Figure 1
Figure 1. PIAS3-Tg mice show an osteopetrotic phenotype and impair osteoclast differentiation. (A) Schematic of the PIAS3 transgene construct containing the TRAP promoter followed by mouse full-length PIAS3 cDNA and poly(A) sequence. ▼ indicates a digestion site by HpaI and NheI for Southern blot analysis. →, ← indicate specific primers for genomic PCR (P1,2) and for generation of Southern blot probe (P3,4). (B) Genomic PCR with tail DNAs from WT and Tg mice (top). Southern blot of tail DNA from WT and PIAS3-Tg mice (bottom). (C) Expression of PIAS3 mRNA by real-time PCR. Total RNA was extracted from BMMs of WT and PIAS3-Tg mice for real-time PCR (right). (D) Radiologic analysis of long bones of TRAP–PIAS3-Tg mice and WT littermates. Right panel shows a maginified view of left panel. (E) Bone mineral density of 20 equal longitudinal divisions of femurs from 8-week-old WT and PIAS3-Tg mice. (F) Histologic analysis of long bone of PIAS3-Tg and WT (top left, Van Gieson; top right, toluidine blue; bottom, TRAP/hematoxylin staining). Arrows indicate the TRAP-positive mature osteoclasts. (G) MicroCT of axial, coronal, and sagittal sections of distal femoral metaphysis from representative 8-week-old PIAS3-Tg and WT. BV/TV (bone volume/tissue volume), Tb.Th (trabecular thickness), Tb.N (trabecular number), and Tb.Sp (trabecular separation) were analyzed by TRY/3D BON. (H) The parameters for osteoclastic bone resorption (ES/BS, N.Oc/B.Pm, and Oc.S/BS) and parameters for osteoblastic bone formation (OV/BV, OS/BS, O.Th, and Ob.S/BS) in the bone morphometrical analysis. ES/BS, eroded surface/bone surface; N.Oc/B.Pm, osteoclast number/bone perimeter; Oc.S/BS, osteoclast surface/bone surface; OV/BV, osteoid volume/bone volume; OS/BS, osteoid surface/bone surface; O.Th, osteoid thickness; Ob.S/BS, osteoblast surface/bone surface. (I) BMMs isolated from PIAS3-Tg and WT mice were cultured for 5 days with 30 ng/mL M-CSF and 50 ng/mL RANKL. Cultured cells were fixed and stained for TRAP. (J) TRAP-positive MNCs having more than 3 nuclei were counted as osteoclast. (K) Expression of PIAS3 and osteoclastogenic markers during RANKL-induced osteoclastogenesis. BMMs isolated from PIAS3-Tg and WT mice were cultured in the presence of 30 ng/mL M-CSF and 50 ng/mL RANKL for 0, 2, and 4 days. Total RNA was extracted from cultured cells, and real-time PCR was performed with primers specific for PIAS3, c-Fos, NFATc1, cathepsin K, and TRAP. Error bars in all panels represent standard deviation (SD). #P < .05; ##P < .01; n.s, not significant.

PIAS3-Tg mice show an osteopetrotic phenotype and impair osteoclast differentiation. (A) Schematic of the PIAS3 transgene construct containing the TRAP promoter followed by mouse full-length PIAS3 cDNA and poly(A) sequence. ▼ indicates a digestion site by HpaI and NheI for Southern blot analysis. →, ← indicate specific primers for genomic PCR (P1,2) and for generation of Southern blot probe (P3,4). (B) Genomic PCR with tail DNAs from WT and Tg mice (top). Southern blot of tail DNA from WT and PIAS3-Tg mice (bottom). (C) Expression of PIAS3 mRNA by real-time PCR. Total RNA was extracted from BMMs of WT and PIAS3-Tg mice for real-time PCR (right). (D) Radiologic analysis of long bones of TRAP–PIAS3-Tg mice and WT littermates. Right panel shows a maginified view of left panel. (E) Bone mineral density of 20 equal longitudinal divisions of femurs from 8-week-old WT and PIAS3-Tg mice. (F) Histologic analysis of long bone of PIAS3-Tg and WT (top left, Van Gieson; top right, toluidine blue; bottom, TRAP/hematoxylin staining). Arrows indicate the TRAP-positive mature osteoclasts. (G) MicroCT of axial, coronal, and sagittal sections of distal femoral metaphysis from representative 8-week-old PIAS3-Tg and WT. BV/TV (bone volume/tissue volume), Tb.Th (trabecular thickness), Tb.N (trabecular number), and Tb.Sp (trabecular separation) were analyzed by TRY/3D BON. (H) The parameters for osteoclastic bone resorption (ES/BS, N.Oc/B.Pm, and Oc.S/BS) and parameters for osteoblastic bone formation (OV/BV, OS/BS, O.Th, and Ob.S/BS) in the bone morphometrical analysis. ES/BS, eroded surface/bone surface; N.Oc/B.Pm, osteoclast number/bone perimeter; Oc.S/BS, osteoclast surface/bone surface; OV/BV, osteoid volume/bone volume; OS/BS, osteoid surface/bone surface; O.Th, osteoid thickness; Ob.S/BS, osteoblast surface/bone surface. (I) BMMs isolated from PIAS3-Tg and WT mice were cultured for 5 days with 30 ng/mL M-CSF and 50 ng/mL RANKL. Cultured cells were fixed and stained for TRAP. (J) TRAP-positive MNCs having more than 3 nuclei were counted as osteoclast. (K) Expression of PIAS3 and osteoclastogenic markers during RANKL-induced osteoclastogenesis. BMMs isolated from PIAS3-Tg and WT mice were cultured in the presence of 30 ng/mL M-CSF and 50 ng/mL RANKL for 0, 2, and 4 days. Total RNA was extracted from cultured cells, and real-time PCR was performed with primers specific for PIAS3, c-Fos, NFATc1, cathepsin K, and TRAP. Error bars in all panels represent standard deviation (SD). #P < .05; ##P < .01; n.s, not significant.

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