Kiss1/Gpr54 Prevents Bone Loss through Src Dephosphorylation by Dusp18 in Osteoclasts


 Osteoclasts were over-activated as we age, which leads to bone loss. Src-deficient mice lead to only one phenotype -severe osteopetrosis due to functional defect in osteoclasts, indicating that Src function is essential in osteoclasts. G-protein-coupled receptors (GPCR) have been targets for ∼35% of approved drugs. However, how Src kinase activity is negatively regulated by GPCRs remains largely elusive. Herein we report that Src is dephosphorylated at Tyr 416 by Dusp18 upon GPR54 activation by its natural ligand Kp-10. Mechanically, both active Src and the Dusp18 phosphatase are recruited by GPR54 through the proline/arginine-rich motif (PR motif) in the C terminus, which is dependent on the Gαq signal pathway. As such, Kiss1, Gpr54, Dusp18 knockout mice all exhibit osteoclast hyperactivation and bone loss. Accordingly, Kp-10 abrogated bone loss by suppressing osteoclasts activity in vivo. Therefore, Kiss1/Gpr54 is a promising therapeutic strategy governing bone resorption through Src dephosphorylation by Dusp18.


INTRODUCTION 14
As we age, bone metabolism and homeostasis shift to favor over-activated osteoclasts, which 15 leads to bone loss, a hallmark of human diseases such as osteoporosis 1,2 . Osteoclasts, which have the 16 only capacity to resorb bone, are formed from bone marrow monocytes induced by macrophage colony-17 stimulating factor (M-CSF) and the receptor activator of nuclear factor-κB ligand (RANKL) 3,4 . 18 Mechanically, both M-CSF and RANKL promote actin remodeling and bone resorption of osteoclasts, 19 which mostly depends upon induction of Src kinase activation (phosphorylation at Y416) 5,6 . Src consists 20 of 4 functional regions: Src homology 4 domain (SH4), SH3, SH2, SH1 (catalytic domain) and is 21 activated through auto-phosphorylation at tyrosine 416 upon SH3 ligand binding [7][8][9] . Two main SH3 22 domain-binding motifs, proline /arginine-rich motif (PR motif), have been identified: R/KxxPxxP (class 23 I) and PxxPxR/K (class II) (where K is lysine and x is any amino acid) [10][11][12][13] . Even though Src is normally 24 3 present in a broad variety of cell types, genetic knockout of the Src gene in mice leads to only 25 one major phenotype -severe osteopetrosis mainly due to impaired osteoclast function. This in vivo 26 phenotype supports the notion that the Src kinase plays an essential biological function in osteoclasts 27 5,[14][15][16][17] . Therefore, inhibition of Src kinase activity has been considered as a useful therapeutic strategy 28 for osteoclast overactivation-mediated bone loss [18][19][20][21][22] . 29 G protein-coupled receptors (GPCRs) are the most important drug targets. It is estimated that 30 ∼35 % of marketed drugs act directly on GPCRs 23,24 . Src was activated by GPCRs through different 31 ways including direct binding with Src through SH3 binding motif in the intracellular domain 25-28 , 32 indirect phosphorylating Src at Y416 by Gαs/i 29 or recruitment of Src via arrestins 30 . However, how 33 Src kinase activity is negatively regulated by GPCRs remains largely elusive. GPR54, also named as 34 KiSS1R (KiSS1 receptor), is a member of the GPCR superfamily 31 . Its natural ligands are Kisspeptins 35 including Kp-54, -14, -13, -10 encoded by KiSS1 gene 32 . Biologically, KiSS1/GPR54 signaling in 36 hypothalamic neurons is the gatekeeper of puberty that controls hormones release via the hypothalamic-37 pituitary-gonadal axis 33,34 . GPR54 activation by Kisspeptins binding triggered signaling cascade 38 including primary Gαq/11-PLCβ (phospholipase C β)-PKC (protein kinase C)/Ca 2+ signal pathways and 39 subsequent signaling that is dependent on β-arrestin-1 and β-arrestin-2 35 . 40 There was a PR motif in the C terminus of human GPR54 (GPR54 CT) 36 . Comparing GPR54 41 with its homologous sequences from other species, we identified that this PR motif emerged and was 42 conserved in terrestrial vertebrates. Furthermore, GPR54 recruited active Src and the phosphatase 43 Dusp18 through this PR motif upon activation by the ligand Kp-10, which is dependent on Gαq signaling.

