Differential activation of p38 mitogen-activated protein kinase and extracellular signal-regulated protein kinases confers cadmium-induced HSP70 expression in 9L rat brain tumor cells

We have reported that treatment with CdCl2 at 40-100 microM induces the heat shock proteins (HSPs) in 9L rat brain tumor cells, during which the activation of heat shock factor (HSF) is essentially involved. By exploiting protein kinase inhibitors, we further analyzed the possible participation of specific protein kinases in the above processes. It was found that induction of HSP70 in cells treated with a high concentration of cadmium (i.e. 100 microM) is preceded by the phosphorylation and activation of p38 mitogen-activated protein kinase (p38(MAPK)), while that in cells treated with a low concentration (60 microM) is accompanied by the phosphorylation and activation of extracellular-regulated protein kinases 1 and 2 (ERK1/2). In 100 microM cadmium-treated cells, both HSP70 induction and HSF1 activation are eliminated in the presence of SB203580, a specific inhibitor of p38(MAPK). By contrast, in 60 microM cadmium-treated cells, the processes are not affected by SB203580 but are significantly suppressed by PD98059, which indirectly inhibits ERK1/2 by acting on MAPK-ERK kinase. Taken together, we demonstrate that p38(MAPK) and ERK1/2 can be simultaneously or independently activated under different concentrations of cadmium and that the signaling pathways participate in the induction of HSP70 by acting on the inducible phosphorylation of HSF1. We thus provide the first evidence that both p38(MAPK) and ERK signaling pathways can differentially participate in the activation of HSF1, which leads to the induction of HSP70 by cadmium.     

Constitutive activation of the 41-/43-kDa mitogen-activated protein kinase signaling pathway in human tumors

The 41-kDa and 43-kDa mitogen-activated protein (MAP) kinases play a pivotal role in the mitogenic signal transduction pathway and are essential components of the MAP kinase cascade, which includes MAP kinase kinase (MEK) and Raf-1. As aberrant activation of signal transducing molecules such as Ras and Raf-1 has been linked with cancer, we examined whether constitutive activation of the 41-/43-kDa MAP kinases is associated with the neoplastic phenotype of 138 tumor cell lines and 102 primary tumors derived from various human organs. Constitutive activation of the MAP kinases was observed in 50 tumor cell lines (36.2%) in a rather tissue-specific manner: cell lines derived from pancreas, colon, lung, ovary and kidney showed especially high frequencies with a high degree of MAP kinase activation, while those derived from brain, esophagus, stomach, liver and of hematopoietic origin showed low frequencies with a limited degree of MAP kinase activation. We also detected constitutive activation of the 41-/43-kDa MAP kinases in a relatively large number of primary human tumors derived from kidney, colon and lung tissues but not from liver tissue. Many tumor cells, in which point mutations of ras genes were detected, showed constitutive activation of MAP kinases, however, there were also many exceptions to this observation. In contrast, the activation of the 41-/43-kDa MAP kinases was accompanied by the activation of Raf-1 in the majority of tumor cells and was completely associated with the activation of MEK and p90rsk in all the tumor cells examined. These results suggest that the constitutive activation of 41-/43-kDa MAP kinases in tumor cells is not due to the disorder of MAP kinases themselves, but is due to the disorder of Raf-1, Ras, or some other signaling molecules upstream of Ras.     

Involvement of heat shock elements and basal transcription elements in the differential induction of the 70-kDa heat shock protein and its cognate by cadmium chloride in 9L rat brain tumor cells

