Bone-Specific Overexpression of PITX1 Induces Senile Osteoporosis in Mice Through Deficient Self-Renewal of Mesenchymal Progenitors and Wnt Pathway Inhibition

Abstract

The cellular and molecular mechanisms underlying senile osteoporosis remain poorly understood. In this study, transgenic mCol1α1-Pitx1 mice overexpressing paired-like homeodomain 1 (PITX1), a homeobox transcription factor, rapidly develop a severe type-II osteoporotic phenotype with significant reduction in bone mass and biomechanical strength similar to that seen in humans and reminiscent of the phenotype previously observed in Sca-1 (Ly6a)-null mice. PITX1 plays a critical role in hind limb formation during fetal development, while loss of expression is associated with primary knee/hip osteoarthritis in aging humans. Through in vivo and in vitro analyses, we demonstrate that Pitx1 directly regulates the self-renewal of mesenchymal progenitors and indirectly regulates osteoclast differentiation through the upregulation of Wnt signaling inhibitors DKK1, SOST, and GSK3-β. This is confirmed by elevated levels of plasma DKK1 and the accumulation of phospho-β-catenin in transgenic mice osteoblasts. Furthermore, overexpressed Pitx1 in mice osteoblasts results in severe repression of Sca-1 (Ly6a) that was previously associated with senile osteoporosis. Our study is the first to demonstrate the novel roles of PITX1 in senile osteoporosis where PITX1 regulates the self-renewal of mesenchymal stem cells or progenitor cells through Sca-1 (Ly6a) repression and, in addition, inhibits the Wnt signaling pathway.

Figure 1

Transgenic Col1α1-Pitx1 mice exhibit growth retardation accompanied with bone loss. (A) Comparative photo of both sexes from 12-week-old transgenic mCol1α1-Pitx1 and wild type mice. (B) Comparative growth curves of both sexes of mCol1α1-Pitx1 mice and their corresponding wild type littermates over a 28-week period. (C) X-ray imaging of 12-week-old female mCol1α1-Pitx1 and wild type mice confirm a very thin and fragile cortical bone in long bones. The femur length of both sexes of transgenic mCol1α1-Pitx1 mice and their corresponding wild type littermates over a 28-week period are also shown. (D) Histological examination of the distal femoral growth plates for the study of growth differences of long bones. Structural abnormalities of growth plates were observed in safranin O-stained sections of transgenic mCol1α1-Pitx1 mice. Mean proteoglycan content in transgenic growth plates was determined by measuring changes in safranin O color intensity using the Image J software. Proteoglycan content of transgenic mouse growth plates showed a reduction of 60% when compared to the wild type mouse growth plate.

Figure 2

Transgenic Col1α1-Pitx1 mice exhibit an altered trabecular microarchitecture. (A–D) Cross-sectional microCT scans of femurs from both sexes of 12-week-old Col1α1-Pitx1 and wild type (WT) mice. (E–H) Quantitative comparison of the trabecular bone parameters: Bone surface (BS)/Bone volume (BV); pattern factor (Pf); thickness (Th); structure model index (SMI). Data are presented as mean ± SD for 5 mice from each sex in each genotype group. Asterisks indicate statistically significant difference (*p < 0.05; **p < 0.005; ***p < 0.0005).

Figure 3

Transgenic Col1α1-Pitx1 mice exhibit altered cortical microarchitecture accompanied with a loss of bone strength. (A–D) Representative cross-sectional microCT scans of femurs from females of 12-week-old transgenic mCol1a1-Pitx1 and wild type mice. (C–F) Quantitation of the cortical bone – Bone volume (BV)/trabecular volume (TV); Bone surface (BS)/BV; Porosity (Th). (G–I) Three-point bending test quantitation – Stiffness; Ultimate Force; Work to ultimate point. Data are presented as mean ± SD for 5 mice from each sex in each genotype group. Asterisks indicate statistically significant difference (*p < 0.05; **p < 0.005; ***p < 0.0005).

Figure 4

Impaired in vivo osteoblast number derived from 12-week-old transgenic mCol1α1-Pitx1 mice. (A–D) Representative Goldner and toluidine blue staining on femur sections reveal a decreased number of osteoblasts and osteoids in transgenic female mCol1α1-Pitx1 mice compared to wild type female mice. (E–G) Histomorphometric quantitation of the osteoid surface (OS/BS), volume (OV/BV), and osteoblast surface (Ob.S BS). Data are presented as mean ± SD for 5 mice from each sex in each genotype group. Asterisks indicate statistically significant difference (*p < 0.05; **p < 0.005).