Kp-10 ameliorated OVX-induced bone loss 225
Our data above showed that Kp-10/Gpr54 negatively regulated osteoclast activity through Src 226 dephosphorylation by Dusp18. Therefore, we intended to examine whether Kp-10 could ameliorate bone 227 loss in vivo. In ovariectomized mice, after intravenous injection of Kp-10 (1, 10, 50 nmol/kg) and bone 228 targeting Kp-10 ((DSS)*6-Kp-10) (1, 10, 50 nmol/kg) twice a week for two months, we observed that 229 mice with 1 or 10 nmol/kg (DSS)*6-Kp-10 showed better bone protection effect than 1 or 10 nmol/kg 230 Kp-10 correspondingly by micro-CT analysis ( Figure S4  and follicle-stimulating hormone (FSH). In addition, FSH and LH prompt the ovaries to begin producing 252 the hormone estrogen and work together to get the testes to begin producing testosterone 31,32 . Both 253 estrogen and androgen have been shown to play a role in bone protection [45][46][47] . Our results showed that 254 Kiss1/Gpr54 negatively regulated osteoclast formation and bone resorption directly. However, whether 255 Kiss1/Gpr54 showed bone protection through modulating osteoblast needs to be explored in the future.    f SPR binding analysis of DUSP18 and mouse Gpr54 ( 336 RVCPCCR 342 ) peptide. The binding affinity was measured at 1.8 µM.
g SPR binding analysis of DUSP18 and mouse Gpr54 ( 339 PCCRQR 344 ) peptide. The binding affinity was measured at 9.2 µM.
h IB of total samples and GST pull-downs using GST proteins purified from E. coli; GST proteins were incubated separately with the WCL of 293T cells transfected with DUSP18-HA.
i IB analysis of total samples and GST pull-downs using His-SRC proteins purified from Sf9 cells, His-DUSP18, and GST proteins purified from E. coli.      Surface plasmon resonance (SPR). SPR was determined using a Biacore T200 instrument (GE). SRC or DUSP18 protein was immobilized on the sensor chip (CM5) using the amine-coupling method according to standard protocols. SRC protein or Dusp18 was diluted in 10 mM acetate buffer, pH 5.5. Immobilization was performed according to the manufacturer's recommendations. The kinetics and affinity assay were examined at 25 °C at a flow rate of 30 µl/minute using PBS buffer. Diluted DUSP18, Src, and GPR54 CT protein were kept at 25 °C and placed into the rack tray before injection. The KD values were calculated with the kinetics and affinity analysis option of Biacore T200 evaluation software. The interaction of GPR54 CT, peptides of PR motifs with Src or DUSP18, and the interaction of DUSP18 and Src were analyzed respectively by regeneration with pH 2.0 Gly-HCl buffer.
Protein expression and purification. The C terminus of human GPR54 (329H-398L) and mouse Gpr54 and C340S mutation) was linked to the C terminus of Src SH3-SH2 domain (G85-V247), which was subcloned into pMCSG7 vector with a 6xHis tag and a TEV protease recognition site at the N terminus before the receptor sequence. The proteins were expressed and purified with the Ni-NTA system as above.
A PD MiniTrap G-25 column was used to remove imidazole. The protein was then treated overnight with His-tagged TEV protease to remove the N-terminal His tag. His-tagged TEV protease cleaved His-tag and uncleaved protein was removed from the sample by passing the sample over an equilibrated nickel-affinity column chromatography. The receptor was then concentrated to 8-13mg/ml with a 30 kDa molecular mass cut-off centrifuge concentrator (Sartorius Stedim).
Protein crystallization and structure determination. For crystallization, the C-terminus of GPR54 (333-356 with mutations C338S and C340S) was inserted into the C-terminus of Src SH3-SH2 domains (G85-V247), this fragment (Src G85-V247 -GPR54 333-356 ) was then subcloned into pMCSG7 vector with a 6xHis tag and a TEV protease recognition site at the N terminus before the chimera sequence. Two cysteines (C338&C340) in the peptide are mutated to serine to avoid potential oxidation during protein purification.
The mutated GPR54 residues, S338 and S340, both do not involve any side-chain interaction with the SH3 domain, except the main chain interaction between 340 and SH3 residue N135. The proteins were expressed and purified with the Ni-NTA system as above. A PD MiniTrap G-25 column was used to remove imidazole. The protein was then treated overnight with His-tagged TEV protease to remove the Nterminal His tag. Uncleaved protein and TEV were removed from the sample by passing the sample over  Table 2. Micro-CT analyses. 3D micro-CT analyses were performed as previously described 46 . We scanned the femur using in vitro X-ray microtomography (Skyscan 1272, Bruker micro CT) at a pixel size of 9 µm, and analyzed the results according to the manufacturer's instructions. Region-of-interest (ROI) was defined from 10 to 110 image slices, where the growth plate slice was defined as 0 mm. The contrast was defined from 68-255; 3D analysis, BMD, and 3D models were analyzed using CTAn software (Bruker micro CT). 3D models were adjusted in CT Vox software (Bruker micro CT).  Table S4. Generation of Dusp18 -/mice was performed using the CRISPR/Cas9 system in the C57BL/6J mouse strain from the Animal Center of East China Normal University (ECNU). Two 20-bp sgRNAs targeting TGCGAGAGGCCTCTGATCGAAGG and GCGACGGGCGCATCGACCACAGG were designed and 164 bp between 406 bp and 569 bp of the Dusp18 gene was deleted. Genotyping was performed by PCR as described in Table S4. Generation of Arrb1 -/and Arrb2 -/mice (strain C57/BL/6) were described previously 48,49 . LysM-Cre mice (strain C57BL/6) were described in reference 50