The EphA3 receptor tyrosine kinase has been implicated in guiding the axons of retinal ganglion cells as they extend in the optic tectum. A repulsive mechanism involving opposing gradients of the EphA3 receptor on retinal axons and its ligands, ephrin-A2 and ephrin-A5, in the tectum influences topographic mapping of the retinotectal projection. To investigate the overall role of the Eph family in patterning of the visual system, we have used in situ hybridization to localize nine Eph receptors in the chicken retina and optic tectum at Embryonic Day 8. Three of the receptors examined correspond to the novel chicken homologs of EphA2, EphA6, and EphA7. Unexpectedly, we found that many Eph receptors are expressed not only in retinal ganglion cells, but also in tectal cells, In particular, EphA3 mRNA is prominently expressed in the anterior tectum, with a pattern reciprocal to that of ephrin-A2 and ephrin-A5. Similarly, ephrin-A5 is expressed not only in tectal cells but also in the nasal retina, with a pattern reciprocal to that of its receptor EphA3 and partially overlapping with that of its other receptor EphA4. Consistent with the even distribution of EphA4 and the polarized distribution of EphA4 ligands in the retina, probing EphA4 immunoprecipitates from different sectors of the retina with anti-phosphotyrosine antibodies revealed spatial differences in receptor phosphorylation. These complex patterns of expression and tyrosine phosphorylation suggest that Eph receptors and ephrins contribute to establishing topography of retinal axons through multiple mechanisms, in addition to playing a role in intraretinal and intratectal organization.     

Requirement of p38 mitogen-activated protein kinase for neuronal differentiation in PC12 cells

Nerve growth factor (NGF) induces sustained activation of classical MAP kinase (MAPK, also known as ERK) and neuronal differentiation in PC12 cells, whereas epidermal growth factor (EGF) induces transient activation of ERK/MAPK and stimulates proliferation of the cells. Although previous studies showed that sustained activation of ERK/MAPK is important for neuronal differentiation of the cells, a recent report revealed that inhibition of the sustained phase of ERK/MAPK activation alone does not block neurite outgrowth caused by NGF. These results suggest requirement for an additional signaling pathway(s) triggered by NGF in neuronal differentiation. Here we show that NGF induces sustained activation of p38, a subfamily member of the MAPK superfamily, and that inhibition of the p38 pathway blocks neurite outgrowth in PC12 cells. Surprisingly, expression of constitutively active MAPK/ERK kinase (MAPKK, also known as MEK) results in p38 activation as well as ERK/MAPK activation, and a p38 inhibitor blocks neurite outgrowth caused by the constitutively active MAPKK/MEK. Moreover, constitutive activation of p38 is able to induce neurite outgrowth when combined with EGF treatment. These results reveal an essential role of p38 in neuronal differentiation in PC12 cells.     

Oxidative stress-induced actin reorganization mediated by the p38 mitogen-activated protein kinase/heat shock protein 27 pathway in vascular endothelial cells

Vascular endothelial cells are constantly in contact with oxyradicals and must be especially well equipped to resist their toxic effects and generate appropriate physiological responses. Despite the importance of oxyradicals in the physiopathology of the vascular endothelium, the mechanisms regulating the oxidative response of endothelial cells are poorly understood. In the present study, we observed that H2O2 in concentrations that induced severe fragmentation of F-actin in fibroblasts rather induced a reorganization of F-actin in primary cultures of human umbilical vein endothelial cells (HUVECs) that was characterized by the accumulation of stress fibers, the recruitment of vinculin to focal adhesions, and the loss of membrane ruffles, H2O2 also induced in these cells a strong (10- to 14-fold) activation of the p38 mitogen-activated protein (MAP) kinase, which resulted in activation of MAP kinase-activated protein kinase-2/3 and phosphorylation of the F-actin polymerization modulator, heat shock protein 27 (HSP27). The MAP kinases extracellular-regulated kinase, and c-Jun N-terminal kinase/stress-activated protein kinase were only slightly increased by these treatments. Inhibiting p38 activity with the highly specific inhibitor SB203580 blocked the H2O2-induced endothelial microfilament responses. Moreover, fibroblasts acquired an endothelium-like SB203580-sensitive actin response when HSP27 concentration was increased by gene transfection to the same high level as found in HUVECs. The results indicate that activation of p38 MAP kinase in cells such as endothelial cells, which naturally express high level of HSP27, plays a central role in modulating microfilament responses to oxidative stress. Consequently, the p38 MAP kinase pathway may participate in the several oxyradical-activated functions of the endothelium that are associated with reorganization of microfilament network.     

