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1.
Ghrelin exhibits its biological effect through binding to the growth hormone secretagogue 1a receptor (GHS-R1a). Recently, it has been reported that ghrelin has an anti-apoptotic effect in several cell types. However, the molecule mechanisms underlying the anti-apoptotic effect of ghrelin remain poorly understood. In this study, we investigated the intracellular mechanisms responsible for anti-apoptotic effect of ghrelin on human umbilical vein endothelial cells (HUVEC). Treatment of HUVEC with ghrelin inhibited high glucose-induced cell apoptosis. Ghrelin stimulated the rapid phosphorylation of mammalian target of rapamycin (mTOR), P70S6K and S6. The GHS-R1a-specific antagonist [D-Lys3]-GHRP-6 abolished the anti-apoptotic effect and inhibited the activation of mTOR, P70S6K, S6 induced by ghrelin. Pretreatment of cells with specific inhibitor of mTOR blocked the anti-apoptotic effect of ghrelin. In addition, ghrelin protected HUVECs against high glucose induced apoptosis by increasing Bcl-2/Bax ratio. Taken together, our results demonstrate that ghrelin produces a protective effect on HUVECs through activating GHS-R1a and mTOR/P70S6K signaling pathway mediates the effect of ghrelin. These observations suggest that ghrelin may act as a survival factor in preventing HUVECs apoptosis caused by high glucose.  相似文献   

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脑肠肽Ghrelin-GHSR信号系统与肿瘤的关系   总被引:1,自引:0,他引:1  
杨敏  陈东  祝世功 《现代生物医学进展》2008,8(9):1746-1749,1738
脑肠肽Ghrelin是一种含有28个氨基酸的生长激素释放肽,为生长激素促分泌素受体(growth hormone secretagogue recep-tor,GHSR)的内源性配体。Ghrelin及其功能性受体GHSR-1a广泛分布于中枢和外周组织。此外,在多种肿瘤组织和癌细胞中发现有Ghrelin及其功能性受体GHSR-1a的表达。我们的前期工作和目前的研究发现Ghrelin可与经典的功能性受体GHSR-1a或新型受体结合,通过激活多条信号转导通路,对肿瘤的生物学行为发挥重要的调控作用。因此,脑肠肽Ghrelin-GHSR信号系统可为肿瘤的临床诊断和预后评估发挥重要作用,并为肿瘤的分子治疗提供新靶点。  相似文献   

4.
The mitogenic and antiapoptotic actions of ghrelin in 3T3-L1 adipocytes   总被引:16,自引:0,他引:16  
Ghrelin, a stomach-derived hormone, induces adiposity when administered to rodents. Because ghrelin receptor is abundantly expressed in adipose tissue, we investigated the role of ghrelin in adipocyte biology. We observed ghrelin receptor expression in 3T3-L1 preadipocytes and adipocytes. Treatment of preadipocytes with ghrelin induced cellular proliferation and differentiation to mature adipocytes, as well as basal and insulin-stimulated glucose transport, but it inhibited adipocyte apoptosis induced by serum deprivation. Exposure of 3T3-L1 cells to ghrelin caused a rapid activation of MAPKs, especially ERK1/2. Chemical inhibition of MAPK blocked the mitogenic and antiapoptotic effects of ghrelin. Ghrelin also stimulated the insulin receptor substrate-associated phosphatidylinositol 3-kinase/Akt pathway in 3T3-L1 preadipocytes and adipocytes, whereas inhibition of this pathway blocked the effects of ghrelin on cell proliferation, antiapoptosis and glucose uptake. These findings suggest that the direct effects of ghrelin on proliferation, differentiation, and apoptosis in adipocytes may play a role in regulating fat cell number. These effects may be mediated via activation of the MAPK and phosphatidylinositol 3-kinase/Akt pathways.  相似文献   

