The Wnt signaling pathway controls cell fate decisions and tissue homeostasis during development and in the adult (Clevers and Nusse, 2012; Nusse and Clevers, 2017). It is also involved in skeletal development and essential for the regulation of bone mass in the adult skeleton (Baron and Kneissel, 2013; Gori and Baron, 2021; Huybrechts et al., 2020). Genetic or therapeutic activation of canonical Wnt signaling is associated with increased bone mass (Baron and Kneissel, 2013) and current therapeutic approaches aim at activating this pathway in patients with osteoporosis or osteogenesis imperfecta for instance (Baron et al., 2020).

Wnt signaling involves a large number of receptors and co-receptors, and of endogenous agonists and antagonists that, together, tightly regulate its activation (Clevers and Nusse, 2012; Nusse and Clevers, 2017; Baron and Kneissel, 2013; Gori and Baron, 2021). Due to this complexity, and even though Wnt signaling has been studied extensively in recent years in bone, several aspects of the mechanisms by which it regulates bone mass remain unclear. Similarly, the specific downstream events regulated by the various components of the Wnt signaling machinery and their interaction with other signaling cascades remain puzzling.

In this context, the fact that several studies have demonstrated that the four Roof plate-specific spondin, R-spondins (Rspo1 to 4), synergize with Wnt ligands to activate Wnt signaling (Gori and Baron, 2021; de Lau et al., 2014; de Lau et al., 2012; Knight and Hankenson, 2014; Lu et al., 2008; Nagano, 2019; Raslan and Yoon, 2019; Shi et al., 2016) raises the question of their potential role in skeletal development and homeostasis. This enhancement of Wnt activity is attributed to the ability of Rspos to prevent the ubiquitination and degradation of the Lrp5/6/Fzd receptor complex via Lgr4-6, closely related orphans of the leucin-rich repeat containing G protein-coupled receptors, and the transmembrane E3 ubiquitin ligases ring finger 43 (Rnf43) and zinc and ring finger 3 (Znrf3), sensitizing cells to Wnt ligands (de Lau et al., 2014; Glinka et al., 2011; Hao et al., 2012; Ruffner et al., 2012; Wang et al., 2013; Xie et al., 2013). Although the role of Lgr receptors in the effects of Rspos is well established, recent reports have shown that Rspo2 and Rspo3 can also enhance Wnt signaling independently of Lgr receptors, possibly by acting as direct antagonist ligands to RNF43 and ZNRF3 (Nagano, 2019; Lebensohn and Rohatgi, 2018; Szenker-Ravi et al., 2018).

Of particular interest is the fact that, in contrast to the many studies that have reported that Rspos co-activate Wnt signaling, studies in Zebrafish have indicated that Rspo3 can also function as a negative regulator of canonical Wnt signaling during dorsoventral and anteroposterior patterning (Rong et al., 2014; Wu et al., 2014). Additionally, it has been shown that Rspos can potentiate non-canonical Wnt cascades, such as the Wnt/PCP signaling (Glinka et al., 2011; Hao et al., 2012) and can function as antagonists of BMPR1 in Xenopus (Lee et al., 2020), two events that could have a negative impact on bone formation and bone mass. Thus, the mechanisms involved in the Rspos/Wnt signaling axis and their influence on skeletal homeostasis appear to be more complex in vivo.

The four Rspos belong to a family of cysteine-rich secreted glycoproteins with high structural similarity and 60% sequence homology (de Lau et al., 2012; Nagano, 2019). Although all Rspos are expressed in bone during development, they have specific and unique functions as reflected by the findings that their deletion leads to different phenotypes (Lu et al., 2008; Shi et al., 2016; Aoki et al., 2007; Neufeld et al., 2012; Ishii et al., 2008; Kazanskaya et al., 2008; Knight et al., 2018; Park et al., 2018; Parma et al., 2006; Wei et al., 2007). Within the Rspo family, Rspo3 is of particular interest for bone because it is highly expressed in skeletal elements during mouse development (Nam et al., 2007; Alhazmi et al., 2021) and several human GWAS studies have shown that RSPO3 SNPs are strongly associated with bone mineral density and risk of fracture (Duncan et al., 2011; Estrada et al., 2012; Lee et al., 2010; Medina-Gomez et al., 2012; Richards et al., 2008; Richards et al., 2012; Nilsson et al., 2021). Not surprisingly, in vitro studies have shown that overexpression of, or treatment with, Rspos can enhance Wnt-ligand mediated osteoblast (OB) differentiation (Lu et al., 2008; Knight et al., 2018; Zhu et al., 2016). However, similar to the Zebrafish and Xenopus studies mentioned above, it has also been reported that Rspo3 may function as a negative regulator of osteoblast differentiation in vitro (Zhang et al., 2017). Thus, taking into account the entire literature raises questions about the true net influence of Rspos, and in particular Rspo3, on skeletal homeostasis. We have recently reported that Rspo3 is expressed in osteoprogenitors in the craniofacial complex in both mice and Zebrafish (Alhazmi et al., 2021). However, Rspo3 disruption in Zebrafish has only mild effects on larval craniofacial cartilage skeleton (Alhazmi et al., 2021) and in mice its haplo-insufficiency does not appear to affect craniofacial development. Nilsson et al. have recently reported that targeted deletion of Rspo3 in osteoblast precursors in mice leads to low bone mass in the axial skeleton (L5) at 12 weeks of age, they examined only the axial skeleton where no changes in cellular and dynamic parameters were observed (Nilsson et al., 2021).