Hyperosmolarity-induced interleukin-8 expression in human bronchial epithelial cells through p38 mitogen-activated protein kinase

The changes in airway osmolarity have been described to contribute to the production of exercise- induced bronchoconstriction (EIB) and the development of the late-phase response (LPR). The mechanism has been investigated; however, the responsiveness of bronchial epithelial cells (BEC) to hyperosmolarity and the intracellular signals leading to cell activation have not been determined. In this study, we examined the effect of hyperosmolar medium on interleukin-8 (IL-8) expression and the role of p38 mitogen-activated protein (MAP) kinase and c-Jun NH2 terminal kinase ( JNK) in human BEC in this response in order to clarify the intracellular signals regulating IL-8 expression in hyperosmolarity-stimulated BEC. The results showed that hyperosmolarity induced IL-8 expression in a concentration dependent manner, p38 MAP kinase phosphorylation and activation, and JNK activation whether NaCl or mannitol was used as the solute. SB 203580 as the specific p38 MAP kinase inhibitor inhibited hyperosmolarity-induced p38 MAP kinase activation and partially inhibited hyperosmolarity-induced IL-8 expression. These results indicate that p38 MAP kinase, at least in part, regulates hyperosmolarity-induced IL-8 expression in BEC. However, other signals such as JNK are possibly also involved. These results provide new evidence on the mechanism responsible for the development of the LPR induced by EIB, and a strategy for treatment with the specific p38 MAP kinase inhibitor.     

Activation of p38 mitogen-activated protein kinase by c-Abl-dependent and -independent mechanisms

The p38 mitogen-activated protein (MAP) kinase defines a subgroup of the mammalian MAP kinases that are induced in response to lipopolysaccharide, hyperosmolarity, and interleukin 1. p38 MAP kinase appears to play a role in regulating inflammatory responses, including cytokine secretion and apoptosis. Here we show that diverse classes of DNA-damaging agents such as cisplatinum, 1-beta-D-arabinofuranosylcytosine, UV light, ionizing radiation, and methyl methanesulfonate activate p38 MAP kinase. We also demonstrate that cells deficient in c-Abl fail to activate p38 MAP kinase after treatment with cisplatinum and 1-beta-D-arabinofuranosylcytosine but not after exposure to UV and methyl methanesulfonate. Reconstitution of c-Abl in the Abl-/- cells restores that response. Similar results were obtained for induction of the Jun-NH2-kinase/stress-activated protein kinase. These findings indicate that p38 MAP and Jun-NH2-kinase/stress-activated protein kinases are differentially regulated in response to different classes of DNA-damaging agents.     

Substance P-induced mitogenesis in human astrocytoma cells correlates with activation of the mitogen-activated protein kinase signaling pathway

The neuropeptide substance P (SP) regulates many biological processes through binding to and activating the SP receptor (NK-1 subtype). Activation of the SP receptor induces mitogenesis in several cell types. In this study, we characterized the mitogenic response induced by SP peptide in the U-373MG astrocytoma cell line and showed that activation of the SP receptor induces [3H]thymidine incorporation into DNA. We also found that SP potently induces c-myc mRNA and protein in the U-373MG cells. Tyrphostin A25, which blocks activity of tyrosine kinases, significantly inhibited SP-induced mitogenesis, suggesting that the mitogenic response induced by SP peptide involves phosphorylation by tyrosine kinases. Furthermore, stimulation of the SP receptor activates tyrosine phosphorylation and enzymatic activity of extracellular signal-regulated kinases (Erk1 and Erk2), also called the mitogen-activated protein kinases (MAPKs). This result suggests that MAPKs participate in the SP peptide-induced signaling pathway. The addition of CP 96,345 ([(2S,3S)-cis-2-(diphenylmethyl)-N-[(2-methoxyphenyl)-methyl]-1 -azabicyclo[2.2.2]octan-3-amine]; an NK-1 receptor antagonist) or PD 098059 (MEK1 inhibitor) inhibited both DNA synthesis and activation of the MAPK pathway, substantiating that SP stimulates mitogenesis by activating the MAPK pathway through receptors of the NK-1 subtype. Our results demonstrate that SP peptide is a strong mitogen in the U-373MG astrocytoma cell line and establish a clear correlation between SP-induced mitogenesis and activation of MAPK signaling pathway.     

