Involvement of p38 mitogen-activated protein kinase and apoptosis signal-regulating kinase-1 in nitric oxide-induced cell death in PC12 cells

Although nitric oxide (NO) plays key signaling roles in the nervous systems, excess NO leads to cell death. In this study, the involvement of p38 mitogen-activated protein kinase (p38 MAPK) and apoptosis signal-regulating kinase-1 (ASK1) in NO-induced cell death was investigated in PC12 cells. NO donor transiently activated p38 MAPK in the wild type parental PC12 cells, whereas the p38 MAPK activation was abolished in NO-resistant PC12 cells (PC12-NO-R). p38 MAPK inhibitors protected the cells against NO-induced death, whereas the inhibitors were not significantly protective against the cytotoxicity of reactive oxygen species. Stable transfection with dominant negative p38 MAPK mutant reduced NO-induced cell death. Stable transfection with dominant negative mutant of ASK1 attenuated NO-stimulated activation of p38 MAPK and decreased NO-induced cell death. These results suggest that p38 MAPK and its upstream regulator ASK1 are involved in NO-induced PC12 cell death.     

Decreased sensitivity of tristetraprolin-deficient cells to p38 inhibitors suggests the involvement of tristetraprolin in the p38 signaling pathway

Treatment of macrophages with pyridinyl imidazole inhibitors of p38 protein kinases can inhibit lipopolysaccharide-stimulated tumor necrosis factor alpha secretion. However, bone marrow-derived macrophages from tristetraprolin (TTP)-deficient mice were less sensitive than normal macrophages to this effect of p38 inhibitors, despite evidence for normal p38 activation in response to lipopolysaccharide. TTP is known to cause decreased stability of tumor necrosis factor alpha and granulocyte-macrophage colony-stimulating factor mRNAs after binding to an AU-rich element in their 3'-untranslated regions. A recombinant TTP fusion protein could be phosphorylated by a recombinant p38 kinase in cell-free assays and was phosphorylated to the same extent by immunoprecipitated p38 derived from normal and TTP-deficient cells stimulated with lipopolysaccharide; in both cases, the enzyme activity was inhibited by the p38 inhibitors. TTP phosphorylation also was increased in intact macrophages after lipopolysaccharide stimulation, an effect that was blocked by the p38 inhibitors. Finally, TTP in mammalian cell extracts bound less well to an AU-rich element RNA probe than did the same amount of TTP following dephosphorylation. These results suggest that TTP may be a component of the signaling cascade, initiated by inflammatory stimuli and mediated in part by activation of p38, that ultimately leads to enhanced secretion of tumor necrosis factor alpha.     

Bcl2, a transcriptional target of p38alpha, is critical for neuronal commitment of mouse embryonic stem cells

Mouse embryonic stem (ES) cells remain pluripotent in vitro when grown in the presence of leukemia inhibitory factor (LIF) cytokine. LIF starvation leads to cell commitment, and part of the ES-derived differentiated cells die by apoptosis together with caspase3-cleavage and p38alpha activation. Inhibition of p38 activity by chemical compounds (PD169316 and SB203580), along with LIF withdrawal, leads to different outcomes on cell apoptosis, giving the opportunity to study the influence of apoptosis on cell differentiation. By gene profiling studies on ES-derived differentiated cells treated or not with these inhibitors, we have characterized the common and specific set of genes modulated by each inhibitor. We have also identified key genes that might account for their different survival effects. In addition, we have demonstrated that some genes, similarly regulated by both inhibitors (upregulated as Bcl2, Id2, Cd24a or downregulated as Nodal), are bona fide p38alpha targets involved in neurogenesis and found a correlation with their expression profiles and the onset of neuronal differentiation triggered upon retinoic acid treatment. We also showed, in an embryoid body differentiation protocol, that overexpression of EGFP (enhanced green fluorescent protein)-BCL2 fusion protein and repression of p38alpha are essential to increase formation of TUJ1-positive neuronal cell networks along with an increase in Map2-expressing cells.     

