence of diverse mechanisms for PTEN expression and Akt activation in fibroblasts and macrophages. RNA interference using double-stranded RNA specific for the PTEN gene reduced both mRNA and protein levels of PTEN in NIH3T3 cells treated or not with LPS. The phosphorylation status of Akt in NIH3T3 cells stimulated with LPS did not change when the PTEN expression had been inhibited by RNA interference. The present results suggest that the up-regulation of PTEN expression by LPS is not involved in the activation of Akt in NIH3T3 cells. PTEN expression might be involved in the diverse inflammatory responses to LPS in fibroblasts and macrophages.     

Critical roles of the p110 beta subtype of phosphoinositide 3-kinase in lipopolysaccharide-induced Akt activation and negative regulation of nitrite production in RAW 264.7 cells

It has been suggested that PI3K participates in TLR signaling. However, identifying specific roles for individual PI3K subtypes in signaling has remained elusive. In macrophages from the p110gamma(-/-) mouse, LPS-induced phosphorylation of Akt occurred normally despite the fact that the action of anaphylatoxin C5a was impaired markedly. In RAW 264.7 cells expressing short hairpin RNA that targets p110beta, LPS-induced phosphorylation of Akt was significantly attenuated. In contrast, the LPS action was not impaired, but was rather augmented in the p110alpha-deficient cells. Previous pharmacologic studies have suggested that a PI3K-Akt pathway negatively regulates TLR-induced inducible NO synthase expression and cytokine production. In the p110beta-deficient cells, inducible NO synthase expression and IL-12 production upon stimulation by LPS were increased, whereas LPS-induced expression of COX-2 and activation of MAPKs were unaffected. Together, the results suggest a specific function of p110beta in the negative feedback regulation of TLR signaling.     

Pre-treatment of recombinant mouse MFG-E8 downregulates LPS-induced TNF-α production in macrophages via STAT3-mediated SOCS3 activation

Milk fat globule-epidermal growth factor factor 8 (MFG-E8) regulates innate immune function by modulating cellular signaling, which is less understood. Herein, we aimed to investigate the direct anti-inflammatory role of MFG-E8 in macrophages by pre-treatment with recombinant murine MFG-E8 (rmMFG-E8) followed by stimulation with LPS in RAW264.7 cells and in peritoneal macrophages, isolated from wild-type (WT) or MFG-E8(-/-) mice. RAW264.7 cells and mouse peritoneal macrophages treated with rmMFG-E8 significantly downregulated LPS-induced TNF-α mRNA by 25% and 24%, and protein levels by 29% and 23%, respectively (P<0.05). Conversely, peritoneal macrophages isolated from MFG-E8(-/-) mice produced 28% higher levels of TNF-α, as compared to WT mice when treated with LPS. In in vivo, endotoxemia induced by intraperitoneal injection of LPS (5 mg/kg BW), at 4 h after induction, serum level of TNF-α was significantly higher in MFG-E8(-/-) mice (837 pg/mL) than that of WT (570 pg/mL, P<0.05). To elucidate the direct anti-inflammatory effect of MFG-E8, we examined STAT3 and its target gene, SOCS3. Treatment with rmMGF-E8 significantly induced pSTAT3 and SOCS3 in macrophages. Similar results were observed in in vivo treatment of rmMFG-E8 in peritoneal cells and splenic tissues. Pre-treatment with rmMFG-E8 significantly reduced LPS-induced NF-κB p65 contents. These data clearly indicated that rmMFG-E8 upregulated SOCS3 which in turn interacted with NF-κB p65, facilitating negative regulation of TLR4 signaling for LPS-induced TNF-α production. Our findings strongly suggest that MFG-E8 is a direct anti-inflammatory molecule, and that it could be developed as a therapy in attenuating inflammation and tissue injury.     

