Diabetic Osteopenia by Decreased β-Catenin Signaling Is Partly Induced by Epigenetic Derepression of sFRP-4 Gene

<div><p>In diabetics, methylglyoxal (MG), a glucose-derived metabolite, plays a noxious role by inducing oxidative stress, which causes and exacerbates a series of complications including low-turnover osteoporosis. In the present study, while MG treatment of mouse bone marrow stroma-derived ST2 cells rapidly suppressed the expression of osteotrophic Wnt-targeted genes, including that of osteoprotegerin (OPG, a decoy receptor of the receptor activator of NF-kappaB ligand (RANKL)), it significantly enhanced that of secreted Frizzled-related protein 4 (sFRP-4, a soluble inhibitor of Wnts). On the assumption that upregulated sFRP-4 is a trigger that downregulates Wnt-related genes, we sought out the molecular mechanism whereby oxidative stress enhanced the sFRP-4 gene. Sodium bisulfite sequencing revealed that the sFRP-4 gene was highly methylated around the sFRP-4 gene basic promoter region, but was not altered by MG treatment. Electrophoretic gel motility shift assay showed that two continuous CpG loci located five bases upstream of the TATA-box were, when methylated, a target of methyl CpG binding protein 2 (MeCP2) that was sequestered upon induction of 8-hydroxy-2-deoxyguanosine, a biomarker of oxidative damage to DNA. These <i>in vitro</i> data suggest that MG-derived oxidative stress (not CpG demethylation) epigenetically and rapidly derepress sFRP-4 gene expression. We speculate that under persistent oxidative stress, as in diabetes and during aging, osteopenia and ultimately low-turnover osteoporosis become evident partly due to osteoblastic inactivation by suppressed Wnt signaling of mainly canonical pathways through the derepression of sFRP-4 gene expression.</p></div

The effect of methylation and oxidation at CpG locus 5 bases upstream of TATA-box on MeCP2 binding.

<p><i>In vitro</i> binding of nuclear protein from HeLa cells to TATA-box and CpGs 5 bases upstream of TATA-box was tested by EMSA. (A) Double-stranded oligonucleotides, unmethylated (UUS/UUA), hemimethylated (UUS/UMA), single-methylated (UMS/UMA), and hemihydroxilated as well as single-methylated (UMS/UOxMA) ones, spanning part of the mouse <i>sFRP-4</i> gene basic promoter region including TATA-box (−57/−29), were subjected to the binding reaction. (B) The unmethylated oligonucleotides show protein-DNA bindings (lanes 1 and 2, TBP binding) which are washed out by the cold consensus TATA-box sequence (lane 3). On the other hand, both hemi- and bi-methylated oligonucleotides show dense and clear protein-DNA binding (lanes 4 and 7, MeCP2 binding) which is block-shifted with an anti-MeCP2 antibody (lanes 5 and 8). At the same time, the protein-DNA bindings appearing at low and high positions (lanes 5 and 8, TBP binding) are washed out by a cold TATA-box competitor (lanes 6 and 9). By inducing a single 8-OHdG, protein-DNA bindings seen in lanes 4 and 7 have disappeared (lane 10), and alternative protein-DNA binding appearing at low and high positions (lanes 10 and 11, TBP binding) are washed out with a cold TATA-box competitor (lane 12). (C) A set of primers was used to amplify 80 bases of DNA containing a CpG dinucleotide, TATA-box and a transcription start site for ChIP assay of TBP and MeCP2 on the mouse sFRP-4 gene promoter. ST2 cells with or without MG treatment were subjected to immunoprecipitation. Input and immunoprecipitated DNA with either anti-TBP or anti-MeCP2 antibody was assessed by PCR using the set of primers as described above. Without MG treatment, a reactive PCR product reflecting mainly endogenous MeCP2 binding is observed. Conversely, a PCR product reflecting TBP binding was found mainly in MG-treated ST2 cells.</p

Status of CpG methylation in mouse sFRP-4 gene promoter region.

