Human leptin promotes survival of human circulating blood monocytes prone to apoptosis by activation of p42/44 MAPK pathway

Leptin, the adipocyte-secreted hormone, is known to function as an immunomodulatory regulator. Thus, we have recently found that human leptin promotes stimulation and proliferation of human peripheral blood mononuclear cells. Besides, we have also demonstrated that leptin triggers PI3K and p42/44 MAPK signaling pathways. In the present work, we sought to study the possible effect of leptin on cell survival and apoptosis, as well as the mechanisms underlying these effects. We have cultured human PBMC in serum-free conditions to assess the effect of leptin on cell survival and apoptosis. We have assayed the early phases of apoptosis by flow cytometric detection of phosphatidylserine expression using fluorescein isothiocyanate (FITC)-labelled Annexin V, simultaneously with dye exclusion of propidium iodide (PI), to discriminate intact cells, apoptotic, and necrotic cells. We have found that leptin promotes dose-dependent cell survival of monocytes after 24-96 h of serum-free culture. This effect of leptin on monocyte survival was completely reversed by blocking p42/44 MAPK activation employing the MEK inhibitor PD98059, whereas it was not affected by PI3K inhibition using Wortmannin. Leptin promotes this survival effect by preventing the apoptosis of monocyte cells, via MAPK activation. Thus, p42/44 MAPK inhibition, using PD98059, but not PI3K inhibition, employing Wortmannin, blocked the protective effect of leptin preventing apoptosis of monocytes cultured in the absence of serum. These data suggest that leptin is a trophic factor for the survival of blood monocytes and this effect is mediated by the p42/44 MAPK pathway.     

Thrombin Enhances NGF-Mediated Neurite Extension via Increased and Sustained Activation of p44/42 MAPK and p38 MAPK

Rapid neurite remodeling is fundamental to nervous system development and plasticity. It involves neurite extension that is regulated by NGF through PI3K/AKT, p44/42 MAPK and p38 MAPK. It also involves neurite retraction that is regulated by the serine protease, thrombin. However, the intracellular signaling pathway by which thrombin causes neurite retraction is unknown. Using the PC12 neuronal cell model, we demonstrate that thrombin utilizes the PI3K/AKT pathway for neurite retraction in NGF-differentiated cells. Interestingly, however, we found that thrombin enhances NGF-induced neurite extension in differentiating cells. This is achieved through increased and sustained activation of p44/42 MAPK and p38 MAPK. Thus, thrombin elicits opposing effects in differentiated and differentiating cells through activation of distinct signaling pathways: neurite retraction in differentiated cells via PI3K/AKT, and neurite extension in differentiating cells via p44/42 MAPK and p38 MAPK. These findings, which also point to a novel cooperative role between thrombin and NGF, have significant implications in the development of the nervous system and the disease processes that afflicts it as well as in the potential of combined thrombin and NGF therapy for impaired learning and memory, and spinal cord injury which all require neurite extension and remodeling.     

Protean agonism of the lysophosphatidic acid receptor-1 with Ki16425 reduces nerve growth factor-induced neurite outgrowth in pheochromocytoma 12 cells

We report here a novel role for the constitutively active lysophosphatidic acid receptor-1 (LPA(1)) receptor in providing Gbetagamma subunits for use by the Trk A receptor. This enhances the ability of nerve growth factor (NGF) to promote signalling and cell response. These conclusions were based on three lines of evidence. Firstly, the LPA(1) receptor was co-immunoprecipitated with the Trk A receptor from lysates, suggesting that these proteins form a complex. Secondly, Ki16425, a selective protean agonist of the LPA(1) receptor, decreased constitutive basal and LPA-induced LPA(1) receptor-stimulated GTPgammaS binding. Ki16425 reduced the LPA-induced activation of p42/p44 mitogen activated protein kinase (MAPK), while acting as a weak stimulator of p42/p44 MAPK on its own, properties typical of a protean agonist. Significantly, Ki16425 also reduced the NGF-induced stimulation of p42/p44 MAPK and inhibited NGF-stimulated neurite outgrowth. Thirdly, the over-expression of the C-terminal GRK-2 peptide, which sequesters Gbetagamma subunits, reduced the NGF-induced activation of p42/p44 MAPK. In contrast, the stimulation of PC12 cells with LPA leads to a predominant G(i)alpha2-mediated Trk A-independent activation of p42/p44 MAPK, where Gbetagamma subunits play a diminished role. These findings suggest a novel role for the constitutively active LPA(1) receptor in regulating NGF-induced neuronal differentiation.     

