Functional analysis of Rptpζ mutations.

<p>(A) qRT-PCR monitoring expression of <i>BGLAP</i> (left) and <i>PTPRZ1</i> (right) in human primary osteoblasts (hObl), SaOS-2 or U2-OS cells at different stages of differentiation. Values represent copy number (cn) relative to <i>GAPDH</i>. Bars represent mean ± SD (n = 3). Asterisks indicate statistical significance vs. day 0 (p<0.05, Kruskal-Wallis followed by Dunn’s post-test). (B) Schematic presentation of the <i>PTPRZ1</i> gene and Rptpζ protein showing the location of the two mutations that have been introduced into a Rptpζ expression plasmid. (C) BrdU incorporation assay with SaOS-2 and U2-OS cells after transfection of wildtype and/or mutant Rptpζ expression plasmids as indicated. Bars represent mean ± SD (n = 6). Asterisks indicate significant differences towards cells transfected with empty vector (ctrl) (p<0.05, one-way ANOVA followed by Dunnett’s post-test).</p

Genes encoding PTPs displaying differential expression during primary osteoblast differentiation.

<p>Given are the Affymetrix signal intensities at two stages of differentiation and the signal log ratios (SLR).</p

The Protein Tyrosine Phosphatase Rptpζ Suppresses Osteosarcoma Development in <i>Trp53</i>-Heterozygous Mice

<div><p>Osteosarcoma (OS), a highly aggressive primary bone tumor, belongs to the most common solid tumors in growing children. Since specific molecular targets for OS treatment remain to be identified, surgical resection combined with multimodal (neo-)adjuvant chemotherapy is still the only way to help respective individuals. We have previously identified the protein tyrosine phosphatase Rptpζ as a marker of terminally differentiated osteoblasts, which negatively regulates their proliferation <i>in vitro</i>. Here we have addressed the question if Rptpζ can function as a tumor suppressor protein inhibiting OS development <i>in vivo</i>. We therefore analyzed the skeletal phenotype of mice lacking <i>Ptprz1</i>, the gene encoding Rptpζ on a tumor-prone genetic background, i.e. <i>Trp53</i>-heterozygosity. By screening a large number of 52 week old <i>Trp53</i>-heterozygous mice by contact radiography we found that <i>Ptprz1</i>-deficiency significantly enhanced OS development with 19% of the mice being affected. The tumors in <i>Ptprz1</i>-deficient <i>Trp53</i>-heterozygous mice were present in different locations (spine, long bones, ribs), and their OS nature was confirmed by undecalcified histology. Likewise, cell lines derived from the tumors were able to undergo osteogenic differentiation <i>ex vivo</i>. A comparison between <i>Ptprz1</i>-heterozygous and <i>Ptprz1</i>-deficient cultures further revealed that the latter ones displayed increased proliferation, a higher abundance of tyrosine-phosphorylated proteins and resistance towards the influence of the growth factor Midkine. Our findings underscore the relevance of Rptpζ as an attenuator of proliferation in differentiated osteoblasts and raise the possibility that activating Rptpζ-dependent signaling could specifically target osteoblastic tumor cells.</p></div

Genes encoding osteogenesis markers displaying differential expression during primary osteoblast differentiation.

<p>Given are the Affymetrix signal intensities at two stages of differentiation and the signal log ratios (SLR).</p

Osteogenic differentiation of OS cell lines.

<p>(A) Alizarin red staining of cells derived from <i>Trp53</i>-heterozygous mice with either one <i>Ptprz1</i> allele (+/-) or with <i>Ptprz1</i>-deficiency (-/-) reveals that both cell lines are able to form a mineralized matrix after 10 and 20 days of differentiation induced by ascorbic acid and ß-glycerophosphate. (B-D) qRT-PCR expression analysis shows that the differentiation is accompanied by increased expression of <i>Bglap</i> (encoding Osteocalcin), <i>Ibsp</i> (encoding Bone sialoprotein) and <i>Phex</i>. Values represent copy number (cn) relative to <i>Gadph</i>. Bars represent mean ± SD (n = 3). Asterisks indicate significant differences towards day 0 of corresponding genotype (p<0.05, Kruskal-Wallis followed by Dunn’s post-test).</p

Proliferation and tyrosine phosphorylation in OS cell lines.

<p>(A) Proliferative capacity of tumor cells derived from <i>Trp53</i>-heterozygous mice with either one <i>Ptprz1</i> allele (+/-) or with <i>Ptprz1</i>-deficiency (-/-). The growth curves (left) and the BrdU incorporation assays (right) demonstrate increased proliferation in the cases of <i>Ptprz1</i>-deficiency. Bars represent mean ± SD (n≥3). Asterisks indicate significant differences between the two genotypes (p<0.05, two-way ANOVA followed by Bonferroni’s post-test (left panel) or two-tailed Student’s <i>t</i>-test (right panel)). (B) SH2 profiling with different SH2 domains reveals differences in tyrosine phosphorylation of specific proteins (indicated by arrowheads). Re-probing of stripped membranes with anti ß-actin mAb served as control for equal loading. Analyses were performed in duplicate with cell extracts harvested at different cell densities.</p

<i>Ptprz1</i> expression by differentiated osteoblasts.

<p>(A) qRT-PCR monitoring <i>Ptprz1</i> expression in brain (Br), femur (Fe), calvaria (Ca), fat (Fa), heart (He), kidney (Ki), liver (Li), lung (Lu) and spleen (Sp). (B) qRT-PCR monitoring <i>Ptprz1</i> expression in primary osteoblasts (Obl) or osteoclasts (Ocl) at different stages of differentiation. Bars represent mean ± SD (n = 3). (C) qRT-PCR monitoring expression of the osteocyte marker <i>Phex</i>. (D) qRT-PCR monitoring expression of the osteoclast marker <i>Acp5</i> (encoding TRAP). Values represent copy number (cn) relative to <i>Gadph</i>. Bars represent mean ± SD (n = 3). Asterisks indicate statistical significance vs. Obl day 0 (p<0.05, Kruskal-Wallis followed by Dunn’s post-test).</p

Effects of Mdk on proliferation of OS cell lines.

<p>(A) BrdU incorporation assays with the indicated OS cell lines performed in the presence of Mdk at different concentrations. Bars represent mean ± SD (n = 8). Asterisks indicate significant differences towards controls without Mdk (p<0.05, one-way ANOVA followed by Dunnett’s post-test). (B) Growth curves of OS cell lines in the presence or absence of Mdk (100 ng/ml). Asterisks indicate significant differences towards controls of <i>Ptprz1</i>^<i>+/-</i> cells without Mdk (p<0.05, two-way ANOVA followed by Bonferroni’s post-test).</p

Skeletal tumors in <i>Ptprz1</i>-deficient <i>Trp53</i>-heterozygous mice.

<p>(A) OS development assessed by screening of 12 month old <i>Trp53</i>-heterozygous mice with the indicated <i>Ptprz1</i> genotypes. The left panel shows the number of analyzed mice (white bars) and the percentage of mice with OS (black bars). The asterisk indicates statistical significance vs. <i>Trp53</i>^<i>+/-</i><i>/Ptprz1</i>^<i>+/+</i> (p<0.05, two-tailed Fishers`s exact test). The right panel shows the total number of tumors and their location. (B) Representative contact Xrays from <i>Ptprz1</i>-deficient <i>Trp53</i>-heterozygous mice with OS in the three different locations. (C) μCT images from the same tumors.</p