Inhibition of MKP-1 expression potentiates JNK related apoptosis in renal cancer cells

PURPOSE: Mitogen-activated protein kinases (MAPKs) comprise 3 subgroups, that is extracellular signal-regulated protein kinase, c-Jun N-terminal kinase (JNK) and p38 MAPK (p38). In this study we analyzed the role of JNK as well as the expression of MAPK phosphatase-1 (MKP-1) in renal cancers. MATERIALS AND METHODS: Four renal cell carcinoma (RCC) cell lines were used. The effects of anisomycin (JNK activator) and Ro-318220 (MKP-1 expression inhibitor) were analyzed by alamar blue assay. Apoptosis was determined by flow cytometric TUNEL analysis, nuclear morphological alternations and the detection of DNA fragmentation. Changes in MKP-1 expression as well as the activation of extracellular signal-regulated protein kinases and JNK were analyzed by Western blotting. RESULTS: All cell lines treated with anisomycin resulted in a transient activation of JNK without inducing apoptosis. Since we hypothesized that elevated MKP-1 expression could possibly prevent persistent JNK activation, Ro-318220 was used. When cells were treated with Ro-318220, MKP-1 expression decreased in Caki-1 and KU 20-01 cells but not in ACHN or 769P cells. Combined treatment of Caki-1 and KU 20-01 cells with anisomycin and Ro-318220 resulted in a decrease in MKP-1 expression concomitant with persistent JNK activation. Apoptosis was induced in each cell line. CONCLUSIONS: These results suggest that prevalent MKP-1 expression in RCC contributes to cancer cell survival by attenuating an apoptosis inducing signal cascade via JNK. Since Ro-318220 potentiated JNK related apoptosis, JNK activation by blocking MKP-1 expression may be an effective therapeutic approach to RCC.     

Mycophenolic acid induces islet apoptosis by regulating mitogen-activated protein kinase activation

Mycophenolic acid (MPA), an inosine monophosphate dehydrogenase inhibitor, is widely used as an immunosuppressive drug after transplantations including those of pancreas islet cells. However, recent reports have indicated that MPA has apoptotic effects on islet cells in vitro. To study the effect of MPA on islet cells and determine its mechanism, we used an insulin secreting cell-line, HIT-T15. We examined mitogen-activated protein kinase (MAPK) activation after MPA treatment, and determining cell death levels using methylthiazdetetrazolium assays. The activations of extracellular signal-regulated protein kinase (ERK), c-jun N-terminal kinase (JNK), and p38 MAPK and caspase-3 cleavage were measured by Western blotting. MPA (1, 10, 30 micromol/L) increased cell death and caspase-3 cleavage within 24 hours. Exogenous 500 micromol/L guanosine reversed the MPA-induced islet cell death, but exogenous adenosine did not. MPA 10 micromol/L induced cell apoptosis and increased the activations of JNK, ERK, and p38 MAPK. Furthermore, exogenous guanosine, but not exogenous adenosine, reversed these effects induced by MPA. This study demonstrated that MPA may induce islet apoptosis in HIT-T15 cells by increasing activations of JNK, ERK, and p38 MAPK in a guanosine-dependent manner.     

Involvement of the mitogen-activated protein kinase family in tetracaine-induced PC12 cell death

