Intracellular Networks of the PI3K/AKT and MAPK Pathways for Regulating Toxoplasma gondii-Induced IL-23 and IL-12 Production in Human THP-1 Cells

Interleukin (IL)-23 and IL-12 are closely related in structure, and these cytokines regulate both innate and adaptive immunity. However, the precise signaling networks that regulate the production of each in Toxoplasma gondii-infected THP-1 monocytic cells, particularly the PI3K/AKT and MAPK signaling pathways, remain unknown. In the present study, T. gondii infection upregulated the expression of IL-23 and IL-12 in THP-1 cells, and both cytokines increased with parasite dose. IL-23 secretion was strongly inhibited by TLR2 monoclonal antibody (mAb) treatment in a dose-dependent manner and by TLR2 siRNA transfection, whereas IL-12 secretion was strongly inhibited by TLR4 mAb treatment dose-dependently and by TLR4 siRNA transfection. IL-23 production was dose-dependently inhibited by the PI3K inhibitors LY294002 and wortmannin, whereas IL-12 production increased dose-dependently. THP-1 cells exposed to live T. gondii tachyzoites underwent rapid p38 MAPK, ERK1/2 and JNK activation. IL-23 production was significantly upregulated by the p38 MAPK inhibitor SB203580 dose-dependently, whereas pretreatment with 10 μM SB203580 significantly downregulated IL-12 production. ERK1/2 inhibition by PD98059 was significantly downregulated IL-23 production but upregulated IL-12 production. JNK inhibition by SP600125 upregulated IL-23 production, but IL-12 production was significantly downregulated dose-dependently. T. gondii infection resulted in AKT activation, and AKT phosphorylation was inhibited dose-dependently after pretreatment with PI3K inhibitors. In T. gondii-infected THP-1 cells, ERK1/2 activation was regulated by PI3K; however, the phosphorylation of p38 MAPK and JNK was negatively modulated by the PI3K signaling pathway. Collectively, these results indicate that IL-23 production in T. gondii-infected THP-1 cells was regulated mainly by TLR2 and then by PI3K and ERK1/2; however, IL-12 production was mainly regulated by TLR4 and then by p38 MAPK and JNK. Our findings provide new insight concerning the intracellular networks of the PI3K/AKT and MAPK signaling cascades for regulating T. gondii-induced IL-23 and IL-12 secretion in human monocytic cells.     

Prevention of TGF-β-induced apoptosis by interlukin-4 through Akt activation and p70S6K survival signaling pathways

In this study, we demonstrate that interleukin-4 (IL-4) protects human hepatocellular carcinoma (HCC) cell line Hep3B from apoptosis induced by transforming growth factor-β (TGF-β). Further investigation of IL-4-transduced signaling pathways revealed that both insulin response substrate 1 and 2 (IRS-1/-2) and extracellular signal-regulated kinase (ERK) pathways were activated after IL-4 stimulation. The IRS-1/-2 activation was accompanied by the activation of phosphotidylinositol-3-kinase (PI3K), leading to Akt and p70 ribosomal protein S6 kinase (p70S6K). Interestingly, a protein kinase C (PKC) inhibitor, Gö6976, inhibited the phosphorylation of Akt, suggesting that the Akt activation was PKC-dependent. Using specific inhibitors for PI3K or ERK, we demonstrated that the PI3K pathway, but not the ERK pathway, was required for protection. The constitutively active form of PI3K almost completely rescued TGF-β-induced apoptosis, further supporting the importance of the PI3K pathway in the protective effect of IL-4. Furthermore, a dominant negative Akt and/or Gö6976 only partially blocked the anti-apoptotic effect of IL-4. Similarly, rapamycin, which interrupted the activation of p70S6K, also only partially blocked the protective effect of IL-4. However, in the presence of both rapamycin and dominant negative Akt with or without Gö6976, IL-4 almost completely lost the anti-apoptotic effect, suggesting that both Akt and p70S6K pathways were required for the protective effect of IL-4 against TGF-β-induced apoptosis.     

