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 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 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

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

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

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

Contribution of <i>FGFR1</i> Variants to Craniofacial Variations in East Asians

<div><p><i>FGFR1</i> plays an important role in the development of the nervous system as well as the regulation of the skeletal development and bone homeostasis. Mutations in <i>FGFR1</i> genes affect skull development, specifically suture and synchondrosis, resulting in craniosynostosis and facial abnormalities. We examined subjects with normal skull morphology for genetic polymorphisms that might be associated with normal craniofacial variations. Genomic DNA was obtained from 216 Japanese and 227 Korean subjects. Four <i>FGFR1</i> SNPs, namely, rs881301, rs6996321, rs4647905, and rs13317, were genotyped. These SNPs were tested for association with craniofacial measurements obtained from lateral and posteroanterior cephalometries, in which principle component analysis was performed to compress the data of the craniofacial measurements. We observed that SNPs rs13317 and rs6996321 were correlated with the overall head size and midfacial development, indicating that <i>FGFR1</i> SNPs played crucial roles in the normal variation of human craniofacial morphology. Subjects with the derived alleles of SNPs rs13317 and rs6996321 had a small face and a facial pattern associated with a retruded midface and relatively wide-set eyes. These facial features were similar to but were milder than those of individuals with Pfeiffer syndrome, which is caused by a dysfunctional mutation in <i>FGFR1</i>.</p></div

Allele frequencies and LD coefficients of <i>FGFR1</i> SNPs.

<p>Allele frequencies and LD coefficients of <i>FGFR1</i> SNPs.</p

PC loading for each PC. (A) PCA of cranial measurements. (B) PCA of mandibular measurements.

<p>PC loading for each PC. (A) PCA of cranial measurements. (B) PCA of mandibular measurements.</p

Lateral and posteroanterior cephalometric tracing showing the landmarks used to obtain craniofacial measurements.

<p>(V) Vertex, (Eu) eunion, (Lo) latero-orbitale, (Or) orbitale, (Zy) zygion, (Cd) condylion, (Ko) Koronoid, (Ma) mastoid, (NC) nasal cavity, (Cr) crista galli, (ANS) anterior nasal spine, (Go) gonion, (Ag) antegonion, (Me) menton, (G) glabella, (N) nasion, (S) sella turcica, (SOr) supra orbitale, (R) rhinion, (KR) key ridge, (Pr) prosthion, (A) point A, (PNS) posterior nasal spine, (Id) infradentale, (Gn) gnathion. The NA plane was used as a reference to measure the anteroposterior position of G, SOr, R, Or, and KR, with positive and negative values indicating whether the landmark is in an anterior and posterior direction, respectively, from the NA plane.</p