BACKGROUND: To explore whether cytotoxicity of local anesthetics is related to apoptosis, the authors examined how local anesthetics affect mitogen-activated protein kinase (MAPK) family members, extracellular signal-regulated kinases (ERKs), c-Jun N-terminal kinases (JNKs)-stress-activated protein kinases, and p38 kinase, which are known to play important roles in apoptosis. METHODS: Cell death was evaluated using PC12 cells. Morphologic changes of cells, cellular membrane, and nuclei were observed. DNA fragmentation was electrophoretically assayed. Western blot analysis was performed to analyze phosphorylation of the MAPK family, cleavage of caspase-3 and poly(adenosine diphosphate-ribose) polymerase. Intracellular Ca2+ concentration was measured using a calcium indicator dye. RESULTS: Tetracaine-induced cell death was shown in a time- and concentration-dependent manner and characterized by nuclear condensation or fragmentation, membrane blebbing, and internucleosomal DNA fragmentation. Caspase-3 activation and phosphorylation of ERK, JNK, and p38 occurred in the cell death. PD98059, an inhibitor of ERK, enhanced tetracaine-induced cell death and JNK phosphorylation, whereas ERK phosphorylation was inhibited. Curcumin, an inhibitor of JNK pathway, attenuated the cell death. Increase of intracellular Ca2+ concentration was detected. In addition to the increase of ERK phosphorylation and the decrease of JNK phosphorylation, two Ca2+ chelators protected cells from death. Neither cell death nor phosphorylation of the MAPK family was caused by tetrodotoxin. Nifedipine did not affect tetracaine-induced apoptosis. CONCLUSIONS: Tetracaine induces apoptosis of PC12 cells via the MAPK family. ERK activation protects cells from death, but JNK plays the opposite role. Toxic Ca2+ influx caused by tetracaine seems to be responsible for the cell death, but blocking of Na+ channels or L-type Ca2+ channels is unlikely involved in the tetracaine's action for apoptosis.     

Estradiol reverses TGF-beta1-induced mesangial cell apoptosis by a casein kinase 2-dependent mechanism

BACKGROUND: The slower rate of progression of chronic renal disease in women than in men is explained in part by the ability of estradiol to reverse the stimulatory effect of transforming growth factor-beta1 (TGF-beta1) on collagen IV synthesis at the level of casein kinase 2 activation. Casein kinase 2 also phosphorylates and activates the pro-apoptotic protein, p53. We hypothesized that estradiol would reverse TGF-beta1-induced mesangial cell apoptosis by antagonizing the stimulatory effects of TGF-beta1 on casein kinase 2 activity, thereby preventing p53 activation. METHODS: The effects of TGF-beta1 on mesangial cell apoptosis, p53 phosphorylation, Bax and Bcl-2 levels, caspase 9 activity, and cleavage of PARP were examined. The abilities of estradiol and a specific inhibitor of CK2 (5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole) (DRB) to modulate the effects of TGF-beta1 on these processes were also examined. RESULTS: TGF-beta1 (2 ng/mL), which up-regulates CK2 activity, induces apoptosis in murine mesangial cells together with p53 serine389 phosphorylation, up-regulation of Bax, suppression of Bcl-2, destabilization of mitochondrial permeability transition pores, stimulation of caspase 9 activity and activation of PARP. TGF-beta1-induced p53 activation and all the intermediary steps leading to mesangial cell apoptosis were reversed by estradiol (10-9 mol/L) and by DRB, potent inhibitors of CK2 activity, but not by inhibitors of the p38 MAPK, ERK or JNK signaling cascades. In contrast, TGF-beta1 failed to induce apoptosis in p53 knockout mesangial cells. CONCLUSIONS: Our data suggest that CK2 mediates the stimulatory effects of TGF-beta1 on mesangial cell apoptosis via a p53-dependent mechanism. The ability of estradiol to reverse TGF-beta1-induced apoptosis may contribute to the protective effects of female gender on the course of chronic renal disease.     

Modulation of vascular smooth muscle cell alignment by cyclic strain is dependent on reactive oxygen species and P38 mitogen-activated protein kinase

PURPOSE: The aim of this study was to investigate the molecular targets of reactive oxygen species (ROS) and to determine whether cyclic strain induces smooth muscle cell (SMC) alignment via the ROS system. We assessed stretch-induced nicotinamide adenine dinucleotide phosphate (NAD(P)H) oxidase activation and the redox sensitivity of cyclic strain-stimulated activation of the mitogen-activated protein kinase (MAPK) family. METHODS: SMCs were seeded on flexible collagen I-coated plates and exposed to cyclic strain. NAD(P)H oxidase activation was measured with lucigenin-enhanced chemiluminescent detection of superoxide. Activation of MAPK was detected by determining phosphorylation of extracellular signal-regulated protein kinase (ERK1/2), c-jun N-terminal kinase (JNK1/2), and p38 MAPK with immunoblotting. In other experiments, SMCs were exposed to diphenylene iodonium (DPI), an NAD(P)H inhibitor, 30 minutes before stretch. MAPK activation and cell orientation were then assessed. RESULTS: Cyclic strain elicits a rapid increase in intracellular NADH/NADPH oxidase in SMCs. There was also a rapid and robust phosphorylation of ERK1/2, JNK1/2, and p38 MAPK. Cyclic strain-induced intracellular NAD(P)H generation was almost completely blocked with DPI. DPI also inhibited the strain-induced phosphorylation of ERK1/2, JNK1/2, and p38 MAPK. Both the p38 MAPK specific inhibitor, SB 202190, and DPI blocked cyclic strain-induced cell alignment, but PD98059, an ERK1/2-specific inhibitor, and SP600125, an anthrazolone inhibitor of JNK, did not. CONCLUSION: Our results provide evidence that p38 MAPK is a critical component of the oxidant stress ROS-sensitive signaling pathway and plays a crucial role in vascular alignment induced by cyclic stain.     

