TLR-4 Agonist Induces IFN-γ Production Selectively in Proinflammatory Human M1 Macrophages through the PI3K-mTOR- and JNK-MAPK-Activated p70S6K Pathway.

IFN-γ, a proinflammatory cytokine produced primarily by T cells and NK cells, activates macrophages and engages mechanisms to control pathogens. Although there is evidence of IFN-γ production by murine macrophages, IFN-γ production by normal human macrophages and their subsets remains unknown. Herein, we show that human M1 macrophages generated by IFN-γ and IL-12- and IL-18-stimulated monocyte-derived macrophages (M0) produce significant levels of IFN-γ. Further stimulation of IL-12/IL-18-primed macrophages or M1 macrophages with agonists for TLR-2, TLR-3, or TLR-4 significantly enhanced IFN-γ production in contrast to the similarly stimulated M0, M2a, M2b, and M2c macrophages. Similarly, M1 macrophages generated from COVID-19-infected patients' macrophages produced IFN-γ that was enhanced following LPS stimulation. The inhibition of M1 differentiation by Jak inhibitors reversed LPS-induced IFN-γ production, suggesting that differentiation with IFN-γ plays a key role in IFN-γ induction. We subsequently investigated the signaling pathway(s) responsible for TLR-4-induced IFN-γ production in M1 macrophages. Our results show that TLR-4-induced IFN-γ production is regulated by the ribosomal protein S6 kinase (p70S6K) through the activation of PI3K, the mammalian target of rapamycin complex 1/2 (mTORC1/2), and the JNK MAPK pathways. These results suggest that M1-derived IFN-γ may play a key role in inflammation that may be augmented following bacterial/viral infections. Moreover, blocking the mTORC1/2, PI3K, and JNK MAPKs in macrophages may be of potential translational significance in preventing macrophage-mediated inflammatory diseases.

Reactivations of Latent Viral Infections Are Associated with an Increased Thr389 p70S6k Phosphorylation in Peripheral Lymphocytes of Renal Transplant Recipients.

Reactivations of BK polyoma virus (BKPyV) and human cytomegalovirus (HCMV) frequently cause life- and graft-threatening complications after renal transplantation. Both viruses are dependent on the mTOR pathway for replication. In this study we investigated the association of viral replication with mTOR activity in peripheral lymphocytes of renal transplant recipients. A flow-cytometry based assay for the measurement of Thr389 p70S6k phosphorylation, a surrogate marker of the mTOR pathway was established. Forty-eight adult renal transplant recipients were recruited to measure p70S6k activity in their peripheral blood mononuclear cells. This data set in conjunction with information concerning previous replication of BKPyV and HCMV was examined for correlations. Episodes of BKPyV replication were significantly associated with increased p70S6k phosphorylation in CD4+ T lymphocytes (p = 0.0002) and CD19+ B lymphocytes (p = 0.0073). HCMV infection of patients with a high-risk HCMV constellation of donor and recipient (D+/R-) was associated with increased p70S6k phosphorylation in CD19+ B lymphocytes (p = 0.0325). These associations were found to be independent of the trough levels of the immunosuppressive drugs. Conclusion: P70S6k phosphorylation in peripheral lymphocytes is associated with BKPyV reactivations and to a lesser extent with HCMV infections in renal transplant recipients.

S6K1/S6 axis-regulated lymphocyte activation is important for adaptive immune response of Nile tilapia.