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.

Wnt signaling is central to skeletal development and homeostasis in health and disease (Baron and Kneissel, 2013). Understanding the biological mechanisms by which this signal operates is, therefore, of both scientific and clinical interest. 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. Unexpectedly though, and as recently reported by Nilsson et al., 2021, we also found that while the axial skeleton is only moderately affected in the haplo-insufficient mice, vertebrae are affected in the opposite manner, that is with a decrease in trabecular bone mass, after OB precursor-targeted deletion of Rspo3. Although it is not possible to identify the mechanism(s) responsible for these differential responses of the skeleton, it is worth mentioning here that axial and appendicular skeleton have different embryonic origin (paraxial mesoderm and later plate mesoderm, respectively) (Berendsen and Olsen, 2015) and that a new population of skeletal stem cells has been recently identified in the vertebrae (vSSC) that is absent from the long bones (Ay et al., 2022), raising the possibility that distinct populations of stem cells and non-stem progenitors in the vertebrae and in the long bones might respond differentially to stimuli. These findings underline the complexity of Wnt signaling regulation of bone homeostasis and support the hypothesis that different bone microenvironments, their embryonic origin, their differential exposure and response to mechanical cues, and the differential expression levels of receptors and co-receptors as well as of agonists and antagonists that balance Wnt signaling activity, might induce specific and distinct skeletal responses. Nevertheless, our findings show that skeletal phenotypes should always be investigated in both the appendicular and axial skeleton, as these two regions may respond differently. 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.

Thus, our studies reveal a novel and unexpected role of Rspo3 in the Wnt signaling machinery and bone homeostasis. Given the unexpected nature of our observations it is important to summarize here the multiple independent in vivo and in vitro experiments that unequivocally support the novel concept that decreasing the levels of Rspo3 can, at least in vitro and in the cellular environment of the appendicular skeleton’s trabecular bone, result in the activation of canonical Wnt signaling, increasing osteoblast differentiation and bone formation, and thereby bone mass: 1/Global haplo-insufficient or Runx2-Cre driven depletion of Rspo3 increased bone mass, OB numbers, and BFR in mice long bones; 2/Ex vivo, haplo-insufficient BMSCs exhibited increased OB differentiation and canonical Wnt signaling; 3/Rspo3 null MEFs also exhibited an enhanced Wnt signaling activity; 4/Basal levels of Erk phosphorylation were high in these cells and inhibition of Erk phosphorylation prevented the activation of Wnt signaling; 5/Rspo3 haplo-insufficiency rescued partially the inhibition of Wnt signaling induced by Dkk1 in vitro and in vivo.

In vitro studies have demonstrated that overexpression of and treatment with Rspo1 or Rspo2 enhance Wnt ligand-mediated OB differentiation (Knight and Hankenson, 2014; Lu et al., 2008; Knight et al., 2018). The literature and our own in vitro studies confirm that Rspo3 can be, as expected, a co-activator of canonical Wnt signaling in the Topflash assay, potentiating Wnt3a-dependent activation of canonical Wnt signaling in cellular assays (Yoon and Lee, 2012 and Figure 6—figure supplement 1). However, the expression of canonical Wnt target genes and the levels of pLrp6, activated β-catenin and Tcf1 were also markedly increased in Rspo3 haplo-insufficient BMSCs and in Rspo3-null MEFs, independent of the addition of Wnt3a to the assays, indicating that, counter-intuitively, the decrease or absence of Rspo3 activates mechanisms that favor β-catenin-dependent signaling. This is in contrast with the data reported by Nilsson et al. who show a decrease in Dkk1 and Sost, but this was done in the vertebrae of Runx2-CreRspo3^fl/fl mice, where BFR is in fact decreased, or a decrease in Tcf7 and Lef1 expression in calvarial OBs (cortical-derived cells Azzolin et al., 2014) treated with and without Tamoxifen to induce Rspo3 deletion in vitro (Nilsson et al., 2021), when cortical bone is in fact not affected. Although we have not analyzed mRNA expression in the vertebrae or calvaria, our extensive set of data (Topflash, Q-RTPCR, Western blot analysis for several Wnt signaling downstream proteins) using different cells (BMSC and MEFs) and long bones clearly show an activation of Wnt signaling with Rspo3 haploinsufficiency or deletion.

Another new finding from our studies is that Rspo3 expression is strongly repressed by Wnt3a and increased by Dkk1. This, together with the results discussed above, suggests that Rspo3 may provide a negative feedback-loop helping to balance canonical Wnt activity. Dkk1 efficacy in blocking Wnt3a-dependent activation of canonical Wnt signaling is significantly impaired in the absence of Rspo3 and Rspo3 haplo-insufficiency antagonizes the inhibition of bone formation induced by OB-targeted expression of Dkk1, confirming in vivo that Rspo3 haplo-insufficiency counteracts the OB-Dkk1 dependent function. Our results show that this is the result of intracellular changes in alternative pathways regulated by Rspo3. Indeed, we found that the Erk signaling pathway is activated and the basal level of Lrp6 phosphorylation enhanced by Rspo3 depletion. Thus, the activation of the Wnt signaling pathway when Rspo3 is expressed at low levels results from intra-cellular changes, distal to the receptor complexes. Our data and that of others (Lee et al., 2020) suggest that there are alternate Rspo3-mediated signaling mechanisms, separate from the Fzd/Lrp/β-catenin Wnt pathway, including the Wnt/PCP signaling (Glinka et al., 2011; Hao et al., 2012) and that these events can in turn regulate Wnt signaling intra-cellularly, such that β-catenin can be stabilized independent of the proximal activation of the canonical Wnt signaling machinery through changes in other signaling pathways (Baron and Kneissel, 2013; Krejci et al., 2012; Azzolin et al., 2014).

