Dimeric ^R25CPTH(1-34) Activates the Parathyroid Hormone-1 Receptor in vitro and Stimulates Bone Formation in Osteoporotic Female Mice

Reviewer #1 (Public review):

Summary:

In this work, the authors investigate the functional difference between the most commonly expressed form of PTH, and a novel point mutation in PTH identified in a patient with chronic hypocalcemia and hyperphosphatemia. The value of this mutant form of PTH as a potential anabolic agent for bone is investigated alongside PTH(1-84), which is a current anabolic therapy. The authors have achieved the aims of the study. Their conclusion that this suggests a "new path of therapeutic PTH analog development" seems unfounded; the benefit of this PTH variant is not clear, but the work is still interesting.

The work does not identify why the patient with this mutation has hypocalcemia and hyperphosphatemia; this was not the goal of the study, but the data is useful for helping to understand it.

Strengths:

The work is novel, as it describes the function of a novel, naturally occurring, variant of PTH in terms of its ability to dimerise, to lead to cAMP activation, to increase serum calcium, and its pharmacological action compared to normal PTH.

Weaknesses:

(1) The use of very young, 10 week old, mice as a model of postmenopausal osteoporosis remains a limitation of this study, but this is now quite clearly described as a limitation,, including justifying the use of the primary spongiosa as a measurement site.

(2) Methods have been clarified. It is still necessary to properly define the micro-CT threshold in mm HA/cc^3. I think it might be at about 200mg HA/cc^3 in this study.

(3) The apparent contradiction between the cortical thickness data (where there is no difference between the two PTH formulations) and the mechanical testing data (where there is a difference) remains unresolved. It is still not clear whether there is a material defect in the bone, which can be partially assessed by reporting the 3 point bending test, corrected for the diameters of the bone (i.e. as stress / strain curves).

(4) It is also puzzling that both dimeric and monomeric PTH lead to a reduction in total bone area (cross sectional area?). This would suggest a reduction in bone growth. This should be discussed in the work.

Author response:

The following is the authors’ response to the original reviews.

Public Reviews:

Reviewer #1 (Public Review):

Summary:

In this work, the authors investigate the functional difference between the most commonly expressed form of PTH, and a novel point mutation in PTH identified in a patient with chronic hypocalcemia and hyperphosphatemia. The value of this mutant form of PTH as a potential anabolic agent for bone is investigated alongside PTH(1-84), which is a previously used anabolic therapy. The authors have achieved the aims of the study. Their conclusion, however, that this suggests a "new path of therapeutic PTH analog development" seems unfounded; the benefit of this PTH variant is not clear, but the work is still interesting.

The work does not identify why the patient with this mutation has hypocalcemia and hyperphosphatemia; this was not the goal of the study, but the data are useful for helping to understand that.

Strengths:

The work is novel, as it describes the function of a novel, naturally occurring, variant of PTH in terms of its ability to dimerise, to lead to cAMP activation, to increase serum calcium, and its pharmacological action compared to normal PTH.

Weaknesses:

(1) The use of very young, 8-10 week old, mice as a model of postmenopausal osteoporosis is a major limitation of this study. At 8 weeks, the effect of ovariectomy leads to lack of new trabecular bone formation, rather than trabecular bone loss due to a defect in bone remodelling. Although the findings here provide a comparison between two forms of PTH, it is unlikely to be of direct relevance to the patient population. For example, the authors find an inhibitory effect of PTH on osteoclast surface, which is very unusual. Adding to this concern is that the authors have not described the regions used for histomorphometry, and from their figures (particularly the TRAP stain), it seems that the primary spongiosa (which is a region of growth) has been used for histomorphometry, rather than the secondary spongiosa (which more accurately reflects bone remodelling). Much further detail is needed to justify the use of this very young model, and a section on the limitations of this model is needed. Please provide that section in the revised manuscript.

Thank you for your crucial comment. We obtained 8-week-old female mice and stabilized them in our facility for 2 weeks. Then, we performed OVX using 10-week-old mice and determined the effects of dimeric ^R25CPTH(1-34) on bone after 8 weeks because of 4 weeks for recovery and 4 weeks for PTH or ^R25CPTH(1-34). Therefore, we sacrificed the mice at 18-week-old mice. We revised the method section on page 18, line 436-441 and page 18, line 442-448 as follows.