Ribosomal protein S6 kinase beta-1 (S6K1) is a serine/threonine kinase downstream of the mechanistic target of rapamycin (mTOR) pathway, and plays crucial roles in immune regulation. Although remarkable progress has been achieved with a mouse model, how S6K1 regulates adaptive immunity is largely unknown in early vertebrates. In this study, we identified an S6K1 from Nile tilapia Oreochromis niloticus (OnS6K1), and further investigated its potential regulatory role on the adaptive immunity of this fish species. Both sequence and structure of OnS6K1 were highly conserved with its homologs from other vertebrates and invertebrates. OnS6K1 was widely expressed in immune tissues, and with a relative higher expression level in the liver, spleen and head kidney. At the adaptive immune stage of Nile tilapia that infected with Aeromonas hydrophila, mRNA expression of OnS6K1 and its downstream effector S6 was significantly up-regulated in spleen lymphocytes. Meanwhile, their phosphorylation level was also enhanced during this process, suggesting that S6K1/S6 axis participated in the primary response of anti-bacterial adaptive immunity in Nile tilapia. Furthermore, after spleen lymphocytes were activated by the T cell-specific mitogen PHA or lymphocytes agonist PMA in vitro, mRNA and phosphorylation levels of S6K1 were elevated, and phosphorylation of S6 was also enhanced. Once S6K1 activity was blocked by a specific inhibitor, both mRNA and phosphorylation levels of S6 were severely impaired. More importantly, blockade of S6K1/S6 axis reduced the expression of T cell activation marker IFN-γ and CD122 in PHA-activated spleen lymphocytes, indicating the essential role of S6K1/S6 axis in regulating T cell activation of Nile tilapia. Together, our study suggests that S6K1 and its effector S6 regulate lymphocyte activation of Nile tilapia, and in turn promote lymphocyte-mediated adaptive immunity. This study enriched the mechanism of adaptive immune response in teleost and provided useful clues to understand the evolution of adaptive immune system.

"Immune TOR-opathies," a Novel Disease Entity in Clinical Immunology.

Primary immunodeficiencies (PIDs) represent a group of mostly monogenic disorders caused by loss- or gain-of-function mutations in over 340 known genes that lead to abnormalities in the development and/or the function of the immune system. However, mutations in different genes can affect the same cell-signaling pathway and result in overlapping clinical phenotypes. In particular, mutations in the genes encoding for members of the phosphoinositide3-kinase (PI3K)/AKT/mTOR/S6 kinase (S6K) signaling cascade or for molecules interacting with this pathway have been associated with different PIDs that are often characterized by the coexistence of both immune deficiency and autoimmunity. The serine/threonine kinase mechanistic/mammalian target of rapamycin (mTOR), which acts downstream of PI3K and AKT, is emerging as a key regulator of immune responses. It integrates a variety of signals from the microenvironment to control cell growth, proliferation, and metabolism. mTOR plays therefore a central role in the regulation of immune cells' differentiation and functions. Here, we review the different PIDs that share an impairment of the PI3K/AKT/mTOR/S6K pathway and we propose to name them "immune TOR-opathies" by analogy with a group of neurological disorders that has been originally defined by PB Crino and that are due to aberrant mTOR signaling (1). A better understanding of the role played by this complex intracellular cascade in the pathophysiology of "immune TOR-opathies" is crucial to develop targeted therapies.

Galectin-9 inhibits TLR7-mediated autoimmunity in murine lupus models.

Uncontrolled secretion of type I IFN, as the result of endosomal TLR (i.e., TLR7 and TLR9) signaling in plasmacytoid DCs (pDCs), and abnormal production of autoantibodies by B cells are critical for systemic lupus erythematosus (SLE) pathogenesis. The importance of galectin-9 (Gal-9) in regulating various autoimmune diseases, including lupus, has been demonstrated. However, the precise mechanism by which Gal-9 mediates this effect remains unclear. Here, using spontaneous murine models of lupus (i.e., BXSB/MpJ and NZB/W F1 mice), we demonstrate that administration of Gal-9 results in reduced TLR7-mediated autoimmune manifestations. While investigating the mechanism underlying this phenomenon, we observed that Gal-9 inhibits the phenotypic maturation of pDCs and B cells and abrogates their ability to mount cytokine responses to TLR7/TLR9 ligands. Importantly, immunocomplex-mediated (IC-mediated) and neutrophil extracellular trap-mediated (NET-mediated) pDC activation was inhibited by Gal-9. Additionally, the mTOR/p70S6K pathway, which is recruited by both pDCs and B cells for TLR-mediated IFN secretion and autoantibody generation, respectively, was attenuated. Gal-9 was found to exert its inhibitory effect on both the cells by interacting with CD44.

