Activation of transient receptor potential vanilloid 4 induces apoptosis in hippocampus through downregulating PI3K/Akt and upregulating p38 MAPK signaling pathways

Transient receptor potential vanilloid 4 (TRPV4) is a calcium-permeable cation channel that is sensitive to cell swelling, arachidonic acid and its metabolites, epoxyeicosatrienoic acids, which are associated with cerebral ischemia. The activation of TRPV4 induces cytotoxicity in many types of cells, accompanied by an increase in the intracellular free calcium concentration. TRPV4 activation modulates the mitogen-activated protein kinase (MAPK) and phosphatidyl inositol 3 kinase (PI3K)/ protein kinase B (Akt) signaling pathways that regulate cell death and survival. Herein, we examined TRPV4-induced neuronal apoptosis by intracerebroventricular (ICV) injection of a TRPV4 agonist (GSK1016790A) and assessed its involvement in cerebral ischemic injury. ICV injection of GSK1016790A dose-dependently induced apoptosis in the mouse hippocampi (GSK-injected mice). The protein level of phosphorylated p38 MAPK (p-p38 MAPK) was markedly increased and that of phosphorylated c-Jun N-terminal protein kinase (p-JNK) was virtually unchanged. TRPV4 activation also decreased Bcl-2/Bax protein ratio and increased the cleaved caspase-3 protein level, and these effects were blocked by a PI3K agonist and a p38 MAPK antagonist, but were unaffected by a JNK antagonist. ICV injection of the TRPV4 antagonist HC-067047 reduced brain infarction after reperfusion for 48 h in mice with middle cerebral artery occlusion (MCAO). In addition, HC-067047 treatment attenuated the decrease in the phosphorylated Akt protein level and the increase in p-p38 MAPK protein level at 48 h after MCAO, while the increase in p-JNK protein level remained unchanged. Finally, the decreased Bcl-2/Bax protein ratio and the increased cleaved caspase-3 protein level at 48 h after MCAO were markedly attenuated by HC-067047. We conclude that activation of TRPV4 induces apoptosis by downregulating PI3K/Akt and upregulating p38 MAPK signaling pathways, which is involved in cerebral ischemic injury.Transient receptor potential vanilloid 4 (TRPV4), a member of the transient receptor potential (TRP) superfamily, is permeable to calcium (Ca²⁺).¹ TRPV4 was first described as a cellular osmotic sensor that detects hypotonic stimulation, and it has now been proven to be activated by multiple stimuli, including mild heat, mechanical stimulation, arachidonic acid (AA) and its metabolites, and exogenous chemical ligands.² TRPV4 is widely expressed in the nervous system and other tissues, including the lungs, bladder and skin.¹ In the central nervous system, TRPV4 is present in neurons and glial cells.^{3, 4} It mediates infrasound- and beta amyloid peptide-induced neuronal impairment, accompanied by an increase in the intracellular free calcium concentration ([Ca²⁺]_i).^{5, 6} Application of a TRPV4 agonist dose-dependently induces hippocampal neuronal death in vivo.⁷ Additionally, a gain-of-function mutant of TRPV4 has been shown to augment Ca²⁺ entry and decrease cell viability in transfected HEK293 cells.⁸ TRPV4 can be activated by cell swelling-induced mechanical stimulation and metabolites of AA that are always associated with cerebral ischemia. The protein level of TRPV4 has been reported to increase with ongoing reperfusion in a mouse model of middle cerebral artery occlusion (MCAO).⁷ Therefore, the over- or hyper-activation of TRPV4 is likely during cerebral ischemia-reperfusion. Blocking of TRPV4 has been shown to exert neuroprotective effects against cerebral ischemic injury in both in vitro and in vivo studies.^{7, 9, 10, 11} Targeting of TRPV4 is attracting more and more attention in the treatment of cerebral ischemia.Cell apoptosis, which is one of the major causes of cerebral ischemic injury, becomes prominent after reperfusion for 24–72 h.¹² It has been reported that excessive Ca²⁺ entry through TRPV4 leads to apoptosis in mouse retinal ganglion cells, which may be due to the activation of Ca²⁺-dependent pro-apoptotic signaling pathways.¹³ Mitogen-activated protein kinase (MAPK) signaling pathways that are involved in cerebral ischemic injury have important roles in regulating cell death and survival through signal translocation pathways related to apoptosis.¹⁴ The activation of phosphatidyl inositol 3-kinase (PI3K)/protein kinase B (Akt) signaling has been reported to inhibit caspase-dependent apoptosis in cultured neurons and a mouse model of Alzheimer''s disease.^{15, 16, 17} Activation of TRPV4 can modulate MAPK and PI3K/Akt signaling pathways in different types of cells.^{7, 18} In this study, we first assessed the effect of TRPV4 activation on neuronal apoptosis in the hippocampus and then explored the mechanisms underlying TRPV4 action. Finally, we examined the involvement of TRPV4-induced apoptosis in MCAO in mice.     

