Involvement of p42/p44 MAPK, p38 MAPK, JNK, and NF-kappaB in IL-1beta-induced VCAM-1 expression in human tracheal smooth muscle cells

Interleukin-1beta (IL-1beta) has been shown to induce the expression of adhesion molecules on airway epithelial and smooth cells and contributes to inflammatory responses. Here, the roles of mitogen-activated protein kinases (MAPKs) and nuclear factor-kappaB (NF-kappaB) pathways for IL-1beta-induced vascular cell adhesion molecule (VCAM)-1 expression were investigated in human tracheal smooth muscle cells (HTSMC). IL-1beta induced expression of VCAM-1 protein and mRNA in a time-dependent manner, which was significantly inhibited by inhibitors of MEK1/2 (U0126 and PD-98059), p38 (SB-202190), and c-Jun NH(2)-terminal kinase (JNK; SP-600125). Consistently, IL-1beta-stimulated phosphorylation of p42/p44 MAPK, p38, and JNK was attenuated by pretreatment with U0126, SB-202190, or SP-600125, respectively. IL-1beta-induced VCAM-1 expression was significantly blocked by the specific NF-kappaB inhibitors helenalin and pyrrolidine dithiocarbamate. As expected, IL-1beta-stimulated translocation of NF-kappaB into the nucleus and degradation of IkappaB-alpha were blocked by helenalin but not by U0126, SB-202190, or SP-600125. Moreover, the resultant enhancement of VCAM-1 expression increased the adhesion of polymorphonuclear cells to a monolayer of HTSMC, which was blocked by pretreatment with helenalin, U0126, SB-202190, or SP-600125 before IL-1beta exposure or by anti-VCAM-1 antibody. Together, these results suggest that in HTSMC, activation of p42/p44 MAPK, p38, JNK, and NF-kappaB pathways is essential for IL-1beta-induced VCAM-1 gene expression. These results provide new insight into the mechanisms of IL-1beta action that cytokines may promote inflammatory responses in airway disease.     

The regulation of Th1 responses by the p38 MAPK

IL-12 is a dimeric cytokine that is produced primarily by APCs. In this study we examined the role that the p38 MAPKs (MAPK/p38) play in regulating IL-12 production. We show that inhibition of p38 dramatically increased IL-12 production upon stimulation, while decreasing TNF-α. This reciprocal effect on these two cytokines following MAPK/p38 inhibition occurred in many different APCs, following a variety of different stimuli. IL-12 production was also increased in macrophages treated with small interfering RNA to limit p38α expression, and in macrophages deficient in MKK3, a kinase upstream of p38. The increase in IL-12 production following MAPK/p38 inhibition appears to be due to enhanced IL-12 (p40) mRNA stability. We show that MAPK/p38 inhibition can promote Th1 immune responses and thereby enhance vaccine efficacy against leishmaniasis. In a mouse model of Leishmania major infection, vaccination with heat-killed L. major plus CpG and SB203580 elicited complete protection against infection compared with heat-killed L. major plus CpG without SB203580. Thus, this work suggests that MAPK/p38 inhibitors may be applied as adjuvants to bias immune responses and improve vaccinations against intracellular pathogens.     

Chk1 inhibition activates p53 through p38 MAPK in tetraploid cancer cells

We have previously shown that tetraploid cancer cells succumb through a p53-dependent apoptotic pathway when checkpoint kinase 1 (Chk1) is depleted by small interfering RNAs (siRNAs) or inhibited with 7-hydroxystaurosporine (UCN-01). Here, we demonstrate that the Chk1 inhibition results in the activating phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK). Depletion of p38 MAPK by transfection with a siRNA targeting the α isoform of p38 MAPK (p38α MAPK) abolishes the phosphorylation of p53 on serines 15 and 46 that is induced by Chk1 knockdown. The siRNA-mediated downregulation and pharmacological inhibition of p38α MAPK (with SB 203580) also reduces cell death induced by Chk1 knockdown or UCN-01. These results underscore the role of p38 MAPK as a pro-apoptotic kinase in the p53-dependant pathway for the therapeutic elimination of polyploidy cells.     