Differential activation of protein kinase B and p70(S6)K by glucose and insulin-like growth factor 1 in pancreatic beta-cells (INS-1)

It has been shown that IGF-1-induced pancreatic beta-cell proliferation is glucose-dependent; however, the mechanisms responsible for this glucose dependence are not known. Adenoviral mediated expression of constitutively active phosphatidylinositol 3-kinase (PI3K) in the pancreatic beta-cells, INS-1, suggested that PI3K was not necessary for glucose-induced beta-cell proliferation but was required for IGF-1-induced mitogenesis. Examination of the signaling components downstream of PI3K, 3-phosphoinositide-dependent kinase 1, protein kinase B (PKB), glycogen synthase kinase-3, and p70-kDa-S6-kinase (p70(S6K)), suggested that a major part of glucose-dependent beta-cell proliferation requires activation of mammalian target of rapamycin/p70(S6K), independent of phosphoinositide-dependent kinase 1/PKB activation. Adenoviral expression of the kinase-dead form of PKB in INS-1 cells decreased IGF-1-induced beta-cell proliferation. However, a surprisingly similar decrease was also observed in adenoviral wild type and constitutively active PKB-infected cells. Upon analysis of extracellular signal-regulated protein kinase 1 and 2 (ERK1/ERK2), an increase in ERK1/ERK2 phosphorylation activation by glucose and IGF-1 was observed in kinase-dead PKB-infected cells, but this phosphorylation activation was inhibited in the constitutively active PKB-infected cells. Hence, there is a requirement for the activation of both ERK1/ERK2 and mammalian target of rapamycin/p70(S6K) signal transduction pathways for a full commitment to glucose-induced pancreatic beta-cell mitogenesis. However, for IGF-1-induced activation, these pathways must be carefully balanced, because chronic activation of one (PI3K/PKB) can lead to dampening of the other (ERK1/2), reducing the mitogenic response.     

Signal pathways involved in activation of p70S6K and phosphorylation of 4E-BP1 following exposure of multiple myeloma tumor cells to interleukin-6

Interleukin-6 (IL-6) is a prominent tumor growth factor for malignant multiple myeloma cells. In addition to its known activation of the Janus tyrosine kinase-STAT and RAS-MEK-ERK pathways, recent work suggests that IL-6 can also activate the phosphatidylinositol 3-kinase (PI3-K)/AKT kinase pathway in myeloma cells. Because activation of the PI3-K/AKT as well as RAS-MEK-ERK pathways may result in downstream stimulation of the p70(S6K) (p70) and phosphorylation of the 4E-BP1 translational repressor, we assessed these potential molecular targets in IL-6-treated myeloma cells. IL-6 rapidly activated p70 kinase activity and p70 phosphorylation. Activation was inhibited by wortmannin, rapamycin, and the ERK inhibitors PD98059 and UO126, as well as by a dominant negative mutant of AKT. The concurrent requirements for both ERK and PI3-K/AKT appeared to be a result of their ability to phosphorylate p70 on different residues. In contrast, IL-6-induced phosphorylation of 4E-BP1 was inhibited by rapamycin, wortmannin, and dominant negative AKT but ERK inhibitors had no effect, indicating ERK function was dispensable. In keeping with these data, a dominant active AKT mutant was sufficient to induce 4E-BP1 phosphorylation but could not by itself activate p70 kinase activity. Prevention of IL-6-induced p70 activation and 4E-BP1 phosphorylation by the mammalian target of rapamycin inhibitors rapamycin and CCI-779 resulted in inhibition of IL-6-induced myeloma cell growth. These results indicate that both ERK and PI3-K/AKT pathways are required for optimal IL-6-induced p70 activity, but PI3-K/AKT is sufficient for 4E-BP1 phosphorylation. Both effects are mediated via mammalian target of rapamycin function, and, furthermore, these effects are critical for IL-6-induced tumor cell growth.     