- ‘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.’

- ‘An ovariectomized (OVX) mouse model was established using 10-week-old C57BL/6N female mice. Following surgery, mice were divided into the following four groups (n = 6 mice/group) as follows: sham, OVX control group, OVX + PTH (1–34) treated group (40 µg/kg/day), and OVX + dimeric ^R25CPTH treated group (40-80 µg/kg/day). OVX mice were allowed to recover for 4 weeks after surgery. Afterward, PTH (1–34) or ^R25CPTH was injected subcutaneously 5 times a week for 4 weeks. Micro-computed tomography (μ-CT) and histological analyses were performed on 4 groups at 18 weeks of age.’

We also appreciate the reviewer's helpful comment on histology analysis. We agree with the reviewer’s comment that the primary spongiosa does not fully reflect bone remodeling. For histomorphometry analysis in young or male mice, we commonly use the secondary spongiosa, which more accurately reflects bone remodeling. However, in aged or OVX-induced osteoporosis mouse models, we use the primary and secondary spongiosa for histomorphometry analysis because of the barely detectable bone in the secondary spongiosa. In the TRAP staining, we observed an inhibitory effect of PTH on the osteoclast surface/bone surface, which was due to an increased bone surface in the PTH treatment group and less bone in the OVX-vehicle group. Serum CTX1 levels showed no significant difference between the OVX+vehicle and OVX+PTH(1-34) groups. We revised the Materials and Methods (page 21, line 502) and Discussion (page 14, line 330) sections as follows.

- ‘In the histomorphometry analysis for TRAP staining, we used the secondary and primary spongiosa for the trabecular ROI because of the barely detectable in the secondary spongiosa of OVX model.’

- ‘This study has several limitations. First, it is urgently necessary to determine whether dimeric ^R25CPTH 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 ^R25CPTH patients and dimeric ^R25CPTH-treated mice, particularly regarding low bone strength. Although the dimeric ^R25CPTH-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 ^R25CPTH treatment decreased circumferential length; it is uncertain if this phenotype is also present in PTH-treated or ^R25CPTH patients. Finally, we did not analyze the ^R25CPTH mutant mouse model, which would allow us to compare phenotypes that most closely resemble those of human patients.’

(2) It is also somewhat concerning that the age range is from 8-10 weeks, increasing the variability within the model. Did the age of mice differ between the groups analysed?

We utilized mice of the same age (10 weeks) across all experiments involving the surgically induced ovariectomy (OVX) model described as above.

(3) Methods are not sufficiently detailed. For example, the regions used for histomorphometry are not described, there is no information on micro-CT thresholds, no detail on the force used for mechanical testing. Please address this request.

Thank you for your comment. Let me address your points step by step.

(1) Thresholds for analysis were determined manually based on grayscale values for each experimental group as follows: trabecular bone: 3000; cortical bone: 5000 for all samples. We utilized an HA (calcium hydroxyapatite) phantom with HA content ranging from 0 to 1200 mg CaHA/cm³ to measure the grayscale values via µ-CT. These measurements were then used to generate a standard curve.

Author response image 1.

(2) Bone parameters and density were analyzed in the region between 0.3–1.755 mm (Voxel size: 9.7um, 150 slices) from the bottom of the growth plate. Analysis of bone structure was performed using adaptive thresholding in a CT Analyser.

Author response image 2.

(3) 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 positioned 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 for the maximum allowable pressure (200N) without interfering with the test (20.0 mm for the femur). A miniature material testing machine (Instron, MA, USA) 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.

(4) As the reviewer’s suggestion, we revised the methods on page 20, line 477 and line 482-486 as follows.

- ‘Bone parameters and density were analyzed in the region between 0.3–1.755 mm (150 slices) from the bottom of the growth plate. Analysis of bone structure was performed using adaptive thresholding in a µ-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.’

- ‘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 (1000N) 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.’

(4) There are three things unclear about the calvarial injection mouse model. Firstly, were the mice injected over the calvariae or with a standard subcutaneous injection (e.g. at the back of the neck)? If they were injected over the calvaria, why were both surfaces measured? Secondly, why was the dose of the R25C-PTH double that of PTH(1-34)? Thirdly, there is no justification for the use of "more intense coloration" as a marker of new bone; this requires calcein labelling to prove it new bone. It would be more reliable to measure and report the thickness of the calvaria. Please address these technical questions.

