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.     

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.     

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 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.     

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.     

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.     

Stimulation of MAPK cascades by insulin and osmotic shock: lack of an involvement of p38 mitogen-activated protein kinase in glucose transport in 3T3-L1 adipocytes

Osmotic shock and insulin stimulate GLUT4 translocation and glucose transport via mechanisms that are for the most part distinct yet convergent. In this article, we investigated the effect of osmotic shock and insulin on the activation of the mitogen-activated protein kinase (MAPK) cascades in differentiated 3T3-L1 adipocytes. The MAPKs are activated by phosphorylation on conserved tyrosine and threonine residues. Both sorbitol and insulin strongly stimulated extracellular regulated kinase (ERK) 1 and 2 phosphorylation (8- and 18-fold, respectively). In contrast, c-jun-NH2-terminal kinase (JNK)/stress-activated protein kinase (SAPK) phosphorylation was stimulated only by sorbitol (sevenfold) and not by insulin. Phosphorylation of p38 MAPK was stimulated strongly by sorbitol (22-fold) but weakly by insulin (2.7-fold). Measurement of intrinsic JNK and p38 MAPK activity confirmed the phosphorylation studies. JNK and p38 MAPK were activated only significantly by sorbitol. The MAPKs are phosphorylated by dual-specificity kinases (mitogen-activated ERK-activating kinase [MEK] or MAPK kinase [MKK]). As expected, sorbitol and insulin both stimulated MEK phosphorylation. MKK4 was phosphorylated only in response to sorbitol, and neither of the stimuli caused phosphorylation of MKK3 or 6. To determine the functional significance of the observed activation of p38 MAPK in response to insulin and osmotic shock, we used three pyridinyl imidazole p38 MAPK inhibitors, SB203580, SB202190, and PD169316. Insulin and osmotic shock-stimulated glucose transport was not inhibited by any inhibitor at concentrations that were shown to block p38 MAPK activity. Furthermore, activation of the p38 MAPK pathway by treatment of cells with anisomycin did not stimulate glucose transport. These results suggest that activation of the p38 MAPK pathway is not involved in the stimulation of glucose transport.     

Low-level laser therapy prevents endothelial cells from TNF-α/cycloheximide-induced apoptosis

Low-level laser therapy (LLLT), widely used in physiotherapy, has been known to enhance wound healing and stimulate cell proliferation, including fibroblast and endothelial cells. Applying LLLT can increase cell proliferation in many kinds of cells including fibroblasts and endothelial cells. However, the protective mechanisms of LLLT on endothelial apoptosis remain unclear. We hypothesized LLLT can protect endothelial cells from inflammation-induced apoptosis. Human endothelial cell line, EA.hy926 cells, and TNF-α/cycloheximide (TNF/CHX) were used to explore the protective effects of LLLT (660 nm) on inflammation-induced endothelial apoptosis. Cell viability, apoptosis, caspase-3/7/8/9 activity, MAPKs signaling, NF-κB activity, and inducible/endothelial nitric oxide synthase (iNOS/eNOS) expression were measured. Our results showed that LLLT increased EA.hy926 cell proliferation, attenuated the TNF/CHX-induced apoptosis, and reduced the TNF/CHX-mediated caspase-3/7/8/9 activation. In addition, LLLT increased ERK MAPK phosphorylation and suppressed the TNF/CHX-increased p38 MAPK, JNK, IKK phosphorylation, NF-κB translocation, and iNOS expression. The caspases-3 cleavage and cell death were not increased in cells treating with ERK inhibitor U0126, which implicated that ERK is not to be responsible for the protective effects of LLLT. After treating with p38 mitogen-activated protein kinase (MAPK) activator, the protection of LLLT in cell apoptosis was no longer existed, showing that LLLT protected the endothelial cells by suppressing p38 MAPK signaling. Our results provide a new insight into the possible molecular mechanisms in which LLLT protects against inflammatory-induced endothelial dysfunction.     

p53 and mitogen‐activated protein kinase pathway protein profiles in fresh and frozen spermatozoa