Neurotoxicity of Lidocaine Involves Specific Activation of the p38 Mitogen-activated Protein Kinase, but Not Extracellular Signal-regulated or c-jun N-Terminal Kinases, and Is Mediated by Arachidonic Acid Metabolites

Background: Pharmacologic inhibition of the p38 mitogen-activated protein kinase (MAPK) leads to a reduction in lidocaine neurotoxicity in vitro and in vivo. The current study investigated in vitro the hypotheses that lidocaine neurotoxicity is specific for dorsal root ganglion cells of different size or phenotype, involves time-dependent and specific activation of the p38 MAPK, that p38 MAPK inhibitors are only effective if applied with local anesthetic, and that p38 MAPK activation triggers activation of lipoxygenase pathways.

Methods: The authors used primary sensory neuron cultures and pheochromocytoma cell line cultures to detect time-dependent activation of the p38 MAPK or related pathways such as extracellular signal-regulated kinases and c-jun N-terminal kinases. Cells were divided by size or by immunoreactivity for calcitonin gene-related peptide or isolectin B4, indicative of nociceptive phenotype. The authors also investigated whether arachidonic acid pathways represent a downstream effector of the p38 MAPK in local anesthetic-induced neurotoxicity.

Results: All types of dorsal root ganglion cells were subject to neurotoxic effects of lidocaine, which were mediated by specific activation of the p38 MAPK but not extracellular signal-regulated kinases or c-jun N-terminal kinases. Neuroprotective efficacy of p38 MAPK inhibitors declined significantly when administered more than 1 h after lidocaine exposure. Activation of p38 MAPK preceded activation of arachidonic acid pathways. Neurotoxicity of lidocaine, specific activation of p38 MAPK, and neuroprotective effects of a p38 MAPK inhibitor were further confirmed in pheochromocytoma cell line cultures.     

Neurotoxicity of lidocaine involves specific activation of the p38 mitogen-activated protein kinase, but not extracellular signal-regulated or c-jun N-terminal kinases, and is mediated by arachidonic acid metabolites

BACKGROUND: Pharmacologic inhibition of the p38 mitogen-activated protein kinase (MAPK) leads to a reduction in lidocaine neurotoxicity in vitro and in vivo. The current study investigated in vitro the hypotheses that lidocaine neurotoxicity is specific for dorsal root ganglion cells of different size or phenotype, involves time-dependent and specific activation of the p38 MAPK, that p38 MAPK inhibitors are only effective if applied with local anesthetic, and that p38 MAPK activation triggers activation of lipoxygenase pathways. METHODS: The authors used primary sensory neuron cultures and pheochromocytoma cell line cultures to detect time-dependent activation of the p38 MAPK or related pathways such as extracellular signal-regulated kinases and c-jun N-terminal kinases. Cells were divided by size or by immunoreactivity for calcitonin gene-related peptide or isolectin B4, indicative of nociceptive phenotype. The authors also investigated whether arachidonic acid pathways represent a downstream effector of the p38 MAPK in local anesthetic-induced neurotoxicity. RESULTS: All types of dorsal root ganglion cells were subject to neurotoxic effects of lidocaine, which were mediated by specific activation of the p38 MAPK but not extracellular signal-regulated kinases or c-jun N-terminal kinases. Neuroprotective efficacy of p38 MAPK inhibitors declined significantly when administered more than 1 h after lidocaine exposure. Activation of p38 MAPK preceded activation of arachidonic acid pathways. Neurotoxicity of lidocaine, specific activation of p38 MAPK, and neuroprotective effects of a p38 MAPK inhibitor were further confirmed in pheochromocytoma cell line cultures. CONCLUSIONS: Specific and time-dependent activation of the p38 MAPK is involved in lidocaine-induced neurotoxicity, most likely followed by activation of lipoxygenase pathways.     