Thus, deletion of Rspo3 enhances Erk signaling which, in turn, stabilizes β−catenin independent of the canonical Wnt signaling receptor complex. In turn, this has a positive effect on OB differentiation (Červenka et al., 2011; Kim et al., 2019; Krejci et al., 2012). Our finding that the increase in Wnt signaling activation and the OB potential of MSCs seen in the absence of Rspo3 is abrogated by blocking Erk signaling confirms that the activation of Erk signaling associated with Rspo3 deficiency is responsible, at least in part, for the observed Wnt signaling activation.

Supporting our findings, in vitro studies have shown that Rspo3 silencing activates Erk signaling downstream of Lgr4, leading to increased OB differentiation of human adipose-derived stem cells (Zhang et al., 2017). This study did not however establish a connection between Erk and Wnt signaling. Although Lgrs function as receptors for Rspos, and Rspos/Lgrs interactions enhance Wnt signaling by inducing the clearance of Rnf43 and Znrf3 (Ruffner et al., 2012; Wang et al., 2013; de Lau et al., 2011), there is also strong evidence that Rspos/Lgrs interaction can activate distinct signaling cascades that can affect bone, including the cAMP/PKA/Creb signaling pathway in Lgr4 null mice (Luo et al., 2009) and the Erk signaling cascade (Zhang et al., 2017; Xu et al., 2016; Lin et al., 2019; Vieira et al., 2015).

Based on our observations, we propose that Rspo3 has a dual mode of action to regulate canonical Wnt signaling and bone formation. This duality is based on the regulation of two distinct signaling cascades and their crosstalk: Rspo3 functions via both the Lgr/Rnf43/Znrf3 and the Lgr/Erk axes, and while activation of the Lgr/Rnf43/Znrf3 axis boosts Wnt signaling strength by the membrane clearance of Rnf43/Znrf3 and subsequent stabilization of Fzd receptors, binding of Rspo3 to Lgr impairs Erk signaling, preventing Erk signaling activation and further stabilization of β−catenin (Figure 10). Thus, haplo-insufficiency and deletion of Rspo3 would dampen Wnt signaling at the cell surface by preventing the Rnf43/Znrf3 effects while intracellularly enhancing pLrp6 and β−catenin stabilization, via Erk phosphorylation, overcompensating the decrease in Rspo3-dependent Lrp5/6 receptors-dependent Wnt activation in OBs and their progenitors. Because activation of the Lgr/Rnf43/Znrf3 cascade is not exclusively dependent on Rspo3, deletion of Rspo3 would only hinder canonical Wnt signaling partially. In contrast, lack of Rspo3 promotes the Lgr/Erk cascade, to not only enhance β−catenin stabilization (Figure 10) but also regulate OB differentiation and bone formation. This model also explains the observed loss of Dkk1 efficacy in inhibiting Wnt signaling: the Erk-dependent stabilization of β−catenin being independent of Wnt receptor activation, Dkk1, which binds to the LRP5/6 receptors, cannot dampen the activation of downstream events as they are independent of the LRP5/6-Fzd receptor complex.

Figure 10

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Supporting the fact that Rspo3 can also regulate Wnt-independent pathways, a recent study has suggested that Rspo3 acts as an antagonist to BMPR1A, inhibiting BMP signaling during development (Lee et al., 2020). Thus, our observations may be due, at least in part, to changes (activation) in BMP signaling, which in turn could lead to the observed increase in pErk (Zhou et al., 2007). Although this remains a possibility, it seems unlikely. First, in contrast to our observations here, BMP activation in the adult skeleton has been linked to activation of non-canonical Wnt signaling, increased Sost expression, and bone resorption (Kiper et al., 2016; Kamiya et al., 2016). Second, several studies have shown that activation of BMP signaling in the osteoblast lineage has a negative impact on bone formation and bone mass (Kamiya et al., 2010; Kamiya et al., 2008; Ko et al., 2017; Shi et al., 2018).

In conclusion, our studies indicate that Rspo3 is required for skeletal homeostasis and show for the first time that axial and appendicular skeleton are differentially regulated by this Wnt signaling regulator. The molecular basis for this differential regulation has not been elucidated but might include different embryologic origin, differences in local micro-environment, or differences in mechanical loading, for instance. Nevertheless, our in vitro studies suggest that Rspo3 regulates bone formation through its interaction not only with the Wnt receptor machinery, as a positive co-activator, but also with other signaling pathways that affect β−catenin stability independent of the receptor complexes. Consequently, its deletion removes a co-activator of Wnt signaling, potentially decreasing bone formation, but also promotes Erk signaling activation, increasing β−catenin stability sufficiently to enhance bone formation and increase bone mass in the appendicular skeleton. Furthermore, because Rspo3 depletion increases Dkk1 and Dkk1 increases Rspo3 expression, this study also reveals a novel Rspo3-dependent negative feedback Wnt signaling regulatory loop. These findings have important implications for understanding the pleiotropic functions of Rspos and Wnt signaling in skeletal homeostasis and the mechanisms that regulate bone mass.

