Indole Derivatives as Potent Inhibitors of Larval Settlement by the Barnacle,Balanus amphitrite

A potent inhibitor of larval settlement by the barnacle, Balanus amphitrite, was isolated as 2,5,6-tribromo-1-methylgraimne from a marine invertebrate. In comparative tests on the activity of related compounds, such compounds as 2-methylgramine and 2-methyl-3-(morpholinomethyl)-indole exhibited potent inhibitory activity. The inhibitory activity toward larval settlement was found not to be due to toxicity but to a repellent effect.     

Differential activation of p38MAPK isoforms by MKK6 and MKK3

All four members of the mammalian p38 mitogen-activated protein kinase (MAPK) family (p38α, p38β, p38γ and p38δ) are activated by dual phosphorylation in the TGY motif in the activation loop. This phosphorylation is mediated by three kinases, MKK3, MKK6 and MKK4, at least in vitro. The role of these MKK in the activation of p38α has been demonstrated in studies using fibroblasts that lack MKK3 and/or MKK6. Nonetheless, the physiological upstream activators of the other p38MAPK isoforms have not yet been reported using MKK knockout cells. In this study, we examined p38β, γ and δ activation by MKK3 and MKK6, in cells lacking MKK3, MKK6 or both. We show that MKK3 and MKK6 are both essential for the activation of p38γ and p38β induced by environmental stress, whereas MKK6 is the major p38γ activator in response to TNFα. In contrast, p38δ activation by ultraviolet radiation, hyperosmotic shock, anisomycin or by TNFα is mediated by MKK3. Moreover, in response to osmotic stress, MKK3 and MKK6 are crucial in regulating the phosphorylation of the p38γ substrate hDlg and its activity as scaffold protein. These data indicate that activation of distinct p38MAPK isoforms is regulated by the selective and synchronized action of two kinases, MKK3 and MKK6, in response to cell stress.     

Activating p38 MAPK: New Tricks for an Old Kinase

Mitogen-activated protein kinases (MAPKs) participate in signaling initiated by a wide variety of extracellular stimuli. MAPKs are most commonly activated by a series of phosphorylation events in which one kinase phosphorylates another, the “MAPK cascade”. The cascade concludes with the dual phosphorylation of MAPKs on a conserved Thr-X-Tyr motif. In the case of the p38 MAPK, an exception to this paradigm has been found when signaling via the T cell antigen receptor (TCR). Rather than trigger the MAPK cascade, TCR-mediated stimulation activates proximal tyrosine kinases, which results in the phosphorylation of p38 on a noncanonical activating residue, Tyr-323. This phosphorylation activates p38 to phosphorylate third party substrates as well as its own Thr-X-Tyr motif. Here we discuss the structural and functional implications of this alternative p38 activation pathway, which may provide a new target for tissue-specific pharmacologic inhibition.     

Inhibitory Effects of Mediterranean Sponge Extracts and Metabolites on Larval Settlement of the Barnacle Balanus amphitrite

One of the most promising alternative technologies to antifouling paints based on heavy metals is the development of coatings whose active ingredients are compounds naturally occurring in marine organisms. This approach is based on the problem of epibiosis faced by all marine organisms and the fact that a great number of them cope with it successfully. The present study investigated the antifouling activity of a series of extracts and secondary metabolites from the epibiont-free Mediterranean sponges Ircinia oros, I. spinosula, Cacospongia scalaris, Dysidea sp., and Hippospongia communis. Antifouling efficacy was evaluated by the settlement inhibition of laboratory-reared Balanus amphitrite Darwin cyprids. The most promising activity was exhibited by the metabolites 2-[24-acetoxy]-octaprenyl-1-4-hydroquinone (8a), dihydrofurospongin II (10), and the alcoholic extract of Dysidea sp.     

