Smad4 mediates activation of mitogen-activated protein kinases by TGF-β in pancreatic acinar cells

2001 ◽  
Vol 281 (1) ◽  
pp. C311-C319 ◽  
Author(s):  
Diane M. Simeone ◽  
Lizhi Zhang ◽  
Kathleen Graziano ◽  
Barbara Nicke ◽  
Trinh Pham ◽  
...  
Keyword(s):  
Map Kinase ◽  
Signaling Pathways ◽  
Acinar Cell ◽  
Acinar Cells ◽  
Dominant Negative ◽  
Pancreatic Acinar Cells ◽  
Kinase Signaling ◽  
Erk Activation ◽  
Smad Proteins ◽  
Mitogen Activated Protein

Transforming growth factor-β (TGF-β) inhibits pancreatic acinar cell growth. In many cell types, TGF-β mediates its growth inhibitory effects by activation of Smad proteins. Recently, it has been reported that Smad proteins may interact with the mitogen-activated protein (MAP) kinase signaling pathways. In this study, we report on the interactions between the TGF-β and MAP kinase signaling pathways in isolated rat pancreatic acinar cells. TGF-β activated the MAP kinases extracellular signal-related kinases (ERKs) and p38 in pancreatic acinar cells, but had no effect on c- junNH2-terminal kinase activity. Activation of MAP kinase by TGF-β was maximal 4 h after treatment. The ability of TGF-β to activate ERKs was concentration dependent and dependent on protein synthesis. TGF-β's stimulation of ERK activation was blocked by PD-98059, an inhibitor of MAP kinase kinase 1, and by adenoviral transfer of dominant negative RasN17. Furthermore, adenoviral-mediated expression of dominant negative Smad4 blocked the ability of TGF-β to activate acinar cell MAP kinase, demonstrating that this activation is downstream of Smads. The biological relevance of ERK activation by TGF-β was indicated by demonstrating that inhibition of ERK signaling by PD-98059 blocked the ability of TGF-β to activate the transcription factor activator protein-1. These studies provide new insight into the signaling mechanisms by which TGF-β mediates biological actions in pancreatic acinar cells.

scholarly journals Epidermal growth factor receptor-mediated cell motility: phospholipase C activity is required, but mitogen-activated protein kinase activity is not sufficient for induced cell movement.

1994 ◽  
Vol 127 (3) ◽  
pp. 847-857 ◽  
Author(s):  
P Chen ◽  
H Xie ◽  
M C Sekar ◽  
K Gupta ◽  
A Wells
Keyword(s):  
Cell Motility ◽  
Map Kinase ◽  
Signaling Pathways ◽  
Phospholipase C ◽  
Cell Lines ◽  
Kinase Activity ◽  
Thymidine Incorporation ◽  
Cell Movement ◽  
Dominant Negative ◽  
Mitogen Activated Protein

We recently have demonstrated that EGF receptor (EGFR)-induced cell motility requires receptor kinase activity and autophosphorylation (P. Chen, K. Gupta, and A. Wells. 1994. J. Cell Biol. 124:547-555). This suggests that the immediate downstream effector molecule contains a src homology-2 domain. Phospholipase C gamma (PLC gamma) is among the candidate transducers of this signal because of its potential roles in modulating cytoskeletal dynamics. We utilized signaling-restricted EGFR mutants expressed in receptor devoid NR6 cells to determine if PLC activation is necessary for EGFR-mediated cell movement. Exposure to EGF (25 nM) augmented PLC activity in all five EGFR mutant cell lines which also responded by increased cell movement. Basal phosphoinositide turnover was not affected by EGF in the lines which do not present the enhanced motility response. The correlation between EGFR-mediated cell motility and PLC activity suggested, but did not prove, a causal link. A specific inhibitor of PLC, U73122 (1 microM) diminished both the EGF-induced motility and PLC responses, while its inactive analogue U73343 had no effect on these responses. Both the PLC and motility responses were decreased by expression of a dominant-negative PLC gamma-1 fragment in EGF-responsive infectant lines. Lastly, anti-sense oligonucleotides (20 microM) to PLC gamma-1 reduced both responses in NR6 cells expressing wild-type EGFR. These findings strongly support PLC gamma as the immediate post receptor effector in this motogenic pathway. We have demonstrated previously that EGFR-mediated cell motility and mitogenic signaling pathways are separable. The point of divergence is undefined. All kinase-active EGFR mutants induced the mitogenic response while only those which are autophosphorylated induced PLC activity. U73122 did not affect EGF-induced thymidine incorporation in these motility-responsive infectant cell lines. In addition, the dominant-negative PLC gamma-1 fragment did not diminish EGF-induced thymidine incorporation. All kinase active EGFR stimulated mitogen-activated protein (MAP) kinase activity, regardless of whether the receptors induced cell movement; this EGF-induced MAP kinase activity was not affected by U73122 at concentrations that depressed the motility response. Thus, the signaling pathways which lead to motility and cell proliferation diverge at the immediate post-receptor stage, and we suggest that this is accomplished by differential activation of effector molecules.