Evaluation of the combination mode and features of p38 MAPK inhibitors: construction of different pharmacophore models and molecular docking

Abnormal expression of tumour necrosis factor-α (TNF-α) can lead to various pathological reactions, such as arthritis, psoriasis, krone disease, etc. p38 mitogen-activated protein kinase (p38 MAPK) is an important signal transduction enzyme that plays important roles in influencing the release of intracellular TNF-α factor. It is very meaningful to study the targeting kinase with specific inhibitors in the treatment of related diseases. In order to achieve a deeper insight, it is necessary to analyse the structural characteristics and the action mode of the p38 MAPK inhibitors in the active site. In the study, a ligand-based common feature pharmacophore model and the receptor structure-based pharmacophore model were constructed, respectively. Their common chemical features consisted of the hydrophobic groups (H) and the hydrogen bond acceptors (A), and kept the consistency of spatial structure distribution. Then, the molecular docking and molecular dynamics simulation were performed with the eight training set compounds. The binding characteristics of molecules binding were described in the topological region of the active site. Finally, the structure–activity relationship (SAR) was obtained by analysing docking results with the different pharmacophore models. This research leads to the proposal of an interaction model in the p38 MAPK active site and provides guidance for the screening and design of more potent and selective p38 MAPK inhibitors.     

p38 MAPK mediates TNF-induced apoptosis in endothelial cells via phosphorylation and downregulation of Bcl-x(L)

The role of p38 mitogen-activated protein kinase (MAPK) in apoptosis is a matter of debate. Here, we investigated the involvement of p38 MAPK in endothelial apoptosis induced by tumor necrosis factor alpha (TNF). We found that activation of p38 MAPK preceded activation of caspase-3, and the early phase of p38 MAPK stimulation did not depend on caspase activity, as shown by pretreatment with the caspase inhibitors z-Val-Ala-Asp(OMe)-fluoromethylketone (zVAD-fmk) and Boc-Asp(OMe)-fluoromethylketone (BAF). The p38 MAPK inhibitor SB203580 significantly attenuated TNF-induced apoptosis in endothelial cells, suggesting that p38 MAPK is essential for apoptotic signaling. Furthermore, we observed a time-dependent increase in active p38 MAPK in the mitochondrial subfraction of cells exposed to TNF. Notably, the level of Bcl-x(L) protein was reduced in cells undergoing TNF-induced apoptosis, and this reduction was prevented by treatment with SB203580. Immunoprecipitation experiments revealed p38 MAPK-dependent serine-threonine phosphorylation of Bcl-x(L) in TNF-treated cells. Exposure to lactacystin prevented both the downregulation of Bcl-x(L) and activation of caspase-3. Taken together, our results suggest that TNF-induced p38 MAPK-mediated phosphorylation of Bcl-x(L) in endothelial cells leads to degradation of Bcl-x(L) in proteasomes and subsequent induction of apoptosis.     

Indole-based heterocyclic inhibitors of p38alpha MAP kinase: designing a conformationally restricted analogue

p38alpha Mitogen Activated Protein Kinase (MAP kinase) is an intracellular soluble serine threonine kinase. p38alpha kinase is activated in response to cellular stresses, growth factors and cytokines such as interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF-alpha). The central role of p38alpha activation in settings of both chronic and acute inflammation has led efforts to find inhibitors of this enzyme as possible therapies for diseases such as rheumatoid arthritis, where p38alpha activation is thought to play a causal role. Herein, we report structure-activity relationship studies on a series of indole-based heterocyclic inhibitors that led to the design and identification of a new class of p38alpha inhibitors.     

Cloning and characterization of two novel zebrafish P2X receptor subunits

Activation of Kupffer cells by lipopolysaccharide (LPS) after ethanol feeding results in overproduction of TNF-alpha, leading to liver injury. Since dilinoleoylphosphatidylcholine (DLPC) protects against liver injury and has antioxidant properties, we investigated whether it alters LPS signaling leading to decreased TNF-alpha production. Kupffer cells were isolated from rats fed alcohol-containing or isocaloric control diets for 3 weeks. With ethanol, cytochrome P4502E1 was upregulated. When stimulated with LPS in culture, Kupffer cells released more TNF-alpha compared to control rats; DLPC diminished the increase. It also reduced ERK1/2 and p38 phosphorylation as well as NF-kappaB activation with decreased nuclear p65 and increased cytosolic IkappaB-alpha expression. ERK1/2 and NF-kappaB activation were abolished by the ERK1/2 inhibitor PD098059. The p38 inhibitor SB203580 abolished p38 activation without affecting NF-kappaB. Both inhibitors reduced TNF-alpha generation. Thus, DLPC diminishes LPS-dependent TNF-alpha generation by inhibiting p38 and ERK1/2 activation; the latter leads to decreased NF-kappaB activation.     

