Disruption of α-actinin-integrin interactions at focal adhesions renders osteoblasts susceptible to apoptosis

2006 ◽  
Vol 291 (5) ◽  
pp. C909-C921 ◽  
Author(s):  
Jason W. Triplett ◽  
Fredrick M. Pavalko
Keyword(s):  
Tyrosine Phosphorylation ◽  
Structural Integrity ◽  
Focal Adhesions ◽  
Bone Cell ◽  
Gfp Expression ◽  
Akt Phosphorylation ◽  
Protein Levels ◽  
Tnf Α ◽  
Survival Signaling ◽  
Induced Apoptosis

Maintenance of bone structural integrity depends in part on the rate of apoptosis of bone-forming osteoblasts. Because substrate adhesion is an important regulator of apoptosis, we have investigated the role of focal adhesions in regulating bone cell apoptosis. To test this, we expressed a truncated form of α-actinin (ROD-GFP) that competitively displaces endogenous α-actinin from focal adhesions, thus disrupting focal adhesions. Immunofluorescence and morphometric analysis of vinculin and tyrosine phosphorylation revealed that ROD-GFP expression dramatically disrupted focal adhesion organization and reduced tyrosine phosphorylation at focal adhesions. In addition, Bcl-2 protein levels were reduced in ROD-GFP-expressing cells, but caspase 3 cleavage, poly(ADP-ribose) polymerase cleavage, histone H2A.X phosphorylation, and cytotoxicity were not increased due to ROD-GFP expression alone. Increases in both ERK and Akt phosphorylation were also observed in ROD-GFP-expressing cells, although inhibition of either ERK or Akt individually or together failed to induce apoptosis. However, we did find that ROD-GFP expression sensitized, whereas α-actinin-GFP expression protected, cells from TNF-α-induced apoptosis. Further investigation revealed that activation of TNF-α-induced survival signals, specifically Akt phosphorylation and NF-κB activation, was inhibited in ROD-GFP-expressing cells. The reduced expression of antiapoptotic Bcl-2 and inhibited survival signaling rendered ROD-GFP-expressing cells more susceptible to TNF-α-induced apoptosis. Thus we conclude that α-actinin plays a role in regulating cell survival through stabilization of focal adhesions and regulation of TNF-α-induced survival signaling.

scholarly journals Tumor Necrosis Factor-α Attenuates Thyroid Hormone-Induced Apoptosis in Vascular Endothelial Cell Line XLgoo Established from Xenopus Tadpole Tails

Endocrinology ◽  
2008 ◽  
Vol 149 (7) ◽  
pp. 3379-3389 ◽  
Author(s):  
Shuuji Mawaribuchi ◽  
Kei Tamura ◽  
Saori Okano ◽  
Shutaro Takayama ◽  
Yoshio Yaoita ◽  
...  
Keyword(s):  
Cell Death ◽  
Thyroid Hormone ◽  
Cell Fate ◽  
Thyroid Hormone Receptor ◽  
Vascular Endothelial Cell ◽  
Vascular Endothelial ◽  
Tnf Α ◽  
Survival Signaling ◽  
Induced Apoptosis ◽  
Thyroid Hormone Receptor Β

Amphibian metamorphosis induced by T3 involves programmed cell death and the differentiation of various types of cells in degenerated and reconstructed tissues. However, the signaling pathway that directs the T3-dependent cell-fate determinations remains unclear. TNF-α is a pleiotropic cytokine that affects diverse cellular responses. Engagement of TNF-α with its receptor (TNFR1) causes intracellular apoptotic and/or survival signaling. To investigate TNF signaling functions during anuran metamorphosis, we first identified Xenopus laevis orthologs of TNF (xTNF)-α and its receptor. We found that xTNF-α activated nuclear factor-κB in X. laevis A6 cells through the Fas-associated death domain and receptor-interacting protein 1. Interestingly, xTNF-α mRNA in blood cells showed prominent expression at prometamorphosis during metamorphosis. Next, to elucidate the apoptotic and/or survival signaling induced by xTNF-α in an in vitro model of metamorphosis, we established a vascular endothelial cell line, XLgoo, from X. laevis tadpole tail. XLgoo cells formed actin stress fibers and elongated in response to xTNF-α. T3 induced apoptosis in these cells, but the addition of xTNF-α blocked the T3-induced apoptosis. In addition, treatment of the cells with T3 for 2 d induced the expression of thyroid hormone receptor-β and caspase-3, and this thyroid hormone receptor-β induction was drastically repressed by xTNF-α. Furthermore, in organ culture of the tail, xTNF-α significantly attenuated the tail degeneration induced by T3. These findings suggested that xTNF-α could protect vascular endothelial cells from apoptotic cell death induced by T3 during metamorphosis and thereby participate in the regulation of cell fate.