Mitogen-activated protein kinase activation and regulation in the pressure-loaded fetal ovine heart

2006 ◽  
Vol 290 (4) ◽  
pp. H1587-H1595 ◽  
Author(s):  
Aaron K. Olson ◽  
Kristin N. Protheroe ◽  
Jeffrey L. Segar ◽  
Thomas D. Scholz
Keyword(s):  
Map Kinase ◽  
Signaling Pathways ◽  
Fetal Heart ◽  
Left Ventricular ◽  
Heart Weight ◽  
Kinase Signaling ◽  
Protein Levels ◽  
Pressure Load ◽  
Map Kinase Signaling ◽  
Mitogen Activated Protein

The mitogen-activated protein (MAP) kinase signaling pathways help to mediate the hypertrophic response of the pressure-loaded adult heart, although their importance in fetal myocardium is less known. The goal of this study was to determine the role the MAP kinase signaling pathways play in regulating the response of the fetal heart to a pressure load. Aortic (Ao) and pulmonary artery (PA) bands were placed in 132-day fetal sheep for 7 days. Protein levels of the total and active (phosphorylated) terminal MAP kinases extracellular signal-regulated kinase (ERK/P-ERK), c-Jun NH2-terminal kinase (JNK/P-JNK), and p38/P-p38 and the MAP kinase phosphatases MKP-1, MKP-2, and MKP-3 were made in the right and left ventricular (RV and LV) free walls. In both Ao- and PA-banded animals, total heart weight normalized to body weight was significantly increased, largely due to an increase in RV free wall mass in the Ao-banded animals and an increase in septal mass in the PA-banded fetuses. Total protein levels of the three terminal kinases and of P-ERK and P-JNK remained stable in both groups of banded animals. However, P-p38 was significantly increased in RV and LV of Ao- and PA-banded fetuses. Whereas MKP-1 and MKP-2 protein levels were unchanged following Ao- and PA-banding, MKP-3 protein levels were significantly increased in the RV of the PA-banded animals. These findings indicate that the MAP kinase signaling pathways are active in the fetal heart and help to modulate the response of prenatal myocardium to a pressure load.

scholarly journals Differential inhibition of signaling pathways by dominant-negative SH2/SH3 adapter proteins.

1995 ◽  
Vol 15 (12) ◽  
pp. 6829-6837 ◽  
Author(s):  
M Tanaka ◽  
R Gupta ◽  
B J Mayer
Keyword(s):  
Protein Kinase ◽  
Signaling Pathways ◽  
Tyrosine Kinases ◽  
Egf Receptor ◽  
Sh3 Domain ◽  
Sh2 Domain ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Erk Activation ◽  
Mitogen Activated Protein

