Osteocytes, not Osteoblasts or Lining Cells, are the Main Source of the RANKL Required for Osteoclast Formation in Remodeling Bone

Abstract

The cytokine receptor activator of nuclear factor kappa B ligand (RANKL), encoded by the Tnfsf11 gene, is essential for osteoclastogenesis and previous studies have shown that deletion of the Tnfsf11 gene using a Dmp1-Cre transgene reduces osteoclast formation in cancellous bone by more than 70%. However, the Dmp1-Cre transgene used in those studies leads to recombination in osteocytes, osteoblasts, and lining cells making it unclear whether one or more of these cell types produce the RANKL required for osteoclast formation in cancellous bone. Because osteoblasts, osteocytes, and lining cells have distinct locations and functions, distinguishing which of these cell types are sources of RANKL is essential for understanding the orchestration of bone remodeling. To distinguish between these possibilities, we have now created transgenic mice expressing the Cre recombinase under the control of regulatory elements of the Sost gene, which is expressed in osteocytes but not osteoblasts or lining cells in murine bone. Activity of the Sost-Cre transgene in osteocytes, but not osteoblast or lining cells, was confirmed by crossing Sost-Cre transgenic mice with tdTomato and R26R Cre-reporter mice, which express tdTomato fluorescent protein or LacZ, respectively, only in cells expressing the Cre recombinase or their descendants. Deletion of the Tnfsf11 gene in Sost-Cre mice led to a threefold decrease in osteoclast number in cancellous bone and increased cancellous bone mass, mimicking the skeletal phenotype of mice in which the Tnfsf11 gene was deleted using the Dmp1-Cre transgene. These results demonstrate that osteocytes, not osteoblasts or lining cells, are the main source of the RANKL required for osteoclast formation in remodeling cancellous bone.

Fig 1. Generation of Sost-Cre transgenic mice.

(A) Diagram of a portion of the Sost-Cre transgene construct used to generate Sost-Cre transgenic mice. UTR, untranslated region, IRES, internal ribosome entry site, GFP::Cre, fusion protein of GFP and the Cre recombinase. (B) Sost-Cre transgene mRNA levels in different tissues of 6-week-old mice male mice as measured by quantitative RT-PCR. n = 4 animals for each tissue.

Fig 2. Anaylsis of Sost-Cre transgenic mice using tdTomato Cre-reporter mice.

Fluorescent images of frozen histological sections of femurs from 2-month-old tdTomato control, Sost-Cre;tdTomato, and 10 kb Dmp1-Cre;tdTomato mice injected with calcein. (A) Confocal image of osteocytes in cortical bone. (B) Epifluorescent image of endocortical surface. (C) Epifluorescent image of cancellous bone. (D) Higher magnification of the cancellous bone from (C). Scale bar, 50 μm. Green: calcein labeling of mineralizing bone; Red: cells that express tdTomato fluorescent protein upon Cre-mediated recombination.

Fig 3. Analysis of Sost-Cre transgenic mice using R26R Cre-reporter mice.

(A) Brightfield microscopy images of X-gal stained frozen histological sections of cortical bone and (B) cancellous bone in the femur of 2-month-old R26R, Sost-Cre;R26R, and 10 kb Dmp1-Cre;R26R mice. (C) Epifluorescent image of the same cancellous bone as in (B) showing calcein labeling. Scale bar, 50 μm.

Fig 4. Analysis of Sost-Cre transgene activity by transmission electron microscopy.

Transmission electron microscopy (TEM) images of electron-dense X-gal deposits in osteocytes (A), osteoblasts (B), and lining cells (C) on the cancellous bone surface of vertebra of 2-month-old R26R, Sost-Cre;R26R, and 10 kb Dmp1-Cre;R26R mice. Size bars are present in each image, with the size indicated.

Fig 5. Deletion of Tnfsf11 using the Sost-Cre transgene increases bone mass.

(A) Quantitative PCR of loxP-flanked Tnfsf11 genomic DNA using genomic DNA isolated from collagenase-digested femoral cortical bone of 6-month-old Sost-Cre;Tnfsf11-f/f (n = 10) and Tnfsf11-f/f (n = 16) littermates. *P < 0.05 using Student’s t-test. Representative images of the teeth (B) and safranin-O-stained histological sections of the lumbar vertebra (Scale bar, 0.1 mm) (C) of 6-month-old Sost-Cre;Tnfsf11-f/f and Tnfsf11-f/f mice. (D) Serial femoral and spinal BMD of Sost-Cre;Tnfsf11-f/f (n = 14), Tnfsf11-f/f (n = 16), wild-type (n = 14), and Sost-Cre (n = 13) littermates measured by DXA. *P < 0.05 versus wild-type, Tnfsf11-f/f, or Sost-Cre mice using one-way ANOVA comparing the 4 genotypes at a given age. All results include data from both male and female mice.

Fig 6. Cancellous bone volume is high in Sost-Cre;Tnfsf11-f/f mice.

(A) Cancellous bone volume over total volume (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), and trabecular separation (Tb.Sp) in the L4 vertebra of 6-month-old Sost-Cre;Tnfsf11-f/f (n = 10) and Tnfsf11-f/f (n = 16) littermates. *P < 0.05 using Student’s t-test. (B) Cancellous BV/TV, trabecular number, trabecular thickness, trabecular separation, and cortical thickness (Ct.Th) in the distal femur of the same mice as in (A). *P < 0.05 using Student’s t-test. Representative μCT images of the L4 vertebra (C) and distal femur (D) of 6-month-old Sost-Cre;Tnfsf11-f/f and Tnfsf11-f/f littermates. All results include data from both male and female mice. Scale bar, 0.5 mm.

Fig 7. Osteoclastogenesis is inhibited in Sost-Cre;Tnfsf11-f/f mice.

(A) Quantitative RT-PCR for Tnfsf11 mRNA in tibia, L5 vertebra, and calvaria of 6-month-old Sost-Cre;Tnfsf11-f/f (n = 10) and Tnfsf11-f/f (n = 16) littermates. *P < 0.05 using Student’s t-test. (B-C) The perimeter of bone surface covered by osteoclasts (Oc.Pm/B.Pm) (B) and the osteoclast number per mm of bone surface (N.Oc/B.Pm) (C) in cancellous bone of the vertebra (left panel) and distal femur (middle) and endocortical bone (right) of 6-month-old Sost-Cre;Tnfsf11-f/f (n = 5) and Tnfsf11-f/f (n = 5) littermates. *P < 0.05 using Student’s t-test. (D) Representative images of osteoclasts in cancellous bone (upper panel) and endocortical surface (lower panel) of 6-month-old Sost-Cre;Tnfsf11-f/f and Tnfsf11-f/f mice. All results include data from both male and female mice.