Phosphorylation of p42/44(MAPK) by various signal transduction pathways activates cytosolic phospholipase A(2) to variable degrees

Arachidonic acid has been implicated to play a role in physiological and pathophysiological processes and is selectively released by the 85-kDa cytosolic phospholipase A(2) (cPLA(2)). The activity of cPLA(2) is regulated by calcium, translocating the enzyme to its substrate, and by phosphorylation by a mitogen-activated protein kinase (MAPK) family member and a MAPK-activated protein kinase. In this study, the signal transduction pathways in growth factor-induced phosphorylation of p42/44(MAPK) and cPLA(2) activation were investigated in Her14 fibroblasts. p42/44(MAPK) in response to epidermal growth factor was not only phosphorylated via the Raf-MEK pathway but mainly through protein kinase C (PKC) or a related or unrelated kinase in which the phosphorylated p42/44(MAPK) corresponded with cPLA(2) activity. Serum-induced phosphorylation of p42/44(MAPK) also corresponded with cPLA(2) activity but is predominantly mediated via Raf-MEK and partly through PKC or a related or unrelated kinase. In contrast, activation of PKC by phorbol ester did not result in increased cPLA(2) activity, while p42/44(MAPK) is phosphorylated, mainly via Raf-MEK and through MEK. Moreover, p42/44(MAPK) phosphorylation is present in quiescent and proliferating cells, and p42/44(MAPK) is entirely phosphorylated via Raf-MEK, but it only corresponds to cPLA(2) activity in the former cells. Collectively, these data show that p42/44(MAPK) in proliferating, quiescent, and stimulated cells is phosphorylated by various signal transduction pathways, suggesting the activation of different populations of p42/44(MAPK) and cPLA(2).     

Assessment of the extracellular and intracellular actions of sphingosine 1-phosphate by using the p42/p44 mitogen-activated protein kinase cascade as a model.

We have investigated the extracellular and intracellular actions of sphingosine 1-phosphate (S1P) by using cultured airway smooth muscle cells. We have demonstrated that exogenous S1P elicited an activation of mitogen-activated protein kinase (p42/p44 MAPK) that was abolished by pertussis toxin (0.1 microg/mL, 24 h), which was used to inactivate Gi. The effect of exogenous S1P might therefore be attributed to an action at a putative Gi-coupled receptor. The regulation of the p42/p44 MAPK cascade by S1P was also shown to include a protein kinase C (PKC)-dependent intermediate step. Platelet-derived growth factor (PDGF) stimulates intracellular S1P formation and was therefore used to evaluate the intracellular action of S1P. This has previously been investigated by others using the sphingosine kinase inhibitors D,L-threo-dihydrosphingosine and N,N-dimethylsphingosine. We have demonstrated here that both inhibitors block the PDGF-dependent activation of p42/p44 MAPK. However, both are also PKC inhibitors, which might account for their effect because PDGF utilises PKC as an intermediate in the regulation of the p42/p44 MAPK cascade. Significantly, sphingosine, which is the substrate of sphingosine kinase and a PKC inhibitor, blocked the activation of p42/p44 MAPK by PDGF with an almost identical concentration dependence compared with D,L-threo-dihydrosphingosine and N,N-dimethylsphingosine. Therefore, the use of so-called sphingosine kinase inhibitors might lead to misleading interpretations because of their additional effect on PKC. Other approaches, such as oligodeoxynucleotide anti-sense against sphingosine kinase, are required to address the intracellular role of S1P.     

Substance P-induced activation of p42/44 mitogen-activated protein kinase associated with cell proliferation in human tracheal smooth muscle cells

Substance P (SP) released from sensory nerve endings in the airways induces several responses including cell proliferation. However, the mechanisms were not completely understood in tracheal smooth muscle cells (TSMCs). We therefore investigated the effect of SP on cell proliferation and activation of p42/p44 mitogen-activated protein kinase (MAPK) in these cells. SP stimulated [3H]thymidine incorporation and p42/p44 MAPK phosphorylation in a time- and concentration-dependent manner in TSMCs. Both DNA synthesis and phosphorylation of MAPK in response to SP were attenuated by pretreatment with pertussis toxin, genistein, D609, U73122, staurosporine, removal of Ca(2+) by BAPTA/AM plus EGTA, PD98059, and SB202190. Furthermore, overexpression of dominant negative mutants, H-Ras-15A and Raf-N4, significantly suppressed p42/p44 MAPK activation induced by SP and PDGF-BB. These results conclude that the mitogenic effect of SP was mediated through the activation of Ras/Raf/MEK/MAPK pathway, which was modulated by PC-PLC, PI-PLC, Ca(2+), and PKC in cultured human TSMCs.     

