Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2025 Jun 30;10(1):199.
doi: 10.1038/s41392-025-02291-y.

The mechanosensitive adhesion G protein-coupled receptor 133 (GPR133/ADGRD1) enhances bone formation

Juliane Lehmann #  1 , Hui Lin #  2   3 , Zihao Zhang #  4 , Maren Wiermann  5 , Albert M Ricken  6 , Franziska Brinkmann  7 , Jana Brendler  6 , Christian Ullmann  5 , Luisa Bayer  5 , Sandra Berndt  5 , Anja Penk  8 , Nadine Winkler  5 , Franz Wolfgang Hirsch  9 , Thomas Fuhs  10 , Josef Käs  10 , Peng Xiao  2 , Torsten Schöneberg  5 , Martina Rauner  7 , Jin-Peng Sun  11   12 , Ines Liebscher  13
Affiliations

The mechanosensitive adhesion G protein-coupled receptor 133 (GPR133/ADGRD1) enhances bone formation

Juliane Lehmann et al. Signal Transduct Target Ther. .

Abstract

Osteoporosis represents an increasing health and socioeconomic burden on aging societies. Current therapeutic options often come with potentially severe side effects or lack long-term efficacy, highlighting the urgent need for more effective treatments. Identifying novel drug targets requires a thorough understanding of their physiological roles. Genome-wide association studies in humans have linked gene variants of the adhesion G protein-coupled receptor 133 (GPR133/ADGRD1) to variations in bone mineral density and body height. In this study, we explore the impact of GPR133/ADGRD1 on osteoblast differentiation and function. Constitutive and osteoblast-specific knockouts of Gpr133/Adgrd1 in mice lead to reduced cortical bone mass and trabecularization in the femurs and vertebrae - features characteristic of osteoporosis. This osteopenic phenotype in receptor-deficient mice is caused by impaired osteoblast function, which, in turn, promotes increased osteoclast activity. At the molecular level, GPR133/ADGRD1 regulates osteoblast function and differentiation through a combined activation mechanism involving interaction with its endogenous ligand, protein tyrosine kinase 7 (PTK7), and mechanical forces. This is demonstrated in vitro through stretch assays and in vivo via a mechanical loading experiment. Further in vitro analysis shows that GPR133/ADGRD1-mediated osteoblast differentiation is driven by cAMP-dependent activation of the β-catenin signaling pathway. Activation of GPR133/ADGRD1 with the receptor-specific ligand AP-970/43482503 (AP503) enhances osteoblast function and differentiation, both in vitro and in vivo, significantly alleviating osteoporosis in a mouse ovariectomy model. These findings position GPR133/ADGRD1 as a promising therapeutic target for osteoporosis and other diseases characterized by reduced bone mass.

