The amino acid sequence of rat chondromodulin-I (rChM-I).

<p>Light gray circles indicate amino acid residues that are not conserved in human ChM-I (hChM-I). Circles in parentheses indicate four amino acid residues that are not conserved in mouse (mChM-I). Four thick bars connecting a pair of Cys residues indicate the intramolecular disulfide bonds, whose arrangements are assumed to be identical with those determined for bChM-I purified from fetal bovine cartilage <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094239#pone.0094239-Neame1" target="_blank">[27]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094239#pone.0094239-Hiraki3" target="_blank">[28]</a>. The putative glycosylation sites are also indicated. The arrowhead indicates the determined cleavage site that gives rise to the 14-kDa form of ChM-I.</p

Effects of 14-kDa ΔN-rhChM-I on the VEGF-A-induced chemotactic migration of HUVECs.

<p>Biological activity of G-rhChM-I and ΔN-rhChM-I was assessed by a modified Boyden chamber assay <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094239#pone.0094239-Miura1" target="_blank">[8]</a>. Serum-starved HUVECs (7×10⁴ cells) were preincubated for 30 min with G-rhChM-I (•) or ΔN-rhChM-I (▴), and seeded onto vitronectin-coated cell culture inserts in serum-free medium. Chemotactic migration of HUVECs was induced by the addition of VEGF-A (20 ng/ml) in the lower chamber, and the cells were allowed to migrate for 4 h toward VEGF-A. The number of cells that had migrated to the bottom surface of the insert was counted. Values are the percentages of migrated cell numbers in the control culture that was stimulated by VEGF-A, and are the means ± SD of a triplicate assay. The data are representative of three independent experiments with similar results.</p

Localization of MMP9 and MMP13 in mouse developing bone at E16.5.

<p>Frozen sections were prepared from legs of an E16.5 mouse embryo. Serial sections of proximal tibia were stained with MMP9 antibody (red; A), MMP13 antibody (red; B), hCHM-05 ChM-I MoAb (green; C) or N-ChM-I Ab (green; D). The nuclei were counterstained with DAPI (blue). Asterisks indicate the hypertrophic cartilage zone. Arrowheads indicate the boundary between cartilage and the invading front of vasculature. Scale bars, 100 μm.</p

Identification and immunoprecipitation of rat 14-kDa ChM-I.

<p>(A) 8 M urea extracts were prepared from male rat ribs (4-week-old) and subjected to immunoblotting with hCHM-05 ChM-I MoAb. Rat 20–25 kDa glycosylated ChM-I (asterisk) was detected as a broad band as well as a 14-kDa band (thick arrow) and a faint 17-kDa band (arrow). (B) Rat ChM-I extracted in 8 M urea buffer was immunoprecipitated with hCHM-05. Immunoprecipitates were resolved by SDS-PAGE and detected by silver staining. For the specification of immunoprecipitated bands (Ex + Ab), cartilage extracts without the antibody (Ex) or 8 M urea buffer with the antibody (Ab) were similarly processed during the immmunoprecipitation. The asterisk and the arrow indicate 20–25 kDa and 14-kDa ChM-I, respectively. (C) Primary cultures of rat costal chondrocytes were cultured to confluence for 3 days and then conditioned in DMEM/F12 medium in the presence of 10% FBS for the indicated periods of time. The collected conditioned medium was concentrated and subjected to immunoblotting with hCHM-05 ChM-I MoAb.</p

Differential distribution of 14-kDa ΔN-ChM-I and 20–25 kDa ChM-I in mouse developing bone at E16.5.

<p>Frozen sections were prepared from legs of an E16.5 mouse embryo. (A) Toluidine blue (TB) staining of the sagittal section of tibia and fibula. (B, C) Immunofluorescent views of the boxed area in panel A. Double immunofluorescent staining of the sagittal section of proximal tibia with CD31 (red) and N-ChM-I Ab (green; B) or hCHM-05 ChM-I MoAb (green; C). (D) Double immunofluorescent staining of the sagittal section of proximal tibia with N-ChM-I Ab (green) and hCHM-05 ChM-I MoAb (red). (E, F) Absorption test for hCHM-05 ChM-I MoAb is shown. The semiserial sections of tibia was immunostained with hCHM-05 ChM-I MoAb (green) preincubated with BSA (E) or G-rhChM-I (F). In panels D, E, and F, the nuclei were counterstained with DAPI (blue). Asterisks in the panels B-D indicate hypertrophic cartilage zone. Scale bars, 100 μm.</p

Staining for β-catenin proteins is increased adjacent to the injured site in the rat Achilles tendon.

