To us, the findings above demonstrate a striking lack of heterogeneity in the pathways studied by the bone remodeling field overall. Moreover, given the heavy enrichment for just a few pathways over the last fifteen years, the lack of diversity may limit paradigm-changing discoveries and restrict novel therapeutic approaches for osteoporosis. In 2018, we called for an expansion into lesser-studied pathways to broaden the collective focus of the field [9]. We additionally highlighted nine pathways for which there was functional evidence to implicate a role in the regulation of bone remodeling in vivo [9]. Among these, two (the AhR and NMU pathways) have received considerably greater attention while four (the AhR, LPA, NMU, and OIP-1 pathways) are subjects of NIH-funded grants. However, relatively little progress has been made in the understanding and/or therapeutic potential of the other pathways we highlighted. Here, we identified an additional twenty pathways for which functional evidence was reported between 2018–2022 implicating a role in bone remodeling in vivo. In the sections below, we highlight several pathways that, to us, are particularly noteworthy as potential “low-hanging fruit” for future study.
CD82
Conditional deletion of the transmembrane scaffolding protein CD82 in the osteoclast lineage leads to increased trabecular bone mass with deficits in osteoclast differentiation [10]. However, it is important to note that a separate study using global knockout of CD82 revealed this protein is involved in both osteoblast and osteoclast compartments and the net effect of knockout is smaller bones with no change in bone mineral density [11]. Thus, translational studies aimed at targeting this factor in bone metabolism may require selectively inhibiting its action in osteoclasts while preserving its function in osteoblasts.
Chemerin
Chemerin, encoded by RARRES2, is an adipokine which signals through chemokine-like receptor 1 (CMKLR1) and is expressed by adipocytes, mesenchymal stem cells, osteoblasts, and osteoclasts [12–14]. Numerous studies in healthy adults or populations with specific diseases/conditions [15–21] [22] and animal models [23, 24] report that serum chemerin levels are inversely related to bone mass [25]. Moreover, chemerin is required for osteoclastic differentiation [26] and targeting chemerin via neutralizing antibodies in vivo results in near complete loss of osteoclastogenesis with correspondingly high bone mass [14]. This raises the possibility of translational studies aimed at pharmacological inhibition of chemerin to increase bone mass. However, it is important to note that CMKLR1 is involved in regulating testosterone production by Leydig Cells in the male gonads [27], which may complicate systemic targeting of chemerin activity and require more targeted strategies.
FAM210A
Several genetic variations at the FAM210A locus in humans are associated with bone mineral density and fracture. And, global loss of FAM210A expression in mice is associated with low bone mass due to reduced bone formation and increased bone resorption [28]. Yet, this factor is not abundantly expressed in the bone microenvironment and skeletal muscle-specific loss of FAM210A also leads to low bone mass [28], suggesting that it may play an indirect role in bone metabolism.
Menin
Menin is a nuclear protein encoded by the MEN1 gene and is involved in transcription regulation and chromatin remodeling, and genome stability. Deleterious mutations in MEN1 are associated with the rare autosomal dominant disorder Multiple Endocrine Neoplasia type 1, which is characterized by the development of tumors in multiple endocrine glands, including the parathyroid glands, pancreatic islet cells, and the anterior pituitary. The location of these tumors and endocrinological interaction with the skeleton underlies symptoms of bone pain and early-onset osteoporosis found in this disease. However, several lines of evidence support a direct role for menin in bone metabolism through regulating osteoblast differentiation and/or activity. For instance, conditional knockout of menin in various stages of mesenchymal stem cells or the osteoblast lineage is associated with intense reduction of bone mass and concurrent decrease in number of osteoblasts [29–32]. These data are consistent with evidence that menin controls expression of type 1 collagen, alkaline phosphatase, and osteocalcin through interaction with the BMP and/or TGF-beta pathways [33–36]. Collectively, these data raise the possibility that strategies aimed at increasing menin production in the osteoblast lineage may hold therapeutic potential for raising bone mass; indeed, mice overexpressing menin in osteoblasts from had increased bone volume [31].
NMU
NMU is an evolutionarily conserved peptide that is expressed in two major molecular forms, both of which are derived from the same mRNA and display similar receptor affinity for the heterotrimeric Gq/11-protein-coupled receptors NMU Receptor 1 (NMUR1) and NMU Receptor 2 (NMUR2) to regulate similar downstream targets [37–39]. NMUR1 is more broadly expressed than NMUR2 (see the Human Protein Atlas, proteinatlas.org) yet both are expressed in the bone microenvironment [39]. Two independent studies in mice implicate NMU as a negative regulator of bone formation [39, 40]. Many of the effects of NMU are attributed to its actions in the hypothalamus; indeed, this was initially proposed to be the mechanism by which NMU regulates bone metabolism since over-activation of the NMU pathway in this location reduces bone remodeling in the appendicular skeleton [40]. However, interpretation of those findings is complicated by the non-wildtype genetic background and “rescue” design of the experiment. Moreover, hypothalamus-specific knockdown of endogenous Nmu expression does not impact bone mass despite > 92% knockdown efficiency [41] and NMU has direct effects on suppressing osteoblastic differentiation of osteoprogenitor cells in vitro [39]. Given that NMU and its receptors are expressed in bone in vivo [39], these findings leave open the possibility that the negative effects of NMU on bone formation are accomplished in the bone microenvironment itself and could be targeted as a potential anabolic therapy for increasing bone mass.
