A 'pure' chemoattractant formylpeptide analogue triggers a specific signalling pathway in human neutrophil chemotaxis

As it has not yet been established whether the second messengers involved in the neutrophil response have identical or specific signalling requirements for each physiological function, protein kinase C (PKC) isoforms and mitogen activated protein kinases (MAPKs) were studied in human chemotaxis triggered by the full agonist for-Met-Leu-Phe-OMe (fMLP-OMe) and the 'pure' chemoattractant for-Thp-Leu-Ain-OMe [Thp1,Ain3] analogue. Experiments were performed in the presence or absence of extracellular Ca2+, known to be an important modulator of second messengers. Our data demonstrate that specific PKC beta1 translocation and p38 MAPK phosphorylation are strongly associated with the chemotactic response of the neutrophils triggered by both peptides, while Ca2+ is not necessary for chemotaxis to occur. PKC and MAPK inhibitors were used in Western blotting assays and in cell locomotion experiments to investigate if the MAPK signalling pathway was controlled by PKC activation. The most important finding emerging from this study is that PKC and MAPK activate the chemotactic function of human neutrophils by two independent pathways.     

OX2R activation induces PKC-mediated ERK and CREB phosphorylation

Deficiencies in brain orexins and components of mitogen activated protein kinase (MAPK) signaling pathway have been reported in either human depression or animal model of depression. Brain administration of orexins affects behaviors toward improvement of depressive symptoms. However, the documentation of endogenous linkage between orexin receptor activation and MAPK signaling pathway remains to be insufficient. In this study, we report the effects of orexin 2 receptor (OX2R) activation on cell signaling in CHO cells over-expressing OX2R and in mouse hypothalamus cell line CLU172. Short-term extracellular signal-regulated kinase (ERK) phosphorylation and long-term cyclic adenosine monophosphate (cAMP) response element binding protein (CREB) phosphorylation were subsequently observed in CHO cells that over-express OX2R while 20 min of ERK phosphorylation was significantly detected in mouse adult hypothalamus neuron cell line CLU172. Orexin A, which can also activate OX2R, mediated ERK phosphorylation was as the same as orexin B in CHO cells. A MAPK inhibitor eliminated ERK phosphorylation but not CREB phosphorylation in CHO cells. Also, ERK and CREB phosphorylation was not mediated by protein kinase A (PKA) or calmodulin kinase (CaMK). However, inhibition of protein kinase C (PKC) by GF 109203X eliminated the phosphorylation of ERK and CREB in CHO cells. A significant decrease in ERK and CREB phosphorylation was observed with 1 μM GF 109203X pre-treatment indicating that the conventional and novel isoforms of PKC are responsible for CREB phosphorylation after OX2R activation. In contrast, ERK phosphorylation induced by orexin B in CLU172 cells cannot be inhibited by 1 μM of protein kinase C inhibitor. From above observation we conclude that OX2R activation by orexin B induces ERK and CREB phosphorylation and orexin A played the same role as orexin B. Several isoforms of PKC may be involved in prolonged CREB phosphorylation. Orexin B induced ERK phosphorylation in mouse hypothalamus neuron cells differs from CHO cell line and cannot be inhibited by PKC inhibitor GF 109203X. And hypothalamus neuron cells may use different downsteam pathway for orexin B induced ERK phosphorylation. This result supports findings that orexins might have anti-depressive roles.     

TrkA signalling pathways in human airway smooth muscle cell proliferation

NGF may play a role in airway inflammation and hyperresponsiveness. We studied its possible involvement in airway remodelling and report here its proliferative effect and its receptor and signalling pathways in human airway smooth muscle cells in culture (HASMC). Proliferation of HASMC induced by NGF (0.1-10 pM) was assessed by the XTT and BrdU techniques with and without kinase inhibitors. Immunoprecipitation and Western blotting were used to study phosphorylation of TrkA and MAPK. NGF caused dose-dependent proliferation of HASMC and induced TrkA phosphorylation, both abolished by the tyrosine-kinase inhibitor K252a. PI3K and JNK inhibitors had no effect. PKC inhibitors partially inhibited NGF-induced proliferation and totally abolished p38 phosphorylation but did not affect ERK1/2 phosphorylation. The rafK inhibitor decreased NGF-induced proliferation, and totally abolished ERK1/2 phosphorylation, but did not affect p38 phosphorylation. This finding was confirmed by the decrease of NGF-induced proliferation after treatment with inhibitors of the p38 or of ERK1/2 pathways. In conclusion, NGF activation of the TrkA receptor involves two distinct signalling pathways: PKC selectively activates p38, and the ras/raf pathway selectively activates ERK1/2. Both are necessary to induce HASMC proliferation.     

