Human immune cells express ppMCH mRNA and functional MCHR1 receptor

The ubiquitously expressed Na(+)/H(+) exchanger (NHE1) plays an important role in the regulation of the intracellular pH. Induction of NHE activity by phorbol esters and inhibition of growth factor-mediated stimulation of the NHE by protein kinase C (PKC) inhibitors suggest an implication of PKCs in the regulation of the NHE. Expression of PKC isotype-specific dominant negative and constitutively active mutants or downregulation of PKC by isotype-specific antisense oligonucleotides revealed that stimulation by epidermal growth factor (EGF) or phorbol ester of the NHE in NIH3T3 cells is a PKC(alpha)-specific effect. Elevation of cytoplasmic calcium by a Ca(2+) ionophore or thapsigargin causes a growth factor-independent stimulation of the NHE predominantly mediated by calcium/calmodulin kinase II. It is concluded that in NIH3T3 cells overexpressing the EGF receptor (EGFR6 cells), EGF requires cPKC(alpha) for the activation of the NHE, while calcium/calmodulin-dependent kinases are essential in thapsigargin induced stimulation of the NHE.     

Arachidonate mobilization is coupled to depletion of intracellular calcium stores and influx of extracellular calcium in differentiated U937 cells

We have previously reported that dimethylsulfoxide-differentiation of U937 cells induced significant A23187-stimulatable arachidonate mobilization, consistent with characteristics of cytosolic phospholipase A₂ (Rzigalinski, B.A. and Rosenthal, M.D. (1994) Biochim. Biophys. Acta 1223, 219–225). The present report demonstrates that differentiated cells attained higher elevations of intracellular free calcium in response to A23187 and thapsigargin, consistent with enhancement of the capacitative calcium influx pathway. Differentiation induced a significant increase in the size of the intracellular calcium stores, as well as in the capacity for store-activated calcium influx. Alterations in the capacitative calcium influx pathway were coupled to differentiation-induced activation of cPLA₂ and mobilization of arachidonate in response to thapsigargin and fMLP stimulation. Although cPLA₂ activity is often associated with influx of extracellular calcium, arachidonate mobilization in response to thapsigargin or fMLP was not simply a consequence of calcium influx. Assessment of intracellular free calcium elevations during thapsigargin or fMLP-induced stimulation suggest that a low level of arachidonic acid release was initiated upon release of intracellular store calcium. This initial release of arachidonate was unaffected by inhibition of calcium influx with nickel, EGTA, or SKF96365. Arachidonate release was observed when extracellular calcium was replaced with extracellular strontium, suggesting activation of the cytosolic PLA₂ rather than secretory PLA₂. Inhibition of PLA₂ with prostaglandin B oligomer prevented both thapsigargin and fMLP-stimulated influx of extracellular calcium. Furthermore, exogenous free arachidonate stimulated influx of extracellular calcium in differentiated U937 cells. These results suggest that cPLA₂-mediated release of free arachidonate may participate in the formation of a calcium influx factor which controls influx of extracellular calcium through store-controlled channels in the plasma membrane.     

B-Raf-dependent regulation of the MEK-1/mitogen-activated protein kinase pathway in PC12 cells and regulation by cyclic AMP.

Growth factor receptor tyrosine kinase regulation of the sequential phosphorylation reactions leading to mitogen-activated protein (MAP) kinase activation in PC12 cells has been investigated. In response to epidermal growth factor, nerve growth factor, and platelet-derived growth factor, B-Raf and Raf-1 are activated, phosphorylate recombinant kinase-inactive MEK-1, and activate wild-type MEK-1. MEK-1 is the dual-specificity protein kinase that selectively phosphorylates MAP kinase on tyrosine and threonine, resulting in MAP kinase activation. B-Raf and Raf-1 are growth factor-regulated Raf family members which regulate MEK-1 and MAP kinase activity in PC12 cells. Protein kinase A activation in response to elevated cyclic AMP (cAMP) levels inhibited B-Raf and Raf-1 stimulation in response to growth factors. Ras.GTP loading in response to epidermal growth factor, nerve growth factor, or platelet-derived growth factor was unaffected by protein kinase A activation. Even though elevated cAMP levels inhibited Raf activation, the growth factor activation of MEK-1 and MAP kinase was unaffected in PC12 cells. The results demonstrate that tyrosine kinase receptor activation of MEK-1 and MAP kinase in PC12 cells is regulated by B-Raf and Raf-1, whose activation is inhibited by protein kinase A, and MEK activators, whose activation is independent of cAMP regulation.     