All data generated or analysed during this study are included in the manuscript and supporting file.

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Decision letter after peer review:

[Editors’ note: the authors submitted for reconsideration following the decision after peer review. What follows is the decision letter after the first round of review.]

Thank you for submitting the paper "R-spondin 3 deletion favors Erk phosphorylation to enhance Wnt signaling and bone formation" for consideration at eLife. Your submission has been assessed by a member of the Board of Reviewing Editors together with two reviewers. Although the work is of interest, we regret to inform you that the findings at this stage are considered too preliminary for further consideration at eLife.

Although both reviewers commended you on the quality of the data presented and the clarity of the presentation, they also had numerous concerns that had to do with what they perceived as a descriptive nature of the work, with discrepancy with other studies published on this topic, and a different interpretation of the DKK1-based experiments.

Reviewer #1:

R-spondins (Rspo1-4) are secreted proteins which have been characterized as positive regulators of the Wnt signaling in several cell types and model organisms. Wnt signaling, through its action on osteoblasts, plays an important role in the acquisition and maintenance of normal bone mass in vertebrates and GWAS studies have linked bone mineral density and fracture risk to the RSPO3 gene in humans. This study aims at determining the role of Rspo3 in osteoblasts using a series of mouse and cellular models. It was found that Rspo3 haplo-insufficiency (Rspo3+/-) in mice results in increase bone formation and bone mass, a result which disagree with the reported role of RSPO3 as a positive regulator of Wnt signaling. Inactivation of Rspo3 specifically in the osteoblast linage also results in a similar phenotype. A series of cell culture experiments shows that RSPO3 inactivation potentiates Wnt signaling possibly by increasing the activation of the ERK pathway.

Strengths of the study:

-Diverse approaches are used, including experiments in vivo (mouse models) and ex vivo taking advantage of various cell culture systems (osteoblasts, MEF).

-Both global and conditional mutants of Rspo3 are studied.

-Extensive bone histomorphometry is conducted to characterize the bone phenotypes of the mutant mice.

-The manuscript is well written.

Weaknesses of the study:

-The impact on bone of Rspo3 haploinsufficiency and osteoblast-specific inactivation presented here is in sharp contrast with a recent study by another group that detected a lower bone mass in mice lacking Rspo3 in osteoblasts (https://pubmed.ncbi.nlm.nih.gov/34389713/). The reason for this apparent discrepancy was not addressed or discussed in the manuscript.

-While histomorphometry is an accepted approach to measure bone density, the findings should be confirmed with another method, i.e., micro-CT. In addition, only one type of bone was examined (tibia) and the impact of Rspo3 deficiency on the axial skeleton was not addressed.

-Most of the mechanistic experiments involve Rspo3-/- MEF. To extend these findings to osteoblasts, these experiments should be repeated with Rspo3 fl/fl; Runx2-Cre BMSC cells.

-A number of important controls are missing. In particular, it will be important to show if recombinant mouse RSPO3 can rescue the cellular phenotypes of the Rspo3-/- cells.

Overall, without these critical missing pieces of data, the results presented do not fully support the claims of the manuscript.

Recommendations for the authors:

1) A recent paper by Nilsson et al. (https://pubmed.ncbi.nlm.nih.gov/34389713/), which is not cited in the current manuscript, shows that Rspo3 inactivation in osteoblasts with the same Runx2-Cre line, causes a low trabecular bone mass phenotype, at least in vertebra. These apparently contradictory results may be indicating that Rspo3 has different impact on bone depending on the skeletal sites. It will therefore be important to examine trabecular bone density in the vertebra in the current study to resolve this question. In addition, micro-CT should be used as an alternative approach to measure BV/TV in long bone and vertebra, since Nilsson et al. used this method.

2) Several experiments are comparing WT or Rspo3-/- MEF, but there is no evidence provided that WT MEF secrete significant amount of Rspo3. In addition, it will be essential to show that the cellular phenotypes of the Rspo3-/- BMSC and MEF presented in Figure 4-7 can be rescued by the exogenous addition of recombinant mouse Rspo3. Otherwise, it is not possible to conclude that the changes in Wnt signaling detected in these cells is really caused by a cell-autonomous effect of the absence of Rspo3. In link with this comment, there are inconsistency between some of the data presented. For example, in figure 5A, exogenous Wnt3a is required to induce Tcf1 promoter, measured using TOPFlash luciferase assay, and the addition of recombinant Rspo3 do not suppressed the induction. In contrast, in figure 5B, Rspo3-/- MEF have increased Tcf1 expression in absence of Wnt3a treatment.

3) Linked to the previous comment, cell culture experiments (Figure 4-7) should be repeated with fl/fl;Runx2-Cre BMSC or with Rspo3 fl/fl calvaria osteoblasts transduced with Cre ex vivo.

4) If the global haploinsufficiency of Rspo3 results in an increased bone mass through a cell-autonomous effect on osteoblasts, then it is expected that an osteoblast specific haploinsufficiency (Rspo3 +/flox ; Runx2-Cre) will also results in a high bone mass phenotype. Yet, this genotype was not analyzed.

