Estradiol and RSPO3 regulate vertebral trabecular bone mass independent of each other

Abstract

Osteoporosis is an age-dependent serious skeletal disease that leads to great suffering for the patient and high social costs, especially as the global population reaches higher age. Decreasing estrogen levels after menopause result in a substantial bone loss and increased fracture risk, while estrogen treatment improves bone mass in women. RSPO3, a secreted protein that modulates WNT signaling, increases trabecular bone mass and strength in the vertebrae of mice, and is associated with trabecular density and risk of distal forearm fractures in humans. The aim of the present study was to determine if RSPO3 is involved in the bone-sparing effect of estrogens. We first observed that estradiol (E2) treatment increases RSPO3 expression in bone of ovariectomized (OVX) mice, supporting a possible role of RSPO3 in the bone-sparing effect of estrogens. As RSPO3 is mainly expressed by osteoblasts in the bone, we used a mouse model devoid of osteoblast-derived RSPO3 (Runx2‑creRspo3flox/flox mice) to determine if RSPO3 is required for the bone-sparing effect of E2 in OVX mice. We confirmed that osteoblast-specific RSPO3 inactivation results in a substantial reduction in trabecular bone mass and strength in the vertebrae. However, E2 increased vertebral trabecular bone mass and strength similarly in mice devoid of osteoblast-derived RSPO3 and control mice. Unexpectedly, osteoblast-derived RSPO3 was needed for the full estrogenic response on cortical bone thickness. In conclusion, although osteoblast-derived RSPO3 is a crucial regulator of vertebral trabecular bone, it is required for a full estrogenic effect on cortical, but not trabecular, bone in OVX mice. Thus, estradiol and RSPO3 regulate vertebral trabecular bone mass independent of each other.

R-spondin 3 deletion induces Erk phosphorylation to enhance Wnt signaling and promote bone formation in the appendicular skeleton

“…Very surprisingly, however, and although the basal expression level of Rspo3 and the efficiency of deletion in long bones and vertebrae were similar ( Figure 3a and Figure 4a ), L5 μCT analysis showed a significant decrease in BV/TV, Tb.N, Tb.Th, and Conn.D accompanied by a marked increase in Tb.Sp. and SMI in Rspo3-OB-cKO compared with their control ( Rspo3 fl ) male and female littermates ( Figure 4b ), confirming a recent report ( Nilsson et al, 2022 ). Bone histomorphometry analysis showed that structural parameters were also significantly decreased but only in Rspo3-OB-cKO males compared with the control group ( Figure 4c–d and Supplementary file 6 ).…”

“…In the current studies, we have further investigated the role of Rspo3 in skeletal homeostasis. We found that if, as recently reported ( Nilsson et al, 2021 ; Nilsson et al, 2022 ), targeted deletion of Rspo3 in Runx2 + cells decreases bone mass in the axial skeleton, both global Rspo3 haplo-insufficiency and targeted deletion of Rspo3 in Runx2 + osteoblast precursors lead to a marked increase in trabecular bone mass in the appendicular skeleton in both male and female mice, mainly as a result of increased bone formation. Mechanistically, we found that Rspo3 deletion leads to increased Erk phosphorylation, and, similar to increased Rspo3, stabilization of β-catenin and, activation of Wnt signaling, revealing a novel Rspo3/Erk/Wnt signaling axis that contributes to the regulation of skeletal homeostasis.…”

“…R-Spondins, classically considered as positive modulators of Wnt signaling, play an important role in normal development of several tissues and organs, including bone, and are implicated in human diseases ( de Lau et al, 2012 ; Knight and Hankenson, 2014 ; Nagano, 2019 ; Raslan and Yoon, 2019 ; Shi et al, 2016 ). Among the 4 R-Spondins, GWAS studies in humans have shown that RSPO3 might be specifically involved in bone homeostasis due to the strong association between RSPO3 common variants and bone mineral density and fracture rate ( Duncan et al, 2011 ; Estrada et al, 2012 ; Medina-Gomez et al, 2012 ; Richards et al, 2008 ; Richards et al, 2012 ; Nilsson et al, 2022 ). Our results demonstrate, through several independent lines of genetic in vivo and in vitro experiments, that, counter-intuitively, decreasing Rspo3 levels globally or specifically in Runx2 + OB precursors leads to increased trabecular bone formation and high bone mass in the appendicular skeleton, mainly driven by increased number of OB progenitors and OBs as well as an increase in their bone forming activity.…”

“…In contrast, Nilsson et al performed their studies only in vertebrae. Furthermore, this is a trabecular bone-rich skeletal site, but the DEXA studies that led to the identification of a link between RSPO3 variants and bone fragility in humans were performed in the distal forearm, a cortical-rich appendicular skeletal site ( Nilsson et al, 2021 ; Nilsson et al, 2022 ). Furthermore, both groups found that cortical bone is not significantly affected by Rspo3 haplo-insufficiency or deletion in mice, confirming that trabecular and cortical bone are differentially regulated ( Movérare-Skrtic et al, 2014 ; Kiper et al, 2016 ) and further underlining the biologic complexity of Wnt signaling regulation of bone homeostasis.…”