The role of p38 MAPK and JNK in Arsenic trioxide-induced mitochondrial cell death in human cervical cancer cells

Previously, we have shown that the release of AIF from mitochondria is required for As2O3-induced cell death in human cervical cancer cells, and that reactive oxygen species (ROS) is necessary for AIF release from mitochondria. In this study, we further investigated the role of MAPKs in ROS-mediated mitochondrial apoptotic cell death triggered by As2O3. As2O3-induced apoptotic cell death in HeLa cells was associated with activation and mitochondrial translocation of Bax, a marked phosphorylation of Bcl-2, reduction of Bcl-2 and Bax interaction, dissipation of mitochondrial membrane potential. Using small interfering RNA, reduced Bax expression effectively attenuated As2O3-induced mitochondrial membrane potential loss and apoptotic cell death. Moreover, the phosphorylation of Bcl-2 induced by As2O3 diminished its ability to bind to Bax. Treatment of cells with As2O3 activated both the p38 MAPK and JNK pathways. Mitochondrial translocation of Bax was completely suppressed in the presence of p38 MAPK inhibitor PD169316 or si-p38 MAPK. The As2O3-induced Bcl-2 phosphorylation was attenuated largely by JNK inhibition using SP600125 or si-JNK and to some extent by p38 MAPK inhibition with PD169316 or si-p38 MAPK. In addition, N-acetyl-L-cystein (NAC), a thiol-containing anti-oxidant, completely blocked As2O3-induced p38 MAPK and JNK activations, mitochondria translocation of Bax, and phosphorylation of Bcl-2. These results support a notion that ROS-mediated activations of p38 MAPK and JNK in response to As2O3 treatment signals activation of Bax and phosphorylation of Bcl-2, resulting in mitochondrial apoptotic cell death in human cervical cancer cells.     

Enhanced cardiomyogenesis of human embryonic stem cells by a small molecular inhibitor of p38 MAPK

Abstract Human embryonic stem cells (hESC) can differentiate to cardiomyocytes in vitro but with generally poor efficiency. Here, we describe a novel method for the efficient generation of cardiomyocytes from hESC in a scalable suspension culture process. Differentiation in serum-free medium conditioned by the cell line END2 (END2-CM) readily resulted in differentiated cell populations with more than 10% cardiomyocytes without further enrichment. By screening candidate molecules, we have identified SB203580, a specific p38 MAP kinase inhibitor, as a potent promoter of hESC-cardiogenesis. SB203580 at concentrations <10 μM, induced more than 20% of differentiated cells to become cardiomyocytes and increased total cell numbers, so that the overall cardiomyocyte yield was approximately 2.5-fold higher than controls. Gene expression indicated that early mesoderm formation was favored in the presence of SB203580. Accordingly, transient addition of the inhibitor at the onset of differentiation only was sufficient to determine the hESC fate. Patch clamp electrophysiology showed that the distribution of cardiomyocyte phenotypes in the population was unchanged by the compound. Interestingly, cardiomyogenesis was strongly inhibited at SB203580 concentrations ≥15 μM. Thus, modulation of the p38MAP kinase pathway, in combination with factors released by END2 cells, plays an essential role in early lineage determination in hESC and the efficiency of cardiomyogenesis. Our findings contribute to transforming human cardiomyocyte generation from hESC into a robust and scalable process.     

Dancing with p53: The role of p38MAPK in mitosis of p53-deficient tetraploid cells

Comment on: Vitale I, et al. Cell Cycle 2010; 9:2823-9.     

Expression and proliferation profiles of PKC,JNK and p38MAPK in physiologically stretched human bladder smooth muscle cells

Objective

To determine protein kinase C (PKC), c-Jun NH2-Terminal Kinase (JNK) and P38 mitogen-activated protein kinases (p38MAPK) expression levels and effects of their respective inhibitors on proliferation of human bladder smooth muscle cells (HBSMCs) when physiologically stretched in vitro.

