Band 4.1 proteins regulate integrin-dependent cell spreading

Integrins link the extracellular matrix (ECM) to the cytoskeleton to control cell behaviors including adhesion, spreading and migration. Band 4.1 proteins contain 4.1, ezrin, radixin, moesin (FERM) domains that likely mediate signaling events and cytoskeletal reorganization via integrins. However, the mechanisms by which Band 4.1 proteins and integrins are functionally interconnected remain enigmatic. Here we have investigated roles for Band 4.1 proteins in integrin-mediated cell spreading using primary astrocytes as a model system. We demonstrate that Proteins 4.1B and 4.1G show dynamic patterns of sub-cellular localization in astrocytes spreading on fibronectin. During early stages of cell spreading Proteins 4.1B and 4.1G are enriched in ECM adhesion sites but become more diffusely localized at later stages of spreading. Combinatorial inactivation of Protein 4.1B and 4.1G expression leads to impaired astrocyte spreading. Furthermore, in exogenous expression systems we show that the isolated Protein 4.1 FERM domain significantly enhances integrin-mediated cell spreading. Protein 4.1B is dispensable for reactive astrogliosis in experimental models of cortical injury, likely due to functional compensation by related Protein 4.1 family members. Collectively, these findings reveal that Band 4.1 proteins are important intracellular components for integrin-mediated cell spreading.     

The inhibition of cell spreading on a cellulose substrate (cuprophan) induces an apoptotic process via a mitochondria-dependent pathway

Cell shape was found to be a strong indicator of whether individual cells grow or die, and may play an important role in controlling apoptosis as well as cell growth. We compared here the behaviour of rounded Swiss 3T3 cells aggregated on a cellulose cuprophan membrane to those cultured on dish polystyrene. We demonstrated that cells aggregated on cellulose substrates for up to 48 h underwent programmed cell death that was associated with phosphatidylserine flipping and caspase 9 and caspase 3 activation, suggesting a mitochondria-dependent apoptotic process. In addition, we found that this phenomenon cannot be entirely explained by disengagement of alpha 5 beta 1 integrin ligation.     

Glucosamine induces autophagy via an mTOR-independent pathway

Autophagy is a cellular process that nonspecifically degrades cytosolic components and is involved in many cellular responses. We found that amino sugars with a free amino group such as glucosamine, galactosamine and mannosamine induced autophagy via an mTOR-independent pathway. Glucosamine-induced autophagy at concentrations of at least 500 μM to over 40 mM. In the presence of 40 mM glucosamine, autophagy induction was initiated at 6 h and reached a plateau at 36 h. Glucosamine-induced autophagy could remove accumulated ubiquitin-conjugated proteins as well as 79-glutamine repeats. Therefore, orally administered glucosamine could contribute to the prevention of neurodegenerative diseases and promotion of antiaging effects.     

Cigarette smoke extract induces endothelial cell injury via JNK pathway

Cigarette smoking is the most crucial factor responsible for chronic obstructive pulmonary disease (COPD). The precise mechanisms of the development of the disease have, however, not been fully understood. Recently, impairment of pulmonary endothelial cells has been increasingly recognized as a critical pathophysiological process in COPD. To verify this hypothesis, we examined how cigarette smoke extract (CSE) damages human umbilical vein endothelial cells (HUVECs). CSE activated c-Jun N-terminal kinase (JNK), and treatment of HUVECs with SP600125, a specific inhibitor of the JNK pathway, significantly suppressed endothelial cell damage by CSE. In contrast, inhibition of the extracellular-regulated kinase or the p38 pathway did not affect the cytotoxicity of CSE. Furthermore, anti-oxidants superoxide dismutase and catalase reduced CSE-induced JNK phosphorylation and endothelial cell injury. These results indicate that CSE damages vascular endothelial cells through the JNK pathway activated, at least partially, by oxidative stress.     

