Low, but Not High, Pulsating Fluid Shear Stress Affects Matrix Extracellular Phosphoglycoprotein Expression, Mainly via Integrin β Subunits in Pre-Osteoblasts
Round 1
Reviewer 1 Report
Comments and Suggestions for Authors
The publication reveals compelling results about the impact of pulsating fluid shear stress (PFSS) on Mepe expression in pre-osteoblasts. However, some sections need significant enhancements to elevate the manuscript's overall quality and influence. Here are particular recommendations for modification.
Major comments
Abstract and Introduction:
- The abstract must deliver a more lucid explanation of the outstanding results.
- Consider summarizing the information to emphasize the principal results and their importance.
- The introduction would be enhanced by a more thorough literature survey. Incorporate latest research examining the function of Mepe and integrins in mechanotransduction and osteogenesis. This context will enhance the justification for your research.
Materials and Methods:
- The techniques section must involve more comprehensive descriptions of the experimental processes.
- Elucidate the reasoning for the selection of particular PFSS magnitudes and the importance of the chosen treatment period.
- Elaborate on the employed statistical methodologies. Elucidate the methodology employed to ascertain the sample size and indicate if a power analysis was performed.
Discussion:
- The discussion should more effectively incorporate your results with the previous literature. Compare your findings with those of previous research, particularly with the varying impacts of low and high PFSS.
- If possible, elucidate the exact mechanisms by which PFSS affects integrin expression and the ensuing cellular signaling pathways. This would clarify the biological significance of your findings.
Conclusion:
- The conclusion should highlight the wider divergences of your findings for bone health and disease. Examine how comprehending the mechanotransduction pathways may guide treatment approaches.
Minor comments
Grammatical Structure and Syntax, for example see the below:
1 – ''The reaction of pre-osteoblasts is linearly correlated with the rate of PFSS.'' Should be ''The reaction of pre-osteoblasts is directly proportional to the rate of PFSS.''
2- ''The role of PFSS magnitude in regulating Mepe expression through the modulation of integrin α and/or β subunit expression in pre-osteoblasts remains uncertain.'' Should be '' The extent to which PFSS influences Mepe expression through the modulation of integrin α and/or β subunit expression in pre-osteoblasts is uncertain.''
Terminology Consistency: Maintain uniformity in terminology throughout the paper, especially when referencing particular proteins or signaling pathways.
1- Inconsistent: The term "pulsating fluid shear stress" may be abbreviated as "PFSS" in certain instances and omitted in others.
2- The term "matrix extracellular phosphoglycoprotein" is referred to as "Mepe" in certain places and as "matrix extracellular protein" in others. Maintain consistency by referring to "matrix extracellular phosphoglycoprotein" (Mepe) throughout the manuscript, using "Mepe" thereafter following its initial reference.
3- Inconsistent: Shear stress may be referred to as "Pa" in one case and "pascals" in another. Consistency: Select a singular format for measuring units and apply it uniformly (e.g., consistently using "Pa" for pascals).
Author Response
The publication reveals compelling results about the impact of pulsating fluid shear stress (PFSS) on Mepe expression in pre-osteoblasts. However, some sections need significant enhancements to elevate the manuscript's overall quality and influence. Here are particular recommendations for modification.
Response
Thank you for your generous assessment of our manuscript.
Comment 1
Abstract and Introduction:
- The abstract must deliver a more lucid explanation of the outstanding results.
Response
We have rewritten the abstract to provide a more lucid explanation of the results as follows:
“Matrix extracellular phosphoglycoprotein (Mepe), present in bone and dentin, plays important multifunctional roles in cell signaling, bone mineralization, and phosphate homeostasis. Mepe expression in bone cells changes in response to pulsating fluid shear stress (PFSS), which is transmitted into cells through integrin-based adhesion sites, i.e., α and β subunits. Whether and to which extent PFSS influences Mepe expression through the modulation of integrin α and/or β subunit expression in pre-osteoblasts is uncertain. Therefore, we aimed to test whether low and/or high PFSS affects Mepe expression via modulation of integrin α and/or β subunit expression. MC3T3-E1 pre-osteoblasts were treated ± 1 h PFSS (magnitude: 0.3 Pa (low PFSS) or 0.7 Pa (high PFSS); frequency: 1 Hz). Single integrin fluorescence intensity in pre-osteoblasts was increased, but single integrin area was decreased by low and high PFSS. Expression of two integrin α subunit-related genes (Itga1 and Itga5 2) was increased by low PFSS, and one (Itga5 2) by high PFSS. Expression of five integrin β subunit genes (Itgb1, Itgb3, Itgb5, Itgb5 13, and Itgb5 123) was increased by low PFSS, and three (Itgb5, Itgb5 13, and Itgb5 123) by high PFSS. Interestingly, Mepe expression in pre-osteoblasts was only modulated by low, but not high PFSS. In conclusion, both low and high PFSS affected integrin α and β subunit expression in pre-osteoblasts, while integrin β subunit expression was more altered by low PFSS. Importantly, Mepe gene expression was only affected by low PFSS. These results might explain the different way how Mepe-induced changes in pre-osteoblast mechanosensitivity may drive signaling pathways of bone cell function at low or high impact loading. This finding might have physiological and biomedical implications, which specifically requires future research addressing the precise role of integrin α or β subunits and Mepe during dynamic loading in bone health and disease.” (Page 1, line 10-30)
- Consider summarizing the information to emphasize the principal results and their importance.
Response
We now summarized and emphasized the principal results and their importance in the abstract as follows:
“In conclusion, both low and high PFSS affected integrin α and β subunit expression in pre-osteoblasts, while integrin β subunit was more altered by low PFSS. Importantly, Mepe gene expression was only affected by low PFSS. These results might explain the different way how Mepe-induced changes in pre-osteoblast mechanosensitivity may drive signaling pathways of bone cell function at low or high impact loading. This finding might have physiological and biomedical implications, which specifically requires future research addressing the precise role of integrin α or β subunits and Mepe during dynamic loading in bone health and disease.” (Page 1, line 23-30)
- The introduction would be enhanced by a more thorough literature survey. Incorporate latest research examining the function of Mepe and integrins in mechanotransduction and osteogenesis. This context will enhance the justification for your research.
