PMC

Expression of Msx2 mRNA in calcified regions of ligament was reduced compared to expression in normal regions in patients with ossification of the posterior longitudinal ligament (PLL). Semiquantitative RT-PCR analysis of Msx2 expression was performed with ligament tissues at the calcified and adjacent normal region of the PLL from the spine of patients with OPLL. Computed tomography (CT) and histological sections stained with hematoxylin and eosin (HE) revealed that a portion of the PLL from each patient had been ossified.

Model for Msx2 function in osteoblasts and ligament fibroblasts. (A) Runx2/Osf2 transcriptional activity is cooperatively repressed by two corepressors, Msx2 and TLE1, recruiting the HDAC complex. Attenuation of Msx2 expression after the middle stage of osteoblast differentiation is important for further Runx2/Osf2 activation, which induces differentiation and maximal matrix mineralization. Msx2 also has a direct function on the cell cycle by regulating target genes such as cyclin D1. (B) In ligament fibroblasts, Msx2 acts as a molecular defense mechanism for preventing ossification, as expression of the Msx2 gene is highly maintained. See the Discussion for details.

Msx2 repressed Runx2/Osf2 transcriptional activity. Transcriptional activity frome the OSE2 sequence of Runx2/Osf2 was assayed with luciferase reporter assays. Each cell line was transfected with p6OSE2-Luc, in which luciferase expression is controlled by six copies of the Runx2 binding OSE2 site of the OCN promoter, followed by the minimal promoter, and the indicated expression constructs. Luciferase activity was determined 24 h after transfection. The relative amount used for the transfection of each construct is indicated as ×N, where N is 1, 2, or 4. Note that the reduction of endogenous Msx2 by transfection with the Msx2 antisense (Msx2 AS) RNA expression plasmid (×2) enhanced Runx2/Osf2 transcriptional activity in PDL-L2 cells (D). See the text for details.

Msx2 and TLE1 cooperatively repress Runx2/Osf2 transcriptional activity by recruiting the HDAC complex. (A to C and F) Transcriptional activity from the OSE2 sequence of Runx2/Osf2 was assayed as in Fig. . Each cell line was transfected with p6OSE2-Luc and the indicated expression constructs. As for Msx2 antisense, the same amount (×2) was used for transfection. (D) Flag-TLE1 and Msx2-Myc expression constructs were cotransfected into COS-1 cells. Lysates of these cells were combined with a GST-Runx2/Osf2 affinity resin and tested for the presence of TLE1 and Msx2 with an anti-Flag and an anti-Myc antibody, respectively. (E) Interaction of Msx2 and HDAC1 was assessed by a GST interaction assay with GST-Msx2 and C3H10T1/2 cell lysates, followed by Western blotting with an anti-HDAC1 antibody. (F) PDL-L2 cells were treated with 500 nM trichostatin A (TSA) at 6 h after transfection and harvested after 18 h of trichostatin A treatment for luciferase and β-galactosidase assays.

Msx2 interacts and colocalizes with Runx2/Osf2. (A) The interaction of Msx2 with Runx2/Osf2 was assessed by GST interaction. Flag-tagged Runx2/Osf2-expressing C3H10T1/2 cell lysates were combined with a GST-Msx2 fusion protein and tested for the presence of Runx2/Osf2 with a Flag antibody. (B) The interaction of Msx2 with Runx2/Osf2 was assessed in coimmunoprecipitation assays. Cell lysates from transfected C2C12 cells expressing the indicated tagged proteins were used for immunoprecipitations (IP), followed by Western blotting (WB) with the indicated antibody (Ab). (C) Schematic representations of Runx2/Osf2 deletion mutants expressed as GST fusion proteins. (D) Flag-tagged Msx2-expressing C3H10T1/2 cell lysates were combined with the various GST-Runx2/Osf2 protein indicated in C. An anti-Flag antibody was used to detect the presence of Msx2. Photographs of Coomassie brilliant blue (CBB)-stained gels are shown to confirm the integrity and nearly equal loading of the fusion proteins. (E) Colocalization of Msx2 with Runx2/Osf2 was assessed by double-label immunofluorescence microscopy. Runx2/Osf2 was cotransfected with Myc-tagged Msx2 into PDL-L2 and C2C12 cells, and the cells were stained with anti-Runx2/Osf2 and anti-Myc antibodies. (F) In vivo expression of Msx2 and Runx2/Osf2 was detected in periodontal ligament fibroblasts of 7-week-old C57BL/6J mice by double-label in situ hybridization. See the text for details.

Expression of Msx2 gene was high and maintained in ligament and tendon fibroblasts. (A) Msx2 expression was determined by semiquantitative RT-PCR analysis. AcT cells, primary cultured Achilles tendon cells; PDL cells, culture of periodontal ligament cells containing a mixed cell population; MC3T3-E1(U), undifferentiated MC3T3-E1 cells; MC3T3-E1(D), fully differentiated osteoblastic MC3T3-E1 cells (mineralization stage); C2C12(U), undifferentiated C2C12 cells; C2C12(D), fully differentiated myoblastic C2C12 cells (myotube stage). AcT, PDL, C3H 10T1/2, PDL-L2, MC3T3-E1(U), and C2C12(U) cells were used at 80% confluence. (B) Expression patterns of Msx2, TLE1, and osteoblast differentiation markers throughout the mineralization experiments were determined by semiquantitative RT-PCR analysis in PDL-L2 and MC3T3-E1 cells. Cells at confluence (0 day) were cultured for 10, 20, or 30 days in differentiation medium (DM). The incubation medium was changed every 3 days. (C) In vivo Msx2 expression was detected in periodontal ligament and tail tendon fibroblasts from 7-week-old C57BL/6J mice by in situ hybridization. Immunofluorescence detection was performed on the tail tendon in order to increase detection sensitivity, as tendon fibroblasts (arrowhead) are sparse and thin in this tissue. Strong, diffuse fluorescent signals represent the sums of several signals coming from above and below the focal plane. D, dentin; C, cementum; L, ligament; B, alveolar bone; HE, hematoxylin-eosin stain. Bar, 40 μm.

Stable modification of Msx2 expression causes an alteration in matrix mineralizing ability. Msx2 antisense and sense expression plasmids were stably transfected into PDL-L2 and MC3T3-E1 cells, respectively. (A) Western blot analysis of Msx2 protein was performed with nuclear extracts (30 μg) of each wild-type (WT) and selected clone. For controls, cell lysates of C3H 10T1/2 cells transfected with the Msx2 expression plasmid were used. (B) For the mineralization assay in each of the wild-type and stable transformants, cells were cultured for 28 days in differentiation medium (DM) in the presence or absence of 250-ng/ml rhBMP-2 and then subjected to alizarin red-S (AR-S) staining. (C) For quantifying the degree of mineralization, each stained culture was subjected to extraction, and aliquots of the alizarin red-S extract were used. Note that PDL-L2-7 cells (reduced Msx2 expression stably) could form mineralized nodules even in differentiation medium (B and C). (D) Expression of osteoblast differentiation markers in PDL-L2 and PDL-L2-7 cells was determined by semiquantitative RT-PCR analysis. Both cell lines were cultured in normal medium (NM) until 80% confluent and then for an additional 20 days in differentiation medium. Total RNA extracts from each cell line were used for analysis. (E) The expression pattern of Msx2 in PDL-L2 cells throughout the mineralization assays was determined by semiquantitative RT-PCR analysis. Cells at confluence were cultured for 6 h, 7 days, 14 days, or 28 days in differentiation medium in the presence or absence of 250-ng/ml rhBMP-2. See the text and Materials and Methods for details.