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Phenotypic Analysis of Dlx5 Overexpression in Post-natal Bone
J. Zhang1,2, ,
J. Zhu1,,
P. Valverde1,
L. Li1,
S. Pageau1,
Q. Tu1,
R. Nishimura3,
T. Yoneda3,
P. Yang2,
W. Zheng4,
W. Ma5 and
J. Chen1,*
1 Division of Oral Biology, Department of General Dentistry, Tufts University School of Dental Medicine, One Kneeland Street, Boston, MA 02111, USA;
2 College of Stomatology, Shandong University, Shandong Province, China;
3 Department of Biochemistry, Osaka University Graduate School of Dentistry, Osaka, Japan;
4 South Genomics Research Center, Guangzhou, 510800, China; and
5 Institute of Genetic Engineering, South Medical University, Guangzhou, 510515, China
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Figure 1. BSP/TVA-driven Dlx5 or mutated Dlx5 overexpression in mineralized tissues and calvarial cultures. Western blot analyses were performed with tissues from BSP/TVA mice 4 days after infection with RCAS-Dlx5 (i.e., on day 9) and calvarial cells 3 days after in vitro infection with RCAS-Dlx5 or RCAS-Dlx5RH. (A) Expression analysis upon in vivo delivery of Dlx5 viral constructs. BSP/TVA mice were infected with RCAS-Dlx5, and expression of overexpressed Dlx5 was detected with an anti-FLAG antibody on day 9. An antibody for β-actin was used as a loading control. Protein lysates were obtained from tibial, mandibular, and calvarial tissues isolated from BSP/TVA mice infected with the RCAS-Dlx5 construct and from a representative soft tissue (i.e., liver). (B) GFP expression analyses upon ex vivo infection of RCAS-GFP into calvarial cells from BSP/TVA transgenic and non-transgenic mice. Calvarial cells from BSP/TVA mice (1,2) or wild-type mice that did not express TVA (2,4) were transfected with the RCAS-GFP construct. We used GFP expression to titer infection efficiency and to confirm the specificity of the overexpression to cells that express TVA 3 days after the infection. The in vitro-infected cells were photographed by fluorescence (1,3) and phase contrast (2,4) microscopy. Green fluorescence was detected in infected cells from BSP/TVA mice (1), but not in those of normal mice, due to the lack of TVA expression (3). (C) Western blot analysis upon ex vivo delivery of viral constructs. The analysis was performed with protein lysates from calvarial cells derived from non-transgenic mice (without TVA expression) before and after being infected ex vivo with RCAS-Dlx5. Calvarial cells from BSP/TVA mice were infected ex vivo with RCAS-Dlx5WT or RCAS-Dlx5RH viral constructs. Expression of exogenous Dlx5 constructs and the loading control β-actin were detected as described above.
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Figure 2. Immunohistochemical analysis of extracellular matrix proteins OPN, BSP, and OC in bone tissues of BSP/TVA overexpressing mutated Dlx5 (RH mutant), wild-type Dlx5 (Wild-type), and empty vector (Control). Nine days after infection of BSP/TVA with viral constructs, bones were isolated, and immunohistochemistry was performed to determine the expression of OPN, BSP, and OC in calvarial and mandibular tissues. Expression levels of OPN, BSP, and OC in mandibles were shown in transverse sections of the developing first and/or second molars and the surrounding tissues at the cementum-enamel junction. In control mandibular tissues, BSP and OCN were both moderately expressed in the cells (osteoblasts and osteocytes) and in bone matrix associated with the alveolar bone surface. BSP expression in mandibular tissues isolated from the wild-type group was up-regulated in the alveolar bone surface area, and moderate expression can be detected in the odontoblast layer. However, BSP expression in mandibular tissues isolated from RH mutant group was down-regulated, and there is no evident BSP expression above background. In contrast, OCN expression in the wild-type group was weaker than that in the RH mutant group. OPN expression in control mandibular tissues can also be detected in the alveolar bone surface (osteoblasts, osteocytes, and bone matrix), as well as the odontoblast layer. OPN expression in the corresponding areas in the wild-type group and the RH mutant group was up-regulated and down-regulated, respectively. Expressions of BSP, OPN, and OCN are all evident in the osteocytes and bone matrix of control calvarial tissues. While BSP and OPN expression levels are increased in the wild-type group and decreased in the RH mutant group, OCN expression level is down-regulated in the wild-type group and up-regulated in the RH mutant group. M1, the first mandibular molar; M2, the second mandibular molar; b, bone.
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Figure 3. Semi-quantitative RT-PCR analysis for markers of osteoblast differentiation, including extracellular matrix proteins and ALP. BSP/TVA mice were infected with RCAS-Dlx5WT (WT), RCAS-Dlx5RH (RH), or RCAS empty vector as a control (CTL). Calvarial tissues were isolated for 1 to 4 wks after infection, and their expression levels of ALP, OPN, BSP, and OC were analyzed by semi-quantitative RT-PCR. Expression of GAPDH was used as a loading control. (A) Representative RT-PCR experiment. (B) Normalized mRNA expression of ALP, OPN, BSP, and OC. Results (in arbitrary units) are expressed as the mean ± SE from 3 different experiments. Values of p < 0.05 were considered significantly different (ap < 0.05 vs. CTL at every specific time point; bp < 0.05 WT vs. RH).
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Figure 4. Effects of RCAS-Dlx5WT, RCAS-Dlx5RH, or empty RCAS vector in bone nodule formation of calvarial cells expressing TVA. Calvarial cells from BSP/TVA mice were infected with RCAS empty vector (Control), wild-type Dlx5 (Dlx5-WT), and mutated Dlx5 (Dlx5-RH). The formation of in vitro mineralization nodules was determined by alizarin red-S histochemical staining, and the number of nodules was counted under a microscope at different time-points (1, 2, and 3 wks). (A) Representative example of alizarin red-S staining 2 wks after infection and osteogenic differentiation. (B) Numbers of nodules obtained 2 and 3 wks after infection and osteogenic differentiation. Results are the mean ± SE from 3 different experiments. Values of p < 0.05 were considered significantly different (ap < 0.05 vs. CTL at every time-point; bp < 0.05 RH vs. WT at every time-point).
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Journal of Dental Research, Vol. 87, No. 1,
45-50 (2008)
DOI: 10.1177/154405910808700107
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