Evidence for ERK1/2 activation by thrombin that is independent of EGFR transactivation

Thrombin is involved in abnormal proliferation of vascular smooth muscle cells (VSMCs) associated with pathogenic vascular remodeling. Thrombin stimulation results in extracellular signal-regulated kinase (ERK)1/2 activation through transactivation of the epidermal growth factor receptor (EGFR). Here, using specific antibodies and inhibitors, we investigated the thrombin-induced phosphorylation of Src family kinases, nonreceptor proline-rich tyrosine kinase (Pyk2), EGFR, and ERK1/2. Our results show that Src and Pyk2 are involved upstream of the EGFR transactivation that is required for ERK1/2 phosphorylation. The investigation of the role of intracellular calcium concentration ([Ca2+]i) and calcium mobilization with the Ca2+ chelator BAPTA and thapsigargin, respectively, indicated that thrombin- and thapsigargin-induced phosphorylation of the EGFR but not ERK1/2 is dependent on an increase in [Ca2+]i. Moreover, only after BAPTA-AM pretreatment was thrombin-induced activation of ERK1/2 partially preserved from the effects of EGFR and PKC inhibition but not Src family kinase inhibition. These results suggest that BAPTA, by preventing [Ca2+]i elevation, unmasks a new pathway of Src family kinase-dependent thrombin-stimulated ERK1/2 phosphorylation that is independent of EGFR and PKC activation.     

Nerve growth factor stimulates a protein kinase in PC-12 cells that phosphorylates microtubule-associated protein-2

Some of the effects of nerve growth factor (NGF) may be mediated by changes in protein phosphorylation. We have identified a protein kinase from PC-12 cells that catalyzes the phosphorylation of pig brain microtubule-associated protein (MAP)-2 in vitro. This activity is stimulated 2-4-fold in extracts from cells treated with NGF or epidermal growth factor (EGF). The partial purification and characterization of this MAP kinase indicate that it is distinct from previously described NGF-stimulated protein kinases. The NGF-stimulated kinase activity is unaffected by direct addition to the assay of the heat-stable cAMP-dependent kinase peptide inhibitor, staurosporine, or K-252A, is slightly stimulated by heparin and is inhibited by sodium fluoride and calcium ions. Treatment of cells with NGF increases the activity of the kinase within 2 min. The activity declines after 10 min, and a second phase of activation is observed at 20-30 min. Comparison of its behavior on gel permeation and sucrose density gradients indicates a molecular mass in the range of 40,000 daltons. The kinase activity is specific for ATP as substrate with a Km of 12 microM. Although the pathway of activation of MAP kinase by NGF is unknown, the stimulation can be reversed by treatment of the enzyme with alkaline phosphatase, suggesting that activation involves phosphorylation of the kinase itself. The properties and hormone sensitivity of the PC-12 MAP kinase suggest that it is similar to the previously identified, growth factor-sensitive MAP kinase from 3T3-L1 adipocytes.     

Activation of MAP kinases in growth responsive pancreatic cancer cells.

The implication of MAP kinases in the proliferation control of pancreatic cancer cells is still unknown. This study was undertaken to examine the contribution of the p44/p42 and p38 MAP kinases in the mitogenic response to epidermal growth factor (EGF) and bombesin in human pancreatic cancer cells, MIA PaCa-2 and PANC-1. Data indicate that EGF and bombesin stimulated growth of both cell lines. In MIA PaCa-2 cells, EGF and bombesin stimulated the in gel activation of p38 while p44/p42 kinases exhibited high basal activity and no response to stimuli. Growth and p38 activation were inhibited by genistein, wortmannin, PD98059 and SB203580, specific inhibitors of tyrosine kinase, phosphatidylinositol 3-kinase, MEK-1 and p38 kinases, respectively. In PANC-1 cells, EGF and bombesin stimulated p42 in gel activation; p44 remained highly activated and unresponsive to stimuli and p38 did not respond. Stimulated growth and p42 activation were inhibited by genistein, wortmannin and PD98059. Estimation of MAPK activities with a specific anti-active MAP kinase antibody indicated, however, that EGF increased the intensity of the bands corresponding to p42 and p44 MAP kinases in both cell lines, indicating that the mitogenic factor can regulate MAP kinase activity. Data also pointed out that ATP is sufficient to increase MAP kinase activity within the in gel assay technique and may thus explain the discrepancies existing between the in gel assay data and those obtained with the anti-active MAP kinase antibody.     