Activation of cAMP-guanine exchange factor confers PKA-independent protection from hepatocyte apoptosis

2004 ◽  
Vol 287 (2) ◽  
pp. G334-G343 ◽  
Author(s):  
Kimberly A. Cullen ◽  
John McCool ◽  
M. Sawkat Anwer ◽  
Cynthia R. L. Webster
Keyword(s):  
Bile Acid ◽  
Protective Effect ◽  
Signaling Pathway ◽  
Fas Ligand ◽  
Kinase Inhibitor ◽  
Pi3 Kinase ◽  
Akt Phosphorylation ◽  
Tnf Α ◽  
Induced Apoptosis

cAMP has previously been shown to promote cell survival in a variety of cell types, but the downstream signaling pathway(s) of this antiapoptotic effect is unclear. Thus the role of cAMP signaling through PKA and cAMP-regulated guanine nucleotide exchange factors (cAMP-GEFs) in cAMP's antiapoptotic action was investigated in the present study. cAMP's protective effect against bile acid-, Fas ligand-, and TNF-α-induced apoptosis in rat hepatocytes was largely unaffected by the selective PKA inhibitor, Rp-8-(4-chlorophenylthio)-cAMP (Rp-cAMP). In contrast, a novel cAMP analog, 8-(4-chlorophenylthio)-2′- O-methyl (CPT-2-Me)-cAMP, which activated cAMP-GEFs in hepatocytes without activating PKA, protected hepatocytes against apoptosis induced by bile acids, Fas ligand, and TNF-α. The role of cAMP-GEF and PKA on activation of Akt, a kinase implicated in cAMP survival signaling, was investigated. Inhibition of PKA with RP-cAMP had no effect on cAMP-mediated Akt phosphorylation, whereas CPT-2-Me-cAMP, which did not activate PKA, induced phosphatidylinositol 3-kinase (PI3-kinase)-dependent activation of Akt. Pretreatment of hepatocytes with the PI3-kinase inhibitor, Ly-294002, prevented CPT-2-Me-cAMP's protective effect against bile acid and Fas ligand, but not TNF-α-mediated apoptosis. Glucagon, CPT-cAMP, and CPT-2-Me-cAMP all activated Rap 1, a downstream effector of cAMP-GEF. These results suggest that a PKA-independent cAMP/cAMP-GEF/Rap pathway exists in hepatocytes and that activation of cAMP-GEFs promotes Akt phosphorylation and hepatocyte survival. Thus a cAMP/cAMP-GEF/Rap/PI3-kinase/Akt signaling pathway may confer protection against bile acid- and Fas-induced apoptosis in hepatocytes.

scholarly journals STAT3-mediated transcription of Bcl-2, Mcl-1 and c-IAP2 prevents apoptosis in polyamine-depleted cells

2005 ◽  
Vol 392 (2) ◽  
pp. 335-344 ◽  
Author(s):  
Sujoy Bhattacharya ◽  
Ramesh M. Ray ◽  
Leonard R. Johnson
Keyword(s):  
Myeloid Cell ◽  
Dominant Negative ◽  
Cell Leukaemia ◽  
Cell Periphery ◽  
Inhibitory Peptide ◽  
Protein Levels ◽  
Tnf Α ◽  
Apoptosis Protein ◽  
Before And After ◽  
Induced Apoptosis

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-α (tumour necrosis factor-α)-induced apoptosis. Polyamine depletion by DFMO (α-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-α. 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-α-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-α 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-α-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-α-induced apoptosis.

scholarly journals HSF1 is involved in suppressing A1 phenotype conversion of astrocytes following spinal cord injury in rats

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Lilan Li ◽  
Yu Li ◽  
Bingqiang He ◽  
Hui Li ◽  
Huiyuan Ji ◽  
...  
Keyword(s):  
Spinal Cord ◽  
Spinal Cord Injury ◽  
Western Blot ◽  
Neuroprotective Activity ◽  
Signal Pathways ◽  
Protein Levels ◽  
Cultured Astrocytes ◽  
Tnf Α ◽  
Cord Injury ◽  
Induced Apoptosis

