Sentrin/sumo Specific Proteases As Novel Tissue-selective Modulators Of Vitamin D Receptor-mediated Signaling

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Depletion of endogenous SENP1 results in a diminished induction of <i>CYP24A</i> mRNA expression by 1,25D.

<p>Caco-2 cells were seeded and co-transfected with 100nM Dharmafect SENP1 siRNA or Non-Targeting (NT) control or as described in methods. Cells were then treated with vehicle or 1,25D (10⁻⁸M) for 24 hours before RNA/protein extraction and subsequent analysis through RT-PCR/qRT-PCR and immunoblotting. <b>A</b>. RT-PCR depicting gene specific PCR products obtained from Caco-2 cells transfected with non-targeting (NT) and SENP1-specific siRNAs and subsequently treated with 1,25D or vehicle control. The lower panels describe Q-PCR analysis of the impact of NT and SENP1-specific siRNAs upon the mRNA expression within 1,25D treated Caco-2 cells of (<b>B</b>) <i>SENP1</i>, (<b>C</b>) <i>CYP24A1</i> and (<b>D</b>) <i>TRPV6</i>. The experiment depicted in <b>A</b> is representative of three independent experiments while <b>B, C</b> and <b>D</b> describe the average of three independent experiments where each data point represents the means (± SD) of triplicate assays (<i>n = </i>3) and where <i>ns</i> p≥0.05, ** p = 0.001–0.01, **** p<0.0001.</p

K91R VDR is associated with an increased level of transactivation by 1,25D.

<p>A. Functional activities of the wild type, K91R and K103R versions of VDR assessed using the pGI3-CYP3A4 and pMCS-24OHase luciferase-based reporter constructs. HEK-293 cells received the appropriate VDR and reporter construct in combination with expression vector for RXRα. The lower panel confirms protein expression of the wild type and mutant forms of VDR within same lysates that generate pMCS-24OHase reporter data. B. Transcriptional responses to ligand exhibited by the wild type, K91R and K103R forms of VDR expressed as Gal4 hybrid proteins and assessed through luciferase activity of the pFLUC reporter vector that contains five copies of the Gal4 response element. Cells were exposed to 1,25D for 24 hours before recording of luciferase values. All data within each figure represents means (± SE) of triplicate assays (<i>n</i> = 3) where <i>ns</i> p≥0.05, ** p = 0.001–0.01, *** p = 0.0001–0.001.</p

Transcriptional activities of VDR and RXRα in MCF-7 cells are differentially modulated by SENPs.

<p><b>A</b>. MCF-7 cells were seeded under conditions defined in methods and received the indicated SENP expression construct or corresponding parent vector control in combination with the pFLUC reporter and pCMVBD-VDRFL or pCMVBD- RXRαFL. <b>B</b>. pSG5-hVDR and pSG5-hRXRα were co-transfected into MCF-7 cells in combination with the pMCS-24OHase reporter and appropriate Flag-SENP expression plasmid or parent vector control. Cells were then dosed for 24 hours with the 1,25D (10⁻⁸M) or 9-<i>cis</i> RA (10⁻⁶M) cognate ligands or vehicle control where indicated. The fold-stimulation (ratio of activity in the presence:absence of ligand) is indicated above each set of bars. The results are presented as means (± SD) from three independent experiments with each data point measured in triplicate (<i>n</i> = 3) where <i>ns</i> p≥0.05, ** p = 0.001–0.01, **** p<0.0001.</p

Members of the SENP family directly interact with VDR.

<p><b>A</b>. Mammalian two-hybrid assay was employed to assess the abilities of VDR to directly associate with SENP 1 or 2. CHO KI cells were co-transfected with the pFR-LUC reporter along with the indicated combination of bait (pCMVBD-SENP1 or 2) and prey (pCMVAD, pCMVAD-VDRLBD) constructs. Cells were incubated with 1,25D (10⁻⁸M), or vehicle control for a period of 24 hours before measurement of luciferase activity. After normalization for transfection efficiency based on the activity of the pRL-TK control, results were expressed as relative luciferase units per well. Data represents the average of three independent experiments run in triplicate, mean ± S.D; where <i>ns</i> = <0.05, * p = 0.01–0.05, ** p = 0.001–0.01. <b>B</b>. Interaction between VDR and SENP1 & 2 was monitored through GST-pulldown assay. GST-hVDR fusion protein, or GST alone, bound to glutathione-coated Sepharose beads were pre-incubated with 10⁻⁶ M 1,25D (D lanes) or ethanol vehicle (Et lanes) for 1 h at 22°C, followed by incubation with 20 µl of [³⁵S]-methionine-labeled SENP or RXRα proteins for 1 hour at 4°C. After washing, coprecipitated SENPs or RXRα was detected by electrophoresis of denatured bead samples followed by autoradiography. Aliquots (5%) of all radiolabeled protein are shown in the input lanes (1 & 2) to assess the level of SENP synthesized in the IVTT reaction. Lanes 9 & 10 detail positive controls employing RXRα, an established VDR-interacting protein. Data is representative of three independent experiments.</p

SENPs facilitate deSUMOylation of VDR.