Vein graft arterialization causes differential activation of mitogen-activated protein kinases

OBJECTIVE: Vascular injury results in activation of the mitogen-activated protein kinases-extracellular-signal regulated kinases, c-jun N-terminal kinase, and p38(MAPK)-which have been implicated in cell proliferation, migration, and apoptosis. The goal of this study was to characterize mitogen-activated protein kinase activation in arterialized vein grafts. METHODS: Carotid artery bypass using reversed external jugular vein was performed in 29 dogs. Vein grafts were harvested after 30 minutes and 3, 8, and 24 hours, and 4, 7, 14, and 28 days. Contralateral external jugular vein and external jugular vein interposition vein-to-vein grafts were used as controls. Vein graft extracts were analyzed for extracellular-signal regulated kinases, c-jun N-terminal kinase, and p38(MAPK) activation. Proliferating cell nuclear antigen expression was investigated as a parameter of cell proliferation. Apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate nick end labeling staining and intimal hyperplasia by morphometric examination of tissue sections. RESULTS: Significant intimal hyperplasia was observed at 28 days. Over the time points studied, vein graft arterialization resulted in bimodal activation of both extracellular-signal regulated kinase and p38(MAPK) (30 minutes through 3 hours; 4 days) but did not induce activation of c-jun N-terminal kinase. Proliferating cell nuclear antigen expression increased from days 1 through 28, and apoptosis increased between 8 and 24 hours. CONCLUSION: Vein graft arterialization induces bimodal activation of extracellular-signal regulated kinase and p38(MAPK); however, in contrast with what is described in arterial injury, it does not induce c-jun N-terminal kinase activation. These results provide the first comprehensive characterization of the mitogen-activated protein kinase signaling pathways activated in vein graft arterialization and identify mitogen-activated protein kinases as potential mediators of vein graft remodeling and subsequent intimal hyperplasia.     

Expression of mitogen-activated protein kinase family in rat renal development

BACKGROUND: Among mitogen-activated protein kinase (MAPK) family members, extracellular signal-regulated kinase (ERK) promotes proliferation or differentiation, whereas c-Jun N-terminal kinase (JNK) and p38 MAPK (p38) are thought to inhibit cell growth and induce apoptosis. MAPK phosphatase-1 (MKP-1) inactivates and modulates MAPKs. During renal development, large scale proliferation and apoptosis occur. We investigated the temporal and spatial expression patterns of MAPKs and MKP-1 in rat kidney during development. METHODS: Western blot analysis and immunohistochemistry were performed in the developing and mature kidney of the rat. RESULTS: The expression of ERK, p38, and MKP-1 were high in developing kidney. On the other hand, JNK was abundantly expressed in adult kidney. Active forms of ERK, p38, and JNK correlated with the protein expression levels. Immunohistochemical studies revealed that ERK was strongly expressed by blastema cells, mesenchymal cells, and ureteric bud tips in nephrogenic zone of embryonic kidney. In neonatal kidney, ERK was more abundant in the deep cortex and the medulla corresponding to tubule maturation. p38 and MKP-1 were detected uniformly in mesenchymal cells, mesangial cells, and ureteric bud epithelia of fetal kidney without an obvious correlation with the occurrence of apoptosis. JNK was expressed by tubular cells and podocytes of adult kidney. CONCLUSIONS: ERK, p38, and MKP-1 are strongly expressed in developing kidney, and JNK is detected predominantly in adult kidney. Both the temporal and spatial expression of ERK coincides with the maturation of the kidney.     

