Dimeric R25C PTH(1-34) Activates the Parathyroid Hormone-1 Receptor in vitro 2 and Stimulates Bone Formation in Osteoporotic Female Mice

Abstract


Introduction
Osteoporosis is a prevalent global bone disorder characterized by low bone mineral density (BMD), causing weakened bones prone leading to fragility fractures, particularly in the spine, hip, and wrist.The development of osteoporosis is influenced by factors including gender, being more prevalent in women; hormonal changes, like decreased estrogen levels during menopause; and age, with heightened susceptibility post-menopause in women contributing to bone loss.Recent metaanalysis of previous studies indicates a global osteoporosis prevalence of 23.1 % among women and 11.7 % among men (1,2).Osteoporosis stands as a noteworthy risk factor that poses challenges to the preservation of autonomous mobility and overall well-being within an aging society.There is thus a pressing need for safe and efficacious therapies for osteoporosis that mitigate fractures, alleviate associated symptoms, and preserve physical functionality.
Anti-resorptive agents (e.g., bisphosphonates, denosumab, and romosozumab) encompass one therapeutic approach that aims to specifically counteract the declines in bone mass by tempering the balance between bone resorption and formation (3)(4)(5).It is pertinent to acknowledge, however, that the prolonged use of most of such agents is limited due to potential long-term side effects.Furthermore, most anti-resorptive therapies cannot stimulate new bone formation (6).In contrast, bone anabolic agents, such as parathyroid hormone (PTH) and its analogs, such as teriparatide (recombinant human PTH ), increase BMD by stimulating bone formation more than bone resorption (7).PTH has an exceptionally short half-life in the blood of approximately 2-4 minutes (8,9), which helps in avoiding excessive increases in blood calcium levels that can otherwise limit the utility of PTH-related medications, while yet inducing a desired anabolic effect on bone.It is also worth noting that studies in rodents reveal that long-term administration of a PTH anabolic agent can lead to bone overgrowth, osteosarcoma, as well as hypercalcemia (10,11).Consequently, a goal of ongoing research is to uncover the underlying molecular mechanisms driving the anabolic and catabolic effects of PTH to thereby secure more effective therapeutic alternatives for osteoporosis (12).
PTH is produced and secreted by the parathyroid glands as a straight-chain monomeric polypeptide of 84 amino acids (13)(14)(15).It plays a vital role in maintaining calcium and phosphate equilibrium by acting on the PTH1R, a class B G protein-coupled receptor (GPCR) (16)(17)(18) that is expressed primarily in cells of bone and kidney (19).The orchestrated downstream effects of PTH in target cells act to ensure optimal bone health and the maintenance of the ambient blood calcium and phosphate concentrations required for proper nerve conduction, muscle activity, and systemic cellular communication, whereas disturbances in this system can lead to multiple disorders.
Central to the PTH signaling cascade is the activation of intracellular G proteins (20), most prominently Gsα, which in turn activates transmembrane adenylyl cyclases, leading to the synthesis of the second messenger cyclic AMP (cAMP), and the activation of cAMP-dependent protein kinase A (PKA) (21,22).PTH can also activate other second messenger cascades, including the Gαq/phospholipase C (PLC) / inositol 1,4,5-trisphosphate (IP3), diacylglycerol (DAG), and protein kinase C (PKC) signaling pathways (23,24), highlighting the diverse biology of PTH and the PTH1R (25).Adding to this, the PTH1R also mediates the actions of parathyroid hormone-related protein (PTHrP), a development protein that acts in the formation of bones and other tissues.PTHrP shares homology with PTH in the first 34 amino acids, which encompass the receptor-binding portions of the two respective ligands.The PTH1R thus has an intrinsic capacity for dual ligand recognition, which opens possibilities for exploring new modes of therapeutic development for diseases such as osteoporosis and hypoparathyroidism (19,20,(26)(27)(28)(29). Pharmacologically, the PTH1R can adopt at least two distinct ligand-binding conformations, RG and R 0 , the selectivity for which can lead to altered modes of signaling in vitro and in vivo for peptides such as PTH, PTHrP, and various hybrid analogs (30)(31)(32)(33)(34) .
The current study extends our prior investigation in which we identified in a patient with chronic hypocalcemia and hyperphosphatemia a mutation that changes the arginine at position 25 in the mature PTH(1-84) polypeptide to cysteine ( R25C PTH) (35,36).Antibody assays revealed the R25C PTH mutant protein to be present in the patient's blood at markedly elevated levels.We now have found that this patient expressing the R25C PTH variant has higher-than-normal BMD.We further characterize the R25C PTH protein and find that it can manifest in two distinct molecular forms: as a monomer and as a dimer, and we demonstrate that a dimeric