Hypertonicity regulates the function of human neutrophils by modulating chemoattractant receptor signaling and activating mitogen-activated protein kinase p38

Excessive neutrophil activation causes posttraumatic complications, which may be reduced with hypertonic saline (HS) resuscitation. We tested if this is because of modulated neutrophil function by HS. Clinically relevant hypertonicity (10-25 mM) suppressed degranulation and superoxide formation in response to fMLP and blocked the activation of the mitogen activated protein kinases (MAPK) ERK1/2 and p38, but did not affect Ca2+ mobilization. HS did not suppress oxidative burst in response to phorbol myristate acetate (PMA). This indicates that HS suppresses neutrophil function by intercepting signal pathways upstream of or apart from PKC. HS activated p38 by itself and enhanced degranulation in response to PKC activation. This enhancement was reduced by inhibition of p38 with SB203580, suggesting that p38 up-regulation participates in HS-induced enhancements of degranulation. HS had similar effects on the degranulation of cells that were previously stimulated with fMLP, but had no effect on its own, suggesting that HS enhancement of degranulation requires another signal. We conclude that depending on other stimuli, HS can suppress neutrophil activation by intercepting multiple receptor signals or augment degranulation by enhancing p38 signaling. In patients HS resuscitation may reduce posttraumatic complications by preventing neutrophil activation via chemotactic factors released during reperfusion.     

Follicle stimulating hormone (FSH) activates the p38 mitogen-activated protein kinase pathway, inducing small heat shock protein phosphorylation and cell rounding in immature rat ovarian granulosa cells

This study investigates the possibility that FSH activates the p38 mitogen-activated protein kinase (MAPK) pathway in immature granulosa cells (GC). FSH induced the phosphorylation (activation) of p38 MAPK as evaluated by immunoprecipitation and by phosphorylation-specific immunoblotting. FSH-induced phosphorylation of p38 MAPK was blocked by pretreatment with the protein kinase A (PKA) inhibitor H89 and mimicked by the cAMP generating agonist forskolin, indicating that FSH-induced cAMP production and PKA activation are necessary and sufficient for the activation of p38 MAPK in GC. The small heat shock protein HSP-27 comprises a downstream phosphorylation target for the p38 MAPK pathway. FSH-induced phosphorylation of HSP-27 was blocked by pretreatment with the p38 MAPK inhibitor SB 203580, indicating that p38 MAPK activation is necessary for FSH-induced HSP-27 phosphorylation. FSH-induced GC rounding/aggregation was blocked by pretreatment with SB 203580 indicating that p38 MAPK activation is necessary for FSH-induced GC cell shape change. The results of these experiments show that the p38 MAPK pathway is activated in GC in response to FSH in a cAMP/PKA-dependent manner, and that p38 MAPK activity is required for FSH-induced HSP-27 phosphorylation as well as rounding/aggregation in GC.     

Stimulation of alpha1A-adrenoceptors in Rat-1 cells inhibits extracellular signal-regulated kinase by activating p38 mitogen-activated protein kinase

We report a patient with cholangiocellular carcinoma with tumor thrombi in the main portal trunk who has survived for 9.5 years after hepatic resection. A 57-year-old woman underwent an extended left lobectomy, and resection of the caudate lobe plus the main portal trunk for a liver tumor that had a portal tumor thrombus in the main portal trunk. The portal vein was reconstructed with an autologous vein graft obtained from the external iliac vein. Histological examination of the resected specimen revealed moderately differentiated tubular adenocarcinoma compatible with cholangiocellular carcinoma. Factors contributing to the patient's long-term survival are discussed. Aggressive surgical resection can be effective even for such an advanced case of cholangiocellular carcinoma.     