Role of reactive oxygen species in TGF-beta1-induced mitogen-activated protein kinase activation and epithelial-mesenchymal transition in renal tubular epithelial cells

Epithelial-mesenchymal transition (EMT) plays an important role in renal tubulointerstitial fibrosis and TGF-beta1 is the key inducer of EMT. Phosphorylation of Smad proteins and/or mitogen-activated protein kinases (MAPK) is required for TGF-beta1-induced EMT. Because reactive oxygen species (ROS) are involved in TGF-beta1 signaling and are upstream signaling molecules to MAPK, this study examined the role of ROS in TGF-beta1-induced MAPK activation and EMT in rat proximal tubular epithelial cells. Growth-arrested and synchronized NRK-52E cells were stimulated with TGF-beta1 (0.2 to 20 ng/ml) or H(2)O(2) (1 to 500 microM) in the presence or absence of antioxidants (N-acetylcysteine or catalase), inhibitors of NADPH oxidase (diphenyleneiodonium and apocynin), mitochondrial electron transfer chain subunit I (rotenone), and MAPK (PD 98059, an MEK [MAP kinase/ERK kinase] inhibitor, or p38 MAPK inhibitor) for up to 96 h. TGF-beta1 increased dichlorofluorescein-sensitive cellular ROS, phosphorylated Smad 2, p38 MAPK, extracellular signal-regulated kinases (ERK)1/2, alpha-smooth muscle actin (alpha-SMA) expression, and fibronectin secretion and decreased E-cadherin expression. Antioxidants effectively inhibited TGF-beta1-induced cellular ROS, phosphorylation of Smad 2, p38 MAPK, and ERK, and EMT. H(2)O(2) reproduced all of the effects of TGF-beta1 with the exception of Smad 2 phosphorylation. Chemical inhibition of ERK but not p38 MAPK inhibited TGF-beta1-induced Smad 2 phosphorylation, and both MAPK inhibitors inhibited TGF-beta1- and H(2)O(2)-induced EMT. Diphenyleneiodonium, apocynin, and rotenone also significantly inhibited TGF-beta1-induced ROS. Thus, this data suggest that ROS play an important role in TGF-beta1-induced EMT primarily through activation of MAPK and subsequently through ERK-directed activation of Smad pathway in proximal tubular epithelial cells.     

Involvement of the Mitogen-activated Protein Kinase Family in Tetracaine-induced PC12 Cell Death

Background: To explore whether cytotoxicity of local anesthetics is related to apoptosis, the authors examined how local anesthetics affect mitogen-activated protein kinase (MAPK) family members, extracellular signal-regulated kinases (ERKs), c-Jun N-terminal kinases (JNKs)-stress-activated protein kinases, and p38 kinase, which are known to play important roles in apoptosis.

Methods: Cell death was evaluated using PC12 cells. Morphologic changes of cells, cellular membrane, and nuclei were observed. DNA fragmentation was electrophoretically assayed. Western blot analysis was performed to analyze phosphorylation of the MAPK family, cleavage of caspase-3 and poly(adenosine diphosphate-ribose) polymerase. Intracellular Ca2+ concentration was measured using a calcium indicator dye.

Results: Tetracaine-induced cell death was shown in a time- and concentration-dependent manner and characterized by nuclear condensation or fragmentation, membrane blebbing, and internucleosomal DNA fragmentation. Casepase-3 activation and phosphorylation of ERK, JNK, and p38 occurred in the cell death. PD98059, an inhibitor of ERK, enhanced tetracaine-induced cell death and JNK phosphorylation, whereas ERK phosphorylation was inhibited. Curcumin, an inhibitor of JNK pathway, attenuated the cell death. Increase of intracellular Ca2+ concentration was detected. In addition to the increase of ERK phosphorylation and the decrease of JNK phosphorylation, two Ca2+ chelators protected cells from death. Neither cell death nor phosphorylation of the MAPK family was caused by tetrodotoxin. Nifedipine did not affect tetracaine-induced apoptosis.     

Homocysteine enhances bone resorption by stimulation of osteoclast formation and activity through increased intracellular ROS generation.