Dimerization of R25C PTH(1-84)
In our previous studies, we showed that synthetic monomeric peptide, R25C PTH(1-34), as compared to PTH , exhibits a moderately diminished PTH1R-binding affinity and decreased cAMP signaling potency in vitro, and that with long-term infusion in mice, R25C PTH(1-34) leads to only minimal calcemic and phosphaturic effects, which corroborates the hypocalcemia seen in the original patient, despite the significantly elevated levels of R25C PTH in the plasma (35).In subsequent analysis of this patient, we found a particularly high BMD (Supplemental figure 1), which prompted us to further characterize the functional properties of R25C PTH, as described herein.We produced recombinant PTH(1-84) with or without the R25C mutation by expression of the corresponding cDNA in HEK293T cells (Figure 1).We considered the possibility that the introduction of a sole new cysteine within the polypeptide chain of R25C PTH(1-84) might induce homologous bimolecular dimerization through a disulfide bond involving the thiol functional group in each monomer (37,38).To specifically investigate this, we designed the cDNA constructs to express either pre-pro-PTH(1-115)-3xFLAG or R56C pre-pro-PTH(1-115)-3xFLAG, such that after intracellular processing and cleavage of the pre-pro regions, the mature PTH(1-84)-3xFLAG or R25C PTH(1-84)-3xFLAG peptides would be generated upon transfection in HEK293T cells (Figure 1C) (39)(40)(41).We performed western blot analysis of total cell lysates and conditioned culture media collected from the transfected cells to specifically assess the possible presence of a disulfide-bonded dimeric form.Each sample was thus prepared in either reduced or non-reduced form and proteins were detected using an anti-flag antibody.The results demonstrated the presence of both a low molecular weight monomeric, and in the non-reduced samples, a higher molecular weight dimeric form of the R25C PTH(1-84)-3XFLAG protein in both the cell lysate and extracellular conditioned medium fractions, and the dimer appeared to be at an elevated proportion in the medium relative to the lysate (Figure 1D).To control for potential artifact effects attributed to the 3xFLAG tag, we utilized plasmid constructs pcDNA3.0-(pre-pro-PTH)-IRESand pcDNA3.0-(R56C prepro-PTH)-IRES encoding non-tagged PTH variants and an anti-PTH(39-84) antibody for Western blot analysis, which again confirmed the presence of the dimer in total HEK293T cell lysates (Supplemental figure 2).
We observed in the above studies that the expression level of R25C PTH(1-84) was higher than that of wild-type PTH(1-84) in both cell lysate and medium, which we considered might be due to an intrinsic enhancement in protein stability and resistance to protein degradation in the dimeric molecule.To address this, we treated the cells with the proteasome inhibitor MG132, which acts by forming a hemiacetal with the hydroxyl groups of active site threonine residues, and compared the expression levels of wild-type PTH(1-84) and R25C PTH(1-84) in the treated vs. untreated cells.The results indicated that while the expression level of wild-type PTH was increased by MG132 treatment, it did not reach the level of R25C PTH, suggesting that the difference in expression is not related to a difference in sensitivity to proteasome-mediated degradation (Supplemental figure 3).
Overall, we have confirmed that R25C PTH(1-84) can form a dimeric structure, and the R25C PTH(1-84) secreted outside the cells predominantly exists in dimeric form.Thus, utilizing dimer R25C PTH(1-84) in the analysis would be more relevant to understanding the actual function of R25C PTH.
Consequently, we aim to conduct further validation using dimeric R25C PTH in our subsequent investigations.