Balance between MKK6 and MKK3 mediates p38 MAPK associated resistance to cisplatin in NSCLC

The p38 MAPK signaling pathway has been proposed as a critical mediator of the therapeutic effect of several antitumor agents, including cisplatin. Here, we found that sensitivity to cisplatin, in a system of 7 non-small cell lung carcinoma derived cell lines, correlated with high levels of MKK6 and marked activation of p38 MAPK. However, knockdown of MKK6 modified neither the response to cisplatin nor the activation of p38 MAPK. Deeper studies showed that resistant cell lines also displayed higher basal levels of MKK3. Interestingly, MKK3 knockdown significantly decreased p38 phosphorylation upon cisplatin exposure and consequently reduced the response to the drug. Indeed, cisplatin poorly activated MKK3 in resistant cells, while in sensitive cell lines MKK3 showed the opposite pattern in response to the drug. Our data also demonstrate that the low levels of MKK6 expressed in resistant cell lines are the consequence of high basal activity of p38 MAPK mediated by the elevated levels of MKK3. This finding supports the existence of a regulatory mechanism between both MAPK kinases through their MAPK. Furthermore, our results were also mirrored in head and neck carcinoma derived cell lines, suggesting our observations boast a potential universal characteristic in cancer resistance of cisplatin. Altogether, our work provides evidence that MKK3 is the major determinant of p38 MAPK activation in response to cisplatin and, hence, the resistance associated with this MAPK. Therefore, these data suggest that the balance between both MKK3 and MKK6 could be a novel mechanism which explains the cellular response to cisplatin.     

A Novel Role of p38α MAPK in Mitotic Progression Independent of Its Kinase Activity

Activation of p38α MAPK triggers G2/M checkpoint, thus inhibiting cell proliferation. In this study we found that depletion of p38α by RNAi also inhibited cell proliferation and caused mitotic arrest. However, treatment with selective small molecule p38 kinase inhibitors had no effect on cell cycle progression, even though the p38 kinase was completely inhibited, revealing p38α functions that are independent of its kinase activity. Indeed, ectopic expression of a kinase negative p38α rescued the lethality caused by RNAi-depletion of the endogenous p38α, thus providing further evidence for a kinase-independent function of p38α. In addition, we showed that overexpression of the wild type or kinase-negative p38α also strongly inhibited cell proliferation, similarly as RNAi depletion of p38α. Together the results demonstrate that, in addition to its kinase-dependent functions, such as in activation of G2/M checkpoint, p38α also has an essential, kinase-independent function.     

Differential involvement of p38 mitogen-activated protein kinase kinases MKK3 and MKK6 in T-cell apoptosis

The p38 mitogen-activated protein kinase (p38MAPK) is activated in response to various stimuli, including cellular stress, inflammatory cytokines and cell surface receptors. The activation of p38MAPK is predominantly mediated by the two upstream MAPK kinases MKK3 and MKK6. To study the role of the p38MAPK pathway in vivo, we generated Mkk6^–/– mice. We examined whether T-cell apoptosis is affected in these mice and in our previously reported Mkk3^–/– mice. Strikingly, in vivo deletion of double positive thymocytes in Mkk6^–/– mice was impaired, whereas Mkk3^–/– mice showed no apparent abnormality. Conversely, CD4⁺T cells from Mkk3^–/– but not from Mkk6^–/– mice were resistant to activation-induced cell death and cytokine-withdrawal-induced apoptosis. In peripheral CD4⁺T cells, MKK3 is induced upon stimulation, whereas MKK6 is downregulated. These results suggest a novel mechanism regulating T-cell apoptosis differentially through the p38MAPK pathway by MKK3 and MKK6.     

Requirement of mitogen-activated protein kinase kinase 3 (MKK3) for activation of p38alpha and p38delta MAPK isoforms by TGF-beta 1 in murine mesangial cells

