Regulation of globular adiponectin-induced apoptosis by reactive oxygen/nitrogen species in RAW264 macrophages

Adiponectin, produced predominantly by differentiating adipocytes, is a protein hormone with antidiabetic and immunosuppressive properties. Here, we report evidence that treatment with globular adiponectin (gAd) induces apoptosis in murine macrophage-like RAW264 cells through the generation of reactive oxygen and/or nitrogen species (ROS/RNS). Treatment with gAd induced apoptosis and enhanced the activities of caspase-3 and -9, but not caspase-8. The gAd stimulation increased ROS generation and significantly reduced the ratio of NADPH to total NADP. Pretreatment with diphenyleneiodonium or apocynin reduced ROS and apoptosis in gAd-treated cells. In addition, transfection with p47(phox)- or gp91(phox)-specific small interfering RNA (siRNA) partially reduced ROS and apoptosis in response to gAd treatment. These results suggest that the administration of gAd induces apoptosis after ROS generation involving activation of NADPH oxidases. The gAd stimulation increased the release of NO into the culture medium, the activity of nitric oxide synthase (NOS), and the expression of inducible NOS (iNOS) mRNA in RAW264 cells. l-NAME reduced gAd-induced apoptotic cell death. In addition, transfection with an iNOS-specific siRNA markedly reduced the generation of NO and the population of apoptotic cells. Taken together, these results demonstrate that the gAd-induced apoptotic process in RAW264 cells involves ROS and RNS, which are generated by NADPH oxidases and iNOS, respectively.     

Globular adiponectin-induced RAW 264 apoptosis is regulated by a reactive oxygen species-dependent pathway involving Bcl-2

Globular adiponectin (gAd), a truncated form of adipocyte-derived cytokine, stimulates RAW 264 cells to produce reactive oxygen species (ROS), which trigger an apoptotic cascade. In this study, we investigated the generation of intracellular and mitochondrial ROS in gAd-stimulated RAW 264 cells. Treatment with gAd efficiently induced the generation of intracellular and mitochondrial ROS, as detected by dichlorodihydrofluorescein diacetate and MitoSOX fluorescence, respectively. Furthermore, gAd treatment significantly increased 8-oxoguanine, a specific indicator of oxidative DNA damage. The transfection of RAW 264 cells with iNOS- and gp91^phox-specific small interfering RNA reduced markedly the generation of intracellular, but not mitochondrial, ROS. Quantitative PCR revealed that the expression ratio of Bcl-2 to Bax was reduced in a time-dependent manner in gAd-treated RAW 264 cells. The overexpression of Bcl-2 markedly inhibited gAd-induced apoptosis in RAW 264 cells and also reduced both the intracellular and the mitochondrial ROS generation induced by gAd treatment. Moreover, the overexpression of Bcl-2 significantly suppressed gAd-induced NO secretion and NOS activity. In addition, the inhibition of NOS activity partially reduced the oxidative DNA damage induced by gAd. Taken together, these results demonstrate that the gAd-induced apoptotic pathway acting via ROS/RNS generation involves Bcl-2.     

Diacylglycerol kinase alpha regulates globular adiponectin-induced reactive oxygen species

It has previously been reported that the globular form of adiponectin (gAd), mature adipocyte-derived cytokine, induced generation of reactive oxygen species (ROS) and nitric oxide (NO) in the murine macrophage cell line RAW 264. This study investigated whether diacylglycerol kinases (DGKs), enzymes functioning in sub-cellular signalling pathways, had a role on gAd-induced ROS generation in RAW 264 cells. Administration of R59022, a specific inhibitor for DGK, reduced gAd-induced ROS generation and NO release. RAW 264 cell expressed DGKα mRNA. Depression of DGKα mRNA by RNA interference significantly reduced the ROS generation in response to gAd treatment. Interestingly, transfection with the DGKα-specific small interfering RNA attenuated the expression level of Nox1 mRNA in gAd-treated RAW 264 cells. In addition, the DGKα knockdown with siRNA suppressed gAd-induced NO release.     