Salmonella overcomes tumor immune tolerance by inhibition of tumor indoleamine 2, 3-dioxygenase 1 expression.

Over the past decades, Salmonella has been proven capable of inhibiting tumor growth. It can specifically target tumors and due to its facultative anaerobic property, can be more penetrative than other drug therapies. However, the molecular mechanism by which Salmonella inhibits tumor growth is still incompletely known. The antitumor therapeutic effect mediated by Salmonella is associated with an inflammatory immune response at the tumor site and a T cell-dependent immune response. Many tumors have been proven to have a high expression of indoleamine 2, 3-dioxygenase 1 (IDO), which is a rate-limiting enzyme that catalyzes tryptophan to kynurenine, thus causing immune tolerance within the tumor microenvironment. With decreased expression of IDO, increased immune response can be observed, which might be helpful when developing cancer immunotherapy. The expression of IDO was decreased after tumor cells were infected with Salmonella. In addition, Western blot analysis showed that the expression levels of phospho-protein kinase B (P-AKT), phospho-mammalian targets of rapamycin (P-mTOR), and phospho-p70 ribosomal s6 kinase (P-p70s6K) in tumor cells were decreased after Salmonella infection. In conclusion, our results indicate that Salmonella inhibits IDO expression and plays a crucial role in anti-tumor therapy, which might be a promising strategy combined with other cancer treatments.

p(7°S6K¹) in the TORC1 pathway is essential for the differentiation of Th17 Cells, but not Th1, Th2, or Treg cells in mice.

The TORC1 pathway is necessary for ribosomal biogenesis and initiation of protein translation. Furthermore, the differentiation of Th1 and Th17 cells requires TORC1 activity. To investigate the role of the TORC1 pathway in the differentiation of Th1 and/or Th17 cells in more detail, we compared the differentiation capacity of naïve T cells from wild type and p70(S6K1) knockout mice. Expression of many of the genes associated with Th17-cell differentiation, such as IL17a, IL17f, and IL-23R, were reduced in p70(S6K1) knockout mice. In contrast, the development of Th1, Th2, and Treg cells was unaffected in the absence of p70(S6K1) . Furthermore, expression of the major transcription factor in Th17-cell differentiation, retinoic acid receptor-related orphan receptor gamma T, remained unchanged. However, the acetylation of histone 3 at the promoters of IL17a and IL17f was reduced in the absence of p70(S6K1) . In accordance with the in vitro data, the kinetics, but not the development, of EAE was affected with the loss of p70(S6K1) expression. Collectively, our findings suggested that both in vitro and in vivo differentiation of Th17 cells were positively regulated by p70(S6K1) .

PLD1 activation mediates Amb a 1-induced Th2-associated cytokine expression via the JNK/ATF-2 pathway in BEAS-2B cells.

The purpose of this study was to identify the role of phospholipase D1 (PLD1) in Amb a 1-induced IL-5 and IL-13 expression. When BEAS-2B cells were stimulated with Amb a 1, PLD activity increased, and knockdown of PLD1 decreased Amb a 1-induced IL-5 and IL-13 expression. Amb a 1 also activated the PLCγ/p70S6K/JNK pathway. Furthermore, Amb a 1-induced PLD activation was also attenuated by PLCγ inhibition, and knockdown of PLD1 decreased Amb a 1-induced activation of P70S6K and JNK. When ATF-2 activity was blocked with ATF-2 siRNA, Amb a 1-induced IL-5 and IL-13 expression was completely abolished, indicating that ATF-2 is a transcriptional factor required for the expression of IL-5 and IL-13 in response to Amb a 1. Taken together, we suggest that PLD1 acts as an important regulator in Amb a 1-induced expression of IL-5 and IL-13 via a PLCγ/p70S6K/JNK/ATF-2 pathway in BEAS-2B cells.