Functional characterization of dimeric R25C PTH(1-34) in vitro
To explore the functional properties of dimeric R25C PTH, we conducted experiments using synthetic peptides of PTH(1-34), R25C PTH(1-34) (monomeric) and disulfide-bonded dimeric R25C PTH .First, we examined the receptor-binding affinity of these ligands by performing competition experiments using membranes prepared from HEK293-derived GP-2.3 cells that stably express the human PTH1R and assay formats designed to assess binding to either the G protein-uncoupled R 0 or G protein-coupled RG receptor conformation.The results revealed that monomeric R25C PTH  bound to both the R 0 and RG conformations with comparable, albeit slightly weaker affinity as compared to PTH(1-34), while dimeric R25C PTH(1-34) bound to each conformation with weaker affinity than did the monomeric form while showing an apparent selectivity for higher binding to the RG vs R 0 conformation of PTH1R (Figure 2A).
To investigate the signaling properties of the ligands, we measured the changes in the increase in intracellular levels of cAMP induced by each ligand in an osteoblastic SaOS-2 derived cell line (SGS-72 cells) that stably expresses the Glosensor cAMP reporter.These assays revealed that each ligand dose-dependently increased the cAMP levels in the cells, detected as an increase in luminescence in an Envision plate reader, and while the potencies were moderately and more substantially reduced for the monomeric and dimeric forms of the ligand, respectively, as compared to PTH , the maximum response attained by each ligand were comparable (Figure 2B).Dimeric R25C PTH(1-34) thus retains signaling functionality at the PTH1R that is characterized by a potency approximately commensurate with its affinity for binding to the RG PTH1R conformation.

Effect of single injection of dimeric R25C PTH(1-34) on calcium and phosphate regulation in mice
To assess whether dimeric R25C PTH can function in vivo, we injected the ligand, and in parallel either vehicle or PTH  (each peptide at a dose of 50 nmol/kg) into CD1 female mice and measured levels of ionized calcium (Ca 2+ ) in the blood (n = 6 mice/group), inorganic phosphate (Pi) in plasma (n = 12 mice/group), and the excretion of Pi into urine (n = 6 mice/group).Blood Ca 2+ levels were measured at serial time points of pre-injection, 1, 2, 4, and 6 hours post-injection.Both PTH  and dimeric R25C PTH(1-34) induced increases in blood Ca 2+ levels that were significant, relative to the levels in vehicle-injected mice, at 1 and 2 hours post-injection, and the levels then returned to the baseline levels of vehicle control mice by 4 hours (Figure 3A).Plasma Pi levels were measured in samples obtained pre-injection, at 6 minutes, and 1, 2, and 6 hours post-injection.PTH(1-34) induced a significant decrease in plasma Pi at 1-hour post-injection, and the levels subsequently returned to baseline by 2 hours.Injection of dimeric R25C PTH(1-34) resulted in a slight decrease in plasma Pi at 2 hours post-injection, but the effect was not significant (Figure 3B).Consistent with this trend, Pi levels in the urine of mice injected with dimeric R25C PTH  were increased significantly at 2 hours postinjection and then gradually returned to baseline levels (Figure 3C).These results thus indicate that dimeric R25C PTH can elicit calcemic and phosphaturic responses in vivo that are fully with those expected for an injected PTH1R agonist ligand.

Effect of dimeric R25C PTH(1-34) on bone calvariae in mice
To initially assess the effects that short-term treatment of dimeric R25C PTH(1-34) can have on bone, we injected 8-week-old male C57BL/6 mice once a day for six days (days 1-6), with either dimeric R25C PTH(1-34), PTH  or vehicle, and after 10 days without treatment (day 16) followed by euthanasia, we isolated the calvariae for histological analysis of new bone formation.Specifically, we examined sections stained with hematoxylin and eosin (H&E) to assess the width of newly formed bone areas along the edge of each sample.These regions exhibited a more vivid coloration compared to the surrounding existing bone tissue, demarcated by a dotted line for clarity (Figure 4A).
Measurements were taken below and above the dissection area where new bone had formed, and these measurements were then utilized to calculate the mean values for further analysis.These analyses revealed that both PTH(1-34) and dimeric R25C PTH(1-34) significantly increased the width of the new bone area by approximately four-fold, as compared to the vehicle group (Figure 4B).These findings thus support a capacity of dimeric R25C PTH(1-34) to induce new bone formation in vivo, similar to PTH, despite molecular and structural changes.