Abstract Background Two activation states of reactive astrocytes termed A1 and A2 subtypes emerge at the lesion sites following spinal cord injury (SCI). A1 astrocytes are known to be neurotoxic that participate in neuropathogenesis, whereas A2 astrocytes have been assigned the neuroprotective activity. Heat shock transcription factor 1 (HSF1) plays roles in protecting cells from stress-induced apoptosis and in controlling inflammatory activation. It is unknown whether HSF1 is involved in suppressing the conversion of A1 astrocytes following SCI. Methods A contusion model of the rat spinal cord was established, and the correlations between HSF1 expression and onset of A1 and A2 astrocytes were assayed by Western blot and immunohistochemistry. 17-AAG, the agonist of HSF1, was employed to treat the primary cultured astrocytes following a challenge by an A1-astrocyte-conditioned medium (ACM) containing 3 ng/ml of IL-1α, 30 ng/ml of TNF-α, and 400 ng/ml of C1q for induction of the A1 subtype. The effects of 17-AAG on the phenotype conversion of astrocytes, as well as underlying signal pathways, were examined by Western blot or immunohistochemistry. Results The protein levels of HSF1 were significantly increased at 4 days and 7 days following rat SCI, showing colocalization with astrocytes. Meanwhile, C3-positive A1 astrocytes were observed to accumulate at lesion sites with a peak at 1 day and 4 days. Distinctively, the S100A10-positive A2 subtype reached its peak at 4 days and 7 days. Incubation of the primary astrocytes with ACM markedly induced the conversion of the A1 phenotype, whereas an addition of 17-AAG significantly suppressed such inducible effects without conversion of the A2 subtype. Activation of HSF1 remarkably inhibited the activities of MAPKs and NFκB, which was responsible for the regulation of C3 expression. Administration of 17-AAG at the lesion sites of rats was able to reduce the accumulation of A1 astrocytes. Conclusion Collectively, these data reveal a novel mechanism of astrocyte phenotype conversion following SCI, and HSF1 plays key roles in suppressing excessive increase of neurotoxic A1 astrocytes.

Dual functional roles of Tie-2/angiopoietin in TNF-α-mediated angiogenesis

2004 ◽  
Vol 287 (1) ◽  
pp. H187-H195 ◽  
Author(s):  
Jian-Xiong Chen ◽  
Ying Chen ◽  
Laura DeBusk ◽  
Wenyu Lin ◽  
Pengnain Charles Lin
Keyword(s):  
Endothelial Cells ◽  
Cell Death ◽  
Low Doses ◽  
Functional Roles ◽  
Akt Phosphorylation ◽  
Dual Functional ◽  
Tnf Α ◽  
Corneal Angiogenesis ◽  
High Doses ◽  
Induced Apoptosis

Inflammation and angiogenesis are associated with pathological disorders. TNF-α is a major inflammatory cytokine that also regulates angiogenesis. TNF-α has been shown to regulate Tie-2 and angiopoietin (Ang) expression, but the functional significance is less clear. In this study, we showed that TNF-α induced a weak angiogenic response in a mouse cornea assay. Systemic overexpression of Ang-1 or Ang-2 dramatically increased corneal angiogenesis induced by TNF-α. In the absence of TNF-α, neither Ang-1 nor Ang-2 promoted corneal angiogenesis. Low doses (0–25 ng/ml) of TNF-α increased vascular branch formation of cultured endothelial cells. Overexpression of Ang-1 or Ang-2 enhanced the effects of TNF-α. These data suggest that Tie-2 signaling synergistically amplifies and participates in TNF-α-mediated angiogenesis. In addition, high doses (≥50 ng/ml) of TNF-α induced apoptosis in endothelial cells, but addition of Ang-1 or Ang-2 significantly reduced cell death. Enhanced endothelial cell survival was correlated with Akt phosphorylation. Collectively, our data reveal dual functional roles of Tie-2: low doses enhance TNF-α-induced angiogenesis, and high doses attenuate TNF-α-induced cell death. The study provides evidence supporting a role for Tie-2 in inflammatory angiogenesis.