p75 neurotrophin receptor is required for constitutive and NGF-induced survival signalling in PC12 cells and rat hippocampal neurones

We have previously shown that nerve growth factor (NGF)-induced activation of nuclear factor-kappaB increased neuronal expression of Bcl-xL, an anti-apoptotic Bcl-2 family protein. In the present study we determined the role of the p75 neurotrophin receptor in constitutive and NGF-induced survival signalling. Treatment of rat pheochromocytoma (PC12) cells with a blocking anti-rat p75 antibody or inhibition of p75 expression by antisense oligonucleotides reduced constitutive and NGF-induced bcl-xL expression. Treatment with the blocking anti-p75 antibody also inhibited NGF-induced activation of the survival kinase Akt. Inhibition of phosphatidylinositol-3-kinase (PI3 kinase) activity or overexpression of a dominant-negative mutant of Akt kinase inhibited NGF-induced nuclear factor-kappaB activation. Activation of Akt kinase by NGF was also observed in PC12nnr5 cells and cultured rat hippocampal neurones which both lack significant TrkA expression. Treatment of hippocampal neurones with the blocking anti-p75 antibody inhibited constitutive and NGF-induced Bcl-xL expression, activation of Akt, and blocked the protective effect of NGF against excitotoxic and apoptotic injury. Our data suggest that the p75 neurotrophin receptor mediates constitutive and NGF-induced survival signalling in PC12 cells and hippocampal neurones, and that these effects are mediated via the PI3-kinase pathway.     

BK-induced cytosolic phospholipase A2 expression via sequential PKC-delta, p42/p44 MAPK, and NF-kappaB activation in rat brain astrocytes

Bradykinin (BK), an inflammatory mediator, has been shown to induce cytosolic phospholipase A2 (cPLA2) expression implicating in inflammatory responses in various cell types. However, the detailed mechanisms underlying BK-induced cPLA2 expression in astrocytes remain unclear. RT-PCR and Western blotting analysis showed that BK induced the expression of cPLA2 mRNA and protein, which was inhibited by Hoe140, suggesting the involvement of B2 BK receptors, confirmed by immunofluorescence staining using anti-B2 BK receptor antibody. BK-induced cPLA2 expression and phosphorylation of p42/p44 MAPK was attenuated by PD98059, indicating the involvement of MEK1/2-p42/p44 MAPK in these responses. BK-induced cPLA2 expression might be due to the translocation of NF-kappaB into nucleus which was inhibited by Hoe140, helenalin, and PD98059, implying the involvement of NF-kappaB. Moreover, BK-induced cPLA2 expression was attenuated by rottlerin, suggesting that PKC-delta might be involved in these responses. This hypothesis was supported by the transfection with a dominant negative plasmid of PKC-delta significantly attenuated BK-induced response. In addition, BK-stimulated translocation of PKC-delta from cytosol to membrane fraction was inhibited by rottlerin but not by PD98059, indicating that PKC-delta might be an upstream component of p42/p44 MAPK. Accordingly, BK-induced phosphorylation of p42/p44 MAPK was attenuated by rottlerin but not by helenalin. These results suggest that in RBA-1 cells, BK-induced cPLA2 expression was sequentially mediated through activation of PKC-delta, p42/p44 MAPK, and NF-kappaB. Understanding the regulation of cPLA2 expression induced by BK in astrocytes might provide a new therapeutic strategy of brain injury and inflammatory diseases.     