Effect of dimeric R25C PTH on bone mass in osteoporotic mice
To more directly assess the impact of dimeric The effects of the treatments on bone strength were assessed by conducting a three-point bending test on femurs isolated from the mice.The maximum load parameter was significantly decreased in the femurs from OVX-control versus Sham mice and was significantly increased, relative to the OVX-controls, by treatment with the PTH(1-34), but not dimeric R25C PTH(1-34) (Figure 5C).The slope parameter was significantly decreased in the femurs from OVX-control versus Sham mice and tended to increase versus OVX-controls by treatment with either PTH(1-34) or R25C PTH(1-34), but the changes were not significant.
We further analyzed the levels of bone metabolism markers in the serum obtained from the mice at the study endpoint (Figure 5D).The levels of serum calcium were within the normal range in all treatment groups, while serum phosphate levels were modestly increased in the OVX mice treated with PTH(1-34) or dimeric R25C PTH(1-34) as compared to with vehicle, but the effect was significant only with PTH(1-34).The serum levels of CTX-1, a bone resorption marker (42), were elevated in each of the OVX groups versus the Sham group, and tended to be lower in the OVX-PTH  treatment group, but the change relative to OVX-vehicle was not significant (Figure 5D).Interestingly, serum P1NP and alkaline phosphatase (ALP) levels were significantly increased in dimeric R25C PTH(1-34)-treated group, compared to the OVX-vehicle group (43,44).Histological staining of proximal tibial sections for tartrate-resistant acid phosphatase (TRAP) activity, a marker of osteoclastmediated bone resorption, revealed an apparent increase in this activity in bones of the OVX mice, as compared to those in Sham control mice, reflecting a heightened rate of bone turnover, as also suggested by the increased levels of serum CTX-1 in the OVX mice, and the TRAP staining appeared to be reduced in the tibiae of the OVX mice treated with PTH(1-34) (Figure 5D and E).Further histomorphometric analysis confirmed a significant increase in the osteoclast surface area relative to bone surface area (Oc.S/BS) in the proximal tibiae of the OVX-vehicle mice, relative to that in the Sham-control mice, and this parameter was significantly decreased by treatment with PTH(1-34), but not with dimeric R25C PTH(1-34) (Figure 5F).
We then analyzed the bone microstructure in the lumbar vertebrae through von Kossa staining of histological sections and histomorphometric quantification (Figure 6A).The trabecular bone volume fraction (Tb BV/TV, %), and trabecular number (Tb N) were significantly reduced in the OVXvehicle group, as compared to the Sham group, and treatment with either PTH(1-34) or dimeric R25C PTH(1-34) resulted in a significant increase in each of these parameters, as well as a concomitant reduction in trabecular separation (Tb Sp), as compared to the respective parameters in the OVXvehicle group (Figure 6B).
Dynamic bone histomorphometry was also performed on the vertebrae to evaluate rates of bone formation (Figure 6C).The trabecular mineral apposition rate (MAR) and cortical MAR were significantly increased in both the OVX-PTH(1-34) and OVX-dimeric R25C PTH(1-34) groups, as compared to in the OVX-vehicle group, and although there was a tendency for an increase in the bone formation rate (BFR/BS) in both the trabecular and cortical bone with either PTH(1-34) or dimeric R25C PTH(1-34) treatment, the differences were not statistically significant, as compared to the OVX control (Figure 6D).
In summary, injection of dimeric R25C PTH into osteoporotic OVX mice resulted in significant increases in cortical bone in the femurs and trabecular bone in the vertebrae, as well as significant increases in osteoblast function and serum markers of bone formation, ALP and P1NP, without inducing excessive bone resorption or hypercalcemia.

