Volume 5, Issue 6, Pages 464-475 (June 2007) Targeted Ablation of Osteocytes Induces Osteoporosis with Defective Mechanotransduction  Sawako Tatsumi, Kiyoaki Ishii, Norio Amizuka, Minqi Li, Toshihiro Kobayashi, Kenji Kohno, Masako Ito, Sunao Takeshita, Kyoji Ikeda  Cell Metabolism  Volume 5, Issue 6, Pages 464-475 (June 2007) DOI: 10.1016/j.cmet.2007.05.001 Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 1 Generation of Transgenic Mice Expressing DT-R Specifically in Osteocytes (A) Schematic representation of the transgenic construct (pDTR-9.6kb). Diphtheria toxin receptor (DT-R, dotted box) with poly(A) signals (dark box) was placed under the control of the 9.6 kb mouse DMP1 promoter (thick line) plus exons 1 and 2 (white bars) and intron 1 between them. The arrows indicate the set of primers used for confirming integration of the transgene (one in exon 2 of the DMP1 gene, and the other in the DT-R cDNA). (B) RT-PCR analysis of transgene (DT-R) expression in DTR-9.6kb transgenic line #2. RNA for bone and dentin was prepared from tibiae/femurs and incisors, respectively. (C–E) Immunohistochemical detection of the transgene product, DT-R, by anti-DT-R (hHB-EGF) antibody in osteocytes of trabecular (C) and cortical bone (D) of the femur of an 8-week-old transgenic mouse. (GP, growth plate; TB, trabecular bone; CB, cortical bone.) Representative osteocytes with prominent staining are indicated by black arrows. A high magnification of a trabecula reveals that osteoblasts on the surface do not express DT-R (red arrows in [E]). Cell Metabolism 2007 5, 464-475DOI: (10.1016/j.cmet.2007.05.001) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 2 Targeted Ablation of Osteocytes in Transgenic Mice (A) Ten-week-old male wild-type (WT, upper panels) and transgenic (Tg, lower panels) mice were injected i.p. with PBS (left panels) or 50 μg/kg body weight DT (right panels), and femoral cortical bone was stained with hematoxylin and eosin at 8 days post DT treatment. Note that empty lacunae are observed only in cortical bone of transgenic mice injected with DT (arrows). Scale bars = 50 μm. (B–E) Osteocyte morphology by electron microscopy showing a normal osteocyte (B), osteocytes with nuclear condensation (C) or fragmentation (D), and an empty lacuna (E) from wild-type mice (B) or transgenic mice injected with DT (C–E) at 8 days. (F) Numbers of empty lacunae (red), lacunae with apoptotic osteocytes (yellow), and lacunae with normal osteocyte morphology (blue) were counted in femoral cortical bone from WT and Tg mice at 8 days following injection of PBS or DT. Mean values are shown as a percentage of all lacunae (n = 3–6). (G) TUNEL staining (arrows) of femoral sections of WT and Tg mice 36 hr after DT administration. Cell Metabolism 2007 5, 464-475DOI: (10.1016/j.cmet.2007.05.001) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 3 Short-Term Effects of Osteocyte Ablation (A) Histological analysis of hematoxylin-and-eosin-stained sections of femurs from 10-week-old WT (left) and Tg (right) mice at 8 days after a single injection of DT. Note the empty lacunae (arrows) and intracortical cavities with vascular invasion. (BV in inset, blood vessel.) (B–D) TRAP (B), ALP (C), and von Kossa (D) staining of femurs of WT (left) and Tg (right) mice injected with DT. Arrows and the asterisk in (B) indicate empty lacunae and intracortical cavities, respectively. The blue staining shown by the red arrows and white arrows in (D) indicates osteoid on the endosteal surface and on the wall of intracortical cavities, respectively. (E) The presence of microfractures (yellow arrows) following osteocyte ablation as revealed by electron microscopy. Red arrows indicate red blood cells in the cracks. The asterisk indicates an empty lacuna. Magnification 1000×. (F) Osteoid surface fraction per bone surface (OS/BS) measured at the trabecular bone of the proximal tibia. In this and all other figures, error bars indicate ±SEM. ∗p < 0.05 (n = 5). (G) Trabecular bone volume/tissue volume (BV/TV) in the proximal tibia as determined by micro-CT was unchanged at 8 days following osteocyte ablation. (H) TRAP staining of the trabecular bone of WT (left) and Tg (right) mice at 8 days after DT administration. (I) Histomorphometric indices of bone formation in the femur at 7 days after injection of DT or PBS in 10-week-old WT or Tg mice. Osteoblast surface (Ob.S) and bone formation rate (BFR) were corrected for bone surface (BS). MAR, mineral apposition rate; Mlt, mineralization lag time. ∗∗p < 0.01 (n = 3 per group). Cell Metabolism 2007 5, 464-475DOI: (10.1016/j.cmet.2007.05.001) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 4 Quantitative RT-PCR Analysis of Molecular Markers of Bone Cells After the bone marrow had been flushed out, RNA was extracted from the femurs and tibiae of 10-week-old DTR-9.6kb transgenic mice injected with PBS (white bars) or DT (black bars) at 2 days. Gene expression was assessed by real-time PCR using a LightCycler system and normalized to EF-1α mRNA. MEPE, matrix extracellular phosphoglycoprotein; Phex, phosphate-regulating gene with homologies to endopeptidases