Activation of STAT3 (signal transducer and activator of transcription 3) plays a crucial role in cell survival and proliferation. The aim of the present study was to clarify the role of STAT3 signalling in the protection of polyamine-depleted intestinal epithelial cells against TNF-alpha (tumour necrosis factor-alpha)-induced apoptosis. Polyamine depletion by DFMO (alpha-difluoromethylornithine) caused phosphorylation of STAT3 at Tyr-705 and Ser-727. Phospho-Tyr-705 STAT3 was immunolocalized at the cell periphery and nucleus, whereas phospho-Ser-727 STAT3 was predominantly detected in the nucleus of polyamine-depleted cells. Sustained phosphorylation of STAT3 at tyrosine residues was observed in polyamine-depleted cells after exposure to TNF-alpha. Inhibition of STAT3 activation by AG490 or cell-membrane-permeant inhibitory peptide (PpYLKTK; where pY represents phospho-Tyr) increased the sensitivity of polyamine-depleted cells to apoptosis. Expression of DN-STAT3 (dominant negative-STAT3) completely eliminated the protective effect of DFMO against TNF-alpha-induced apoptosis. Polyamine depletion increased mRNA and protein levels for Bcl-2, Mcl-1 (myeloid cell leukaemia-1) and c-IAP2 (inhibitor of apoptosis protein-2). Significantly higher levels of Bcl-2 and c-IAP2 proteins were observed in polyamine-depleted cells before and after 9 h of TNF-alpha treatment. Inhibition of STAT3 by AG490 and DN-STAT3 decreased Bcl-2 promoter activity. DN-STAT3 decreased mRNA and protein levels for Bcl-2, Mcl-1 and c-IAP2 in polyamine-depleted cells. siRNA (small interfering RNA)-mediated inhibition of Bcl-2, Mcl-1 and c-IAP2 protein levels increased TNF-alpha-induced apoptosis. DN-STAT3 induced the activation of caspase-3 and PARP [poly(ADP-ribose) polymerase] cleavage in polyamine-depleted cells. These results suggest that activation of STAT3 in response to polyamine depletion increases the transcription and subsequent expression of anti-apoptotic Bcl-2 and IAP family proteins and thereby promotes survival of cells against TNF-alpha-induced apoptosis.

The activity of Src kinases appears to play a role in both assembly and disassembly of tight junction. However, the role of a specific isoform of Src kinase in regulation of tight junction is not known. In the present study the role of c-Src in regulation of epithelial tight junction was investigated in Caco-2 cell monolayers. Oxidative stress (xanthine oxidase ؉ xanthine) induced an activation and membrane translocation of c-Src. The oxidative stress-induced decrease in transepithelial electrical resistance, increase in inulin permeability, and redistribution of occludin and ZO-1 from the intercellular junctions were prevented by PP2. The rates of oxidative stress-induced activation of c-Src, tyrosine phosphorylation of ZO-1 and ␤-catenin, decrease in resistance, increase in permeability to inulin, and redistribution of occludin and ZO-1 were significantly greater in cells transfected with wild type c-Src, whereas it was low in cells transfected with kinaseinactive c-SrcK297R mutant, when compared with those in empty vector-transfected cells. The rates of recovery of resistance, increase in barrier to inulin, and reorganization of occludin and ZO-1 into the intercellular junctions during the calcium-induced reassembly of tight junction were much greater in Caco-2 cells transfected with c-SrcK297R as compared with those in cells transfected with empty vector or wild type c-Src. These results show that the dominant-negative expression of kinase-inactive c-Src delays the oxidative stress-induced disruption of tight junction and accelerates calciuminduced assembly of tight junction in Caco-2 cells and demonstrate that oxidative stress-induced disruption of tight junction is mediated by the activation of c-Src. The tight junction (TJ)1 forms a barrier to the diffusion of toxins, allergens, and pathogens across the epithelial tissue.Three types of transmembrane proteins, occludin, claudins, and junction adhesion molecule, have been identified at the TJ (1-3). The intracellular domains of these transmembrane proteins interact with a number of plaque proteins, which in turn anchors TJ protein complex to the actin cytoskeleton (4, 5). Although the specific interactions among TJ proteins are yet to be delineated, there is evidence to support occludin interaction with zonula occludens (ZO)-1, ZO-2, and ZO-3 (5-7). These protein-protein interactions are crucial for the assembly of TJ and the maintenance of epithelial barrier functions (6). A significant body of evidence indicates that TJ is under dynamic regulation by intracellular signaling molecules. Although only little is known about the specific interactions of signaling molecules with the TJ proteins, a number of signaling molecules, including c-Src, c-Yes, protein kinase C, and G-proteins, appear to be localized at the vicinity of TJ (1). A recent study indicates that signaling molecules such as phosphatidylinositol 3-kinase, c-Yes, and protein kinase C may be associated with the Cterminal tail of occludin (8). Additionally, pharmacologic modulations of the activity...