Absence of Appl2 sensitizes endotoxin shock through activation of PI3K/Akt pathway

Background

The adapter proteins Appl1 (adaptor protein containing pleckstrin homology domain, phosphotyrosine domain, and leucine zipper motif 1) and Appl2 are highly homologous and involved in several signaling pathways. While previous studies have shown that Appl1 plays a pivotal role in adiponectin signaling and insulin secretion, the physiological functions of Appl2 are largely unknown.

Results

In the present study, the role of Appl2 in sepsis shock was investigated by using Appl2 knockout (KO) mice. When challenged with lipopolysaccharides (LPS), Appl2 KO mice exhibited more severe symptoms of endotoxin shock, accompanied by increased production of proinflammatory cytokines. In comparison with the wild-type control, deletion of Appl2 led to higher levels of TNF-α and IL-1β in primary macrophages. In addition, phosphorylation of Akt and its downstream effector NF-κB was significantly enhanced. By co-immunoprecipitation, we found that Appl2 and Appl1 interacted with each other and formed a complex with PI3K regulatory subunit p85α, which is an upstream regulator of Akt. Consistent with these results, deletion of Appl1 in macrophages exhibited characteristics of reduced Akt activation and decreased the production of TNFα and IL-1β when challenged by LPS.

Conclusions

Results of the present study demonstrated that Appl2 is a critical negative regulator of innate immune response via inhibition of PI3K/Akt/NF-κB signaling pathway by forming a complex with Appl1 and PI3K.

     

Kinetic analysis of the MAPK and PI3K/Akt signaling pathways

Computational modeling of signal transduction is currently attracting much attention as it can promote the understanding of complex signal transduction mechanisms. Although several mathematical models have been used to examine signaling pathways, little attention has been given to crosstalk mechanisms. In this study, an attempt was made to develop a computational model for the pathways involving growth-factor-mediated mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3'-kinase/protein kinase B (PI3K/Akt). In addition, the dynamics of the protein activities were analyzed based on a set of kinetic data. The simulation approach integrates the information on several levels and predicts systems behavior. The in-silico analysis conducted revealed that the Raf and Akt pathways act independently.     

3',4'-didemethylnobiletin induces phase II detoxification gene expression and modulates PI3K/Akt signaling in PC12 cells

Oxidative stress is considered a major cause of neurodegenerative disorders. In this work, we investigated the cytoprotective effects and mechanisms of the citrus flavonoid nobiletin (NOB) and its metabolite, 3',4'-didemethylnobiletin (3',4'-dihydroxy-5,6,7,8-tetramethoxyflavone; DTF), in PC12 cells. Both NOB and DTF exhibited strong potency in attenuating serum withdrawal- and H(2)O(2)-caused cell death and increased intracellular GSH level via upregulation of both catalytic and modifier subunits of glutamate-cysteine ligase (GCL). However, only DTF suppressed intracellular ROS accumulation in H(2)O(2)-treated cells, induced heme oxygenase-1 (HO-1) expression, and enhanced nuclear factor E2-related factor 2 (Nrf2) binding to the ARE. Nevertheless, DTF-mediated HO-1 upregulation was independent of Nrf2 activation because knockdown of Nrf2 expression by siRNA did not affect its expression. DTF suppressed NF-κB activation, and addition of NF-κB inhibitor, pyrrolidine dithiocarbamate or Bay 11-7082, synergistically enhanced DTF-mediated HO-1 expression, indicating that HO-1 induction is associated with NF-κB suppression. NOB and DTF also activated the ERK, JNK, and Akt pathways in PC12 cells that had undergone serum starvation. Addition of pharmacological kinase inhibitors, U0126, SP600125, and LY294002, caused cytotoxicity and the last significantly attenuated NOB- and DTF-mediated antiapoptotic actions, indicating the involvement of PI3K/Akt signaling in their cytoprotective effects. In conclusion, HO-1 and GCL upregulation and intrinsic ROS-scavenging activity may contribute to DTF-mediated cytoprotection. Furthermore, modulation of PI3K/Akt signaling is involved in channeling the DTF stimulus for cell survival against oxidative insults.     