Hyperhomocystinemia is a modifiable risk factor for osteoporosis and fracture. Physiologic concentrations of Hcy directly activate osteoclast formation and activity through stimulation of p38 MAPK and integrin beta3. The effects of Hcy were mediated by generation of intracellular ROS. INTRODUCTION: Hyperhomocysteinemia is a modifiable risk factor for osteoporosis and its related bone fractures. It has been reported that bone resorption and turnover rate were increased in hyperhomocystinemia. Using mouse bone marrow cells, we examined the direct effects of homocysteine (Hcy) on osteoclast formation and activity. MATERIALS AND METHODS: Osteoclast formation was determined by TRACP staining and TRACP activity. Intracellular reactive oxygen species (ROS) generation was measured using a fluorescent probe, dichlorodihydrofluorescein diacetate. Intracellular signaling cascades of p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and NF-kappaB were measured by Western blotting. Integrin beta3 mRNA levels were measured by RT-PCR. Actin ring formation and bone resorption assays were also performed. RESULTS: Physiologic concentrations of Hcy upregulated TRACP+ multinucleated cells and TRACP activity, stimulated actin ring formation, and increased the number of nuclei per cell and the level of expression of integrin beta3 mRNA. In addition, Hcy increased bone resorption and stimulated p38 MAPK activity and intracellular reactive oxygen species (ROS) generation. All of these Hcy-induced changes were blocked by pretreatment with the antioxidant, N-acetyl cysteine. CONCLUSIONS: Hcy directly activates osteoclast formation and activity through increased generation of intracellular ROS. These findings suggest that, in individuals with mild to moderate hyperhomocystinemia, increased bone resorption by osteoclasts may contribute to osteoporosis and that an antioxidant may attenuate bone loss in these individuals.     

Activation of mitogen-activated protein kinases during preparation of vein grafts and modulation by a synthetic inhibitor

OBJECTIVE: Long-term durability of saphenous vein grafts used for coronary artery bypass grafting is limited by neointimal formation. Arterial vascular injury is known to activate intracellular mitogen-activated protein kinases, including extracellular signal-regulated kinases and c-jun N-terminal kinases, that affect cell differentiation, proliferation, migration, and apoptosis. This study tests the hypothesis that these mitogen-activated protein kinases are activated in saphenous veins during preparation for coronary artery bypass grafting. METHODS: Saphenous veins were harvested from 10 patients undergoing coronary artery bypass grafting. A specimen from each vein was placed in ice-cold lysis buffer immediately after harvesting (t = 0). The remaining tissue was incubated at room temperature in normal saline, 0.1% dimethylsulfoxide (vehicle), or 50 mmol/L PD98059 (mitogen-activated protein kinase kinase-1/2 inhibitor) until the vein was grafted (mean 50 minutes). To study kinetics of intracellular signaling pathways, canine saphenous veins were harvested, and mitogen-activated protein kinases and PI-3 kinase pathways were studied after different incubation time intervals. Extracted proteins were analyzed by Western blotting or in vitro kinase assay. RESULTS: The human saphenous veins showed elevated levels of active extracellular signal-regulated kinase after harvesting (t = 0) and prior to implant (t = 1). Incubation with PD98059 resulted in decreased activation of extracellular signal-regulated kinase. Kinetics of canine saphenous veins showed extracellular signal-regulated kinase and c-jun N-terminal kinase activation, in a time-dependent manner, along with activation of the growth factor-regulated PI3 kinase pathway. CONCLUSIONS: This study characterizes activation of extracellular signal-regulated kinases and c-jun N-terminal kinases during vein graft preparation and demonstrates the ability to inhibit extracellular signal-regulated kinase activation by simple incubation with a specific inhibitor. Further studies are needed to evaluate the significance of these findings with respect to graft durability.     