Discussion
In this study, we show the introduction of a cysteine mutation at the 25th amino acid position of mature parathyroid hormone ( R25C PTH) facilitates the formation of homodimers comprised of the resulting dimeric R25C PTH peptide in vitro.This dimerization surprisingly was compatible with receptor binding affinity and led to relatively minor deviations in functional behavior as assessed in our cellbased assays and compared to the standard monomeric control PTH peptide.The homozygous R25C PTH mutation was identified in patients who presented with hypocalcemia and hyperphosphatemia, despite elevated PTH levels (35,45), and the mutation was found to impact the bioactive region of PTH (35,(46)(47)(48)(49). Our initial research on this R25C PTH mutant focused on the monomeric state of the peptide, and these studies revealed relatively moderate decreases in PTH1R binding affinity and cAMP-stimulating potency in vitro and moderately impaired calcemic effects upon infusion in mice.Additional patient observations, however, revealed the patients to have a higher BMD than anticipated for age-matched averages.This elevated bone mass, coupled with the elevated serum PTH levels, prompted our further investigations into the properties of the mutant PTH, as described herein.
Our investigations brought to light the capacity of the cysteine-25 mutation to induce dimer formation in the otherwise monomeric PTH polypeptide as produced in transfected cells.This result was established by comparing western blots of transfected cell lysates analyzed under reducing versus non-reducing conditions of gel electrophoresis.Subsequently, we employed synthetic peptides to further explore the functional properties of dimeric R25C PTH .The results of our current studies show some divergence from our previous findings obtained using the monomeric counterpart, R25C PTH  .Compared to the monomer, dimeric R25C PTH(1-34) exhibited a more preferential binding affinity for the RG versus R 0 PTH1R conformation, despite a diminished affinity for either conformation.We also observed that the potency of cAMP production in cells was lower for dimeric R25C PTH as compared to the monomeric R25C PTH, in accordance with a lower PTH1R-binding affinity.
Previous reports indicated that a mutation at the 25th position of PTH results in the loss of calcium ion allosteric effects on monomeric R25C PTH, leading to faster ligand dissociation, rapid receptor recycling, and a shorter cAMP time course (50).Correspondingly, the weaker receptor affinity and reduced cAMP production observed in dimeric R25C PTH suggest a possibility that the formation of a disulfide bond at the 25th position significantly alters the function of PTH as a PTH1R ligand.While these structural effects are not yet fully understood and need to be investigated further.
We further pursued in vivo applications in mice.We assessed the calcemic and phosphatemic responses to a single injection of synthetic peptides of either PTH(1-34) or dimeric R25C PTH(1-34) in intact mice.We found the dimer could induce increases in plasma calcium levels that were at least as robust and as sustained as those induced by PTH(1-34) and were similarly phosphaturic (Figure 3).(34,(51)(52)(53)(54).The precise binding mode used by dimeric R25C PTH to the PTH1R is unknown, but it may be anticipated that it differs to some extent from that used by the monomeric peptide, due, for example, to the changes in bulk molecular size and display of accessible functional groups.Consequently, the receptor conformational changes and the modes of coupling to downstream effectors may differ for the monomeric versus dimeric ligands, which could potentially lead to altered signaling and biological responses in vivo.Whether such changes account for the increased bone density observed in the patient with the homozygous R25C PTH mutation is unknown, but cannot be presently ruled out.

Activation of the canonical
The results of practical assessments of dimeric Interestingly, the recent identification of a young patient in Denmark displaying homozygous R25C PTH has opened avenues for observing the direct impacts of R25C PTH within the human biological system (45).The continual monitoring and observation of patients will contribute to a more profound comprehension of the long-term consequences associated with R25C PTH exposure.This extensive observation is crucial in delineating the extended effects of this compound on individuals.
Consequently, by conducting thorough investigations to confirm the potential bone anabolic effect of R25C PTH, we hope to develop a novel bone anabolic agent with a targeted focus on the PTH1R.

Plasmid construction
The coding sequences (CDS) of pre-pro-PTH and the mutated form, R56C pre-pro-PTH[ R25C PTH], were amplified using primers containing the attB site.These CDS fragments were obtained from pcDNA3.0-(hpre-pro-PTH)-IRES and pcDNA3.0-(hR56C pre-pro-PTH)-IRES, which were used in the previous research conducted by Lee, et  NaCl, pH 7.5) and then washed three times for 10 min each with tris-buffered saline with 0.05 % tween-20 (TBST).The membranes were incubated with primary antibody for 1 hour at RT, then washed three times for 10 min each with TBST.If needed, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody for 1 hour at RT after primary antibody incubation, then washed three times for 10 min each with TBST.After washing, the membranes were rinsed and soaked in TBS.To develop a blot image, the membranes were treated with a chemiluminescent substrate solution (Merck Millipore, Immobilon ECL Ultra Western HRP Substrate, Cat No. WBULS0500, USA) according to the manufacturer's instruction.The blot images were obtained by LAS 4000 mini (Cytiva, ImageQuant™ LAS 4000 mini, USA).The dilution condition for the anti-FLAG with hHRP conjugated antibody (Sigma-Aldrich, Monoclonal ANTI-FLAG® M2-Peroxidase (HRP) antibody produced in mouse, Cat No. A8592, USA) was 1:2,000.The dilution condition for the anti-HSP90 primary antibody (Santa Cruz Biotechnology Inc., HSP 90 Antibody (AC-16), Cat No. sc-101494, USA) was 1:5,000.The dilution condition for the anti-mouse secondary antibody was 1:5,000.Each antibody was diluted in TBST with 1 % BSA solution.