Thank you for your valuable feedback on the calvarial injection mouse model. Below are our responses to the specific points mentioned:

(1) Injection method and measurement sites: The injections were administered subcutaneously above the calvaria, rather than at the standard subcutaneous site such as the back of the neck. This approach was chosen to ensure direct delivery of the peptide to the target area, enhancing the localized effects on bone formation. Measurements were taken at two different parts of the calvaria to account for any variation in the spread and absorption of the administered substance following injection. By analyzing both surfaces, we aimed to provide a comprehensive assessment of the impact on calvarial bone thickness.

(2) Dose of ^R25CPTH compared to PTH(1-34): The dose of ^R25CPTH used in our study was determined based on molecular weight calculations. The molecular weight of the dimeric ^R25CPTH(1-34) is approximately twice that of the monomeric PTH(1-34). Therefore, to maintain a consistent molar concentration and ensure comparable biological effects, the dose of ^R25CPTH was adjusted accordingly.

(3) Use of "more intense coloration" as a marker of new bone: We acknowledge that calcein labeling would provide a more reliable and quantifiable way to identify new bone formation. The use of “more intense coloration” was intended as a qualitative indicator in this study, and we recognize the technical limitations of this approach.

(5) The presentation of mechanical testing data is not sufficient. Example curves should be shown, and data corrected for bone size needs to be shown. The difference in mechanical behaviour is interesting, but does it stem from a difference in the amount of bone, or two a difference in the quality of the bone? Please explain this matter better in the manuscript.

Thank you for your comment.

As a reviewer's comment, we provided example curves for the rat femur three-point bending test as shown below.

Author response image 3.

(1) The cortical bone area was decreased in the OVX-Vehicle and OVX-^R25CPTH(1-34) groups but not in the OVX-PTH(1-34) group compared to the Sham group. However, the total bone area was decreased in the PTH(1-34) and ^R25CPTH(1-34) treated groups, with no significant difference in the OVX-Vehicle group compared to the Sham group. Collectively, there was an increase in cortical thickness which resulted in a narrowing of the bone marrow space in OVX-^R25CPTH(1-34) groups. Accordingly, we revised Fig 5B with the addition of Tt.Ar and Ct.Ar.

(2) As the reviewer’s suggestion, we revised the results on page 10, line 220-228 s follows.

- ‘Quantitative micro-computed 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% (Figure 5A and 5B). Treatment with dimeric ^R25CPTH(1-34) had similar effects on the femoral cortical bone parameters, as it increased Ct.BMD by 104%, Ct.BV/TV by 125%, Ct.Th by 107%, and Ct.Ar/Tt.Ar by 116%, while decreased Tt.Ar 86% (Figure 5). 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 ^R25CPTH(1-34) but not with PTH(1-34), whereas an increase in femoral trabecular bone was only observed with PTH(1-34).’

(6) The micro-CT analysis of the cortical bone in the OVX model is insufficient. Please indicate whether cross-sectional area has increased. Is there an increase in the size of the bones, or is the increase in cortical thickness due to a narrowing of the marrow space? This may help resolve the apparent contradiction between the cortical thickness data (where there is no difference between the two PTH formulations) and the mechanical testing data (where there is a difference). Please explain this matter better in the manuscript.

Thank you for your comment.

(1) The cortical bone area was decreased in the OVX-Vehicle and OVX-^R25CPTH(1-34) groups but not in the OVX-PTH(1-34) group compared to the Sham group. However, the total bone area was decreased in the PTH(1-34) and ^R25CPTH(1-34) treated groups, with no significant difference in the OVX-vehicle group compared to the Sham group. Taken together, there was an increase in cortical thickness due to a narrowing of the bone marrow space in OVX-^R25CPTH(1-34) groups. Therefore, we revised as above.

(2) As the reviewer’s suggestion, we revised the results on page 10, line 220-228 as follows.