<p><b>(A, B)</b> The Achilles tendon (AT) between calcaneum (C) and gastrocnemius (G) was punctuated with a 14-gauge needle (N) at the injured site (IS). Scale bar = 2 mm. <b>(C, D)</b> Hematoxylin-eosin staining of sagittal sections of injured <b>(C)</b> and sham-operated tendons <b>(D)</b> on postoperative day 14. Position of IS with abundant inflammatory cells is indicated by a double-headed arrow in <b>C</b>. Tendons are placed with the distal side on the left and the proximal side on the right. Scale bar = 200 μm. <b>(E-G)</b> High magnification of the areas indicated in <b>C</b> and <b>D</b>. <b>(E)</b> An image distant from IS and close to the calcaneum. <b>(F</b>) An image adjacent to IS and near the center of the tendon. <b>(G)</b> An image in sham-operated tendon. Scale bar = 200 μm. <b>(H)</b> Immunostaining for β-catenin (green) with DAPI (blue). Scale bar = 5 μm. <b>(I)</b> The ratio of β-catenin-positive cells in the field of ~36,000 μm² is indicated by mean and SD (<i>n</i> = 3 rats each). The numbers of cells counted in the ~36,000 μm²-image field for Distal to IS, Proximal to IS, and Sham tendon are 21–56 cells, 29–72 cells, and 28–55 cells, respectively. The number of β-catenin positive cells is divided by the number of DAPI-positive cells in the field. <b>(J)</b> Mean and SD (<i>n</i> = 3 rats each) of intensities of total cellular and nuclear β-catenin signals of the tendon cells indicated in <b>(I)</b>. Each intensity is normalized by the number of DAPI-positive cells. <i>p</i> < 0.05 by one-way ANOVA. *<i>p</i> < 0.05, **<i>p</i> < 0.01 by Tukey-Kramer post-hoc test.</p

TGF-β signaling has no significant effect on <i>MKX</i> and <i>TNMD</i> expressions in <i>SCX</i>-programmed tendon progenitors (hMSC-Scx cells).

<p><b>(A)</b> Quantitative RT-PCR of <i>SMAD2</i> and <i>SMAD3</i> expressions in hMSC-Scx cells treated with 1 μM BIO for 24 hrs. Each mRNA expression is normalized by <i>GAPDH</i> mRNA. <b>(B)</b> Western blotting for Smad2, Smad3, and phosphorylated Smad2/3 (p-Smad2/3) in hMSC-Scx cells treated with 1 μM BIO for 48 hrs, as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182051#pone.0182051.g004" target="_blank">Fig 4A and 4B</a>. Band intensities are normalized by β-actin or total-Smad2/3. <b>(C, D)</b> Relative expressions of <i>AXIN2</i>, <i>MKX</i>, and <i>TNMD</i> in hMSC-Scx cells treated with 0 to 8 ng/ml TGF-β1 <b>(C)</b>, or 0 to 8 μM SD208 (an inhibitor of TGF-β signaling) <b>(D)</b>, for 48 hrs, as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182051#pone.0182051.g003" target="_blank">Fig 3</a>. <b>(E)</b> Quantitative RT-PCR of <i>MKX</i> and <i>TNMD</i> expressions in hMSC-Scx cells treated with 1 μM BIO with or without 2 ng/ml TGF-β1 for 48 hrs. Each mRNA expression is normalized by <i>GAPDH</i> mRNA. All samples in <b>A</b>, <b>B</b>, and <b>E</b> were incubated under 0.002% DMSO. <b>(A, B)</b> **<i>p</i> < 0.01 by unpaired Student’s <i>t</i>-test. <b>(C-E)</b> Tukey-Kramer post-hoc test (*<i>p</i> < 0.05, **<i>p</i> < 0.01) is performed only when <i>p</i> < 0.05 by one-way ANOVA. <b>(A-E)</b> Mean and SD of three independent wells are indicated. <b>(F)</b> A schematic showing induction and suppression of gene expressions of <i>Scx</i>, <i>Mkx</i>, and <i>Tnmd</i> by TGF-β signaling and Wnt/β-catenin in tendon cells. Wnt/β-catenin suppresses expression of <i>Tnmd</i> via <i>Scx</i> and <i>Mkx</i>. Direct suppression of <i>Tnmd</i> by Wnt/β-catenin remain to be determined. Arrows from <i>Scx</i> and <i>Mkx</i> to <i>Tnmd</i> are based on a previous report observed in mouse bone marrow-derived mesenchymal stem cells (BMMSCs) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182051#pone.0182051.ref038" target="_blank">38</a>].</p