Inhibition of RIP2/RICK/CARDIAK activity by pyridinyl imidazole inhibitors of p38 MAPK

Pyridinyl imidazole inhibitors of p38 mitogen-activated protein kinase (MAPK) have been used extensively in vitro and in vivo to investigate the role of p38 in physiological processes. As with other pharmacological inhibitors, non-specific targets of the p38 inhibitors have been reported. We have found that the protein kinase receptor interacting protein-2 (RIP2) is another target for the family of p38 inhibitors. The autophosphorylation of RIP2 was inhibited in vitro by the p38 inhibitors SB220025, SB203580 and PD169316 at concentrations comparable to those used to inhibit p38. We also identified two new in vitro substrates for RIP2, myelin basic protein and histone H3 with apparent K_m values of 2.1 M and 0.65 M, respectively. The ability of RIP2 to phosphorylate these two substrates was sensitive to the p38 inhibitors as well. As was shown for p38, a conserved threonine in the kinase domain of RIP2 is required for sensitivity to the inhibitors, indicating that the mechanism of inhibition of RIP2 is similar to that of p38. These results demonstrate that the pyridinyl imidazole inhibitors block RIP2 as well as p38 kinase activity. (Mol Cell Biochem 268: 129–140, 2005)     

Regulation of TGFbeta1-mediated collagen formation by LOX-1: studies based on forced overexpression of TGFbeta1 in wild-type and lox-1 knock-out mouse cardiac fibroblasts

Transforming growth factor beta(1) (TGFbeta(1)) activation leads to tissue fibrosis. Here, we report on the role of LOX-1, a lectin-like 52-kDa receptor for oxidized low density lipoprotein, in TGFbeta(1)-mediated collagen expression and underlying signaling in mouse cardiac fibroblasts. TGFbeta(1) was overexpressed in wild-type (WT) and LOX-1 knock-out mouse cardiac fibroblasts by transfection with adeno-associated virus type 2 vector carrying the active TGFbeta(1) moiety (AAV/TGFbeta (ACT)(1)). Transfection of WT mouse cardiac fibroblasts with AAV/TGFbeta (ACT)(1) markedly enhanced the expression of NADPH oxidases (p22(phox), p47(phox), and gp91(phox) subunits) and LOX-1, formation of reactive oxygen species, and collagen synthesis, concomitant with an increase in the activation of p38 and p44/42 mitogen-activated protein kinases (MAPK). The TGFbeta(1)-mediated increase in collagen synthesis was markedly attenuated in the LOX-1 knock-out mouse cardiac fibroblasts as well as in WT mouse cardiac fibroblasts treated with a specific anti-LOX-1 antibody. Treatment with anti-LOX-1 antibody also reduced NADPH oxidase expression and MAPK activation. The NADPH oxidase inhibitors and gp91phox small interfering RNA reduced LOX-1 expression, MAPK activation, and collagen formation. The p38 MAPK inhibitors as well as the p44/42 MAPK inhibitors reduced collagen formation without affecting LOX-1 expression in cardiac fibroblasts. These observations suggest that collagen synthesis in cardiac fibroblasts involves a facilitative interaction between TGFbeta(1)-NADPH oxidase and LOX-1. Further, the activation of MAPK pathway appears to be downstream of TGFbeta(1)-reactive oxygen species-LOX-1 cascade.     

Inhibition of the protein kinase MK-2 protects podocytes from nephrotic syndrome-related injury

While mitogen-activated protein kinase (MAPK) activation has been implicated in the pathogenesis of various glomerular diseases, including nephrotic syndrome (NS), its specific role in podocyte injury is not known. We hypothesized that MK-2, a downstream substrate of p38 MAPK, mediates the adverse effects of this pathway and that inhibition of MK-2 would protect podocytes from NS-related injury. Using cultured podocytes, we analyzed 1) the roles of MK-2 and p38 MAPK in puromycin aminonucleoside (PAN)-induced podocyte injury; 2) the ability of specific MK-2 and p38 MAPK inhibitors to protect podocytes against injury; 3) the role of serum albumin, known to induce podocyte injury, in activating p38 MAPK/MK-2 signaling; and 4) the role of p38 MAPK/MK-2 signaling in the expression of Cox-2, an enzyme associated with podocyte injury. Treatment with protein kinase inhibitors specific for both MK-2 (C23, a pyrrolopyridine-type compound) or p38 MAPK (SB203580) reduced PAN-induced podocyte injury and actin cytoskeletal disruption. Both inhibitors reduced baseline podocyte p38 MAPK/MK-2 signaling, as measured by the degree of phosphorylation of HSPB1, a downstream substrate of MK-2, but exhibited disparate effects on upstream signaling. Serum albumin activated p38 MAPK/MK-2 signaling and induced Cox-2 expression, and these responses were blocked by both inhibitors. Given the critical importance of podocyte injury to both NS and other progressive glomerular diseases, these data suggest an important role for p38 MAPK/MK-2 signaling in podocyte injury and identify MK-2 inhibition as a promising potential therapeutic strategy to protect podocytes in various glomerular diseases.     