<p>(A) The basic promoter region of the sFRP-4 gene has TATA-box located 38 bases upstream of the transcription start site, around which CpG dinucleotides are clustered (shown in boldface). (B) Twelve independent bisulfite mappings. Corresponding to the repression of the <i>sFRP-4</i> gene expression in steady-state ST2 cells, CpGs within the mouse <i>sFRP-4</i> gene basic promoter, especially upstream of TATA-box, are highly methylated. (C) The effect of 2 nM of 5-aza-dC treatment for 72 hr on steady-state <i>sFRP-4</i> expression. Although not markedly prominent, quantitative RT-PCR showed that the steady-state expression of <i>sFRP-4</i> was, albeit two-fold at most, significantly restored by 5-aza-dC treatment. The statistical significance was determined by Student’s t test, *<i>P<0.05</i>.</p

Confirmation of the effect of methylglyoxal on the expression of selected genes.

<p>(A) The effect of methylglyoxal (MG) treatment on the mRNA expression of sFRP-4, OPG, RANKL, Trx1, Runx2, OPN, BAMBI, PPARgamma, aP2, p16^INK4a, and Casp-3 in ST2 cells. Administration of 100 µM of MG resulted in a 3.88- and a 4.63-fold increase in sFRP-4 and RANKL mRNA expression, respectively, but a decrease in OPG expression to 10% of its basal level. Quantitative real-time reverse transcription PCR (Q-real-time RT-PCR) confirmed reciprocal change in the expression levels between sFRP-4 and OPG. Furthermore, MG treatment increased p16^INK4a and caspase 3 expression, leading to cell cycle arrest and apoptosis, while markers of osteoblastic differentiation (Runx2, BAMBI, and OPN) increased slightly, and those for adipocytic differentiation (PPARγαµµα and aP2) decreased. The statistical significance was determined by Student’s t test, *<i>P<0.05</i>. (B) <i>In vitro</i> MG treatment for 12 hr, a model for the acute phase of oxidative stress on stromal cells, showed rapid induction of sFRP-4 protein in the cytoplasm of ST2 cells (right, x200, counterstained with phalloidin and DAPI), while little sFRP-4 protein was observed in the control (left, x200, counterstained with phalloidin and DAPI).</p

Effect of methylation at two CpG loci upstream of TATA-box on MeCP2 binding.

<p><i>In vitro</i> binding of nuclear protein from HeLa cells to TATA-box and two CpG dinucleotides upstream of TATA-box was tested by EMSA. (A) Double-stranded oligonucleotides, unmethylated (UUS/UUA), single-methylated (UMS/UMA or MUS/MUA), and double-methylated (MMS/MMA) were subjected to the binding reaction. (B) Both single-methylated and double-methylated oligonucleotides showed prominent protein-DNA binding at a high position (black arrowheads, lanes 3, 5, and 7), which was block-shifted by the anti-MeCP2 antibody (lanes 4, 6, and 8). The sequence date has been quoted from GenBank (accession number AF364906.1) by Wong VK <i>et al </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102797#pone.0102797-Wong1" target="_blank">[41]</a>.</p

A schematic model of gene activation mechanism through 8-OHdG modification at methylated CpG locus upstream of TATA-box.

<p>When the CpG locus is not methylated, TBP gains access to TATA-box and transactivates the gene. (B) By the addition of single methylation at the CpG locus, the sequence becomes the target of MeCP2. As a result, TBP binding to TATA-box is disturbed, and the gene becomes inactive. (C) Guanine modification by oxidative stress typically results in the induction of hydroxylation at position 8, which sequesters MeCP2 binding to the target DNA. (D) As a consequence, TBP binding to TATA-box is restored, and the gene is reactivated.</p

Western blotting for proteins related to canonical and non-canonical Wnt-signaling.

