Krishna Pada Sarker

Anandamide induces apoptosis of PC‐12 cells: involvement of superoxide and caspase‐3

Anandamide (arachidonoylethanolamide), an endogenous cannabinoid receptor ligand has been suggested to have physiological role in mammalian nervous system. However, little is known about the role of anandamide on neuronal cells. Here, we demonstrate that anandamide causes death of PC‐12 cells, showing marked DNA condensation and fragmentation, appearance of cells at sub‐G0/G1 and redistribution of phosphatidyl serine, the hallmark features of apoptosis. Anandamide raised intracellular superoxide level and CPP32‐like protease activity in PC‐12 cells markedly. Furthermore, antioxidant N‐acetyl cysteine prevented anandamide‐induced superoxide anion formation and cell death, implying that intracellular superoxide is a novel mediator of anandamide‐induced apoptosis of PC‐12 cells.

Polymyxin B binds to anandamide and inhibits its cytotoxic effect

Anandamide (ANA), an endogenous cannabinoid, can be generated by activated macrophages during endotoxin shock and is thought to be a paracrine contributor to hypotension. We discovered that ANA in saline/ethanol solution and in serum was efficiently adsorbed in a polymyxin B (PMB)‐immobilized beads column and eluted with ethanol. We confirmed the direct binding of PMB to ANA by using surface plasmon resonance. The adsorption of ANA by PMB may abolish the diverse effects of ANA such as hypotension, immunosuppression, and cytotoxicity, and may suggest a new therapeutic strategy for endotoxin shock.

Anandamide induces cell death independently of cannabinoid receptors or vanilloid receptor 1: possible involvement of lipid rafts.

Abstract: Anandamide triggers various cellular activities by binding to cannabinboid (CB1/CB2) receptors or vanilloid receptor 1 (VR1). However, the role of these receptors in anandamide-induced apoptosis remains largely unknown. Here, we show that SR141716A, a specific inhibitor of cannabinoid receptor (CB1-R), did not block anandamide-induced cell death in endogenously CB1-R expressing cells. In addition, CB1-R-lacking Chinese hamster ovary (CHO) cells underwent cell death after anandamide treatment. SR144528, a specific inhibitor of CB2-R also failed to block anandamide-induced cell death in HL-60 cells. Capsazepine, a specific antagonist of VR1 could not prevent anandamide-induced cell death in constitutively and endogenously VR1 expressing PC12 cells. Moreover, anandamide noticeably triggered cell death in VR1-lacking human embryonic kidney (HEK) cells. In contrast, methyl-β cyclodextrin (MCD), a membrane cholesterol depletor, completely blocked anandamide-induced cell death in a variety of cells, including PC12, C6, Neuro-2a, CHO, HEK, SMC, Jurkat and HL-60 cells. MCD also blocked anandamide-induced superoxide generation, phosphatidyl serine exposure and p38 MAPK/JNK activation. Thus, our data imply a novel role for of membrane lipid rafts in anandamide-induced cell death.

ASK1–p38 MAPK/JNK signaling cascade mediates anandamide-induced PC12 cell death

Anandamide is a neuroimmunoregulatory molecule that triggers apoptosis in a number of cell types including PC12 cells. Here, we investigated the molecular mechanisms underlying anandamide‐induced cell death in PC12 cells. Anandamide treatment resulted in the activation of p38 mitogen‐activated protein kinase (MAPK), c‐Jun N‐terminal kinase (JNK), and p44/42 MAPK in apoptosing cells. A selective p38 MAPK inhibitor, SB203580, or dn‐JNK, JNK1(A‐F) or SAPKβ(K‐R), blocked anandamide‐induced cell death, whereas a specific inhibitor of MEK‐1/2, U0126, had no effect, indicating that activation of p38 MAPK and JNK is critical in anandamide‐induced cell death. An important role for apoptosis signal‐regulating kinase 1 (ASK1) in this event was also demonstrated by the inhibition of p38 MAPK/JNK activation and death in cells overexpressing dn‐ASK1, ASK1 (K709M). Conversely, the constitutively active ASK1, ASK1ΔN, caused prolonged p38 MAPK/JNK activation and increased cell death. These indicate that ASK1 mediates anandamide‐induced cell death via p38 MAPK and JNK activation. Here, we also found that activation of p38 MAPK/JNK is accompanied by cytochrome c release from the mitochondria and caspase activation (which can be inhibited by SB203580), suggesting that anandamide triggers a mitochondrial dependent apoptotic pathway. The caspase inhibitor, zVAD, and the mitochondrial pore opening inhibitor, cyclosporine A, blocked anandamide‐induced cell death but not p38 MAPK/JNK activation, suggesting that activation of these kinases may occur upstream of mitochondrial associated events.