BK-induced COX-2 expression via PKC-delta-dependent activation of p42/p44 MAPK and NF-kappaB in astrocytes

Bradykinin (BK) is an inflammatory mediator, elevated levels in the region of several brain injury and inflammatory diseases. It has been shown to induce cyclooxygenase-2 (COX-2) expression implicating in inflammatory responses in various cell types. However, the signaling mechanisms underlying BK-induced COX-2 expression in astrocytes remain unclear. First, RT-PCR and Western blotting analysis showed that BK induced the expression of COX-2 mRNA and protein, which was inhibited by B(2) BK receptor antagonist Hoe140, suggesting the involvement of B(2) BK receptors. BK-induced COX-2 expression and translocation of PKC-delta from cytosol to membrane fraction were inhibited by rottlerin, suggesting that PKC-delta might be involved in these responses. This hypothesis was further supported by the transfection with a dominant negative plasmid of PKC-delta significantly blocked BK-induced COX-2 expression. BK-stimulated p42/p44 MAPK phosphorylation, COX-2 mRNA expression, and prostaglandin E(2) (PGE(2)) release were attenuated by PD98059, indicating the involvement of MEK/p42/p44 MAPK in this pathway. Accordingly, BK-stimulated phosphorylation of p42/p44 MAPK was attenuated by rottlerin, indicating that PKC-delta might be an upstream component of p42/p44 MAPK. Moreover, BK-induced COX-2 expression might be mediated through the translocation of NF-kappaB into nucleus which was blocked by helenalin, rottlerin and PD98059, implying the involvement of NF-kappaB. These results suggest that in RBA-1 cells, BK-induced COX-2 expression and PGE(2) release was sequentially mediated through PKC-delta-dependent activation of p42/p44 MAPK and NF-kappaB. Understanding the regulation of COX-2 expression and PGE(2) release induced by BK in astrocytes might provide a new therapeutic strategy of brain injury and inflammatory diseases.     

Regulation of cholinergic gene expression by nerve growth factor depends on the phosphatidylinositol-3'-kinase pathway

Nerve growth factor (NGF) exerts anti-apoptotic, trophic and differentiating actions on sympathetic neurons and cholinergic cells of the basal forebrain and activates the expression of genes regulating the synthesis and storage of the neurotransmitter acetylcholine (ACh). We have been studying the intracellular signaling pathways involved in this process. Although, in the rat pheochromocytoma cell line PC12, NGF strongly activates the mitogen-activated protein kinase (MAPK) pathway, prolonged inhibition of MAPK kinase (MEK) activity by PD98059 or U0126 did not affect the ability of NGF to up-regulate choline acetyltransferase (ChAT) or to increase intracellular ACh levels. In contrast, the treatment with the phosphatidylinositol 3'-kinase (PI3K) inhibitor LY294002, but not with its inactive analogue LY303511, completely abolished the NGF-induced production of ACh. Inhibition of PI3K also eliminated the NGF effect on the intracellular ACh level in primary cultures of septal neurons from E18 mouse embryos. Blocking the PI3K pathway prevented the activation of cholinergic gene expression, as demonstrated in RT/PCR assays and in transient transfections of PC12 cells with cholinergic locus promoter-luciferase reporter constructs. These results indicate that the PI3K pathway, but not the MEK/MAPK pathway, is the mediator of NGF-induced cholinergic differentiation.     

p42/p44 mitogen-activated protein kinase activation is involved in prostaglandin F2alpha-induced interleukin-6 synthesis in osteoblasts.