Transforming growth factor-beta1 (TGF-beta1) is a potent inducer of extracellular matrix (ECM) synthesis that leads to renal fibrosis. Intracellular signaling mechanisms involved in this process remain incompletely understood. Mitogen-activated protein kinase (MAPK) is a major stress signal-transducing pathway, and we have previously reported activation of p38 MAPK by TGF-beta1 in rat mesangial cells and its role in the stimulation of pro-alpha1(I) collagen. In this study, we further investigated the mechanism of p38 MAPK activation by TGF-beta1 and the role of MKK3, an upstream MAPK kinase of p38 MAPK, by examining the effect of targeted disruption of the Mkk3 gene. We first isolated glomerular mesangial cells from MKK3-null (Mkk3-/-) and wild-type (Mkk3+/+) control mice. Treatment with TGF-beta1 induced rapid phosphorylation of MKK3 as well as p38 MAPK within 15 min in cultured wild-type (Mkk3+/+) mouse mesangial cells. In contrast, TGF-beta1 failed to induce phosphorylation of either MKK3 or p38 MAPK in MKK3-deficient (Mkk3-/-) mouse mesangial cells, indicating that MKK3 is required for TGF-beta1-induced p38 MAPK activation. TGF-beta1 selectively activated the p38 MAPK isoforms p38alpha and p38delta in wild-type (Mkk3+/+) mesangial cells, but not in MKK3-deficient (Mkk3-/-) mesangial cells. Thus, activation of p38alpha and p38delta is dependent on the activation of upstream MKK3 by TGF-beta1. Furthermore, MKK3 deficiency resulted in a selective disruption of TGF-beta1-stimulated up-regulation of pro-alpha1(I) collagen expression but not TGF-beta1 induction of fibronectin and PAI-1. These data demonstrate that the MKK3 is a critical component of the TGF-beta1 signaling pathway, and its activation is required for subsequent p38alpha and p38delta MAPK activation and collagen stimulation by TGF-beta1.     

Coordinated regulation of autophagy by p38α MAPK through mAtg9 and p38IP

Autophagy, a lysosomal degradation pathway, is essential for homeostasis, development, neurological diseases, and cancer. Regulation of autophagy in human disease is not well understood. Atg9 is a transmembrane protein required for autophagy, and it has been proposed that trafficking of Atg9 may regulate autophagy. Mammalian Atg9 traffics between the TGN and endosomes in basal conditions, and newly formed autophagosomes in response to signals inducing autophagy. We identified p38IP as a new mAtg9 interactor and showed that this interaction is regulated by p38α MAPK. p38IP is required for starvation‐induced mAtg9 trafficking and autophagosome formation. Manipulation of p38IP and p38α alters mAtg9 localization, suggesting p38α regulates, through p38IP, the starvation‐induced mAtg9 trafficking to forming autophagosomes. Furthermore, we show that p38α is a negative regulator of both basal autophagy and starvation‐induced autophagy, and suggest that this regulation may be through a direct competition with mAtg9 for binding to p38IP. Our results provide evidence for a link between the MAPK pathway and the control of autophagy through mAtg9 and p38IP.     