Neurotensin-induced Erk1/2 phosphorylation and growth of human colonic cancer cells are independent from growth factors receptors activation

Neurotensin (NT) promotes the proliferation of human colonic cancer cells by undefined mechanisms. We already demonstrated that, in the human colon adenocarcinoma cell line HT29, the effects of NT were mediated by a complex formed between the NT receptor-1 (NTSR1) and-3 (NTSR3). Here we examined cellular mechanisms that led to NT-induced MAP kinase phosphorylation and growth factors receptors transactivation in colonic cancer cells and proliferation in HT29 cells. With the aim to identify upstream signaling involved in NT-elicited MAP kinase activation, we found that the stimulatory effects of the peptide were totally independent from the activation of the epidermal growth factor receptor (EGFR) both in the HT29 and the HCT116 cells. NT was unable to promote phosphorylation of EGFR and to compete with EGF for its binding to the receptor. Pharmacological approaches allowed us to differentiate EGF and NT signaling in HT29 cells since only NT activation of Erk1/2 was shown to be sensitive to PKC inhibitors and since only NT increased the intracellular level of calcium. We also observed that NT was not able to transactivate Insulin-like growth factor receptor.Our findings indicate that, in the HT29 and HCT116 cell lines, NT stimulates MAP kinase phosphorylation and cell growth by a pathway which does not involve EGF system but rather NT receptors which transduce their own intracellular effectors. These results indicate that depending on the cell line used, blocking EGFR is not the general rule to inhibit NT-induced cancer cell proliferation.     

Acetylcholine muscarinic m1 receptor regulation of cyclic AMP synthesis controls growth factor stimulation of Raf activity.

Acetylcholine muscarinic m2 receptors (m2R) couple to heterotrimeric Gi proteins and activate the Ras/Raf/mitogen-activated protein kinase pathway and phosphatidylinositol 3-kinase in Rat 1a cells. In contrast to the m2R, stimulation of the acetylcholine muscarinic m1 receptor (m1R) does not activate the Ras/Raf/mitogen-activated protein kinase regulatory pathway in Rat 1a cells but rather causes a pronounced inhibition of epidermal growth factor and platelet-derived growth factor receptor activation of Raf. In Rat 1a cells, m1R stimulation of phospholipase C beta and the marked rise in intracellular calcium stimulated cyclic AMP (cAMP) synthesis, resulting in the activation of protein kinase A. Stimulation of protein kinase A inhibited Raf activation in response to growth factors. Platelet-derived growth factor receptor stimulation of phosphatidylinositol 3-kinase activity was not affected by either m1R stimulation or protein kinase A activation in response to forskolin-stimulated cAMP synthesis. GTP loading of Ras in response to growth factors was unaffected by protein kinase A activation but was partially inhibited by carbachol stimulation of the m1R. Therefore, protein kinase A action at the Ras/Raf activation interface selectively inhibited only one branch of the signal transduction network initiated by tyrosine kinases. Specific adenylyl cyclases responding to different signals, including calcium, with enhanced cAMP synthesis will regulate Raf activation in response to Ras.GTP. Taken together, the data indicate that G protein-coupled receptors can positively and negatively regulate the responsiveness of tyrosine kinase-stimulated mitogenic response pathways.     