- ‘Quantitative micro-computed 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% (Figure 5A and 5B). Treatment with dimeric ^R25CPTH(1-34) had similar effects on the femoral cortical bone parameters, as it increased Ct.BMD by 104%, Ct.BV/TV by 125%, Ct.Th by 107%, and Ct.Ar/Tt.Ar by 116%, while decreased Tt.Ar 86% (Figure 5B). 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 ^R25CPTH(1-34) but not with PTH(1-34), whereas an increase in femoral trabecular bone was only observed with PTH(1-34).’

(7) The evidence that dimeric PTH has a different effect to monomeric PTH is very slim; I am not sure this is a real effect. Such differences take a long time to sort out (e.g. the field is still trying to determine whether teriparatide and abaloparatide are different). I think the authors need to look more carefully at their data - almost all effects are the same. Ultimately, the statement that dimeric PTH may be a more effective anabolic therapy than monomeric PTH are not supported by the data, and this should be removed. There is little to no difference found between normal PTH and the variant in their effects on calcium and phosphate homeostasis or on bone mass. However, the analysis has been somewhat cursory, with insufficient mechanical testing or cortical data presented. Many of the effects seem to be the same (e.g. cortical thickness, P1NP, ALP, vertebral BV/TV and MAR), but the way it is written it sounds like there is a difference. Please remove some of the unfounded claims that you have made in this manuscript.

Thank you for your insightful comments. We strongly agree with your conclusion that PTH and dimeric ^R25CPTH indeed exhibit similar activities. We have toned-down our statement, however, there are still some elements showing statistical significance that need to be clearly stated. Specifically, when we changed the statistical method from t-test to one-way ANOVA, the significance of bone formation markers were only observed in dimeric PTH treated samples, and we have revised the manuscript of Results section on page 9, line 206-212 as follows to reflect the change.

- ‘These analyses revealed that both PTH(1-34) and dimeric ^R25CPTH(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 ^R25CPTH(1-34) to induce new bone formation in vivo, similar to PTH, despite molecular and structural changes.’

Although it is unclear whether ^R25CPTH circulate as dimeric form or mutant monomeric form, the absence of bone resorption associated with long-term PTH exposure in the patients suggests the potential for a bone anabolic drug without side effects. Also, continued observation of the recently reported young patient in Denmark is expected to clarify this effect further. However, we acknowledge that our current data alone are insufficient to claim that ^R25CPTH may be a more effective anabolic therapy than wild type PTH, and we have adjusted our tone accordingly.

(8) Statistical analysis used multiple t-tests. ANOVA would be more appropriate.

We agree with your suggestion. To compare the means among three or more groups, ANOVA is more appropriate than the t-test. Accordingly, we performed new statistical analyses using one-way and two-way ANOVA. One-way ANOVA was applied to figure 4, 5, and 6 (In previous, figure 5, 6, and 7), and two-way ANOVA was applied to Figure 3, considering both time and treatment variables. We revised some of the figures and descriptions to reflect the changes in significance.

Thank you for Reviewer #1’s thorough and thoughtful review. We greatly appreciate the suggestions and will incorporate them to enhance the quality of our paper.

Reviewer #2 (Public Review):

Summary:

The study conducted by Noh et al. investigated the effects of parathyroid hormone (PTH) and a dimeric PTH peptide on bone formation and serum biochemistry in ovariectomized mice as a model for postmenopausal osteoporosis. The authors claimed that the dimeric PTH peptide has pharmacological benefits over PTH in promoting bone formation, despite both molecules having similar effects on bone formation and serum Ca2+. However, after careful evaluation, I am not convinced that this manuscript adds a significant contribution to the literature on bone and mineral research.

Strengths:

Experiments are well performed, but strengths are limited to the methodology used to evaluate bone formation and serum biochemical analysis.

Weaknesses:

(1) Limited significance of this study:

• This study follows a previous study (not cited) reporting the effect of the dimeric R25CPTH(1-34) on bone regeneration in an osteoporotic dog (Beagle) model (Jeong-Oh Shin et al., eLife 13:RP93830, 2024). It's unclear why the authors tested the dimeric R25C-PTH peptide on a rodent animal model, which has limitations because the healing mechanism of human bone is more similar in dogs than in mice.