Response
We now included the latest literature on the function of Mepe and integrins in mechanotransduction and osteogenesis as follows:
“This leads to the next question: What is the relationship between Mepe and mechanical loading? Four-point bending has been shown to increase Mepe gene expression in tibia of female Wistar rats, which might regulate bone mineralization and phosphate homeostasis [14].”(Page 2, line 53-56)
And
“Integrins, as bridges, mediate mechanical signals between the ECM and the cytoskeleton via focal adhesion kinases, are able to sense matrix features, e.g. texture, stiffness, and external forces, and transfer these signals into biochemical or biological signals [27], [28]. This transduction process is called “mechanical signal transduction” [27]). When integrins are active, they recruit different integrin-associated proteins (IAP) near the cell membrane, e.g. talin, vinculin, and kindlin. The IAPs enhance the number of specific cell surface receptors which have a similar function with integrins to amplify the effect, such as opening of mechanically-gated channels (e.g. PIEZO1 channels) [29], [30]. IAPs convert the signal received by integrins to the cytoskeleton (e.g. actin filaments) which undergo contraction, changing the tension of the phospholipid bilayer on the membrane, thereby leading to the opening of mechanically-gated channels [27], [29], [30] (Page 2, line 79-890
And
“… regulating osteogenesis [31], [32]. MC3T3-E1 pre-osteoblast proliferation is decreased when the integrin gene is knocked out. Furthermore, integrins (e.g. β1 integrin) increase osteogenic differentiation and ECM production by mechanically loaded osteoblasts, indicating that integrins are important for ECM integrity, biomechanical properties, and bone quality in vivo [33].” (Page 2, line 93-97)
References
[14] A. K. Nepal, H. W. van Essen, C. M. A. Reijnders, P. Lips, and N. Bravenboer, “Mechanical loading modulates phosphate related genes in rat bone,” PLoS One, vol. 18, no. 3, pp. e0282678–e0282689, Mar. 2023.
[27] S. Ding, Y. Chen, C. Huang, L. Song, Z. Liang, and B. Wei, “Perception and response of skeleton to mechanical stress,” Phys. Life Rev., vol. 49, pp. 77–94, Jul. 2024.
[28] T. Iskratsch, H. Wolfenson, and M. P. Sheetz, “Appreciating force and shape-the rise of mechanotransduction in cell biology,” Nat. Rev. Mol. Cell Biol., vol. 15, no. 12, pp. 825–833, 2014.
[29] D. Cheng, Z. Zhou, J. Li, M. Yao, and C. D. Cox, “PIEZO1 channels as regulators of integrin-mediated focal adhesions in cardiac fibroblasts,” Biophys. J., vol. 123, no. 3, p. 68a, Feb. 2024.
[30] D. Cheng, J. Wang, M. Yao, and C. D. Cox, “Joining forces: Crosstalk between mechanosensitive PIEZO1 ion channels and integrin-mediated focal adhesions,” Biochem. Soc. Trans., vol. 51, no. 5, pp. 1897–1906, Oct. 2023.
[31] H. Pommerenke et al., “The mode of mechanical integrin stressing controls intracellular signaling in osteoblasts,” J. Bone Miner. Res., vol. 17, no. 4, pp. 603–611, Apr. 2002.
[32] L. Yang et al., “Research progress on the regulatory mechanism of integrin‐mediated mechanical stress in cells involved in bone metabolism,” J. Cell. Mol. Med., vol. 28, no. 7, pp. e18183–e18199, Apr. 2024.
[33] L. Qin et al., “Osteocyte β1 integrin loss causes low bone mass and impairs bone mechanotransduction in mice,” J. Orthop. Transl., vol. 34, pp. 60–72, 2022.
Comment 2
Materials and Methods
- The techniques section must involve more comprehensive descriptions of the experimental processes.
Response
We now provide more comprehensive descriptions of the experimental processes as follows:
“One day before mechanical loading by PFSS, MC3T3-E1 pre-osteoblasts were seeded at 1 × 103 cells/cm2 (small glass slide: 24 × 24 × 0.15 mm (length × width × height)) or 3 × 103 cells/cm2 (big glass slide: 36 × 76 × 1 mm (length × width × height)) on poly-L-lysine-coated (50 μg/ml; poly-L-lysine hydrobromide; Sigma-Aldrich) small and big glass slides. A “small chamber” (14 × 14 × 0.2 mm (length × width × height); inner dimension) was used for integrin observation by confocal microscopy. A “big chamber” (58 × 32 × 0.3 mm (length × width × height); inner dimension) was used for observing cell morphology, and measuring gene expression. In both chambers, cells were treated with low PFSS (magnitude: 0.3 Pa) or high PFSS (magnitude: 0.7 Pa) at 1 Hz for 1 h at 37°C. The PFSS magnitude was calculated as follows [38]:
τ=6.Q.µ / (b.h2)
τ: shear stress magnitude; Q: flow rate (low PFSS: 7 ml/min; high PFSS: 20 ml/min); µ: viscosity (0.0078 dynes.sec/cm2); b: slit width; h: channel height.”
Static control cultures were kept in a Petridish under similar conditions as experimental cultures, i.e., α-MEM with 10% FBS, 300 μg/ml penicillin, 250 μg/ml streptomycin, and 1.25 μg/ml fungizone, as well as 1 h incubation at 37°C. (Page 4, line 136-151)
- Elucidate the reasoning for the selection of particular PFSS magnitudes and the importance of the chosen treatment period.