Sperm or testicular tissue cryopreservation is performed in cases of male infertility as a treatment for the preservation of fertility. When these sperm cells are used in assisted reproductive techniques, fertilisation rates, developmental and implantation potential of embryos decrease and the abortion rates increase. In the present work, differences of both phosphorylation and expression levels of p53 and Mitogen‐activated protein kinases (MAPK) proteins were analysed in 61 individual sperm samples before and after cryopreservation. We observed that p53 protein residue at Ser 15 was phosphorylated after cryopreservation. Because MAPK pathway activations may be involved in p53 phosphorylation, MAPK/ERK, Stress‐activated protein kinases (SAPK)/JNK and p38MAPK proteins were also investigated. Analysis showed that p38MAPK phosphorylations increased significantly. However, ERK and JNK expressions and phosphorylations decreased, although the differences were not statistically significant. According to our results, it may be suggested that cryopreservation process activates p53 via p38 MAPK pathway that subsequently causes apoptosis, which may be related to sperm parameters.     

Mitogen-activated protein kinases signal inhibition of apoptosis in lipopolysaccharide-stimulated neutrophils.

BACKGROUND: Neutrophil (PMN) apoptosis is critical to the resolution of infection and the limitation of inflammation. Bacterial endotoxin (lipopolysaccharide [LPS]) inhibits PMN apoptosis and activates the p38 mitogen-activated protein kinase (MAPK) signal cascade. The role of p38 and other MAPKs (ERK and SAPK/JNK) in regulating PMN apoptosis after LPS stimulation is unknown. We hypothesize that MAPK activation by LPS signals inhibition of PMN apoptosis. METHODS: PMNs were isolated from the blood of healthy human volunteers and incubated with PD98059 (ERK inhibitor), SB203580 (p38 inhibitor), or 0.1% dimethyl sulfoxide (vehicle) for 1 hour before treatment with LPS (0, 10, or 1000 ng/mL). Neutrophil MAPK activation was determined by Western blot analysis for phosphorylated p38, ERK, and SAPK/JNK. Apoptosis was quantified by flow cytometry with use of propidium iodide and annexin V. RESULTS: LPS inhibited PMN apoptosis and activated p38 and ERK in a dose- and time-dependent fashion. SAPK/JNK was not activated by LPS. Treatment of cells with ERK inhibitor before LPS stimulation abrogated LPS signaled inhibition of PMN apoptosis. Conversely, p38 inhibition with SB203580 augmented inhibition of apoptosis by LPS. CONCLUSIONS: These data demonstrate opposing roles of MAPKs in mediating PMN apoptosis after LPS stimulation. We conclude that LPS signal transduction by ERK inhibits PMN apoptosis while activation of p38 promotes apoptosis.     

Evidence for JNK-dependent up-regulation of proteoglycan synthesis and for activation of JNK1 following cyclical mechanical stimulation in a human chondrocyte culture model

OBJECTIVE: To examine the expression of mitogen-activated protein kinases (MAPKs) in human chondrocytes, to investigate whether selective activation of MAPKs is involved in up-regulation of proteoglycan (PG) synthesis following cyclical mechanical stimulation (MS), and to examine whether MS is associated with integrin-dependent or independent activation of MAPKs. METHODS: The C-28/I2 and C-20/A4 human chondrocyte cell lines were mechanically stimulated in monolayer cell culture. PG synthesis was assessed by [(35)S]-sulphate incorporation in the presence and absence of the p38 inhibitor SB203580, and the extracellular-regulated kinase (ERK1/2) inhibitor PD98059. Kinase expression and activation were assessed by Western blotting using phosphorylation status-dependent and independent antibodies, and by kinase assays. The Jun N-terminal kinase (JNK) inhibitor SP600125 and the anti-beta(1) integrin (CD29) function-blocking antibody were used to assess JNK activation and integrin dependence, respectively. RESULTS: Increased PG synthesis following 3 h of cyclic MS was abolished by pretreatment with 10 microM SB203580, but was not affected by 50 microM PD98059. The kinases p38, ERK1/ERK2 and JNKs were expressed in both stimulated and unstimulated cells. Phosphorylated p38 was detected at various time points following 0.5, 1, 2 and 3 h MS in C-28/I2, but not detected in C-20/A4 cell lines. Phosphorylation of ERK1 and ERK2 was not significantly affected by MS. Phosphorylation of the 54 and 46 kDa JNKs increased following 0.5, 1, 2 and 3 h of MS, and following CO(2) deprivation. MS-induced JNK phosphorylation was inhibited by SB203580 at concentrations > or =5 microM and activation of JNK1 following MS was blocked by SP600125 and partially inhibited by anti-CD29. CONCLUSIONS: The data suggest JNK, rather than p38 or ERK dependent increases in PG synthesis, and selective, partially integrin-dependent, activation of JNK kinases in human chondrocyte cell lines following cyclical MS. JNK activation is also very sensitive to changes in CO(2)/pH in this chondrocyte culture model.     

