Mycobacterium tuberculosis-induced expression of Leukotactin-1 is mediated by the PI3-K/PDK1/Akt signaling pathway

Chemokines function in the migration of circulating leukocytes to regions of inflammation, and have been implicated in chronic inflammatory conditions including mycobacterial infection. We investigated whether Leukotactin-1 (Lkn-1), a novel member of the CC-chemokines, is involved in the immune response of macrophages against Mycobacterium tuberculosis (MTB). In PMA-differentiated THP-1 cells, MTB infection increased mRNA expression of Lkn-1 in a dose-dependent manner. Lkn-1 induction peaked 12 h after infection, then declined gradually and returned to its basal level at 72 h. Secretion of Lkn-1 was elevated by MTB infection. The increase in expression and secretion of Lkn-1 caused by MTB was reduced in cells treated with inhibitors of phosphatidylinositol 3-kinase (PI3-K), 3-phosphoinositide-dependent kinase 1 (PDK1) and Akt. MTB-induced Akt phosphorylation was blocked by treatment with a PI3-K inhibitor or a PDK1 inhibitor, implying that PI3-K, PDK1, and Akt are associated with the signaling pathway that up-regulates Lkn-1 in response to MTB. These results suggest that Lkn-1 is novel member of the group of chemokines that is released by macrophages infected with MTB.     

The differential interaction of p38 MAP kinase and tumor necrosis factor-alpha in human alveolar macrophages and monocytes induced by Mycobacterium tuberculois

Cellular signaling by TNF-alpha is mediated through activation of mitogen activated protein (MAP) kinases. In particular, p38 MAP kinase is activated in mononuclear phagocytes and may be important in sustaining TNF-alpha activity. Here, we compared the activation and mutual regulation of p38 MAP kinase and TNF-alpha by MTB in human alveolar macrophages (AM) and blood monocytes (MN). AM and autologous MN were prepared, and stimulated by MTB at 1:1 (bacteria/cell). MAP kinase activation was assessed by immunoprecipitation and kinase activity. TNF-alpha mRNA was assessed by real-time RT-PCR, and TNF-alpha immunoreactivity was assessed by ELISA. MTB-induced p38MAP kinase rapidly in AM as compared to MN, and inhibition of p38 MAP kinase by SB203580 reduced both TNF-alpha mRNA and protein. Activation of ERK (1/2) by MTB followed similar kinetics in both AM and MN. TNF-alpha produced by MTB sustained p38 MAP kinase activation in MN only. These data suggest that interaction of resident pulmonary macrophages and the more immature MN with MTB differ with regard to both p38 MAP kinase activation and TNF-alpha expression.     

Mechanisms of serum potentiation of GM-CSF production by human airway smooth muscle cells

Inflammation and vascular leakage are prevalent in asthma. This study aimed to elucidate the mechanisms involved in serum potentiation of cytokine-induced granulocyte macrophage colony stimulating factor (GM-CSF) production by human airway smooth muscle cells and to identify possible factors responsible. Serum-deprived cells at low density were stimulated with TNF-alpha and IL-1beta for 24 h. Human AB serum (10%), inhibitors of RNA and protein synthesis or specific signaling molecules, or known smooth muscle mitogens were then added for 24 h. Culture supernatants were analyzed for GM-CSF levels, and cells were harvested to assess viability, cell cycle progression, GM-CSF-specific mRNA content, and p38 phosphorylation. Serum potentiated GM-CSF release when added before, together with (maximal), or after the cytokines. The potentiation involved both new GM-CSF-specific mRNA production and protein synthesis. The mitogens IGF, PDGF, and thrombin all potentiated GM-CSF release, and neutralizing antibodies for EGF, IGF, and PDGF reduced the serum potentiation. Inhibitor studies ruled as unlikely the involvement of p70(S6kinase) and the MAPK p42/p44, two signaling pathways implicated in proliferation, and the involvement of the MAPK JNK, while establishing roles for p38 MAPK and NF-kappaB in the potentiation of GM-CSF release. Detection of significant p38 phosphorylation in response to serum stimulation, through Western blotting, further demonstrated the involvement of p38. These studies have provided evidence to support p38 being targeted to interrupt the cycle of inflammation, vascular leakage and cytokine production in asthma.     

