Regulation of bone formation and remodeling by G-protein-coupled receptor 48 - PubMed (original) (raw)
. 2009 Aug;136(16):2747-56.
doi: 10.1242/dev.033571. Epub 2009 Jul 15.
Wei Zhou, Xin Zhou, Dali Li, Jinsheng Weng, Zhengfang Yi, Sung Gook Cho, Chenghai Li, Tingfang Yi, Xiushan Wu, Xiao-Ying Li, Benoit de Crombrugghe, Magnus Höök, Mingyao Liu
Affiliations
- PMID: 19605502
- PMCID: PMC2730404
- DOI: 10.1242/dev.033571
Regulation of bone formation and remodeling by G-protein-coupled receptor 48
Jian Luo et al. Development. 2009 Aug.
Abstract
G-protein-coupled receptor (GPCR) 48 (Gpr48; Lgr4), a newly discovered member of the glycoprotein hormone receptor subfamily of GPCRs, is an orphan GPCR of unknown function. Using a knockout mouse model, we have characterized the essential roles of Gpr48 in bone formation and remodeling. Deletion of Gpr48 in mice results in a dramatic delay in osteoblast differentiation and mineralization, but not in chondrocyte proliferation and maturation, during embryonic bone formation. Postnatal bone remodeling is also significantly affected in Gpr48(-/-) mice, including the kinetic indices of bone formation rate, bone mineral density and osteoid formation, whereas the activity and number of osteoclasts are increased as assessed by tartrate-resistant acid phosphatase staining. Examination of the molecular mechanism of Gpr48 action in bone formation revealed that Gpr48 can activate the cAMP-PKA-CREB signaling pathway to regulate the expression level of Atf4 in osteoblasts. Furthermore, we show that Gpr48 significantly downregulates the expression levels of Atf4 target genes/proteins, such as osteocalcin (Ocn; Bglap2), bone sialoprotein (Bsp; Ibsp) and collagen. Together, our data demonstrate that Gpr48 regulates bone formation and remodeling through the cAMP-PKA-Atf4 signaling pathway.
Figures
Fig. 1.
Growth retardation of Gpr48 mutant mice and expression of Gpr48 in embryonic skeletal and bone cells. (A-C) Decrease in embryonic (A) and postnatal (B) body weight and long-bone length (C) in_Gpr48-/-_ (HO) mice. (D-J) Whole-mount lacZ staining of E12.5 (D,E) and E16.5 (F,G) wild-type (WT) (D,F) and_Gpr48+/-_ (E,G) mouse embryos. Tissue sections of femurs from E16.5 wild-type (H) and Gpr48+/- (I,J) mice. The wild-type bone does not show any lacZ staining (D,F,H). (K) RT-PCR analysis of Gpr48 expression in osteoblast and chondrocyte cells, showing bone marrow stromal cell (BMSC), calvarial osteoblast (CO), osteoblast cell line MC3T3-E1, primary cultured chondrocyte (CC) and chondrocyte cell line ATDC5 with Gpr48-/- calvarial osteoblast (CO) and chondrocyte (CC) as negative controls.
Fig. 2.
Bone formation is delayed in _Gpr48_-/- embryos. (A-H) Whole skeletal preparation of wild-type (A,C,E,G) and_Gpr48_-/- mutant (B,D,F,H) mice at E14.5 (A,B), E16.5 (C,D) and E18.5 (E-H). Arrows indicate the delayed Alizarin Red staining (of bone) in the skull, jaw, sternum, clavicle, phalange and limb in_Gpr48-/-_ embryos. (I-L) Effects of Gpr48 on intramembranous bone formation and skull ossification. Top view of the mouse skull at E16.5 (I,J) and P0 (K,L) for wild-type (I,K) and_Gpr48_-/- (J,L) mice after Alcian Blue/Alizarin Red staining. Arrows indicate the widening of cranial sutures and the opened fontanelles in Gpr48-/- newborn mice. (M-R) von Kossa staining of femur of wild-type (M,O,Q) and _Grp48_-/- (N,P,R) embryos at E14.5 (M,N) (20×), E16.5 (O,P) (5×) and E18.5 (Q,R) (5×). Arrows indicate that wild-type mice already show von Kossa signal at E14.5, but this is absent from Gpr48-/- mice.
Fig. 3.
Deletion of Gpr48 has little effect on chondrocyte maturation, but delays osteoblast differentiation in embryonic bone. (A-D′) In situ hybridization analysis (A-B′,D,D′) and immunohistochemistry (C,C′) for chondrocyte differentiation and proliferation markers in wild-type and Gpr48_-/- femur at E16.5. Probes are Col2a1 (A,A′), Ihh (B,B′),Col10a1 (D,D′) and Sox9 antibody (C,C′). (E-G′) In situ hybridization analysis of the chondrocyte and osteoblast differentiation markers Col10a1(E,E′),Col1a1 (F,F′) and Runx2 (G,G′) in E14.5 femur. (H-L′) In situ hybridization analysis of the osteoblast differentiation markers Col1a1 (H,H′), Runx2 (I,I′), Atf4 (J,J′), Bsp (K,K′) and_Ocn (L,L′) in E16.5 femur. (M) Alkaline phosphatase (ALP) assays of primary cultured cavarial osteoblasts from wild-type (WT) and_Gpr48-/-_ (HO) mice at day (D) 5, 10 and 18.**P<0.01.
Fig. 4.