Thank you for your interest in our research. To address the paper by Shin et al. (2024, DOI:10.7554/eLife.93830.1), we would like to clarify that our research on dimeric ^R25CPTH(1-34) was conducted first. Initially, we confirmed dimerization under in vitro conditions and observed its effects in a mouse model. Recognizing the need for additional animal models, we collaborated with Shin et al.'s team. Due to delays during the submission process, our paper was submitted later, which seems to have led to this misunderstanding. However, Shin et al. (2024) cited our pre-print article on bioRxiv (Noh, M., Che, X., Jin, X., Lee, D. K., Kim, H. J., Park, D. R., ... & Lee, S. (2024). Dimeric R25CPTH (1-34) Activates the Parathyroid Hormone-1 Receptor in vitro and Stimulates Bone Formation in Osteoporotic Female Mice. bioRxiv, 2024-03.DOI: 10.1101/2024.03.13.584815). Both Shin et al., and our mouse work supports the action of dimeric R25CPTH(1-34) on regulating bone metabolism.

• The authors should clarify why they tested the effects of dimeric ^R25CPTH(1-34) and not dimeric ^R25CPTH(1-84)?

Thank you for your valid comments. Here are several reasons why we used the 1-34 fragment peptide in our experiment. Currently, PTH analog peptides for medical purposes include human parathyroid hormone fragment 1-34 (PTH(1-34)) and full-length recombinant human parathyroid hormone (rhPTH(1-84)). PTH(1-34) is used as a bone anabolic agent, while rhPTH(1-84) is used for PTH replacement therapy in hypoparathyroid patients with hypocalcemia. We aimed to compare the bone formation effects of R25CPTH with wild-type PTH, for which PTH(1-34) was deemed more appropriate. Additionally, previous studies have shown that both PTH(1-34) and PTH(1-84) possess equal ligand binding affinity for the PTH1 receptor. Key sites within the first 34 N-terminal amino acids of PTH are critical for high-affinity interactions and receptor activation. Alterations in the N-terminal sequence of PTH(1-84) significantly reduce receptor binding, while truncations at the C-terminal end do not affect receptor affinity. The peptide used in our experiment was synthetic, and if the length does not affect affinity to its receptor affinity, the shorter length of PTH(1-34) made its synthesis more reasonable. Consequently, we tested the effects of PTH(1-34) and dimeric R25CPTH(1-34) due to its known efficacy on bone anabolic effect and relevance in receptor interactions. However, we aim to conduct functional analysis of the dimeric R25CPTH(1-84) in further study.

• The study is descriptive with no mechanism.

We recognize that your concern is legitimate. While our study includes descriptive elements, it extends beyond mere observation. The R25CPTH research, which began with a case report, has evolved to utilize molecular techniques to better understand the unique physiological phenomena observed in patients. We have validated the peptide’s dimerization caused by mutations in vitro and assessed their effects in both in vitro cell line models and in vivo mouse models. Although we have not yet confirmed whether ^R25CPTH exists as a dimer or monomer in patient blood, we anticipate it may exist in dimeric form at least some fractions and are currently conducting mass spectrometry on patient blood samples to determine this. Therefore, this paper serves as the first report on this PTH mutant suggesting that it may form a homodimer. Importantly, we are actively investigating the molecular mechanisms and downstream signaling pathways that differentiate normal PTH from dimeric ^R25CPTH. This includes analyzing differences in proteome and transcriptome induced by PTH and dimeric ^R25CPTH and examining the direct molecular characteristics and structural changes responsible for these mutations. Through this comprehensive approach, we aim to provide a detailed mechanistic understanding of ^R25CPTH in the subsequent publication.

(2) Statistics are inadequately described or performed for the experimental design:

• The statistical analysis in Figure 5 needs to be written in a way that makes it clearer how statistics were done; t-test or one-way ANOVA?

Sorry for the inconvenience and thank you for your thorough review. Initially, we conducted the statistical analysis using a t-test. However, during the revision process, we performed a new statistical analysis using one-way ANOVA, as it is more appropriate for comparing the means among three or more groups. Despite this change, there were no differences in statistical significance, so the descriptions remained unchanged.

• Statistics in Figures 6 and 7 should be performed by one-way ANOVA to compare the mean values of one variable among three or more groups, and not t-test.