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Diverse stimuli initiate the activation of apoptotic signaling pathways that often causes nuclear DNA fragmentation. Here, we report a new antiapoptotic protein, a caspase-activated DNase (CAD) inhibitor that interacts with ASK1 (CIIA). CIIA, by binding to apoptosis signal-regulating kinase 1 (ASK1), inhibits oligomerization-induced ASK1 activation. CIIA also associates with CAD and inhibits the nuclease activity of CAD without affecting caspase-3-mediated ICAD cleavage. Overexpressed CIIA reduces H2O2- and tumor necrosis factor-alpha-induced apoptosis. CIIA antisense oligonucleotides, which abolish expression of endogenous CIIA in murine L929 cells, block the inhibitory effect of CIIA on ASK1 activation, deoxyribonucleic acid fragmentation, and apoptosis. These findings suggest that CIIA is an endogenous antagonist of both ASK1- and CAD-mediated signaling.  相似文献   

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In the present study, we explore the role of apoptosis signal-regulating kinase 1 (ASK1) in denbinobin-induced apoptosis in human lung adenocarcinoma (A549) cells. Denbinobin-induced cell apoptosis was attenuated by an ASK1 dominant-negative mutant (ASK1DN), two antioxidants (N-acetyl-L-cysteine (NAC) and glutathione (GSH)), a c-Jun N-terminal kinase (JNK) inhibitor (SP600125), and an activator protein-1 (AP-1) inhibitor (curcumin). Treatment of A549 cells with denbinobin caused increases in ASK1 activity and reactive oxygen species (ROS) production, and these effects were inhibited by NAC and GSH. Stimulation of A549 cells with denbinobin caused JNK activation; this effect was markedly inhibited by NAC, GSH, and ASK1DN. Denbinobin induced c-Jun phosphorylation, the formation of an AP-1-specific DNA-protein complex, and Bim expression. Bim knockdown using a bim short interfering RNA strategy also reduced denbinobin-induced A549 cell apoptosis. The denbinobin-mediated increases in c-Jun phosphorylation and Bim expression were inhibited by NAC, GSH, SP600125, ASK1DN, JNK1DN, and JNK2DN. These results suggest that denbinobin might activate ASK1 through ROS production to cause JNK/AP-1 activation, which in turn induces Bim expression, and ultimately results in A549 cell apoptosis.  相似文献   

9.
Ghrelin is a well-characterized hormone that has protective effects on endothelial cells. Elevated HCY (homocysteine) can be a cardiovascular risk factor, but it is not known whether ghrelin can inhibit HCY-induced dysfunction and inflammatory response in rat CMECs (cardiac microvascular endothelial cells). We found that HCY treatment for 24 h inhibited proliferation and NO (nitric oxide) secretion, but with increased cell apoptosis and secretion of cytokines in CMECs. In contrast, ghrelin pretreatment significantly improved proliferation and NO secretion, and inhibited cell apoptosis and secretion of cytokines in HCY-induced CMECs. In addition, Western blot assay showed that NF-κB (nuclear factor κB) and cleaved-caspase 3 expression were elevated, and PCNA (proliferating cell nuclear antigen) and eNOS (endothelial nitric oxide synthase) expression were decreased after treatment with HCY, which was significantly reversed by pretreatment with ghrelin. The data suggest that ghrelin inhibits HCY-induced CMEC dysfunction and inflammatory response, probably mediated by inhibition of NF-κB activation.  相似文献   