Selective activation of Fyn/PI3K and p38 MAPK regulates IL-4 production in BMMC under nontoxic stress condition

Mast cells have the ability to react to multiple stimuli, implicating these cells in many immune responses. Specific signals from the microenvironment in which mast cells reside can activate different molecular events that govern distinct mast cells responses. We previously demonstrated that hydrogen peroxide (H(2)O(2)) promotes IL-4 and IL-6 mRNA production and potentates FcepsilonRI-induced cytokine release in rat basophilic leukemia RBL-2H3 cells. To further evaluate the effect of an oxidative microenvironment (which is physiologically present in an inflammatory site) on mast cell function and the molecular events responsible for mast cell cytokine production in this environment, we analyzed the effect of H(2)O(2) treatment on IL-4 production in bone marrow-derived, cultured mast cells. Our findings show that nanomolar concentrations of H(2)O(2) induce cytokine secretion and enhance IL-4 production upon FcepsilonRI triggering. Oxidative stimulation activates a distinct signal transduction pathway that induces Fyn/PI3K/Akt activation and the selective phosphorylation of p38 MAP kinase. Moreover, H(2)O(2) induces AP-1 and NFAT complexes that recognize the IL-4 promoter. The absence of Fyn and PI3K or the inhibition of p38 MAPK activity demonstrated that they are essential for H(2)O(2)-driven IL-4 production. These findings show that mast cells can respond to an oxidative microenvironment by initiating specific signals capable of eliciting a selective response. The findings also demonstrate the dominance of the Fyn/p38 MAPK pathway in driving IL-4 production.     

MNAR plays an important role in ERa activation of Src/MAPK and PI3K/Akt signaling pathways

Estrogens play a critical role in the regulation of cellular proliferation, differentiation, and apoptosis. Evidence indicates that this regulation is mediated by a complex interface of direct control of gene expression (so-called "genomic action") and by regulation of cell-signaling/phosphorylation cascades (referred to as the "non-genomic", or "extranuclear" action). However, the mechanisms of the non-genomic action of estrogens are not well defined. We have recently described the identification of a novel scaffold protein termed MNAR (modulator of non-genomic action of estrogen receptor), that couples conventional steroid receptors with extranuclear signal transduction pathways, thus potentially providing additional and tissue- or cell-specific level of steroid hormone regulation of cell functions. We have demonstrated that the MNAR is required for ER alpha (ERa) interaction with p60(src) (Src), which leads to activation of Src/MAPK pathway. Our new data also suggest that activation of cSrc in response to E2 leads to MNAR phosphorylation, interaction with p85, and activation of the PI3 and Akt kinases. These data therefore suggest that MNAR acts as an important scaffold that integrates ERa action in regulation of important signaling pathways. ERa non-genomic action has been suggested to play a key role in estrogen-induced cardio-, neuro-, and osteo-protection. Therefore, evaluation of the molecular crosstalk between MNAR and ERa may lead to development of functionally selective ER modulators that can separate between beneficial, prodifferentiative effects in bone, the cardiovascular system and the CNS and the "detrimental", proliferative effects in reproductive tissues and organs.     

Targeting RET to induce medullary thyroid cancer cell apoptosis: an antagonistic interplay between PI3K/Akt and p38MAPK/caspase-8 pathways