Aquatic birnavirus induces necrotic cell death via the mitochondria-mediated caspase pathway

Aquatic birnavirus induces necrotic cell death by an ill-understood process. Presently, we demonstrate that infectious pancreatic necrosis virus (IPNV) induces post-apoptotic necrotic cell death through loss of mitochondrial membrane potential (MMP) followed by caspase-3 activation in CHSE-214 cells. Progressive phosphatidylserine externalization was observed at 6 h post-infection (p.i.). This was followed by the development of bulb-like vesicles (bleb formation) at 8 h p.i. Progressive loss of MMP was also observed in IPNV-infected CHSE-214 cells beginning at 6 h p.i. At 8 h and 12 h p.i., IPNV-infected cells demonstrated a dramatic increase in MMP loss, rapid entry into necrotic cell death, and activation of caspase-9 and -3. Additionally, treatment with an inhibitor of MMP loss, bongkrekic acid, an adenine nucleotide translocase inhibitor, blocked IPNV-induced PS exposure and MMP loss, as well as reduced the activation of caspase-3. Taken together, our results suggest that IPNV induces apoptotic cell death via loss of MMP, thereby triggering secondary necrosis and caspases-3 activation. Furthermore, this death-signaling pathway is disrupted by bongkrekic acid in fish cells, indicating that this drug may serve to modulate IPNV-induced pathogenesis.     

Peroxynitrite induces integrin-dependent adhesion of human neutrophils to endothelial cells via activation of the Raf-1/MEK/Erk pathway.

Accumulating evidence suggests that enhanced peroxynitrite (ONOO-) formation occurs during inflammation. We have studied the impact and the mechanisms of ONOO- action on expression of adhesion molecules on human neutrophils and coronary artery endothelial cells (HCAEC) and binding of neutrophils to HCAEC. Addition of ONOO- (0.1 to 200 5M) to isolated neutrophils resulted in a concentration-dependent down-regulation of L-selectin expression, and up-regulation of CD11b/CD18 expression. ONOO- stimulation of Erk activity was accompanied by activation of Ras, Raf-1 and MEK (mitogen-activated protein kinase kinase), and was sensitive to the MEK inhibitor PD 98059. We have observed a tight association between Erk activation and changes in CD11b/CD18 expression. ONOO- also evoked activation of neutrophil p38 MAPK. Neither ONOO--induced up-regulation of CD11b/CD18 expression nor Erk activation was affected by SB 203580, a selective inhibitor of p38 MAPK. ONOO- by itself had little effect on expression of ICAM-1 and E-selectin on HCAEC, whereas it markedly enhanced attachment of neutrophils to lipopolysaccharide-activated HCAEC only when it was added together with neutrophils. Increases in neutrophil adhesion evoked by ONOO- were blocked by an anti-CD18 monoclonal antibody. These data suggest that ONOO- activates Erk in neutrophils via the Ras/Raf-1/MEK signal transduction pathway, leading to up-regulation of surface expression of CD11b/CD18 and consequently to increased neutrophil adhesion to endothelial cells.     

Phospholipase C-gamma1 potentiates integrin-dependent cell spreading and migration through Pyk2/paxillin activation

Phospholipase C-gamma1 (PLC-gamma1), which generates two second messengers, namely, inositol-1, 4, 5-trisphosphate and diacylglycerol, is implicated in growth factor-mediated chemotaxis. However, the exact role of PLC-gamma1 in integrin-mediated cell adhesion and migration remains poorly understood. In this study, we demonstrate that PLC-gamma1 is required for actin cytoskeletal organization and cell motility through the regulation of Pyk2 and paxillin activation. After fibronectin stimulation, PLC-gamma1 directly interacted with the cytoplasmic tail of integrin beta1. In PLC-gamma1-silenced cells, integrin-induced Pyk2 and paxillin phosphorylation were significantly reduced and PLC-gamma1 potentiated the integrin-induced Pyk2/paxillin activation in its enzymatic activity-dependent manner. In addition, specific knock-down of PLC-gamma1 resulted in a failure to form focal adhesions dependent on fibronectin stimulation, which appeared to be caused by the suppression of Pyk2 and paxillin phosphorylation. Interestingly, PLC-gamma1 potentiated the activations of Rac, thus integrin-induced lamellipodia formation was up-regulated. Consequently, the strength of cell-substratum interaction and cell motility were profoundly up-regulated by PLC-gamma1. Taken together, these results suggest that PLC-gamma1 is a key player in integrin-mediated cell spreading and motility achieved by the activation of Pyk2/paxillin/Rac signaling.     