Gpr48 regulates bone formation postnatally. (A-D) H&E staining of tibia of wild-type (A,C) (10×) and_Gpr48_-/- (B,D) (10×) mice at P18. There is much less trabecular bone in Gpr48-/- than wild-type mice, and the cortical bone is much thinner in the mutant tibia (arrows). (E-H) Transverse (E,F) and frontal (G,H) microCT sections of distal and midfemoral diaphyses of 1-month-old wild-type and _Gpr48_-/- mice. (I,J) von Kossa staining of L3 vertebral bodies from 1-month-old wild-type and Gpr48_-/- mice. There is a significant reduction in bone perimeter, bone area and trabecular width in_Gpr48-/- mice. (K,L) Osteoid synthesis defect in _Gpr48_-/- mice demonstrated with Goldner staining (osteoid is stained red, yellow arrows). Deletion of Gpr48 significantly reduced osteoid formation (compare K with L). (M,N) Masson-Trichrome staining of osteoid (blue).
Fig. 5.
Quantitative analysis of the osteoblast defect in_Gpr48_-/- mice. (A) Both trabecular bone mineral density (BMD) and cortical BMD dramatically decreased in_Gpr48-/-_ mice as assessed on microCT sections. (B) Deletion of Gpr48 leads to a reduction in bone volume/tissue volume (BV/TV), trabecular thickness, trabecular number, and increased trabecular separation as assessed by histomorphometric analysis. (C)Gpr48 regulates the kinetic indices of mineral apposition (MAR), bone formation rates (BFR/BS) and mineralizing surface (MS/BS). (D) Deletion of Gpr48 causes significant defects in osteoid characteristics, including decreased osteoid thickness (O.Th), osteoid surface (OS/BS) and osteoid volume (OV/BV). In each case, results show mean±s.d.,_n_=3, age and sex matched; **P<0.01 in A-D.
Fig. 6.
Activity of osteoclasts is increased in _Gpr48_-/- mice as assessed by TRAP staining. (A,B) TRAP staining of E18.5 tibia. TRAP-positive osteoclast cells (arrows) are in red. (C,D) TRAP staining of L3 vertebrae of 1-month-old mice. (E) The number of TRAP-positive osteoclasts per mm2 tissue area (N.Oc/mm2) is comparable in wild-type and_Gpr48_-/- mice. (F) Rankl and Opg expression levels in wild-type and _Gpr48_-/- osteoblasts as measured by Q-PCR.
Fig. 7.
Regulation of Atf4 expression by Gpr48 through the cAMP-PKA-CREB signaling pathway. (A) Phosphorylated CREB (at Ser133) protein is decreased in Gpr48_-/- calvarial osteoblasts; total CREB provides a loading control. (B,C) Deletion of Gpr48 decreases the expression level of Atf4 in calvarial osteoblasts at P4 as assessed by Q-PCR (B) and western blot (C). **P<0.01. (D) Decrease in Atf4 protein in Gpr48-/- mice at E16.5 as assessed by immunohistochemistry with specific anti-Atf4 antibody. (E,F) Overexpression of Gpr48 and its constitutively active form, T755I, increased the Atf4 expression level as measured by Q-PCR (E) and western blot (F). (G-I) Gpr48 regulates the binding of CREB to the CRE site in the Atf4 promoter as assessed by EMSA. (G) Direct binding of CREB transcription factor to the CRE site in the Atf4 promoter. F, free probe; Hot, hot probe; Cold 50×, cold competitors at a 50-fold excess; Mut Cold, mutant cold competitors; Ab, CREB antibody. (H) Deletion of_Gpr48 decreased CREB binding to the Atf4 promoter in osteoprogenitor cell nuclear extracts as measured by EMSA. (I) Overexpression of Gpr48 and T755I increases CREB binding to the Atf4 promoter in_Gpr48_-transfected cell nuclear extracts. (J) Activation of the_Atf4_ promoter by Gpr48 and T755I through the PKA pathway. A constitutively active PKA subunit was used as a strong activator of the promoter. A specific inhibitor of PKA, H89, abolished the Gpr48-activated promoter activity. V, vector control.
Fig. 8.
Gpr48 regulates Atf4 downstream target genes. (A,B) Downregulation of the Atf4 target genes Ocn and_Bsp_ in Gpr48-/- osteoblasts as measured by Q-PCR (A) and western blot (B) analyses. **P<0.01. (C) Activation of the Ocn promoter by Gpr48 is abolished by a mutation at the Atf4 site (OG2mOSE1). (D) Gpr48 activation of Atf4 using p6OSE1-Luc as a reporter (100 ng). Atf4 alone, or co-expression of PKA and Atf4, strongly activated this reporter. (E) Collagen fibrils in_Gpr48_ wild-type, Gpr48+/- and_Gpr48_-/- E16.5 femur as revealed by van Gieson staining. Reduced numbers of van Gieson-stained collagen-rich trabeculae were observed in Gpr48+/- and Gpr48-/- (arrowheads). (F) Model of Gpr48-mediated signaling cascades in bone formation and remodeling. Gpr48 can activate the cAMP-PKA-CREB pathway to regulate Atf4 expression through CREB binding to the Atf4 promoter. PKA can also phosphorylate and activate Atf4 protein directly. Upregulation and activation of Atf4 lead to the expression of its downstream bone matrix target genes_Ocn_ and Bsp and to collagen synthesis in bone formation.
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