Regulation of the phosphatidylinositol 3-kinase-Akt and the mitogen-activated protein kinase pathways by ursolic acid in human endometrial cancer cells

The phosphatidylinositol 3-kinase-Akt (PI3K-Akt) pathway and the mitogen activated protein kinase (MAPK) pathway are important in the development and proliferation of various human cancers. It has been found recently that ursolic acid treatment affects growth and apoptosis in cancer cells. We sought to determine whether prominent signaling pathways, including the PI3K-Akt pathway and the MAPK (JNK, P38, and P44/42) pathway mediate these effects. Endometrial cancer cells often have high levels of phosphorylated Akt seen in conjunction with a PTEN mutation or deletion. Elevation in Akt protects the cancer cell from apoptosis. Ursolic acid treatment moderately decreased PI3K levels in SNG-II cells. Treatment also decreased phospho-Akt and phospho-P44/42 in a dose- and time-dependent fashion, dramatically in SNG-II cells and moderately in HEC108 cells. This effect was most pronounced following treatment with 50 mum ursolic acid for 72 h. Our study found inhibition of both the PI3K-Akt pathway and the MAPK pathway in two endometrial cancer cell lines, SNG-II and the poorly differentiated HEC108 cell line.     

Positive and negative roles of p85 alpha and p85 beta regulatory subunits of phosphoinositide 3-kinase in insulin signaling

Class IA phosphoinositide (PI) 3-kinase is composed of a p110 catalytic subunit and a p85 regulatory subunit and plays a pivotal role in insulin signaling. To explore the physiological roles of two major regulatory isoforms, p85 alpha and p85 beta, we have established brown adipose cell lines with disruption of the Pik3r1 or Pik3r2 gene. Pik3r1-/- (p85 alpha-/-) cells show a 70% reduction of p85 protein and a parallel reduction of p110. These cells have a 50% decrease in PI 3-kinase activity and a 30% decrease in Akt activity, leading to decreased insulin-induced glucose uptake and anti-apoptosis. Pik3r2-/- (p85 beta-/-) cells show a 25% reduction of p85 protein but normal levels of p85-p110 and PI 3-kinase activity, supporting the fact that p85 is more abundant than p110 in wild type. p85 beta-/- cells, however, exhibit significantly increased insulin-induced Akt activation, leading to increased anti-apoptosis. Reconstitution experiments suggest that the discrepancy between PI 3-kinase activity and Akt activity is at least in part due to the p85-dependent negative regulation of downstream signaling of PI 3-kinase. Indeed, both p85 alpha-/- cells and p85 beta-/- cells exhibit significantly increased insulin-induced glycogen synthase activation. p85 alpha-/- cells show decreased insulin-stimulated Jun N-terminal kinase activity, which is restored by expression of p85 alpha, p85 beta, or a p85 mutant that does not bind to p110, indicating the existence of p85-dependent, but PI 3-kinase-independent, signaling pathway. Furthermore, a reduction of p85 beta specifically increases insulin receptor substrate-2 phosphorylation. Thus, p85 alpha and p85 beta modulate PI 3-kinase-dependent signaling by multiple mechanisms and transmit signals independent of PI 3-kinase activation.     

Glucocorticoid conditioning of myeloid progenitors enhances TLR4 signaling via negative regulation of the phosphatidylinositol 3-kinase-Akt pathway

The immunomodulatory effects of glucocorticoids (GCs) have been described as bimodal, with high levels of GCs exerting immunosuppressive effects and low doses of GCs being immunopermissive. While the mechanisms used by GCs to achieve immunosuppression have been investigated intensely, the molecular mechanisms underlying the permissive effects of GCs remain uncharacterized. Herein, we demonstrate that GC conditioning during the differentiation of myeloid progenitors into macrophages (Mphis) results in their enhanced LPS responsiveness, demonstrated by an overexpression of the inflammatory cytokines TNF-alpha, IL-6, and IL-12. Inflammatory cytokine overexpression resulted from an increased activation of NF-kappaB and the MAPK signaling cascade and a reduced activation of the PI3K-Akt pathway following LPS stimulation. GC conditioning during Mphi differentiation induced an increase in the expression of SHIP1, a phosphatase that negatively regulates the PI3K signaling pathway. Small interfering RNA-mediated knockdown of SHIP1 expression increased PI3K-dependent Akt activation and subsequently decreased inflammatory cytokine expression, suggesting GC-mediated up-regulation of SHIP1 expression is responsible for the augmentation in inflammatory cytokine production following LPS stimulation. We also show that splenic Mphis purified from normal mice that were implanted with timed-release GC pellets exhibited an enhanced LPS responsiveness and increased SHIP1 expression, indicating that GCs can regulate SHIP1 expression in vivo. Our results suggest that minor fluctuations in physiological levels of endogenous GCs can program endotoxin-responsive hemopoietic cells during their differentiation by regulating their sensitivity to stimulation.     