Protein Kinase Activity of Phosphoinositide 3-Kinase Regulates Cytokine-Dependent Cell Survival

The dual specificity protein/lipid kinase, phosphoinositide 3-kinase (PI3K), promotes growth factor-mediated cell survival and is frequently deregulated in cancer. However, in contrast to canonical lipid-kinase functions, the role of PI3K protein kinase activity in regulating cell survival is unknown. We have employed a novel approach to purify and pharmacologically profile protein kinases from primary human acute myeloid leukemia (AML) cells that phosphorylate serine residues in the cytoplasmic portion of cytokine receptors to promote hemopoietic cell survival. We have isolated a kinase activity that is able to directly phosphorylate Ser585 in the cytoplasmic domain of the interleukin 3 (IL-3) and granulocyte macrophage colony stimulating factor (GM-CSF) receptors and shown it to be PI3K. Physiological concentrations of cytokine in the picomolar range were sufficient for activating the protein kinase activity of PI3K leading to Ser585 phosphorylation and hemopoietic cell survival but did not activate PI3K lipid kinase signaling or promote proliferation. Blockade of PI3K lipid signaling by expression of the pleckstrin homology of Akt1 had no significant impact on the ability of picomolar concentrations of cytokine to promote hemopoietic cell survival. Furthermore, inducible expression of a mutant form of PI3K that is defective in lipid kinase activity but retains protein kinase activity was able to promote Ser585 phosphorylation and hemopoietic cell survival in the absence of cytokine. Blockade of p110α by RNA interference or multiple independent PI3K inhibitors not only blocked Ser585 phosphorylation in cytokine-dependent cells and primary human AML blasts, but also resulted in a block in survival signaling and cell death. Our findings demonstrate a new role for the protein kinase activity of PI3K in phosphorylating the cytoplasmic tail of the GM-CSF and IL-3 receptors to selectively regulate cell survival highlighting the importance of targeting such pathways in cancer.     

Human intestinal epithelial crypt cell survival and death: Complex modulations of Bcl-2 homologs by Fak, PI3-K/Akt-1, MEK/Erk, and p38 signaling pathways

To investigate the mechanisms responsible for survival and apoptosis/anoikis in normal human intestinal epithelial crypt cells, we analyzed the roles of various signaling pathways and cell adhesion on the expression of six Bcl-2 homologs (Bcl-2, Bcl-XL, Mcl-1, Bax, Bak, Bad) in the well established HIEC-6 cell model. Pharmacological inhibitors and/or dominant-negative constructs were used to inhibit focal adhesion kinase (Fak) and p38 isoforms, as well as the phosphatidylinositol 3'-kinase (PI3-K)/Akt-1 and mitogen-activated protein kinase [MAPK] kinase (MEK)/extracellular regulated kinases (Erk) pathways. Cell adhesion was disrupted by antibody-inhibition of integrin binding or forced cell suspension. The activation levels of studied kinase pathways were also analyzed. Herein, we report that beta1 integrins, Fak, and the PI3-K/Akt-1 pathway, but not beta4 integrins or the MEK/Erk pathway, are crucial for the survival of HIEC-6 cells. Conversely, p38beta, but not p38alpha or gamma, is required for the induction of apoptosis/anoikis in HIEC-6 cells. However, each of the signaling molecules/pathways analyzed were found to affect distinctively the individual expression of the Bcl-2 homologs studied. For example, the inhibition of the PI3-K/Akt-1 pathway down-regulated Bcl-XL, Mcl-1, and Bad, while at the same time up-regulating Bax, whereas the inhibition of Fak up-regulated both Bax and Bak, down-regulated Bad, and did not affect the other Bcl-2 homologs analyzed. These results indicate that integrins, Fak, PI3-K/Akt-1, MEK/Erk, and p38 isoforms perform distinct roles in the regulation of HIEC-6 cell survival and/or death. In addition, our data show that the functions performed by these molecules/pathways in promoting cell survival or apoptosis/anoikis translate into complex, differential modulations of individual Bcl-2 homologs.     

A positive role of the PI3-K/Akt signaling pathway in PC12 cell differentiation

Activation of phosphatidylinositol 3-kinase (PI3-K) is considered to be a key event upon stimulation of cells with growth factors. Akt is known to be a downstream target of PI3-K when it is activated by nerve growth factor (NGF). NGF induces cell differentiation of PC12 cells as indicated by neurite outgrowth. In order to investigate the role of PI3-K/Akt in NGF-induced differentiation of PC12 cells, we generated cells ectopically expressing constitutively activated (CA), wild type (WT) and dominant negative (DN) forms of Akt. NGF-induced neurite outgrowth was greatly accelerated in the cells expressing CA-Akt, and dramatically inhibited in those expressing DN-Akt. Pre-treatment with an Akt inhibitor, ML-9 [1-(5-chloronaphthalene-1-sulfonyl)-1H- hexahydro-1,4-diazepine], inhibited NGF-induced Akt phosphorylation as well as neurite outgrowth but did not markedly affect the activities of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK). The PI3-K inhibitors wortmannin and LY294002 blocked NGF-induced Akt phosphorylation as well as neurite outgrowth. These results indicate that PI3-K/Akt is a positive regulator of NGF-induced neuronal differentiation in PC12 cells.     