Response
We have now mentioned why we have applied PFSS of certain magnitudes and duration of application as follows:
“Pre-osteoblasts were treated during 1 h with 0.3 Pa (low PFSS) or 0.7 Pa (high PFSS), at 1 Hz frequency. Both low and high PFSS were used to treat the cells, since we have found earlier that the response of pre-osteoblasts is linearly dependent on the rate of PFSS, which depends on the amplitude and frequency of stress [39], [40]. High PFSS affects cell body volume, nuclear volume, and paxillin expression in pre-osteoblasts [41]. Moreover, low PFSS affects cytoskeleton-related gene expression in these cells (unpublished data). Moreover, 1 h PFSS was applied since this is long enough to allow maximal activation of signaling molecule production, such as nitric oxide and prostaglandins, which are known parameters for bone cell activation as they are early mediators of mechanical loading-induced bone formation [38], [41].” (Page 8, line 287-296)
References
[38] A. D. Bakker, K. Soejima, J. Klein-Nulend, and E. H. Burger, “The production of nitric oxide and prostaglandin E2 by primary bone cells is shear stress dependent,” J. Biomech., vol. 34, no. 5, pp. 671–677, May 2001.
[39] R. G. Bacabac, T. H. Smit, M. G. Mullender, S. J. Dijcks, J. J. W. . Van Loon, and J. Klein-Nulend, “Nitric oxide production by bone cells is fluid shear stress rate dependent,” Biochem. Biophys. Res. Commun., vol. 315, no. 4, pp. 823–829, Mar. 2004.
[40] R. G. Bacabac et al., “Dynamic shear stress in parallel-plate flow chambers,” J. Biomech., vol. 38, no. 1, pp. 159–167, Jan. 2005.
[41] J. Jin, R. T. Jaspers, G. Wu, J. A. M. Korfage, J. Klein-Nulend, and A. D. Bakker, “Shear stress modulates osteoblast cell and nucleus morphology and volume,” Int. J. Mol. Sci., vol. 21, no. 21, pp. 1–22, Nov. 2020.
- Elaborate on the employed statistical methodologies. Elucidate the methodology employed to ascertain the sample size and indicate if a power analysis was performed.
Response
We elaborated on the employed statistical methodologies as follows:
“… (cell morphology: 3 independent experiments with 9 glass slides (n=3); integrin structure: 3 independent experiments with 9 glass slides (n=3); integrin number, fluoresence, and area: 15 pictures from 3 independent experiments with 9 glass slides (n=3); gene expression: 4 independent experiments with 12 glass slides (n=4).” (Page 5, line 217-220)
And
“… (experimental design: no matching or pairing; assume gaussian distribution of residuals: yes, use ANOVA; assume equal SDs: yes, use ordinary ANOVA test; no repeated measures; multiple comparisons (follow up tests): compare the mean of each column with the mean of every other column).” (Page 5, line 221-224)
Sample size: In general, an n=3-4 (per group; as in our study) is acceptable for in vitro studies, as our study. A The total sample size in our study was 9 and 12 (3 groups (n=3-4/group): 1 control, 2 experimental groups). A G*Power calculated sample size is never used to predict the sample size in in-vitro studies (but it is for the number of patients/animals to be included in clinical/animal studies).
Comment 3
Discussion
- The discussion should more effectively incorporate your results with the previous literature. Compare your findings with those of previous research, particularly with the varying impacts of low and high PFSS.
Response
We have now included more discussion on the relationship between our findings and those from previous research as follows:
“Additionally, it has been shown that mouse long bone cells subjected to lower streaming potentials and chemotransport, but the same high shear stress, exhibit a similar response, as revealed by the release of nitric oxide production and prostaglandin E2 in the flow medium [38]. Moreover, nitric oxide production induced by high PFSS is accompanied by parallel alignment of actin stress fibers in osteoblasts [44]. Prostaglandin E2 induced by high PFSS is related to loading stimulation of focal adhesions which are formed after disrupting the cytoskeleton in osteoblasts, indicating that the cytoskeleton (or cell morphology) is related to the release of nitric oxide production and prostaglandin E2 induced by mechanical loading [44]. In this study, cell morphology was not affected by low and high PFSS, which is not consistent with the findings by McGarry et al. [44]. However, this study is consistent with our previous findings, showing that high PFSS does not affect filament actin and microtubules [26], [41].” (Page 9, line 316-328)
And
“Our this study investigated different types of integrins. The heterogeneity of integrin mechanical properties has been shown to determine the response of osteoblasts to mechanical loading [55]. Thus measurement of different integrin dimers is highly important for a better understanding of mechanotransduction dynamics [55]. Additionally, integrin α5β1 related gene expression is enhanced by dynamic loading [56]. However, in our study, we did not investigate the combined integrin α and β related genes under low or high PFSS. Therefore, future studies should also address other types of integrin and the combined integrin α and β without or with low or high PFSS.” (Page 10, line 380-385)
And
“Srinivasan et al. has shown that low-magnitude loading with 10 sec of rest between each load cycle significantly increases the osteogenic potential of the regimen [59]. This supports our findings that osteoblasts might be more sensitive to low PFSS.” (Page 11, line 415-418)
References
[26] J. Jin et al., “Pulsating fluid flow affects pre-osteoblast behavior and osteogenic differentiation through production of soluble factors,” Physiol. Rep., vol. 9, no. 12, pp. e14917–e14934, Jun. 2021.
[41] J. Jin, R. T. Jaspers, G. Wu, J. A. M. Korfage, J. Klein-Nulend, and A. D. Bakker, “Shear stress modulates osteoblast cell and nucleus morphology and volume,” Int. J. Mol. Sci., vol. 21, no. 21, pp. 1–22, Nov. 2020.
[38] A. D. Bakker, K. Soejima, J. Klein-Nulend, and E. H. Burger, “The production of nitric oxide and prostaglandin E2 by primary bone cells is shear stress dependent,” J. Biomech., vol. 34, no. 5, pp. 671–677, May 2001.
