Lysyl oxidase knockdown prevents osteoblast development of C3H10T1/2 cells.

<p>C3H10T1/2 cells transduced with two different lysyl oxidase shRNAs or empty virus were grown to confluence and then induced to differentiate as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100669#s2" target="_blank">Materials and Methods</a>. At intervals, cells were fixed and stained with alizarin red. Fixed and stained cultures were then photographed. This experiment was performed twice with the same outcomes.</p

Lysyl oxidase (LOX) regulates C3H10T1/2 cell growth.

<p>C3H10T1/2 cells were transduced with lentiviral particles containing LOX shRNA1850, LOX shRNA 1852, or control shRNA. Non-transduced cells were eliminated using puromycin, a selective reagent. Transduced cells were seeded at 20,000 cells per well in 12-well plates for the growth curve analysis. Total RNA was also collected from these cells to measure lysyl oxidase mRNA levels by real-time PCR. <b>A)</b> The growth curves were plotted for cells transduced with either LOX shRNA 1852 or control shRNA. Data are presented as means ± SD (n = 4). <b>B)</b> The growth curves were plotted for cells transduced with either LOX shRNA 1850 or control shRNA. Data are presented as means ± SD (n = 4). <b>C)</b> The chart shows lysyl oxidase mRNA levels in LOX knockdown and control cells. Two independent LOX shRNA with different shRNA sequences were used in this experiment. Data are presented as means ± SD (n = 3*, p<0.05; Student's t-test).</p

Primers for Site-directed Mutagenesis of the Lysyl Oxidase Promoter.

<p>Primers for Site-directed Mutagenesis of the Lysyl Oxidase Promoter.</p

Wnt3a transcriptionally up-regulates lysyl oxidase mRNA levels in C3H10T1/2 pluripotent progenitor cells.

<p>Serum-depleted cells were treated with Wnt3a- or control-conditioned medium for 24 hours. Total RNA and protein were extracted and subjected to real time PCR and Western blotting analyses. <b>A)</b> The bar graph presents lysyl oxidase mRNA levels in response to Wnt3a in C3H10T1/2 cells (n = 6), mouse primary bone marrow stromal cells (BMSCs, n = 3), rat primary calvarial osteoblasts (n = 9), and a mouse pre-osteoblast cells (MC3T3-E1, n = 3). Data presented for C3H10T1/2 cells, BMSCs and primary calvarial osteoblasts were pooled from two independent experiments; data from MC3T3-E1 cells were from one experiment performed in triplicate. Data shown are means ± SD (*, p<0.05, N.S, not significant; Student's t-test). <b>B)</b> The bar graph shows the fold change of lysyl oxidase transcriptional activity in response to Wnt3a in C3H10T1/2 cells (n = 3). Data are from one representative experiment performed in triplicate of three independent experiments, all showing significantly increased lysyl oxidase transcription after Wnt3a treatment. Data shown are means ± SD (*, p<0.05; Student's t-test). <b>C)</b> The bar graph presents lysyl oxidase protein levels in response to Wnt3a in C3H10T1/2 cells (n = 3). Data shown are means ± SD (*, p<0.05; Student's t-test). <b>D)</b> Lysyl oxidase protein levels in response to Wnt3a in rat primary calvarial cells (n = 6). Data shown are means ± SD N.S, not significant; Student's t-test).</p

A potential cis-element for Wnt3a regulation of lysyl oxidase is located at −1321 to −1328 bp upstream of lysyl oxidase translation start site.

<p>Three putative TCF/LEF cis-elements within the first 1.5 kbp of the murine lysyl oxidase promoter (pLOXFL) were mutated by site-directed mutagenesis individually, in pairs, and all three together. C3H10T1/2 cells were transfected with a Renilla luciferase thymidine kinase (pRL-TK) and either wild type pLOXFFL or mutant pLOXFFL reporter constructs. After 24 hours, transfected cells were serum starved and treated with Wnt3a- or control-conditioned media for 24 hours. Luciferase activity was assessed as explained in Experimental Procedures. The fold change of lysyl oxidase transcriptional activity of wild-type and mutated reporter constructs in response to Wn3a are presented as means ± SD. Data are pooled from three independent experiments (n = 9; *, p<0.05; Student's t-test).</p

TNF-α post-transcriptionally attenuates Wnt3a-stimulated lysyl oxidase mRNA levels.