SH2/SH3 adapters are thought to function in signal transduction pathways by coupling inputs from tyrosine kinases to downstream effectors such as Ras. Members of the mitogen-activated protein kinase family are known to be activated by a variety of mitogenic stimuli, including tyrosine kinases such as Abl and the epidermal growth factor (EGF) receptor. We have used activation of the mitogen-activated protein kinase Erk-1 as a model system with which to examine whether various dominant-negative SH2/SH3 adapters (Grb2, Crk, and Nck) could block signaling pathways leading to Erk activation. Activation of Erk-1 by oncogenic Abl was effectively inhibited by Grb2 with mutations in either its SH2 or SH3 domain or by Crk-1 with an SH3 domain mutation. The Crk-1 SH2 mutant was less effective, while Nck SH2 and SH3 mutants had little or no effect on Erk activation. These results suggest that both Crk and Grb2 may contribute to the activation of Erk by oncogenic Abl, whereas Nck is unlikely to participate in this pathway. Next we examined whether combinations of these dominant-negative adapters could inhibit Erk activation more effectively than each mutant alone. When combinations of Crk-1 and Grb2 mutants were analyzed, the combination of the Crk-1 SH3 mutant plus the Grb2 SH3 mutant gave a striking synergistic effect. This finding suggests that in Abl-transformed cells, more than one class of tyrosine-phosphorylated sites (those that bind the Grb2 SH2 domain and those that bind the Crk SH2 domain) can lead to Ras activation. In contrast to results with Abl, Erk activation by EGF was strongly inhibited only by Grb2 mutants; Crk and Nck mutants had little or no effect. This finding suggests that Grb2 is the only adapter involved in the activation of Erk by EGF. Dominant-negative adaptors provide a novel means to identify binding interactions important in vivo for signaling in response to a variety of stimuli.

scholarly journals Proteolytic Inhibition of Salmonella enterica Serovar Typhimurium-Induced Activation of the Mitogen-Activated Protein Kinases ERK and JNK in Cultured Human Intestinal Cells

2002 ◽  
Vol 70 (1) ◽  
pp. 86-95 ◽  
Author(s):  
Tracey L. Mynott ◽  
Ben Crossett ◽  
S. Radhika Prathalingam
Keyword(s):  
Map Kinase ◽  
Signaling Pathways ◽  
Salmonella Enterica ◽  
Map Kinases ◽  
Inflammatory Responses ◽  
Cysteine Proteases ◽  
Kinase Signaling ◽  
Map Kinase Signaling ◽  
Serovar Typhimurium ◽  
Mitogen Activated Protein

ABSTRACT Bromelain, a mixture of cysteine proteases from pineapple stems, blocks signaling by the mitogen-activated protein (MAP) kinases extracellular regulated kinase 1 (ERK-1) and ERK-2, inhibits inflammation, and protects against enterotoxigenic Escherichia coli infection. In this study, we examined the effect of bromelain on Salmonella enterica serovar Typhimurium infection, since an important feature of its pathogenesis is its ability to induce activation of ERK-1 and ERK-2, which leads to internalization of bacteria and induction of inflammatory responses. Our results show that bromelain dose dependently blocks serovar Typhimurium-induced ERK-1, ERK-2, and c-Jun NH2-terminal kinase (JNK) activation in Caco-2 cells. Bromelain also blocked signaling induced by carbachol and anisomycin, pharmacological MAP kinase agonists. Despite bromelain inhibition of serovar Typhimurium-induced MAP kinase signaling, it did not prevent subsequent invasion of the Caco-2 cells by serovar Typhimurium or alter serovar Typhimurium -induced decreases in resistance across Caco-2 monolayers. Surprisingly, bromelain also did not block serovar Typhimurium-induced interleukin-8 (IL-8) secretion but synergized with serovar Typhimurium to enhance IL-8 production. We also found that serovar Typhimurium does not induce ERK phosphorylation in Caco-2 cells in the absence of serum but that serovar Typhimurium-induced invasion and decreases in monolayer resistance are unaffected. Collectively, these data indicate that serovar Typhimurium-induced invasion of Caco-2 cells, changes in the resistance of epithelial cell monolayers, and IL-8 production can occur independently of the ERK and JNK signaling pathways. Data also confirm that bromelain is a novel inhibitor of MAP kinase signaling pathways and suggest a novel role for proteases as inhibitors of signal transduction pathways in intestinal epithelial cells.

scholarly journals c-Jun/AP-1 is required for CCK-induced pancreatic acinar cell dedifferentiation and DNA synthesis in vitro

