Connexin hemichannels with prostaglandin release in anabolic function of bone to mechanical loading

  1. Dezhi Zhao
  2. Manuel A Riquelme
  3. Teja Guda
  4. Chao Tu
  5. Huiyun Xu
  6. Sumin Gu
  7. Jean X Jiang

  • Curated by eLife

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    Evaluation Summary:

    The authors have used two transgenic mouse models expressing dominant negative Cx43 mutants to evaluate the role of Cx43 hemichannels in mechanical loading response in bone. While understanding the molecular mechanisms by which osteocytes respond to mechanical strain is of interest in the skeletal biology arena, the conclusions of this study are not fully supported by experimental data and are of only incremental in nature.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript.The reviewers remained anonymous to the authors.)

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Abstract

Mechanical stimulation, such as physical exercise, is essential for bone formation and health. Here, we demonstrate the critical role of osteocytic Cx43 hemichannels in anabolic function of bone in response to mechanical loading. Two transgenic mouse models, R76W and Δ130–136, expressing dominant-negative Cx43 mutants in osteocytes were adopted. Mechanical loading of tibial bone increased cortical bone mass and mechanical properties in wild-type and gap junction-impaired R76W mice through increased PGE 2 , endosteal osteoblast activity, and decreased sclerostin. These anabolic responses were impeded in gap junction/hemichannel-impaired Δ130–136 mice and accompanied by increased endosteal osteoclast activity. Specific inhibition of Cx43 hemichannels by Cx43(M1) antibody suppressed PGE 2 secretion and impeded loading-induced endosteal osteoblast activity, bone formation and anabolic gene expression. PGE 2 administration rescued the osteogenic response to mechanical loading impeded by impaired hemichannels. Together, osteocytic Cx43 hemichannels could be a potential new therapeutic target for treating bone loss and osteoporosis.

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  1. eLife

    Author Response

    Reviewer #1 (Public Review):

    I'm not sure why the authors are not seeing Evans Blue dye entry into the osteocytes of loaded bone from D130-136 transgenic mice. The Augusta, GA very nicely (and it has since been repeated) that osteocyte membrane disruptions occur with much milder loading (e.g., treadmill running) and allow in EB dye. These membrane tears have nothing to do with channel or hemichannel activity. So it is very hard to understand why the D130-136 mice would be spared from membrane tears that should allow copious amounts of EB into the cells. Do certain mutations in connexin prevent membrane tears? If the R76W mutation enhances hemichannel function, and the conclusions of the paper are correct that the hemichannels are controlling the response to loading, then why were the R76W mutants not more responsive than WT to mechanical loading?

    There are uncertainties regarding how common the phenotypes of membrane tears occurred in the cells in the referred study since no specific inhibitor and underlying mechanism are currently available. Hemichannels have been investigated for years and they are selective channels that allow molecular weight less than 1 kDa to pass through. In addition to specific hemichannel-blocking antibodies, and other hemichannel blockers, such as chemicals, such as carboxlone and connexin extracellular mimetic peptides showed the inhibition of smaller dye (i.e. EB, EtBr, Lucifer yellow) uptake, but not bigger dyes (i.e. rhodamine dextran (~10 kDa) both in vitro and in vivo.

    It is true that R76W mutation has enhanced hemichannel function and some anabolic bone responses as compared to WT are indeed enhanced in R76W mice including bone volume fraction, trabecular thickness and BMD, although not as dramatic as expected. It is possible that certain threshold of hemichannel activity is required for the anabolic function in response to mechanical loading and excess hemichannel activity can be attenuated by a feedback inhibition mechanism. Our earlier study showed that prolonged activity of osteocytic Cx43 hemichannels increases extracellular PGE2 level and excess extracellular PGE2 acting in an autocrine manner activates EP2/4 receptors, leading to MAPK activation. MAPK directly phosphorylates Cx43 and closes hemichannels (Riquelme et al., 2015). This mechanism could similarly regulate the activity of R76W, resulting in comparable anabolic responses to mechanical loading as WT. We have included the above in the Discussion.

    Fig 3: How is it justified to say that the D130-D136 mice had increased bone formation response to loading on the periosteum when the relative change between loaded and nonloaded look to be about the same in all three genotypes? Are the authors not adjusting for the higher or lower control leg bone formation measurements?

    In WT and R76W mice, the bone formation in both periosteal and endosteal surface were increased by tibial loading and this increase is correlated with the increase of bone area fractions and cortical thickness. In D130-136 mice, only bone formation on the periosteal surface increased, but not on the endosteal surface. In addition, we observed the increased osteoclast number in endosteal surface. The net effect in D130-136 is a decreased bone area fraction and cortical thickness. The data presented in this study include the higher or lower control leg formation measurements. We have revised the text in the Discussion to make it clear.

    Reviewer #2 (Public Review):

    This study examines the effects of mechanical loading on the bones of two transgenic mouse models of connexin 43 overexpression, one mutant which impairs both gap junction intercellular communication (GJIC) and hemichannel activity ( 130-136) and another that supports only enhanced hemichannel activity but not GJIC (R76W). The authors conclude that hemichannels but not GJIC facilitate the effects of mechanical loading on bone via the secretion of PGE2 through the hemichannels.

    While provocative, the data fall short of being convincing of the interpretation.