Methylome Analysis in Chickens Immunized with Infectious Laryngotracheitis Vaccine.

In this study we investigated the methylome of chickens immunized with Infectious laryngotracheitis (ILT) vaccine derived from chicken embryos. Methyl-CpG binding domain protein-enriched genome sequencing (MBD-Seq) method was employed in the detection of the 1,155 differentially methylated regions (DMRs) across the entire genome. After validation, we ascertained the genomic DMRs distribution and annotated them regarding genes, transcription start sites (TSS) and CpG islands. We found that global DNA methylation decreased in vaccinated birds, presenting 704 hypomethylated and 451 hypermethylated DMRs, respectively. Additionally, we performed an enrichment analysis detecting gene networks, in which cancer and RNA post-transcriptional modification appeared in the first place, followed by humoral immune response, immunological disease and inflammatory disease. The top four identified canonical pathways were EIF2 signaling, regulation of EIF4 and p70S6K signaling, axonal guidance signaling and mTOR signaling, providing new insight regarding the mechanisms of ILT etiology. Lastly, the association between DNA methylation and differentially expressed genes was examined, and detected negative correlation in seventeen of the eighteen genes.

IL-37b suppresses T cell priming by modulating dendritic cell maturation and cytokine production via dampening ERK/NF-κB/S6K signalings.

Interleukin 37b (IL-37b) plays a key role in suppressing immune responses, partially by modulating the function of dendritic cells (DCs). However, the precise mechanisms are still largely unknown. Here, we investigated the effects of IL-37b on DC maturation and T cell responses induced by DCs, and explored the involved signaling pathways. It was found that IL-37b down-regulated the expressions of co-stimulatory molecules CD80 and CD86 on DCs in vitro. At the same time, the expressions of pro-inflammatory cytokines, such as TNF-α and IL-6, were suppressed, while the expression of the T cell inhibitory cytokine TGF-ß was increased in IL-37b-treated DCs. In addition, the activation effect of DCs on T cells was impaired by IL-37b. We further revealed that extracellular single-regulated kinase (ERK), nuclear factor-κB (NF-κB), and mTOR-S6K signaling pathways were involved in the inhibition of DCs induced by IL-37b. This was confirmed by the similarly suppressive effect of chemical inhibitors against NF-κB, ERK, and S6K on the expressions of IL-6 and TNF-α in DCs. In conclusion, these results demonstrated that IL-37b suppressed DC maturation and immunostimulatory capacity in T cell priming by involving in ERK, NF-κB, and S6K-based inhibitory signaling pathways.

Expression of S6K1 in human visceral adipose tissue is upregulated in obesity and related to insulin resistance and inflammation.

The ribosomal protein S6 kinase 1 (S6K1) is a component of the insulin signalling pathway that has been proposed as a key molecular factor in insulin resistance development under conditions of nutrient overload. The aim was to evaluate the involvement of S6K1 in obesity as well as to explore their association with visceral adipose tissue (VAT) inflammation. Samples obtained from 40 subjects were used. Gene expression levels of RPS6KB1 and key inflammatory markers were analysed in VAT. The effect of insulin on transcript levels of RPS6KB1 in human differentiated adipocytes was also explored. RPS6KB1 mRNA levels in VAT were increased (P < 0.05) in obese patients. Insulin treatment significantly enhanced (P < 0.01) gene expression levels of RPS6KB1 and a positive association (P < 0.05) of RPS6KB1 expression with different markers of insulin resistance was observed. Moreover, RPS6KB1 gene expression levels were positively correlated with VAT gene expression levels of the inflammatory markers CCL2, CD68, MMP2, MMP9, VEGFA and CHI3L1 as well as with mRNA levels of MTOR and MAPK8, representative players involved in signalling pathways related to S6K1. The increased levels of S6K1 in obesity and its positive association with insulin resistance and inflammation suggest a role for this protein in the changes that take place in VAT in obesity establishing a link between inflammation and a higher risk for the development of metabolic diseases.