Nerve growth factor activation of the extracellular signal-regulated kinase pathway is modulated by Ca(2+) and calmodulin

Nerve growth factor is a member of the neurotrophin family of trophic factors that have been reported to be essential for the survival and development of sympathetic neurons and a subset of sensory neurons. Nerve growth factor exerts its effects mainly by interaction with the specific receptor TrkA, which leads to the activation of several intracellular signaling pathways. Once activated, TrkA also allows for a rapid and moderate increase in intracellular calcium levels, which would contribute to the effects triggered by nerve growth factor in neurons. In this report, we analyzed the relationship of calcium to the activation of the Ras/extracellular signal-regulated kinase pathway in PC12 cells. We observed that calcium and calmodulin are both necessary for the acute activation of extracellular signal-regulated kinases after TrkA stimulation. We analyzed the elements of the pathway that lead to this activation, and we observed that calmodulin antagonists completely block the initial Raf-1 activation without affecting the function of upstream elements, such as Ras, Grb2, Shc, and Trk. We have broadened our study to other stimuli that activate extracellular signal-regulated kinases through tyrosine kinase receptors, and we have observed that calmodulin also modulates the activation of such kinases after epidermal growth factor receptor stimulation in PC12 cells and after TrkB stimulation in cultured chicken embryo motoneurons. Calmodulin seems to regulate the full activation of Raf-1 after Ras activation, since functional Ras is necessary for Raf-1 activation after nerve growth factor stimulation and calmodulin-Sepharose is able to precipitate Raf-1 in a calcium-dependent manner.     

Basal cPLA(2) phosphorylation is sufficient for Ca(2+)-induced full activation of cPLA(2) in A549 epithelial cells

The release of [(3)H] arachidonic acid (AA) and its connection with the triggering of the MAP kinase cascade were studied in the human A549 epithelial cell line upon stimulation with thapsigargin. Thapsigargin can increase AA release along with the increase of intracellular calcium concentration, phosphorylation, and activation of extracellular regulated kinase (ERK) and cytosolic phospholipase A(2) (cPLA(2)). Both ERK and cPLA(2) phosphorylation in response to thapsigargin were inhibited by PD 98059, a specific inhibitor of MAP kinase kinase of the ERK group (MEK), and EGTA. cPLA(2) phosphorylation was not affected by Ro 31-8220 (an inhibitor of all PKC isoforms) or LY 379196 (a PKCbeta selective inhibitor), while both of them indeed attenuated ERK activation. On the other hand, rottlerin (the selective PKCdelta inhibitor), SB 203580 (the selective p38 MAPK inhibitor), and wortmannin (the PI 3-kinase inhibitor) can affect neither cPLA(2) nor ERK phosphorylation. In A549 cells, PKC activator PMA cannot increase either the basal or thapsigargin-induced (3)H-AA release, while it can induce the phosphorylation of ERK and cPLA(2.) The PMA-induced ERK phosphorylation was inhibited by Ro 31-8220, LY 379196, rottlerin, and PD 98059, but unaffected by SB 203580 and wortmannin. Moreover, the phosphorylation by PMA was non-additive with that of thapsigargin. This implies that intracellular Ca(2+) level is the key factor for induction of cPLA(2) activity and thapsigargin-elicited ERK activation itself is substantially sufficient for cPLA(2) activation upon intracellular Ca(2+) increase.     

Activation of bradykinin B2 receptors increases calcium entry and intracellular mobilization in C9 liver cells.

In C9 rat liver cells bradykinin and kallidin increased (approximately 2-fold) the intracellular concentration of calcium, but the B1 agonist, des-Arg9-bradykinin did not. The effect of bradykinin was inhibited by the B2 antagonists, Hoe 140 and N-alpha-adamantaneacetyl-D-Arg-[Hyp3, Thi5,8, D-Phe7]-bradykinin, but not by the B1 antagonist, des-Arg9-[Leu8]-bradykinin. The action of bradykinin was diminished, but not abolished, in medium without calcium. The peptide was able to increase intracellular calcium concentration in cells treated with thapsigargin. Bradykinin action was not observed in cells previously stimulated with this local mediator: however, under the same conditions, angiotensin II induced a clear increase in intracellular calcium concentration. Our data indicate that activation of bradykinin B2 receptors increase intracellular calcium concentrations by inducing both gating of the cation and intracellular mobilization in C9 liver cells. In addition, homologous desensitization was observed.     