Prostaglandin F2alpha (PGF2alpha) significantly induced p42/p44 mitogen-activated protein (MAP) kinase activity in osteoblast-like MC3T3-E1 cells. PD98059, a selective inhibitor of MAP kinase kinase, inhibited PGF2alpha-induced interleukin-6 (IL-6) synthesis as well as PGF2alpha-induced p42/p44 MAP kinase activation. PD98059 suppressed the IL-6 synthesis induced by 12-O-tetradecanoylphorbol-13-acetate (TPA), a protein kinase C (PKC) activator, or NaF, an activator of heterotrimeric GTP-binding protein, as well as the p42/p44 MAP kinase activation by TPA or NaF. Calphostin C, a highly potent and specific inhibitor of PKC, inhibited the PGF2alpha-induced p42/p44 MAP kinase activity. These results strongly suggest that PKC-dependent p42/p44 MAP kinase activatioin is involved in PGF2alpha-induced IL-6 synthesis in osteoblasts.     

Activation of mitogen-activated protein kinases p42/44, p38, and stress-activated protein kinases in myelo-monocytic cells by Treponema lipoteichoic acid

We have shown previously that phenol/water extracts derived from two novel Treponema species, Treponema maltophilum, and Treponema brennaborense, resembling lipoteichoic acid (LTA), induce cytokines in mononuclear cells. This response was lipopolysaccharide binding-protein (LBP)-dependent and involved Toll-like receptors (TLRs). Here we show that secretion of tumor necrosis factor-alpha induced by Treponema culture supernatants and extracted LTA was paralleled by an LBP-dependent phosphorylation of mitogen-activated protein kinases (MAPKs) p42 and p44, and p38, as well as the stress-activated protein kinases c-Jun N-terminal kinases 1 and 2. Phosphorylation of p42/44 correlated with an increase of activity, and tumor necrosis factor-alpha levels were significantly reduced by addition of inhibitors of p42/44 and p38, PD 98059 and SB 203580, respectively. Treponeme LTA differed from bacterial lipopolysaccharide regarding time course of p42/44 phosphorylation, exhibiting a prolonged activation of MAPKs. Furthermore, MAPK activation and cytokine induction failed to be strictly correlated. Involvement of TLR-4 for phosphorylation of p42/44 was shown employing the neutralizing anti-murine TLR-4 antibody MTS 510. In TLR-2-negative U373 cells, the compounds studied differed regarding MAPK activation with T. maltophilum leading to a stronger activation. In summary, the data presented here show that treponeme LTA are able to activate the MAPK and stress-activated protein kinase pathway involving LBP and TLR-4.     

Thyroid hormones regulate DNA-synthesis and cell-cycle proteins by activation of PKCalpha and p42/44 MAPK in chick embryo hepatocytes

The molecular mechanism by which thyroid hormones exert their effects on cell growth is still unknown. In this study, we used chick embryo hepatocytes at different stages of development as a model to investigate the effect of the two thyroid hormones, T3 and T4, and of their metabolite T2, on the control of cell proliferation. We observed that T2 provokes increase of DNA-synthesis as well as T3 and T4, independently of developmental stage. We found that this stimulatory effect on the S phase is reverted by specific inhibitors of protein kinase C (PKC) and p42/44 mitogen-activated protein kinase (p42/44 MAPK), Ro 31-8220 or PD 98059. Furthermore, the treatment with thyroid hormones induces the activation of PKCalpha and p42/44 MAPK, suggesting their role as possible downstream mediators of cell response mediated by thyroid hormones. The increase of DNA-synthesis is well correlated with the increased levels of cyclin D1 and cdk4 that control the G1 phase, and also with the activities of cell-cycle proteins involved in the G1 to S phase progression, such as cyclin E/A-cdk2 complexes. Interestingly, the activity of cyclin-cdk2 complexes is strongly repressed in the presence of PKC and p42/44 MAPK inhibitors. In conclusion, we demonstrated that the thyroid hormones could modulate different signaling pathways that are able to control cell-cycle progression, mainly during G1/S transition.     