Diacylglycerol kinase alpha regulates globular adiponectin-induced reactive oxygen species

Abstract:

It has previously been reported that the globular form of adiponectin (gAd), mature adipocyte-derived cytokine, induced generation of reactive oxygen species (ROS) and nitric oxide (NO) in the murine macrophage cell line RAW 264. This study investigated whether diacylglycerol kinases (DGKs), enzymes functioning in sub-cellular signalling pathways, had a role on gAd-induced ROS generation in RAW 264 cells. Administration of R59022, a specific inhibitor for DGK, reduced gAd-induced ROS generation and NO release. RAW 264 cell expressed DGKα mRNA. Depression of DGKα mRNA by RNA interference significantly reduced the ROS generation in response to gAd treatment. Interestingly, transfection with the DGKα-specific small interfering RNA attenuated the expression level of Nox1 mRNA in gAd-treated RAW 264 cells. In addition, the DGKα knockdown with siRNA suppressed gAd-induced NO release.     

Inhibition of JAK2/STAT3 signalling induces colorectal cancer cell apoptosis via mitochondrial pathway

Abnormalities in the JAK2/STAT3 pathway are involved in the pathogenesis of colorectal cancer (CRC), including apoptosis. However, the exact mechanism by which dysregulated JAK2/STAT3 signalling contributes to the apoptosis has not been clarified. To investigate the role of both JAK2 and STAT3 in the mechanism underlying CRC apoptosis, we inhibited JAK2 with AG490 and depleted STAT3 with a small interfering RNA. Our data showed that inhibition of JAK2/STAT3 signalling induced CRC cellular apoptosis via modulating the Bcl-2 gene family, promoting the loss of mitochondrial transmembrane potential (Δψm) and the increase of reactive oxygen species. In addition, our results demonstrated that the translocation of cytochrome c (Cyt c), caspase activation and cleavage of poly (ADP-ribose) polymerase (PARP) were present in apoptotic CRC cells after down-regulation of JAK2/STAT3 signalling. Moreover, inhibition of JAK2/STAT3 signalling suppressed CRC xenograft tumour growth. We found that JAK2/STAT3 target genes were decreased; meanwhile caspase cascade was activated in xenograft tumours. Our findings illustrated the biological significance of JAK2/STAT3 signalling in CRC apoptosis, and provided novel evidence that inhibition of JAK2/STAT3 induced apoptosis via the mitochondrial apoptotic pathway. Therefore, JAK2/STAT3 signalling may be a potential target for therapy of CRC.     

Blockade of JAK2 activity suppressed accumulation of β‐catenin in leukemic cells

The Wnt/β‐catenin pathway has been implicated in leukemogenesis. We found β‐catenin abnormally accumulated in both human acute T cell leukemia Jurkat cells and human erythroleukemia HEL cells. β‐Catenin can be significantly down‐regulated by the Janus kinase 2 specific inhibitor AG490 in these two cells. AG490 also reduces the luciferase activity of a reporter plasmid driven by LEF/β‐catenin promoter. Similar results were observed in HEL cells infected with lentivirus containing shRNA against JAK2 gene. After treatment with 50 µM AG490 or shRNA, the mRNA expression levels of β‐catenin, APC, Axin, β‐Trcp, GSK3α, and GSK3β were up‐regulated within 12–16 h. However, only the protein levels of GSK3β and β‐Trcp were found to have increased relative to untreated cells. Knockdown experiments revealed that the AG490‐induced inhibition of β‐catenin can be attenuated by shRNA targeting β‐TrCP. Taken together; these results suggest that β‐Trcp plays a key role in the cross‐talk between JAK/STAT and Wnt/β‐catenin signaling in leukemia cells. J. Cell. Biochem. 111: 402–411, 2010. © 2010 Wiley‐Liss, Inc.     

Involvement of Ca in globular adiponectin-induced reactive oxygen species

Globular adiponectin (gAd) induces the generation of reactive oxygen species (ROS) and nitric oxide (NO) in the murine macrophage cell line RAW 264. We investigated the role of Ca²⁺ in gAd-induced ROS and NO generation. Pretreatment with BAPTA-AM, a selective chelator of intracellular Ca²⁺ ([Ca²⁺]_i), partially reduced gAd-induced generation of ROS and NO in gAd-treated RAW 264 cells. The lowest [Ca²⁺]_i occurred 30 min after gAd treatment, after which [Ca²⁺]_i increased continually and exceeded the initial level. The mitochondrial Ca²⁺ ([Ca²⁺]_m) detected by Rhod-2 fluorescence started to increase at 6 h after gAd treatment. Pretreatment with a NAD(P)H oxidase inhibitor, diphenyleneiodonium, prevented the reduction of [Ca²⁺]_i in the early phase after gAd treatment. Calcium depletion by BAPTA-AM had no effect on the gAd-induced [Ca²⁺]_m oscillation. The administration of a specific calmodulin inhibitor, calmidazolium, significantly suppressed gAd-induced ROS and NO generation and NOS activity.     