Phospholipase Cγ Activation, Phosphotidylinositol Hydrolysis, and Calcium Mobilization are Not Required for FGF Receptor-Mediated Chemotaxis

Basic fibroblast growth factor (FGF) is a potent angiogenic factor that stimulates several cell types to migrate along a chemotactic gradient. Most chemoartractant receptors appear to share a common mechanism that involves activation of phospholipase C (PLC), hydrolysis of phosphotidylinositol, and mobilization of intracellular calcium. We transf ected two different cell lines with either human FGF receptor-1 cDNA or chimeric FGF receptor cDNA. Ligand stimulation induced chemotaxis, activation of PLOγ, phosphotidylinositol hydrolysis, and calcium mobilization in both wild-type receptor cell lines. No such response was elicited in control cells. Mutation of the two fibroblast growth factor receptors at residue 766, replacing tyrosine with phenylalanine, made the receptors incapable of associating with and activating PLCγ following ligand stimulation. These mutant receptors also failed to mediate phosphotidylinositol hydrolysis and calcium mobilization. However, cells transfected with the mutant fibroblast growth factor receptors were as chemotactically responsive to the appropriate ligand as were cells transfected with the wild-type receptors. These findings demonstrate that the ability of the fibroblast growth factor receptor to promote chemotaxis is not dependent on increased activation of PLCγ, increased hydrolysis of phosphotidylinositol, or increased global mobilization of calcium.     

Phospholipase Cγ Activation,Phosphotidylinositol Hydrolysis,and Calcium Mobilization are Not Required for FGF Receptor-Mediated Chemotaxis

Basic fibroblast growth factor (FGF) is a potent angiogenic factor that stimulates several cell types to migrate along a chemotactic gradient. Most chemoartractant receptors appear to share a common mechanism that involves activation of phospholipase C (PLC), hydrolysis of phosphotidylinositol, and mobilization of intracellular calcium. We transf ected two different cell lines with either human FGF receptor-1 cDNA or chimeric FGF receptor cDNA. Ligand stimulation induced chemotaxis, activation of PLOγ, phosphotidylinositol hydrolysis, and calcium mobilization in both wild-type receptor cell lines. No such response was elicited in control cells. Mutation of the two fibroblast growth factor receptors at residue 766, replacing tyrosine with phenylalanine, made the receptors incapable of associating with and activating PLCγ following ligand stimulation. These mutant receptors also failed to mediate phosphotidylinositol hydrolysis and calcium mobilization. However, cells transfected with the mutant fibroblast growth factor receptors were as chemotactically responsive to the appropriate ligand as were cells transfected with the wild-type receptors. These findings demonstrate that the ability of the fibroblast growth factor receptor to promote chemotaxis is not dependent on increased activation of PLCγ, increased hydrolysis of phosphotidylinositol, or increased global mobilization of calcium.     

Thyrotropin-induced mitogenesis is Ras dependent but appears to bypass the Raf-dependent cytoplasmic kinase cascade.

Cellular growth control requires the coordination and integration of multiple signaling pathways which are likely to be activated concomitantly. Mitogenic signaling initiated by thyrotropin (TSH) in thyroid cells seems to require two distinct signaling pathways, a cyclic AMP (cAMP)-dependent signaling pathway and a Ras-dependent pathway. This is a paradox, since activated cAMP-dependent protein kinase disrupts Ras-dependent signaling induced by growth factors such as epidermal growth factor and platelet-derived growth factor. This inhibition may occur by preventing Raf-1 protein kinase from binding to Ras, an event thought to be necessary for the activation of Raf-1 and the subsequent activation of the mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK) kinases (MEKs) and MAP kinase (MAPK)/ERKs. Here we report that serum-stimulated hyperphosphorylation of Raf-1 was inhibited by TSH treatment of Wistar rat thyroid cells, indicating that in this cell line, as in other cell types, increases in intracellular cAMP levels inhibit activation of downstream kinases targeted by Ras. Ras-stimulated expression of genes containing AP-1 promoter elements was similarly inhibited by TSH. On the other hand, stimulation of thyroid cells with TSH resulted in stimulation of DNA synthesis which was Ras dependent but both Raf-1 and MEK independent. We also show that Ras-stimulated DNA synthesis required the use of this kinase cascade in untreated quiescent cells but not in TSH-treated cells. These data suggest that in TSH-treated thyroid cells, Ras might be able to signal through effectors other than the well-studied cytoplasmic kinase cascade.     