Materials and methods

HBSMCs were grown on silicone membrane and stretch was applied under varying conditions; (equibiaxial elongation: 2.5%, 5%, 10%, 15%, 20%, 25%), (frequency: 0.05, 0.1, 0.2, 0.5, 1 Hz). Optimal physiological stretch was established by assessing proliferation with 5-Bromo-2-deoxyuridine (BrdU) assay and flow cytometry. PKC, JNK and p38 expression levels were analyzed by Western blot. Specificity was maintained by employing specific inhibitors; (GF109203X for PKC, SP600125 for JNK and SB203580 for p38MAPK), in some experiments.

Results

Optimum proliferation was observed at 5% equibiaxial stretch (BrdU: 0.837 ± 0.026 (control) to 1.462 ± 0.023)%, (P < 0.05) and apoptotic cell death rate decreased from 16.4 ± 0.21% (control) to 4.5 ± 0.13% (P < 0.05) applied at 0.1 Hz. Expression of PKC was upregulated with slight increase in JNK and no change in p38MAPK after application of stretch. Inhibition had effects on proliferation (1.075 ± 0.024, P < 0.05 GF109203X); (1.418 ± 0.021, P > 0.05 SP600125) and (1.461 ± 0.01, P > 0.05 SB203580). These findings show that mechanical stretch can promote magnitude-dependent proliferative modulation through PKC and possibly JNK but not via p38MAPK in hBSMCs.     

Leptin enhances TNF-alpha production via p38 and JNK MAPK in LPS-stimulated Kupffer cells

Leptin is now recognized as a proinflammatory cytokine and thought to be a progressive factor for non-alcoholic steatohepatitis (NASH). Here we showed the effects of leptin on the production of TNF-alpha (tumor necrosis factor-alpha) by Kupffer cells (KCs) with signal transduction. Leptin enhanced TNF-alpha production accompanied by a dose-dependent increase of MAPK activity in lipopolysaccharide (LPS)-stimulated KCs. SB203580 and JNK inhibitor I, specific inhibitors of P38 and JNK, inhibited TNF-alpha production in KCs but PD98059, an inhibitor of the ERK pathway, did not affect TNF-alpha production by KCs. Recombinant constitutively active adenovirus (Ad)-MKK6 and-MKK7 increased TNF-alpha production in KCs with activation of P38 and JNK without any change by Ad-MEK1 delivery. On the other hand, KCs isolated from the Zucker rat (fa/fa), a leptin receptor-deficient rat, showed reduced production of TNF-alpha on stimulation with LPS. The delivery of Ad-MKK6 and-MKK7, but not Ad-MEK1, increased TNF-alpha production in KCs of Zucker rats with activation of P38 and JNK. Addition of leptin to normal rats increased LPS-induced hepatic TNF-alpha production in vivo and leptin receptor-deficient Zucker rats showed reduced hepatic TNF-alpha production on addition of LPS in vivo. These findings indicate that P38 and JNK pathways are involved in the signal transduction of leptin enhancement of LPS-induced TNF-alpha production.     

Mixed-lineage kinase control of JNK and p38 MAPK pathways

Mixed-lineage kinases (MLKs) are serine/threonine protein kinases that regulate signalling by the c-Jun amino-terminal kinase (JNK) and p38 mitogen-activated-protein kinase (MAPK) pathways. MLKs are represented in the genomes of both Caenorhabditis elegans and Drosophila melanogaster. The Drosophila MLK Slipper regulates JNK to control dorsal closure during embryonic morphogenesis. In mammalian cells, MLKs are implicated in the control of apoptosis and are potential drug targets for many neurodegenerative diseases.     

Prevention of kidney ischemia/reperfusion-induced functional injury and JNK, p38, and MAPK kinase activation by remote ischemic pretreatment