10.
In addition to regulating growth hormone release from the pituitary, ghrelin receptors also influence cell proliferation and apoptosis. By studying mitogen-activated protein kinase activity in human embryonic kidney 293 cells over-expressing ghrelin receptors, we aimed to identify the specific cell signalling pathways used by ghrelin receptors, and to determine if the truncated ghrelin receptor polypeptide had any influence on the functional activity of ghrelin receptors. We found that ghrelin activated extracellular signal-regulated kinases 1/2 with an EC50 value of 10 nM, and that this response was inhibited by the ghrelin receptor antagonists D-Lys3-GHRP-6 and [D-Arg1,D-Phe5,D-Trp(7,9),Leu11]-substance P. Ghrelin had little or no effect on the activity of c-Jun N-terminal kinase, p38 kinase or Akt. Ghrelin appeared to activate extracellular signal-regulated kinases 1/2 through a calcium-independent novel protein kinase C isoform which may utilize diacylglycerol derived from hydrolysis of phosphatidylcholine rather than from phosphatidylinositol. Ghrelin-stimulated extracellular signal-regulated kinases 1/2 activity was independent of transactivation of epidermal growth factor receptors, and even when ghrelin receptor internalization was blocked by concanavalin A or a beta-arrestin mutant, there was no decrease in phosphorylated extracellular signal-regulated kinases 1/2, suggesting this is a G protein-dependent process. The truncated ghrelin receptor polypeptide had no effect on ghrelin receptor signalling to extracellular signal-regulated kinases 1/2, but decreased the constitutive activation of phosphatidylinositol-specific phospholipase C by ghrelin receptors. In conclusion, our results suggest that any up-regulation of the truncated ghrelin receptor polypeptide might preferentially attenuate functional activity dependent on the constitutive activation of ghrelin receptors, while leaving ghrelin-dependent signalling unaffected.  相似文献   

11.
Nitric oxide is an endogenous thiol-reactive molecule that modulates the functions of many regulatory proteins by a thiol-redox mechanism. NO has now been shown to inhibit the activation of apoptosis signal-regulating kinase 1 (ASK1) in murine fibrosarcoma L929 cells through such a mechanism. Exposure of L929 cells to interferon-gamma resulted in the endogenous production of NO and in inhibition of the activation of ASK1 by hydrogen peroxide. The interferon-gamma-induced inhibition of ASK1 activity was blocked by N(G)-nitro-l-arginine, an inhibitor of NO synthase. Furthermore, the NO donor S-nitro-N-acetyl-dl-penicillamine (SNAP) inhibited ASK1 activity in vitro, and this inhibition was reversed by thiol-reducing agents such as dithiothreitol and beta-mercaptoethanol. SNAP did not inhibit the kinase activities of MKK3, MKK6, or p38 in vitro. The inhibition of ASK1 by interferon-gamma was not changed by 1H- (1,2,4)oxadiazolo[4,3-alpha]quinoxalin-1-one, an inhibitor of guanylyl cyclase nor was it mimicked by 8-bromo-cyclic GMP. Site-directed mutagenesis revealed that replacement of cysteine 869 of ASK1 by serine rendered this protein resistant to the inhibitory effects both of interferon-gamma in intact cells and of SNAP in vitro. Co-immunoprecipitation data showed that NO production inhibited a binding of ASK1, but not ASK1(C869S), to MKK3 or MKK6. Moreover, interferon-gamma induced the S-nitrosylation of endogenous ASK1 in L929 cells. Together, these results suggest that NO mediates the interferon-gamma-induced inhibition of ASK1 in L929 cells through a thiolredox mechanism.  相似文献   

12.
Stress-induced cardiomyocyte apoptosis plays an important role in the pathogenesis of a variety of cardiovascular diseases. Our early studies showed that HSP70 effectively inhibited apoptosis, but the underlying mechanism remained unclear. Fas-associated factor 1 (FAF1) is a member of the Fas death-inducing signaling complex (Fas-DISC) that acts upstream of caspase-8. We investigated the interactions among FAF1, HSP70, and FAS in stressed cardiomyocytes to elucidate the protective mechanism of HSP70. FAS and caspase-3/8 activity was higher in cardiomyocytes undergoing stress-induced apoptosis in restraint-stressed rats compared with cardiomyocytes in non-stressed rats, which indicated that the Fas signaling pathway was activated after restraint stress. Geranylgeranylacetone (GGA) induced an increase in HSP70 expression, which reduced stress-induced apoptosis. Additionally, overexpression of HSP70 via transfection with the pEGFP-rHSP70 plasmid attenuated norepinephrine (NE)-induced apoptosis. FAF1 expression increased during stress-induced apoptosis, and overexpression of FAF1 exacerbated NE-induced apoptosis. We also found that HSP70 interacted with FAF1. Overexpression of HSP70 inhibited the binding of FAF1 to FAS in H9C2 cells, which indicated that HSP70 suppressed NE-induced apoptosis by competitively binding to FAF1. An N-terminal deletion mutant of HSP70 (HSP70-△N) was unable to interact with FAF1. After HSP70-△N was transfected into H9C2 cells, the cells were unable to attenuate the NE-induced increases in caspase-8 and apoptosis. These results indicate that the 1–120 sequence of HSP70 binds to FAF1, which alters the interactions between FAS and FAF1 and inhibits the activation of the Fas signaling pathway and apoptosis.