Role of Protein Kinase C, Ca2+/Calmodulin-dependent Protein Kinase II, and Mitogen-activated Protein Kinases in Volatile Anesthetic-induced Relaxation in Newborn Rabbit Pulmonary Artery

Background: This study examined the responsiveness of skinned pulmonary arteries from newborn rabbit to volatile anesthetics and the role of protein kinase C (PKC), Ca2+/calmodulin-dependent protein kinase II (CaMKII), and the downstream effectors, mitogen-activated protein kinases (ERK1/2 and p38).

Methods: Pulmonary arterial strips from 9- to 12-day-old rabbits were mounted on force transducers and treated with saponin ("skinned" strips). The skinned strips were activated by pCa 6.3 until force reached a steady state (control). Isoflurane or halothane was then administered. The result (test) was expressed as a percentage of the control. Inhibitors included bisindolylmaleimide (Ca2+-dependent and -independent PKC), Go6976 (Ca2+-dependent PKC), CKIINtide (CaMKII), KN-93 (CaMKII), PD98059 (MEK/ERK1/2), and SB203580 (p38).

Results: The anesthetics dose-dependently decreased pCa-induced force (4-32% for 1-5% isoflurane; 17-76% for 1-3% halothane). The inhibitors of PKC (bisindolylmaleimide and Go6976) and MEK/ERK1/2 (PD98059) completely prevented the relaxation induced by 3% isoflurane and partially prevented that induced by 2% and 3% halothane with the same effective inhibitor concentrations. In contrast, the effective concentration of CaMKII inhibitors was a direct function of the anesthetic concentration for different inhibitors (KN-93 for isoflurane and CKIINtide for halothane), and that of the p38 inhibitor (SB20358) was a direct function of both anesthetics.     

Fibrogenic stresses activate different mitogen-activated protein kinase pathways in renal epithelial, endothelial or fibroblast cell populations

Fibrogenic stresses promote progression of renal tubulointerstitial fibrosis, disparately affecting survival, proliferation and trans-differentiation of intrinsic renal cell populations through ill-defined biomolecular pathways. We investigated the effect of fibrogenic stresses on the activation of cell-specific mitogen-activated protein kinase (MAPK) in renal fibroblast, epithelial and endothelial cell populations. The relative outcomes (cell death, proliferation, trans-differentiation) associated with activation or inhibition of extracellular-regulated protein kinase (ERK) or stress activated/c-Jun N terminal kinase (JNK) were analysed in each renal cell population after challenge with oxidative stress (1 mmol/L H2O2), transforming growth factor-beta1 (TGF-beta1, 10 ng/mL) or tumour necrosis factor-alpha (TNF-alpha, 50 ng/mL) over 0-20 h. Apoptosis increased significantly in all cell types after oxidative stress (P < 0.05). In fibroblasts, oxidative stress caused the activation of ERK (pERK) but not JNK (pJNK). Inhibition of ERK by PD98059 supported its role in a fibroblast death pathway. In epithelial and endothelial cells, oxidative stress-induced apoptosis was preceded by early induction of pERK, but its inhibition did not support a pro-apoptotic role. Early ERK activity may be conducive to their survival or promote the trans-differentiation of epithelial cells. In epithelial and endothelial cells, oxidative stress induced pJNK acutely. Pretreatment with SP600125 (JNK inhibitor) verified its pro-apoptotic activity only in epithelial cells. Transforming growth factor-beta1 did not significantly alter mitosis or apoptosis in any of the cell types, nor did it alter MAPK activity. Tumor necrosis factor-alpha caused increased apoptosis with no associated change in MAPK activity. Our results demonstrate renal cell-specific differences in the activation of ERK and JNK following fibrotic insult, which may be useful for targeting excessive fibroblast proliferation in chronic fibrosis.     