PubMed Disclaimer

Conflict of interest statement

Competing interests: The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Constitutive Gpr133/Adgrd1 deficiency in male mice results in trabecular and cortical bone loss. a–d LacZ staining of the entire spine of 3-months-old male Gpr133/Adgrd1 homozygous (Homo) knockout (KO) and wild-type (WT) mice. Scale bar: 1 mm (a/c) and 2 mm (b/d). e Gpr133/Adgrd1 mRNA expression in different bone and organs of 23-weeks-old male WT mice using qPCR (n = 5). f–j Femora from 23-weeks-old male WT, heterozygous (Het), or homozygous (Homo) Gpr133/Adgrd1 KO mice were examined by µCT. f Representative 3D reconstructions of the trabecular compartment and the femoral midshaft. g Cortical thickness (Ct.Th), (h) trabecular number (Tb.N), (i) bone volume/total volume (BV/TV) and (j) cortical bone mineral density (BMD) were measured at the femoral midshaft. (n = 9 per group). k Basal Gpr133/Adgrd1 mRNA expression levels determined by qPCR (ΔCT) in undifferentiated bone marrow-derived mesenchymal stromal cells (BM-MSCs), differentiated primary osteoblasts (OBs), MC3T3 cells (osteoblast-like cell line), MLO-Y4 cells (osteocyte-like cell line), RAW264.7 cells (osteoclast-like cell line), undifferentiated bone marrow macrophages (BMMs), and differentiated primary osteoclasts (OCs). (n = 5). l–p Femora from 23-weeks-old male Cre-control (Cre-Ctrl), Gpr133/Adgrd1tm1c (+/+) and osteoblast-precursor-specific Gpr133/Adgrd1tm1d KO (fl/fl) mice were examined by µCT. l Representative 3D reconstructions of the trabecular compartment and the femoral midshaft. (m) Ct. Th, (n) Tb.N, (o) BV/TV and (p) BMD were measured at the femoral midshaft. (n = 9 per group). q A three-point bending test was performed on femora as an indicator of bone strength and stiffness. Figure for experimental design was created with BioRender. (n = 9 per group). The maximum load (Fmax) tolerated by femora of (r) constitutive or (s) osteoblast-precursor-specific Het and Homo Gpr133/Adgrd1 KO compared to WT controls were determined. (n = 9 per group). Data information: All data are presented as mean ± SD, each dot indicates an individual mouse. Statistical analysis was performed using one-way ANOVA: *p < 0.05; **p < 0.01; ***p < 0.001 comparing Het/ Homo vs. WT and fl/fl vs Cre-Ctrl or +/+
Fig. 2
Fig. 2
Bone depletion in constitutive and osteoblast precursor-specific Gpr133/Adgrd1 knockout mice is mainly driven by osteoblast dysfunction. a–n 23-weeks-old male wild-type (WT), heterozygous (Het), or homozygous (Homo) Gpr133/Adgrd1 knockout (KO) and Cre-control (Cre-Ctrl), Gpr133/Adgrd1tm1c (+/+), and osteoblast-precursor-specific Gpr133/Adgrd1tm1d KO (fl/fl) mice were examined. a/b Serum concentrations of the bone formation marker type 1 procollagen amino-terminal propeptide (P1NP) and (c/d) the bone resorption marker cross-linked C-telopeptide of type I collagen (CTX) were measured by ELISA. (e/f) Bone formation rate per bone surface (BFR/BS) assessed by calcein double labeling of tibial bone slides and compared between groups. Number of (g/h) osteoblasts per bone perimeter (N.Ob./B.pm), (i/j) osteocytes per bone perimeter (N.Ocy./B.pm), and (k/l) osteoclasts per bone perimeter (N.Oc./B.pm) were determined by Tartrate-resistant acid phosphatase (TRAP) staining of femoral bone slides. m/n Heat map of mRNA expression levels (fold change over WT/Cre-Ctrl mice) of osteoblast differentiation markers osterix (Osx), alkaline phosphatase (Alp), osteocalcin (Ocn), and the coupling factors osteoprotegerin (Opg) and receptor activator of NF-κB ligand (Rankl) in tibiae. (n = 9 per group). o Gpr133/Adgrd1 mRNA expression during osteogenic differentiation of bone marrow-derived mesenchymal stromal cells was analyzed by qPCR. Results were calculated using the ΔΔCT method, normalized to β-actin mRNA, and presented as x-fold changes compared to d0. (n = 5). p Real-time PCR analysis was used to measure the mRNA expression of collagen type I alpha 1 (Col1a1), Alp, runt-related transcription factor 2 (Runx2), Osx, and Ocn in primary murine osteoblasts at days 0, 7, 14, 21, and 28 of differentiation. Results were calculated using the ΔCT method and