Electronic supplementary material

The online version of this article (doi:10.1007/s12192-015-0589-9) contains supplementary material, which is available to authorized users.  相似文献   

13.
Apoptosis signal-regulating kinase (ASK) 1 was recently identified as a mitogen-activated protein (MAP) kinase kinase kinase which activates the c-Jun N-terminal kinase (JNK) and p38 MAP kinase pathways and is required for tumor necrosis factor (TNF)-alpha-induced apoptosis; however, the mechanism regulating ASK1 activity is unknown. Through genetic screening for ASK1-binding proteins, thioredoxin (Trx), a reduction/oxidation (redox)-regulatory protein thought to have anti-apoptotic effects, was identified as an interacting partner of ASK1. Trx associated with the N-terminal portion of ASK1 in vitro and in vivo. Expression of Trx inhibited ASK1 kinase activity and the subsequent ASK1-dependent apoptosis. Treatment of cells with N-acetyl-L-cysteine also inhibited serum withdrawal-, TNF-alpha- and hydrogen peroxide-induced activation of ASK1 as well as apoptosis. The interaction between Trx and ASK1 was found to be highly dependent on the redox status of Trx. Moreover, inhibition of Trx resulted in activation of endogenous ASK1 activity, suggesting that Trx is a physiological inhibitor of ASK1. The evidence that Trx is a negative regulator of ASK1 suggests possible mechanisms for redox regulation of the apoptosis signal transduction pathway as well as the effects of antioxidants against cytokine- and stress-induced apoptosis.  相似文献   

14.
Recent studies have suggested that ghrelin plays a direct role in controlling female reproduction. The aim of the present study was to investigate the mRNA and protein expression of ghrelin and its receptor (via real time PCR, Western blot and immunohistochemistry analysis, respectively) in porcine corpora lutea (CL) collected during early (CL1: 1-2 days after ovulation), middle (CL2: 7-10 after ovulation), and late luteal phase (CL3: 13-15 after ovulation). Ghrelin expression and concentration of both acylated and unacylated forms of ghrelin significantly increased during CL development. Immunohistochemistry analysis shown localization of ghrelin protein in the cytoplasm of large luteal cells. No changes in the expression of the ghrelin receptor were observed. Direct in vitro effects of ghrelin on progesterone (P4) secretion and 3-beta-hydroxysteroid dehydrogenase (3β-honestly significant difference (HSD)) activity, which were measured by the conversion of pregnenolone (P5) to P4, and 3β-HSD protein expression were then analyzed. To assess 3β-HSD activities, mature luteal cells were first cultured for 24 h with ghrelin at 100, 250, 500 and 1000 pg/mL with P5, or with aminoglutethimide (AMG). AMG is an inhibitor of CYP11A1-mediated hydroxylation; an addition of AMG and P5 enabled P4 production to serve as an index of 3β-HSD activity. Inhibitory effects of ghrelin on P4 secretion, 3β-HSD activity and protein expression were observed. In conclusion, the presence of ghrelin and its receptor in porcine corpora lutea and the direct inhibitory effects of ghrelin on luteal P4 secretion and 3β-HSD suggest potential auto/paracrine regulation by ghrelin in the luteal phase of ovary function.  相似文献   

