Differential expression of prohibitin is correlated with dual action of Vitamin D as a proliferative and antiproliferative hormone in breast epithelial cells

Abstract

Our previous microarray analysis showed that N-methyl-N-nitrosourea (MNU) transformed MCF12F breast epithelial cells exhibited upregulation of several genes, including prohibitin, which was reversed by 1alpha-hydroxyvitamin D(5) (1alpha(OH)D(5)) treatment. The in silico screening for putative transcription factor binding sites identified two VDR/RXR binding sites in the 1kb promoter region of prohibitin. Other binding sites for EGR and GR which are also Vitamin D target genes were identified in this region, indicating that prohibitin is a potential target gene for Vitamin D. The combination of multiple binding sites also provides a basis for a possible dual regulation of prohibitin by Vitamin D. Prohibitin upregulation by 1alpha(OH)D(5) treatment at both transcription and translation level was observed in Vitamin D sensitive BT474 breast cancer cells, in which 1alpha(OH)D(5) significantly inhibited cell proliferation in normal culture condition. On the other hand, prohibitin down-regulation accompanied with Vitamin D mediated maintenance of proliferation of breast epithelial cells was observed under stressed condition. These results demonstrated that Vitamin D mediated antiproliferative activity in unstressed condition and growth maintaining activity under stressed condition involve differential expression of prohibitin.

Figure 1

Prohibitin is a target gene of vitamin D. (A), In silico analysis of potential VDR/RXR binding sites in the promoter region of prohibitin gene. The core sequence (in bold face) of the putative transcription binding sites for VDR/RXR, EGR and GR with high identity to the authentic core and matrix sequences as identified by MatInspector V2.2 and Promo are underlined. Nucleotides are numbered negatively to the left of the sequence with nucleotide + 1 corresponding to the transcription start site. (B) and (C) Upregulation of prohibitin by vitamin D correlates with its inhibition of cell proliferation in BT474 cells. (B), Western blot analysis of cell lysates from BT474 cells treated with 0.5 μM 1α(OH)D₅ for 24h. 1α(OH)D₅ treatment significantly increased prohibitin expression. β-actin was used as an internal control. (C), MTT assay of BT474 cell proliferation after 4 day treatment with 1α(OH)D₅ (0.5 μM) and 1,25(OH)₂D₃ (100 nM). Results are expressed as mean ± SEM of three independent experiments with 8 wells per treatment in each experiment. *** p<0.001 compared to control.

Figure 2

Downregulation of prohibitin by 1α,25(OH)₂D₃ or siRNA in stressed MCF-7 cells correlates with enhanced proliferative activity. (A), cells were transfected with siCon and incubated in the medium containing low serum to cause stress and then treated with 100 nM 1α,25(OH)₂D₃ for 48h. (B), transfection of siPHB served as a control for prohibitin downregualtion. Both 1α,25(OH)₂D₃ and siPHB transfection downregualted prohibitin under the experimental condition, which was accompanied by increased BrdU incorporation in MCF-7 cells. (A), representative FACS analysis of BrdU incorporation in siCon transfected MCF-7 cells with ETOH (lfet) or 1α,25(OH)₂D₃ (right) treatment. (B), BrdU incorporation analysis of siPHB transfected MCF-7. (C), Western blot analysis of prohibitin expression corresponding to above treatments; lane 1 siCon transfection with ETOH treatment; lane 2, siCon transfection with 1α,25(OH)₂D₃ treatment; lane 3, siPHB transfection without treatment.

Figure 3

Prohibitin downregulation by 1α,25(OH)₂D₃ in stressed MCF12F cells correlates to enhanced proliferative activity. (A), dual regulation of cell proliferation by 1α,25(OH)₂D₃ (100 nM) in normal culture condition (left) and stressed condition (low serum without supplements) (right). Cells were treated for 4 days and subjected to MTT assay. Results are expressed as mean ± SEM, *** p<0.001 compared to control. (B), prohibitin regulation by 1α,25(OH)₂D₃ in normal culture condition and stressed condition (2%FBS). MCF12F cells were treated for 48h and subjected to western blot analysis; lane 1 and 3, ETOH (control); lane 2 and 4, 1α,25(OH)₂D₃. (C), Schematic model of dual regulation of prohibitin by active vitamin D analog (VD) in relation to its proliferation-regulatory effects. Under stressed condition, active vitamin D plays a protective role by downregulating prohibitin and maintaining cell proliferation; while in normal culture condition, vitamin D upregulates or fine-tunes prohibitin which is involved in its antiproliferative effect.