Thank you for your thorough review, and I apologize for any inconvenience. I agree with your suggestion that ANOVA is more appropriate than the t-test for comparing means among three or more groups. Accordingly, we performed new statistical analyses using one-way ANOVA. When we changed the statistical method from t-test to one-way ANOVA, the significance of bone formation markers, P1NP and ALP, appeared only in dimeric R25CPTH and not in wild-type PTH. We have reflected these findings in the text.

(3) Misleading and confused discussion:

• The first paragraph lacks clarity in the PTH nomenclature and the authors should provide a clear statement that the PTH mutant found in patients is likely a monomeric R25CPTH(1-84), considering that there has been no proof of a dimeric form.

Thank you for your insightful comments. I agree that there was some ambiguity in the nomenclature used in the first paragraph of the Discussion section. However, we do not believe that no proof of a dimeric form of the ^R25CPTH(1-84) mutant necessarily indicates that the PTH mutant in the blood is solely monomeric. Identifying the in vivo structure of ^R25CPTH(1-84) is one of the goals of our ongoing project. While the exact form of ^R25CPTH(1-84) in patients is still elusive, we are investigating the possibility that some fraction may exist as a dimer. On page 12, line 274-276, we have revised the content to address this issue and improve clarity as follows.

- ‘In this study, we show the introduction of a cysteine mutation at the 25th amino acid position of mature parathyroid hormone (^R25CPTH) facilitates the formation of homodimers comprised of the resulting dimeric R25CPTH peptide in vitro.’

• Moreover, the authors should discuss the study by White et al. (PNAS 2019), which shows that there are defective PTH1R signaling responses to monomeric R25CPTH(1-34). This results in faster ligand dissociation, rapid receptor recycling, a short cAMP time course, and a loss of calcium ion allosteric effect.

Sorry for the inconvenience and thank you for your thorough review. The authors were aware of the referenced paper and deeply apologize for its omission during the writing and editing process. Citing this paper will enhance the credibility of our findings. We have now included this citation and made the necessary adjustments to the manuscript of Discussion section on page 12, line 295-296 as follows.

- ‘We also observed that the potency of cAMP production in cells was lower for dimeric ^R25CPTH as compared to the monomeric ^R25CPTH, 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 ^R25CPTH, 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 ^R25CPTH suggest a possibility that the formation of a disulfide bond at the 25th position significantly alters the function of PTH as a PTH1R ligand. These structural effects are not yet fully understood and need to be investigated further.’

• The authors should also clarify what they mean by "the dimeric form of R25CPTH can serve as a new peptide ...(lines 328-329)" The dimeric R25CPTH(1-34) induces similar bone anabolic effects and calcemic responses to PTH(1-34), so it is unclear what the new benefit of the dimeric PTH is.

We apologize for any confusion in our previous description. We concur that, as you mentioned, PTH and dimeric ^R25CPTH indeed exhibit similar activities. We have toned-down our statement, however, there are still some elements showing statistical significance that need to be clearly stated. Specifically, when we changed the statistical method from t-test to one-way ANOVA, the significance of bone formation markers was only observed in dimeric PTH treated samples, and we have revised the manuscript of Results section on page 9, line 206-212 as follows to reflect the change.

- ‘These analyses revealed that both PTH(1-34) and dimeric ^R25CPTH(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 ^R25CPTH(1-34) to induce new bone formation in vivo, similar to PTH, despite molecular and structural changes.’

Although it is unclear whether ^R25CPTH circulate as dimeric form or mutant monomeric form, the absence of bone resorption associated with long-term PTH exposure in the patients suggests the potential for a bone anabolic drug without side effects. Also, continued observation of the recently reported young patient in Denmark is expected to clarify this effect further. However, we acknowledge that our current data alone are insufficient to claim that ^R25CPTH may be a more effective anabolic therapy than wild type PTH, and we have adjusted our tone accordingly.

Thank you for Reviewer #2’s comprehensive and considerate review. We are grateful for the ideas, and we have revised our manuscript accordingly them to improve our paper.

Reviewer #1 (Recommendations For The Authors):

(1) Figure 1D lacks molecular weight markers.

Thank you for your thorough review. We added protein molecular weight markers in the figure.