Role of the phosphatidylinositol 3 kinase-Akt pathway in the regulation of IL-10 and IL-12 by Porphyromonas gingivalis lipopolysaccharide

Stimulation of the APC by Porphyromonas gingivalis LPS has been shown to result in the production of certain pro- and anti-inflammatory cytokines. However, the signaling pathways that regulate these processes are currently unknown. In the present study, the role of the phosphatidylinositol 3 kinase (PI3K)-Akt pathway in regulating P. gingivalis LPS-induced production of IL-10, IL-12 p40, and IL-12 p70 by human monocytes was investigated. P. gingivalis LPS selectively activates the PI3K-Akt pathway via Toll-like receptor 2, and inhibition of this pathway results in an abrogation of extracellular signal-regulated kinase 1/2 phosphorylation, whereas the activation of p38 and c-Jun N-terminal kinase 1/2 kinases were unaffected. Analysis of cytokine production following stimulation of monocytes with P. gingivalis LPS revealed that inhibition of the PI3K pathway differentially regulated IL-10 and IL-12 synthesis. IL-10 production was suppressed, whereas IL-12 levels were enhanced. Inhibition of P. gingivalis LPS-mediated activation of the PI3K-Akt pathway resulted in a pronounced augmentation of NF-kappaB p65 that was independent of IkappaB-alpha degradation. Furthermore, the ability of the PI3K-Akt pathway to modulate IL-10 and IL-12 production appears to be mediated by the selective suppression of extracellular signal-regulated kinase 1/2 activity, as the MEK1 inhibitor PD98059 closely mimicked the effects of wortmannin and LY294002 to differentially regulate IL-10 and IL-12 production by P. gingivalis LPS-stimulated monocytes. These studies provide new insight into how engagement of the PI3K-Akt pathway by P. gingivalis LPS affects the induction of key immunoregulatory cytokines that control both qualitative and quantitative aspects of innate and adaptive immunity.     

Phosphatase of Regenerating Liver 2 (PRL2) Deficiency Impairs Kit Signaling and Spermatogenesis

The Phosphatase of Regenerating Liver (PRL) proteins promote cell signaling and are oncogenic when overexpressed. However, our understanding of PRL function came primarily from studies with cultured cell lines aberrantly or ectopically expressing PRLs. To define the physiological roles of the PRLs, we generated PRL2 knock-out mice to study the effects of PRL deletion in a genetically controlled, organismal model. PRL2-deficient male mice exhibit testicular hypotrophy and impaired spermatogenesis, leading to decreased reproductive capacity. Mechanistically, PRL2 deficiency results in elevated PTEN level in the testis, which attenuates the Kit-PI3K-Akt pathway, resulting in increased germ cell apoptosis. Conversely, increased PRL2 expression in GC-1 cells reduces PTEN level and promotes Akt activation. Our analyses of PRL2-deficient animals suggest that PRL2 is required for spermatogenesis during testis development. The study also reveals that PRL2 promotes Kit-mediated PI3K/Akt signaling by reducing the level of PTEN that normally antagonizes the pathway. Given the strong cancer susceptibility to subtle variations in PTEN level, the ability of PRL2 to repress PTEN expression qualifies it as an oncogene and a novel target for developing anti-cancer agents.     