TL1, a novel tumor necrosis factor-like cytokine, induces apoptosis in endothelial cells. Involvement of activation of stress protein kinases (stress-activated protein kinase and p38 mitogen-activated protein kinase) and caspase-3-like protease

TL1 is a recently discovered novel member of the tumor necrosis factor (TNF) cytokine family. TL1 is abundantly expressed in endothelial cells, but its function is not known. The present study was undertaken to explore whether TL1 induces apoptosis in endothelial cells and, if so, to explore its mechanism of action. Cultured bovine pulmonary artery endothelial cells (BPAEC) exposed to TL1 showed morphological (including ultrastructural) and biochemical features characteristic of apoptosis. TL1-induced apoptosis in BPAEC was a time- and concentration-dependent process (EC50 = 72 ng/ml). The effect of TL1 was not inhibited by soluble TNF receptors 1 or 2. TL1 up-regulated Fas expression in BPAEC at 8 and 24 h after treatment, and significantly activated stress-activated protein kinase (SAPK) and p38 mitogen-activated protein kinase (p38 MAPK). The peak activities of SAPK and p38 MAPK in TL1-treated BPAEC were increased by 9- and 4-fold, respectively. TL1-induced apoptosis in the BPAEC was reduced by expression of a dominant-interfering mutant of c-Jun (62.8%, p < 0.05) or by a specific p38 inhibitor, SB203580 (1-10 microM) dose-dependently. TL1 also activated caspases in BPAEC, and TL1-induced apoptosis in BPAEC was significantly attenuated by the caspase inhibitor, ZVAD-fluromethyl-ketone. The major component activated by TL1 in BPAEC was caspase-3, which was based on substrate specificity and immunocytochemical analysis. These findings suggest that TL1 may act as an autocrine factor to induce apoptosis in endothelial cells via activation of multiple signaling pathways, including stress protein kinases as well as certain caspases.     

Sustained activation of the mitogen-activated protein kinase pathway. A mechanism underlying receptor tyrosine kinase specificity for matrix metalloproteinase-9 induction and cell migration

Activation of the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) pathway is required for ligand-dependent regulation of numerous cellular functions by receptor tyrosine kinases. We have shown previously that although many receptor tyrosine kinase ligands are mitogens for keratinocytes, cell migration and induction of the 92-kilodalton gelatinase/matrix metalloproteinase (MMP)-9 are selectively regulated by the epidermal growth factor and scatter factor/hepatocyte growth factor receptors. In this report we present evidence of an underlying mechanism to account for these observed differences in receptor tyrosine kinase-mediated response. Ligands that are mitogenic, but do not induce MMP-9 or colony dispersion, transiently activate the p42/p44 ERK/MAP kinases. In contrast, ligands that stimulate MMP-9 induction and colony dispersion induced sustained activation of these kinases. The functional significance of sustained MAPK activation was demonstrated by inhibition of the MAP kinase kinase MEK1. Disruption of the prolonged signal by addition of the MEK1 inhibitor PD 98059 up to 4 h after growth factor stimulation substantially impaired ligand-dependent colony dispersion and MMP-9 induction. These findings support the conclusion that duration of MAPK activation is an important determinant for certain growth factor-mediated functions in keratinocytes.     

Analysis of the Gs/mitogen-activated protein kinase pathway in mutant S49 cells

Heterotrimeric G protein-coupled receptors can activate the mitogen-activated protein kinase (MAPK) cascade. Recent studies using pharmacological inhibitors or dominant-negative mutants of signaling molecules have advanced our understanding of the pathways from G protein-coupled receptors to MAPK. However, molecular genetic analysis of these pathways is inadequate in mammalian cells. Here, using the well characterized Gsalpha- and protein kinase A-deficient S49 mouse lymphoma cells, we provide the molecular genetic evidence that Gsalpha is responsible for transducing the beta-adrenergic receptor signal to MAPK in a protein kinase A-dependent pathway involving Rap1 and Raf (but not Ras) molecules.