Excretory/secretory products from plerocercoids of Spirometra erinaceieuropaei suppress interleukin-1beta gene expression in murine macrophages

The present study shows that ES products from plerocercoids of Spirometra erinaceieuropaei suppressed interleukin-1beta mRNA expression in lipopolysaccharide-stimulated RAW 264.7 macrophages in the absence or presence of a cyclic AMP analogue, dibutyryl cyclic AMP. Investigation using the inhibitors of mitogen-activated protein kinase (MAPK) pathways revealed that extracellular signal-regulated protein kinase 1/2 and p38 mitogen-activated protein kinase pathways are crucial for full induction of interleukin-1beta mRNA expression. ES products additionally suppressed interleukin-1beta mRNA expression in the cells treated with p38 mitogen-activated protein kinase inhibitor (SB203580) or extracellular signal-regulated protein kinase 1/2 inhibitor (PD98059). Western blot analysis showed that dibutyryl cyclic AMP enhanced lipopolysaccharide-induced phosphorylation of extracellular signal-regulated protein kinase 1/2, p38 mitogen-activated protein kinase and cyclic AMP responsive element binding protein (CREB) and, in turn, we demonstrated that ES products reduced the lipopolysaccharide and dibutyryl cyclic AMP-induced phosphorylation of extracellular signal-regulated protein kinase 1/2 and p38 mitogen-activated protein kinase, but not cyclic AMP responsive element binding protein. These data demonstrate that ES products from the plerocercoids of S. erinaceieuropaei may evade induction of interleukin-1beta mRNA by inhibiting extracellular signal-regulated protein kinase 1/2 and p38 mitogen-activated protein kinase pathways in lipopolysaccharide and/or dibutyryl cyclic AMP-stimulated macrophages.     

Curcumin inhibits 19-kDa lipoprotein of Mycobacterium tuberculosis induced macrophage apoptosis via regulation of the JNK pathway

Recently, synthetic curcumin analogs are reported as potential active compounds against Mycobacterium tuberculosis (MTB). During the process of MTB infection, macrophages show increased apoptosis. The candidate virulence factors such as 19-kDa lipoprotein secreted by the MTB (P19) strongly influences macrophages by activation of Toll-like receptor 2 (TLR2) and mitogen-activated protein kinases (MAPKs). It has been reported that curcumin could affect the apoptosis of tumor cells via regulation of MAPKs. However, its effect on the P19-induced apoptosis of macrophages is unclear. This study investigates the effect of curcumin on the MAPKs signaling and apoptosis in human macrophages. The results showed that curcumin and P19 induced macrophage apoptosis in a time- and dose-dependent manner Low doses of curcumin (10 and 20 μM) protected macrophages from P19 induced apoptosis, accompanied by decreased production of cytokines and reduced activation of the c-Jun amino-terminal kinase (JNK) and p38 MAPK. The protective effect of curcumin on P19 induced apoptosis of macrophages were enhanced by treatment with the JNK-specific inhibitors, whereas SB203580, the inhibitor of p38 MAPK had no effect. Curcumin had no effect on the activity of extracellular signal-regulated protein kinase (ERK). Taken together, our data show that the JNK pathway, but not the p38 or ERK pathway, plays an important role in the protective effect of curcumin against P19 induced macrophage apoptosis, and regulation of the JNK pathway may partially elucidate the anti-tuberculosis activity of curcumin.     