Inducible costimulator facilitates T-dependent B cell activation by augmenting IL-4 translation.

The inducible costimulator (ICOS) is highly expressed in follicular helper T (Tfh) cells, a subset of CD4 T cells that migrate into the B cell zone and facilitate germinal center reactions. Although ICOS is known to play a critical role in forming the Tfh cell population during immune responses, its contribution to the effector functions of Tfh cells remains unclear. Using activated mouse splenic CD4 T cells we demonstrate that ICOS assists TCR-mediated signal transduction by potentiating the PI3K-AKT-mTOR signaling cascade that leads to hyper-phosphorylation of p70S6K and 4E-BP1, events that are known to augment cap-dependent mRNA translation. Consequently, ICOS costimulation promotes the formation of polysomes on IL-4 mRNA in a PI3K-dependent manner. Furthermore, we show that the supply of IL-4 becomes a limiting factor for T-dependent B cell activation during in vitro co-culture when the ICOS-PI3K signaling axis is disrupted in T cells. This ICOS costimulation-dependent translational control may ensure targeted delivery of IL-4 to cognate B cells during T-B collaborations in the germinal center.

Jurkat cell proliferation is suppressed by Chlamydia (Chlamydophila) pneumoniae infection accompanied with attenuation of phosphorylation at Thr389 of host cellular p70S6K.

Chlamydia (Chlamydophila) pneumoniae infects T lymphocytes and multiplies within them. Our previous studies have indicated that C. pneumoniae infection suppresses proliferation of peripheral blood mononuclear cells stimulated with Staphylococcus-enterotoxin B; however, the mechanism of suppression was unclear. In this study, we explored the molecular mechanism involved in C. pneumoniae infection by using human acute T cell leukemia cell line, Jurkat E6-1. Proliferation of Jurkat cells was suppressed in an m.o.i.-dependent manner by C. pneumoniae infection. The suppression by the infection was particularly evident during the initial 24h of the infection, and down modulation of cyclin D3 protein levels were observed at the same time period by immunoblot analysis. The suppression of the Jurkat cell proliferation and the down modulation of cyclin D3 protein level were only induced by viable C. pneumoniae infection, not by exposure to UV-killed or heat-killed C. pneumoniae. Phosphorylations at Thr308 and Ser473 of AKT were induced by C. pneumoniae infection; however, phosphorylation at Thr389 of the downstream kinase, p70S6K was inhibited by unidentified mechanism associated with C. pneumoniae infection. Taking into account that G1 arrest of the C. pneumoniae infected Jurkat cells were not observed and that p70S6K is one of the most important regulators of protein synthesis, it was suggested that the suppression of Jurkat cell proliferation by C. pneumoniae was at least in part mediated by down modulation of protein synthesis through attenuation of Thr389 phosphorylation of p70S6K.

The mTOR signaling pathway in the prefrontal cortex is compromised in major depressive disorder.