(2) The lack of change in plasma cAMP is very surprising, particularly given that there is no difference in the effect of the two forms of PTH on serum calcium or phosphate, or urinary phosphate. This data is somewhat of a distraction since no effort has been made to assess the difference in the effects of these PTH forms on kidney function. I suggest removing this data and spending time working on the origin of this difference.

Thank you for your insightful comments and valuable suggestions on our manuscript. We also could not precisely explain the discrepancy between the cell line and animal model experiments. However, since the results were consistently observed, we included them in the paper as they may be significant. We acknowledge that in the context of our current research, these data lack sufficient correlation with other findings. Therefore, we have removed the data about the lack of change in plasma cAMP by PTH injection (Figure 4. Effect of cAMP production by PTH injection in CD1 female mice) and revised the manuscript accordingly (Page 8, line 188-194; page 12, line 301-306; page 19, line 454-456). We are currently conducting further research with multiomics data analysis to elucidate potential differences in the sub-signaling pathways between PTH and dimeric R25CPTH, to identify the specific functions affected by these variations, and to understand the underlying mechanisms. The lack of changes in plasma cAMP levels in vivo will be addressed in a subsequent publication detailing our findings.

(3) Introduction, line 61. The authors state that "most" anti-resorptive therapies cannot stimulate new bone formation. I don't believe that ANY anti-resorptive therapies stimulate new bone formation! If there is one, this should be referenced.

Thank you for pointing out important aspects. Romosozumab, a humanized monoclonal anti-sclerostin antibody, has a dual effect by enhancing bone formation and inhibiting bone resorption. Sclerostin, a protein produced by osteocytes, plays a role in the regulation of bone metabolism. It promotes osteoclast differentiation, which is associated with bone resorption, and suppresses osteoblast activity, which is crucial for bone formation. By binding to sclerostin, Romosozumab prevents it from blocking the signaling pathways necessary for osteogenesis. Consequently, Romosozumab therapy not only regulates bone resorption but also affects new bone formation. We added the references to that information.

(4) The authors tend to include a lot of methods in the results section (e.g. describing the number of replicates, and details of histological analysis). This should be minimized.

Thank you for your thorough review, and sorry for the inconvenience. We have minimized the methodological details in the results section, ensuring that only essential information for understanding the findings and the procedures remain.

(5) Lines 302-305: If retaining the blood cAMP data, please provide references for the assertion that renal PTH receptors mediate this response.

PTH exerts its effects primarily through the PTH1 receptor (PTH1R), a G protein-coupled receptor present in various tissues, including bone and kidney (Chase et al., 1968, Chase et al., 1970). When activated by PTH, this receptor stimulates the production of cyclic AMP (cAMP), with the kidneys playing a significant role in this process (Maeda et al., 2013). In the initial manuscript, the importance of renal PTH receptors in mediating the blood cAMP response may have been overemphasized. We appreciate your feedback on this point, and we have provided references to support this assertion. However, by process following the former ‘Recommendations for the Authors’, we removed the data about the lack of change in plasma cAMP by PTH injection, the description of the renal PTH receptors mediate this response of blood cAMP also removed.

- Chase, Lewis R., and G. D. Aurbach. "Renal adenyl cyclase: anatomically separate sites for parathyroid hormone and vasopressin." Science 159.3814 (1968): 545-547.DOI:10.1126/science.159.3814.545

- Chase, Lewis R., and G. D. Aurbach. "The effect of parathyroid hormone on the concentration of adenosine 3', 5'-monophosphate in skeletal tissue in vitro." Journal of Biological Chemistry 245.7 (1970): 1520-1526.DOI:10.1016/S0021-9258(19)77126-9

- Maeda, Akira, et al. "Critical role of parathyroid hormone (PTH) receptor-1 phosphorylation in regulating acute responses to PTH." Proceedings of the National Academy of Sciences 110.15 (2013): 5864-5869.DOI: 10.1073/pnas.1301674110

(6) Eosin stains bone pink and haematoxylin stains cells purple. This has been incorrectly described in the manuscript.

Thank you for your thorough review, and I apologize for any confusion caused by the poor description. It appears that the terms were used interchangeably during the editing process. We have corrected the description in the manuscript and will ensure such mistakes do not occur again in the future.

