Characterization of a Human 12/15-Lipoxygenase Promoter Variant Associated with Atherosclerosis Identifies Vimentin as a Promoter Binding Protein

<div><h3>Background</h3><p>Sequence variation in the human 12/15 lipoxygenase (ALOX15) has been associated with atherosclerotic disease. We functionally characterized an ALOX15 promoter polymorphism, rs2255888, previously associated with carotid plaque burden.</p> <h3>Methodology/Principal Findings</h3><p>We demonstrate specific <em>in vitro</em> and <em>in vivo</em> binding of the cytoskeletal protein, vimentin, to the ALOX15 promoter. We show that the two promoter haplotypes carrying alternate alleles at rs2255888 exhibit significant differences in promoter activity by luciferase reporter assay in two cell lines. Differences in i<em>n-vitro</em> vimentin-binding to and formation of DNA secondary structures in the polymorphic promoter sequence are also detected by electrophoretic mobility shift assay and biophysical analysis, respectively. We show regulation of ALOX15 protein by vimentin.</p> <h3>Conclusions/Significance</h3><p>This study suggests that vimentin binds the ALOX15 promoter and regulates its promoter activity and protein expression. Sequence variation that results in changes in DNA conformation and vimentin binding to the promoter may be relevant to ALOX15 gene regulation.</p> </div

In vitro and In vivo Binding of Vimentin to Human ALOX15 Promoter Variants.

<p>(<b>A</b>) Oligonucleotide sequences used for the gel shift assay experiments corresponding to promoters P1 and P2, which differ at rs2255888 (indicated by arrow). (<b>B</b>) Identification and analysis of Vimentin, from magnetic bead DNA-protein pull-down assay, by LC-MS/MS. Proteins were identified by database search of the fragment spectra against the National Center for Biotechnology Information nonredundant protein database (NCBInr) using Mascot (version 2.2, Matrix Science, London, UK). The sequence match for one of the assigned peptides is shown with the fragment ions identified as y(n), b(m) to indicate the y and b ions, respectively. The table shows the top-ranked peptide match and the scores for the next 9 for that spectrum. (<b>C</b>) Chromatin immunoprecipitation (ChIP) assay of P1 luciferase transfected NIH3T3 cells with anti-vimentin antibody (C20).</p

Higher-Order Structure in Duplex and Single Stranded Oligonucleotides.

<p>(<b>A</b>) 15% non-denaturing polyacrylamide gel electrophoresis of duplex oligos in the presence of 40 mM NaCl. The oligos were incubated in binding buffer (Materials and Methods) for 40 min and 20 min at 4°C and 25°C respectively. 29G* (Lanes1 and 3), 29A* (Lanes 2 and 4) are at 4°C and 25°C respectively. (<b>B</b>) 1D proton NMR spectra (imino, amino and aromatic signal regions) of oligonucleotides 29G (top) and 29A (bottom) in the presence of 40 mM Na+, 10 mM phosphate, pH 7.0. (<b>C</b>) Denaturation profiles of single stranded oligos 29G (red) and 29A (blue) at 295 nm wavelength. Experimental conditions: 40 mM Na+, 10 mM phosphate, pH 7.0.</p

Promoter activities of ALOX15 variants in NIH3T3 cells and MCF-7 cells.

<p>Vimentin binds to ALOX15 promoter (<b>A</b>) ALOX15 promoter sequence. Primers used for this study and alternative alleles of rs2255888 are shown in bold. (G/A) in bold denotes SNP rs2255888 position. (<b>B</b>) Qualitative measurement of human ALOX15 (upper panel) and GAPDH (lower panel) gene expression by RT-PCR in NIH3T3 cells. (<b>C</b>) Luciferase activities of P1 and P2 transfected NIH3T3 cells was measured after 24 h. *denotes p = 2×10⁻⁵ vs. P2. The results are the average of two independent transfections performed in triplicate ±S.E. (<b>D</b>) Human ALOX15 (upper panel) and GAPDH (lower panel) gene expression by RT-PCR in MCF-7 cells in presence of vimentin. (<b>E</b>) Luciferase activities of P1 and P2 transfected MCF-7 cells were measured after 24 h. The experiments were done in triplicates. **denotes p<0.0001 between the experimental sets. Transfection of NIH3T3 cells and MCF-7 cells with human ALOX15 cDNA for 24 hr was performed separately as control in gene expression experiments. pRL and pTK luciferase constructs were co-transfected in NIH3T3 and MCF-7 respectively. Luciferase activity was normalized using pRL and pTK luciferase activity in NIH3T3 and MCF-7 respectively. The results are the average of three independent transfections performed in triplicate ± S.E. (<b>F</b>) Western blot of vector (CMV) and vimentin cDNA transfected BPH-1 cells. The cells were harvested after 48 h. The cells were lysed with lysis buffer and loaded on to the SDS-PAGE Gel. The blot was probed with anti-ALOX15 antibody (1∶2000), anti vimentin antibody (1∶1000) and anti-β-actin antibody (1∶7000).</p

Specific <i>in vitro</i> Binding of Vimentin to Promoter Variants.