Recent studies demonstrate that rapid antidepressant response to ketamine is mediated by activation of the mammalian target of rapamycin (mTOR) signaling pathway, leading to increased synaptic proteins in the prefrontal cortex (PFC) of rats. Our postmortem studies indicate robust deficits in prominent postsynaptic proteins including N-methyl-d-aspartate (NMDA) receptor subunits (NR2A, NR2B), metabotropic glutamate receptor subtype 5 (mGluR5) and postsynaptic density protein 95kDa (PSD-95) in the PFC in major depressive disorder (MDD). We hypothesize that deficits in the mTOR-dependent translation initiation pathway contribute to the molecular pathology seen in the PFC of MDD subjects, and that a rapid reversal of these abnormalities may underlie antidepressant activity. The majority of known translational regulation occurs at the level of initiation. mTOR regulates translation initiation via its downstream components: p70-kDa ribosomal protein S6 kinase (p70S6K), and eukaryotic initiation factors 4E and 4B (eIF4E and eIF4B). In this study, we examined the expression of mTOR and its core downstream signaling targets: p70S6K, eIF4E, and eIF4B in the PFC of 12 depressed subjects and 12 psychiatrically healthy controls using Western blot. Levels of eIF4E phosphorylated at serine 209 (p-eIF4E-Ser209) and eIF4B phosphorylated at serine 504 (p-eIF4B-Ser504) were also examined. Adjacent cortical tissue samples from both cohorts of subjects were used in our previous postmortem analyses. There was a significant reduction in mTOR, p70S6K, eIF4B and p-eIF4B protein expression in MDD subjects relative to controls. No group differences were observed in eIF4E, p-eIF4E or actin levels. Our findings show deficits in mTOR-dependent translation initiation in MDD particularly via the p70S6K/eIF4B pathway, and indicate a potential association between marked deficits in synaptic proteins and dysregulation of mTOR signaling in MDD.

mTOR signaling is activated by FLT3 kinase and promotes survival of FLT3-mutated acute myeloid leukemia cells.

Activating mutations of the FLT3 gene mediate leukemogenesis, at least in part, through activation of PI3K/AKT. The mammalian target of rapamycin (mTOR)-Raptor signaling pathway is known to act downstream of AKT. Here we show that the mTOR effectors, 4EBP1, p70S6K and rpS6, are highly activated in cultured and primary FLT3-mutated acute myeloid leukemia (AML) cells. Introduction of FLT3-ITD expressing constitutively activated FLT3 kinase further activates mTOR and its downstream effectors in BaF3 cells. We also found that mTOR signaling contributes to tumor cell survival, as demonstrated by pharmacologic inhibition of PI3K/AKT/mTOR, or total silencing of the mTOR gene. Furthermore, inhibition of FLT3 kinase results in downregulation of mTOR signaling associated with decreased survival of FLT3-mutated AML cells. These findings suggest that mTOR signaling operates downstream of activated FLT3 kinase thus contributing to tumor cell survival, and may represent a promising therapeutic target for AML patients with mutated-FLT3.

Inactivation of mammalian target of rapamycin increases STAT1 nuclear content and transcriptional activity in alpha4- and protein phosphatase 2A-dependent fashion.

Target of rapamycin (TOR) is a highly conserved serine/threonine kinase that controls cell growth, primarily via regulation of protein synthesis. In Saccharomyces cerevisiae, TOR can also suppress the transcription of stress response genes by a mechanism involving Tap42, a serine/threonine phosphatase subunit, and the transcription factor Msn2. A physical association between mammalian TOR (mTOR) and the transcription factor signal transducer and activator of transcription-1 (STAT1) was recently identified in human cells, suggesting a similar role for mTOR in the transcription of interferon-gamma-stimulated genes. In the current study, we identified a macromolecular protein complex composed of mTOR, STAT1, the Tap42 homologue alpha4, and the protein phosphatase 2A catalytic subunit (PP2Ac). Inactivation of mTOR enhanced its association with STAT1 and increased STAT1 nuclear content in PP2Ac-dependent fashion. Depletion of alpha4, PP2A, or mTOR enhanced the induction of early (i.e. IRF-1) and late (i.e. caspase-1, hiNOS, and Fas) STAT1-dependent genes. The regulation of IRF-1 or caspase-1 by mTOR was independent of other known mTOR effectors p70 S6 kinase and Akt. These results describe a new role for mTOR and alpha4/PP2A in the control of STAT1 nuclear content, and the expression of interferon-gamma-sensitive genes involved in immunity and apoptosis.

Sphingosine-1-phosphate induced mTOR-activation is mediated by the E3-ubiquitin ligase PAM.