Effects of insulin, contraction, and phorbol esters on mitogen-activated protein kinase signaling in skeletal muscle from lean and ob/ob mice

Effects of diverse stimuli, including insulin, muscle contraction, and phorbol 12-myristate-13-acetate (PMA), were determined on phosphorylation of mitogen-activated protein kinase (MAPK) signaling modules (c-Jun NH(2)-terminal kinase [JNK], p38 MAPK, and extracellular signal-related kinase [ERK1/2]) in skeletal muscle from lean and ob/ob mice. Insulin increased phosphorylation of JNK, p38 MAPK, and ERK1/2 in isolated extensor digitorum longus (EDL) and soleus muscle from lean mice in a time- and dose-dependent manner. Muscle contraction and PMA also elicited robust effects on these parallel MAPK modules. Insulin action on JNK, p38 MAPK, and ERK1/2 phosphorylation was significantly impaired in EDL and soleus muscle from ob/ob mice. In contrast, muscle contraction-mediated JNK, p38 MAPK, and ERK1/2 phosphorylation was preserved. PMA effects on phosphorylation of JNK and ERK1/2 were normal in ob/ob mice, whereas effects on p38 MAPK were abolished. In conclusion, insulin, contraction, and PMA activate MAPK signaling in skeletal muscle. Insulin-mediated responses on MAPK signaling are impaired in skeletal muscle from ob/ob mice, whereas the effect of contraction is generally well preserved. In addition, PMA-induced phosphorylation of JNK and ERK1/2 are preserved, whereas p38 MAPK pathways are impaired in skeletal muscle from ob/ob mice. Thus, appropriate MAPK responses can be elicited in insulin-resistant skeletal muscle via an insulin-independent mechanism.     

Low-level Er:YAG laser irradiation enhances osteoblast proliferation through activation of MAPK/ERK

Although the use of high-level Er:YAG laser irradiation has been increasing in periodontal and peri-implant therapy, the effects of low-level Er:YAG laser on surrounding tissues and cells remain unclear. In the present study, the effects of low-level Er:YAG laser irradiation on osteoblast proliferation were investigated. Cells of the osteoblastic cell line MC3T3-E1 were treated with low-level Er:YAG laser irradiation with various combinations of laser settings (fluence 0.7–17.2 J/cm²) and in the absence or presence of culture medium during irradiation. On day 1 and/or day 3, cell proliferation and death were determined by cell counting and by measurement of lactate dehydrogenase (LDH) levels. Further, the role of mitogen-activated protein kinase (MAPK) pathways in laser-enhanced cell proliferation was investigated by inhibiting the MAPK pathways and then measuring MAPK phosphorylation by Western blotting. Higher proliferation rates were found with various combinations of irradiation parameters on days 1 and 3. Significantly higher proliferation was also observed in laser-irradiated MC3T3-E1 cells at a fluence of approximately 1.0–15.1 J/cm², whereas no increase in LDH activity was observed. Further, low-level Er:YAG irradiation induced the phosphorylation of extracellular signal-regulated protein kinase (MAPK/ERK) 5 to 30 min after irradiation. Although MAPK/ERK 1/2 inhibitor U0126 significantly inhibited laser-enhanced cell proliferation, activation of stress-activated protein kinases/Jun N-terminal kinase (SAPK/JNK) and p38 MAPK was not clearly detected. These results suggest that low-level Er:YAG laser irradiation increases osteoblast proliferation mainly by activation of MAPK/ERK, suggesting that the Er:YAG laser may be able to promote bone healing following periodontal and peri-implant therapy.     