15.
Acetylshikonin (ASK) is a natural naphthoquinone derivative of traditional Chinese medicine Lithospermum erythrorhyzon. It has been reported that ASK has bactericidal, anti‐inflammatory and antitumour effects. However, whether ASK induces apoptosis and autophagy in acute myeloid leukaemia (AML) cells and the underlying mechanism are still unclear. Here, we explored the roles of apoptosis and autophagy in ASK‐induced cell death and the potential molecular mechanisms in human AML HL‐60 cells. The results demonstrated that ASK remarkably inhibited the cell proliferation, viability and induced apoptosis in HL‐60 cells through the mitochondrial pathway, and ASK promoted cell cycle arrest in the S‐phase. In addition, the increased formation of autophagosomes, the turnover from light chain 3B (LC3B) I to LC3B II and decrease of P62 suggested the induction of autophagy by ASK. Furthermore, ASK significantly decreased PI3K, phospho‐Akt and p‐p70S6K expression, while enhanced phospho‐AMP‐activated protein kinase (AMPK) and phospho‐liver kinase B1(LKB1) expression. The suppression of ASK‐induced the conversion from LC3B I to LC3B II caused by the application of inhibitors of AMPK (compound C) demonstrated that ASK‐induced autophagy depends on the LKB1/AMPK pathway. These data suggested that the autophagy induced by ASK were dependent on the activation of LKB1/AMPK signalling and suppression of PI3K/Akt/mTOR pathways. The cleavage of the apoptosis‐related markers caspase‐3 and caspase‐9 and the activity of caspase‐3 induced by ASK were markedly reduced by inhibitor of AMPK (compound C), an autophagy inhibitor 3‐methyladenine (3‐MA) and another autophagy inhibitor chloroquine (CQ). Taken together, our data reveal that ASK‐induced HL‐60 cell apoptosis is dependent on the activation of autophagy via the LKB1/AMPK and PI3K/Akt‐regulated mTOR signalling pathways.  相似文献   

16.
Inducible heat shock protein70 (HSP70) is one of the most important HSPs for maintenance of cell integrity during normal cellular growth as well as pathophysiological conditions. Apoptosis signal-regulating kinase (ASK) 1, a mammalian MAPKKK, activates the JNK and p38 pathways. Here we report a novel function of HSP70 in regulating TNF-α-induced cell apoptosis. Our study demonstrated that HSP70 physically interacted with ASK1 and promoted the ubiquitin-dependent proteasomal degradation of ASK1. CHIP (carboxyl terminus of the HSC70-interacting protein) which acted as a co-chaperone of HSP70 cooperated with HSP70 in regulating ASK1. We also found that TNF-α stimulated HSP70/CHIP/ASK1 association and through cooperating with CHIP, HSP70 inhibits TNF-α-induced cell apoptosis both in over-expression and RNAi conditions. Structural analysis indicated that C-terminal domain of HSP70 was necessary for ASK1 degradation, and N- terminal domain of ASK1 was essential for its binding to HSP70. All these findings indicated that HSP70 and CHIP association is important for HSP70 in interacting with ASK1. Through forming the complex of HSP70/CHIP/ASK1, HSP70 promotes ASK1 proteasomal degradation and prevents TNF-α-induced cell apoptosis.  相似文献   

17.
Murine protein serine/threonine kinase 38 (MPK38) is a member of the AMP-activated protein kinase-related serine/threonine kinase family that plays an important role in various cellular processes, including cell cycle, signaling pathways, and self-renewal of stem cells. Here we demonstrate a functional association between MPK38 and apoptosis signal-regulating kinase 1 (ASK1). The physical association between MPK38 and ASK1 was mediated through their carboxyl-terminal regulatory domains and was increased by H(2)O(2) or tumor necrosis factor alpha treatment. The use of kinase-dead MPK38 and ASK1 mutants revealed that MPK38-ASK1 complex formation was dependent on the activities of both kinases. Ectopic expression of wild-type MPK38, but not kinase-dead MPK38, stimulated ASK1 activity by Thr(838) phosphorylation and enhanced ASK1-mediated signaling to both JNK and p38 kinases. However, the phosphorylation of MKK6 and p38 by MPK38 was not detectable. In addition, MPK38-mediated ASK1 activation was induced through the increased interaction between ASK1 and its substrate MKK3. MPK38 also stimulated H(2)O(2)-mediated apoptosis by enhancing the ASK1 activity through Thr(838) phosphorylation. These results suggest that MPK38 physically interacts with ASK1 in vivo and acts as a positive upstream regulator of ASK1.  相似文献   