[44] J. G. McGarry, J. Klein-Nulend, and P. J. Prendergast, “The effect of cytoskeletal disruption on pulsatile fluid flow-induced nitric oxide and prostaglandin E2 release in osteocytes and osteoblasts,” Biochem. Biophys. Res. Commun., vol. 330, no. 1, pp. 341–348, 2005.
[55] A. Shuaib, D. Motan, P. Bhattacharya, A. McNabb, T. M. Skerry, and D. Lacroix, “Heterogeneity in the mechanical properties of integrins determines mechanotransduction dynamics in bone osteoblasts,” Sci. Rep., vol. 9, no. 1, pp. 13113–13126, Sep. 2019.
[56] T. Kurakawa et al., “Functional impact of integrin α5β1 on the homeostasis of intervertebral discs: A study of mechanotransduction pathways using a novel dynamic loading organ culture system,” Spine J., vol. 15, no. 3, pp. 417–426, 2015.
[59] S. Srinivasan, D. A. Weimer, S. C. Agans, S. D. Bain, and T. S. Gross, “Low-magnitude mechanical loading becomes osteogenic when rest is inserted between each load cycle,” J. Bone Miner. Res., vol. 17, no. 9, pp. 1613–1620, 2002.
- If possible, elucidate the exact mechanisms by which PFSS affects integrin expression and the ensuing cellular signaling pathways. This would clarify the biological significance of your findings.
Response
We now elucidate on the possible mechanisms by which PFSS affects integrin expression and ensuing signaling pathways as follows:
“A possible mechanism by which low and high PFSS differentially affect integrin α and β related gene expression might be related to the opening of ion channels (e.g. calcium, and PIEZO1) on the cell membrane. Integrins are located on the cell membrane as well. Since the magnitude of low and high PFSS is different, the cells will receive different mechanical signals possibly resulting in opening or closing of ion channels.” (Page 10, line 387-392)
And
“A possible mechanism relating Mepe gene expression to integrin α and β expression without/with low and high PFSS is provided by the focal adhesion kinases. The cells receive different mechanical signals, and might form different focal adhesion kinases which directly connect with integrin α and β, and then with extracellular matrix protein (e.g. Mepe). On the other side, the focal adhesion kinases directly or indirectly connect with the cytoskeleton and the nucleus, resulting in changes in cell function, e.g. Mepe gene or protein expression.” (Page 11, line 418-424)
Comment 4
Conclusion:
- The conclusion should highlight the wider divergences of your findings for bone health and disease. Examine how comprehending the mechanotransduction pathways may guide treatment approaches.
Response
Thank you for the remark. We included the following text to highlight the wider divergences of our findings:
“This finding might have physiological and biomedical implications, which specifically requires future research addressing the precise role of integrin α or β subunits and Mepe during dynamic loading in bone health and disease.” (Page 11, line 439-441)
Comment 5
Minor comments
Grammatical Structure and Syntax, for example see the below:
1 – ''The reaction of pre-osteoblasts is linearly correlated with the rate of PFSS.'' Should be ''The reaction of pre-osteoblasts is directly proportional to the rate of PFSS.''
Response
Thank you for your comment. However, the sentence “The reaction of pre-osteoblasts is linearly correlated with the rate of PFSS.” does not exist in our manuscript. You are probably referring to the following sentence: “The response of pre-osteoblasts is linearly dependent on the rate of PFSS.” We corrected this sentence as follows:
“The response of pre-osteoblasts is directly proportional to the rate of PFSS.” (Page 8, line 289-290)
2- ''The role of PFSS magnitude in regulating Mepe expression through the modulation of integrin α and/or β subunit expression in pre-osteoblasts remains uncertain.'' Should be '' The extent to which PFSS influences Mepe expression through the modulation of integrin α and/or β subunit expression in pre-osteoblasts is uncertain.''
Response
Thank you for your comment. However, the sentence ''The role of PFSS magnitude in regulating Mepe expression through the modulation of integrin α and/or β subunit expression in pre-osteoblasts remains uncertain.'' does not exist in our manuscript. You are probably referring to the following sentence: “Whether PFSS magnitude controls Mepe expression via modulation of integrin α and/or β subunit expression in pre-osteoblasts is unknown.” (abstract). We corrected this sentence as follows:
“Whether and to which extent PFSS influences Mepe expression through the modulation of integrin α and/or β subunit expression in pre-osteoblasts is uncertain.” (Page 1, line 13-15)
Terminology Consistency: Maintain uniformity in terminology throughout the paper, especially when referencing particular proteins or signaling pathways.
1- Inconsistent: The term "pulsating fluid shear stress" may be abbreviated as "PFSS" in certain instances and omitted in others.
Response
The term “pulsating fluid shear stress” is abbreviated as “PFSS” when “pulsating fluid shear stress” appears for the first time in the abstract (Page 1, line 12) as well as in the main body of the manuscript (Page 3, line 110).
2- The term "matrix extracellular phosphoglycoprotein" is referred to as "Mepe" in certain places and as "matrix extracellular protein" in others. Maintain consistency by referring to "matrix extracellular phosphoglycoprotein" (Mepe) throughout the manuscript, using "Mepe" thereafter following its initial reference.
Response
The term "matrix extracellular phosphoglycoprotein" is referred to as "Mepe" in this manuscript. However, the term “matrix extracellular proteins” appears 2 times in this manuscript, which does not refer to "matrix extracellular phosphoglycoprotein". The first time “extracellular matrix proteins” appears on Page 1 line 38, which refers to the general extracellular matrix proteins, including osteopontin, dentin matrix proteins, bone sialoprotein, dentin sialophosphoprotein, and matrix extracellular phosphoglycoprotein (Page 1, line 38-40). The second time “extracellular matrix proteins” appears on Page 3 line 103, which also refers to the general extracellular matrix proteins, such as osteopontin, fibronectin, fibrinogen, and vitronectin. (Page 3, line 103-104)
Additionally, the term “matrix extracellular phosphoglycoprotein” is abbreviated as “Mepe” when “matrix extracellular phosphoglycoprotein” appears for the first time in the abstract (Page 1, line 10) and in the main body of the manuscript (Page 1, line 35).