normalized to β-actin mRNA. Data are presented as ΔCT values. (n = 6 per group). q ALP activity was assessed at days 0, 7, and 14 of differentiation. (n = 6). r Gpr133/Adgrd1 mRNA expression during osteoclast (OC) differentiation in primary OCs was analyzed by qPCR. Results were calculated using the ΔΔCT method, normalized to β-actin mRNA, and presented as x-fold changes compared to d0. (n = 6). s TRAP positive multinucleated cells were identified as osteoclasts and counted. (n = 8). t Real-time PCR analysis was used to measure the mRNA expression of C-terminal Src family kinase (Csk), nuclear factor-activated T cells c1 (Nfatc1) and Tartrate-resistant acid phosphatase (Trap) in primary mouse osteoclasts at days 0, 3, 5, 7, 10 and 14 of differentiation. Results were calculated using the ΔCT method and normalized to β-actin mRNA. Data are presented as ΔCT values. (n = 8). u WT bone marrow-derived macrophages were cultured and differentiated into OC in the presence of WT or homozygous Gpr133/Adgrd1 KO-derived conditioned medium (CM) of 7 days differentiated osteoblasts (OBs). Figure for experimental design was created with BioRender. v TRAP positive multinucleated cells were identified as osteoclasts and counted. (n = 8). w Real-time PCR analysis was used to measure the mRNA expression of Csk, Nfatc1 and Trap in primary murine osteoclasts at days 0, 3, 5, 7, and 10 of differentiation. Results were calculated using the ΔCT method and normalized to β-actin mRNA. Data are presented as ΔCT values. (n = 8). Data information: All data are presented as the mean ± SD. Each dot represents an individual mouse. Statistical analysis was performed using (a–n, q, u) one-way ANOVA: *p < 0.05; **p < 0.01; ***p < 0.001 Het/Homo KO vs. WT control or fl/fl vs. Cre-Ctrl and +/+ and (p, t, v, w) two-way ANOVA: *p < 0.05; **p < 0.01; ***p < 0.001 WT vs. Homo KO or WT CM vs. Homo KO CM
Fig. 3
Fig. 3
Osteoblast function is governed by GPR133/ADGRD1-mediated increase in cAMP-dependent ß-catenin signaling. a cAMP accumulation in 7-day differentiated primary bone marrow mesenchymal stem cells (BM-MSCs) derived from wild-type (WT) and homozygous (Homo) Gpr133/Adgrd1 knockout (KO) mice. Cells were treated with different concentrations of AP503 or 1 mM Stachel-derived peptide pGPR133. (n = 5). b Real-time PCR analysis of mRNA expression levels of collagen type I alpha 1 (Col1a1), alkaline phosphatase (Alp), runt-related transcription factor 2 (Runx2), osterix (Osx), osteocalcin (Ocn), osteoprotegerin (Opg), and receptor activator of NF-κB ligand (Rankl) in BM-MSCs derived from WT and Homo Gpr133/Adgrd1 KO mice at days 7, 14, and 21 of differentiation. Results were calculated using the ΔCT method, normalized to β-actin mRNA. (n = 6). c ALP activity was assessed at days 0, 7, and 14 of differentiation. (n = 6). d Mineralization capacity of osteoblasts was determined by Alizarin red staining at days 7, 14, 21, and 28 of differentiation. (n = 6). e The mRNA expression of Wnt signaling genes Wnt3a, Wnt5a, transcription factor 7 (Tcf7), Axin2, and Wnt inhibitors Dickkopf-1 (Dkk1) and sclerostin (Sost) in tibial bone tissue obtained from 23-weeks-old male WT, Het, or Homo Gpr133/Adgrd1 KO mice was quantified by real-time PCR. Data are represented as the mean ± SD of ΔCT, normalized to β-actin mRNA. (n = 6). (f/g) Relative total and phosphorylated (Thr41/Ser45) β-catenin protein levels of 7-day differentiated primary BM-MSCs from WT and homozygous Gpr133/Adgrd1 KO mice were determined by a cell-based ELISA. Cells were stimulated with AP503 (1 µM) and signaling pathways were probed using phosphodiesterase inhibitor IBMX, adenylyl cyclase activator forskolin, protein kinase A inhibitor H89 or Glycogen synthase kinase-3 inhibitor CHIR-99021 all at given concentrations. (n = 5). Data information: Data are presented as the mean ± SD, performed in triplicate per group. Statistical analysis was performed using two-way ANOVA: *p < 0.05; **p < 0.01; ***p < 0.001 Homo/Het KO vs. WT, and #/°p < 0.05; ##/°°p < 0.01; ###/°°°p < 0.001 basal vs stimulated. n.s. no significance
Fig. 4
Fig. 4
