Table 1.
Oligonucleotides used in the study as indicated in Material and Methods.
Table 2.
Primers used for quantitative real-time PCR (qRT-PCR).
Figure 1.
p54nrb promoter activity is controlled by MIA/CD-RAP.
(A) Luciferase reporter assays revealed strong activity of the murine p54nrb (mp54nrb) promoter in primary murine mesenchymal stem cells (mMSCs). In MIA/CD-RAP-deficient (MIA-/-) mMSCs, the promoter activity was reduced (***p<0.001). The values represent the mean ± s.d. from four independent experiments. (B) Murine p54nrb promoter activity analyzed using reporter gene assays was decreased in the wild-type (WT) mMSCs transfected with siRNA against MIA/CD-RAP compared with the control (*p<0.05). The values represent the mean ± s.d. from three experiments. (C) The knockdown efficiency of MIA/CD-RAP in WT mMSCs was validated using qRT-PCR. The MIA/CD-RAP mRNA expression was successfully inhibited compared to the siControl-transfected cells (*p<0.05). The values represent the mean ± s.d. from three independent experiments. (D) The human p54nrb promoter analyzed using reporter gene assays was highly active in WT mMSCs and showed reduced activity in MIA-/- mMSCs (***p<0.001). The values represent the mean ± s.d. from eleven experiments.
Figure 2.
Mapping the p54nrb promoter region that mediates the effects of MIA/CD-RAP on p54nrb transcription.
The human p54nrb promoter luciferase constructs ranging from residues −8436 to −7037 bp are indicated on the left. The luciferase activities resulting from transient transfections into WT (white bars) or MIA-/- mMSCs (black bars) are indicated in the middle. The ratios of human p54nrb promoter activities comparing WT and MIA-/- mMSCs are shown on the right for each luciferase construct. The ratio of the human p54nrb promoter activity comparing WT and MIA-/- mMSCs significantly decreased in deletions involving the region between −7079 and −7037 bp, identifying this 42 bp DNA region as the mediator of MIA/CD-RAP up-regulation of p54nrb promoter activity (*p<0.05). The values represent the mean ± s.d. from three experiments.
Figure 3.
EMSA competition experiments and reporter gene assays demonstrated binding ability to one specific region in the p54nrb promoter.
(A) Schematic overview of the MIA/CD-RAP-dependent region between −7079 and −7038 bp, highlighted in grey, of the human p54nrb promoter-luciferase reporter. The oligos used for the EMSA assay were labeled as follows: 5′-, 3′-hp54nrb and 5′-hp54nrb Mut I and II. The mutated regions are shown in bold letters. The numbers represent the locations with respect to the ATG translation start codon. (B) EMSA analysis revealed two strong DNA-protein complexes interacting with the 5′-hp54nrb oligo in the nuclear extracts from mMSCs (lane 3). Binding capacity of MIA-/- mMSC (lane 4) nuclear extracts was diminished. No binding was observed with the 3′-hp54nrb oligo (lane 5 and 6). (C) Incubation of nuclear extracts from WT mMSCs (lane 4) with the 5′-hp54nrb oligo confirmed the formation of two strong DNA-protein complexes that were diminished when incubated with MIA-/- mMSC nuclear extracts (lane 7). Mutations I and II in the 5′-hp54nrb oligo abolished the formation of DNA-protein complexes in the nuclear extracts from WT cells and MIA-/- mMSCs (lane 5, 6 and 8, 9). The contents of the reaction mixtures are marked in the tables below the images of the gels. (D) For competition experiments, the gel shifts were assessed in competition mixtures containing three different concentrations (x-fold) of unlabeled 5′-hp54nrb oligo (lanes 3–5) or unlabeled mutated oligos (Mut I and II) (lanes 6–8 and 9–11, respectively). In contrast to incubation with unlabeled 5′-hp54nrb oligo, incubation with the unlabeled mutated oligos did not lead to competition of the complexes formed by the binding of the labeled 5′-hp54nrb oligo to WT mMSC nuclear extracts (lane 2). (E) On the left, human p54nrb promoter-luciferase constructs ranging from residues −7079 to −7037 are shown. Luciferase activities resulting from transient transfections are presented in the center. The ratios of human p54nrb promoter activities in the WT and MIA-/- mMSCs are shown to the right of each luciferase construct. Mutation of site I or II in the p54nrb promoter significantly abolished the MIA/CD-RAP-mediated p54nrb promoter activation as measured using the reporter gene assay (*p<0.05; **p<0.01). The values represent the mean ± s.d. from three independent experiments each.
Figure 4.
MIA/CD-RAP activates the p54nrb promoter via the transcription factor YBX1.
(A) YBX1 activity as measured using a reporter gene assay revealed a significant decrease in YBX1 promoter activity in MIA-/- mMSCs compared with WT mMSCs (III) (**p<0.01). The values represent the mean ± s.d. from eight independent experiments. (B) Luciferase reporter assays revealed the significant inhibition of YBX1Luc II activity in MIA-/- mMSCs compared with WT mMSCs (**p<0.01). The values represent the mean ± s.d. from four experiments. (C) Detection of p54nrb and phospho-S102 YBX1 protein levels in WT and MIA-/- mMSCs analyzed by western blot analysis. Consistent with the reduced p54nrb protein levels, the levels of phospho-S102 YBX1 were also considerably reduced in the MIA/CD-RAP-negative cell system compared with the control. (D) Western blot analysis revealed elevated phospho-S102 YBX1 protein levels during differentiation of WT mMSC. (E) Immunohistochemical staining of YBX1 in rips of embryos at different developmental stages. At stage 14.5 days post coitum no YBX1 staining was detected. Scattered and slightly stained chondrocytes were assessed in ribs of 15.5dpc embryos. At 16.5dpc, strong YBX1 expression was detected in chondrocytes and hypertrophic chondrocytes. (F) Immunostaining of YBX1 in tibial growth plates revealed its strong expression in chondrocytes of the proliferative and hypertrophic chondrocyte zone and bone marrow (I). Positive signals were also detected in mouse (II) and human (II) cartilage. The black arrows mark positive stained cells. RZ: resting chondrocyte zone; PZ: proliferative chondrocyte zone; HZ: hypertrophic chondrocyte zone; TB: trabecular bone.
Figure 5.
The YBX1 site is conserved in genes associated with cartilage differentiation.
(A) Gene expression differences in the genes with promoters containing the regulatory region GATTGG, which spans the −7075 to −7070 bp region relative to the ATG in the p54nrb promoter region. Genes are distributed along the x-axis, and the chondrocytic samples are distributed along the y-axis (GSE16464). Chondrocyte differentiation correlates with MIA/CD-RAP expression levels [4], [3], [29]. The relative gene expression levels are color coded to indicate higher (yellow) and lower (blue) expression levels. The genes are distributed along the y-axis, and the cell data are distributed along the x-axis. (B) The mRNA expression levels of Gnas measured by qRT-PCR were significantly reduced in different lots of differentiated MIA-/- mMSCs compared with WT mMSCs The values represent the mean ± s.d. from five experiments. (*p<0.05).