3- Inconsistent: Shear stress may be referred to as "Pa" in one case and "pascals" in another. Consistency: Select a singular format for measuring units and apply it uniformly (e.g., consistently using "Pa" for pascals).
Response
We fully agree with you that one should be consistent throughout a manuscript. We have been consistent and only used “Pa” throughout our manuscript, and not pascals.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsDear Authors,
I read your manuscript titled “Low But Not High Pulsating Fluid Shear Stress Affects Matrix Extracellular Phosphoglycoprotein Expression Mainly Via Integrin β Subunits in Pre-Osteoblasts”.
The manuscript is well-organized, and readable, but raises some questions about the connection between integrins and Mepe expression as well as the choice of the model system.
In my opinion, the abstract is too long, with some unclear parts such as the first sentence. Could you please rephrase the first sentence to improve clarity?
The introduction is a characteristic section for the authors to write what is the problem regarding the aim of the study by presenting objective evidence, then what are its biological mechanisms in general terms, and at the end the justification of the objective of the study.
The Introduction section is too long as well, indicating that the authors gathered a lot of information regarding the theme they were dealing with. Maybe it would be advisable to shorten the part explaining integrins. It is an important part of the Introduction, but I believe that the explanation given in the Discussion is more important.
The main question for me is the choice of model system. Could you please explain why you used MC3T3-E1 pre-osteoblasts? How did you decide for how long to treat the cells? Did you expect some changes in cell morphology?
Low and high PFSS increased the fluorescence intensity of integrins of importance but did not affect integrin area per cell. Could you explain that?
Did you expect that only low PFSS decreased Mepe expression in preosteoblasts? Could you please explain a possible mechanism? Is it via modulation integrin subunits expression in pre-osteoblast as you proposed, or you still don’t have a definite conclusion (as you said in limitations: we did not investigate the exact (mechanistic) relationship between integrin and Mepe expression in a bone cell under mechanical loading).
The aims at the end of the Introduction and the beginning of the Discussion are not quite the same. Could you please explain what was your major aim and did you accomplished it?
The expression of genes included in osteogenic differentiation didn’t change. Did you expect to change? It wasn’t enough time?
I don’t understand the sentence in Discussion: lines 341-342. I can’t find the facts in the manuscript to approve the sentence lines 341-342. Is it based on your previous work? Could you please explain that? Maybe the reference is missing? Is it just an assumption?
The conclusion is not separated from the Discussion. The conclusion section does not contain clear information to the readers.
It was necessary to set limitations. You very well noticed what are the major imperfections of the manuscript.
Best regards
Author Response
I read your manuscript titled “Low But Not High Pulsating Fluid Shear Stress Affects Matrix Extracellular Phosphoglycoprotein Expression Mainly Via Integrin β Subunits in Pre-Osteoblasts”. The manuscript is well-organized, and readable, but raises some questions about the connection between integrins and Mepe expression as well as the choice of the model system.
Response
Thank you for the thorough review of our manuscript.
Comment 1
In my opinion, the abstract is too long, with some unclear parts such as the first sentence. Could you please rephrase the first sentence to improve clarity?
Response
For clarity we now changed the first sentences:
“The secreted calcium binding matrix extracellular phosphoglycoprotein (Mepe) is a member of the small integrin binding ligand, N-linked glycoprotein (SIBLING) family of proteins in the extracellular matrix of bone and dentin. It plays important multifunctional roles in cell signaling, bone mineralization, and phosphate homeostasis.”
Into
“Matrix extracellular phosphoglycoprotein (Mepe), present in bone and dentin, plays important multifunctional roles in cell signaling, bone mineralization, and phosphate homeostasis.” (Page 1, line 10-11)
Comment 2
The introduction is a characteristic section for the authors to write what is the problem regarding the aim of the study by presenting objective evidence, then what are its biological mechanisms in general terms, and at the end the justification of the objective of the study.
Response
We fully agree with you. Firstly, the problem regarding the aim of the study by presenting objective evidence is described (“However, the exact role of Mepe in the regulation of mineralization and bone formation as well as the underlying mechanisms are still unclear.” Page 1, line 41-43; “However, the molecular mechanism by which mechanical forces are converted into a bone anabolic response, i.e., osteogenesis or bone formation, still needs to be further unraveled (e.g., force rate or magnitude) in (pre)osteoblasts.” Page 2, line 72-75; and “However, little is known about how Mepe expression is associated with changes in integrin α and/or β subunit expression in response to mechanical loading of different magnitude in bone cells. More specifically, whether the extent to which pulsating fluid shear stress (PFSS) influences Mepe expression through the modulation of integrin α and/or β subunit expression in osteoblasts is uncertain” Page 3, line 107-112). Then we introduced the possible biological information about mechanical forces, integrin α and β subunits. At the end the justification of the objective of the study is presented (“we aimed to test whether low and/or high PFSS affects Mepe expression via modulation of integrin α and/or β subunit expression in MC3T3-E1 pre-osteoblasts.” Page 3, line 113-114). In order to more fluently present the introduction, we added the following text:
“This leads to the next question: What is the relationship between Mepe and mechanical loading? Four-point bending has been shown to increase Mepe gene expression in tibia of female Wistar rats, which might regulate bone mineralization and phosphate homeostasis [14].” (Page 2, line 53-56)
And
“Integrins, as bridges, mediate mechanical signals between the ECM and the cytoskeleton via focal adhesion kinases, are able to sense matrix features, e.g. texture, stiffness, and external forces, and transfer these signals into biochemical or biological signals [27], [28]. This transduction process is called “mechanical signal transduction” [27]. When integrins are active, they recruit different integrin associated proteins (IAP) near the cell membrane, e.g. talin, vinculin, and kindlin. The IAPs enhance the number of specific cell surface receptors which have a similar function with integrins to amplify the effect, such as opening of mechanically-gated channels (e.g. PIEZO1 channels) [29], [30]. IAPs convert the signal received by integrins to the cytoskeleton (e.g. actin filaments) which undergo contraction, changing the tension of the phospholipid bilayer on the membrane, thereby leading to the opening of mechanically-gated channels [27], [29], [30].” (Page 2, line 79-90)
And
““… regulating osteogenesis [31], [32]. MC3T3-E1 pre-osteoblast proliferation is decreased when the integrin gene is knocked out. Furthermore, integrins (e.g. β1 integrin) increase osteogenic differentiation and ECM production by mechanically loaded osteoblasts , indicating that integrins are important for ECM integrity, biomechanical properties, , and bone quality in vivo [33].” (Page 2, line 93-97)
References
[14] A. K. Nepal, H. W. van Essen, C. M. A. Reijnders, P. Lips, and N. Bravenboer, “Mechanical loading modulates phosphate related genes in rat bone,” PLoS One, vol. 18, no. 3, pp. e0282678–e0282689, Mar. 2023.