Mutations in REarranged during Transfection (RET) receptor tyrosine, followed by the oncogenic activation of RET kinase is responsible for the development of medullary thyroid carcinoma (MTC) that responds poorly to conventional chemotherapy. Targeting RET, therefore, might be useful in tailoring surveillance of MTC patients. Here we showed that theaflavins, the bioactive components of black tea, successfully induced apoptosis in human MTC cell line, TT, by inversely modulating two molecular pathways: (i) stalling PI3K/Akt/Bad pathway that resulted in mitochondrial transmembrane potential (MTP) loss, cytochrome-c release and activation of the executioner caspases-9 and -3, and (ii) upholding p38MAPK/caspase-8/caspase-3 pathway via inhibition of Ras/Raf/ERK. Over-expression of either constitutively active myristoylated-Akt-cDNA (Myr-Akt-cDNA) or dominant-negative-caspase-8-cDNA (Dn-caspase-8-cDNA) partially blocked theaflavin-induced apoptosis, while co-transfection of Myr-Akt-cDNA and Dn-caspase-8-cDNA completely eradicated the effect of theaflavins thereby negating the possibility of existence of other pathways. A search for the upstream signaling revealed that theaflavin-induced disruption of lipid raft caused interference in anchorage of RET in lipid raft that in turn stalled phosphorylation of Ras and PI3Kinase. In such anti-survival cellular micro-environment, pro-apoptotic signals were triggered to culminate into programmed death of MTC cell. These findings not only unveil a hitherto unexplained mechanism underlying theaflavin-induced MTC death, but also validate RET as a promising and potential target for MTC therapy.     

Leptin Inhibits Glucose Intestinal Absorption via PKC,p38MAPK,PI3K and MEK/ERK

The role of leptin in controlling food intake and body weight is well recognized, but whether this is achieved by modulating nutrient absorption is still a controversial issue. The aim of this work was to investigate the direct effect of luminal leptin on glucose intestinal absorption and elucidate for the first time its signaling pathway. Fully differentiated Caco-2 cells grown on transwell filters were used for glucose transport studies. Leptin caused a significant reduction in glucose absorption. Individual and simultaneous inhibition of ERK, p38MAPK, PI3K or PKC abrogated completely the inhibitory effect of leptin. Activating PKC, lead to a stimulatory effect that appeared only when ERK, p38MAPK, or PI3K was inactive. Moreover, leptin increased the phosphorylation of ERK, Akt and p38MAPK. This increase changed into a decrease when p38MAPK and PKC were inactivated individually. Inhibiting ERK maintained the leptin-induced up-regulation of p-Akt and p-p38MAPK while inhibiting PI3K reduced the level of p-ERK and p-Akt but maintained the increase in p-p38MAPK. These results suggest that leptin reduces glucose absorption by activating PKC. Although the latter modulates glucose absorption via a stimulatory and an inhibitory pathway, only the latter is involved in leptin’s action. Active PKC leads to a sequential activation of p38MAPK, PI3K and ERK which exerts an inhibitory effect on glucose absorption. The results reveal a modulatory role of leptin in nutrient absorption in addition to its known satiety inducing effect.     

Interplay between PI3K/Akt and MAPK signaling pathways in DNA-damaging drug-induced apoptosis

In order to elucidate the role of the mitogen-activated protein kinases, including JNK, p38 MAPK and ERK, as well as the survival-associated PI3K/Akt signaling pathway, in the response to chemotherapy, we have conducted a comparative study regarding the effects of doxorubicin on these pathways. Doxorubicin was determined to elicit the apoptosis of NIH3T3 cells in a dose-dependent manner. Prior to cell death, both Akt and p38 MAPK were transiently activated, and subsequently inactivated almost wholly, whereas ERK and JNK evidenced sustained activations in response to the drug treatment. The inhibition of PI3K/Akt and p38 MAPK both accelerated and enhanced doxorubicin-induced apoptosis and ERK inhibition apparently exerted negative effect on apoptosis. The modulation of PI3K/Akt activation by treatment of LY294002 or expression of Akt mutants such as Akt-DN or Myr-Akt exerted a significant effect on the activation of ERK1/2. We also observed that PI3K/Akt and sustained ERK activation were associated intimately with the etoposide-induced apoptosis. Taken together, our results clearly suggest that the differential regulation of the PI3K/Akt, ERK1/2, and p38 MAPK signaling pathways are crucial in the context of DNA-damaging drug-induced apoptosis, and this has compelled us to propose that the sustained activation of ERK1/2 pathway may be generally involved in the apoptosis induced by anticancer DNA-damaging drugs, including doxorubicin and etoposide.     

