Ihh and Runx2/Runx3 Signaling Interact to Coordinate Early Chondrogenesis: A Mouse Model

<div><p>Endochondral bone formation begins with the development of a cartilage intermediate that is subsequently replaced by calcified bone. The mechanisms occurring during early chondrogenesis that control both mesenchymal cell differentiation into chondrocytes and cell proliferation are not clearly understood in vertebrates. Indian hedgehog (Ihh), one of the hedgehog signaling molecules, is known to control both the hypertrophy of chondrocytes and bone replacement; these processes are particularly important in postnatal endochondral bone formation rather than in early chondrogenesis. In this study, we utilized the maternal transfer of 5E1 to E12.5 in mouse embryos, a process that leads to an attenuation of Ihh activity. As a result, mouse limb bud chondrogenesis was inhibited, and an exogenous recombinant IHH protein enhanced the proliferation and differentiation of mesenchymal cells. Analysis of the genetic relationships in the limb buds suggested a more extensive role for Ihh and Runx genes in early chondrogenesis. The transfer of 5E1 decreased the expression of <em>Runx2</em> and <em>Runx3</em>, whereas an exogenous recombinant IHH protein increased <em>Runx2</em> and <em>Runx3</em> expression. Moreover, a transcription factor Gli1 in hedgehog pathway enhances the direct induction of both Runx2 and Runx3 transcription. These findings suggested that Ihh signaling plays an important role in chondrocyte proliferation and differentiation via interactions with Runx2 and Runx3.</p> </div

Microarray and RT-qPCR.

<p>(A) A table showing the fold change in the known chondrogenesis-related genes in limb between the groups injected with either PBS or 5E1 at E12.5. As expected, <i>Gli1, Hhip,</i> and <i>Ptch1</i> were down-regulated. Interestingly, <i>Runx2</i>, <i>Runx3, and Bmp5</i> were down-regulated. <i>Sox9</i>, which is the master gene in chondrogenesis, was not altered by 5E1 treatment. (B) The results of the RT-qPCR are consistent with the microarray data. The amount of each of the RT-qPCR products was normalized using β-2-microglobulin (B2m) as an internal control. Student’s t-test was used for statistical analysis with the level of statistical significance set at (*) <i>p</i><0.01 or (**) <i>p</i><0.05.</p

Skeletal staining with alcian blue and alizarin red and microCT of limb length.

<p>Skeletal staining was attempted with the PBS- and 5E1-injected mice. (A) 6-week old mice exposed to 5E1 at E12.5 exhibit limb, skull, tail and trunk bones that are reduced in size and length, but mice exposed to PBS show the same bone length as wild-type mice. (B) The forelimbs and hindlimbs of E14.5, one-day-old, one-week-old, three-week-old and six-week-old mice were stained with alcian blue and alizarin red. Mice injected with 5E1 have both shorter forelimbs and hindlimbs, and their bones grow similarly to those of PBS-injected mice. (C) The length of the humerus, ulna, and radius in 5E1-injected mice are shorter than in the PBS-injected mice at one-week old, three-week old, and 6-week old; the skeletal staining results agree with those of the microCT analysis. Student’s t-test was employed for statistical analysis, with the level of statistical significance set at (*) <i>p</i><0.01 or (**) <i>p</i><0.05 (Sc; Scapula, S; Stylopod, Z; Zeugopod, A; Autopod, scale bar = 3 mm).</p

The exogenous IHH protein induced proliferation and differentiation of E12.5 limb bud mesenchymal cells and ectopic expression of <i>Runx2</i> and <i>Runx3</i>.

<p>(A, B) Cells were cultured at a density of 2×10⁷ cells/ml with 500 ng/ml of the IHH protein and 130 µg/ml of 5E1. The addition of exogenous IHH protein leads to the production of more cartilage nodules, whereas 5E1 treatment leads to a slight decrease in the formation of cartilage nodules. The value was quantified by measuring the absorbance of the bound alcian blue at 570 nm. (C) After the beads soaked in 1 mg/ml of IHH were implanted into 1.5×10⁶ mesenchymal cell pellets of limb buds at E12.5, followed by incubation for 3 or 24 hours, ectopic <i>Gli1</i>, <i>Runx2</i> and <i>Runx3</i> expression appear strongly around the IHH protein-soaked beads. (D) The <i>Gli1</i>, <i>Runx2</i> and <i>Runx3</i> expression levels are up-regulated by the exogenous IHH protein. Student’s t-test was employed for statistical analysis, with the level of statistical significance set at (*) <i>p</i><0.01 or (**) <i>p</i><0.005 (scale bar = 1 mm in the whole view, and scale bar = 100 µm in the section view of the cell pellets).</p

The Ihh-Gli pathway positively induces Runx2 and Runx3.