IL-3 and IL-4 activate cyclic nucleotide phosphodiesterases 3 (PDE3) and 4 (PDE4) by different mechanisms in FDCP2 myeloid cells

In FDCP2 myeloid cells, IL-4 activated cyclic nucleotide phosphodiesterases PDE3 and PDE4, whereas IL-3, granulocyte-macrophage CSF (GM-CSF), and phorbol ester (PMA) selectively activated PDE4. IL-4 (not IL-3 or GM-CSF) induced tyrosine phosphorylation of insulin-receptor substrate-2 (IRS-2) and its association with phosphatidylinositol 3-kinase (PI3-K). TNF-alpha, AG-490 (Janus kinase inhibitor), and wortmannin (PI3-K inhibitor) inhibited activation of PDE3 and PDE4 by IL-4. TNF-alpha also blocked IL-4-induced tyrosine phosphorylation of IRS-2, but not of STAT6. AG-490 and wortmannin, not TNF-alpha, inhibited activation of PDE4 by IL-3. These results suggested that IL-4-induced activation of PDE3 and PDE4 was downstream of IRS-2/PI3-K, not STAT6, and that inhibition of tyrosine phosphorylation of IRS molecules might be one mechnism whereby TNF-alpha could selectively regulate activities of cytokines that utilized IRS proteins as signal transducers. RO31-7549 (protein kinase C (PKC) inhibitor) inhibited activation of PDE4 by PMA. IL-4, IL-3, and GM-CSF activated mitogen-activated protein (MAP) kinase and protein kinase B via PI3-K signals; PMA activated only MAP kinase via PKC signals. The MAP kinase kinase (MEK-1) inhibitor PD98059 inhibited IL-4-, IL-3-, and PMA-induced activation of MAP kinase and PDE4, but not IL-4-induced activation of PDE3. In FDCP2 cells transfected with constitutively activated MEK, MAP kinase and PDE4, not PDE3, were activated. Thus, in FDCP2 cells, PDE4 can be activated by overlapping MAP kinase-dependent pathways involving PI3-K (IL-4, IL-3, GM-CSF) or PKC (PMA), but selective activation of PDE3 by IL-4 is MAP kinase independent (but perhaps IRS-2/PI3-K dependent).     

Apoptosis-signal regulating kinase-1 is involved in the low potassium-induced activation of p38 mitogen-activated protein kinase and c-Jun in cultured cerebellar granule neurons

Previously, we reported that p38, which belongs to the mitogen-activated protein kinase (MAPK) superfamily, has an important role in the induction of apoptosis of cultured cerebellar granule neurons. However, the molecular mechanisms upstream of p38 activation remain unclear. Apoptosis signal-regulating kinase-1 (ASK1), a MAPK kinase kinase (MAPKKK) protein, is known to activate both c-Jun N-terminal kinase (JNK) and p38 via MAPK kinase (MKK) 4/7 and MKK3/6, respectively. Here, we examined whether ASK1 is involved in the activation of p38 in the low potassium (LK)-induced apoptosis of cerebellar granule neurons. We found that ASK1 was activated after a change to LK medium. In addition, the expression of ASK1-KM, a dominant-negative form of ASK1, using an adenovirus system was found to inhibit the activation of p38 and c-Jun and to prevent apoptosis. On the other hand, the expression of ASK1-DeltaN, a constitutively active form of ASK1, activated p38 and c-Jun, but not JNK, another possible downstream target of ASK1. Furthermore, we examined the relationship between phosphatidylinositol 3-kinase (PI3-K) and ASK1. The addition of LY294002, a specific inhibitor of PI3-K, enhanced the ASK1 activity. These results indicate that ASK1 works downstream of PI3-K to regulate the p38-c-Jun pathway and apoptosis in cultured cerebellar granule neurons.     

A recombinant human G-CSF/GM-CSF fusion protein from E. coli showing colony stimulating activity on human bone marrow cells

Granulocyte-Macrophage colony stimulating factor (GM-CSF) and Granulocyte colony stimulating factor (G-CSF) are cytokines involved in the differentiation of bone marrow progenitor cells into myeloid cells. They also activate mature myeloid cells to mediate a variety of antimicrobial activities and inflammatory responses. Recombinant GM-CSF and G-CSF proteins have been used to treat various diseases including cancer and hematopoietic diseases and to isolate peripheral blood progenitor cells for bone marrow transplantation. A plasmid construct expressing recombinant human G-CSF/GM-CSF fusion protein has now been prepared by linking the human G-CSF and GM-CSF coding regions and the recombinant fusion protein has been successfully expressed in E. coli. The recombinant human G-CSF/GM-CSF fusion protein was extracted and purified from the cellular inclusion and refolded into the biologically active form to show colony stimulating activity. The recombinant fusion protein exhibited colony stimulating activity on human bone marrow cell cultures, indicating that the linkage of GM-CSF and G-CSF by a linker peptide may not interrupt activities of the cytokines in the fusion protein. The colony forming unit of the fusion protein was also higher than those of the cultures treated with the same molar numbers of the recombinant human GM-CSF and G-CSF separately, which suggests that the fusion protein presumably retains both G-CSF and GM-CSF activities.