BMP-2 Induced Expression of Alx3 That Is a Positive Regulator of Osteoblast Differentiation

<div><p>Bone morphogenetic proteins (BMPs) regulate many aspects of skeletal development, including osteoblast and chondrocyte differentiation, cartilage and bone formation, and cranial and limb development. Among them, BMP-2, one of the most potent osteogenic signaling molecules, stimulates osteoblast differentiation, while it inhibits myogenic differentiation in C2C12 cells. To evaluate genes involved in BMP-2-induced osteoblast differentiation, we performed cDNA microarray analyses to compare BMP-2-treated and -untreated C2C12 cells. We focused on <i>Alx3</i> (aristaless-like homeobox 3) which was clearly induced during osteoblast differentiation. <i>Alx3</i>, a homeobox gene related to the <i>Drosophila</i><i>aristaless</i> gene, has been linked to developmental functions in craniofacial structures and limb development. However, little is known about its direct relationship with bone formation. In the present study, we focused on the mechanisms of <i>Alx3</i> gene expression and function during osteoblast differentiation induced by BMP-2. In C2C12 cells, BMP-2 induced increase of <i>Alx3</i> gene expression in both time- and dose-dependent manners through the BMP receptors-mediated SMAD signaling pathway. In addition, silencing of <i>Alx3</i> by siRNA inhibited osteoblast differentiation induced by BMP-2, as showed by the expressions of alkaline phosphatase (<i>Alp</i>), <i>Osteocalcin</i>, and <i>Osterix</i>, while over-expression of <i>Alx3</i> enhanced osteoblast differentiation induced by BMP-2. These results indicate that <i>Alx3</i> expression is enhanced by BMP-2 via the BMP receptors mediated-Smad signaling and that Alx3 is a positive regulator of osteoblast differentiation induced by BMP-2.</p> </div

BMP-2 induced <i>Alx3</i> expression through the SMAD signaling pathway.

<p>C2C12 cells were pretreated with <i>Smad4</i> siRNA and 100 nM of Dorsomorphin, followed by treatment with or without BMP-2 for 3 days. (A) The expression of <i>Smad4</i> was examined by real-time PCR and Western blotting. (B, C) The expression of <i>Alx3</i> was examined by real-time PCR. ** <i>p</i> < 0.01 by Student’s <i>t</i> test.</p

Effect of Alx3 over-expression on BMP-2-induced osteoblast differentiation.

<p>(A) Western blot analysis of Alx3 was performed using C2C12 cells transfected with empty (<i>Mock</i>) or <i>Alx3-FLAG</i> expression (<i>Alp</i>) vectors. Equal protein loading was documented by blotting for β-actin. (B) The expressions of <i>Alp</i> and <i>Ocn</i> were quantified by real-time PCR. (C, D) Measurement of ALP activity and ALP staining. ** <i>p</i> < 0.01, * <i>p</i> < 0.05 by Student’s <i>t</i> test. (E) Schematic diagram of upstream region of mouse <i>Alp</i> gene showing locations of putative Alx3-binding sites tested in ChIP analyses. Arrowheads indicate the positions of the primers used for ChIP analysis. ChIP analyses were performed using DNA fragments immunoprecipitated with a FLAG antibody or isotype-specific control antibody. Immunoprecipitates were PCR amplified with primers flanking the putative Alx3-binding region. Ab, antibody; BS, binding site.</p

BMP-2-induced <i>Alx3</i> gene expression during osteoblast differentiation in C2C12 cells.

<p>(A) Semi-quantitative RT-PCR analyses of <i>Alp</i>, <i>Ocn</i>, and <i>Myogenin</i> gene expressions. (B) Double staining for α-MHC (<i>red; arrowheads</i>) and ALP (<i>blue</i>) as markers of differentiation for mature myotubuls and osteoblasts, respectively. (C) Semi-quantitative RT-PCR analyses of <i>Alx3</i>, <i>Cart1</i> and <i>Alx4</i> gene expressions.</p

BMP-2 induced <i>Alx3</i> expression in time- and dose- dependent manners through the Smad signaling pathway.

<p>(A) Dose effects of BMP-2 on <i>Alx3</i> expression. C2C12 cells were treated with 10, 30, 100, 300, or 1000 ng/ml BMP-2 for 3 days. (B) Time course analysis of BMP-2 effects on <i>Alx3</i> expression. C2C12 cells were treated with or without 300 ng/ml of BMP-2 for 1, 2, 3, or 4 days.</p

Effect of <i>Alx3</i> siRNA knockdown on BMP-2-induced osteoblast differentiation.

<p>(A) C2C12 cells were pretreated with <i>Alx3</i> siRNA, followed by treatment with or without BMP-2 for 3 days. The expressions of <i>Alx3</i>, <i>Alp</i>, <i>Ocn</i> (<i>Osteocalcin</i>), and <i>Osx</i> (<i>Osterix</i>) were quantified by real-time PCR. (B) Measurement of ALP activity and ALP staining. ** <i>p</i> < 0.01 by Student’s <i>t</i> test. (C) Effect of Alx3 siRNA knockdown on BMP-2-induced phosphorylation of Smad1/5. C2C12 cells were pretreated with <i>Alx3</i> siRNA, followed by treatment with BMP-2 for 15, 30, and 60 minutes.</p