4) Whether global Rspo3 haploinsufficiency results in decreased expression of Rspo3 in osteoblasts and in bone was not shown.

5) An important control to be included in Figure 3 is the +/+ ; Runx2-Cre mice to exclude any potential non-specific effect of this Cre line on bone mass.

6) A description of how the Rspo3 floxed mice used in figure 3 were generated is not included in the material and methods and no reference is provided in link with this line. How this model compares to the one published by Nilsson et al. (see point 1)?

7) The rational for using recombinant human Wnt3a, Dkk1 and Rspo3 on mouse osteoblast and MEF cultures is not clear, since the equivalent mouse proteins are available from R&D System.

8) The interpretation of the data presented in Figure 6.D-E (Rspo3+/-;TgDkk1) is challenging. Since these two mouse lines have opposite phenotypes in term of bone mass, the normalization of the bone density in the double transgenic does not necessarily provide a clear demonstration that the two genes act in the same pathway. Moreover, the TgDkk1 overexpress Dkk1 in osteoblasts (col1a1 promoter) while the haploinsufficiency of Rspo3 is in all cells, further complicating the interpretation of this genetic experiment.

9) In Figure 6A there is only n=2 per group. Although one can run ANOVA or t test on n=2, it will be more appropriate to have additional replicates.

Reviewer #2:

The work entitled "R-spondin 3 deletion favors Erk phosphorylation to enhance Wnt signaling and bone formation" by Nagano et al. investigated the role of RSPO3 in skeletal homeostasis. The authors analyzed bone formation and bone mass in RSPO3 haplo-insufficient mice and in mice lacking RSPO3 in osteoblasts. They found an increased bone mass. They propose that this phenotype is driven by an increase WNT and ERK signaling in osteoblast progenitor cells. The in vivo data clearly demonstrate that Rspo3 controls bone formation and high bone mass. However, in its current status, the work is descriptive and lacks the levels of novelty and mechanistic insights that are expected by readers of eLife. Indeed the regulation of both DKK/WNT and ERK signaling by RSPO3 has been previously described. The authors confirmed these observations without providing additional mechanistic insights into this regulation. As result the work appears quite descriptive/preliminary. Importantly, the in vivo data showing that Rspo3 haplo-insufficiency counteracted the effect of Dkk1 overexpression in vivo, clearly indicate that Dkk1 is not involved in RSPO3 phenotype, since Dkk1 overexpression does not have any effect on RSPO3 high bone mass phenotype. Thus most likely, Dkk1 does not play significant roles in RSPO3-mediated regulation of bone mass.

In addition, the link between ERK and DKK1 signaling regulation by RSPO3 has not been explored, so it is unclear whether these two pathways act in parallel or not.

The authors propose that the high bone mass phenotype observed in Rspo3 haplo-insufficient mice wes due to an increase in bone marrow precursor cells (mesenchymal stromal cells (MSC) population (Lin-CD45-CD31-CD51+Sca-1+)). This phenotype was mirrored by the RUNX2-Cre mediated deletion of Rspo3. Do MSC cells express Runx2-Cre? This should be verified by the authors.

[Editors’ note: the authors resubmitted a revised version of the paper for consideration. What follows is the authors’ response to the first round of review.]

Although both reviewers commended you on the quality of the data presented and the clarity of the presentation, they also had numerous concerns that had to do with what they perceived as a descriptive nature of the work, with discrepancy with other studies published on this topic, and a different interpretation of the DKK1-based experiments.

Reviewer #1:

R-spondins (Rspo1-4) are secreted proteins which have been characterized as positive regulators of the Wnt signaling in several cell types and model organisms. Wnt signaling, through its action on osteoblasts, plays an important role in the acquisition and maintenance of normal bone mass in vertebrates and GWAS studies have linked bone mineral density and fracture risk to the RSPO3 gene in humans. This study aims at determining the role of Rspo3 in osteoblasts using a series of mouse and cellular models. It was found that Rspo3 haplo-insufficiency (Rspo3+/-) in mice results in increase bone formation and bone mass, a result which disagree with the reported role of RSPO3 as a positive regulator of Wnt signaling. Inactivation of Rspo3 specifically in the osteoblast linage also results in a similar phenotype. A series of cell culture experiments shows that RSPO3 inactivation potentiates Wnt signaling possibly by increasing the activation of the ERK pathway.

Strengths of the study:

-Diverse approaches are used, including experiments in vivo (mouse models) and ex vivo taking advantage of various cell culture systems (osteoblasts, MEF).

-Both global and conditional mutants of Rspo3 are studied.

-Extensive bone histomorphometry is conducted to characterize the bone phenotypes of the mutant mice.

-The manuscript is well written.

Weaknesses of the study:

-The impact on bone of Rspo3 haploinsufficiency and osteoblast-specific inactivation presented here is in sharp contrast with a recent study by another group that detected a lower bone mass in mice lacking Rspo3 in osteoblasts (https://pubmed.ncbi.nlm.nih.gov/34389713/). The reason for this apparent discrepancy was not addressed or discussed in the manuscript.

-While histomorphometry is an accepted approach to measure bone density, the findings should be confirmed with another method, i.e., micro-CT. In addition, only one type of bone was examined (tibia) and the impact of Rspo3 deficiency on the axial skeleton was not addressed.