Nerve growth factor activates brain-derived neurotrophic factor promoter IV via extracellular signal-regulated protein kinase 1/2 in PC12 cells

Brain-derived neurotrophic factor (BDNF) is a neuromodulator of nociceptive responses in the dorsal root ganglia (DRG) and spinal cord. BDNF synthesis increases in response to nerve growth factor (NGF) in trkA-expressing small and medium-sized DRG neurons after inflammation. Previously we demonstrated differential activation of multiple BDNF promoters in the DRG following peripheral nerve injury and inflammation. Using reporter constructs containing individual promoter regions, we investigated the effect of NGF on the multiple BDNF promoters, and the signaling pathway by which NGF activates these promoters in PC12 cells. Although all the promoters were activated 2.4-7.1-fold by NGF treatment, promoter IV gave the greatest induction. The p38 mitogen-activated protein kinase (MAPK) inhibitor, SB203580, phosphatidylinositol 3-kinase (PI-3K) inhibitor, LY294003, protein kinase A (PKA) inhibitor, H89, and protein kinase C (PKC) inhibitor, chelerythrine, had no effect on activation of promoter IV by NGF. However, activation was completely abolished by the MAPK kinase (MEK) inhibitors, U0126 and PD98059. In addition, these inhibitors blocked NGF-induced phosphorylation of extracellular signal-regulated protein kinase (ERK) 1/2. Taken together, these results suggest that the ERK1/2 pathway activates BDNF promoter IV in response to NGF independently of NGF-activated signaling pathways involving PKA and PKC.     

Evidence for a role of MEK and MAPK during signal transduction by protein kinase C zeta.

Protein kinase C zeta (zeta PKC) is critically involved in the control of a number of cell functions, including proliferation and nuclear factor kappa B (NF-kappa B) activation. Previous studies indicate that zeta PKC is an important step downstream of Ras in the mitogenic cascade. The stimulation of Ras initiates a kinase cascade that culminates in the activation of MAP kinase (MAPK), which is required for cell growth. MAPK is activated by phosphorylation by another kinase named MAPK kinase (MEK), which is the substrate of a number of Ras-activated serine/threonine kinases such as c-Raf-1 and B-Raf. We show here that MAPK and MEK are activated in vivo by an active mutant of zeta PKC, and that a kinase-defective dominant negative mutant of zeta PKC dramatically impairs the activation of both MEK and MAPK by serum and tumour necrosis factor (TNF alpha). The stimulation of other kinases, such as stress-activated protein kinase (SAPK) or p70S6K, is shown here to be independent of zeta PKC. The importance of MEK/MAPK in the signalling mechanisms activated by zeta PKC was addressed by using the activation of a kappa B-dependent promoter as a biological read-out of zeta PKC.     

Mitogen-activated protein kinase signalling pathways triggered by the hepatitis C virus envelope protein E2: implications for the prevention of infection