<p>Depicted are cell-based SUMOylation assays performed as described in materials and methods using the following experimental conditions: A. HEK-293 cells received expression constructs for V5-VDR, His-SUMO2 and UBC9 or parent control. Cell lysates from each treatment group were incubated with V5-agarose beads and the resulting precipitated material subjected to western blot analysis with antibodies specific for 6x His (upper panel) or V5 (lower panel) epitope tags. The arrowheads indicate unconjugated and SUMO2-modified versions of V5-VDR. B. Where indicated, cells were co-transfected with V5-VDR, His-SUMO2, UBC-9 in combination with FLAG-SENP-1 or FLAG-SENP-2 with (-) denoting inclusion of appropriate parent vector control. The upper panel describes detection of SUMO2-conjugated VDR within precipitated lysates with expression of V5-VDR and Flag-SENP within the appropriate treatment groups confirmed in the lower panel. C. Cells received the indicated combination of V5-VDR, His-SUMO2, UBC-9 along and FLAG-SENP-2 or parent vector control. Upper panel depicts detection of SUMO2 conjugated VDR and reversal of this modification by SENP2. Lower panels confirm the expression status of unconjugated SUMO2.</p

SENP1 selectively potentiates the transcriptional activities of VDR and RXRα.

<p>HEK-293 cells were transfected with the pFLUC reporter vector in combination with the appropriate pCMVBD-based expression vector for each Gal4-nuclear receptor (LBD) hybrid protein under evaluation. Where indicated, cells also received the SENP1 expression plasmid, pFLAG-CMV-SENP1 (200 ng), or an equivalent amount of parent vector (minus SENP1 insert) as control. The total amount of DNA in each transfection was kept at a constant value through inclusion of the appropriate amount of empty expression vector. Treated cells were dosed with the appropriate cognate ligand or vehicle control for a period of 24 hours before measurement of luciferase activity. All ligands were used at a concentration of 10⁻⁶M, except 1,25D (10⁻⁸M). After normalization for transfection efficiency based on the activity of the pRL-TK control, results were expressed as relative luciferase units per well. The fold-stimulation (ratio of activity in the presence:absence of ligand) is indicated above each set of bars. Data represents analysis of at least three independent experiments with each treatment run in triplicate (<i>n = </i>3) mean ± SD; where <i>ns</i> = p≥0.05, ** p = 0.001–0.01, *** p = 0.0001–0.001 and **** p<0.0001.</p

Identification of a possible SUMO acceptor site within VDR.

<p>Top: Predicted SUMO sites within VDR using the SUMOplot™ tool. A. Depicted are HEK-293 based SUMOylation assays of VDR mutant variants containing K to R substitutions at potential SUMO acceptor sites present within the C-terminal extension (left panel) and ubiquitin (K399R) and acetylation sites (middle panel). The asterisks denote SUMO2 modified VDR forms migrating at 72, 95 and 135 KD. B. SUMOylation assay of the wild type and K91R forms of VDR using HeLa cells that stably express SUMO2. All experiments included the expression construct for UBC9. Lower panels to both A and B confirm the equivalent expression of each mutant and individual components included in the assay.</p

SENPs potentiate transactivation of the full length VDR protein.

<p><b>A</b>. HEK-293 cells received pCMVBD-VDRFL that encodes Gal4DBD fused to full length human VDR, in combination with the pFR-LUC reporter and the indicated SENP expression construct or parent vector control. The lower panel depicts an immunoblot analysis in which combined cellular lysates from each treatment group were probed with the antibodies specific for VDR (9A7) and β-actin. <b>B</b>. pSG5-hVDR that expresses full length human VDR were co-transfected into HEK-293 cells in combination with the reporter construct pMCS-24OHase, pSG5-hRXRα and the appropriate Flag-SENP expression plasmid or parent vector control. Transfected cells were incubated with 1,25D (10⁻⁸M) or vehicle for 24 hours before measurement of luciferase activity. The lower panel depicts immunoblot analysis of cell lysates transfected with the pFlag-SENP1 or pFlag-SENP2 expression constructs and then probed with the Flag or β-actin specific antibodies, with WCE representing the untransfected whole cell lysate control. The fold stimulation are expressed as means (± SD) and results presented are the average of three independent experiments, where <i>n = 3</i> in each assay. <b>C</b>. Caco-2 cells were co-transfected with pCMVBD-VDRFL and pFR-LUC reporter in combination with the appropriate SENP expression constructs or parent vector control. <b>D</b>. Caco-2 cells received pSG5-hVDR+pSG5-hRXRα, the pMCS-24OHase reporter, together with the indicated SENP expression plasmid or parent vector control. Transfected cells were then incubated with 1,25D (10⁻⁸M) or vehicle control for 24 hours before measurement of luciferase activity. All depicted data represents an average of four independent experiments with values expressed as means (± SD) of triplicate assays (<i>n</i> = 3) where ** p = 0.001–0.01, *** p = 0.0001 - 0.001, **** p<0.0001.</p

SENP1 potentiates the hormone responsiveness of an endogenous vitamin D target gene.

<p>Caco-2 cells were plated as described in methods and co-transfected with pSG5hVDR and, where indicated, pFLAG-CMV-SENP1 or corresponding parent vector control. Following incubation for a period of 24 hours with 1,25D (10 nM) or vehicle control, total RNA was isolated from cells, converted to cDNA and real time PCR analysis performed. The fold-stimulation of <i>CYP24A1</i> mRNA expression achieved through the presence of 1,25D is indicated above the black bars. The depicted data represents an average of three independent experiments with each data point a means (± SD) of triplicate assays (<i>n = </i>3) and **** p<0.0001.</p