Mechanism of zinc-induced phosphorylation of p70 S6 kinase and glycogen synthase kinase 3beta in SH-SY5Y neuroblastoma cells

We have previously reported an aberrant accumulation of activated protein kinase B (PKB), glycogen synthase kinase (GSK)-3beta, extracellular signal-regulated kinase (ERK1/2), c-Jun N-terminal kinase (JNK), p38 and p70 S6 kinase (p70S6K) in neurons bearing neurofibrillary tangles (NFTs) in Alzheimer's disease (AD). However, the mechanism by which these tau candidate kinases are involved in the regulation of p70S6K and GSK-3beta phosphorylation is unknown. In the current study, 100 microM zinc sulfate was used, and influences of various components of phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways on p70S6K and GSK-3beta phosphorylation have been investigated in serum-deprived SH-SY5Y neuroblastoma cells. We found that zinc could induce an increase of phosphorylated (p) p70S6K, p-PKB, p-GSK-3beta, p-ERK1/2, p-JNK and p-p38, especially in long-term treatment (4-8 h). Treatment with different inhibitors including rapamycin, wortmannin, LY294002, and U0126, and their combinations, indicated that phosphorylation of p70S6K and GSK-3beta is regulated by rapamycin-dependent, PI3K and MAPK pathways. Furthermore, phosphorylation of p70S6K and GSK-3beta affected levels of tau unphosphorylated at the Tau-1 site and phosphorylated at the PHF-1 site, and p70S6K phosphorylation affected the total tau level. Thus, 100 microM zinc might activate PKB, GSK-3beta, ERK1/2, JNK, p38 and p70S6K, that are consequently involved in tau changes in SH-SY5Y cells.     

Extracellular ATP-induced proliferation of adventitial fibroblasts requires phosphoinositide 3-kinase, Akt, mammalian target of rapamycin, and p70 S6 kinase signaling pathways

Extracellular nucleotides are increasingly recognized as important regulators of growth in a variety of cell types. Recent studies have demonstrated that extracellular ATP is a potent inducer of fibroblast growth acting, at least in part, through an ERK1/2-dependent signaling pathway. However, the contributions of additional signaling pathways to extracellular ATP-mediated cell proliferation have not been defined. By using both pharmacologic and genetic approaches, we found that in addition to ERK1/2, phosphatidylinositol 3-kinase (PI3K), Akt, mammalian target of rapamycin (mTOR), and p70 S6K-dependent signaling pathways are required for ATP-induced proliferation of adventitial fibroblasts. We found that extracellular ATP acting in part through G(i) proteins increased PI3K activity in a time-dependent manner and transient phosphorylation of Akt. This PI3K pathway is not involved in ATP-induced activation of ERK1/2, implying activation of independent parallel signaling pathways by ATP. Extracellular ATP induced dramatic increases in mTOR and p70 S6K phosphorylation. This activation of the mTOR/p70 S6 kinase (p70 S6K) pathway in response to ATP is because of independent contributions of PI3K/Akt and ERK1/2 pathways, which converge on the level of p70 S6K. ATP-dependent activation of mTOR and p70 S6K also requires additional signaling inputs perhaps from pathways operating through Galpha or Gbetagamma subunits. Collectively, our data demonstrate that ATP-induced adventitial fibroblast proliferation requires activation and interaction of multiple signaling pathways such as PI3K, Akt, mTOR, p70 S6K, and ERK1/2 and provide evidence for purinergic regulation of the protein translational pathways related to cell proliferation.     

Porphyromonas gingivalis lipopolysaccharide regulates interleukin (IL)-17 and IL-23 expression via SIRT1 modulation in human periodontal ligament cells