Engulfment of Axon Debris by Microglia Requires p38 MAPK Activity

The clearance of debris after injuries to the nervous system is a critical step for restoration of the injured neural network. Microglia are thought to be involved in elimination of degenerating neurons and axons in the central nervous system (CNS), presumably restoring a favorable environment after CNS injuries. However, the mechanism underlying debris clearance remains elusive. Here, we establish an in vitro assay system to estimate phagocytosis of axon debris. We employed a Wallerian degeneration model by cutting axons of the cortical explants. The cortical explants were co-cultured with primary microglia or the MG5 microglial cell line. The cortical neurites were then transected. MG5 cells efficiently phagocytosed the debris, whereas primary microglia showed phagocytic activity only when they were activated by lipopolysaccharide or interferon-β. When MG5 cells or primary microglia were co-cultured with degenerated axons, p38 mitogen-activated protein kinase (MAPK) was activated in these cells. Engulfment of axon debris was blocked by the p38 MAPK inhibitor SB203580, indicating that p38 MAPK is required for phagocytic activity. Receptors that recognize dying cells appeared not to be involved in the process of phagocytosis of the axon debris. In addition, the axons undergoing Wallerian degeneration did not release lactate dehydrogenase, suggesting that degeneration of the severed axons and apoptosis may represent two distinct self-destruction programs. We observed regrowth of the severed neurites after axon debris was removed. This finding suggests that axon debris, in addition to myelin debris, is an inhibitory factor for axon regeneration.Axon degeneration is an active, tightly controlled, and versatile process of axon segment self-destruction. The lesion-induced degeneration process was first described by Waller (1) and has since been known as Wallerian degeneration (2, 3). This degeneration involves rapid blebbing and fragmentation of an entire axonal stretch into short segments, which are then removed by locally activated phagocytic cells. Phagocytic removal of damaged axons and their myelin sheaths distal to the injury is important for creating a favorable environment for axonal regeneration in the nervous system. Although the debris of degenerated axons and myelin is cleared by phagocytes in the peripheral nervous system (PNS), the debris is removed very slowly in the central nervous system (CNS)³ (4, 5). This is considered to be one of the obstacles for regeneration of the injured axons in the CNS.Apoptotic neurons are also engulfed by activated phagocytic cells. Apoptosis is very well documented in the CNS where a significant proportion of neurons undergo programmed cell death (6). To prevent the diffusion of damaging degradation products into surrounding tissues, dying neurons are phagocytosed. In the brain, apoptotic cells are engulfed mainly by the resident population of phagocytes known as microglia. Microglia are generally considered to be immune cells of the CNS (7). They respond to any kind of pathology with a reaction termed “microglial activation.” After injuries to the CNS, microglia react within a few hours with a migratory response toward the lesion site.Although insight into the mechanism of phagocytosis of dying cells by microglia has improved, little is known about the mechanism of clearance of degenerated axons and myelin debris by microglia after axonal injury in the CNS. Interestingly, the axons undergoing Wallerian degeneration do not seem to possess detectable activation of the caspase family (8), suggesting that Wallerian degeneration and apoptosis may represent two distinct self-destruction programs. Thus, the mechanism of microglial phagocytosis of dying cells might be different from that of axon/myelin debris. We aimed to elucidate the mechanism of debris clearance by microglia after an axonal injury. We established an in vitro assay system to estimate phagocytosis of degenerated axon debris. We found that p38 mitogen-activated protein kinase (MAPK) was critical for the phagocytic activity of microglia. Treatment with lipopolysaccharide (LPS) or interferon-β (IFN-β) was necessary for the primary microglia to become phagocytic. In addition, clearance of degenerated axon debris allowed axonal growth from the severed neurites, suggesting that removal of the axon debris provides a favorable environment for axonal regeneration.     

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.     

Methylglyoxal induces apoptosis through activation of p38 MAPK in rat Schwann cells

The formation of glucose-derived methylglyoxal (MG), a highly reactive dicarbonyl compound, is accelerated under diabetic conditions. We examined whether MG was capable of inducing apoptosis in Schwann cells (SCs), since recent studies have suggested a potential involvement of apoptotic cell death in the development of diabetic neuropathy. MG induced apoptosis in SCs in a dose-dependent manner, accompanied by a reduction of intracellular glutathione content and activation of the p38 MAPK. Inhibiting the p38 MAPK activation by SB203580 successfully suppressed the MG-induced apoptosis in SCs. Aminoguanidine and N-acetyl-l-cysteine also inhibited the MG-induced p38 MAPK activation and apoptosis along with restoration of the intracellular glutathione content. These results suggest a potential role for MG in SC injury through oxidative stress-mediated p38 MAPK activation under diabetic conditions, and it may serve as a novel insight into therapeutic strategies for diabetic neuropathy.     

MKK6/3 and p38 MAPK pathway activation is not necessary for insulin-induced glucose uptake but regulates glucose transporter expression