2012 ◽  
Vol 302 (12) ◽  
pp. G1381-G1396 ◽  
Author(s):  
Lili Guo ◽  
Maria Dolors Sans ◽  
Yanan Hou ◽  
Stephen A. Ernst ◽  
John A. Williams
Keyword(s):  
Dna Synthesis ◽  
Cyclin D1 ◽  
Signaling Pathways ◽  
Acinar Cell ◽  
Acinar Cells ◽  
Pancreatic Acinar Cell ◽  
Dominant Negative ◽  
Cell Dedifferentiation ◽  
Cell Markers

Endogenous CCK plays an important role in pancreatic regeneration after pancreatitis. We used primary culture of mouse pancreatic acinar cells to evaluate the effect of CCK on acinar cell morphology and gene expression and to determine signaling pathways required for proliferation of acinar cells in vitro. Over 4 days in culture, cells grew out from acini and formed patches of monolayer, which displayed a reduced expression of acinar cell markers including digestive enzymes and Mist1 and an increased expression of ductal and embryonic markers, including cytokeratin 7, β-catenin, E-cadherin, pdx-1, and nestin. There was no appearance of stellate cell markers. CCK enhanced cellular spreading, DNA synthesis, and cyclin D1 expression. When signaling pathways were evaluated, CCK stimulation increased c-Jun expression, JNK and ERK activity, and AP-1 activation. Chemical inhibitors of JNK and ERK pathways, dominant-negative JNK and c-Jun, and c-Jun shRNA significantly inhibited CCK-induced DNA synthesis, CCK-induced AP-1 activation, and cyclin D1 expression. Furthermore, dominant-negative c-Jun reduced the increased expression of β-catenin and the decreased expression of amylase during culture. These results show that MAPK/c-Jun/AP-1 pathway plays an important role in pancreatic acinar cell dedifferentiation and proliferation in culture. Monolayer culture can serve as a model to study acinar cell proliferation similar to regeneration after pancreatitis in vivo.

scholarly journals Differentiation of central nervous system neuronal cells by fibroblast-derived growth factor requires at least two signaling pathways: roles for Ras and Src.

1997 ◽  
Vol 17 (8) ◽  
pp. 4633-4643 ◽  
Author(s):  
W L Kuo ◽  
K C Chung ◽  
M R Rosner
Keyword(s):  
Growth Factor ◽  
Map Kinase ◽  
Signaling Pathways ◽  
Hippocampal Neurons ◽  
Alternative Pathway ◽  
Neuronal Cells ◽  
Neuronal Cell ◽  
Mek Inhibitor ◽  
Dominant Negative ◽  
Mitogen Activated Protein

To evaluate the role of mitogen-activated protein (MAP) kinase and other signaling pathways in neuronal cell differentiation by basic fibroblast-derived growth factor (bFGF), we used a conditionally immortalized cell line from rat hippocampal neurons (H19-7). Previous studies have shown that activation of MAP kinase kinase (MEK) is insufficient to induce neuronal differentiation of H19-7 cells. To test the requirement for MEK and MAP kinase (ERK1 and ERK2), H19-7 cells were treated with the MEK inhibitor PD098059. Although the MEK inhibitor blocked the induction of differentiation by constitutively activated Raf, the H19-7 cells still underwent differentiation by bFGF. These results suggest that an alternative pathway is utilized by bFGF for differentiation of the hippocampal neuronal cells. Expression in the H19-7 cells of a dominant-negative Ras (N17-Ras) or Raf (C4-Raf) blocked differentiation by bFGF, suggesting that Ras and probably Raf are required. Expression of dominant-negative Src (pcSrc295Arg) or microinjection of an anti-Src antibody blocked differentiation by bFGF in H19-7 cells, indicating that bFGF also signals through a Src kinase-mediated pathway. Although neither constitutively activated MEK (MEK-2E) nor v-Src was sufficient individually to differentiate the H19-7 cells, coexpression of constitutively activated MEK and v-Src induced neurite outgrowth. These results suggest that (i) activation of MAP kinase (ERK1 and ERK2) is neither necessary nor sufficient for differentiation by bFGF; (ii) activation of Src kinases is necessary but not sufficient for differentiation by bFGF; and (iii) differentiation of H19-7 neuronal cells by bFGF requires at least two signaling pathways activated by Ras and Src.