Increased interleukin (IL)-17 and IL-23 levels exist in the gingival tissue of periodontitis patients, but the precise molecular mechanisms that regulate IL-17 and IL-23 production remain unknown. The aim of this study was to explore the role of SIRT1 signaling on Porphyromonas gingivalis lipopolysaccharide (LPS)-induced IL-17 and IL-23 production in human periodontal ligament cells (hPDLCs). IL-17 and IL-23 production was significantly increased in LPS-treated cells. LPS treatment also led to the upregulation of SIRT1 mRNA and protein expression. LPS-induced IL-17 and IL-23 upregulation was attenuated by pretreatment with inhibitors of phosphoinositide 3-kinase (PI3K), p38, extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), mitogen-activated protein kinase (MAPK), and NF-κB, as well as neutralizing antibodies against Toll-like receptors (TLRs) 2 and 4. Sirtinol treatment (a known SIRT1 inhibitor) or SIRT1 knockdown by small interfering RNA blocked LPS-stimulated IL-17 and IL-23 expression. Further investigation showed that LPS decreased osteoblast markers (i.e., ALP, OPN, and BSP) and concomitantly increased osteoclast markers (i.e., RANKL and M-CSF). This response was attenuated by inhibitors of the PI3K, p38, ERK, JNK, NF-κB, and SIRT1 pathways. These findings, for the first time, suggest that human periodontopathogen P. gingivalis LPS is implicated in periodontal disease bone destruction and may mediate IL-17 and IL-23 release from hPDLCs. This process is dependent, at least in part, on SIRT1-Akt/PI3K-MAPK-NF-κB signaling.     

L-leucine increases [3H]-thymidine incorporation in chicken hepatocytes: involvement of the PKC, PI3K/Akt, ERK1/2, and mTOR signaling pathways

This study examined how L-leucine affected DNA synthesis and cell cycle regulatory protein expression in cultured primary chicken hepatocytes. L-Leucine promoted DNA synthesis in a dose- and time-dependent manner, with concomitant increases in cyclin D1 and cyclin E expression. Phospholipase C (PLC) and protein kinase C (PKC) mediated the L-leucine-induced increases in [3H]-thymidine incorporation and cyclin D1/CDK4 and cyclin E/CDK2 expression, as U73122 (a PLC inhibitor) or bisindolylmaleimide I (a PKC blocker) inhibited these effects. L-Leucine also increased PKC phosphorylation and intracellular Ca2+ levels. L-Leucine-mediated increases in [3H]-thymidine incorporation and cyclin/CDK expression were sensitive to LY 294002 (PI3K inhibitor), Akt inhibitor, PD 98059 (MEK inhibitor). It was also observed that L-leucine-induced increases of cyclin/CDK expression were inhibited by PI3K siRNA and ERK siRNA; L-leucine increased extracellular signal-regulated kinases 1/2 (ERK1/2) and Akt phosphorylation levels. Bisindolylmaleimide I attenuated L-leucine-induced phosphorylation of ERK1/2 but did not influence Akt phosphorylation, and PI3K siRNA and LY 294002 inhibited L-leucine-induced ERK1/2 phosphorylation, suggesting some cross-talk between the PKC and ERK1/2 or PI3K/Akt and ERK1/2 pathways. L-Leucine also increased the levels of phosphorylated molecular target of rapamycin (mTOR) and two of its targets, ribosomal protein S6 kinase (p70S6K), and 4E binding protein 1 (4E-BP1); furthermore, rapamycin (an mTOR inhibitor) blocked all of the mitogenic effects of L-leucine. In addition, Akt inhibitor blocked L-leucine-induced mTOR phosphorylation. In conclusion, L-leucine stimulated DNA synthesis and promoted cell cycle progression in primary cultured chicken hepatocytes through PKC, ERK1/2, PI3K/Akt, and mTOR.     

Signal transduction pathways involved in phosphorylation and activation of p70S6K following exposure to UVA irradiation

