Inactivation of the human vitamin D receptor by caspase-3

Area of Science:

• Molecular Biology
• Cell Biology
• Cancer Research

Background:

• Calcitriol, the active form of vitamin D, exerts its effects through the vitamin D receptor (VDR), a nuclear transcription factor.
• Calcitriol plays a role in cell growth inhibition, cell cycle arrest, and apoptosis, particularly in cancer cells.
• Previous observations in LNCaP prostate cancer cells suggested VDR inactivation during apoptosis induced by staurosporine (STS).

Purpose of the Study:

• To investigate whether the human VDR is a direct target of caspases during apoptosis.
• To identify the specific caspase cleavage site(s) within the human VDR.
• To determine the functional consequences of VDR caspase cleavage on its activity.

Main Methods:

• Site-directed mutagenesis was used to create mutations at a putative caspase-3 cleavage site (D195MMD198S) in the human VDR.
• Wild-type (WT) VDR and mutant VDRs were expressed in COS-7 cells and treated with STS to induce apoptosis.
• In vitro cleavage assays using purified caspase-3, -6, and -7, as well as analysis of cell extracts from STS-treated LNCaP cells and fibroblasts, were performed.
• The effect of the proteasome inhibitor MG-132 on VDR cleavage fragments was assessed.

Main Results:

• STS treatment induced cleavage of WT VDR and a S199A mutant, but not D195A or D198A mutants, in transfected COS-7 cells.
• In vitro assays confirmed that caspase-3, -6, and -7 cleave WT VDR, while D195A and D198A mutants were resistant.
• The rat VDR, lacking the identified human caspase-3 site, was not cleaved by STS.
• MG-132 protected VDR caspase cleavage fragments from proteasomal degradation.

Conclusions:

• The human VDR is a direct target of caspase-3, -6, and -7, with a critical cleavage site at D195MMD198.
• Caspase-mediated cleavage of VDR likely contributes to the inactivation of VDR signaling during apoptosis.
• These findings reveal a novel mechanism by which apoptosis can modulate VDR activity.