on the X chromosome; RANKL, receptor activator of NF-κB ligand; OPG, osteoprotegerin; ALP, alkaline phosphatase; OC, osteocalcin. ∗∗p < 0.01; ∗p < 0.05 (n = 3 mice per group). Cell Metabolism 2007 5, 464-475DOI: (10.1016/j.cmet.2007.05.001) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 5 Osteoporotic Changes as Long-Term Consequences of Osteocyte Ablation (A and B) Representative tibial sections of 18-week-old WT and Tg mice at 40 days following a single DT administration. Note the thinning of the cortical bone and accumulation of adipose tissue in the marrow space following osteocyte ablation. Scale bars = 200 μm. Part of the cortical bone in (A) is highlighted in (B) to illustrate intracortical porosity (arrows). (C and D) Intracortical porosity area per cortical area (Po.Ar/Ct.Ar) (C) and marrow adiposity (% fat volume/marrow volume) (D) of femurs at 40 days after DT administration. ∗∗p < 0.01 (n = 5–6 per group). (E and F) Representative micro-CT images of lumbar vertebrae of WT and Tg mice at 40 days following DT administration (E) with microstructural parameters (BV/TV, 3D bone volume fraction per tissue volume; Conn-Dens., connectivity density; SMI, structure model index; Tb.Th, trabecular thickness) derived from micro-CT analysis (F). ∗∗p < 0.01 (n = 5 per group). (G and H) Recovery of bone volume (G) and bone strength (H) along with osteocyte regeneration. Trabecular bone volume and bone strength were determined by micro-CT and the four-point bending test, respectively, on the femurs of transgenic mice injected with PBS (white bars) or DT (black bars) at 40 and 90 days. ∗p < 0.05; ∗∗p < 0.01 (n = 5 per group). Cell Metabolism 2007 5, 464-475DOI: (10.1016/j.cmet.2007.05.001) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 6 Osteocyte-Ablated Mice Are Resistant to Unloading-Induced Bone Loss (A) Experimental schedule with representative micro-CT images. Eighteen-week-old WT and Tg mice were subjected to skeletal unloading by tail suspension (TS) for 7 days. Mice on the ground (Gr) served as controls. DT was administered 1 day prior to the initiation of TS (−1). (B) Changes in 3D bone volume at the tibial metaphysis following 7 day unloading by tail suspension (black bars). Mice on the ground (white bars) served as controls. ∗∗p < 0.01 (n = 5 per group). (C) Number of osteoclasts (N.Oc/BS) and bone formation rate (BFR/BS) in TS (black bars) and ground control (white bars) mice. ∗p < 0.05 (n = 5 per group). (D) Quantitative RT-PCR analysis. RNA was extracted from tibiae and femurs of WT and DTR-9.6kb Tg mice injected with DT (red bars) or PBS (pale green bars) at 3 days following the initiation of TS, after bone marrow had been flushed out. Transgenic mice on the ground (Gr) served as controls (white and black bars). ∗p < 0.05; ∗∗p < 0.01 (n = 3 per group). Cell Metabolism 2007 5, 464-475DOI: (10.1016/j.cmet.2007.05.001) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 7 Absence of Osteocytes Does Not Affect Bone Gain during Reloading (A) Experimental schedule for reloading after tail suspension (TS→Gr) and representative micro-CT images. Eighteen-week-old WT and DTR-9.6kb Tg mice were subjected to TS for 7 days and were then left ambulatory for the following 14 days. DT (4) or PBS as vehicle (3) was injected before initiation of reloading so that osteocytes were ablated specifically during the reloading period (4). Mice on the ground throughout the experimental period (1) and mice subjected to TS only (2) served as controls. (B and C) Changes in 3D bone volume at the tibial metaphysis of WT and Tg mice with or without DT administration (B) and histomorphometric indices for osteoclast number (N.Oc) and bone formation rate (BFR) corrected for bone surface (BS) (C) after reloading (TS→Gr). ∗∗p < 0.01; ∗p < 0.05 (n = 5 per group). (1)–(4) correspond to the four experimental groups shown in (A). (D) Proposed roles of osteocytes under different mechanical conditions. Based on the current results, under normal loading conditions (Mechanical input +), osteocytes function to keep osteoclastic (Oc) bone resorption in check and to maintain mineralization by osteoblasts (Ob). Thus, when osteocytes are ablated, aberrantly elevated bone resorption with impaired mineralization takes place. In response to unloading (Mechanical input −), osteocytes execute the stimulation of bone resorption and suppression of bone formation, resulting in marked bone loss and microstructural deterioration in a short period. Thus, when osteocytes are ablated specifically during tail suspension, those changes do not take place, and bone is resistant to disuse-induced atrophy. However, based on the results of the reloading experiments following unloading (Mechanical input − → +), it is suggested that osteocytes are dispensable for this recovery phase and that osteoclasts (Oc) and osteoblasts (Ob) respond to the reloading stimulus, bypassing osteocytes, with reversal of elevated bone resorption and release from suppressed bone formation, respectively. Cell Metabolism 2007 5, 464-475DOI: (10.1016/j.cmet.2007.05.001) Copyright © 2007 Elsevier Inc. Terms and Conditions