MAPK activities, including JNK, p38, and ERK, are markedly enhanced after ischemia in vivo and chemical anoxia in vitro. The relative extent of JNK, p38, or ERK activation has been proposed to determine cell fate after injury. A mouse model was established in which prior exposure to ischemia protected against a second ischemic insult imposed 8 or 15 days later. In contrast to what was observed after 30 min of bilateral ischemia, when a second period of ischemia of 30- or 35-min duration was imposed 8 days later, there was no subsequent increase in plasma creatinine, decrease in glomerular filtration rate, or increase in fractional excretion of sodium. A shorter period of prior ischemia (15 min) was partially protective against subsequent ischemic injury 8 days later. Unilateral ischemia was also protective against a subsequent ischemic insult to the same kidney, revealing that systemic uremia is not necessary for protection. The ischemia-related activation of JNK and p38 and outer medullary vascular congestion were markedly mitigated by prior exposure to ischemia, whereas preconditioning had no effect on post-ischemic activation of ERK1/2. The phosphorylation of MKK7, MKK4, and MKK3/6, upstream activators of JNK and p38, was markedly reduced by ischemic preconditioning, whereas the post-ischemic phosphorylation of MEK1/2, the upstream activator of ERK1/2, was unaffected by preconditioning. Pre- and post-ischemic HSP-25 levels were much higher in the preconditioned kidney. In summary, post-ischemic JNK and p38 (but not ERK1/2) activation was markedly reduced in a model of kidney ischemic preconditioning that was established in the mouse. The reduction in JNK and p38 activation can be accounted for by reduced activation of upstream MAPK kinases. The post-ischemic activation patterns of MAPKs may explain the remarkable protection against ischemic injury observed in this model.     

Corticosterone-induced rapid phosphorylation of p38 and JNK mitogen-activated protein kinases in PC12 cells

The present study showed that corticosterone (B) could induce a rapid activation of p38 and c-Jun NH(2)-terminal protein kinase (JNK) in PC12 cells. The dose-response and time-response curves were bell-shaped with maximal activation at 10(-9) M and at 15 min. RU38486 had no effect, and bovine serum albumin-coupled B could induce the activation. Genistein failed to block the phosphorylation, suggesting the pathway was not involved in tyrosine kinase activity. Phorbol 12-myristate 13-acetate could mimic, while G?6976 could abolish the actions. These results demonstrated that B might act via a putative membrane receptor to activate p38 and JNK rapidly through a protein kinase C-dependent pathway.     

Myocardial improvement with human embryonic stem cell-derived cardiomyocytes enriched by p38MAPK inhibition

Background aimsWe have shown previously that inhibition of the p38 mitogen-activated protein kinase (p38MAPK) directs the differentiation of human embryonic stem cell (hESC)-derived cardiomyocytes (hCM). We investigated the therapeutic benefits of intramyocardial injection of hCM differentiated from hESC by p38MAPK inhibition using closed-chest ultrasound-guided injection at a clinically relevant time post-myocardial infarction (MI) in a mouse model.MethodsMI was induced in mice and the animals treated at day 3 with: (a) hCM, (b) human fetal fibroblasts (hFF) as cell control, or (c) medium control (n = 10 animals/group). Left ventricular ejection fraction (LVEF) was evaluated post-MI prior to therapy, and at days 28 and 60 post-cell therapy. Hearts were analyzed at day 60 for infarct size, angiogenesis, cell fate and teratoma formation.ResultsLVEF was improved in the hCM-treated animals compared with both hFF and medium control-treated animals at day 28 (39.03 ± 1.79% versus 27.89 ± 1.27%, P < 0.05, versus 32.90 ± 1.46%, P < 0.05, respectively), with sustained benefit until day 60. hCM therapy resulted in significantly smaller scar size, increased capillary bed area, increased number of arterioles, less native cardiomyocyte (CM) apoptosis, and increased CM proliferation compared with the other two groups. These benefits were achieved despite a very low retention rate of the injected cells at day 60, as assessed by immunohistochemistry and quantitative real-time polymerase chain reaction (qPCR). Therapy with hCM did not result in intramyocardial teratoma formation at day 60.ConclusionsThis study demonstrates that hCM derived from p38MAPK-treated hESC have encouraging therapeutic potential.     