Effects of exercise and insulin on mitogen-activated protein kinase signaling pathways in rat skeletal muscle

1996 ◽  
Vol 271 (2) ◽  
pp. E403-E408 ◽  
Author(s):  
L. J. Goodyear ◽  
P. Y. Chang ◽  
D. J. Sherwood ◽  
S. D. Dufresne ◽  
D. E. Moller
Keyword(s):  
Skeletal Muscle ◽  
Protein Kinase ◽  
Map Kinase ◽  
Signaling Pathways ◽  
Mammalian Cells ◽  
Mitogen Activated Protein Kinase ◽  
Kinase Signaling ◽  
Rat Skeletal Muscle ◽  
Map Kinase Signaling ◽  
Mitogen Activated Protein

Studies in mammalian cells have established the existence of at least three distinct mitogen-activated protein kinase (MAP kinase) signaling pathways that are activated by a variety of growth factors and/or environmental stressors. We determined whether physical exercise, a physiological stressor, and insulin, a metabolic stimulator and growth factor, activate the c-jun NH2-terminus kinase (JNK), the p38 kinase, and/or the extracellular regulatory kinases (ERK; p42MAPK and p44MAPK) signaling pathways in rat skeletal muscle. Animals were studied immediately after running on a motorized treadmill for 10-60 min (20 m/min, 10% grade) or 5-30 min after an intraperitoneal injection of insulin (20 U/rat). Exercise increased skeletal muscle JNK activity by two- to threefold throughout the time course studied, whereas insulin did not significantly increase JNK activity. The p38 activity was slightly stimulated by exercise and not by insulin. The ERK kinase pathway, as assessed by ribosomal S6 kinase-2 activity assays and phosphospecific p42MAPK/p4NAPK immunoblotting, was stimulated by both exercise and insulin. These data are the first demonstration of exercise stimulating multiple intracellular signaling pathways in skeletal muscle. Activation of these MAP kinase signaling pathways may mediate changes in skeletal muscle growth and metabolism that occur in response to exercise.

Different Signaling Pathways Are Involved in CCKB Receptor-Mediated MAP Kinase Activation in COS-7 Cells

2000 ◽  
Vol 381 (8) ◽  
pp. 763-768 ◽  
Author(s):  
M. Zieger ◽  
W. Oehrl ◽  
R. Wetzker ◽  
P. Henklein ◽  
G. Nowak ◽  
...  
Keyword(s):  
Map Kinase ◽  
Signaling Pathways ◽  
Significant Inhibition ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Mitogenic Signaling ◽  
Catalytic Subunits ◽  
Kinase Activation ◽  
Erk Activation ◽  
Erk Activity

Abstract Recently, the involvement of the MAP kinase ERK in mitogenic signaling of cholecystokininB (CCKB) receptors has been shown. However, the intracellular effector systems involved in this signaling pathway are poorly defined. In this study, we used COS-7 cells transiently transfected with the human CCKB receptor to investigate cholecystokinin-induced MAP kinase activation. CCK-8 induced activation of ERK2 which is associated with its phosphorylation and localization in the nucleus. The CCK-8-dependent ERK stimulation is sensitive to wortmannin an inhibitor of phosphoinositide 3-kinases (PI3Ks) indicating the involvement of PI3K activity. To identify the PI3K species involved in mitogenic signaling of the CCKB receptor several dominant-negative mutants of PI3K regulatory and catalytic subunits were transiently expressed. Surprisingly, different catalytically inactive mutants of the G protein-sensitive PI3Kγ did not affect ERK stimulation induced by CCK, whereas a dominant-negative mutant of the regulatory p85 subunit induced significant inhibition of CCK-dependent ERK activity. These results indicate an involvement of PI3K class 1A species α, β or/and δ in signal transduction via CCKB receptors. In addition, protein kinase C (PKC)-dependent signaling pathways contribute to CCKB-mediated MAP kinase signaling as shown by inhibition of CCK-8-induced ERK activation by the PKC inhibitor bisindolylmaleimide.

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