Proteasome inhibition assay
HEK293T cells were seeded in culture dishes at approximately 60 % confluence, and they were allowed to grow for about 20 to 24 hours prior to transfection.The transfection of pcDNA3.1-(pre-pro-PTH)-3xFLAG-V5and pcDNA3.1-(R56C pre-pro-PTH)-3xFLAG-V5 was conducted following the method mentioned earlier.After 24 hours of transfection, MG132, dissolved in DMSO, was added to the cells to achieve a final concentration of 10 µM.For the mock treatment, DMSO alone was added.
The cells were then incubated for an additional 24 hours after MG132 treatment.Both the culture medium and cell lysate were prepared for western blot analysis to assess the restored protein levels.
The western blot procedure was carried out as described in the previous section.

PTH1R competition binding assay
The binding of PTH and its analogs to G protein-uncoupled PTH1R (R 0 conformation) and G protein-coupled PTH1R (RG conformation) was assessed using a competition method with membranes prepared from GP-2.3 cells (HEK-293 cells stably expressing the hPTH1R).For tracer radioligands, we utilized

cAMP assay
To measure intracellular cAMP production, SGS-72 cells, derived from SaOS-2 cells and stably expressing the Glosensor cAMP reporter, were utilized to measure intracellular cAMP production.The detection of cAMP-dependent expression was performed using an Envision plate reader (PerkinElmer, Waltham, MA, USA), based on luciferase-based luminescence, as previously described by Maeda, et al. (56).Each measurement was replicated four times.

Animal model used in the study
CD1 female mice were purchased from Charles River Laboratories (Massachusetts, USA), and all animal care and experimental procedures were conducted under the guidelines set by the Institutional Animal Care and Use Committee (IACUC) at Massachusetts General Hospital (MGH).
The mice were housed in a specific pathogen-free environment, with 4-5 mice per cage, under a 12hour light cycle at a temperature of 22±2°C.
Eight-week-old C57BL/6N female mice were purchased from KOATECH (Gyeonggi-do, Republic of Korea), and stabilized mice for 2 weeks.All animal care and experimental procedures were conducted under the guidelines set by the Institutional Animal Care and Use Committees of Kyungpook National University (KNU-2021-0101).The mice were housed in a specific pathogen-free environment, with 4-5 mice per cage, under a 12-h light cycle at 22 ± 2°C.They were provided with standard rodent chow and water ad libitum.

Acute injections
The peptides PTH(1-34) and dimeric R25C PTH(1-34) were diluted in a solution comprising 0.05% Tween 80, 10 mM citric acid, and 150 mM NaCl at a pH of 5.0.Intravenous injections of these peptides were administered at doses ranging from 50-100 μg/kg into 9-week-old CD1 female mice.As a control, mice received only the vehicle.Levels of ionized calcium (Ca2+) in the blood (n = 6 mice/group) were measured at serial time points of pre-injection, 1, 2, 4, and 6 hours post-injection.
Inorganic phosphate (Pi) in plasma (n = 12 mice/group), and the excretion of Pi into urine (n = 6 mice/group) were measured in samples obtained at serial time points of pre-injection, at 6 minutes, and 1, 2, and 6 hours post-injection.
On the sixteenth day, the mice were sacrificed, and their bone tissues were harvested and fixed in 10 % formaldehyde at 4°C.The fixed bone tissues were then decalcified in PBS (pH 7.4) containing 0.5 moles of ethylenediaminetetraacetic acid (EDTA).Following decalcification, the tissues were embedded in paraffin, and paraffinized tissues were sectioned to a thickness of 5-7 µm.Histological analysis was performed using the sectioned tissue slides stained with hematoxylin and eosin (H&E).
The area of new bone formation, which displays a more intense coloration compared to the existing bone tissue, was examined.