Timed inhibition of p38MAPK directs accelerated differentiation of human embryonic stem cells into cardiomyocytes

Background aimsHeart failure therapy with human embryonic stem cell (hESC)-derived cardiomyocytes (hCM) has been limited by the low rate of spontaneous hCM differentiation. As others have shown that p38 mitogen-activated protein kinase (p38MAPK) directs neurogenesis from mouse embryonic stem cells, we investigated whether the p38MAPK inhibitor, SB203580, might influence hCM differentiation.MethodsWe treated differentiating hESC with SB203580 at specific time-points, and used flow cytometry, immunocytochemistry, quantitative real-time (RT)–polymerase chain reaction (PCR), teratoma formation and transmission electron microscopy to evaluate cardiomyocyte formation.ResultsWe observed that the addition of inhibitor resulted in 2.1-fold enrichment of spontaneously beating human embryoid bodies (hEB) at 21 days of differentiation, and that 25% of treated cells expressed cardiac-specific α-myosin heavy chain. This effect was dependent on the stage of differentiation at which the inhibitor was introduced. Immunostaining and teratoma formation assays demonstrated that the inhibitor did not affect hESC pluripotency; however, treated hESC gave rise to hCM exhibiting increased expression of sarcomeric proteins, including cardiac troponin T, myosin light chain and α-myosin heavy chain. This was consistent with significantly increased numbers of myofibrillar bundles and the appearance of nascent Z-bodies at earlier time-points in treated hCM. Treated hEB also demonstrated a normal karyotype by array comparative genomic hybridization and viability in vivo following injection into mouse myocardium.ConclusionsThese studies demonstrate that p38MAPK inhibition accelerates directed hCM differentiation from hESC, and that this effect is developmental stage-specific. The use of this inhibitor should improve our ability to generate hESC-derived hCM for cell-based therapy.     

High glucose induces mitochondrial p53 phosphorylation by p38 MAPK in pancreatic RINm5F cells

Pancreatic β-cell death in type 2 diabetes has been related to p53 subcellular localisation and phosphorylation. However, the mechanisms by which p53 is phosphorylated and its activation in response to oxidative stress remain poorly understood. Therefore, the aim of this study was to investigate mitochondrial p53 phosphorylation, its subcellular localisation and its relationship with apoptotic induction in RINm5F cells cultured under high glucose conditions. Our results show that p53 phosphorylation in the mitochondrial fraction was greater at ser392 than at ser15. This increased phosphorylation correlated with an increase in reactive oxygen species, a decrease in the Bcl-2/Bax ratio, a release of cytochrome c and an increase in the rate of apoptosis. We also observed a decline in ERK 1/2 phosphorylation over time, which is an indicator of cell proliferation. To identify the kinase responsible for phosphorylating p53, p38 mitogen-activated protein kinase (MAPK) activation was analysed. We found that high glucose induced an increase in p38 MAPK phosphorylation in the mitochondria after 24–72 h. Moreover, the phosphorylation of p53 (ser392) by p38 MAPK in mitochondria was confirmed by colocalisation studies with confocal microscopy. The addition of a specific p38 MAPK inhibitor (SB203580) to the culture medium during high glucose treatment blocked p53 mobilisation to the mitochondria and phosphorylation; thus, the release of cytochrome c and the apoptosis rate in RINm5F cells decreased. These results suggest that mitochondrial p53 phosphorylation by p38 MAPK plays an important role in RINm5F cell death under high glucose conditions.     

Pro-survival effects of JNK and p38 MAPK pathways in LPS-induced activation of BV-2 cells

Identifying MAPK pathways and understanding their role in microglial cells may be crucial for understanding the pathogenesis of neurodegenerative diseases since activated microglia could contribute to the progressive nature of neurodegeneration. In this study we show that the JNK pathway plays an important role in the survival of resting microglia BV-2 cells, as evidenced by Annexin-V positive staining and caspase-3 activation in cells treated with the specific JNK inhibitor SP600125. During LPS-induced activation of BV-2 cells inhibition of the p38 and JNK pathways with SB203580 and SP600125, respectively, results in apoptosis as detected by apoptotic markers. In the presence SP600125 the phosphorylation of p38 was significantly increased both in control and LPS-activated BV-2 cells. This suggests that the pro-survival role of JNK is possible due to its abrogation of a potentially apoptotic signal mediated by p38 MAPK pathway. Furthermore, inhibition of the p38 MAPK pathway during LPS-induced activation of BV-2 cells resulted in an increased phosphorylation of c-Jun, suggesting that the pro-survival effect of p38 MAPK during inflammatory conditions involves the JNK pathway. In conclusion, the results of this study demonstrate that both the JNK and p38 MAPK pathways possess anti-apoptotic functions in the microglial cell line BV-2 during LPS-induced activation.