Akt protects mouse hepatocytes from TNF-α- and Fas-mediated apoptosis through NK-κB activation

2001 ◽  
Vol 281 (6) ◽  
pp. G1357-G1368 ◽  
Author(s):  
Etsuro Hatano ◽  
David A. Brenner
Keyword(s):  
Transcriptional Activity ◽  
Primary Cultures ◽  
Nuclear Factor Κb ◽  
Protective Role ◽  
Binding Activity ◽  
Dominant Negative ◽  
Akt Phosphorylation ◽  
Tnf Α ◽  
Mouse Hepatocytes ◽  
Induced Apoptosis

To determine the role of phosphatidylinositol 3-kinase (PI3K)/Akt and nuclear factor-κB (NF-κB) in protecting hepatocytes from tumor necrosis factor-α (TNF-α)- and Fas-mediated apoptosis, we pretreated primary cultures of mouse hepatocytes with pharmacological and adenovirus-mediated inhibitors of the PI3K/Akt and NF-κB pathways followed by treatment with TNF-α or Jo2, an anti-Fas antibody. Jo2 and, to a lesser extent, TNF-α phosphorylate Akt. The PI3K inhibitor LY-294002 blocks TNF-α- and Fas-mediated Akt phosphorylation. LY-294002 pretreatment reduces NF-κB binding activity and transcriptional activity and NF-κB-responsive gene expression by TNF-α or Jo2. LY-294002 promotes apoptosis after TNF-α or Jo2. The expression of dominant-negative Akt blocks NF-κB activation and sensitizes hepatocytes to TNF-α- and Fas-mediated apoptosis. The expression of constitutively active Akt rescues LY-294002-pretreated cells from TNF-α- and Fas-mediated apoptosis. Active Akt induces NF-κB transcriptional activity but not NF-κB binding activity or IκB degradation. Furthermore, LY-294002 pretreatment blocks TNF-α- and Jo2-induced Bcl-xL levels in hepatocytes, with no effect on the phosphorylation levels of Bad. Bcl-xL overexpression protects hepatocytes from Fas- but not TNF-α-induced apoptosis after sensitization by actinomycin D or the IκB superrepressor. Together, the PI3K/Akt pathway has a protective role in Fas-mediated apoptosis, which requires NF-κB activation, partially through the subsequent induction of Bcl-xL.

scholarly journals Pearl extract protects HaCaT cells from UV radiation-induced apoptosis through mitochondrial pathway regulation

2020 ◽  
Author(s):  
Zhixiong Chen ◽  
jing wang ◽  
Anquan Yang ◽  
Lihua Zhang ◽  
Yaojia Lu ◽  
...  
Keyword(s):  
Protein Expression ◽  
Caspase 3 ◽  
Hacat Cells ◽  
Caspase 9 ◽  
Expression Levels ◽  
Protein Levels ◽  
Bax Protein ◽  
Reactive Oxygen Species Content ◽  
Tnf Α ◽  
Induced Apoptosis

Abstract Background: Previous studies have demonstrated that pearl extract (PE) promotes wound healing and skin whitening. However, it remains unclear whether PE can inhibit ultraviolet (UV)-photodamage in HaCaT cells. In this study, an in vitro photoaging cell model was established to observe the effect of PE on UV-induced damage and the apoptosis of HaCaT cells. The aim of this study was to provide a reference for the future development of natural sunscreens.Results: PE concentrations of 0.1 and 1 μg/mL were considered the most effective and safe concentrations. Compared to that in the control group, superoxide dismutase and glutathione peroxidase activity in the photoaging group was significantly reduced, whereas malondialdehyde and reactive oxygen species content, along with tumour necrosis factor-alpha (TNF-α) and interleukin (IL)-10 mRNA and protein levels, were markedly increased. In contrast, Bcl-2 protein expression was significantly decreased, whereas caspase-3, caspase-9, and Bax protein expression levels were significantly increased. Compared to that in the photoaging group, HaCaT cell proliferation was significantly increased in the PE group. Both PE concentrations significantly increased superoxide dismutase and glutathione peroxidase activity in cells, reduced malondialdehyde and reactive oxygen species content, decreased TNF-α and IL-10 mRNA expression in cells, and reduced TNF-α and IL-10 protein levels in the supernatant. Additionally, Bcl-2 protein expression levels were significantly increased, whereas caspase-3, caspase-9, and Bax protein expression levels were significantly reduced by PE treatment.Conclusions: PE can inhibit UV-induced apoptosis by inhibiting mitochondria-mediated apoptosis and regulating TNF-α and IL-10 expression.