Kaposi's Sarcoma-Associated Herpesvirus-Encoded G Protein-Coupled Receptor ORF74 Constitutively Activates p44/p42 MAPK and Akt via Gi and Phospholipase C-Dependent Signaling Pathways

The G protein-coupled receptor encoded by Kaposi's sarcoma-associated herpesvirus, also referred to as ORF74, has been shown to stimulate oncogenic and angiogenic signaling pathways in a constitutively active manner. The biochemical routes linking ORF74 to these signaling pathways are poorly defined. In this study, we show that ORF74 constitutively activates p44/p42 mitogen-activated protein kinase (MAPK) and Akt via G(i)- and phospholipase C (PLC)-mediated signaling pathways. Activation of Akt by ORF74 appears to be phosphatidylinositol 3-kinase (PI3-K) dependent but, interestingly, is also mediated by activation of protein kinase C (PKC) and p44/p42 MAPK. ORF74 may signal to Akt via p44/p42 MAPK, which can be activated by G(i), through activation of PI3-K or through PKC via the PLC pathway. Signaling of ORF74 to these proliferative and antiapoptotic signaling pathways can be further modulated positively by growth-related oncogene (GROalpha/CXCL1) and negatively by human gamma interferon-inducible protein 10 (IP-10/CXCL10), thus acting as an agonist and an inverse agonist, respectively. Despite the ability of the cytomegalovirus-encoded chemokine receptor US28 to constitutively activate PLC, this receptor does not increase phosphorylation of p44/p42 MAPK or Akt in COS-7 cells. Hence, ORF74 appears to signal through a larger diversity of G proteins than US28, allowing it to couple to proliferative and antiapoptotic signaling pathways. ORF74 can therefore be envisioned as an attractive target for novel treatment of Kaposi's sarcoma.     

Effect of adenosine triphosphate on phosphate uptake in renal proximal tubule cells: involvement of PKC and p38 MAPK

ATP has been known to act as an extracellular signal and to be involved in various functions of kidney. Renal proximal tubular reabsorption of phosphate (Pi) contributes to the maintenance of phosphate homeostasis, which is regulated by Na+/Pi cotransporter. However, the effects of ATP on Na+/Pi cotransporters were not elucidated in proximal tubule cells (PTCs). Thus, the effects of ATP on Na+/Pi cotransporter and its related signal pathways are examined in the primary cultured renal PTCs. In the present study, ATP inhibited Pi uptake in a time (> 1 h) and dose (>10(-6)M) dependent manner. ATP-induced inhibition of Pi uptake was correlated with the decrease of type II Na+/Pi cotransporter mRNA. ATP-induced inhibition of Pi uptake may be mediated by P2Y receptor activation, since suramin (non-specific P2 receptor antagonist) and RB-2 (P2Y receptor antagonist) blocked it. ATP-induced inhibition of Pi uptake was blocked by neomycin, U73122 (phospholipase C (PLC) inhibitors), bisindolylmaleimide I, H-7, and staurosporine (protein kinase C (PKC) inhibitors), suggesting the role of PLC/PKC pathway. ATP also increased inositol phosphates (IPs) formation and induced PKC translocation from cytosolic fraction to membrane fraction. In addition, ATP-induced inhibition of Pi uptake was blocked by SB 203580 [a p38 mitogen activated protein kinase (MAPK) inhibitor], but not by PD 98059 (a p44/42 MAPK inhibitor). Indeed, ATP induced phosphorylation of p38 MAPK, which was not blocked by PKC inhibitor. In conclusion, ATP inhibited Pi uptake via PLC/PKC as well as p38 MAPK in renal PTCs.     

Sphingosine-1-phosphate induces COX-2 expression via PI3K/Akt and p42/p44 MAPK pathways in rat vascular smooth muscle cells

Sphingosine 1-phosphate (S1P) has been shown to regulate smooth muscle cell proliferation, migration, and vascular maturation. S1P increases the expression of several proteins including COX-2 in vascular smooth muscle cells (VSMCs) and contributes to arteriosclerosis. However, the mechanisms regulating COX-2 expression by S1P in VSMCs remain unclear. Western blotting and RT-PCR analyses showed that S1P induced the expression of COX-2 mRNA and protein in a time- and concentration-dependent manner, which was attenuated by inhibitors of MEK1/2 (U0126) and PI3K (wortmannin), and transfection with dominant negative mutants of p42/p44 mitogen-activated protein kinases (ERK2) or Akt. These results suggested that both p42/p44 MAPK and PI3K/Akt pathways participated in COX-2 expression induced by S1P in VSMCs. In accordance with these findings, S1P stimulated phosphorylation of p42/p44 MAPK and Akt, which was attenuated by U0126, LY294002, or wortmannin, respectively. Furthermore, this up-regulation of COX-2 mRNA and protein was blocked by a selective NF-kappaB inhibitor helenalin. Consistently, S1P-stimulated translocation of NF-kappaB into the nucleus was revealed by immnofluorescence staining. Moreover, S1P-stimulated activation of NF-kappaB promoter activity was blocked by phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 and helenalin, but not by U0126, suggesting that involvement of PI3K/Akt in the activation of NF-kappaB. COX-2 promoter assay showed that S1P induced COX-2 promoter activity mediated through p42/p44 MAPK, PI3K/Akt, and NF-kappaB. These results suggested that in VSMCs, activation of p42/p44 MAPK, Akt and NF-kappaB pathways was essential for S1P-induced COX-2 gene expression. Understanding the mechanisms involved in S1P-induced COX-2 expression on VSMCs may provide potential therapeutic targets in the treatment of arteriosclerosis.     