Differential contribution of three mitogen-activated protein kinases to PDGF-BB-induced mesangial cell proliferation and gene expression

This study examined the role of mitogen-activated protein (MAP) kinase in PDGF-BB-induced proliferation and gene expression of human mesangial cells (MC). PDGF-BB stimulation of MC increased mRNA for transforming growth factor-beta1 (TGF-beta1), monocyte chemoattractant protein-1 (MCP-1), and plasminogen activator inhibitor-1 (PAI-1) and increased the cell numbers. To inhibit activation of extracellular signal-regulated kinase (ERK), c-Jun amino-terminal kinase (JNK), and p38, MC were infected with recombinant adenovirus containing dominant-negative mutants of ERK, JNK, and p38 (Ad-DN-ERK, Ad-DN-JNK, Ad-DN-p38, respectively), respectively. Infection of MC with Ad-DN-ERK or Ad-DN-JNK inhibited PDGF-BB-induced increase in [(3)H]thymidine incorporation and cell numbers, whereas Ad-DN-p38 did not. Ad-DN-ERK inhibited MCP-1 and PAI-1 mRNA expression in MC, but not TGF-beta1. Ad-DN-JNK and Ad-DN-p38 inhibited TGF-beta1 and MCP-1 mRNA expression, but not PAI-1. The inhibition of activator protein-1 (AP-1) in MC, by adenovirus containing dominant-negative mutant of c-Jun (Ad-DN-c-Jun), inhibited PDGF-BB-induced cell proliferation and TGF-beta1, MCP-1, and PAI-1 expressions. Furthermore, Ad-DN-JNK or Ad-DN-p38, but not Ad-DN-ERK, attenuated PDGF-BB-induced AP-1 activation in MC, indicating the involvement of JNK and p38 in AP-1 activation. Our results indicated that ERK and JNK, but not p38, participated in PDGF-BB-induced MC proliferation. PDGF-BB-induced expression of TGF-beta1 was mediated by JNK and p38, MCP-1 expression was through ERK, JNK, and p38, whereas PAI-1 expression was due to only ERK. AP-1 activation, which was partially due to JNK and p38 activations, was involved in MC proliferation and these three gene expressions. Thus, three MAP kinases seem to contribute to progression of glomerular disease via different molecular mechanisms.     

Activation of ERK and p38 kinase mediated keloid fibroblast apoptosis after flashlamp pulsed-dye laser treatment

BACKGROUND AND OBJECTIVES: Flashlamp pulsed-dye lasers (PDLs) revealed effective regression or arrest in patients with keloids in our clinical studies [Kuo YR et al., Laser Surg Med 2004;34:104-108]. In this study, we further investigated whether the induction of keloid regression seen with PDL treatment through activation in mitogen-activated protein (MAP) kinase and caspase promotes cell apoptosis and reduces fibroblast proliferation. STUDY DESIGN/MATERIALS AND METHODS: Keloid tissues were obtained from 10 patients with intralesional or punch biopsies prior to and 7 days after PDL treatments [fluence per pulse was 10-18 J/cm2 (mean 14 J/cm2)]. Prior to and after PDL treatments, the proliferating fibroblasts in keloid tissue were immunohistochemically detected by proliferating cell nuclear antigen (PCNA) expression. The apoptotic cell was detected by terminal deoxynucleotidyl transferase dUTP-nick end labeling (TUNEL) staining and fragmented caspase-3 expression. MAP kinase activation as represented by extracellular signal-regulated kinase (ERK), p38 kinase (p38), and c-Jun N-terminal kinase (JNK) expression of keloid tissues was investigated by immunohistochemical (IHC) staining, respectively. RESULTS: IHC staining indicated that PCNA expression of fibroblasts was significantly reduced in keloid tissue after PDL irradiation. TUNEL assay revealed lower apoptotic cells expression in the keloid tissue prior to laser treatment. Following laser treatment, apoptotic cells with relatively strong DNA damage and fragmentation were seen in all keloid biopsy samples, especially in the keloid fibroblast population. The activation of ERK and p38 MAP kinase increased significantly in keloid tissue after PDL treatment. JNK was shown to be unchanged. CONCLUSIONS: The PDL treatment is shown to induce keloid regression through suppression of keloid fibroblast proliferation, induction of apoptosis, and upregulation of ERK and p38 MAP kinase activity.     