μ-CT analysis
Mouse femurs were fixed in a 4 % paraformaldehyde solution for 24 hours at 4°C.In μ-CT, we used the Quantum FX μ-CT (Perkin Elmer, Waltham, MA, USA).The images were acquired at a 9.7 μm voxel resolution, with settings of 90 kV and 200 μA, a 10 mm field of view, and a 3-minute exposure time.Serial cross-sectional images were reconstructed using the Analyze 12.0 software (Overland Park, KS, USA).To ensure consistent analysis, identical regions of interest (ROIs) were selected for the trabecular and cortical bones.Bone parameters and density were analyzed in the region between 0.3-1.755mm (150 slices) from the bottom of the growth plate.Analysis of bone structure was performed using adaptive thresholding in CT Analyser.Thresholds for analysis were determined manually based on grayscale values for each experimental group: trabecular bone: 3000; cortical bone: 5000 for all samples.All bone parameters were evaluated according to the guidelines of the American Society for Bone and Mineral Research (57).

Three-point bending test
The left femur of the mouse was immersed in 0.9 % NaCl solution, wrapped in gauze, and stored at −20°C until ready for a three-point bending test.In this test, we placed the mouse femurs horizontally with the anterior surface facing upwards, centered on the supports, and the compressive force was applied vertically to the mid-shaft.The pressure sensor was positioned at a distance that allowed maximum allowable pressure (200N) without interfering with the test (20.0 mm for the femur).
A miniature material testing machine (Instron, MA, U.S.A.) was used for this test.The crosshead speed was decreased to 1 mm/min until failure.During the test, force-displacement data were collected to determine the maximum load and slope of the bones.

Serum biochemistry analysis
Serum bone resorption and osteogenesis marker levels, specifically the C-terminal telopeptide of type I collagen (CTX) and procollagen type I N-terminal propeptide (P1NP), were assessed in mice from the Sham, OVX-control, PTH , and dimeric R25C PTH(1-34) groups by using the enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's instructions.
Additionally, their concentrations were determined using specific mouse CTX-1 and P1NP ELISA kits (Cloud Clone, Wuhan, China) respectively.

Bone histological analyses
The tibiae were initially fixed in 4% paraformaldehyde at 4°C overnight.The following day, samples were decalcified using 10% ethylenediaminetetraacetic acid (EDTA) solution (pH 7.4) for 4 weeks at 4°C.The decalcified tibiae were then embedded in paraffin and sectioned at 3 μm thick.For TRAP staining, dehydrated paraffin sections were fixed in an acetone/ethanol mixture (1:1) for 1 minute, followed by complete air-drying at RT for 20 minutes.Thereafter, the sections were immersed in TRAP reagent for 30 minutes at 37°C.In the histomorphometry analysis for TRAP staining, we used the primary spongiosa for the trabecular ROI because of the barely detectable in the secondary spongiosa of OVX model.Osteoclast surface per bone surface (Oc.S/BS) and Osteoclast number per bone surface (Oc.N/BS) analysis followed the ASBMR guidelines (58).

Dynamic bone histomorphometric analysis
To conduct dynamic histomorphometry analysis, we injected the mice with 25 mg/kg body weight of calcein (Sigma-Aldrich) or Alizarin Red S (Sigma-Aldrich) intraperitoneally before sacrifice, with 3-or 10-day intervals between injections as previously described (59,60).Briefly, femurs or vertebrates were fixed in 4 % paraformaldehyde solution for 24 hours at 4°C.The following day, the samples were washed with phosphate-buffered saline (PBS) solution and then dehydrated using a gradient of ethanol (50 %, 70 %, 85 %, 90 %, and 100 %).Subsequently, we embedded the dehydrated femurs or vertebrates in methyl methacrylate (Sigma) to prepare resin blocks.The resin blocks were sectioned at 6 μm thick by using a Leica SP1600 microtome (Leica Microsystems, Germany).The fluorescence signals of calcein (green) and Alizarin Red S (red) from ROIs were captured using a fluorescence microscope (Leica, Wetzlar, Germany).For vertebral bone analysis, bone mineralization was evaluated by von Kossa staining.The sections were placed in 2methoxyethyl acetate (Sigma-Aldrich) for 20 minutes, followed by rehydration with serial ethanol solutions (100 %, 90 %, 80 %, 70 %, and 50 %) and distilled water for 2 minutes each.The sections were subsequently dipped in a 1 % AgNO 3 solution (Sigma-Aldrich) for 5 minutes under ultraviolet (UV) light photons, washed in distilled water for 5 minutes, and dipped in 5 % sodium thiosulfate solution for 5 minutes to remove nonspecific binding.Finally, we covered the sections with mounting solution and captured images by using a Leica microscope.The parameters of dynamic bone histomorphometry were analyzed using the Bioquant Osteo 2019ME program (Bioquant Osteo, Nashville, TN, USA).