AMPK induces vascular smooth muscle cell senescence via LKB1 dependent pathway

Vascular cells have a limited lifespan with limited cell proliferation and undergo cellular senescence. The functional changes associated with cellular senescence are thought to contribute to age-related vascular disorders. AMP-activated protein kinase (AMPK) has been discussed in terms of beneficial or harmful effects for aging-related diseases. However, the detailed functional mechanisms of AMPK are largely unclear. An aging model was established by stimulating vascular smooth muscle cell (VSMC) with adriamycin. Adriamycin progressively increased the mRNA and protein expressions of AMPK. The phosphorylation levels of LKB1 and acetyl-CoA carboxylase (ACC), the upstream and downstream of AMPK, were dramatically increased by adriamycin stimulation. The expressions of p53 and p21, which contribute to vascular senescence, were also increased. Inhibition of AMPK diminished senescence-associated β-galactosidase (SA-β-gal) staining, and restored VSMC proliferation. Cytosolic translocation of LKB1 by adriamycin could be a mechanism for AMPK activation in senescence. Furthermore, p53 siRNA and p21 siRNA transfection attenuated adriamycin-induced SA-β-gal staining. These results suggest that LKB1 dependent AMPK activation elicits VSMC senescence and p53–p21 pathway is a mediator of LKB1/AMPK-induced senescence.     

Crambene induces pancreatic acinar cell apoptosis via the activation of mitochondrial pathway

We investigated the apoptotic pathway activated by crambene (1-cyano-2-hydroxy-3-butene), a plant nitrile, on pancreatic acinar cells. As evidenced by annexin V-FITC staining, crambene treatment for 3 h induced the apoptosis but not necrosis of pancreatic acini. Caspase-3, -8, and -9 activities in acini treated with crambene were significantly higher than in untreated acini. Treatment with caspase-3, -8, and -9 inhibitors inhibited annexin V staining, as well as caspase-3 activity, pointing to an important role of these caspases in crambene-induced acinar cell apoptosis. The mitochondrial membrane potential was collapsed, and cytochrome c was released from the mitochondria in crambene-treated acini. Neither TNF-alpha nor Fas ligand levels were changed in pancreatic acinar cells after crambene treatment. These results provide evidence for the induction of pancreatic acinar cell apoptosis in vitro by crambene and suggest the involvement of mitochondrial pathway in pancreatic acinar cell apoptosis.     

GAS1 induces cell death through an intrinsic apoptotic pathway

Growth Arrest Specific 1 (GAS1) is a protein expressed when cells are arrested and during development. When ectopically expressed, GAS1 induces cell arrest and apoptosis of different cell lines, and we have previously demonstrated that the apoptotic process set off by GAS1 is caused by its capacity inhibiting the GDNF-mediated intracellular survival signaling. In the present work, we have dissected the molecular pathway leading to cell death. We employed the SH-SY5Y human neuroblastoma cell line that expresses GAS1 when deprived of serum. We observed, as we have previously described, that the presence of GAS1 reduces RET phosphorylation and inhibits the activation of AKT. We have now determined that the presence of GAS1 also triggers the dephosphorylation of BAD, which, in turn, provokes the release of Cytochrome-c from the mitochondria to the cytosol activating caspase-9, prompting the activity of caspase-3 and resulting in apoptosis of the cells. The apoptotic process is intrinsic, because there is no activation of caspase-8, thus this is consistent with apoptosis induced by the lack of trophic support. Interestingly, in cells where GAS1 has been silenced there is a significant delay in the onset of apoptosis.     