Ligation of microglial CD40 results in p44/42 mitogen-activated protein kinase-dependent TNF-alpha production that is opposed by TGF-beta 1 and IL-10

Recently, it has been demonstrated that the CD40 receptor is constitutively expressed on cultured microglia at low levels. Ligation of CD40 by CD40 ligand on these cells results in microglial activation, as measured by TNF-alpha production and neuronal injury. However, the intracellular events mediating this effect have yet to be investigated. We report that ligation of microglial CD40 triggers activation of p44/42 mitogen-activated protein kinase (MAPK). This effect is evident 30 min posttreatment, and progressively declines thereafter (from 30 to 240 min). Phosphorylated p38 MAPK is not observed in response to ligation of microglial CD40 across the time course examined. Inhibition of the upstream activator of p44/42 MAPK, mitogen-activated protein/extracellular signal-related kinase kinase 1/2, with PD98059, decreases phosphorylation of p44/42 MAPK and significantly reduces TNF-alpha release following ligation of microglial CD40. Furthermore, cotreatment of microglial cells with CD40 ligand and TGF-beta1 or IL-10, or both, inhibits CD40-mediated activation of p44/42 MAPK and production of TNF-alpha in a statistically interactive manner. Taken together, these data show that ligation of microglial CD40 triggers TNF-alpha release through the p44/42 MAPK pathway, an effect that can be opposed by TGF-beta1 and IL-10.     

One-way cross-talk between p38(MAPK) and p42/44(MAPK). Inhibition of p38(MAPK) induces low density lipoprotein receptor expression through activation of the p42/44(MAPK) cascade.

In this paper, we report that SB202190 alone, a specific inhibitor of p38(MAPK), induces low density lipoprotein (LDL) receptor expression (6-8-fold) in a sterol-sensitive manner in HepG2 cells. Consistent with this finding, selective activation of the p38(MAPK) signaling pathway by expression of MKK6b(E), a constitutive activator of p38(MAPK), significantly reduced LDL receptor promoter activity. Expression of the p38(MAPK) alpha-isoform had a similar effect, whereas expression of the p38(MAPK) betaII-isoform had no significant effect on LDL receptor promoter activity. SB202190-dependent increase in LDL receptor expression was accompanied by induction of p42/44(MAPK), and inhibition of this pathway completely prevented SB202190-induced LDL receptor expression, suggesting that p38(MAPK) negatively regulates the p42/44(MAPK) cascade and the responses mediated by this kinase. Cross-talk between these kinases appears to be one-way because modulation of p42/44(MAPK) activity did not affect p38(MAPK) activation by a variety of stress inducers. Taken together, these findings reveal a hitherto unrecognized one-way communication that exists between p38(MAPK) and p42/44(MAPK) and provide the first evidence that through the p42/44(MAPK) signaling cascade, the p38(MAPK) alpha-isoform negatively regulates LDL receptor expression, thus representing a novel mechanism of fine tuning cellular levels of cholesterol in response to a diverse set of environmental cues.     

Confluence of vascular endothelial cells induces cell cycle exit by inhibiting p42/p44 mitogen-activated protein kinase activity

Like other cellular models, endothelial cells in cultures stop growing when they reach confluence, even in the presence of growth factors. In this work, we have studied the effect of cellular contact on the activation of p42/p44 mitogen-activated protein kinase (MAPK) by growth factors in mouse vascular endothelial cells. p42/p44 MAPK activation by fetal calf serum or fibroblast growth factor was restrained in confluent cells in comparison with the activity found in sparse cells. Consequently, the induction of c-fos, MAPK phosphatases 1 and 2 (MKP1/2), and cyclin D1 was also restrained in confluent cells. In contrast, the activation of Ras and MEK-1, two upstream activators of the p42/p44 MAPK cascade, was not impaired when cells attained confluence. Sodium orthovanadate, but not okadaic acid, restored p42/p44 MAPK activity in confluent cells. Moreover, lysates from confluent 1G11 cells more effectively inactivated a dually phosphorylated active p42 MAPK than lysates from sparse cells. These results, together with the fact that vanadate-sensitive phosphatase activity was higher in confluent cells, suggest that phosphatases play a role in the down-regulation of p42/p44 MAPK activity. Enforced long-term activation of p42/p44 MAPK by expression of the chimera DeltaRaf-1:ER, which activates the p42/p44 MAPK cascade at the level of Raf, enhanced the expression of MKP1/2 and cyclin D1 and, more importantly, restored the reentry of confluent cells into the cell cycle. Therefore, inhibition of p42/p44 MAPK activation by cell-cell contact is a critical step initiating cell cycle exit in vascular endothelial cells.     

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.