ERK activation is only one role of PKC in TCR-independent cytotoxic T cell granule exocytosis

Cytotoxic T cells (CTLs) kill target cells by releasing lytic agents via regulated exocytosis. Three signals are known to be required for exocytosis: an increase in intracellular Ca²⁺, activation of protein kinase C (PKC) and activation of extracellular signal regulated signal kinase (ERK). ERK activation required for exocytosis depends on activity of PKC. The simplest possibility is that the sole effect of PKC required for exocytosis is ERK activation. Testing this requires dissociating ERK and PKC activation. We did this using TCR-independent stimulation of TALL-104 human leukemic CTLs. When cells are stimulated with thapsigargin and PMA, agents that increase intracellular Ca²⁺ and activate PKC, respectively, PKC-dependent ERK activation is required for lytic granule exocytosis. Expressing a constitutively active mutant MAP kinase kinase activates ERK independent of PKC. However, activating ERK without PKC does not support lytic granule exocytosis, indicating that there are multiple effects of PKC required for granule exocytosis.     

p42/mitogen-activated protein kinase as a converging target for different growth factor signaling pathways: use of pertussis toxin as a discrimination factor.

Mitogen-activated protein (MAP) kinase is a 42-kDa serine/threonine-specific protein kinase that requires phosphorylation on both tyrosine and threonine residues for activity. This enzyme is rapidly and transiently activated in quiescent cells after addition of various agonists, including insulin, epidermal growth factor, platelet-derived growth factor, and phorbol esters. We show here that addition of the growth factors thrombin or basic fibroblast growth factor to CCL39 fibroblasts rapidly induces tyrosine phosphorylation of the p42 MAP kinase protein and concomitantly stimulates MAP kinase enzymatic activity. To elucidate the signaling pathways utilized in this activation, we took advantage of the sensitivity of CCL39 cells to the toxin of bordetella pertussis, which ADP-ribosylates two Gi proteins in this cell system. We show that pretreatment of cells with the toxin inhibited thrombin stimulation of MAP kinase by greater than 75% but had no detectable effect on the stimulation induced by basic fibroblast growth factor. We also demonstrate that these two growth factors that synergize for mitogenicity are able to cooperate in activation of MAP kinase and that this synergism is partially sensitive to pertussis toxin. Finally, we describe a 44-kDa protein, the tyrosine phosphorylation of which appears to be coregulated with p42 MAP kinase. We conclude that p42 MAP kinase (and the pp44 protein) are at or are downstream from a point of convergence of two different receptor-induced signaling pathways and might well play a key role in integrating those signals.     

Cyclic nucleotides and intracellular-calcium homeostasis in human platelets.

The relationship between agonist-sensitive calcium compartments and those discharged by the Ca(2+)-ATPase inhibitor thapsigargin were studied in human platelets. In this context, calcium mobilization from intracellular pools and manganese influx was investigated in relation to the effect of altered cyclic-nucleotide levels. For maximal calcium release from intracellular stores, thapsigargin, compared to a receptor agonist like thrombin, requires the platelet's self-amplification mechanism, known to generate thromboxane A2. With this lipid mediator formed, thapsigargin released calcium and stimulated manganese influx in a manner similar to thrombin. Blocking the thromboxane receptor by addition of sulotroban (BM13.177) or, alternatively, increasing platelet cAMP or cGMP using prostacyclin or sodium nitroprusside, dramatically reduced the ability of thapsigargin to release calcium from intracellular compartments. The same experimental conditions significantly reduced the rate of manganese influx initiated by thapsigargin compared to thrombin. The experiments indicate that thapsigargin-sensitive compartments play only a minor role in inducing manganese influx compared to the receptor-sensitive compartment. Cyclic nucleotides accelerate the redistribution of an agonist-elevated platelet calcium into the thapsigargin-sensitive compartment, from which calcium can be released by inhibition of the Ca(2+)-ATPase. In human platelets, thapsigargin-induced calcium increase and influx were responsible for only part the calcium release resulting from inhibition of the corresponding ATPase; another part results from the indirect effect of thapsigargin acting via thromboxane-A2-receptor activation. Cyclic nucleotides are therefore an interesting regulatory device which can modify the thapsigargin response by not allowing the self-amplification mechanism of platelets to operate.