Inhibition of p38 mitogen activated protein kinase increases lipopolysaccharide induced inhibition of apoptosis in neutrophils by activating extracellular signal-regulated kinase

BACKGROUND: Prolonged polymorphonuclear neutrophil (PMN) survival has been implicated in tissue injury after sepsis. Previously we reported that lipopolysaccharide (LPS) inhibits PMN apoptosis via the activation of the extracellular signal-regulated kinase (ERK). Conversely, the p38 mitogen activated protein kinase (MAPK) pathway is involved in the spontaneous apoptosis of PMNs. The interaction between these 2 pathways and their ability to regulate apoptosis during sepsis remain largely undefined. We hypothesize that there is interaction between the ERK and p38 pathways during sepsis. METHODS: PMNs were isolated from healthy volunteers by Ficoll gradient centrifugation and red blood cell sedimentation. Cells were then pretreated for 1 hour with the ERK inhibitor (PD98059, 10 micromol/L), p38 inhibitor (SB203580, 1 micromol/L), or vehicle. After treatment with LPS, apoptosis and MAPK activity were correlated. RESULTS: LPS stimulation significantly inhibits PMN apoptosis compared with unstimulated cells. Furthermore, inhibition of ERK significantly abrogates this effect, whereas inhibition of p38 augments LPS induced inhibition of apoptosis. Elk-1 phosphorylation (ERK target) is significantly increased by LPS alone and by inhibition of the p38 pathway during LPS stimulation. This correlates with ERK phosphorylation by Western blot. CONCLUSIONS: These data show that p38 inhibition enhances ERK activity during endotoxemia. Furthermore, these data suggest that cooperation between ERK and p38 MAPK pathways dictates the apoptotic potential of PMNs during inflammatory states.     

Extracellular signal-regulated kinase inhibition slows disease progression in mice with polycystic kidney disease

The expression of mitogen-activated protein kinases (MAPK) in DBA/2-pcy/pcy (pcy) mice, a murine model of polycystic kidney disease was investigated. Proliferating cell nuclear antigen-positive cells were recognized in cyst epithelium from embryonic day 14.5 to 25 wk of age. Extracellular signal-regulated kinase (ERK) was expressed in the renal tubules of control and pcy mice, but stronger immunostaining was observed in cyst epithelium. Phosphorylated ERK was detected only in pcy mice and was localized predominantly in the cysts. p38 MAPK (p38) was no longer expressed after birth in controls but was detected in the cyst epithelium and in occasional tubular cells of pcy mice at all stages examined. c-Jun N-terminal kinase (JNK) was expressed in all tubular segments of controls after neonatal day 7, whereas in pcy kidneys, tubules became positive for JNK after 8 wk, and the cysts expressed little JNK. Administration of an oral MAP/ERK kinase inhibitor, PD184352, 400 mg/kg per d, to 10-wk-old pcy mice daily for the first week and then every third day for 6 additional weeks significantly decreased BP, kidney weight, serum creatinine level, and water intake and significantly increased urine osmolality. The cystic index and expression of phosphorylated ERK and ERK were significantly lower in PD184352-treated pcy mice. These results demonstrate that the expression of MAPK is dysregulated in cyst epithelium and that inhibition of ERK slowed the progression of renal disease in pcy mice.     

Molecular mechanisms of cell death of mycophenolic acid-treated primary isolated rat islets: implication of mitogen-activated protein kinase activation

Optimal immunosuppression after pancreas islet transplantation has not yet been established to achieve long-term graft survival. Mycophenolic acid (MPA) is widely used as an immunosuppressive drug after transplantation including among recipients of pancreas islet cells. Previously, we reported MPA-induced islet apoptosis in the HIT-T15 cell line. In this study, we confirmed the effects of MPA on cell death and its potential implications on the mitogen-activated protein kinase (MAPK) family expression levels in primary isolated rat islets. Lewis islets isolated by collagenase digestion were purified by the density gradient method. Cell death was analyzed by methylthiazoletetrazolium assay. Activation of MAPK kinase 4 (MKK4), c-jun N-terminal protein kinase (JNK), p38 MAPK, and caspase-3 cleavage was examined by Western blot analyses. MPA treatments (>25 μmol/L) increased cell death significantly at 24 hours and in a dose-dependent manner activated MKK4, JNK, and p38 MAPK at 20 hours. Caspase-3 cleavage was also increased by MPA treatment. These results suggested that MPA induced significant cell death among primary isolated rat islets by activation of MKK4, JNK, and p38 MAPK, as well as caspase-3 cleavage.