Continuous elevation of PTH increases the number of osteoblasts via both osteoclast-dependent and -independent mechanisms

Abstract

Sustained parathyroid hormone (PTH) elevation stimulates bone remodeling (ie, both resorption and formation). The former results from increased RANKL synthesis, but the cause of the latter has not been established. Current hypotheses include release of osteoblastogenic factors from osteoclasts or from the bone matrix during resorption, modulation of the production and activity of osteoblastogenic factors from cells of the osteoblast lineage, and increased angiogenesis. To dissect the contribution of these mechanisms, 6-month-old Swiss-Webster mice were infused for 5 days with 470 ng/h PTH(1-84) or 525 ng/h soluble RANKL (sRANKL). Both agents increased osteoclasts and osteoblasts in vertebral cancellous bone, but the ratio of osteoblasts to osteoclasts and the increase in bone formation was greater in PTH-treated mice. Cancellous bone mass was maintained in mice receiving PTH but lost in mice receiving sRANKL, indicating that maintenance of balanced remodeling requires osteoblastogenic effects beyond those mediated by osteoclasts. Consistent with this contention, PTH, but not sRANKL, decreased the level of the Wnt antagonist sclerostin and increased the expression of the Wnt target genes Nkd2, Wisp1, and Twist1. Furthermore, PTH, but not sRANKL, increased the number of blood vessels in the bone marrow. Weekly injections of the RANKL antagonist osteoprotegerin at 10 µg/g for 2 weeks prior to PTH infusion eliminated osteoclasts and osteoblasts and prevented the PTH-induced increase in osteoclasts, osteoblasts, and blood vessels. These results indicate that PTH stimulates osteoclast-dependent as well as osteoclast-independent (Wnt signaling) pro-osteoblastogenic pathways, both of which are required for balanced focal bone remodeling in cancellous bone.

Fig. 1

Experimental design. (A) Female Swiss Webster mice were treated as indicated to distinguish osteoclast (Oc)–dependent and –independent mechanisms for increasing osteoblast number during PTH-stimulated bone remodeling. (B) The decline in osteoclasts and osteoblasts in vertebral bone (L₅) following a single injection of 10 µg/g of OPG-Fc on day 0 was monitored using quantitative PCR (qPCR) at the indicated times to determine expression of the osteoclast-specific gene cathepsin K and the osteoblast-specific gene osteocalcin, relative to GAPDH (n = 6 to 8/group).

Fig. 2

sRANKL, but not PTH, decreases vertebral cancellous bone mass. Vertebral bone (L₄) was analyzed by µCT. (A) Representative µCT images. (B) Measurements of cancellous bone volume per tissue volume (BV/TV), BMD, trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), and trabecular number (Tb.N). n = 8 to 10/group. *p < .05 versus vehicle (Veh) control.

Fig. 3

Reduced osteoblast number in response to sRANKL compared with PTH and blockade of PTH effects by OPG-Fc. (A) Histomorphometric determination of osteoclast (Oc) and osteoblast (Ob) numbers was done in vertebral cancellous bone sections (L₁–L₃). Owing to the severe reduction in osteoblasts and osteoclasts in bone of mice pretreated with OPG, the ratio of Ob to Oc was not calculated (NC) in these specimens. (B) Photomicrographs of osteoblasts (arrows) in animals receiving vehicle, PTH, or sRANKL. Nondecalcified sections, original magnification ×400. Osteoid perimeter (O.Pm) was determined as in panel A. (C) Prevalence of osteoblast apoptosis as determined by ISEL. Osteoblast apoptosis was not calculated (NC) in mice pretreated with OPG owing to the very low number of osteoblasts available for inspection. The photomicrograph shows an apoptotic osteoblast (black arrow) among a group of viable osteoblasts (white arrows) in a bone section from a sRANKL-treated mouse. n = 4 to 6/group. *p < .05 versus vehicle (Veh) control; ^†p < .05 versus PTH.

Fig. 4

Reduced osteoblast activity in mice receiving sRANKL compared with PTH and blockade of PTH effects by OPG-Fc. (A) Histomorphometric determination of osteoid width (O.Wi), alizarin red (AR)–labeled perimeter, double-labeled surface (dL.Pm), and bone-formation rate (BFR) using the same sections as in Fig. 3. Osteocalcin transcripts were measured by qPCR of RNA preparations from vertebral bone (L₄). Serum osteocalcin was measured by RIA. Osteoid width was not measured (NM) in mice pretreated with OPG-Fc. n = 4 to 6/group. *p < .05 versus vehicle (Veh) control; ^†p < .05 versus PTH. (B) Double labeling with tetracycline and alizarin red in vertebral bone sections from mice infused with vehicle, PTH, or sRANKL. Original magnification ×400.

Fig. 5

PTH, but not sRANKL, attenuates sclerostin expression and activates Wnt signaling. (A) Sclerostin was extracted from tibias and quantified by Western blotting. Each lane represents one animal. Blots of three of the six samples in each group are shown. *p < .05 versus vehicle (Veh) control. (B) Expression of Nkd2, Wisp1, and Twist1 by qPCR of RNA from lumbar vertebrae (L₄) relative to GAPDH. n = 8 to 10/group; *p < .05 versus vehicle (Veh) control; ^†p < .05 versus PTH. (C) Luciferase activity (relative light units, RLUs) in UAMS-32P and UMR 106 cells transfected with a TCF-Luc reporter construct. Activity was measured 24 hours after addition of vehicle (0.01% acetic acid), 50 nM PTH(1-34), or 100 ng/mL of sRANKL. n = 3/group. *p < .05 versus vehicle.

Fig. 6

PTH, but not sRANKL, increases marrow vascularity. (A) Photomicrograph of blood vessels in the marrow of secondary spongiosa of lumbar vertebrae. Vessels were recognized by CD34⁺ endothelial cells (arrows) of vessels containing red blood cells. Original magnification ×200. (B) Histomorphometric determination of blood vessels in the marrow space adjacent to the secondary spongiosa. n = 4 to 6/group; *p < .05 versus vehicle (Veh) control; ^†p < .05 versus PTH.

Fig. 7

PTH-stimulated osteoclast-dependent and -independent mechanisms involved in the control of osteoblast number during bone remodeling. See text for details. HSC = hematopoietic stem cell progenitors of osteoclasts; Oc = osteoclasts; MSC = mesenchymal stem cell progenitors of osteoblasts; Ob = osteoblasts; ECP = endothelial cell progenitors; GF = growth factors.