The polyamines spermidine and spermine and their precursor putrescine are intimately involved in and are required for cell growth and proliferation. This study examines the mechanism by which polyamines modulate cell growth, cell cycle progression, and signal transduction cascades. IEC-6 cells were grown in the presence or absence ofdl-α-difluoromethylornithine (DFMO), a specific inhibitor of ornithine decarboxylase, which is the first rate-limiting enzyme for polyamine synthesis. Depletion of polyamines inhibited growth and arrested cells in the G1 phase of the cell cycle. Cell cycle arrest was accompanied by an increase in the level of p53 protein and other cell cycle inhibitors, including p21Waf1/Cip1 and p27Kip1. Induction of cell cycle inhibitors and p53 did not induce apoptosis in IEC-6 cells, unlike many other cell lines. Although polyamine depletion decreased the expression of extracellular signal-regulated kinase (ERK)-2 protein, a sustained increase in ERK-2 isoform activity was observed. The ERK-1 protein level did not change, but ERK-1 activity was increased in polyamine-depleted cells. In addition, polyamine depletion induced the stress-activated protein kinase/c-Jun NH2-terminal kinase (JNK) type of mitogen-activated protein kinase (MAPK). Activation of JNK-1 was the earliest event; within 5 h after DFMO treatment, JNK activity was increased by 150%. The above results indicate that polyamine depletion causes cell cycle arrest and upregulates cell cycle inhibitors and suggest that MAPK and JNK may be involved in the regulation of the activity of these molecules.

The modABC gene products constitute the molybdate-specific transport system in Escherichia coli. Another operon coding for two proteins which diverges from the modABCD operon has been identified. The first gene of this operon codes for a 262-amino-acid protein, designated ModE (28 kDa The presence of molybdenum is required for the activity of several enzymes found in animals, plants, and bacteria, such as sulfite oxidase, xanthine dehydrogenase, nitrate reductase, formate dehydrogenase, and nitrogenase (46). In these organisms, the molybdenum is present in the molybdoenzymes in the form of a pterin-containing molybdenum cofactor (MoCo), with the exception of that found in nitrogenase, which has an ironmolybdenum cofactor (FeMoCo) (1,16,34).In Escherichia coli, the successful production of molybdoenzymes relies upon the efficient uptake of molybdate via the molybdate-specific transporter encoded by the modABCD operon (formerly known as the chlD locus) (18,26,35,42). The E. coli molybdate transport machinery, in which modA encodes a molybdate-specific periplasmic binding protein, modB encodes an integral membrane channel-forming protein, and modC encodes an ATP-binding energizer, closely resembles the established ATP-binding cassette (ABC) transporter motif (15, 26). Homologous molybdate transport systems have also been described for Azotobacter vinelandii, Rhodobacter capsulatus, and Haemophilus influenzae (9,23,45).Initial studies of the characterization of mod mutants revealed that strains harboring these mutations were incapable of producing nitrate reductase or formate dehydrogenase activity without molybdate supplementation (11,14,27,36,39,41,43). There are data to suggest that when mod mutants are grown in molybdate-supplemented media, molybdate enters the cells by means of the sulfate transport system as well as other nonspecific anion transporters (22,36). Some of the mod mutants were also analyzed for their molybdate uptake kinetics and were shown to transport molybdate at a much lower rate than mod ϩ strains did (5, 14). Further investigations of mod mutants have demonstrated that the modABCD operon is regulated by the intracellular concentration of molybdate. Specifically, high levels of molybdate reduced the level of transcription of the transport genes (27,35,36). This reduction in the level of transcription of the modABCD operon is most likely mediated by a molybdate-activated repressor protein.An analysis of the modABCD DNA sequence revealed a region in between the transcription and translation start sites of the modA gene which contains an 8-base inverted repeat (TAAC ⅐ GTTA) flanked by two CAT (CA) boxes (26,35,36). There are indications that either the CAT boxes or the inverted repeat or both are involved in the binding of the mod operon repressor (35). The inverted repeat found in this region is analogous to the inverted-repeat sequences implicated as the target binding sites for both the Met and Trp repressor (MetJ and TrpR) proteins (21,32,33).In our laboratory, a derivative of strain SE2069 ...

The polyamines spermidine, spermine, and their precursor putrescine are essential for cell growth and the regulation of the cell cycle. Recent studies suggest that excessive accumulation of polyamines favors either malignant transformation or apoptosis, depending on the cell type and the stimulus. This study examines the involvement of polyamines in the induction of apoptosis by the DNA topoisomerase I inhibitor, camptothecin. In IEC-6 cells, camptothecin induced apoptosis within 6 h, accompanied by detachment of cells. Detached cells showed DNA laddering and caspase 3 induction, characteristic features of apoptosis. Depletion of putrescine, spermidine, and spermine by DL-alpha-difluoromethylornithine (DFMO), a specific inhibitor of ornithine decarboxylase (ODC) that is the first rate-limiting enzyme for polyamine biosynthesis, decreased the apoptotic index. Delayed apoptosis was accompanied by a decrease in caspase 3 activity in polyamine-depleted cells. Addition of putrescine restored the induction of apoptosis as indicated by an increase in the number of detached cells and caspase 3 activity. Polyamine depletion did not change the level of caspase 3 protein. Inhibition of S-adenosylmethionine decarboxylase by a specific inhibitor [diethylglyoxal bis-(guanylhydrazone); DEGBG] led to depletion of spermidine and spermine with a significant accumulation of putrescine and induction of ODC. The DEGBG-treated cells showed an increase in apoptosis, suggesting the importance of putrescine in the apoptotic process. Addition of putrescine to DFMO-treated cell extracts did not increase caspase 3 activity. The above results indicate that polyamine depletion delays the onset of apoptosis in IEC-6 cells and confers protection against DNA damaging agents, suggesting that polyamines might be involved in the caspase activating signal cascade.