<p><b>A)</b> The effect of TNF-α on Wnt3a-stimulated lysyl oxidase mRNA levels in C3H10T1/2 cells was examined by treating cells with Wnt3a- or control-conditioned media in the presence or absence of increasing concentrations of TNF-α. Lysyl oxidase mRNA analysis of total RNA extracted from these cells was performed by real time PCR. Lysyl oxidase mRNA levels were normalized to the levels of GAPDH mRNA. Data are means ± SD (n = 3; *, p<0.05; Student's t-test). <b>B)</b> Luciferase reporter was used to functionally assess lysyl oxidase transcriptional activity in response to TNF-α. Data are means ± SD (n = 6, N.S, not significant; Student's t-test). Data shown were pooled from two independent experiments.</p

TNF-α reduces Wnt3a-stimulated lysyl oxidase mRNA stability.

<p>Serum starved C3H10T1/2 were pre-treated with Wnt3a- or conditioned media for 16 hours and then treated with or without TNF-α (20 ng/ml) in the presence of dichlorobenzimidazole riboside (an inhibitor of mRNA transcription) for various intervals. Total RNA was isolated from cell lysates and subjected to real time PCR. Data were plotted as percent log remaining lysyl oxidase mRNA levels (normalized to 18 s rRNA mRNA levels) vs time. The TNF-α induced loss of lysyl oxidase mRNA stability was calculated from the relative slopes of the lines of best fit. Student's t-test statistical analyses were performed by comparing the slopes and intercepts of these two lines and showed a P value of 0.006 (GraphPad Prism 5). Data are means ± SD (n = 3 for each time point) and are from one of two representative experiments with the same outcome.</p

Wild-type and Mutant Sequences of TCF/LEF Elements in the Lysyl Oxidase Promoter.

<p><b>TCF/LEF consensus sequence: CTTTG(A/T)(A/T).</b></p

Analysis of miR203 functionality to down-regulate lysyl oxidase mRNA levels.

<p><b>A)</b> miR203 down-regulates lysyl oxidase mRNA levels: C3H10T1/2 cells were transfected with miR203 mimic or non-specific control micro RNA. Cell lysates were collected after five days post-transfection and total extracted RNA subjected to real time PCR analysis for lysyl oxidase and 18S rRNA mRNA for normalization. Data are presented as means ± SD (n = 3; *, p<0.05; Student's t-test). Data are from one of two independent experiments with the same outcome. <b>B</b>) To functionally evaluate TNF-α up-regulation of functional miR203 in C3H10T1/2 cells, cells were transfected with the miR203 reporter plasmid miR203-RenSPL and R01-RenSPL (control) constructs. 24 Hours post-transfection, cells were serum starved and pre-treated with (i) Wnt3a- or (ii) control conditioned media for 24 hours. Cells were then treated with or without TNF-α (20 ng/ml) for 24 hours. Cell lysates were collected and subjected to a luciferase activity assay. Panel (iii) is from cells treated only with or without Wnt3a showing that this reporter for miR203 activity is not affected by Wmt3a alone, as expected. Data are presented as means ± SD (n = 6; *, p<0.05; N.S, not significant; Student's t-test). Data shown are from one of two independent experiments with the same outcomes.</p

Micro RNAs Increased More Than 4-fold by TNF-α in Wnt3a-stimulated C3H10T1/2 Cells.

<p>Micro RNAs Increased More Than 4-fold by TNF-α in Wnt3a-stimulated C3H10T1/2 Cells.</p