Sirtuin-6
Sirtuin-6 is a sirtuin family protein that plays a vital role in genomic stability, aging, metabolism, and stress response via NAD+-dependent deacetylase activity. Functional evidence has indicated Sirt-6 plays a vital role in promoting osteoblastogenesis and preventing bone resorption. Several in vitro experiments have revealed that microRNAs – specifically miR-545-3p, miR-186, and miR-128 – bind to Sirt-6-3’UTR to downregulate its activity, resulting in decreased expression of osteoblastic markers and increased expression of osteoclastic markers [42–44]. Additionally, osteoblast/osteocyte-specific knockout of Sirtuin-6 has been shown to promote the phosphorylation of NF-kappaB, increasing RANKL-induced osteoclastogenesis, bone resorption, and expression of inflammatory cytokines as well as decreasing mature osteoblast function [45–49]. Restoration of Sirt-6 expression significantly reverses the increased expression of RANKL and proinflammatory cytokines [47]. In vivo experiments also revealed decreased trabecular bone mass/cortical bone thickness in homozygous Sirt-6 mutant mice [50], cavitation of femoral heads in mice with decreased Sirt-6 expression due to glucocorticoid-induced osteonecrosis [51], and decreased bone mass and apoptosis in aged Sirt-6 knockout mice. Reintroduction of Sirt-6 expression in the aforementioned Sirt-6 knockout models restores apoptosis of osteoclasts [52], increases osteoblast viability [53], and is associated with improved bone formation and calcification of tissues [49]. These studies call to mind studies aimed at increasing expression of Sirtuin-6 as an anabolic strategy to increase bone mass.
Tas1R
At least two of the Taste Receptor Tas1R family of heterotrimeric G-protein coupled receptors, which function in non-gustatory tissues as nutritional sensors, regulates bone metabolism as knock-out of Tas1R2 or Tas1R3 leads to high bone mass [54, 55]. These two receptors are capable of heterodimerizing with one another as a means of monitoring extracellular glucose levels and, given that the serum marker of osteoclastic activity CTx is dramatically reduced in Tas1R3 knockout mice[55], is possible that pharmacological targeting of this pathway could disrupt osteoclast function.
Vangl2
Vang-like protein 2 (Vangl2) is an essential component of the planar cell polarity (PCP) signaling pathway and is involved in various developmental and physiological functions. Vangl2 mutations are associated with skeletal patterning defects including malformed digit development [56]. Additionally, a specific role for Vangl2 in the negative regulation of osteoblasts comes from a report involving conditional deletion of this factor in the embryonic limb bud, which is associated with enhanced osteoblast differentiation of precursors and high bone mass in the postnatal skeleton [57]. This model also revealed increased bone formation rate in the absence of Vangl2 expression with no defects in bone resorption, suggesting that strategies aimed at limiting this factor in the osteoblastic lineage may hold promise as an anabolic therapy for increasing bone mass.
Miscellaneous
In addition to the above pathways, there are several others for which functional evidence indicates an endogenous role in bone remodeling and potential opportunity for therapeutic modulation. For instance, galectin-8 mediates coupling between osteoclasts and osteoblasts and global loss of this factor results in accelerated age-related bone loss [58]. Similarly, conditional deletion of the gene encoding Lysine (K)-specific demethylase 4B (KDM4B) in the embryonic limb bud is associated with enhanced age-related bone loss with selective defects in osteoblast activity (but not osteoclast activity) and increased bone marrow adipocity [59]. On the other hand, global loss of expression of Adaptor protein containing pleckstrin homology domain, phosphotryosine binding domain and leucine zipper motif (APPL1) – which plays an important role in intracellular signaling and vesicle trafficking and is an adaptor protein of the adiponectin receptor – leads to high bone mass associated with higher number of osteoblasts and reduced bone marrow adipocity [60].
Engulfment and Cell Motility Protein 1 (ELMO1) plays a significant role in osteoclast function as global loss of this factor reduces bone resorption in two mouse models of osteoporosis [61]; importantly, this study provided details on a peptide capable of disrupting ELMO1 function and reducing osteoclast activity in vitro, thus providing rationale for future studies examining this or other inhibitors as a means of dampening bone resorption. Finally, global loss of Transient Receptor Potential Cation Channel, Subfamily C, Member 6 (TRPC6) expression results in low bone mass with increased osteoclastic activity [62].