Zinc Inhibits H2O2-Induced MC3T3-E1 Cells Apoptosis via MAPK and PI3K/AKT Pathways

Zinc has been shown to increase bone mass and promote bone cell proliferation and differentiation. We, therefore, hypothesized that zinc might be cytoprotective for bone cells during oxidative stress. The cells were divided into H(2)O(2), zinc and zinc+H(2)O(2) groups. In the present study, zinc was found to inhibit H(2)O(2)-induced apoptosis in MC3T3-E1 cells, as shown by analysis of Annexin V/PI double staining. Western blot data showed that in zinc+H(2)O(2)-treated cells, zinc decreased the levels of AIF, Bax and active caspase-9 and -3, which are pro-apoptotic factors. And zinc inhibited release of cytochrome c from mitochondria to cytosol in zinc+H(2)O(2)-treated cells. Further investigation shows that protection is via activation of PI3K/Akt/mTor and MAPK /ERK pathways and inhibition of MAPK/P38 and MAPK/JNK pathways. Protecting osteoblast cells from oxidative damage presents a potential application in the treatment of osteoporosis.     

Axl is essential for VEGF-A-dependent activation of PI3K/Akt

Herein, we report that vascular endothelial growth factor A (VEGF-A) engages the PI3K/Akt pathway by a previously unknown mechanism that involves three tyrosine kinases. Upon VEGF-A-dependent activation of VEGF receptor-2 (VEGFR-2), and subsequent TSAd-mediated activation of Src family kinases (SFKs), SFKs engage the receptor tyrosine kinase Axl via its juxtamembrane domain to trigger ligand-independent autophosphorylation at a pair of YXXM motifs that promotes association with PI3K and activation of Akt. Other VEGF-A-mediated signalling pathways are independent of Axl. Interfering with Axl expression or function impairs VEGF-A- but not bFGF-dependent migration of endothelial cells. Similarly, Axl null mice respond poorly to VEGF-A-induced vascular permeability or angiogenesis, whereas other agonists induce a normal response. These results elucidate the mechanism by which VEGF-A activates PI3K/Akt, and identify previously unappreciated potential therapeutic targets of VEGF-A-driven processes.     

PI3K/Akt信号传导通路与肿瘤

信号转导通路的异常激活是肿瘤细胞的发生、发展重要步骤,PI3K/Akt 信号通路在人类绝大多数恶性肿瘤中被异常激活,其在肿瘤的增殖、存活、细胞运动、抵抗凋亡、血管发生和转移以及对化疗耐药、放疗抗拒中发挥了重要作用.因此,通过对PI3K/Akt 通路的研究进一步了解肿瘤的发生、发展机制,并寻求抗肿瘤药物的新靶点,本文就 PI3K/Akt 信号转导通路的结构特点、与肿瘤发生、发展的关系及其时放化疗的影响作一综述.     

ApoA-I/SR-BI modulates S1P/S1PR2-mediated inflammation through the PI3K/Akt signaling pathway in HUVECs

Endothelial dysfunction plays a vital role during the initial stage of atherosclerosis. Oxidized low-density lipoprotein (ox-LDL) induces vascular endothelial injury and vessel wall inflammation. Sphingosine-1-phosphate (S1P) exerts numerous vasoprotective effects by binding to diverse S1P receptors (S1PRs; S1PR1-5). A number of studies have shown that in endothelial cells (ECs), S1PR2 acts as a pro-atherosclerotic mediator by stimulating vessel wall inflammation through the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway. Scavenger receptor class B member I (SR-BI), a high-affinity receptor for apolipoprotein A-I (apoA-I)/high-density lipoprotein (HDL), inhibits nuclear factor-κB (NF-κB) translocation and decreases the plasma levels of inflammatory mediators via the PI3K/Akt pathway. We hypothesized that the inflammatory effects of S1P/S1PR2 on ECs may be regulated by apoA-I/SR-BI. The results showed that ox-LDL, a pro-inflammatory factor, augmented the S1PR2 level in human umbilical vein endothelial cells (HUVECs) in a dose- and time-dependent manner. In addition, S1P/S1PR2 signaling influenced the levels of inflammatory factors, including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and IL-10, aggravating inflammation in HUVECs. Moreover, the pro-inflammatory effects induced by S1P/S1PR2 were attenuated by SR-BI overexpression and enhanced by an SR-BI inhibitor, BLT-1. Further experiments showed that the PI3K/Akt signaling pathway was involved in this process. Taken together, these results demonstrate that apoA-I/SR-BI negatively regulates S1P/S1PR2-mediated inflammation in HUVECs by activating the PI3K/Akt signaling pathway.