(7) Sodium thiosulphate is a fixative for Von Kossa staining, not an agent that removes nonspecific binding.

Thank you for your careful review. However, there seems to be a misunderstanding of sodium formaldehyde as sodium thiosulfate. A 5% sodium thiosulfate solution is a critical in vitro diagnostic agent used in various staining kits. As a reducing agent, it effectively removes excess silver ions in staining kits based on silver impregnation techniques. In our experiment, sodium thiosulfate was specifically used to remove residual silver ions in Von Kossa staining. For more details, please refer to the following link: https://www.morphisto.de/en/shop/detail/d/Natriumthiosulfat_5//12825/.

Reviewer #2 (Recommendations For The Authors):

Moderate-to-Minor points:

• Line 73: it's either class B GPCR or secretin receptor family but not class B GPCR family.

Thank you for your thorough review, and I apologize for any confusion in our previous description. We corrected the description in the manuscript as class B GPCR.

• Line 79: correct "adenylate cyclase" to "transmembrane adenylate cyclases"

Thank you for your thorough review, and I apologize for any confusion in our previous description. We corrected the description in the manuscript as transmembrane adenylate cyclases.

• Line 89: should "hypothyroidism" be "hypoparathyroidism"?

Thank you for your thorough review, and I apologize for any confusion in our previous description. We corrected the description in the manuscript as hypoparathyroidism.

• Line 159: all agonists display higher binding affinities when their receptors are coupled to G proteins, so it's unclear why the higher affinity of the dimeric ^R25CPTH(1-34) for the RG state seems to be important for the authors.

Thank you for your insightful comments. First of all, comparing the binding affinities of the R0 (G protein-uncoupled) and RG (G protein-coupled) conformations of the receptor is inappropriate. This is because the form and size of the radio-label ligand bound to each conformation differ, which consequently affects their binding affinities and, in turn, influences the binding strength of target ligands such as PTH, monomeric ^R25CPTH, and dimeric ^R25CPTH. Therefore, it is preferable to compare how the binding strengths of test ligands differ for each conformation. Additionally, the fact that significant binding affinity is lost for R⁰ while remaining high for the RG conformation of PTH1R is important because typical PTH exhibits high binding affinity for R0, whereas PTHrP shows higher affinity for the RG conformation. This suggests that dimeric ^R25CPTH may possess distinct molecular characteristics and potentially induce different downstream signaling pathways compared to typical PTH.

• Line 169-170 and Fig. 2: According to the theory of receptor pharmacology established in the 60s' for native receptors (Arch. Int. Pharmacodyn. 127:459-478 (1960); Arch. Int. Pharmacodyn. 136:385-413 (1962)) and verified later in the 80-90's for recombinant GPCRs, the activity constant (Kact or EC50) value of hormone actions in various tissues or cells is equal to the dissociation constant (Kd) of the hormone when receptors are not overexpressed (EC50 = Kd). When receptors are overexpressed (presence of spare receptors), then EC50 < Kd. Assuming that after Cheng-Prussof correction for data in Fig. 2, IC50 < Ki = Kd, how do the authors explain that IC50 values for RG are about 1-Log lower than EC50s (i.e., EC50 > Kd)?

We appreciate your insightful comment and fully acknowledge the established theory of receptor pharmacology, which states that Kd equals EC50, and when the receptor is overexpressed, EC50 is less than Kd. After having read your comments, we have revisited this paper Okazaki et al, PNAS, 2008 to better understand the PTH interaction with PTH1R. While our data might appear to contradict this theory, we believe that a direct comparison between the IC50 of RG and the EC50 in Figure 2 may not be entirely appropriate for the following reasons. First, the IC50 was determined from membrane preparations of a receptor-overexpressing cell line (GP-2.3), whereas the EC50 was calculated based on the cAMP response in SaOS-2 cells. These different experimental conditions contribute to the observed discrepancies. Second, the peptides used in the competition assays differ. R⁰ utilized radiolabeled PTH(1-34), while RG employed M-PTH(1-15) with several amino acid substitutions and a shorter length. This further complicates a direct comparison between the EC50 and IC50 values in our study.

Thank you for all the reviewers’ thorough and thoughtful reviews. We greatly appreciate your suggestions and have addressed all the issues to enhance the quality of our paper.