SnoN Is a Cell Type-specific Mediator of Transforming Growth Factor-β Responses

The transforming growth factor-β (TGF-β) family of secreted proteins have pleiotropic functions that are critical to normal development and homeostasis. However, the intracellular mechanisms by which the TGF-β proteins elicit cellular responses remain incompletely understood. The Smad proteins provide a major means for the propagation of the TGF-β signal from the cell surface to the nucleus, where the Smad proteins regulate gene expression leading to TGF-β-dependent cellular responses including the inhibition of cell proliferation. Recent studies have suggested that a nuclear Smad-interacting protein termed SnoN, when overexpressed in cells, suppresses TGF-β-induced Smad signaling and TGF-β inhibition of cell proliferation. However, the physiologic function of endogenous SnoN in TGF-β-mediated biological responses remained to be elucidated. Here, we determined the effect of genetic knock-down of SnoN by RNA interference on TGF-β responses in mammalian cells. Unexpectedly, we found that SnoN knock-down specifically inhibited TGF-β-induced transcription in the lung epithelial cell line Mv1Lu but not in HeLa or HaCaT cells. SnoN knock-down was also found to block TGF-β-dependent cell cycle arrest in Mv1Lu cells. Collectively, these data indicate that rather than suppressing TGF-β-induced responses, endogenous SnoN acts as a positive mediator of TGF-β-induced transcription and cell cycle arrest in lung epithelial cells. Our study also shows that SnoN couples the TGF-β signal to gene expression in a cell-specific manner.

Ebselen inhibits NO-induced apoptosis of differentiated PC12 cells via inhibition of ASK1-p38 MAPK-p53 and JNK signaling and activation of p44/42 MAPK and Bcl-2

Ebselen, a selenium‐containing heterocyclic compound, prevents ischemia‐induced cell death. However, the molecular mechanism through which ebselen exerts its cytoprotective effect remains to be elucidated. Using sodium nitroprusside (SNP) as a nitric oxide (NO) donor, we show here that ebselen potently inhibits NO‐induced apoptosis of differentiated PC12 cells. This was associated with inhibition of NO‐induced phosphatidyl Serine exposure, cytochrome c release, and caspase‐3 activation by ebselen. Analysis of key apoptotic regulators during NO‐induced apoptosis of differentiated PC12 cells showed that ebselen blocks the activation of the apoptosis signaling‐regulating kinase 1 (ASK1), and inhibits phosphorylation of p38 mitogen‐activated protein kinase (MAPK) and c‐jun N‐terminal protein kinase (JNK). Moreover, ebselen inhibits NO‐induced p53 phosphorylation at Ser15 and c‐Jun phosphorylation at Ser63 and Ser73. It appears that inhibition of p38 MAPK and p53 phosphorylation by ebselen occurs via a thiol‐redox‐dependent mechanism. Interestingly, ebselen also activates p44/42 MAPK, and inhibits the downregulation of the antiapoptotic protein Bcl‐2 in SNP‐treated PC12 cells. Together, these findings suggest that ebselen protects neuronal cells from NO cytotoxicity by reciprocally regulating the apoptotic and antiapoptotic signaling cascades.

Sumoylated SnoN Represses Transcription in a Promoter-specific Manner

The transcriptional modulator SnoN controls a diverse set of biological processes, including cell proliferation and differentiation. The mechanisms by which SnoN regulates these processes remain incompletely understood. Recent studies have shown that SnoN exerts positive or negative regulatory effects on transcription. Because post-translational modification of proteins by small ubiquitin-like modifier (SUMO) represents an important mechanism in the control of the activity of transcriptional regulators, we asked if this modification regulates SnoN function. Here, we show that SnoN is sumoylated. Our data demonstrate that the SUMO-conjugating E2 enzyme Ubc9 is critical for SnoN sumoylation and that the SUMO E3 ligase PIAS1 selectively interacts with and enhances the sumoylation of SnoN. We identify lysine residues 50 and 383 as the SUMO acceptor sites in SnoN. Analyses of SUMO “loss-of-function” and “gain-of-function” SnoN mutants in transcriptional reporter assays reveal that sumoylation of SnoN contributes to the ability of SnoN to repress gene expression in a promoter-specific manner. Although this modification has little effect on SnoN repression of the plasminogen activator inhibitor-1 promoter and only modestly potentiates SnoN repression of the p21 promoter, SnoN sumoylation robustly augments the ability of SnoN to suppress transcription of the myogenesis master regulatory gene myogenin. In addition, we show that the SnoN SUMO E3 ligase, PIAS1, at its endogenous levels, suppresses myogenin transcription. Collectively, our findings suggest that SnoN is directly regulated by sumoylation leading to the enhancement of the ability of SnoN to repress transcription in a promoter-specific manner. Our study also points to a physiological role for SnoN sumoylation in the control of myogenin expression in differentiating muscle cells.