OBJECTIVE: Hepatitis C virus (HCV) is a major pathogenic factor of liver diseases. During HCV infection, interaction of the envelope protein E2 of the virion, with target cells, is a crucial process for viral penetration into the cell and its propagation. We speculate that such interaction may trigger early signalling events required for HCV infection. MATERIALS AND METHODS: Human liver cell line L-02 was treated with HCV E2. The kinase phosphorylation levels of mitogen-activated protein kinase (MAPK) signalling pathways in the treated cells were analyzed by Western blotting. The proliferation of the E2-treated cells was evaluated by MTT assay. RESULTS: HCV E2 was shown to be an efficient activator for MAPK pathways. Levels of phosphorylation of upstream kinases Raf-1 and MEK1/2 were seen to be elevated following E2 treatment and similarly, phosphorylation levels of downstream kinases MAPK/ERK and p38 MAPK also increased in response to E2 treatment, and specificity of kinase activation by E2 was confirmed. E2-induced MAPK/ERK activation was inhibited by the MEK1/2 inhibitor U0126 in a concentration-dependent manner. Blockage of relevant cellular receptors reduced activation of Raf-1, MEK1/2, MAPK/ERK and p38 MAPK by E2, indicating efflux of the E2 signal from extracellular to the intracellular spaces. Thus, kinase cascades of MAPK pathways were continuously affected by E2 presence. Moreover, enhancement of cell proliferation by E2 appeared to be associated with the dynamic phosphorylation of MAPK/ERK and p38 MAPK. CONCLUSION: These results suggest that MAPK signalling pathways triggered by E2 may be a potential target for prevention of HCV infection.     

Borrelia burgdorferi BBB07 interaction with integrin alpha3beta1 stimulates production of pro-inflammatory mediators in primary human chondrocytes

Borrelia burgdorferi , the causative agent of Lyme disease, activates multiple signalling pathways leading to induction of pro-inflammatory mediators at sites of inflammation. Binding of B. burgdorferi to integrin α₃β₁ on human chondrocytes activates signalling leading to release of several pro-inflammatory mediators, but the B. burgdorferi protein that binds integrin α₃β₁ and elicits this response has remained unknown. A search of the B. burgdorferi genome for a canonical integrin binding motif, the RGD (Arg–Gly–Asp) tripeptide, revealed several candidate ligands for integrins. In this study we show that one of these candidates, BBB07, binds to integrin α₃β₁ and inhibits attachment of intact B. burgdorferi to the same integrin. BBB07 is expressed during murine infection as demonstrated by recognition by infected mouse sera. Recombinant purified BBB07 induces pro-inflammatory mediators in primary human chondrocyte cells by interaction with integrin α₃β₁. This interaction is specific, as P66, another integrin ligand of B. burgdorferi , does not activate signalling through α₃β₁. In summary, we have identified a B. burgdorferi protein, BBB07, that interacts with integrin α₃β₁ and stimulates production of pro-inflammatory mediators in primary human chondrocyte cells.     

Oxidative stress-induced intestinal epithelial cell apoptosis is mediated by p38 MAPK

Free oxygen radicals are involved in the pathogenesis of necrotizing enterocolitis (NEC) in premature infants. The stress-activated p38 mitogen-activated protein kinase (MAPK) has been implicated in gut injury. Here, we found that phosphorylated p38 was detected primarily in the villus tips of normal intestine, whereas it was expressed in the entire mucosa in NEC. H(2)O(2) treatment resulted in a rapid phosphorylation of p38 MAPK and subsequent apoptosis of rat intestinal epithelial (RIE)-1 cells; this induction was attenuated by treatment with SB203580, a selective p38 MAPK inhibitor, or transfection with p38alpha siRNA. Moreover, SB203580 also blocked H(2)O(2)-induced PKC activation. In contrast, the PKC inhibitor (GF109203x) did not affect p38 activation, indicating that p38 MAPK activation occurs upstream of PKC activation in H(2)O(2)-induced apoptosis. H(2)O(2) treatment also decreased mitochondrial membrane potential; pretreatment with SB203580 attenuated this response. Our study demonstrates that the p38 MAPK/PKC pathway plays an important role as a pro-apoptotic cellular signaling during oxidative stress-induced intestinal epithelial cell injury.     

