Therapeutics targeting osteoclasts are commonly used treatments for bone metastasis; however, whether and how osteoclasts regulate premetastatic niche and bone tropism are largely unknown. In this study, we report that osteoclast precursors (OPs) can function as a premetastatic niche component that facilitates breast cancer (BCa) bone metastasis at early stages. At the molecular level, unbiased GPCR ligand/agonist screening in BCa cells suggested that R-spondin 2 (RSPO2) and RANKL, through interaction with their receptor LGR4, promoted osteoclastic premetastatic niche formation and enhanced BCa bone metastasis. This was achieved by RSPO2/RANKL-LGR4 signal modulating the WNT inhibitor DKK1 through Gα_q and β-catenin signaling. DKK1 directly facilitated OP recruitment through suppression of its receptor LDL receptor–related protein 5 (LRP5) but not LRP6, upregulating Rnasek expression via inhibition of canonical WNT signaling. In clinical samples, RSPO2, LGR4, and DKK1 expression showed a positive correlation with BCa bone metastasis. Furthermore, soluble LGR4 extracellular domain (ECD) protein, acting as a decoy receptor for RSPO2 and RANKL, significantly alleviated bone metastasis and osteolytic lesions in a mouse bone metastasis model. These findings provide unique insights into the functional role of OPs as key components of the premetastatic niche for BCa bone metastasis and identify RSPO2/RANKL-LGR4 signaling as a promising target for inhibiting BCa bone metastasis.

Inflammatory diseases decrease the extracellular environmental pH. However, whether proton-activated G protein-coupled receptors (GPCRs) can regulate the development of osteoarthritis (OA) is largely unknown. In this study, we report that proton-activated GPR4 is essential for OA development. We found a marked increase in expression of the proton-activated GPR4 in human and mouse OA cartilage. Lentivirus-mediated overexpression of GPR4 in mouse joints accelerated the development of OA, including promotion of articular cartilage damage, synovial hyperplasia, and osteophyte formation, while Gpr4 knockout effectively attenuated the development of posttraumatic and aging-associated OA in mice. We also found that inhibition of GPR4 with the antagonist NE52-QQ57 ameliorated OA progression in mice, promoted extracellular matrix (ECM) production, and protected cartilage from degradation in human articular cartilage explants. Moreover, GPR4 overexpression upregulated matrix-degrading enzymes’ expression and inflammation factors under pro-inflammatory and slightly acidic conditions. Mechanistically, GPR4 suppressed chondrocyte differentiation and upregulated cartilage homeostasis through NF-κB/MAPK signaling activation by regulating CXCR7/CXCL12 expression. Together, our results take the lead to illustrate that proton-activated GPCR acts as a key regulator for OA pathogenesis in vivo, and support that GPR4 could be a promising therapeutic target for OA treatment.

Adhesion GPCR Gpr126/Adgrg6 regulates mouse body length and bone mass by controlling osteoblast differentiation.

Background: The chondrogenic differentiation of mesenchymal stem cells (MSCs) to enhance cartilage repair and regeneration is a promising strategy to alleviate osteoarthritis (OA) progression. Method: The potency of JD-312 in inducing chondrogenic differentiation of MSCs was assessed and verified. The efficacy of JD-312-treated MSCs was evaluated using a Sprague-Dawley rat DMM model. Additionally, the capacity of JD-312 to successfully recruit bone marrow-derived mesenchymal stem cells (BMSCs) for the treatment of OA in vitro was confirmed via intra-articular injection. The repair status of the articular cartilage was analyzed in vivo through histological examination. Result: In this study, we identify JD-312 as a novel non-toxic small molecule that can promote chondrogenic differentiation in human umbilical cord-derived MSCs (hUCMSCs) and human bone marrow MSCS (hBMSCs) in vitro. We also show that transient differentiation of MSCs with JD-312 prior to in vivo administration remarkably improves the regeneration of cartilage and promotes Col2a1 and Acan expression in rat models of DMM, in comparison to kartogenin (KGN) pre-treatment or MSCs alone. Furthermore, direct intra-articular injection of JD-312 in murine model of OA showed reduced loss of articular cartilage and improved pain parameters. Lastly, we identified that the effects of JD-312 are at least in part mediated via upregulation of genes associated with the focal adhesion, PI3K-Akt signaling and the ECM-receptor interaction pathways, and specifically cartilage olig-omeric matrix protein (COMP) may play a vital role. Conclusion: Our study demonstrated that JD-312 showed encouraging repair effects for OA in vivo. The translational potential of this article: Together, our findings demonstrate that JD-312 is a promising new therapeutic molecule for cartilage regeneration with clinical potential.

Adhesion G protein–coupled receptors (GPCRs) play crucial roles in numerous physiological and pathological conditions. However, the functions of adhesion GPCRs remain poorly understood because of the lack of effective modulators. Here, we used the adhesion GPCR D1 (ADGRD1/GPR133) as a model to unveil a strategy for finding exogenous agonists that target adhesion GPCRs while revealing previously unidentified functions of ADGRD1. We identified the small molecule GL64 as a selective agonist of ADGRD1. GL64 activates ADGRD1 by mimicking the stachel sequence. Using GL64 as a chemical tool, we demonstrated that ADGRD1 negatively regulates bone loss by inhibiting osteoclastogenesis. The cAMP-PKA-NFATC1 pathway was identified as the downstream signaling pathway of ADGRD1 in osteoclasts. Furthermore, administering GL64 prevented bone loss and suppressed osteoclast activity in the osteoporosis mouse model induced by ovariectomy. Our findings provide mechanistic insights into the activation of adhesion GPCRs by exogenous agonists and underscore the therapeutic potential of targeting ADGRD1 in osteoclast-related diseases.

Background/Objectives: Mesenchymal stem cells (MSCs) possess the remarkable ability to differentiate into various cell types, including osteoblasts. Understanding the molecular mechanisms governing MSC osteogenic differentiation is crucial for advancing clinical applications and our comprehension of complex disease processes. However, the key biological molecules regulating this process remain incompletely understood. Methods: In this study, we conducted systematic re-analyses of published high-throughput transcriptomic datasets to identify and validate key biological molecules that dynamically regulate MSC osteogenic differentiation. Our approach involved a comprehensive analysis of gene expression patterns across human tissues, followed by the rigorous experimental validation of the identified candidates. Results: Through integrated analytical and experimental approaches, we utilized high-throughput transcriptomics to identify four critical regulators of MSC osteogenic differentiation: PTBP1, H2AFZ, BCL6, and TTPAL (C20ORF121). Among these, PTBP1 and H2AFZ functioned as positive regulators, while BCL6 and TTPAL acted as negative regulators in osteogenesis. The regulatory roles of these genes in osteogenesis were further validated via overexpression experiments. Conclusions: Our findings advance our understanding of MSC differentiation fate determination and open new therapeutic possibilities for bone-related disorders. The identification of these regulators provides a foundation for developing targeted interventions in regenerative medicine.