Ultraviolet light A (UVA) plays an important role in the etiology of human skin cancer, and UVA-induced signal transduction has a critical role in UVA-induced skin carcinogenesis. The upstream signaling pathways leading to p70(S6K) phosphorylation and activation are not well understood. Here, we observed that UVA induces phosphorylation and activation of p70(S6K). Further, UVA-stimulated p70(S6K) activity and phosphorylation at Thr(389) were blocked by wortmannin, rapamycin, PD98059, SB202190, and dominant negative mutants of phosphatidylinositol (PI) 3-kinase p85 subunit (DNM-Deltap85), ERK2 (DNM-ERK2), p38 kinase (DNM-p38), and JNK1 (DNM-JNK1) and were absent in Jnk1-/- or Jnk2-/- knockout cells. The p70(S6K) phosphorylation at Ser(411) and Thr(421)/Ser(424) was inhibited by rapamycin, PD98059, or DNM-ERK2 but not by wortmannin, SB202190, DNM-Deltap85, or DNM-p38. However, Ser(411), but not Thr(421)/Ser(424) phosphorylation, was suppressed in DNM-JNK1 and abrogated in Jnk1-/- or Jnk2-/- cells. In vitro assays indicated that Ser(411) on immunoprecipitated p70(S6K) proteins is phosphorylated by active JNKs and ERKs, but not p38 kinase, and Thr(421)/Ser(424) is phosphorylated by ERK1, but not ERK2, JNKs, or p38 kinase. Moreover, p70(S6K) co-immunoprecipitated with PI 3-kinase and possibly PDK1. The complex possibly possessed a partial basal level of phosphorylation, but not at MAPK sites, which was available for its activation by MAPKs in vitro. Thus, these results suggest that activation of MAPKs, like PI 3-kinase/mTOR, may be involved in UVA-induced phosphorylation and activation of p70(S6K).     

Activation of phosphatidylinositol 3-kinase, Akt, and mammalian target of rapamycin is necessary for hypoxia-induced pulmonary artery adventitial fibroblast proliferation.

In contrast to cell types in which exposure to hypoxia causes a general reduction of metabolic activity, a remarkable feature of pulmonary artery adventitial fibroblasts is their ability to proliferate in response to hypoxia. Previous studies have suggested that ERK1/2, phosphatidylinositol 3-kinase (PI3K), Akt, and mammalian target of rapamycin (mTOR) are activated by hypoxia and play a role in a variety of cell responses. However, the pathways involved in mediating hypoxia-induced proliferation are largely unknown. Using pharmacological inhibitors, we established that PI3K-Akt, mTOR-p70 ribosomal protein S6 kinase (p70S6K), and EKR1/2 signaling pathways play a critical role in hypoxia-induced adventitial fibroblast proliferation. We found that exposure of serum-starved fibroblasts to 3% O2 resulted in a time-dependent activation of PI3K and transient phosphorylation of Akt. However, activation of PI3K was not required for activation of ERK1/2, implying a parallel involvement of these pathways in the proliferative response of fibroblasts to hypoxia. We found that hypoxia induced significant increases in mTOR, p70S6K, 4E-BP1, and S6 ribosomal protein phosphorylation, as well as dramatic increases in p70S6K activity. The activation of p70S6K/S6 pathway was sensitive to inhibition by rapamycin and LY294002, indicating that mTOR and PI3K/Akt are upstream signaling regulators. However, the magnitude of hypoxia-induced p70S6K activity and phosphorylation suggests involvement of additional signaling pathways. Thus our data demonstrate that hypoxia-induced adventitial fibroblast proliferation requires activation and interaction of PI3K, Akt, mTOR, p70S6K, and ERK1/2 and provide evidence for hypoxic regulation of protein translational pathways in cells exhibiting the capability to proliferate under hypoxic conditions.     

TLR5-mediated phosphoinositide 3-kinase activation negatively regulates flagellin-induced proinflammatory gene expression

