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

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 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

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

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

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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

SPIN90 phosphorylation modulates its association with PSD scaffolding proteins.

<p>(<b>A</b>) <i>In vitro</i> binding assays carried out with GST-SPIN90 WT, YE or YF agarose beads and lysates from crude synaptosomal fraction. The bound proteins were verified using anti-PSD95 and anti-SPIN90 antibodies. (<b>B</b>) The interaction between SPIN90 and PSD95 was analyzed in HEK293T cells co-transfected with HA-PSD95 plus GFP-SPIN90 WT, YF or YE. (<b>C</b>) In BiFC assays, HEK293T cells and cultured hippocampal neurons co-expressing VN-PSD95 and VC-SPIN90 WT, VC-SPIN90 YE or VC-SPIN90 YF were monitored for the venus signal (green). Cells were also immunostained with anti-Flag antibody to detect VN-PSD95, and with anti-HA antibody to detect VC-SPIN90. Scale bars, 10 µm in HEK293T cells and 20 µm in neurons. (<b>D</b>) The interaction between SPIN90 and Shank1b was analyzed in HEK293T cells co-transfected with HA-Shank1b plus GFP-SPIN90 WT, YF or YE.</p

SPIN90 is phosphorylated by Src kinase.

<p>(<b>A</b>) Src-catalyzed SPIN90 phosphorylation was tested in SYF (deficient in Src, Yes, Fyn) and c-Src recovery cells in SYF cell lines. (<b>B</b>) COS7 cells were transfected with empty vector, Src KD, Src WT or Src CA together with GFP-SPIN90 and immunoprecipitated with anti-GFP antibody. (<b>C</b>) Schematic diagram of SPIN90 showing the positions of tyrosine phosphorylated residues. The indicated domains are as follows: SH3, Src homology 3; PRD, proline-rich domain. (<b>D</b>) <i>In vitro</i> phosphorylation of GST-SPIN90 proteins. Bacterially overexpressed GST, GST-SPIN90 domains or mutant proteins were incubated with recombinant Src (5 U) for 10 min at 30°C. Phosphorylated products were revealed by autoradiography.</p

Phosphorylated SPIN90 is localized to spines in neurons.

<p>(<b>A</b>,<b>B</b>) Rat hippocampal neurons transfected with GFP-SPIN90 at DIV 10-12 were fixed at DIV 19–21 and labeled with the indicated antibodies. (<b>C</b>) Mouse brain lysates were fractionated to generate crude synaptosomal (P2) and cytosolic fractions (S2), and equal amount of protein was examined by immunoblotting. (<b>D</b>) Rat cortical neurons (DIV 18) were incubated with or without PP2 (10 µM for 30 min), after which SPIN90 phosphorylation was assessed using anti-phosphotyrosine antibody (pY).</p