ET-1 induces cortical spreading depression via activation of the ETA receptor/phospholipase C pathway in vivo

Recently, it has been shown that brain topical superfusion of endothelin (ET)-1 at concentrations around 100 nM induces repetitive cortical spreading depressions (CSDs) in vivo. It has remained unclear whether this effect of ET-1 is related to a primary neuronal/astroglial effect, such as an increase in neuronal excitability or induction of interastroglial calcium waves, or a penumbra-like condition after vasoconstriction. In vitro, ET-1 regulates interastroglial communication via combined activation of ET(A) and ET(B) receptors, whereas it induces vasoconstriction via single activation of ET(A) receptors. We have determined the ET receptor profile and intracellular signaling pathway of ET-1-induced CSDs in vivo. In contrast to the ET(B) receptor antagonist BQ-788 and concentration dependently, the ET(A) receptor antagonist BQ-123 completely blocked the occurrence of ET-1-induced CSDs. The ET(B) receptor antagonist did not increase the efficacy of the ET(A) receptor antagonist. Direct stimulation of ET(B) receptors with the selective ET(B) agonist BQ-3020 did not trigger CSDs. The phospholipase C (PLC) antagonist U-73122 inhibited CSD occurrence in contrast to the protein kinase C inhibitor G?-6983. Our findings indicate that ET-1 induces CSDs through ET(A) receptor and PLC activation. We conclude that the induction of interastroglial calcium waves is unlikely the primary cause of ET-1-induced CSDs. On the basis of the receptor profile, likely primary targets of ET-1 mediating CSD are either neurons or vascular smooth muscle cells.     

Lycorine induces cell death in the amitochondriate parasite, Trichomonas vaginalis, via an alternative non-apoptotic death pathway

In this study, the mechanism of action of the pro-apoptotic alkaloid lycorine on an amitochondriate cell, the parasite Trichomonas vaginalis, was investigated. The cytotoxicity of lycorine against T. vaginalis was studied from 2.5 to 1000 μM and several important ultrastructural alterations were observed by electron microscopy. Lycorine arrested the T. vaginalis cell cycle, although no hallmarks of apoptosis, such as apoptotic bodies, were observed. Consequently, the underlying mechanism of action fails to completely fulfill the criteria for apoptosis. However, some similarities to paraptotic cell death were observed.     

Shear stress induces cell apoptosis via a c-Src-phospholipase D-mTOR signaling pathway in cultured podocytes

The glomerular capillary wall, composed of endothelial cells, the glomerular basement membrane and the podocytes, is continually subjected to hemodynamic force arising from tractional stress due to blood pressure and shear stress due to blood flow. Exposure of glomeruli to abnormal hemodynamic force such as hyperfiltration is associated with glomerular injury and progressive renal disease, and the conversion of mechanical stimuli to chemical signals in the regulation of the process is poorly understood in podocytes. By examining DNA fragmentation, apoptotic nuclear changes and cytochrome c release, we found that shear stress induced cell apoptosis in cultured podocytes. Meanwhile, podocytes exposed to shear stress also stimulated c-Src phosphorylation, phospholipase D (PLD) activation and mammalian target of rapamycin (mTOR) signaling. Using the antibodies against c-Src, PLD(1), and PLD(2) to perform reciprocal co-immunoprecipitations and in vitro PLD activity assay, our data indicated that c-Src interacted with and activated PLD(1) but not PLD(2). The inhibition of shear stress-induced c-Src phosphorylation by PP(2) (a specific inhibitor of c-Src kinase) resulted in reduced PLD activity. Phosphatidic acid, produced by shear stress-induced PLD activation, stimulated mTOR signaling, and caused podocyte hypertrophy and apoptosis.     