[27] S. Ding, Y. Chen, C. Huang, L. Song, Z. Liang, and B. Wei, “Perception and response of skeleton to mechanical stress,” Phys. Life Rev., vol. 49, pp. 77–94, Jul. 2024.
[28] T. Iskratsch, H. Wolfenson, and M. P. Sheetz, “Appreciating force and shape-the rise of mechanotransduction in cell biology,” Nat. Rev. Mol. Cell Biol., vol. 15, no. 12, pp. 825–833, 2014.
[29] D. Cheng, Z. Zhou, J. Li, M. Yao, and C. D. Cox, “PIEZO1 channels as regulators of integrin-mediated focal adhesions in cardiac fibroblasts,” Biophys. J., vol. 123, no. 3, p. 68a, Feb. 2024.
[30] D. Cheng, J. Wang, M. Yao, and C. D. Cox, “Joining forces: Crosstalk between mechanosensitive PIEZO1 ion channels and integrin-mediated focal adhesions,” Biochem. Soc. Trans., vol. 51, no. 5, pp. 1897–1906, Oct. 2023.
[31] H. Pommerenke et al., “The mode of mechanical integrin stressing controls intracellular signaling in osteoblasts,” J. Bone Miner. Res., vol. 17, no. 4, pp. 603–611, Apr. 2002.
[32] L. Yang et al., “Research progress on the regulatory mechanism of integrin‐mediated mechanical stress in cells involved in bone metabolism,” J. Cell. Mol. Med., vol. 28, no. 7, pp. e18183–e18199, Apr. 2024.
[33] L. Qin et al., “Osteocyte β1 integrin loss causes low bone mass and impairs bone mechanotransduction in mice,” J. Orthop. Transl., vol. 34, pp. 60–72, 2022.
Comment 3
The Introduction section is too long as well, indicating that the authors gathered a lot of information regarding the theme they were dealing with. Maybe it would be advisable to shorten the part explaining integrins. It is an important part of the Introduction, but I believe that the explanation given in the Discussion is more important.
Response
We moved the following text from the introduction section into the discussion section:
“In mammals, each integrin is composed of an α and β subunit in a noncovalent complex. Eighteen α subunits and eight β subunits generate 24 unique heterodimeric transmembrane receptors, excluding spice- and glycosylation variants [53]. Each α and β subunit contains a short cytosolic tail, single-span helical transmembrane domain, and large ectodomain, except the β4 subunit [54]. Most α subunits only form one kind of complex with one β subunit. However, the α4 and αv subunits interact with more than one β subunit, e.g., α4β1, α4β7, αvβ1, αvβ3, αvβ5, αvβ6, and αvβ8 [35]. The β1 subunit can form heterodimeric complexes with 12 α subunits, but β4, β5, β6, and β8 only interact with one α subunit [35].” (Page 10, line 370-378)
References
[35] X. Pang et al., “Targeting integrin pathways: mechanisms and advances in therapy,” Signal Transduct. Target. Ther., vol. 8, no. 1, pp. s41392–s41433, Jan. 2023.
[53] R. O. Hynes, “Integrins,” Cell, vol. 110, no. 6, pp. 673–687, Sep. 2002.
[54] Y. Zheng and K. Leftheris, “Insights into protein–ligand interactions in integrin complexes: Advances in structure determinations,” J. Med. Chem., vol. 63, no. 11, pp. 5675–5696, Jun. 2020.
Comment 4
The main question for me is the choice of model system. Could you please explain why you used MC3T3-E1 pre-osteoblasts? How did you decide for how long to treat the cells? Did you expect some changes in cell morphology?
Response
Could you please explain why you used MC3T3-E1 pre-osteoblasts?
We used MC3T3-E1 pre-osteoblasts since this study follows our previous work published in the International Journal of Molecular Sciences and in Physiological Reports, where we used the same cell type and high PFSS. We did refer to these 2 publications (reference 25 and 26) as follows:
“Osteoblasts sense mechanical forces and translate them into biochemical signals to regulate cell shape and function via extracellular matrix, gap junctions, integrins, ion channels, focal adhesion kinases, and the cytoskeleton (e.g., actin filaments, microtubules, and intermediate filaments) [25], [26].” (Page 2, line 76-79)
And
“Mechanical force also induces substantial changes in osteoblast cell body and nucleus volume, which are accompanied by changes in integrin and paxillin [26].” (Page 2, line 90-91)
How did you decide for how long to treat the cells?
We added the following text into the discussion section:
“Moreover, 1 h PFSS was applied since this is long enough to allow maximal activation of signaling molecule production, such as nitric oxide and prostaglandins, which are known parameters for bone cell activation as they are early mediators of mechanical loading-induced bone formation [38], [41].” (Page 8, line 292-296)
And
“Additionally, alterations in osteoblast cytoskeletal structure in response to shear stress occur within minutes [26], [40],[43], [44]. Therefore, we have chosen 1 h PFF as an end point for our investigations.” (Page 8, line 302; Page 9, line 303-304)
References
[26] J. Jin et al., “Pulsating fluid flow affects pre-osteoblast behavior and osteogenic differentiation through production of soluble factors,” Physiol. Rep., vol. 9, no. 12, pp. e14917–e14934, Jun. 2021.