The signaling pathways that are regulated by sphingosine-1-phosphate (S1P) and mammalian target of rapamycin (mTOR) modulate cell growth, mitogenesis and apoptosis in various cell types and are of major interest for the development of new cancer therapeutics. Previous reports show that S1P can cross-activate the mTOR pathway although the mechanisms that connect both pathways are still unknown. We found that S1P-treatment activates mTOR in several cancer cell lines and primary cells. The activation was independent of ERK, Akt and PI3-kinase, but instead was mediated by the E3 ubiquitin ligase Protein Associated with Myc (PAM). Increased intracellular PAM concentrations facilitated S1P- and insulin-induced mTOR activation as well as p70S6K and 4EBP1 phosphorylation while genetic deletion of PAM decreased S1P- and insulin-induced mTOR activation. PAM activated by facilitating the GDP/GTP-exchange of Rheb which is an activator of mTOR. In conclusion we show that PAM is a novel regulator of the mTOR pathway and that PAM may directly activate Rheb as a guanosine exchange factor (GEF).

Toll-like receptor-mediated induction of type I interferon in plasmacytoid dendritic cells requires the rapamycin-sensitive PI(3)K-mTOR-p70S6K pathway.

Robust production of type I interferon (IFN-alpha/beta) in plasmacytoid dendritic cells (pDCs) is crucial for antiviral immunity. Here we show involvement of the mammalian target of rapamycin (mTOR) pathway in regulating interferon production by pDCs. Inhibition of mTOR or its 'downstream' mediators, the p70 ribosomal S6 protein kinases p70S6K1 and p70S6K2, during pDC activation by Toll-like receptor 9 (TLR9) blocked the interaction of TLR9 with the adaptor MyD88 and subsequent activation of the interferon-regulatory factor IRF7, which resulted in impaired IFN-alpha/beta production. Microarray analysis confirmed that inhibition of mTOR by the immunosuppressive drug rapamycin suppressed antiviral and anti-inflammatory gene expression. Consistent with this, targeting rapamycin-encapsulated microparticles to antigen-presenting cells in vivo resulted in less IFN-alpha/beta production in response to CpG DNA or the yellow fever vaccine virus strain 17D. Thus, mTOR signaling is crucial in TLR-mediated IFN-alpha/beta responses by pDCs.

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.

Functional characterization of a maize ribosomal S6 protein kinase (ZmS6K), a plant ortholog of metazoan p70(S6K).

Ribosomal protein S6 (S6rp) is phosphorylated by the p70S6K enzyme in mammals, under mitogen/IGF regulation. This event has been correlated with an increase in 5'TOP mRNA translation. In this research, a maize S6 kinase (ZmS6K) was isolated from maize (Zea mays L.) embryonic axes by human p70S6K antibody immunoprecipitation. This enzyme, a 62 kDa peptide, proved to be specific for S6rp phosphorylation, as revealed by in vivo and in vitro kinase activity using either the 40S ribosomal subunit or the RSK synthetic peptide as the substrates. ZmS6K activation was achieved by phosphorylation on serine/threonine residues. Specific phospho-Threo recognition by the p70S6K antibody directed to target phospho-Threo residue 389 correlated with ZmS6K activation. The ZmS6K protein content remained almost steady during maize seed germination, whereas the ZmS6K activity increased during this process, consistent with Zm6SK phosphorylation. Addition of insulin to germinating maize axes proved to increase ZmS6K activity and the extent of S6rp phosphorylation. These events were blocked by rapamycin, an inhibitor of the insulin signal transduction pathway in mammals, at the TOR (target of rapamycin) enzyme level. We conclude that ZmS6K is a kinase, structurally and functionally ortholog of the mammalian p70S6K, responsible for in vivo S6rp phosphorylation in maize. Its activation is induced by insulin in a TOR-dependent manner by phosphorylation on conserved serine/threonine residues.