Discovery of a novel class of non-ATP site DFG-out state p38 inhibitors utilizing computationally assisted virtual fragment-based drug design (vFBDD)

Discovery of a new class of DFG-out p38α kinase inhibitors with no hinge interaction is described. A computationally assisted, virtual fragment-based drug design (vFBDD) platform was utilized to identify novel non-aromatic fragments which make productive hydrogen bond interactions with Arg 70 on the αC-helix. Molecules incorporating these fragments were found to be potent inhibitors of p38 kinase. X-ray co-crystal structures confirmed the predicted binding modes. A lead compound was identified as a potent (p38α IC(50)=22 nM) and highly selective (≥ 150-fold against 150 kinase panel) DFG-out p38 kinase inhibitor.     

Kinetics of small molecule inhibitor binding to p38 kinase.

p38 mitogen-activated protein kinase (MAPK) (p38/p38-alpha/CSBP2/RK) has been implicated in the regulation of many proinflammatory pathways. Because of this, it has received much attention as a potential drug target for controlling diseases such as rheumatoid arthritis, endotoxic shock, inflammatory bowel disease, osteoporosis, and many others. A number of small molecule inhibitors of this kinase have been described, and in this paper we have used surface plasmon resonance to directly measure and quantitate their binding to p38. Despite the relatively low molecular mass (approximately 400 Da) of these inhibitors, specific binding can be observed. For the two most potent inhibitors studied, SB 203580 and RWJ 67657, dissociation constants, K(d)'s, of 22 and 10 nm, respectively, were obtained. These values closely match the IC(5)0 values observed in a cell-based TNF alpha release assay implying that p38 plays a major role in TNF alpha release. The association and dissociation rates for the binding of these inhibitors to p38 have also been quantitated. SB 203580 and RWJ 67657 have very similar association rates of around 8 x 10(5) m(-1) x s(-1), and the differences in affinity are determined by different dissociation rates. The weaker binding compounds have dissociation rates similar to SB 203580, but the association rates vary by an order of magnitude or more. The direct measurement of compounds binding to p38 may help in understanding the difference between potency and efficacy for these inhibitors. This in turn may yield clues on how to develop better inhibitors.     

Benzimidazolone p38 inhibitors

The synthesis and in vitro p38 alpha activity of a novel series of benzimidazolone inhibitors is described. The p38 alpha SAR is consistent with a mode of binding wherein the benzimidazolone carbonyl serves as the H-bond acceptor to Met109 of p38 alpha in a manner analogous to the pyridine nitrogen of prototypical pyridylimidazole p38 inhibitors. Potent p38 alpha activity comparable to that of several previously reported p38 inhibitors is observed for this novel chemotype.     

A selective inhibitor of p38 MAP kinase, SB202190, induced apoptotic cell death of a lipopolysaccharide-treated macrophage-like cell line, J774.1

A selective p38 MAP kinase (p38 MAPK) inhibitor, SB202190, induced apoptotic cell death of a macrophage-like cell line, J774.1, in the presence of lipopolysaccharide (LPS), as judged by DNA nicks revealed by terminal deoxy transferase (TdT)-mediated dUTP nick end labeling (TUNEL), activation of caspase-3, and subsequent release of lactate dehydrogenase. This cytotoxicity was dependent on both LPS and SB202190, and such inhibitors of the upstream LPS-signaling cascade as polymyxin B and TPCK blocked this macrophage cell death. SB202190 suppressed the kinase activity of p38, leading to inhibition of activation of MAPKAPK2 and then the subsequent phosphorylation of hsp27 in LPS-treated macrophages both in vitro and in vivo, but an inactive analog of SB202190, SB202474, did not. There was a threshold of the time of addition of SB202190 to LPS-treated macrophages to induce apoptosis, which was before full transmission of p38 activity to a direct downstream kinase, MAPKAPK2. Besides, localization of phosphorylated hsp27 in Golgi area of the LPS-treated macrophages was suppressed by SB202190, while it was not by SB202474. These results suggest that selective inhibition of p38 MAPK activity in LPS-induced MAP kinase cascade leads to apoptosis of macrophages.     