Thank you for your comment and for calling our attention on the manuscript by Nilsson et al. Note however that this paper was published while our manuscript was in review, explaining that we did not mention or discuss their findings. It is however important to note that the Ohlsson group reported only axial skeleton (vertebrae) data and at only one time point (12 week), when we had reported only appendicular skeleton (tibia) data, which showed consistent increase in bone mass at least 3 different ages.

Thus, in response to your comments and Ohlsson group’s paper, we now have analyzed the vertebrae in the same animals for which we reported tibia data. This led to the very surprising finding that if in long bones the Runx2-cre driven deletion of Rspo3 led to high bone mass, the vertebrae of the same animals showed a lower bone mass, in agreement with the findings reported by Nilsson et al. Thus, looking at the same part of the skeleton as Nilsson et al., we find the same results. Importantly, Nilsson et al. did not examine the appendicular skeleton and our data, confirmed in several in vivo models and in vitro studies, confirms that Rspo3 haploinsufficiency and its deletion in osteoblast progenitor can also lead to increased bone formation and bone mass. Notably also, examination of the vertebrae in Rspo3^+/- mice, where we found high bone mass in tibiae, showed no significant changes, despite a trend to increased bone formation. Thus, the effects of Rspo3 global deletion or its targeted deletion in osteoprogenitors differ between the axial and the appendicular skeleton.

It is not easy to explain such a surprising finding, and few papers in the literature examined both sites in animal studies. We have now added this data and discussed various possibilities that could explain the differences: crosstalk with load bearing (vertebrae are less loaded than limbs), the differential expression levels of receptors and co-receptors, distinct embryonic origin as well as of agonists and antagonists and the presence of distinct populations of stem cells and non-stem progenitors in the vertebrae and in the long bones, are examples of potential explanations. Yet, these findings do not invalidate our very strong and abundant set of data showing that, at least in vitro and in vivo in long bones, Rspo3 haploinsufficiency and OB-targeted deletion can also lead to high bone formation and bone mass.

On your other point, we had previously collected microCT data and have now increased this data to include both long bones and vertebrae.

-Most of the mechanistic experiments involve Rspo3-/- MEF. To extend these findings to osteoblasts, these experiments should be repeated with Rspo3 fl/fl; Runx2-Cre BMSC cells.

We show that BMSCs isolated from Rspo3 haploinsufficient mice have increased canonical Wnt signaling (new Figure 5) and increased CFU-F and CFU-OB potential (Figure 2). We believe that this set of experiments together with our in vivo data in Rspo3^+/- mice and targeted deletion in Runx2⁺ cells, establish a cell autonomous role of Rspo3. We do not think that to repeat these studies in the Runx2-Cre model would add much.

-A number of important controls are missing. In particular, it will be important to show if recombinant mouse RSPO3 can rescue the cellular phenotypes of the Rspo3-/- cells.

Overall, without these critical missing pieces of data, the results presented do not fully support the claims of the manuscript.

Thank you for this useful suggestion. We have now performed such rescue experiments and show now (new Figure 2) that recombinant Rspo3 prevents (rescues) the increase in CFU-OB number seen with Rspo3 haploinsufficiency, confirming that Rspo3 can also have a negative impact on OB differentiation.

Recommendations for the authors:

1) A recent paper by Nilsson et al. (https://pubmed.ncbi.nlm.nih.gov/34389713/), which is not cited in the current manuscript, shows that Rspo3 inactivation in osteoblasts with the same Runx2-Cre line, causes a low trabecular bone mass phenotype, at least in vertebra. These apparently contradictory results may be indicating that Rspo3 has different impact on bone depending on the skeletal sites. It will therefore be important to examine trabecular bone density in the vertebra in the current study to resolve this question. In addition, micro-CT should be used as an alternative approach to measure BV/TV in long bone and vertebra, since Nilsson et al. used this method.

These points have now been addressed (see above).

2) Several experiments are comparing WT or Rspo3-/- MEF, but there is no evidence provided that WT MEF secrete significant amount of Rspo3. In addition, it will be essential to show that the cellular phenotypes of the Rspo3-/- BMSC and MEF presented in Figure 4-7 can be rescued by the exogenous addition of recombinant mouse Rspo3. Otherwise, it is not possible to conclude that the changes in Wnt signaling detected in these cells is really caused by a cell-autonomous effect of the absence of Rspo3. In link with this comment, there are inconsistency between some of the data presented. For example, in figure 5A, exogenous Wnt3a is required to induce Tcf1 promoter, measured using TOPFlash luciferase assay, and the addition of recombinant Rspo3 do not suppressed the induction. In contrast, in figure 5B, Rspo3-/- MEF have increased Tcf1 expression in absence of Wnt3a treatment.

Levels of Rspo3 in MEF are presented in Figure 6-supplement 1b and by RT-QPCR cycle number for Rspo3 in MEF is between 22 and 24. We show now that recombinant Rspo3 prevents (rescues) the increase in CFU-OB number seen with Rspo3 haploinsufficiency (new Figure 2).