PTK7 and mechanical strain are important for Gpr133/Adgrd1-mediated osteoblast differentiation. a Experimental design for PTK7 coating and Gpr133/Adgrd1 knockdown (KD) in MC3T3 cells (referring to b–d). Figure created with BioRender. b cAMP accumulation in 3- and 7-day differentiated negative control- (NC) and Gpr133/Adgrd1 siRNA-transfected MC3T3 cells. (n = 6 per group). c Real-time PCR analysis of mRNA expression levels of collagen type I alpha 1 (Col1a1), alkaline phosphatase (Alp), runt-related transcription factor 2 (Runx2), osterix (Osx), and osteocalcin (Ocn) in NC- and Gpr133/Adgrd1 siRNA-transfected MC3T3 cells at days 7 and 14 of differentiation. Results were calculated using the ΔCT method, normalized to β-actin mRNA. (n = 6 per group). d ALP activity was assessed at days 7 and 14 of differentiation in NC- and Gpr133/Adgrd1 siRNA-transfected MC3T3 cells. (n = 6 per group). e Experimental design for mechanical strain and Gpr133/Adgrd1 KD in MC3T3 cells (referring to f–i). Figure created with BioRender. f cAMP accumulation in 3- and 7-day differentiated NC- and Gpr133/Adgrd1 siRNA-transfected MC3T3 cells subjected to 8 h of 10% or 0% cyclic tensile strain. (n = 6 per group). g mRNA expression levels of Col1a1, Alp, Runx2, Osx, and Ocn were measured in NC- and Gpr133/Adgrd1 siRNA-transfected MC3T3 cells subjected to 8 h of 10% or 0% cyclic tensile strain at day 7 of differentiation. Results were calculated using the ΔCT method, normalized to β-actin mRNA. (n = 6 per group). h cAMP accumulation in 3- and 7-day differentiated NC- and Ptk7 siRNA-transfected MC3T3 cells subjected to 8 h of 10% or 0% cyclic tensile strain. (n = 6 per group). i mRNA expression levels of Col1a1, Alp, Runx2, Osx, and Ocn were measured in NC- and Ptk7 siRNA-transfected MC3T3 cells subjected to 8 h of 10% or 0% cyclic tensile strain at day 7 of differentiation. Results were calculated using the ΔCT method, normalized to β-actin mRNA. (n = 6 per group). Data information: All data are presented as mean ± SD, performed in triplicate per group. Statistical analysis was performed using two-way ANOVA: *p < 0.05; **p < 0.01; ***p < 0.001 basal vs stimulated (PTK7 coating or stretch), and #p < 0.05; ##p < 0.01; ###p < 0.001 NC-siRNA vs. KD. n.s. no significance
Fig. 5
Fig. 5
AP503 increases bone mass in male mice via GPR133/ADGRD1. a Experimental scheme for in vivo treatment (referring to data in b-h). Male wild-type (WT), heterozygous (Het), or homozygous (Homo) Gpr133/Adgrd1 knockout (KO) mice were examined. 5-weeks-old WT, Het or Homo mice were intraperitoneally injected with vehicle or AP503 (2 mg/kg) every day for 4 weeks. Figure created with BioRender. b–h Femora from 9-weeks-old male mice were examined. b Representative µCT images of distal femurs (cross sections and longitudinal sections). c Bone volume/total volume (BV/TV) and (d) trabecular number (Tb.N.) were assessed in the distal femur using µCT. e Number of osteoblasts per bone perimeter (N.Ob./B.pm), (f) number of osteocytes per bone perimeter (N.Ocy./B.pm), and (g) number of osteoclasts per bone perimeter (N.Oc./B.pm) were determined by Tartrate-resistant acid phosphatase (TRAP) staining of femoral bone slides. h Bone formation rate per bone surface (BFR/BS) was assessed by calcein double labeling of tibial bone slides and compared between groups. i Experimental scheme for mechanical load model (referring to data in j-o). 4-weeks-old WT male mice were divided into 4 groups: sedentary control (Sed), Sed+AP503, exercise (Exe) and Exe+AP503. Mice in the Exe and Exe+AP503 group underwent treadmill running following a 5-day-per-week acclimatization protocol at 9 a.m., beginning with a 1-week pre-exercise phase, followed by a 4-week formal exercise period. Mice in the Sed and Exe groups were intraperitoneally injected daily with an equal vehicle, while those in the Sed+AP503 and Exe+AP503 groups received AP503 (2 mg/kg) daily. Figure created with BioRender. j–o Femora from 9-weeks-old male mice were examined. j Representative µCT images of distal femurs (cross sections and longitudinal sections). k Fold change of BV/TV, Tb.N, Tb.Th, and Tb.Sp compared to the Sed group. l N.Ob./B.pm, (m), N.Ocy./B.pm (n), and N.Oc./B.pm were determined by TRAP staining of femoral bone slides. o BFR/BS was assessed by calcein double labeling of tibial bone slides and compared between groups. p Serum calcium and (q) phosphate concentrations from control and AP503 treated male WT, Het or Homo Gpr133/Adgrd1 KO mice. r Serum calcium and (s) phosphate concentrations from male WT mice following exercise and/or AP503 treatment. Data information: The data are presented as the mean ± SEM values, n = 5-14 mice per group. Each dot represents an individual mouse. The data were analyzed via one-way ANOVA with Tukey’s test. *p < 0.05, **p < 0.01, ***p < 0.001, #p < 0.05, ###p < 0.001, $p < 0.05