Activation of the Rb/E2F1 pathway by the nonproliferative p38 MAPK during Fas (APO1/CD95)-mediated neuronal apoptosis

Aberrant activation of the Rb/E2F1 pathway in cycling cells, in response to mitogenic or nonmitogenic stress signals, leads to apoptosis through hyperphosphorylation of Rb. To test whether in postmitotic neurons the Rb/E2F1 pathway can be activated by the nonmitogenic stress signaling, we examined the role of the p38 stress-activated protein kinase (SAPK) in regulating Rb phosphorylation in response to Fas (CD95/APO1)-mediated apoptosis of cultured cerebellar granule neurons (CGNs). Anti-Fas antibody induced a dramatic and early activation of p38. Activated p38 was correlated with the induction of hyperphosphorylation of both endogenous and exogenous Rb. The p38-selective inhibitor, SB203580, attenuated such an increase in pRb phosphorylation and significantly protected CGNs from Fas-induced apoptosis. The cyclin-dependent kinase-mediated Rb phosphorylation played a lesser role in this neuronal death paradigm, since cyclin-dependent kinase inhibitors, such as olomoucine, roscovitine, and flavopiridol, did not significantly prevent anti-Fas antibody-evoked neuronal apoptosis. Hyperphosphorylation of Rb by p38 SAPK resulted in the release of Rb-bound E2F1. Increased E2F1 modulated neuronal apoptosis, since E2F1-/- CGNs were significantly less susceptible to Fas-mediated apoptosis in comparison with the wild-type CGNs. Taken together, these studies demonstrate that neuronal Rb/E2F1 is modulated by the nonproliferative p38 SAPK in Fas-mediated neuronal apoptosis.     

Monitoring mouse serotonin transporter internalization in stem cell-derived serotonergic neurons by confocal laser scanning microscopy

In the central nervous system serotonergic neurotransmission is terminated by the rapid removal of serotonin (5-hydroxytryptamine, 5HT) out of the extra-cellular space back into the presynaptic neuron. This task is fulfilled by a specific serotonin transporter (SERT) protein which controls the concentration of extra-cellular 5HT. Consequently, one mechanism to regulate the efficacy of serotonergic neurotransmission is via modulation of the density of SERT molecules on the cell membrane.In this regard it has been shown, that chronic activation of the p38 mitogen-activated protein kinase (p38 MAPK) leads to enhanced SERT surface expression whereas activation of protein kinase C (PKC) reduces SERT surface expression. In addition, it has been reported that exposure to selective serotonin re-uptake inhibitors (SSRIs) leads to a down-regulation of SERT expression in vivo and in vitro in different cellular systems. Here, we have studied interactions between kinase- and SSRI-induced SERT internalization in mouse stem cell-derived serotonergic neurons expressing the native SERT allele in its natural surroundings. Therefore we established a method to quantify the amount of cell surface-expressed SERT molecules on individual cells by antibody detection combined with confocal laser scanning microscopy.Using this methodology we could show that activation of PKC, inhibition of the p38 MAPK as well as exposure to the SSRI citalopram each induced a significant reduction of cell surface-expressed SERT over time. Combinations of PKC activation, p38 MAPK inhibition and SSRI exposure led to a more pronounced down-regulation of SERT surface expression depending on the time of drug exposure.     

Structure-based virtual screening approach to the discovery of p38 MAP kinase inhibitors

p38 Mitogen-activated protein kinase (MAPK) has been considered to be a promising target for the development of therapeutics for various immunologic diseases. Herein we report an example for a successful application of the virtual screening with protein-ligand docking to identify the novel inhibitors of p38α MAPK. These inhibitors were screened for having desirable physicochemical properties as a drug candidate and compound 1-3 revealed a moderate inhibitory activity with IC(50) values ranging from 0.7 to 20 μM. Therefore, they deserve a consideration for further development by structure-activity relationship (SAR) studies to optimize the inhibitory activities. Structural features relevant to the stabilization of the newly identified inhibitors in the ATP-binding site of p38 MAPK are addressed in detail.