18.
ASK1 activates JNK and p38 mitogen-activated protein kinases and constitutes a pivotal signaling pathway in cytokine- and stress-induced apoptosis. However, little is known about the mechanism of how ASK1 executes apoptosis. Here we investigated the roles of caspases and mitochondria in ASK1-induced apoptosis. We found that benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVAD-fmk), a broad-spectrum caspase inhibitor, mostly inhibited ASK1-induced cell death, suggesting that caspases are required for ASK1-induced apoptosis. Overexpression of ASK1DeltaN, a constitutively active mutant of ASK1, induced cytochrome c release from mitochondria and activation of caspase-9 and caspase-3 but not of caspase-8-like proteases. Consistently, caspase-8-deficient (Casp8 (-/-)) cells were sensitive to ASK1-induced caspase-3 activation and apoptosis, suggesting that caspase-8 is dispensable for ASK1-induced apoptosis, whereas ASK1 failed to activate caspase-3 in caspase-9-dificient (Casp9 (-/-)) cells. Moreover, mitochondrial cytochrome c release, which was not inhibited by zVAD-fmk, preceded the onset of caspase-3 activation and cell death induced by ASK1. ASK1 thus appears to execute apoptosis mainly by the mitochondria-dependent caspase activation.  相似文献   

19.
Heat shock protein 72 (Hsp72) is thought to protect cells against cellular stress. The protective role of Hsp72 was investigated by determining the effect of this protein on the stress-activated protein kinase signaling pathways. Prior exposure of NIH 3T3 cells to mild heat shock (43 degrees C for 20 min) resulted in inhibition of H(2)O(2)-induced activation of apoptosis signal-regulating kinase 1 (ASK1). Overexpression of Hsp72 also inhibited H(2)O(2)-induced activation of ASK1 as well as that of downstream kinases in the p38 mitogen-activated protein kinase (MAPK) signaling cascade. Recombinant Hsp72 bound directly to ASK1 and inhibited ASK1 activity in vitro. Furthermore, coimmunoprecipitation analysis revealed a physical interaction between endogenous Hsp72 and ASK1 in NIH 3T3 cells exposed to mild heat shock. Hsp72 blocked both the homo-oligomerization of ASK1 and ASK1-dependent apoptosis. Hsp72 antisense oligonucleotides prevented the inhibitory effects of mild heat shock on H(2)O(2)-induced ASK1 activation and apoptosis. These observations suggest that Hsp72 functions as an endogenous inhibitor of ASK1.  相似文献   

20.
The expression of heat shock proteins (HSPs) is known to be increased via activation of heat shock factor 1 (HSF1), and excess expression of HSPs exerts feedback inhibition of HSF1. However, the molecular mechanism to modulate such relationships between HSPs and HSF1 is not clear. In the present study, we show that stable transfection of either Hsp25 or inducible Hsp70 (Hsp70i) increased expression of endogenous HSPs such as HSP25 and HSP70i through HSF1 activation. However, these phenomena were abolished when the dominant negative Hsf1 mutant was transfected to HSP25 or HSP70i overexpressed cells. Moreover, the increased HSF1 activity by either HSP25 or HSP70i was found to result from dephosphorylation of HSF1 on serine 307 that increased the stability of HSF1. Either HSP25 or HSP70i inhibited ERK1/2 phosphorylation because of increased MKP1 phosphorylation by direct interaction of these HSPs with MKP1. Treatment of HOS and NCI-H358 cells, which showed high expressions of endogenous HSF1, with small interfering RNA (siRNA) of either HSP27 (siHSP27)or HSP70i (siHSP70i) inhibited both HSP27 and HSP70i proteins; this was because of increased ERK1/2 phosphorylation and serine phosphorylation of HSF1. The results, therefore, suggested that when the HSF1 protein level was high in cancer cells, excess expression of HSP27 or HSP70i strongly facilitates the expression of HSP proteins through HSF1 activation, resulting in severe radio- or chemoresistance.  相似文献   

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