Apoptosis is a widespread phenomenon and its dysregulation may result in a variety of human
pathologies, such as cancer, autoimmune diseases and neurodegenerative disorders. CXXC-type zinc finger
protein 5 (CXXC5) is commonly considered as a tumor suppressor undergoing deregulation or deletion in
hematonosis. But it has implied involvement in apoptosis indirectly and the molecular mechanism remains
unknown. In this study, we investigated CXXC5-induced apoptosis as well as its underlying mechanism. A
fluorescence resonance energy transfer (FRET) assay suggested that CXXC5 induced cell death and
caspase-3 activity in primary rat cortical neurons. Further colorimetric TUNEL assay, Hoechst staining and flow
cytometric assay indicated a time-dependent apoptosis in which the activities of caspase-8 and caspase-3
were both regulated via CXXC5 according to enzymatic activity assay, Hoechst staining and Western blotting.
Transcription reporter assay and Western blotting showed that CXXC5 resulted in activation of tumor necrosis
factor-α (TNF-α), initiated the extrinsic apoptosis pathway and cross-linked with the intrinsic mitochondrial
pathway. Being a bone morphogenetic protein 4 (BMP-4) downstream regulator, and also a transcription
factor, cellular co-localization and co-immunoprecipitation results indicate that CXXC5 co-localized and
interacted with Smads. Western blotting and nuclear fraction extraction implied that CXXC5 facilitated Smad3
phosphorylation and Smad4 nuclear translocation, and that co-expression of Smad together with CXXC5
resulted in higher TNF-α reporter activity. In sum, CXXC5 appears to regulate the TNF-α apoptosis pathway by
associating with Smads.
Keywords: Apoptosis, Caspase-3, CXXC5, FRET, Smads, TNF-α.
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