The reviewer is correct in mentioning that in the old Figure 5a (revised Figure 6-supplement 1b), Wnt3a induces the Tcf1 promoter and that Rspo3 functions as a potentiator of Wnt3a activity in wt MEFs. It is important to note however that Rspo3 by itself does not lead to Wnt signaling activation in the absence of Wnt3a. Here, our studies in BMSCs, in long bones and in MEFs, shown in the new Figure 5, 6 and 8, clearly and unexpectedly demonstrate that absence of Rspo3 results by itself in a significant activation in Wnt signaling at steady state, as showed by an increase in Tcf1 and Axin2 expression levels and active β-catenin and pLrp6 protein levels. We are aware that this is the opposite of what one could expect when knocking down or deleting Rspo3, but this is what makes it the key point of this paper. Several of our independent in vivo and in vitro experiments demonstrate that this is indeed the case, making this paper most novel and interesting. This is detailed in our discussion.

3) Linked to the previous comment, cell culture experiments (Figure 4-7) should be repeated with fl/fl;Runx2-Cre BMSC or with Rspo3 fl/fl calvaria osteoblasts transduced with Cre ex vivo.

These points have now been addressed (see above).

4) If the global haploinsufficiency of Rspo3 results in an increased bone mass through a cell-autonomous effect on osteoblasts, then it is expected that an osteoblast specific haploinsufficiency (Rspo3 +/flox ; Runx2-Cre) will also results in a high bone mass phenotype. Yet, this genotype was not analyzed.

Please note that in the long bones Runx2creRspo^fl induces only a 50% decrease in Rspo3 gene expression, in fact mimicking a haplo-insufficiency.

4) Whether global Rspo3 haploinsufficiency results in decreased expression of Rspo3 in osteoblasts and in bone was not shown.

Thank you for your comment. These control data have now been added in Figure 1a and in Supplemental Figure 1-supplement 1a.

5) An important control to be included in Figure 3 is the +/+ ; Runx2-Cre mice to exclude any potential non-specific effect of this Cre line on bone mass.

As shown in the new Figure 3 and 4 – Supplement 1, microCT analysis shows no significant differences in BV/TV, Tb.N, Th.th. and Tb.S in both L5 and Femur between Runx2Cre, wt and Rspo3^fl mice.

6) A description of how the Rspo3 floxed mice used in figure 3 were generated is not included in the material and methods and no reference is provided in link with this line. How this model compares to the one published by Nilsson et al. (see point 1)?

A description of the strategy used to generate the Rspo3 mutant mice is reported in reference#27 (Kazanskaya O, Ohkawara B, Heroult M, Wu W, Maltry N, Augustin HG, Niehrs C. The Wnt signaling regulator R-spondin 3 promotes angioblast and vascular development. Development) Reference 27 was and is mentioned in the methods in the Animal section.

Floxed mice and global het mice were generated using the same targeting strategy.

Briefly, as detailed in Suppl. Figure S2 in reference # 27, the Rspo3 mutant mice used in our studies were generated by flanking exon1 with loxP sequences. The fragment flanked by the 2 loxP genes was then removed by cre-recombinase to obtain a Rspo3 haploinsufficient mouse colony used to generate flox mice for targeted deletion. The Rspo3 flox mice used by Nilsson et al. were generated by flanking exons 2-4 with Loxp sequences and recombined by Cre recombinase.

7) The rational for using recombinant human Wnt3a, Dkk1 and Rspo3 on mouse osteoblast and MEF cultures is not clear, since the equivalent mouse proteins are available from R&D System.

In response to this comment, there was not specific rationale to use human or mouse recombinant proteins. Both have been shown to work in mouse cells.

8) The interpretation of the data presented in Figure 6.D-E (Rspo3+/-;TgDkk1) is challenging. Since these two mouse lines have opposite phenotypes in term of bone mass, the normalization of the bone density in the double transgenic does not necessarily provide a clear demonstration that the two genes act in the same pathway. Moreover, the TgDkk1 overexpress Dkk1 in osteoblasts (col1a1 promoter) while the haploinsufficiency of Rspo3 is in all cells, further complicating the interpretation of this genetic experiment.

Thank you for this comment. We agree with the reviewer that interpretation of such genetic experiments can be complex. Nonetheless our data show clearly that Rspo3 haploinsufficiency can rescue the low bone mass phenotype of mice overexpressing Dkk1 in osteoblasts, indicating that Rspo3 deletion effects occur in the osteoblast micro-environment, findings strongly supported by our in vitro data showing that when Rspo3 is deleted the efficiency of Dkk1 in blocking Wnt signaling activation is decreased.

9) In Figure 6A there is only n=2 per group. Although one can run ANOVA or t test on n=2, it will be more appropriate to have additional replicates.

As requested, we have added 1 extra experiment and we have now n=3 (now in the new Figure 7a). Note that in Figure 7a, the statistical analysis performed is Student T-test. This is reflected in the legend.

Reviewer #2:

The work entitled "R-spondin 3 deletion favors Erk phosphorylation to enhance Wnt signaling and bone formation" by Nagano et al. investigated the role of RSPO3 in skeletal homeostasis. The authors analyzed bone formation and bone mass in RSPO3 haplo-insufficient mice and in mice lacking RSPO3 in osteoblasts. They found an increased bone mass. They propose that this phenotype is driven by an increase WNT and ERK signaling in osteoblast progenitor cells. The in vivo data clearly demonstrate that Rspo3 controls bone formation and high bone mass. However, in its current status, the work is descriptive and lacks the levels of novelty and mechanistic insights that are expected by readers of eLife. Indeed the regulation of both DKK/WNT and ERK signaling by RSPO3 has been previously described. The authors confirmed these observations without providing additional mechanistic insights into this regulation. As result the work appears quite descriptive/preliminary. In addition, the link between ERK and DKK1 signaling regulation by RSPO3 has not been explored, so it is unclear whether these two pathways act in parallel or not.

