Unconjugated
Proteins that directly regulate tumor necrosis factor (TNF) signaling have critical roles in determining cell death and survival. Previously we characterized ubiquitously expressed transcript (UXT)-V2 as a novel transcriptional cofactor to regulate nuclear factor-κB in the nucleus. Here we report that another splicing isoform of UXT, UXT-V1, localizes in cytoplasm and regulates TNF-induced apoptosis. UXT-V1 knockdown cells are hypersensitive to TNF-induced apoptosis. We demonstrated that UXT-V1 is a new component of TNF receptor signaling complex. We found that UXT-V1 binds to TNF receptor-associated factor 2 and prevents TNF receptor-associated death domain protein from recruiting Fas-associated protein with death domain. More importantly, UXT-V1 is a short-half-life protein, the degradation of which facilitates the formation of the apoptotic receptor complex II in response to TNF treatment. This study demonstrates that UXT-V1 is a novel regulator of TNF-induced apoptosis and sheds new light on the underlying molecular mechanism of this process.
PURPOSE:
We investigated apoptosis induced by hydroxycamptothecin (HCPT) in human Tenon's capsule using fibroblasts cultured from human Tenon's capsule (HTFs), and the mechanism of induction.
METHODS:
HTFs were treated with 0-4 mg/L HCPT for 24 hours. Cell proliferation was measured by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay, and apoptotic cells were analyzed by Hoechst 33258 stain. The mRNA and protein levels of caspase-3, -8, and -9 were detected by RT-PCR and Western blotting.
RESULTS:
By MTT assay, HCPT induced apoptosis in HTFs in a concentration- and time-dependent manner. Hoechst 33258 staining and transmission electron microscopy showed typical apoptotic morphology, such as condensed chromatin, irregular nuclei, and apoptotic body formation. The mRNA and protein levels of caspase-3 and caspase-9 were upregulated, while caspase-8 was unchanged. Z-VAD-FMK, a caspase inhibitor, inhibited the apoptosis of fibroblasts induced by HCPT. The expression levels of caspase-3 and caspase-9 were down-regulated after Z-VAD-FMK treatment.
CONCLUSIONS:
Caspase-3 and caspase-9 are important elements in regulating HCPT-induced apoptosis in HTFs.