Epithelial cells detect motile pathogens via TLR5 ligation of flagellin, resulting in rapid induction of antibacterial/proinflammatory gene expression. Although such flagellin-induced gene expression is quite transient, likely to avoid the negative consequences of inflammation, little is known regarding the molecular mechanisms that mediate its shutdown. We hypothesized that, analogous to the case for TLR4, phosphoinositide 3-kinase (PI3K) might negatively regulate TLR5 signaling. However, because PI3K is an essential positive mediator of some pathways of TLR-mediated gene expression, the opposite hypothesis was also considered. Herein, we observed that flagellin stimulation of epithelial cells indeed induced rapid (<30 min) PI3K activation, as evidenced by Akt phosphorylation, via a TLR5-mediated mechanism. Blockade of PI3K with wortmannin resulted in marked enhancement of flagellin-induced gene expression as assessed by measuring levels of inducible NO synthase, IL-6, and IL-8. Such enhancement of gene expression by PI3K inhibition correlated with prolonged activation of MAPK (p38 and ERK1/2) and was ablated under MAPK inhibition. Such effect of inhibiting PI3K with wortmannin was mimicked by the PI3K inhibitor LY294002, and, conversely, a constitutively active PI3K prevented p38 activation in response to flagellin. Last, to test the significance of these results in vivo, we measured flagellin-induced gene expression in PI3K knockout mice. PI3K-null mice displayed increased levels of flagellin-induced serum IL-6, KC (IL-8 homolog), and nitrite as compared with heterozygous littermates. Thus, TLR5's rapid activation of PI3K serves to limit MAPK signaling, thus limiting proinflammatory gene expression and reducing the potential negative consequences of proinflammatory gene expression.     

L-threonine regulates G1/S phase transition of mouse embryonic stem cells via PI3K/Akt, MAPKs, and mTORC pathways

Although amino acids can function as signaling molecules in the regulation of many cellular processes, mechanisms surrounding L-threonine involvement in embryonic stem cell (ESC) functions have not been explored. Thus, we investigated the effect of L-threonine on regulation of mouse (m)ESC self-renewal and related signaling pathways. In L-threonine-depleted mESC culture media mRNA of self-renewal marker genes, [(3)H]thymidine incorporation, expression of c-Myc, Oct4, and cyclins protein was attenuated. In addition, resupplying L-threonine (500 μM) after depletion restores/maintains the mESC proliferation. Disruption of the lipid raft/caveolae microdomain through treatment with methyl-β-cyclodextrin or transfection with caveolin-1 specific small interfering RNA blocked L-threonine-induced proliferation of mESCs. Addition of L-threonine induced phosphorylation of Akt, ERK, p38, JNK/SAPK, and mTOR in a time-dependent manner. This activity was blocked by LY 294002 (PI3K inhibitor), wortmannin (PI3K inhibitor), or an Akt inhibitor. L-threonine-induced activation of mTOR, p70S6K, and 4E-BP1 as well as cyclins and Oct4 were blocked by PD 98059 (ERK inhibitor), SB 203580 (p38 inhibitor) or SP 600125 (JNK inhibitor). Furthermore, L-threonine induced phosphorylation of raptor and rictor binding to mTOR was completely inhibited by 24 h treatment with rapamycin (mTOR inhibitor); however, a 10 min treatment with rapamycin only partially inhibited rictor phosphorylation. L-threonine induced translocation of rictor from the membrane to the cytosol/nuclear, which blocked by pretreatment with rapamycin. In addition, rapamycin blocked L-threonine-induced increases in mRNA expressions of trophoectoderm and mesoderm marker genes and mESC proliferation. In conclusion, L-threonine stimulated ESC G(1)/S transition through lipid raft/caveolae-dependent PI3K/Akt, MAPKs, mTOR, p70S6K, and 4E-BP1 signaling pathways.     

Homocysteine induces smooth muscle cell proliferation through differential regulation of cyclins A and D1 expression