<p>ST2 cells with serial periods of MG treatment were subjected to Western blotting. Hela cell lysate was used as a control. While Wnt5a/b, Ror1, Ror2, whole- and phospho-SAPK/JNK, whole- and phospho-c-jun (factors related to non-canonical Wnt-signaling) were almost constant, the β-catenin protein level decreased and the phospho-β-catenin protein level increased reciprocally at 12 hr after treatment.</p

OPN expression and co-localization with antimicrobial proteins in COPD lung tissues.

<p>(A) Multiple cells in the bronchiolar epithelium expressed OPN (brown-colored DAB). (I) Goblet cells, the inset shows OPN expression in the goblet cell mucus. (II) Bronchial submucosal glands (Br = bronchial epithelium. (III) OPN expression in some basal cells (arrowhead). (IV) Flask-shaped cells (arrowhead). Primary antibodies from non-immunized animals resulted in loss of labeling (not shown). Scale bars: (I) = 15 μm; (II) = 70 μm; (III, IV) = 20 μm. (B) Immunohistochemical staining for OPN and the AMPs lactoferrin, lysozyme, and SLPI performed on parallel sections of lung tissue obtained from a patients with COPD (GOLD stage IV). Immunoreactivity is visualized by a brown-colored DAB staining in the bronchiolar epithelium and also in the airway lumen of COPD lungs. OPN, lactoferrin, and SLPI are all detected both in bronchiolar epithelium (Ep) and in cellular debris and mucus of the lumen (Lu) while lysozyme is present only in the lumen and to a lesser extent in the airway epithelium. The latter is explained by its preferential expression in the submucosal glands of large airways (not shown). Cell nuclei are counterstained with Mayer’s hematoxylin (blue stain). The scale bar in the right panel of the bottom figure is 100 μm.</p

Impairment of the bactericidal activity of AMPs from the OPN-fragment VSS60 that is generated by elastase of <i>P</i>. <i>aeruginosa</i>.

<p>(A) Amino acid sequence of full length OPN and the VSS60-peptide (highlighted in green) which is generated by elastase of <i>P</i>. <i>aeruginosa</i>. The full-length OPN has an RGD-motif providing a binding site for integrins (highlighted in orange). (B) To investigate whether the VSS60-peptide retains the AMP-neutralizing properties of full-length OPN, <i>P</i>. <i>aeruginosa</i> were incubated in either buffer alone, with AMPs (3 μM), or AMPs pre-incubated with VSS60 peptide at a molecular ratio of 1:1 before addition to bacteria and incubated for one hour at 37°C. The antimicrobial activity was determined using viable counts. The VSS60-peptide reduced the bactericidal activity of SLPI, hBD-3, and TSLP. A similar but lower inhibitory activity compare with full length OPN. The histograms represent mean and standard deviation from three separate experiments. Two-way ANOVA with Sidak’s multiple comparisons test was used for statistical analysis. *<i>P</i>≤0.05, and ****<i>P</i> ≤ 0.0001. (C) Biophysical properties of OPN and peptide VSS60 derived from OPN showing number of amino acids, molecular weight, net charge and pI.</p

OPN does not influence the muramidase and protease inhibitory functions of lysozyme and SLPI respectively.

<p>(A) Effect of OPN on muramidase activity of lysozyme. Lysozyme was pre-incubated with or without OPN at equimolar concentrations and fluorogenic substrate was added to the mixture and incubated for 1 h at 37°C. The lysozyme activity was determined by the development of fluorescence, which is represented as relative fluorescence units (RFU). (B) Neutrophil elastase inhibitory property of SLPI was investigated in presence of OPN. Equimolar concentrations of OPN and SLPI were pre-incubated for 1 h at 37°C. This mixture was incubated with human neutrophil elastase (NE) (0.05 U/ml) for 20 min at RT. The NE activity was determined by a chromogenic substrate solution by recording the absorbance at 405 nm. The above experiments suggest that OPN cannot influence the enzymatic activities of lysozyme and SLPI.</p