Evidence that inhibition of p44/42 mitogen-activated protein kinase signaling is a factor in proteasome inhibitor-mediated apoptosis

The proteasome is emerging as a target for cancer therapy because small molecule inhibitors of its catalytic activity induce apoptosis in both in vitro and in vivo models of human malignancies and are proving to have efficacy in early clinical trials. To further elucidate the mechanism of action of these inhibitors, their impact on signaling through the p44/42 mitogen-activated protein kinase (MAPK) pathway was studied. Proteasome inhibition with either carbobenzoxy-leucyl-leucyl-phenylalaninal or lactacystin led to a loss of dually phosphorylated, activated p44/42 MAPK in A1N4-myc human mammary and MDA-MB-231 breast carcinoma cells in a dose- and time-dependent fashion. This correlated with an induction of the dual specificity MAPK phosphatases (MKP)-1 and -2, and blockade of MKP induction using either actinomycin D or Ro-31-8220 significantly decreased loss of activated p44/42 MAPK. Inhibition of p44/42 MAPK signaling by use of the MAPK kinase inhibitors PD 98059 or U0126, or by use of a dominant negative MAPK construct, enhanced proteasome inhibitor-mediated apoptosis. Conversely, activation of MAPK by epidermal growth factor, or use of a mutant MAPK resistant to MKP-mediated dephosphorylation, inhibited apoptosis. These studies support a role for inactivation of signaling through the p44/42 MAPK pathway in proteasome inhibitor-mediated apoptosis.     

Sustained phosphorylation of mitogen-activated protein kinase is required for basic fibroblast growth factor-mediated axonal branch formation in cultured rat hippocampal neurons

Basic fibroblast growth factor (bFGF) has been reported to promote the formation of axonal branches in cultured brain neurons. In the present study, we investigated whether the mitogen-activated protein kinase (MAPK) cascade was involved in this action of bFGF in cultured rat hippocampal neurons. Recombinant human bFGF (0.1-10 ng/ml) induced phosphorylation of p44/42 MAPK in a concentration and time-dependent manner. The phosphorylation of p44/42 MAPK occurred rapidly within 5 min after addition of bFGF, and lasted for 48 h. The bFGF-induced phosphorylation of p44/42 MAPK and axonal branch formation were both blocked by simultaneous addition of U0126 and PD98059, specific inhibitors of MAPK kinases. Furthermore, when U0126 and PD98059 were added 24 h after bFGF, phosphorylation of p44/42 m MAPK was decreased, and axonal branch formation was stopped. These results suggest that sustained activation of the MAPK cascade is required for bFGF-mediated axonal branch formation.     

Urocortin protects the heart from reperfusion injury via upregulation of p42/p44 MAPK signaling pathway

Reperfusion of ischemic myocardium is essential for tissue salvage but paradoxically contributes to cell death. We hypothesized that activation of potential survival pathways such as p42/p44 MAPK may prevent lethal reperfusion injury. Urocortin is a peptide factor that affects the p42/p44 MAPK signaling pathway. Both isolated and in vivo rat heart models were used to examine the potential for urocortin to prevent reperfusion injury. Isolated rat hearts underwent 35-min regional ischemia and 2-h reperfusion, with urocortin perfused for 20 min from the onset of reperfusion. In the in vivo study, urocortin was administered as an intravenous bolus 3 min before reperfusion with a protocol of 25-min regional ischemia and 2-h reperfusion. Blockade of the p42/p44 MAPK pathway with the inhibitor PD-98059 was used in both models. Urocortin attenuated lethal reperfusion-induced injury both in vitro and in vivo via a p42/p44 MAPK-dependent mechanism. Furthermore, Western blot analysis demonstrated the ability of urocortin to directly upregulate this signaling pathway. In conclusion, we believe that the p42/p44 MAPK-dependent signaling pathway represents an important survival mechanism against reperfusion injury.