Anti-apoptotic effect of quercetin: intervention in the JNK- and ERK-mediated apoptotic pathways

BACKGROUND: Bioflavonoid quercetin inhibits hydrogen peroxide (H2O2)-induced apoptosis via intervention in the activator protein 1 (AP-1)-mediated apoptotic pathway. In this report, we investigated molecular events involved in the anti-apoptotic effect of quercetin, focusing especially on its effects on the family of mitogen-activated protein (MAP) kinases. METHODS: Cultured mesangial cells were exposed to H2O2, and activation of c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinases (ERKs), and p38 MAP kinase was evaluated in the presence or absence of quercetin. Using pharmacological and genetic inhibitors, the roles for individual MAP kinases in H2O2-induced apoptosis were examined. Involvement of ERKs in the induction and activation of AP-1 was also investigated using Northern blot analysis and a reporter assay. RESULTS: Mesangial cells exposed to H2O2 exhibited rapid phosphorylation of JNK, ERKs, and p38 MAP kinase. Quercetin abrogated the activation of all three MAP kinases in response to H2O2. Pretreatment with MAP kinase kinase inhibitor PD098059 or JNK-c-Jun/AP-1 inhibitor curcumin attenuated the H2O2-induced apoptosis. In contrast, the p38 MAP kinase inhibitor SB203580 did not improve the cell survival. Consistently, transfection with dominant-negative mutants of ERK1 and ERK2 or a dominant-negative mutant of JNK inhibited H2O2-induced apoptosis. Transfection with a dominant-negative p38 MAP kinase did not attenuate the apoptotic process. Inhibition of ERKs by PD098059 suppressed induction of c-fos without affecting early induction of c-jun, leading to attenuated activation of AP-1 in response to H2O2. CONCLUSIONS: These results suggested that (1) activation of JNK and ERKs, but not p38 kinase, is required for the H2O2-induced apoptosis; and (2) suppression of the JNK-c-Jun/AP-1 pathway and the ERK-c-Fos/AP-1 pathway is involved in the anti-apoptotic effect of quercetin.     

Modulation of phorbol ester-induced regulation of matrix metalloproteinases and tissue inhibitors of metalloproteinases by SB203580, a specific inhibitor of p38 mitogen-activated protein kinase

OBJECT: Expression of matrix metalloproteinases (MMPs) has been postulated to play a central role in brain tumor invasion; however, its underlying mechanism is not yet fully understood. In the present study, by assessing the effect of a specific p38 mitogen-activated protein kinase (MAPK) inhibitor, SB203580, on the secretion of MMPs and in vitro invasion of various glioma cells, the authors attempt to define the role of the p38 MAPK pathway in the regulation of MMPs and tissue inhibitors of metalloproteinases (TIMPs) activated by phorbol ester (phorbol-12-myristate-13-acetate [PMA]) in the D54 human glioblastoma cell line. METHODS: The activation of MAPKs was determined using Western blot analysis after addition of phospho-specific antibodies against these kinases, the status of MMPs and TIMPs was analyzed using gelatin zymography and Western blot analysis, and the invasion rate of D54 cells and other glioma cells was analyzed using a modified Boyden chamber assay. Treatment of D54 cells with PMA activated two distinct MAPKs, extracellular signal-regulated kinase (ERK) 1/2 and p38 MAPK, but not c-Jun N-terminal kinase/stress-activated protein kinase. Induction of MMP-9 production and MMP-2 activation by PMA were blocked by SB203580, a specific inhibitor of p38 MAPK, but not by PD98059, a specific inhibitor of ERK 1/2. In addition, PMA-induced downregulation of TIMP-1 and TIMP-2 secretion and upregulation of the membrane type I MMP, a major activator of MMP-2 on the cell surface, were reversed by SB203580 in these cells; the PMA-induced increase of invasion in vitro decreased when SB203580 was added to the top compartment of a modified Boyden chamber; and the inhibitor also reduced the MMP secretion and PMA-induced in vitro invasion in various glioma cell lines. CONCLUSIONS: These results indicate that activation of p38 MAPK by PMA plays a central role in the regulation of MMPs and TIMPs in D54 cells, which has a major influence in tumor invasion and metastasis. Furthermore, inhibition of p38 MAPK by SB203580 blocked the secretion of MMPs and in vitro invasion of various glioma cells, underscoring a possible role of p38 MAPK inhibitors as antiinvasive and/or antimetastatic agents of malignant gliomas.