SPARC expression induces cell cycle arrest via STAT3 signaling pathway in medulloblastoma cells

Dynamic cell interaction with ECM components has profound influence in cancer progression. SPARC is a component of the ECM, impairs the proliferation of different cell types and modulates tumor cell aggressive features. We previously reported that SPARC expression significantly impairs medulloblastoma tumor growth in vivo. In this study, we demonstrate that expression of SPARC inhibits medulloblastoma cell proliferation. MTT assay indicated a dose-dependent reduction in tumor cell proliferation in adenoviral mediated expression of SPARC full length cDNA (Ad-DsRed-SP) in D425 and UW228 cells. Flow cytometric analysis showed that Ad-DsRed-SP-infected cells accumulate in the G2/M phase of cell cycle. Further, immunoblot and immunoprecipitation analyses revealed that SPARC induced G2/M cell cycle arrest was mediated through inhibition of the Cyclin-B-regulated signaling pathway involving p21 and Cdc2 expression. Additionally, expression of SPARC decreased STAT3 phosphorylation at Tyr-705; constitutively active STAT3 expression reversed SPARC induced G2/M arrest. Ad-DsRed-SP significantly inhibited the pre-established orthotopic tumor growth and tumor volume in nude-mice. Immunohistochemical analysis of tumor sections from mice treated with Ad-DsRed-SP showed decreased immunoreactivity for pSTAT3 and increased immunoreactivity for p21 compared to tumor section from mice treated with mock and Ad-DsRed. Taken together our studies further reveal that STAT3 plays a key role in SPARC induced G2/M arrest in medulloblastoma cells. These new findings provide a molecular basis for the mechanistic understanding of the effects of SPARC on medulloblastoma tumor cell proliferation.     

Armepavine oxalate induces cell death on CCRF-CEM leukemia cell line through an apoptotic pathway

Drug-induced cell death can occur as a result of DNA damage, which in turn may lead to the reduction of bcl-2 expression and activation of caspase-3 expression. In the present study, we investigated the effect of armepavines and atherosperminine on the cell survival rate and expression of bcl-2 and caspase-3 in CCRF-CEM cells. Our data have revealed that armepavine oxalate reduced the survival rate of CCRF-CEM cells in a dose- and time-dependent manner by MTT assay. However, no significant effects of armepavine MeI and atherosperminine N-oxide on the survival rate of the CCRF-CEM cell were observed. Armepavine oxalate-induced cell death was considered to be apoptotic on the basis of observed formation of the DNA ladder and the typical apoptotic morphological change by Hoechst 33258 staining. The expression of bcl-2 protein in CCRF-CEM cells treated with 30 microM armepavine oxalate was significantly decreased in western blotting analysis. In contrast, the expression of active caspase-3 in the cells was increased by armepavine oxalate in a dose-dependent manner. These findings indicate the involvement of bcl-2 and caspase-3 in the apoptotic process of CCRF-CEM cells induced by armepavine oxalate. The increased expression of active caspase-3 as well as decreased expression of bcl-2 support the assumption the armepavine oxalate-treated cells may be capable to complete the entire apoptotic process ending in cell fragmentation.     

Ephrin-A5 modulates cell adhesion and morphology in an integrin-dependent manner

The ephrins are membrane-tethered ligands for the Eph receptor tyrosine kinases, which play important roles in patterning of the nervous and vascular systems. It is now clear that ephrins are more than just ligands and can also act as signalling-competent receptors, participating in bidirectional signalling. We have recently shown that ephrin-A5 signals within caveola-like domains of the plasma membrane upon engagement with its cognate Eph receptor, leading to increased adhesion of the cells to fibronectin. Here we show that ephrin-A5 controls sequential biological events that are consistent with its role in neuronal guidance. Activation of ephrin-A5 induces an initial change in cell adhesion followed by changes in cell morphology. Both effects are dependent on the activation of beta1 integrin involving members of the Src family of protein tyrosine kinases. The prolonged activation of ERK-1 and ERK-2 is required for the change in cell morphology. Our work suggests a new role for class A ephrins in specifying the affinity of the cells towards various extracellular substrates by regulating integrin function.