Inhibition of mycobacterial infection by the tumor suppressor PTEN

The tumor suppressor PTEN is a lipid phosphatase that is frequently mutated in various human cancers. PTEN suppresses tumor cell proliferation, survival, and growth mainly by inhibiting the PI3K-Akt signaling pathway through dephosphorylation of phosphatidylinositol 3,4,5-triphosphate. In addition to it role in tumor suppression, the PTEN-PI3K pathway controls many cellular functions, some of which may be important for cellular resistance to infection. Currently, the intersection between tumorigenic signaling pathways and cellular susceptibility to infection is not well defined. In this study we report that PTEN signaling regulates infection of both noncancerous and cancerous cells by multiple intracellular mycobacterial pathogens and that pharmacological modulation of PTEN signaling can affect mycobacterial infection. We found that PTEN deficiency renders multiple types of cells hyper-susceptible to infection by Mycoplasma and Mycobacterium bovis Bacillus Calmette-Guérin (BCG). The lipid phosphatase activity of PTEN is required for attenuating infection. Furthermore, we found mycobacterial infection activates host cell Akt phosphorylation, and pharmacological inhibition of Akt or PI3K activity reduced levels of intracellular infection. Intriguingly, inhibition of mTOR, one of the downstream components of the Akt signaling and a promising cancer therapeutic target, also lowered intracellular Bacillus Calmette-Guérin levels in mammary epithelial cancer MCF-7 cells. These findings demonstrate a critical role of PTEN-regulated pathways in pathogen infection. The relationship of PTEN-PI3K-Akt mTOR status and susceptibility to mycobacterial infection suggests that the interaction of mycobacterial pathogens with cancer cells may be influenced by genetic alterations in the tumor cells.     

Molecular Balance between the Regulatory and Catalytic Subunits of Phosphoinositide 3-Kinase Regulates Cell Signaling and Survival

Class Ia phosphoinositide (PI) 3-kinase is a central component in growth factor signaling and is comprised of a p110 catalytic subunit and a regulatory subunit, the most common family of which is derived from the p85alpha gene (Pik3r1). Optimal signaling through the PI 3-kinase pathway depends on a critical molecular balance between the regulatory and catalytic subunits. In wild-type cells, the p85 subunit is more abundant than p110, leading to competition between the p85 monomer and the p85-p110 dimer and ineffective signaling. Heterozygous disruption of Pik3r1 results in increased Akt activity and decreased apoptosis by insulin-like growth factor 1 (IGF-1) through up-regulated phosphatidylinositol (3,4,5)-triphosphate production. Complete depletion of p85alpha, on the other hand, results in significantly increased apoptosis due to reduced PI 3-kinase-dependent signaling. Thus, a reduction in p85alpha represents a novel therapeutic target for enhancing IGF-1/insulin signaling, prolongation of cell survival, and protection against apoptosis.     

Pik3ip1 Modulates Cardiac Hypertrophy by Inhibiting PI3K Pathway

Cardiac hypertrophy is an adaptive response to various physiological and pathological stimuli. Phosphoinositide-3 kinase (PI3K) is a highly conserved lipid kinase involved in physiological cardiac hypertrophy (PHH). PI3K interacting protein1 (Pik3ip1) shares homology with the p85 regulatory subunit of PI3K and is known to interact with the p110 catalytic subunit of PI3K, leading to attenuation of PI3K activity in liver and immune cells. However, the role of Pik3ip1 in the heart remains unknown. In the present study, the effects of Pik3ip1 on cardiac hypertrophy were examined. We found that the expression level of Pik3ip1 was markedly higher in cardiomyocytes than in fibroblasts. The interaction of Pik3ip1 with the p110a subunit of PI3K in the heart was identified by immunoprecipitation using neonatal rat cardiomyocytes (NRCM). Approximately 35% knockdown of Pik3ip1 was sufficient to induce myocardial hypertrophy. Pik3ip1 deficiency was shown to lead to activation of PI3K/protein kinase B (AKT)/ mammalian target of rapamycin (mTOR) signaling pathway, increasing protein synthesis and cell size. However, adenovirus-mediated overexpression of Pik3ip1 attenuated PI3K-mediated cardiac hypertrophy. Pik3ip1 was upregulated by PHH due to swimming training, but not by pathological cardiac hypertrophy (PAH) due to pressure-overload, suggesting that Pik3ip1 plays a compensatory negative role for PHH. Collectively, our results elucidate the mechanisms for the roles of Pik3ip1 in PI3K/AKT signaling pathway.