scholarly journals The Adipokine Chemerin Induces Apoptosis in Cardiomyocytes

2015 ◽  
Vol 37 (1) ◽  
pp. 176-192 ◽  
Author(s):  
Diego Rodríguez-Penas ◽  
Sandra Feijóo-Bandín ◽  
Vanessa García-Rúa ◽  
Ana Mosquera-Leal ◽  
Darío Durán ◽  
...  
Keyword(s):  
Western Blot ◽  
Vital Staining ◽  
Cardiac Cells ◽  
Neonatal Rat ◽  
Caspase 9 ◽  
Rat Cardiomyocytes ◽  
Akt Phosphorylation ◽  
Protein Levels ◽  
Tnf Α ◽  
Hoechst Dye

Background: The adipokine chemerin has been associated with cardiovascular disease. We investigated the effects of chemerin on viability and intracellular signalling in murine cardiomyocytes, and the effects of insulin and TNF-α on cardiomyocyte chemerin production. Methods: Hoechst dye vital staining and cell cycle analysis were used to analyse the viability of murine cardiac cells in culture. Western blot was used to explore the phosphorylation of AKT and caspase-9 activity in neonatal rat cardiomyocytes and HL-1 cells. Finally, RT-qPCR, ELISA and western blot were performed to examine chemerin and CMKLR1 expression after insulin and TNF-α treatment in cardiac cells. Results: Chemerin treatment increased apoptosis, reduced phosphorylation of AKT at Thr308 and increased caspase-9 activity in murine cardiomyocytes. Insulin treatment lowered chemerin and CMKLR1 mRNA and protein levels, and the amount of chemerin in the cell media, while TNF-α treatment increased chemerin mRNA and protein levels but decreased expression of the CMKLR1 gene. Conclusion: Chemerin induces apoptosis, reduces AKT phosphorylation and increases the cleavage of caspase-9 in murine cardiomyocytes. The expression of chemerin is regulated by important metabolic (insulin) and inflammatory (TNF-α) mediators at cardiac level. Our results suggest that chemerin could play a role in the physiopathology of cardiac diseases.

AAnti-Inflammatory Effects of Plasma Circulating Exosomes Obtained from Normal-Weight and Obese Subjects on Hepatocytes

2020 ◽  
Vol 20 ◽  
Author(s):  
Reza Afrisham ◽  
Sahar Sadegh-Nejadi ◽  
Reza Meshkani ◽  
Solaleh Emamgholipour ◽  
Molood Bagherieh ◽  
...  
Keyword(s):  
Inflammatory Cytokines ◽  
Hepg2 Cells ◽  
Human Liver ◽  
Liver Cells ◽  
Normal Weight ◽  
Protein Levels ◽  
Human Liver Cells ◽  
Tnf Α ◽  
Anti Inflammatory

Introduction: Obesity is a disorder with low-grade chronic inflammation that plays a key role in the hepatic inflammation and steatosis. Moreover, there are studies to support the role of exosomes in the cellular communications, the regulation of metabolic homeostasis and immunomodulatory activity. Accordingly, we aimed to evaluate the influence of plasma circulating exosomes derived from females with normal-weight and obesity on the secretion of inflammatory cytokines in human liver cells. Methods: Plasma circulating exosomes were isolated from four normal (N-Exo) and four obese (O-Exo) women. The exosomes were characterized and approved for CD63 expression (common exosomal protein marker) and morphology/size using the western blot and TEM methods, respectively. The exosomes were used for stimulation of HepG2 cells in vitro. After 24 h incubation, the protein levels of TNF-α,IL-6, and IL-1β were measured in the culture supernatant of HepG2 cells using the ELISA kit. Results: The protein levels of IL-6 and TNF-α in the cells treated with O-Exo and N-Exo reduced significantly in comparison with control group (P=0.039 and P<0.001 respectively), while significance differences were not found between normal and obese groups (P=0.808, and P=0.978 respectively). However, no significant differences were found between three groups in term of IL-1β levels (P=0.069). Based on the correlation analysis, the protein levels of IL-6 were positively correlated with TNF-α (r 0.978, P<0.001). Conclusion: These findings suggest that plasma circulating exosomes have probably anti-inflammatory properties independently from body mass index and may decrease the secretion of inflammatory cytokines in liver. However, further investigations in vitro and in vivo are needed to address the anti-inflammatory function of N-Exo and O-Exo in human liver cells and/or other cells.

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