The mechanism of homocysteine‐induced cell proliferation in human vascular smooth muscle cells (SMCs) remains unclear. We investigated the molecular mechanisms by which homocysteine affects the expression of cyclins A and D1 in human umbilical artery SMCs (HUASMCs). Homocysteine treatment induced proliferation of HUASMCs and increased the expression levels of cyclins A and D1. Knocking down either cyclin A or cyclin D1 by small interfering RNA (siRNA) inhibited homocysteine‐induced cell proliferation. Furthermore, treatment with extracellular signal‐related kinase (ERK) inhibitor (PD98059) and dominant negative Ras (RasN17) abolished homocysteine‐induced cyclin A expression; and treatment with phosphatidylinositol 3‐kinase (PI3K) inhibitor (LY294002) and mammalian target of rapamycin (mTOR) inhibitor (rapamycin) attenuated the homocysteine‐induced cyclin D1 expression. Homocysteine also induced transient phosphorylation of ERK, Akt, and p70 ribosomal S6 kinase (p70S6K). Neutralizing antibody and siRNA for β1 integrin blocked cell proliferation, expression of cyclins A and D1, and phosphorylation of ERK and Akt. In conclusion, homocysteine‐induced differential activation of Ras/ERK and PI3K/Akt/p70S6K signaling pathways and consequent expression of cyclins A and D1 are dependent on β1 integrin. Homocysteine may accelerate progression of atherosclerotic lesions by promoting SMC proliferation. J. Cell. Physiol. 226: 1017–1026, 2011. © 2010 Wiley‐Liss, Inc.     

Distinct cell cycle timing requirements for extracellular signal-regulated kinase and phosphoinositide 3-kinase signaling pathways in somatic cell mitosis

Mitogen-activated protein (MAP) kinase and phosphoinositide 3-kinase (PI3K) pathways are necessary for cell cycle progression into S phase; however the importance of these pathways after the restriction point is poorly understood. In this study, we examined the regulation and function of extracellular signal-regulated kinase (ERK) and PI3K during G(2)/M in synchronized HeLa and NIH 3T3 cells. Phosphorylation and activation of both the MAP kinase kinase/ERK and PI3K/Akt pathways occur in late S and persist until the end of mitosis. Signaling was rapidly reversed by cell-permeable inhibitors, indicating that both pathways are continuously activated and rapidly cycle between active and inactive states during G(2)/M. The serum-dependent behavior of PI3K/Akt versus ERK pathway activation indicates that their mechanisms of regulation differ during G(2)/M. Effects of cell-permeable inhibitors and dominant-negative mutants show that both pathways are needed for mitotic progression. However, inhibiting the PI3K pathway interferes with cdc2 activation, cyclin B1 expression, and mitotic entry, whereas inhibiting the ERK pathway interferes with mitotic entry but has little effect on cdc2 activation and cyclin B1 and retards progression from metaphase to anaphase. Thus, our study provides novel evidence that ERK and PI3K pathways both promote cell cycle progression during G(2)/M but have different regulatory mechanisms and function at distinct times.     

Signaling in lipopolysaccharide-induced stabilization of formyl peptide receptor 1 mRNA in mouse peritoneal macrophages

To identify the TLR4-initiated signaling events that couple to formyl peptide receptor (FPR)1 mRNA stabilization, macrophages were treated with LPS along with a selection of compounds targeting several known signaling pathways. Although inhibitors of protein tyrosine kinases, MAPKs, and stress-activated kinases had little or no effect on the response to LPS, LY294002 (LY2) and parthenolide (an IkappaB kinase inhibitor) were both potent inhibitors. LY2 but not parthenolide blocked the LPS-induced stabilization of FPR1 mRNA. Although both LY2 and wortmannin effectively blocked PI3K activity, wortmannin had little effect on FPR1 expression and did not modulate the decay of FPR1 mRNA. Moreover, although LY2 was demonstrated to be a potent inhibitor of PI3K activity, a structural analog of LY2, LY303511 (LY3), which did not inhibit PI3K, was equally effective at preventing LPS-stimulated FPR1 expression. The mammalian target of rapamycin activity (measured as phospho-p70S6 kinase) was activated by LPS but not significantly blocked by LY2. In addition, although rapamycin blocked mTOR activity, it did not inhibit FPR1 mRNA expression. Finally, the mechanisms involved in stabilization of FPR1 by LPS could be distinguished from those involved in stabilization of AU-rich mRNAs because the prolonged half-life of FPR1 mRNA was insensitive to the inhibition of p38 MAPK. These findings demonstrate that LY2/LY3 targets a novel TLR4-linked signaling pathway that selectively couples to the stabilization of FPR1 mRNA.