<p>(<b>A</b>) Biotin-labeled duplex oligos and 10 µg NIH3T3 nuclear extract were used for the experiments. Arrow indicates protein binding to the oligos. (<b>B</b>) Determination of specificity of duplex 29G* by completion with unlabeled duplex oligos. (<b>C</b>) Specific binding of vimentin to 29G* and 29A* oligos was demonstrated with anti-vimentin antibody.</p

Proteomics Profiling of Exosomes from Primary Mouse Osteoblasts under Proliferation versus Mineralization Conditions and Characterization of Their Uptake into Prostate Cancer Cells

Osteoblasts communicate both with normal cells in the bone marrow and with tumor cells that metastasized to bone. Here we show that osteoblasts release exosomes, we termed osteosomes, which may be a novel mechanism by which osteoblasts communicate with cells in their environment. We have isolated exosomes from undifferentiated/proliferating (D0 osteosomes) and differentiated/mineralizing (D24 osteosomes) primary mouse calvarial osteoblasts. The D0 and D24 osteosomes were found to be vesicles of 130–140 nm by dynamic light scattering analysis. Proteomics profiling using tandem mass spectrometry (LC–MS/MS) identified 206 proteins in D0 osteosomes and 336 in D24 osteosomes. The proteins in osteosomes are mainly derived from the cytoplasm (∼47%) and plasma membrane (∼31%). About 69% of proteins in osteosomes are also found in Vesiclepedia, and these canonical exosomal proteins include tetraspanins and Rab family proteins. We found that there are differences in both protein content and levels in exosomes isolated from undifferentiated and differentiated osteoblasts. Among the proteins that are unique to osteosomes, 169 proteins are present in both D0 and D24 osteosomes, 37 are unique to D0, and 167 are unique to D24. Among those 169 proteins present in both D0 and D24 osteosomes, 10 proteins are likely present at higher levels in D24 than D0 osteosomes based on emPAI ratios of >5. These results suggest that osteosomes released from different cellular state of osteoblasts may mediate distinct functions. Using live-cell imaging, we measured the uptake of PKH26-labeled osteosomes into C4-2B4 and PC3-mm2 prostate cancer cells. In addition, we showed that cadherin-11, a cell adhesion molecule, plays a role in the uptake of osteosomes into PC3-mm2 cells as osteosome uptake was delayed by neutralizing antibody against cadherin-11. Together, our studies suggest that osteosomes could have a unique role in the bone microenvironment under both physiological and pathological conditions

Proteomics Profiling of Exosomes from Primary Mouse Osteoblasts under Proliferation versus Mineralization Conditions and Characterization of Their Uptake into Prostate Cancer Cells

Osteoblasts communicate both with normal cells in the bone marrow and with tumor cells that metastasized to bone. Here we show that osteoblasts release exosomes, we termed osteosomes, which may be a novel mechanism by which osteoblasts communicate with cells in their environment. We have isolated exosomes from undifferentiated/proliferating (D0 osteosomes) and differentiated/mineralizing (D24 osteosomes) primary mouse calvarial osteoblasts. The D0 and D24 osteosomes were found to be vesicles of 130–140 nm by dynamic light scattering analysis. Proteomics profiling using tandem mass spectrometry (LC–MS/MS) identified 206 proteins in D0 osteosomes and 336 in D24 osteosomes. The proteins in osteosomes are mainly derived from the cytoplasm (∼47%) and plasma membrane (∼31%). About 69% of proteins in osteosomes are also found in Vesiclepedia, and these canonical exosomal proteins include tetraspanins and Rab family proteins. We found that there are differences in both protein content and levels in exosomes isolated from undifferentiated and differentiated osteoblasts. Among the proteins that are unique to osteosomes, 169 proteins are present in both D0 and D24 osteosomes, 37 are unique to D0, and 167 are unique to D24. Among those 169 proteins present in both D0 and D24 osteosomes, 10 proteins are likely present at higher levels in D24 than D0 osteosomes based on emPAI ratios of >5. These results suggest that osteosomes released from different cellular state of osteoblasts may mediate distinct functions. Using live-cell imaging, we measured the uptake of PKH26-labeled osteosomes into C4-2B4 and PC3-mm2 prostate cancer cells. In addition, we showed that cadherin-11, a cell adhesion molecule, plays a role in the uptake of osteosomes into PC3-mm2 cells as osteosome uptake was delayed by neutralizing antibody against cadherin-11. Together, our studies suggest that osteosomes could have a unique role in the bone microenvironment under both physiological and pathological conditions