Identification by immunoblotting of a 14-kDa fragment of ChM-I in mouse cartilage extracts.

<p>(A) Cartilage extracts of 14-week-old male mice were prepared from ribs of wild-type (wt) and <i>Chm1</i> (-/-) null mice, and analyzed by immunoblotting with N-ChM-I Ab or hCHM-05 ChM-I MoAb. N-ChM-I Ab recognizes mouse 22-kDa glycosylated mature ChM-I (asterisks) as a broad immunoreactive band, whereas hCHM-05 ChM-I MoAb indicated the presence of a 14-kDa band (thick arrow) as well as a faint 17-kDa band (arrow). None of these bands were detected in the extracts from cartilage of <i>Chm1</i> (-/-) null mice. (B) Mouse tissue extracts were prepared from E11.5 whole embryos, and limbs dissected out at E13.5 and E16.5 as well as rib cartilage from 14-week-old male mice, and analyzed by immunoblotting with hCHM-05 ChM-I MoAb or β-actin antibody as a loading control. Note that the loading amount of rib cartilage extracts was reduced to 2.5 μg protein to give a quantitative range of signals in the same blot, while 10 μg protein was loaded per each lane for embryonic tissue extracts.</p

Activation of TGF-β signaling increases mRNA expression of <i>Scx</i>.

<p>Relative expressions of <i>Scx</i>, <i>Mkx</i>, <i>Tnmd</i>, and <i>Axin2</i> in TDCs treated with 0 to 8 ng/ml TGF-β1 <b>(A)</b> or 0 to 8 μM SD208 (an inhibitor of TGF-β signaling) <b>(B)</b> for 72 hrs. Each mRNA expression is normalized by <i>Gapdh</i> mRNA. Mean and SD are indicated (<i>n</i> = 3 wells each). Tukey-Kramer post-hoc test (*<i>p</i> < 0.05, **<i>p</i> < 0.01) is indicated only when <i>p</i> < 0.05 by one-way ANOVA.</p

Activation of Wnt/β-catenin signaling reduces total and phosphorylated Smad2/3 proteins, and suppresses <i>Scx</i> expression in TDCs.

<p><b>(A, C)</b> Western blotting for Smad2, Smad3, and phosphorylated Smad2/3 (p-Smad2/3) proteins. Rat TDCs were treated with 4 μM BIO for 48 hrs <b>(A)</b> or with 4 μM BIO for 48 hrs followed by treatment with 2 ng/ml TGF-β1 for 30 min <b>(C)</b>. The mean and SD of band intensities of three independent wells are indicted. Band intensities are normalized by β-actin or total-Smad2/3. <b>(B)</b> Quantitative RT-PCR of <i>Smad2</i> and <i>Smad3</i> in TDCs treated with 4 μM BIO for 24 hrs. Mean and SD are indicated (<i>n</i> = 3 wells each). <b>(D)</b> Quantitative RT-PCR of <i>Scx</i>, <i>Mkx</i>, and <i>Tnmd</i> in TDCs treated with 4 μM BIO with or without 2 ng/ml TGF-β1 for 48 hrs. Each mRNA expression is normalized by <i>Gapdh</i> mRNA. Mean and SD are indicated (<i>n</i> = 3 wells each). All samples in <b>A-D</b> were added with 0.008% DMSO, because BIO was dissolved in DMSO. <b>(A, B)</b> *<i>p</i> < 0.05, ** <i>p</i> < 0.01 by unpaired Student’s <i>t</i>-test. <b>(C, D)</b> <i>p</i> < 0.05 by one-way ANOVA for all groups. *<i>p</i> < 0.05, **<i>p</i> < 0.01 by Tukey-Kramer post-hoc test.</p