Neuropeptide Release from Dental Pulp Cells by RgpB via Proteinase-Activated Receptor-2 Signaling

Dental pulp inflammation often results from dissemination of periodontitis caused mostly by Porphyromonas gingivalis infection. Calcitonin gene-related peptide and substance P are proinflammatory neuropeptides that increase in inflamed pulp tissue. To study an involvement of the periodontitis pathogen and neuropeptides in pulp inflammation, we investigated human dental pulp cell neuropeptide release by arginine-specific cysteine protease (RgpB), a cysteine proteinase of P. gingivalis, and participating signaling pathways. RgpB induced neuropeptide release from cultured human pulp cells (HPCs) in a proteolytic activity-dependent manner at a range of 12.5–200 nM. HPCs expressed both mRNA and the products of calcitonin gene-related peptide, substance P, and proteinase-activated receptor-2 (PAR-2) that were also found in dental pulp fibroblast-like cells. The PAR-2 agonists, SLIGKV and trypsin, also induced neuropeptide release from HPCs, and HPC PAR-2 gene knockout by transfection of PAR-2 antisense oligonucleotides inhibited significantly the RgpB-elicited neuropeptide release. These results indicated that RgpB-induced neuropeptide release was dependent on PAR-2 activation. The kinase inhibitor profile on the RgpB-neuropeptide release from HPC revealed a new PAR-2 signaling pathway that was mediated by p38 MAPK and activated transcription factor-2 activation, in addition to the PAR-2-p44/42 p38MAPK and -AP-1 pathway. This new RgpB activity suggests a possible link between periodontitis and pulp inflammation, which may be modulated by neuropeptides released in the lesion.

L6 myoblast differentiation is modulated by Cdk5 via the PI3K-AKT-p70S6K signaling pathway.

Cdk5 regulates myogenesis but the signaling cascade through which Cdk5 modulates this process remains to be characterized. Here, we investigated whether PI3K, Akt, p70S6K, p38 MAPK, p44/42 MAPK, and Egr-1 serve as upstream regulators of Cdk5 during L6 myoblast differentiation. Upon serum reduction, we found that besides elevated expression of Cdk5 and its activator, p35, and increased Cdk5/p35 activity, Egr-1, Akt, p70S6K, and p38 MAPK activity were upregulated in differentiating L6 cells. However, p44/42 MAPK was downregulated and SAPK/JNK was unaffected. LY294002, a PI3K inhibitor, blocked the activation of Akt and p70S6K, indicating that Akt and p70S6K activation is linked to PI3K activation. The lack of LY294002 effect on p38 MAPK suggests that p38 MAPK activation is not associated with PI3K activation. Rapamycin, a specific inhibitor of FRAP/mTOR (the upstream kinase of p70S6K), also blocked p70S6K activation, indicating the involvement of FRAP/mTOR activation. LY294002 and rapamycin also blocked the enhancement of Egr-1 level, Cdk5 activity, and myogenin expression, suggesting that upregulation of these factors is coupled to PI3K–p70S6K activation. Overexpression of dominant-negative-Akt also reduced Cdk5/p35 activity and myogenin expression, indicating that the PI3K–p70S6K–Egr-1–Cdk5 signaling cascade is linked to Akt activation. SB2023580, a p38 MAPK inhibitor, had no effect on p70S6K, Egr-1, or Cdk5 activity, suggesting that p38 MAPK activation lies in a pathway distinct from the PI3K–Akt–p70S6K–Egr-1 pathway that we identify as the upstream modulator of Cdk5 activity during L6 myoblast differentiation.

ING2 as a Novel Mediator of Transforming Growth Factor-β-dependent Responses in Epithelial Cells

Members of the ING (inhibitor of growth) family of chromatin modifying proteins (ING1-ING5) have emerged as critical regulators of gene expression and cellular responses, suggesting that the ING proteins may impinge on specific signal transduction pathways and their mediated effects. Here, we demonstrate a role for the protein ING2 in mediating responses by the transforming growth factor (TGF)-β-Smad signaling pathway. We show that ING2 promotes TGF-β-induced transcription. Both gain-of-function and RNA interference-mediated knockdown of endogenous ING2 reveal that ING2 couples TGF-β signals to the induction of transcription and cell cycle arrest. We also find that the Smad-interacting transcriptional modulator SnoN interacts with ING2 and promotes the assembly of a protein complex containing SnoN, ING2, and Smad2. Knockdown of endogenous SnoN blocks the ability of ING2 to promote TGF-β-dependent transcription, and conversely expression of SnoN augments ING2 enhancement of the TGF-β response. Collectively, our data suggest that ING2 collaborates with SnoN to mediate TGF-β-induced Smad-dependent transcription and cellular responses.