[40] J. Jin, R. T. Jaspers, G. Wu, J. A. M. Korfage, J. Klein-Nulend, and A. D. Bakker, “Shear stress modulates osteoblast cell and nucleus morphology and volume,” Int. J. Mol. Sci., vol. 21, no. 21, pp. 1–22, Nov. 2020.
[43] J. D. Gardinier, S. Majumdar, R. L. Duncan, and L. Wang, “Cyclic hydraulic pressure and fluid flow differentially modulate cytoskeleton re-organization in MC3T3 osteoblasts,” Cell. Mol. Bioeng., vol. 2, no. 1, pp. 133–143, Mar. 2009.
[44] J. G. McGarry, J. Klein-Nulend, and P. J. Prendergast, “The effect of cytoskeletal disruption on pulsatile fluid flow-induced nitric oxide and prostaglandin E2 release in osteocytes and osteoblasts,” Biochem. Biophys. Res. Commun., vol. 330, no. 1, pp. 341–348, 2005.
Did you expect some changes in cell morphology?
The answer is no, we did not expect changes in cell morphology. However, we observed changes in integrins. Earlier we published cell morphology data after mechanical loading in Physiological Reports [26] and in the International Journal of Molecular Sciences [41], showing that MC3T3-E1 pre-osteoblast morphology is not affected by 1 h pulsating fluid shear stress. We added the following text to the discussion section:
“The observed morphology of pre-osteoblasts seeded on glass slides indicated that the cells spread well. One hour low or high PFSS did not change the cell morphology as observed from a top view using light microscopy at the cell (or micro) scale. These bone cells sense and transmit (called mechanotransduction) [41] the physical signals to the inside of the cells, and to adjacent cells. This process of mechanotransduction in bone cells might change the cell structure (at the nanoscale), but not the cell morphology (at the microscale). This data in bone cells is different with earlier observations in muscle cells by Haroon et al., where PFSS did modulate the morphology and number of muscle stem cells [45]. This difference in results between the current study and the study by Haroon et al. lays in the type of cell, pre-osteoblasts and muscle cells. Pre-osteoblasts are much more flat than muscle stem cells, and adhere more strongly to the glass slide. As a result, changes in cell morphology and number might depend on cell type.” (Page 9, line 305-316)
References
[26] J. Jin et al., “Pulsating fluid flow affects pre-osteoblast behavior and osteogenic differentiation through production of soluble factors,” Physiol. Rep., vol. 9, no. 12, pp. e14917–e14934, Jun. 2021
[41] J. Jin, R. T. Jaspers, G. Wu, J. A. M. Korfage, J. Klein-Nulend, and A. D. Bakker, “Shear stress modulates osteoblast cell and nucleus morphology and volume,” Int. J. Mol. Sci., vol. 21, no. 21, pp. 1–22, Nov. 2020.
[45] M. Haroon et al., “Reduced growth rate of aged muscle stem cells is associated with impaired mechanosensitivity,” Aging (Albany. NY)., vol. 14, no. 1, pp. 28–53, Jan. 2022.
Comment 5
Low and high PFSS increased the fluorescence intensity of integrins of importance but did not affect integrin area per cell. Could you explain that?
Response
We now added the following explanation for this observation:
“Previously we have shown that high PFSS increases integrin fluorescence, number, area, and size in MC3T3-E1 pre-osteoblasts [41]. In the current study, low and high PFSS increased integrin fluorescence per integrin, decreased single integrin area, but did not affect integrin number. This might be explained by a difference in integrin staining intensity. In our previous study, we used α5 rat monoclonal IgG-2a (Abcam) for integrin staining, while this study used rabbit recombinant monoclonal integrin α5 antibody (Abcam), since α5 rat monoclonal IgG-2a could not be purchased anymore. Confocal microscopy revealed that there was no significant difference in fluorescence intensity and area between low and high PFSS-treated cells. This might be explained by the fact that we have tested the effect of PFSS of two single magnitudes within the physiological range, but not PFSS resembling disuse or overuse, since this will cause bone cell apoptosis and cell death [48]. The physical signals are sensed by the cells from the extracellular matrix into the cytoskeleton. Additionally, with respect to the decreased single integrin area, the height of integrins might be increased by low and high PFSS.” (Page 9, line 332-346)
References
[41] J. Jin, R. T. Jaspers, G. Wu, J. A. M. Korfage, J. Klein-Nulend, and A. D. Bakker, “Shear stress modulates osteoblast cell and nucleus morphology and volume,” Int. J. Mol. Sci., vol. 21, no. 21, pp. 1–22, Nov. 2020.
[48] S. D. Tan, T. J. de Vries, A. M. Kuijpers-Jagtman, C. M. Semeins, V. Everts, and J. Klein-Nulend, “Osteocytes subjected to fluid flow inhibit osteoclast formation and bone resorption,” Bone, vol. 41, no. 5, pp. 745–751, Nov. 2007.
In addition, in order to present the integrin results more clearly, we replaced figure 3 by the following new figure:
(Page 6, line 245)
Comment 6
Did you expect that only low PFSS decreased Mepe expression in preosteoblasts? Could you please explain a possible mechanism? Is it via modulation integrin subunits expression in pre-osteoblast as you proposed, or you still don’t have a definite conclusion (as you said in limitations: we did not investigate the exact (mechanistic) relationship between integrin and Mepe expression in a bone cell under mechanical loading).
Response
Did you expect that only low PFSS decreased Mepe expression in preosteoblasts?
The answer is no. We did not expect that only low PFSS decreased Mepe expression in pre-osteoblasts.