Constitutive JAK2/STAT1 activation regulates endogenous BACE1 expression in neurons

The protease BACE1 (β-site APP-cleaving enzyme 1) is essential for the generation of amyloid beta (Aβ) from amyloid precursor protein (APP). Although BACE1 is expressed primarily in neurons, which are a principal source of Aβ in the brain, the mechanism that underlies basal expression of BACE1 in neurons has not been studied thoroughly. In the present study, we found that endogenous BACE1 expression was mediated by constitutive JAK2/STAT1 activation in neurons. Inhibition of the JAK2/STAT1 signaling pathway, using AG490 (a JAK2 inhibitor), a dominant-negative form of STAT1, and SOCS1 and SOCS3 overexpression, reduced levels of BACE1 promoter activity, expression of endogenous BACE1, and generation of Aβ. These results were recapitulated in the SH-SY5Y neuronal cell line, primary cultured neurons, and mouse brains. Therefore, we propose that constitutive JAK2/STAT1 activation mediates endogenous BACE1 expression in neurons and that inhibition of JAK2/STAT1 signaling abrogates basal levels of BACE1 expression and Aβ generation.     

BBR induces apoptosis in HepG2 cell through an Akt‐ASK1‐ROS‐p38MAPKs‐linked cascade

Berberine (BBR) has indicated significant antimicrobial activity against a variety of organisms including bacteria, viruses, and fungi. The mechanism by which BBR initiates apoptosis remains poorly understood. In the present study, we demonstrated that BBR exhibited significant cytotoxicity in human hepatoma HepG2 cells. Herein, we investigated cytotoxicity mechanism of BBR in HepG2 cells. The results showed that the induction of apoptosis in HepG2 cells by BBR was characterized by DNA fragmentation, an increased percentage of annexin V, and the activation of caspase‐3. The expressions of Bcl‐2 protein and pro‐caspase‐3 were reduced by BBR in HepG2 cells. However, Bax protein was increased in the cells. BBR‐induced apoptosis was preceded by increased generation of reactive oxygen species (ROS). NAC treatment, a scavenger of ROS, reversed BBR‐induced apoptosis effects via inhibition of Bax activation and Bcl‐2 inactivation. BBR‐induced, dose‐dependent induction of apoptosis was accompanied by sustained phosphorylation of MAP Kinases (JNK and p38 MAPK), ASK1, Akt, and p53. Furthermore, SB203580, p38 inhibitor, reduced the apoptotic effect of BBR, and blocks the generation of ROS and NO as well as activation of Bax. We found that the treatment of HepG2 cells with BBR triggers generation of ROS through Akt phosphorylation, resulting in dissociation of the ASK1‐mediated activation of JNK and p38 pathways. J. Cell. Biochem. 109: 329–338, 2010. © 2009 Wiley‐Liss, Inc.     

Differential regulation by fucoidan of IFN‐γ‐induced NO production in glial cells and macrophages

Fucoidan has shown numerous biological actions; however, the molecular bases of these actions have being issued. We examined the effect of fucoidan on NO production induced by IFN‐γ and the molecular mechanisms underlying these effects in two types of cells including glia (C6, BV‐2) and macrophages (RAW264.7, peritoneal primary cells). Fucoidan affected IFN‐γ‐induced NO and/or iNOS expression both in macrophages and glial cells but in a contrast way. Our data showed that in C6 glioma cells both JAK/STAT and p38 signaling positively regulated IFN‐γ‐induced iNOS, which were inhibited by fucoidan. In contrast, in RAW264.7 cells JAK/STAT is a positive regulator whereas p38 is a negative regulator of NO/iNOS production. In RAW264.7 cells, fucoidan enhanced p38 activation and induced TNF‐α production. We also confirmed the dual regulation of p38 in BV‐2 microglia and primary peritoneal macrophages. From these results, we suggest that fucoidan affects not only IFN‐γ‐induced NO/iNOS production differently in brain and peritoneal macrophages due to the different roles of p38 but the effects on TNF‐α production in the two cell types. These novel observations including selective and cell‐type specific effects of fucoidan on IFN‐γ‐mediated signaling and iNOS expression raise the possibility that it alters the sensitivity of cells to the p38 activation. J. Cell. Biochem. 111: 1337–1345, 2010. © 2010 Wiley‐Liss, Inc.