The main novelty of our report is that deletion of Rspo3 would be expected, based on current knowledge, to decrease bone formation and bone mass by removing a Wnt co-activator. In contrast, we demonstrate instead that decreased levels of Rspo3 do increase bone formation and bone mass, and this is in several independent in vivo genetic and in vitro models. Moreover, to our knowledge there are no previous data showing that in vivo haploinsufficiency of Rspo3 can rescue the low bone mass seen in mice with osteoblast targeted overexpression of Dkk1.

Our signaling findings also add to the current models. As mentioned in the manuscript, previous reports showed that Rspo3 deletion in vitro (siRNA) in adipose precursor cells leads to increased pERK phosphorylation and increased cell differentiation. We confim here activation of pERK by low Rspo3 levels but the novelty of our studies lies in that we report a direct link to Wnt signaling, establishing a novel Rspo3/ERK/Wnt axis.

We show that in the absence of Rspo3 the increase in pERK phosphorylation contributes to Wnt signaling activation as indicated by the findings that blocking ERK signaling significantly impairs Wnt signaling activation seen in absence of Rspo3. Therefore, our studies add Wnt to the Rspo3/ERK axis.

Importantly, the in vivo data showing that Rspo3 haplo-insufficiency counteracted the effect of Dkk1 overexpression in vivo, clearly indicate that Dkk1 is not involved in RSPO3 phenotype, since Dkk1 overexpression does not have any effect on RSPO3 high bone mass phenotype.

Thus most likely, Dkk1 does not play significant roles in RSPO3-mediated regulation of bone mass.

We respectfully disagree with this comment. Our data show (1) haploinsufficiency of Rspo3 rescues significantly the low bone mass phenotype seen in TgDkk1 mice and (2) However, in contrast to what is stated by the reviewer (“Dkk1 overexpression does not have any effect on RSPO3 high bone mass phenotype”) overexpression of Dkk1 in osteoblasts decreased significantly the high bone mass phenotype seen in Rspo3 het mice, validating our interpretation of the results.

The authors propose that the high bone mass phenotype observed in Rspo3 haplo-insufficient mice wes due to an increase in bone marrow precursor cells (mesenchymal stromal cells (MSC) population (Lin-CD45-CD31-CD51+Sca-1+)). This phenotype was mirrored by the RUNX2-Cre mediated deletion of Rspo3. Do MSC cells express Runx2-Cre? This should be verified by the authors.

The reviewer is correct: we show that global haploinsufficiency of Rspo3 leads to an increase in BM osteoblast precursors and as shown by bone histomorphometry, to an increase in the number of osteoblasts in Rspo3 het mice. The population of MSC in Runx2creRspo3^fl mice was not investigated by flow cytometry. However, deletion of Rspo3 in Runx2⁺ cells also led to a significant increase in the number of osteoblasts by bone histomorphometry.

Author details

1. Kenichi Nagano

   School of Dental Medicine, Harvard University, Boston, United States

   Contribution

   Data curation, Formal analysis, Investigation, Methodology, Writing – original draft, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-3145-4841

2. Kei Yamana

   School of Dental Medicine, Harvard University, Boston, United States

   Contribution

   Data curation, Formal analysis, Methodology, Writing – original draft

   Competing interests

   No competing interests declared

3. Hiroaki Saito

   School of Dental Medicine, Harvard University, Boston, United States

   Contribution

   Data curation, Formal analysis, Investigation, Methodology

   Competing interests

   No competing interests declared

4. Riku Kiviranta

   School of Dental Medicine, Harvard University, Boston, United States

   Contribution

   Data curation, Formal analysis, Investigation, Methodology

   Competing interests

   No competing interests declared

5. Ana Clara Pedroni

   School of Dental Medicine, Harvard University, Boston, United States

   Contribution

   Data curation, Formal analysis, Methodology

   Competing interests

   No competing interests declared

6. Dhairya Raval

   School of Dental Medicine, Harvard University, Boston, United States

   Contribution

   Data curation, Formal analysis, Methodology

   Competing interests

   No competing interests declared

7. Christof Niehrs

   1. German Cancer Research Center, DKFZ-ZMBH Alliance, Heidelberg, Germany
   2. Institute of Molecular Biology (IMB), Mainz, Germany

   Contribution

   Resources, Writing – review and editing

   Competing interests

   No competing interests declared

8. Francesca Gori

   School of Dental Medicine, Harvard University, Boston, United States

   Contribution

   Conceptualization, Resources, Data curation, Formal analysis, Supervision, Funding acquisition, Investigation, Methodology, Writing – original draft, Project administration, Writing – review and editing

   For correspondence

   francesca_gori@hsdm.harvard.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-5685-8303

9. Roland Baron

   1. School of Dental Medicine, Harvard University, Boston, United States
   2. Department of Medicine, Harvard Medical School, Boston, United States
   3. Endocrine Unit, Massachusetts General Hospital, Boston, United States

   Contribution

   Conceptualization, Resources, Supervision, Funding acquisition, Validation, Investigation, Writing – original draft, Project administration, Writing – review and editing

   For correspondence

   Roland_baron@hsdm.harvard.edu

   Competing interests

   No competing interests declared

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