p38 mitogen-activated protein kinase (MAPK), which is situated downstream of MAPK kinase (MKK) 6 and MKK3, is activated by mitogenic or stress-inducing stimuli, as well as by insulin. To clarify the role of the MKK6/3-p38 MAPK pathway in the regulation of glucose transport, dominant negative p38 MAPK and MKK6 mutants and constitutively active MKK6 and MKK3 mutants were overexpressed in 3T3-L1 adipocytes and L6 myotubes using an adenovirus-mediated transfection procedure. Constitutively active MKK6/3 mutants up-regulated GLUT1 expression and down-regulated GLUT4 expression, thereby significantly increasing basal glucose transport but diminishing transport induced by insulin. Similar effects were elicited by chronic (24 h) exposure to tumor necrosis factor alpha, interleukin-1beta, or 200 mm sorbitol, all activate the MKK6/3-p38 MAPK pathway. SB203580, a specific p38 MAPK inhibitor, attenuated these effects, further confirming that both MMK6 and MMK3 act via p38 MAPK, whereas they had no effect on the increase in glucose transport induced by a constitutively active MAPK kinase 1 (MEK1) mutant or by myristoylated Akt. In addition, suppression of p38 MAPK activation by overexpression of a dominant negative p38 MAPK or MKK6 mutant did not diminish insulin-induced glucose uptake by 3T3-L1 adipocytes. It is thus apparent that activation of p38 MAPK is not essential for insulin-induced increases in glucose uptake. Rather, p38 MAPK activation leads to a marked down-regulation of insulin-induced glucose uptake via GLUT4, which may underlie cellular stress-induced insulin resistance caused by tumor necrosis factor alpha and other factors.     

APPL1 mediates adiponectin-stimulated p38 MAPK activation by scaffolding the TAK1-MKK3-p38 MAPK pathway

The adaptor protein APPL1 mediates the stimulatory effect of adiponectin on p38 mitogen-activated protein kinase (MAPK) signaling, yet the underlying mechanism remains unclear. Here we show that, in C(2)C(12) cells, overexpression or suppression of APPL1 enhanced or suppressed, respectively, adiponectin-stimulated p38 MAPK upstream kinase cascade, consisting of transforming growth factor-β-activated kinase 1 (TAK1) and mitogen-activated protein kinase kinase 3 (MKK3). In vitro affinity binding and coimmunoprecipitation experiments revealed that TAK1 and MKK3 bind to different regions of APPL1, suggesting that APPL1 functions as a scaffolding protein to facilitate adiponectin-stimulated p38 MAPK activation. Interestingly, suppressing APPL1 had no effect on TNFα-stimulated p38 MAPK phosphorylation in C(2)C(12) myotubes, indicating that the stimulatory effect of APPL1 on p38 MAPK activation is selective. Taken together, our study demonstrated that the TAK1-MKK3 cascade mediates adiponectin signaling and uncovers a scaffolding role of APPL1 in regulating the TAK1-MKK3-p38 MAPK pathway, specifically in response to adiponectin stimulation.     

抑制p38MAPK信号通路对Bpiv3复制的影响

由牛副流感病毒3型(Bovine parainfluenza virus type 3,Bpiv3)感染引起的牛副流感病已成为各国牛场最重要的传染病之一,每年都会给世界养牛业造成巨大的经济损失,但关于该病致病的分子机制研究较少。本研究通过观察Bpiv3感染对MDBK细胞中丝裂原活化蛋白激酶(MKK3)及其下游分子p38丝裂酶原活化的蛋白激酶(p38MAPK)的表达的影响,探讨相关的信号转导机制,对p38 MAPK通路在Bpiv3感染过程中的作用进行了初步研究。Bpiv3感染细胞后,采用Western Blot检测MKK3,p38 MAPK在蛋白水平的表达变化,并采用ELISA法检测细胞上清中IL-6,IL-8,IL-13和TNF-α的水平变化,采用SPSS 12软件进行统计学分析。结果表明,Bpiv3在感染后能够诱导MKK3的激活以及p38的磷酸化,激活了p38 MAPK信号通路。而且p38 MAPK信号通路参与了Bpiv3的复制过程。ELISA检测Bpiv3感染后以及使用抑制剂SB202190处理后的细胞上清中IL-6、IL-8、IL-13和TNF-α的水平发现,p38 MAPK信号通路参与了Bpiv3诱导的炎症反应。研究证实Bpiv3感染能够激活p38 MAPK通路,显著上调MKK3的表达并诱导p38发生磷酸化,进一步激活下游分子发挥生物学活性,促进Bpiv3的复制及诱导促炎细胞因子的产生。p38 MAPK信号通路的激活可能是Bpiv3感染诱发炎症反应的机制之一。