<p>(A) Compared to the pGL3-basic, treatment with 10 ng of the Gli1 expression vector leads to a significant increase of wild-type Runx2 containing Gli-binding site (pGL3-Runx2 WT, 500 ng), but the luciferase activity of Runx2 containing mutated Gli-binding site (pGL2-Runx2 Mut, 500 ng) is decreased, compared to pGL3-Runx2 WT. (B) pGL3-Runx3 WT luciferase activity increases significantly with the Gli expression vector, but pGL3-Runx3 Mut luciferase activity decreases than pGL3-Runx3 WT. Student’s t-test was employed for statistical analysis, with the level of statistical significance set at (**) <i>p</i><0.005.</p

Expression of <i>Ihh</i>, <i>Ptch1</i>, <i>Gli1, Runx2, Runx3</i> and <i>Sox9.</i>

<p>(A and A’) Ihh expressed in prehypertrophic chondrocytes. (B and B’) The <i>Ihh</i> expression pattern was not altered when hedgehog signaling one day of 5E1 injection at E12.5. The <i>Ihh</i>-expressing regions in second and third phalanges were connected following the injection of 5E1, but not after injecting PBS. (C and C’, E and E’) <i>Ptch1</i> and <i>Gli1</i> were expressed in the mesenchymal cells surrounding the prehypertrophic chondrocytes. (D and D’, F and F’) <i>Ptch1</i> and <i>Gli1</i> were down-regulated due to blocking the activity of the IHH protein. (G and G’, I and I’) <i>Runx2</i> and <i>Runx3</i> were expressed in mesenchymal cells, similar to <i>Ptch1</i> and <i>Gli1</i>. (H and H’, J and J’) <i>Runx2</i> and <i>Runx3</i> were down-regulated when 5E1 was transferred. (K–L’) <i>Sox9</i> expression did not differ between the PBS- and 5E1-treated groups. p1 (green box), condensation of phalange 1; p2/3 (red and yellow box), unseparated primordium of phalanges 2 and 3; p2/3* (orange box), connecting region of Ihh expression; m, metacarpals; A, anterior; D, distal part of the limb; (A–L, scale bar = 1 mm. A’–L’; longitudinal section view, scale bar = 100 µm.).</p

Frequent Amplification of CENPF, GMNN and CDK13 Genes in Hepatocellular Carcinomas

<div><p>Genomic changes frequently occur in cancer cells during tumorigenesis from normal cells. Using the Illumina Human NS-12 single-nucleotide polymorphism (SNP) chip to screen for gene copy number changes in primary hepatocellular carcinomas (HCCs), we initially detected amplification of 35 genes from four genomic regions (1q21–41, 6p21.2–24.1, 7p13 and 8q13–23). By integrated screening of these genes for both DNA copy number and gene expression in HCC and colorectal cancer, we selected <em>CENPF</em> (centromere protein F/mitosin), <em>GMNN</em> (geminin, DNA replication inhibitor), <em>CDK13</em> (cyclin-dependent kinase 13), and <em>FAM82B</em> (family with sequence similarity 82, member B) as common cancer genes. Each gene exhibited an amplification frequency of ∼30% (range, 20–50%) in primary HCC (n = 57) and colorectal cancer (n = 12), as well as in a panel of human cancer cell lines (n = 70). Clonogenic and invasion assays of NIH3T3 cells transfected with each of the four amplified genes showed that <em>CENPF</em>, <em>GMNN</em>, and <em>CDK13</em> were highly oncogenic whereas <em>FAM82B</em> was not. Interestingly, the oncogenic activity of these genes (excluding <em>FAM82B</em>) was highly correlated with gene-copy numbers in tumor samples (correlation coefficient, r>0.423), indicating that amplifications of <em>CENPF</em>, <em>GMNN</em>, and <em>CDK13</em> genes are tightly linked and coincident in tumors. Furthermore, we confirmed that <em>CDK13</em> gene copy number was significantly associated with clinical onset age in patients with HCC (<em>P = </em>0.0037). Taken together, our results suggest that coincidently amplified <em>CDK13</em>, <em>GMNN</em>, and <em>CENPF</em> genes can play a role as common cancer-driver genes in human cancers.</p> </div

Detection of four amplified genes with high mRNA expression common to both liver and colon cancer.

<p>Among 35 amplified genes in HCC, 8 genes were initially selected as HCC-specifically amplified and overexpressed genes and then four genes (<i>CENPF</i>, <i>GMNN</i>, <i>CDK13</i>, and <i>FAM82B</i>) were ultimately selected as amplified and overexpressed cancer genes common to both liver and colon cancer.</p

Screening of gene amplification in primary tumor tissues and a panel of human cancer cell lines.

<p>The number of gene copies in primary liver tumor tissues (n = 57) and colon tumor tissues (n = 12) was examined using the TaqMan Copy Number Assay System, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043223#s4" target="_blank">Materials and Methods</a>. All paired nontumor tissues had a normal number of gene copies (2 copies) in both HCC and colorectal cancer patients. However, a various numbers of gene copies, especially gene amplification, were detected in the primary liver tumor tissues (A) and colon tumor tissues (B). In addition, copy numbers were also examined in a panel of human cancer cell lines (n = 70) (C).</p

Oncogenic effects of <i>CENPF</i>, <i>GMNN</i> and <i>CDK13</i> after stable transfection in NIH3T3 cells.

<p>Clonogenic assays and invasion assays were performed using stably transfected NIH3T3 cells with <i>CENPF</i>, <i>GMNN</i>, <i>CDK13</i> or <i>FAM82B</i> gene. The images of clonogenic assays (A) and migration assays (B) in a representative experiment were taken at day 14 and 24 h after treatment, respectively, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043223#s4" target="_blank">Materials and Methods</a>. Data representing means and standard deviations of triplicate experiments are shown for (C) clonogenic assays and (D) invasion assays as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043223#s4" target="_blank">Materials and Methods</a> (*<i>P<</i>0.05, **<i>P</i><0.01 versus control group; t-test).</p