Statistical analysis
Statistical analysis was performed in GraphPad Prism 10.1.2.The data are presented as the mean ± standard error of the means (SEM).Statistically significant differences between the two groups were determined using an analysis of variance (ANOVA).A p-value less than 0.05 was considered statistically significant.
Considering the proven bone-anabolic capacity of several established PTH agonist ligands, and the need for safe, long-term treatments for skeletal disorders, our studies on dimeric R25C PTH(1-    34)suggest alternative strategies to consider in such drug development programs.
R25CPTHsynthetic peptide retains agonistic properties on the PTH1R that are driven by a moderate selectivity for the RG vs. R 0 receptor conformation.Finally, we demonstrate in mice that R25C PTH(1-34) can induce skeletal responses that are similar to those induced by PTH, but without triggering an excessive hypercalcemic response.
Ct.BV/TV by 125%, Ct.Th by 107%, and Ct.Ar/Tt.Ar by 116%, while decreased Tt.Ar 86% (Figure56).Considering the reduction of Tt.Ar and no change of Ct.Ar compared to the OVX+vehicle controls, the increase of Ct.Ar/Tt.Ar indicates a decrease in bone marrow space.The increase in cortical bone BMD was significant with dimeric R25CPTHon bone mass, we administered it to ovariectomized (OVX) mice, which serve as a well-established model for postmenopausal osteoporosis.The OVX mice were injected 5 times per week for 4 weeks with either the dimeric ligand (OVX + dimeric R25C PTH(1-34), PTH(1-34) (OVX + PTH(1-34)) or vehicle (OVX + vehicle, OVXcontrols), and sham-operated (Sham) mice were used as further controls.Mice were euthanized at the end of the injection period and tissue samples were isolated for analysis.Quantitative microcomputed tomography (μ-CT) analysis of the femurs obtained from each group revealed that, as compared to OVX + vehicle controls, treatment with PTH(1-34) increased femoral trabecular bone volume fraction (Tb.BV/TV) by 121%, cortical bone volume fraction (Ct.BV/TV) by 128%, cortical thickness (Ct.Th) by 115%, cortical area (Ct.Ar) by 110%, and cortical area fraction (Ct.Ar/Tt.Ar) by 118%, while decreased total tissue area (Tt.Ar) by 93% (Figure5A and 5B).Treatment with dimeric R25C PTH(1-34) had similar effects on the femoral cortical bone parameters, as it increased Ct.BMD by 104%, R25C PTH(1-34) but not with PTH(1-34), whereas an increase in femoral trabecular bone was only observed with PTH(1-34).
PTH, decreased bone resorption markers such as TRAP staining and CTX-1 levels, suggesting different effects of wild-type monomeric PTH and dimeric R25C PTH.In this study, the increase of bone resorption markers of dimeric R25C PTH compared to PTH may be one reason for low influences on bone metabolism alike to wild-type PTH.In OVX mice, PTH and dimeric R25C PTH significantly increased bone formation markers P1NP and ALP.However, wild-type PTH, but not dimeric R25C R25C PTH is present in human patient serum.Second, TRAP staining showed an inhibitory effect of PTH treatment on the primary spongiosa area.However, the secondary spongiosa, which more accurately reflects bone remodeling (55), was not examined due to the barely detectable bone in this area in OVX-induced osteoporosis mouse models.Third, it is unclear whether similar bone phenotypes exist between human R25C PTH patients and dimeric R25C PTH-treated mice, particularly regarding low bone strength.Although the dimeric R25C PTH-treated group showed higher cortical BMD compared to WT-Sham or PTH groups, there was no difference in bone strength compared to the osteoporotic mouse model.Fourth, our study showed that PTH or R25C PTH treatment decreased circumferential length; it is uncertain if this phenotype is also present in PTH-treated or R25C PTH patients.Finally, we did not analyze the R25C PTH mutant mouse model, which would allow us to compare phenotypes that most closely resemble those of human patients.
was collected and used for western blot as a secreted protein sample.The rest of the cells were lysed by RIPA buffer (Thermo Scientific™, RIPA Lysis and Extraction Buffer, Cat No. 89900, USA) following the manufacturer's instruction and used for western blot as total cell lysate sample.