Could you please explain a possible mechanism? Is it via modulation integrin subunits expression in pre-osteoblast as you proposed, or you still don’t have a definite conclusion (as you said in limitations: we did not investigate the exact (mechanistic) relationship between integrin and Mepe expression in a bone cell under mechanical loading).
Yes, we added a possible mechanism to the discussion section (see below). Like you mentioned, we assume that it is via modulation of integrin subunit expression in pre-osteoblasts as we proposed.
“Srinivasan et al has shown that low-magnitude loading with 10 sec of rest between each load cycle significantly increases the osteogenic potential of the regimen [59]. This supports our findings that osteoblasts might be more sensitive to low PFSS. A possible mechanism relating Mepe gene expression to integrin α and β expression without/with low and high PFSS is provided by the focal adhesion kinases. The cells receive different mechanical signals, and might form different focal adhesion kinases which directly connect with integrin α and β, and then with extracellular matrix protein (e.g. Mepe). On the other side, the focal adhesion kinases directly or indirectly connect with the cytoskeleton and the nucleus, resulting in changes in cell function, e.g. Mepe gene or protein expression.” (Page 11, line 415-424)
Comment 7
The aims at the end of the Introduction and the beginning of the Discussion are not quite the same. Could you please explain what was your major aim and did you accomplished it?
Response
The aim (introduction) was: “In this study, we aimed to test whether low and/or high PFSS affects Mepe expression via modulation of integrin α and/or β subunit expression in pre-osteoblasts”. The aim (discussion) was: “This study aimed to test whether low and/or high PFSS treatment affects Mepe expression via modulation of integrin α and/or β subunit expression in MC3T3-E1 pre-osteoblasts”.
These aims (introduction and discussion) have been rephrased to one identical aim as follows:
“In this study, we aimed to test whether low and/or high PFSS affects Mepe expression via modulation of integrin α and/or β subunit expression in MC3T3-E1 pre-osteoblasts.” (Page 3, line 113-114; Page 8, line 277-278)
Did you accomplish your aim?
Partly. As you mentioned in comment 6: “as you said in limitations: we did not investigate the exact (mechanistic) relationship between integrin and Mepe expression in a bone cell under mechanical loading”. Since we accomplished part of our aim, we got a new scientific question (i.e. to investigate the exact (mechanistic) relationship between integrin and Mepe expression in a bone cell under mechanical loading), as mentioned in our limitations of the study. On the other hand, we hope that this work will give some inspiration to the readers.
Comment 8
The expression of genes included in osteogenic differentiation didn’t change. Did you expect to change? It wasn’t enough time?
Response
No, we did not expect a change in osteogenic gene expression. One hour PFSS is sufficient to cause a.o. changes in mitochondria, which likely directly or indirectly affect osteogenic differentiation of pre-osteoblasts. We added the following text to the discussion section:
“This data meets our expectation. However, one hour PFSS affects mitochondrial biogenetic related Pgc-1α gene expression (unpublished data), which might affect osteogenic differentiation of pre-osteoblasts. Furthermore, one to six hour post incubation after 1 h PFSS treatment significantly increases osteogenic differentiation of pre-osteoblasts [26].” (Page 10, line 400-404)
Reference
[26] J. Jin et al., “Pulsating fluid flow affects pre-osteoblast behavior and osteogenic differentiation through production of soluble factors,” Physiol. Rep., vol. 9, no. 12, pp. e14917–e14934, Jun. 2021.
Comment 9
I don’t understand the sentence in Discussion: lines 341-342. I can’t find the facts in the manuscript to approve the sentence lines 341-342. Is it based on your previous work? Could you please explain that? Maybe the reference is missing? Is it just an assumption?
Response
We thank the reviewer for pointing out this unclarity and omission of a missing reference. We now corrected the sentence and added the reference:
“Interestingly, we found that low PFSS decreased Mepe expression. This might indicate that low PFSS enhances osteogenic differentiation in pre-osteoblasts, since targeted disruption of Of45 (osteoblast/osteocyte factor 45 gene) has been shown to enhance bone formation [8].” (Page 10, line 404-407)
Reference
[8] L. C. Gowen et al., “Targeted disruption of the osteoblast/osteocyte factor 45 gene (Of45) results in increased bone formation and bone mass,” J. Biol. Chem., vol. 278, no. 3, pp. 1998–2007, 2003.
Comment 10
The conclusion is not separated from the Discussion. The conclusion section does not contain clear information to the readers.
Response
We separated the conclusion from the Discussion with a subtitle “5. Conclusion”. (Page 11, line 434)
We rephrased the conclusion from
“In conclusion, this study revealed that Mepe expression in pre-osteoblasts was modulated by low but not high PFSS. Integrin α subunit expression was altered by low and high PFSS, while integrin β subunit was more altered by low PFSS. This might explain the different way how Mepe-induced changes in pre-osteoblast mechanosensitivity may drive signaling pathways of bone cell function at low or high impact loading.”
Into
“In this study, both low and high PFSS affected integrin α and β subunit expression in pre-osteoblasts, while integrin β subunit was more altered by low PFSS. Importantly, Mepe gene expression was only affected by low PFSS. These results might explain the different way how Mepe-induced changes in pre-osteoblast mechanosensitivity may drive signaling pathways of bone cell function at low or high impact loading. This finding might has physiological and biomedical implications, which specifically requires future research addressing the precise role of integrin α or β subunits and Mepe during dynamic loading in in bone health and disease.” (Page 11, line 435-441)
Comment 11
It was necessary to set limitations. You very well noticed what are the major imperfections of the manuscript.
Response
Thank you for your commitment. We like our work. We know what we did. We also know what we are going to do.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsThe authors have responded to all the concerns raised by the reviewer, and the article is now accepted and ready for publication.
Reviewer 2 Report
Comments and Suggestions for AuthorsDear Authors,
You made a hugh efforts to answer my questions. The answers are thorough and precise. I am completly satisfied with provided answers.
Best regards