Data availability
Full scans of the gels and blots are available in Supplementary Fig. 8. All relevant data are available from the corresponding author.  Figure 1 Kiss1/Gpr54 governs osteoclasts formation and bone resorption mainly through Src dephosphorylation. a IB analysis of WCL derived from BMMs isolated from eight-week-old wild-type (WT) and Gpr54 -/-mice. BMMs were starved in serum-free α-MEM for 4 hours and treated with 100 ng/mL RANKL for another 30   (336RVCPCCR342) peptide. The binding a nity was measured at 1.8 μM. g SPR binding analysis of DUSP18 and mouse Gpr54 (339PCCRQR344) peptide. The binding a nity was measured at 9.2 μM. h IB of total samples and GST pull-downs using GST proteins puri ed from E. coli; GST proteins were incubated separately with the WCL of 293T cells transfected with DUSP18-HA. i IB analysis of total samples and GST pull-downs using His-SRC proteins puri ed from Sf9 cells, His-DUSP18, and GST proteins puri ed from E. coli.

Figure 4
Src was dephosphorylated by DUSP18 when GPR54 was activated by Kp-10 a SPR binding analysis of DUSP18 and Src. The binding a nity of DUSP18 and Src was measured at 5.9 nM. b IB analysis of WCL and anti-Flag IP derived from 293T cells transfected with HA-Src and either Dusp18-Flag or Dusp18 (C104S)-Flag constructs. c Dusp18 dephosphorylated Src at Y416 in vitro. His-Dusp18 and His-Dusp18 (C103S) proteins puri ed from E. coli and SRC proteins puri ed from Sf9 insect cells were incubated in the phosphatase buffer at 30oC for 30 minutes. IB analysis of the protein phosphatase reaction products with antibodies as indicated. d IF staining of RAW264.7 cells treated with 10 nM Kp-10 for 20 minutes was carried out using the indicated antibodies. e IB analysis of WCL and anti-Flag IP derived from 293T cells transfected with HA-Src, GPR54-myc, and either Dusp18-Flag or Dusp18 (C104S)-Flag constructs. f IB analysis of WCL and anti-Src IP derived from RAW264.7 cells treated with or without Kp-10 for 20 minutes. g IB analysis of WCL a derived from Dusp18-/-RAW264.7 cells treated with or without Kp-10 for 20 minutes. h BMMs isolated from eight-week-old WT and Dusp18-/-mice were cultured in the absence of serum and with indicated doses of Kp-10 for 20 minutes, and then treated with 100 ng/ml RANKL for 30 minutes. Cells were lysed and blots probed with indicated antibodies. i BMMs isolated from eight- week-old WT and Dusp18 -/-mice were seeded on bone slices and stimulated with M-CSF (10 ng/ml) and RANKL (50 ng/ml) for 5-7 days. Pits were scanned by confocal microscopy (XY and z section). Scale bars, 125 μm. j Pit depths were measured by confocal microscopy. Mean ± SEM; ** P< 0.01; *** P< 0.01; ns, not signi cant.