Article Figures & Data
Figures
- Fig. 1.
(A) The GM550 element contains a composite site consisting of overlapping NFAT and VDR binding sites (nVDRE) and a proximal AP-1 site. Also depicted is a 30-bp synthetic oligonucleotide that contains the 7-bp nVDRE core sequence in the context of a random nonspecific sequence (nVDRE/random). (B) VDR binds the 7-bp nVDRE independent of flanking GM550 sequences. A comparison of VDR binding to GM550 and nVDRE/random is depicted. Purified VDR, ranging from 20 to 100 ng, was incubated with 12 fmol of either radiolabeled GM550 oligonucleotide or nVDRE/random.
- Fig. 2.
VDR and NFAT1 do not co-occupy the GM550 element. (A) Mutually exclusive binding of VDR and NFATXS to GM550. Fifteen femtomoles of radiolabeled GM550 element was incubated with the indicated amounts of recombinant VDR and NFATXS, where the order of addition was reversed, as indicated (lanes 1 to 6 and 7 to 12). At excess concentrations of DNA, NFATXS binding is independent of VDR. (B) VDR and NFATXS compete for binding to the GM550 site at limiting DNA concentrations. Increasing amounts of VDR were incubated with a constant amount of NFATXS (30 ng) in a binding reaction in which the radiolabeled GM550 element was limiting (5 fmol). The quantitation of the NFATXS-shifted species below the gel indicates that 120 ng of VDR resulted in a >50% decrease in NFATXS binding.
- Fig. 3.
Overexpression of NFAT1 in T cells partially relieves 1,25(OH)2D3-mediated repression of the N3GMCSF reporter. Jurkat cells were transiently transfected with the reporter plasmid depicted above the histogram, together with VDR and NFAT1 overexpression plasmids under the control of the CMV promoter at the indicated ratios. A 0 indicates the endogenous level of the respective factor. Cells were activated with PMA and PHA in the presence or absence of 1,25(OH)2D3 for 8 h; cells were then harvested, and luciferase activity was assayed. Activation levels independent of 1,25(OH)2D3 treatment were set to 100%. Shown are results of a representative experiment carried out in triplicate and repeated five times. All values were normalized to protein concentration as well as to β-galactosidase activity produced off the internal control plasmid. LUC, luciferase-encoding sequence.
- Fig. 4.
VDR stabilizes the AP-1 complex on the GM550 element. (A) VDR stabilizes the Jun-Fos-GM550 ternary complex in the presence or absence of NFATXS. Twelve nanograms of c-Jun protein (J) and 6 ng of c-Fos protein (F) were used in lanes 2, 3, 5, and 6 to 8. Ten nanograms of NFATXS (N) was used in lanes 1, 3, 5, and 6 to 8, and 30 ng of VDR (V) was used in lanes 4 and 5. A titration of VDR was performed (lanes 6 to 8) in an attempt to detect an enhancement in the mobility shift of the upper complex. (B) VDR is present in the Jun-Fos DNA-bound complex. The top panel shows the DNA binding profile of the indicated purified proteins in a complex with a GM550 probe, using the amounts used for panel A. The asterisks denote those complexes which were excised as gel fragments, eluted, and run in an SDS-polyacrylamide gel. The bottom panel shows an immunoblot analysis of the SDS gel probed with an anti-VDR monoclonal antibody (Affinity BioReagents). Lane numbers correspond to the same lanes on the gel mobility shift from which the complexes were excised. +, purified VDR loaded directly onto the SDS gel.
- Fig. 5.
Overexpression of Jun prevents 1,25(OH)2D3-mediated transrepression of the GM-CSF locus. Jurkat cells were transiently transfected with the indicated plasmids and were activated with PMA and PHA in the presence or absence of 10−8 M 1,25(OH)2D3. (A) A total of 250 ng of CMV-VDR leads to 50% repression. (Note that this is half the amount of VDR that was used in Fig. 3.) (B) CMV-Jun, but not CMV-Fos, blocks 1,25(OH)2D3-mediated repression. The indicated amount of plasmid DNAs were used in each transfection. Note that overall activation levels are decreased in the presence of overexpressed c-Jun and c-Fos plasmids, most likely due to general squelching (compare the average luciferase (LUC) units in panel B, 15,000, with that in panel A, 30,000). All values were normalized to protein concentration as well as to β-galactosidase activity produced off the internal control plasmid.
- Fig. 6.
VDR interacts directly with c-Jun, but not c-Fos, in an in vitro interaction assay. (A) VDR interactions with c-Jun. Lanes 1 to 4, glutathione-agarose beads containing immobilized GST-VDR coincubated with 50 to 300 ng of c-Jun; lane 5, input c-Jun protein; lanes 6 to 9, immobilized GST incubated with identical amounts of added purified c-Jun protein; lane 10, input c-Jun. (B) VDR does not interact with c-Fos. Lanes 1 to 6, immobilized GST-VDR incubated with 50 to 1,000 ng of c-Fos protein; lane 7, input c-Fos protein; lanes 8 to 13, immobilized GST incubated with identical amounts of added purified c-Fos protein; lane 14, 100 ng of input c-Fos. Visualization of c-Jun and c-Fos proteins was by Western blot analysis with specific antibodies raised against the two proteins. (Note that detection of c-Fos required greater amounts of protein than detection of c-Jun due to its lower-affinity antibody; therefore, the c-Fos titration was taken out to 1,000 ng.) (C) GST-VDR (lane 1) and GST (lane 2) baits shown at the amounts used in both pull-down series (2.5 μg).
- Fig. 7.
Nuclear extract derived from 1,25(OH)2D3-treated cells demonstrates that VDR is bound to the GM550 element. (A) 1,25(OH)2D3alters an endogenous GM550 binding complex in nuclear extracts from activated Jurkat T cells. Nuclear extracts were prepared from activated T cells which were untreated or treated with 1,25(OH)2D3. Three milligrams of nuclear extract was incubated with 12 fmol of radiolabeled GM550. Complexes were separated on 4% nondenaturing acrylamide gels supplemented with glycerol. (B) VDR binds to the GM550 element in extracts from activated cells in the presence of 1,25(OH)2D3. Jurkat cells were transfected with FLAG-VDR and were either mock treated (lanes 1 to 3) or treated with 1,25(OH)2D3(lanes 7 to 9), PHA-tetradecanoyl phorbol acetate (TPA) in the absence of ligand (lanes 4 to 6), or PHA-tetradecanoyl phorbol acetate in the presence of 1,25(OH)2D3 (lanes 10 to 12). Nuclear extracts were adjusted for FLAG-VDR expression levels (as determined by a Western blot quantitation in panel C) and were incubated with an end-labeled GM550 DNA element and either an anti-FLAG antibody (lanes 3, 6, 9, and 12), a nonspecific antibody (lanes 2, 5, 8, and 11), or no antibody (lanes 1, 4, 7, and 10). Protein-DNA-antibody complexes were precipitated with agarose A beads, washed, and counted. Precipitated counts per minute are shown; values are the means of three independent experiments carried out in triplicate. (C) Western blot analysis of adjusted FLAG-VDR expression levels in nuclear extracts as used for panel B.
- Fig. 8.
A two-hit mechanism for repression of GM-CSF-activated transcription by VDR.
