cytochrome-c-t and thymosin-beta(4)

Thymosin β4 (Tβ4) has been suggested to regulate multiple cell signal pathways and a variety of cellular functions such as cell migration, proliferation, survival, and angiogenesis. Here, we investigated the effect of Tβ4 on endothelial progenitor cells (EPCs) apoptosis induced by serum deprivation and the corresponding signal transduction pathways involved in this process. Circulating EPCs, isolated from healthy volunteers, were cultured in the absence or presence of Tβ4 and various signal cascade inhibitors. Apoptosis was evaluated with Annexin V immunostaining and cytosolic cytochrome c expression. Incubation of EPCs with Tβ4 caused a concentration dependent increase in cell viability and proliferation activity. It also caused an inhibitory effect on EPCs apoptosis, which was abolished by PI3K inhibitors (either LY294002 or Wortmannin) or JNK MAPK inhibitor SP600125. In addition, the expression and activity of caspase-3 and -9 were decreased by Tβ4, which markedly increased the Bcl-2/Bax ratio within EPCs. Furthermore, Tβ4 was immunoprecipitated with integrin-linked kinase (ILK), accompanied by augmentation of ILK activity. Transfection of EPCs with ILK-siRNA resulted in abolishment of the activation of ILK-Akt and the ameliorative effect on apoptosis by Tβ4. Together, Tβ4 mediated inhibitory effect on EPCs apoptosis under serum deprivation can be attributed, at least in part, to ILK-Akt activation. The activation of JNK MAPK might also be involved in this process.

Topics: Androstadienes; Annexin A5; Anthracenes; Apoptosis; bcl-2-Associated X Protein; Caspase 3; Caspase 9; Cell Proliferation; Cell Survival; Cells, Cultured; Chromones; Culture Media, Serum-Free; Cytochromes c; Endothelial Cells; Enzyme Inhibitors; Humans; Morpholines; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; Stem Cells; Thymosin; Wortmannin

The purpose of this study was to determine the effect of thymosin beta 4 (Tbeta(4)) treatment on human corneal epithelial cells exposed to ethanol in vitro. The efficacy of Tbeta(4) in preventing mitochondrial disruption and in inhibiting caspase-mediated apoptosis was examined.. Nontransformed human corneal epithelial cells (HCECs) at passage 4 were untreated or treated with ethanol (20% for 20 seconds) or a combination of ethanol and Tbeta(4). The cells were allowed to recover from ethanol treatment for 24 hours. Mitochondrial membrane integrity and the release of cytochrome c to the cytoplasm were assessed using microscopy, Western blot, and ELISA. Bcl-2 expression and cell proliferation were measured using ELISA. Colorimetric activity assays were completed for caspase-2, -3, -8, and -9.. Tbeta(4) treatment decreased deleterious mitochondrial alterations, significantly decreased cytochrome c release from mitochondria, and increased Bcl-2 expression in ethanol-exposed human corneal epithelial cells. In ethanol-exposed corneal epithelium Tbeta(4) treatment inhibited caspase-2, -3, -8, and -9 activity, with caspase-8 showing the most significant inhibition. Tbeta(4) treatment resulted in no significant effect on the proliferation of human corneal epithelial cells after ethanol exposure.. Tbeta(4) plays an antiapoptotic role under conditions of epithelial cell challenge with an external stress such as exposure to ethanol. Tbeta(4) may function as an antiapoptotic agent by inhibiting the release of cytochrome c from mitochondria and by suppressing the activation of caspases.

Topics: Apoptosis; Blotting, Western; Caspase Inhibitors; Caspases; Cell Division; Cell Line; Cytochromes c; Enzyme-Linked Immunosorbent Assay; Epithelium, Corneal; Ethanol; Humans; Microfilament Proteins; Mitochondria; Proto-Oncogene Proteins c-bcl-2; Thymosin