Fig. 6
Fig. 6
AP503 alleviates osteoporosis burden in vivo. a Overview of the experimental procedures for the ovariectomy mouse model. 8-weeks-old females were subjected to bilateral oophorectomy or sham operation, followed by 4 weeks of surgical recovery. After the recovery period, these mice with bilateral oophorectomy were intraperitoneally injected with vehicle or AP503 (2 mg/kg/day, i.p.) for 4 weeks before µCT test. Figure created with BioRender. b-m Femora from 16-weeks-old female mice were examined. b Representative µCT images of the trabecular compartment from 16-weeks-old sham, OVX and OVX + AP503 females. (c) Bone volume/total volume (BV/TV), (d) cortical bone mineral density (BMD), (e) cortical thickness (Ct.Th), (f) trabecular thickness (Tb.Th), (g) trabecular separation (Tb.Sp), and (h) trabecular number (Tb.N) of the femoral midshaft were assessed in the distal femur using µCT. i Number of osteoblasts per bone perimeter (N.Ob./B.pm), (j) number of osteoclasts per bone perimeter (N.Oc./B.pm), and (k) number of osteocytes per bone perimeter (N.Ocy./B.pm) were determined by Tartrate-resistant acid phosphatase (TRAP) staining of femoral bone slides. l The maximum load (Fmax) and (m) elastic modulus (Emod) were determined by 3-point bending test. n Summary of the proposed molecular mechanism through which GPR133/ADGRD1 controls bone formation. 1) Activation of GPR133/ADGRD1 through interaction with PTK7, mechanical forces or small molecule (AP503) induces osteoblasts differentiation through a cAMP-dependent ß-catenin pathway. 2) As a result osteoblasts secret an increased amount of osteoprotegerin (OPG), which masks receptor activator of nuclear factor-κB ligand (RANKL), thereby 3) reducing osteoclast (OC) activation. An additional suppression of OC activation is likely mediated through GPR133/ADGRD1-dependent cAMP-dependent inhibition of OC function. These combined effects mediate bone formation. 4) In the absence of a functional GPR133/ADGRD1 osteoblast differentiation and function is impaired, resulting among others in reduced expression of OPG, allowing now 5) for increased activation of OCs. Loss of GPR133/ADGRD1 in OCs leads to further activation, which taken together results in an osteopenic phenotype. Figure created with BioRender. Data information: The data are presented as the mean ± SEM values, n = 8 mice per group. Each dot indicates an individual mouse. The data were analyzed via one-way analysis of variance (ANOVA) with Tukey’s test. *p < 0.05, **p < 0.01, ***p < 0.001
See this image and copyright information in PMC

References

    1. Hernlund, E. et al. Osteoporosis in the European Union: medical management, epidemiology and economic burden. A report prepared in collaboration with the International Osteoporosis Foundation (IOF) and the European Federation of Pharmaceutical Industry Associations (EFPIA). Arch. Osteoporos.8, 136 (2013). - DOI - PMC - PubMed
    1. Manolagas, S. C. Birth and death of bone cells: basic regulatory mechanisms and implications for the pathogenesis and treatment of osteoporosis. Endocr. Rev.21, 115–137 (2000). - PubMed
    1. Compston, J. E., McClung, M. R. & Leslie, W. D. Osteoporosis. Lancet393, 364–376 (2019). - DOI - PubMed
    1. Walker, M. D. & Shane, E. Postmenopausal Osteoporosis. N. Engl. J. Med.389, 1979–1991 (2023). - DOI - PubMed
    1. Burge, R. et al. Incidence and Economic Burden of Osteoporosis-Related Fractures in the United States, 2005–2025*. J. Bone Min. Res.22, 465–475 (2009). - DOI - PubMed

Publication types

Substances

Associated data