Figure 5

Impaired in vitro osteoblast differentiation derived from 12-week-old transgenic mCol1α1-Pitx1 mice when compared to their wild type littermates. (A,B) Alizarin red and ALP staining of osteoblast cultures after 14 days of induction in osteogenic medium. (C). Real time PCR analysis of Pitx1 and osteogenic markers (Runx2, Osx, Alp1, Ocn and Spp1) in wild type (Wt) and transgenic (mCol1α1-Pitx1) osteoblasts on day 14 of osteogenic induction. Expression was normalized with that of wild type. Asterisks indicate statistically significant difference (*P < 0.05; **P < 0.005). (D) Alizarin red staining of osteoblast culture after transfection with either siRNA targeting Pitx1 (siRNA Pitx1) or scrambled sequence siRNA (Neg) used as a control. (E) Real time PCR analysis of Pitx1 and osteogenic markers (Runx2, Osx, Alp1 and Spp1) in mouse transgenic osteoblasts untransfected (NT), transfected with Pitx1 siRNA or with a scramble sequence siRNA (Neg). Expression was normalized with that of untransfected transgenic osteoblasts. Asterisks indicate statistically significant difference (*P < 0.05; **P < 0.005). (F) Alizarin red staining of osteoblast culture after infection with either lentivirus coding for Pitx1 or DsRed as a control. (G) Real time PCR analysis of Pitx1 and osteogenic markers (Runx2, Osx, Alp1 and Spp1) in wild type mouse osteoblasts uninfected (NT), infected with either lentivirus coding for Pitx1 or DsRed as a control. Expression was normalized with that of uninfected wild type osteoblasts. Asterisks indicate statistically significant difference (*P < 0.05; **P < 0.005; ***P < 0.0005).

Figure 6

Mesenchymal stem cells (MSC) derived from 12-week-old transgenic mCol1α1-Pitx1 mice display impaired osteoblast differentiation in vitro when compared to their wild type littermates. (A,B,D) Analysis of the colony-forming unit (CFU) number and osteogenic CFU-F number (ALP + CFU-F). (C) Alizarin red staining of MSC cultures after 14 days of induction in osteogenic media. (E) Real time PCR analysis of osteogenic markers (Runx2, Osx, Alp1, Ocn and Spp1) in MSCs. (F) Alizarin red staining of MSC culture after transfection with either siRNA targeting Pitx1 or scrambled sequence siRNA (used as a control). H. Alizarin red staining of MSC culture after infection with either lentivirus coding for Pitx1 or DsRed (control). (G,I) Real time PCR analysis of osteogenic markers (Runx2, Osx, Alp1, and Spp1) in MSCs. Expression was normalized with that of wild type.

Figure 7

MSCs derived from 12-week-old transgenic mCol1α1-Pitx1 mice displayed impaired adipocyte differentiation in vitro when compared to their wild type littermates. (A) Oil red ‘O’ staining of MSC cultures after 11 days of induction in adipogenesis media. (B) Real time PCR analysis of adipocytic markers (Pparγ, Cebp, Adipoq and aP2) in MSCs. Expression in real time PCR analysis was normalized in comparison with wild type. Asterisks indicate statistically significant difference (*p < 0.05; **p < 0.005).

Figure 8

Expression analysis of Pitx1, Sca-1(Ly6a), Tnfsf11 and Tnfrsf11b gene in transgenic mCol1α1-Pitx1 and wild type mouse primary osteoblasts. Real time qPCR analysis of Pitx1 (A), Sca-1 (Ly6a) (B), Tnsfs11 (C) and Tnfrsf11b (D) in transgenic mCol1α1-Pitx1 and wild type mouse specimens. Expression was normalized with that of wild type. Data are presented as mean+/− SD for five mice from each genotype.

Figure 9

Impaired in vivo osteoclast number and function in Col1α1-Pitx1 mice. (A–C) TRAP activity staining and quantitation in femur sections of both sexes from 12-week-old Col1α1-Pitx1 and wild type mice. (D) Histomorphometric quantitation of the osteoclast surface (Oc.S BS) within trabecular bone areas. (E–G) Measurements of plasma levels of RANKL and OPG. Data are presented as mean ± SD for 5 mice from each sex in each genotype group. Asterisks indicate statistically significant difference (*p < 0.05, **p < 0.005).

Figure 10

Inactivation of the Wnt canonical pathway in Col1α1-Pitx1 mice. (A) Real time PCR analysis of Wnt canonical pathway markers sclerostin (SOST), Dickkopf-related protein 1 (DKK1), and glycogen synthase kinase-3β (GSK3β). Expression was normalized with the same proteins in wild type mice. (B) Mean plasma levels of SOST and DKK1 in Col1α1-Pitx1 transgenic mice compared to that of wild type littermates. Asterisks indicate statistically significant difference (**p < 0.005; ***p < 0.0005). (C) Western blot of β-catenin vs phospho β-catenin obtained from protein extracts of cultured osteoblasts from Col1α1-Pitx1 transgenic mice compared with their wild type littermates. (D) Immunolocalization of β-catenin by confocal microscopy with anti-β-catenin antibodies in wild type (Wt) and transgenic (Tg) mouse osteoblasts. (E) Detection of phospho-β-catenin by the same method with anti-phospho-β-catenin antibodies. Original uncropped blots corresponding to panel C are shown in Supplementary Fig. S4. (F) Real time qPCR expression analysis of Axin-2, a known Wnt target, was carried-out in transgenic mCol1α1-Pitx1 and wild type mouse primary osteoblasts. Expression was normalized with that of wild type. Data are presented as mean+/− SD for five mice (females) from each genotype.