The signalling profile of recombinant human orexin-2 receptor

Orexin-A and orexin-B orchestrate their diverse central and peripheral effects via two G-protein coupled receptors, OX1R and OX2R, which activate multiple G-proteins. In many tissues, orexins activate extracellular signal-regulated kinase (ERK(1/2)) and p38 mitogen-activated protein kinase (MAPK); however, the mechanism by which OX2R alone mediates MAPK activation is not understood. This study describes the intracellular signalling pathways involved in OX2R-mediated ERK(1/2) and p38 MAPK activation. In HEK-293 cells stably over-expressing recombinant human OX2R, orexin-A/B resulted in a rapid, dose and time dependent increase in activation of ERK(1/2) and p38 MAPK, with maximal activation at 10 min for ERK(1/2) and 30 min for p38 MAPK. Using dominant-negative G-proteins and selective inhibitors of intracellular signalling cascades, we determined that orexin-A and orexin-B induced ERK(1/2) and p38 MAPK activation through multiple G-proteins and different intracellular signalling pathways. ERK(1/2) activation involves Gq/phospholipase C (PLC)/protein kinase C (PKC), Gs/adenylyl cyclase (AC)/cAMP/protein kinase A (PKA) and Gi cascades; however, the Gq/PLC/PKC pathway, as well as PKA is not required for OX2R-mediated p38 MAPK activation. Interestingly, orexin-B-induced ERK(1/2) activation is predominantly mediated through the Gq/PLC/PKC pathway. In conclusion, this is the first comprehensive signalling study of the human OX2R recombinant receptor, showing ERK(1/2) and p38 MAPK activation are regulated by differential signalling pathways in HEK-293 cells, and that the ERK(1/2) activation is severely affected by naturally occurring mutants associated with narcolepsy. Moreover, it is evident that the human OX2R has ligand specific effects, with orexin-B being more potent in this transfected system and this distinct modulation of the MAPKs through OX2R, may translate to the regulation of diverse biological actions of orexins.     

Borrelia burgdorferi lipoprotein BmpA activates pro-inflammatory responses in human synovial cells through a protein moiety

Borrelia burgdorferi invasion of mammalian joints results in genesis of Lyme arthritis. Other than spirochete lipids, existence of protein antigens, which are abundant in joints and participate in B. burgdorferi-induced host inflammatory response, is unknown. Here, we report that major products of the B. burgdorferi basic membrane protein (bmp) A/B operon that are induced in murine and human joints, possess inflammatory properties. Compared to the wild type B. burgdorferi, an isogenic bmpA/B mutant induced significantly lower levels of pro-inflammatory cytokines TNF-alpha and IL-1beta in cultured human synovial cells, which could be restored using bmpA/B-complemented mutants, and more directly, upon addition of recombinant BmpA, but not BmpB or control spirochete proteins. Non-lipidated and lipidated versions of BmpA induced similar levels of cytokines, and remained unaffected by treatment with lipopolysaccharide inhibitor, polymyxin B. The bmpA/B mutant was also impaired in the induction of NF-kappaB and p38 MAP kinase signaling pathways in synovial cells, which were activated by non-lipidated BmpA. These results show that a protein moiety of BmpA can induce cytokine responses in synovial cells via activation of the NF-kappaB and p38 MAP kinase pathways and thus, could potentially contribute to the genesis of Lyme arthritis.     

Intracellular signal transduction pathways induced by leptin in C2C12 cells

As experimental evidence suggests that leptin may have direct effects on peripheral tissues, we investigated some of the transductional molecules induced by leptin in C2C12 cells. In immunoprecipitation experiments using anti-p85 antibodies (a regulatory subunit of phosphatidylinositol-3-kinase; PI3K), we observed a significant increase in PI3K activity. Immunoblot analyses showed that Akt, GSK3, ERK1, ERK2, and p38 mitogen-activated protein kinase (p38 MAPK) phosphorylation significantly increased after leptin treatment. Protein kinase C (PKC)-zeta was also activated by leptin, as documented by an immunocomplex kinase assay and immunoblotting experiments. The treatment of C2C12 cells with Wortmannin before leptin administration inhibited induction of the phosphorylation of ERKs (extracellular signal-regulated kinases) but not that of p38 MAPK, whereas pre-treatment with a PKC-zeta inhibitor partially decreased ERK phosphorylation. Taken together, our in vitro results further support the hypothesis that leptin acts acutely on skeletal muscle tissue through some of the components of insulin signalling, including PKC-zeta.