    A major concern is the statistical approaches used to evaluate data. The conclusions obligate that each group of animals (WT, R76W and 130-136 mice with or without loading) be compared to each other to determine differences in their ability to mount a response of bone to a mechanical load. The correct statistical test is a two way ANOVA when there are multiple variables (genotype and load). However, multiple t-tests are used to support major conclusions. Since primary data was supplied by the authors in the supplement, we checked this using statistical software. Many of the statistical analyses do not hold up when run through the appropriate statistical test. Thus, the primary findings reported are not supported.

    By working closely with a biostatistician expert, in the revision, we have thoroughly reanalyzed the data with statistical analyses. To determine the mechanical responses, the major analysis should be the paired comparison within each genotype group, WT, R76W and D130-136. Therefore, paired student T-test is an appropriate statistical approach. We agree that one-way ANOVA is improper to compare multiple variables and comparison with multiple variable (genotype and load) would provide irrelevant information regarding the treatment responses. In this study, we focus on the comparison between loaded and contralateral, unloaded tibias within each genotype using paired student T-test.

    Two additional significant weaknesses affect the potential quality and impact of this study.

    1. No convincing evidence is presented that the phenotype was rescued by PGE2. In Figure 8 and the corresponding supplement, vehicle treated and PGE2 treated unloaded controls are not shown and are critical to the appropriate interpretation of the experiment. Meaningful bone parameters including bone area and cortical thickness are not affected by the PGE2. Trabecular bone was completely unaffected by PGE2 or even the M1 antibody. Also, a oneway ANOVA is the incorrect measure with which to assess these changes. There are many variables in these mice: treatment with or without M1 antibody, loading or unloading (although not included) and treatment with or without PGE2. These are not accounted for with the statistical models used to assess the data.

    The significant reduction of bone area fraction, a key parameter by M1 was ablated with PGE2 treatment as well bone marrow area and cortical thickness. As the reviewer pointed out, the rescue by PGE2 in cortical bone was not shown in trabecular bone. We are not certain for the difference between cortical and trabecular bones. A recent paper has also shown more beneficial osteogenic responses of combined treatment of PTH(1-34) and mechanical loading to cortical bone than trabecular bone (Roberts et al., 2020). As discussed in this paper, one of the possibilities could be related to the higher strain levels experienced by cortical bone compared to trabecular bone. We have included the above in the Discussion. We have reanalyzed the data and comparison. The major comparison should be paired student-T test by comparing vehicle and M1 treated within each group, Control and PGE2.

    1. No convincing evidence that PGE2 secretion through connexin 43 hemichannels is shown. Instead, Figure 4C shows that a protein (COX2) responsible for producing PGE2 is reduced in the cells that produce PGE2 in the D130-136 mice. Several papers have shown that connexin 43 regulates ptgs2 and could affect PGE2 abundance independent of the ability to pass through connexin 43 hemichannels and others show that PGE2 also regulates connexin 43 abundance and gap junctional communication.

    Our earlier study has showed that Cx43 hemichannels in osteocytes serve as a direct portal for the release of PGE2 (Cherian et al., 2005). In this study, we showed that increased PGE2 in tibia bone by mechanical loading was totally attenuated in D130-136 mice (Fig. 4A) and M1 treated mice (Fig. 7A). Moreover, we have previous reported that the inhibition of Cx43 hemichannels does not affect intracellular PGE2 level (Siller-Jackson et al., 2008), suggesting the reduced PGE2 biosynthesis by COX2 since COX2 is the enzyme subtype responding to mechanical loading. Indeed, in this study, we showed the attenuated upregulation of COX2 expression and reduction of PGE2 level in D130-136 mice as well as in M1 treated mice. We did not find any previous papers raised by the reviewer regarding “connexin 43 regulates ptgs2 and could affect PGE2 abundance independent of the ability to pass through connexin 43 hemichannels”. We and others have shown that PGE2 can increase Cx43 expression and gap junction communication in cultured osteoblasts (Civitelli et al., 1998) and osteocytes (Cheng et al., 2001), but has no effect in oral-derived human osteoblasts (Adamo et al., 2001). Additionally, increasing Cx43 expression enhances PGE2-dependent β-catenin signaling activation in osteoblast cells (Gupta et al., 2019). Cx43 overexpression in rabbit and human synovial fibroblast cell lines increased PTGS2 gene expression (Gupta et al., 2014). Moreover, increased extracellular PGE2 could serve as a feedback inhibitor that activates MAPK, phosphorylates Cx43 and closes Cx43 hemichannels (Riquelme et al., 2015). The outcomes of this study will help establish hemichannels as a potential de novo drug target for treating bone loss and osteoporosis. We have included the above in the Discussion.

  2. eLife

    Evaluation Summary:

    The authors have used two transgenic mouse models expressing dominant negative Cx43 mutants to evaluate the role of Cx43 hemichannels in mechanical loading response in bone. While understanding the molecular mechanisms by which osteocytes respond to mechanical strain is of interest in the skeletal biology arena, the conclusions of this study are not fully supported by experimental data and are of only incremental in nature.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript.The reviewers remained anonymous to the authors.)

  3. eLife

    Reviewer #2 (Public Review):

    This study examines the effects of mechanical loading on the bones of two transgenic mouse models of connexin 43 overexpression, one mutant which impairs both gap junction intercellular communication (GJIC) and hemichannel activity (130-136) and another that supports only enhanced hemichannel activity but not GJIC (R76W). The authors conclude that hemichannels but not GJIC facilitate the effects of mechanical loading on bone via the secretion of PGE2 through the hemichannels.

    While provocative, the data fall short of being convincing of the interpretation.

    A major concern is the statistical approaches used to evaluate data. The conclusions obligate that each group of animals (WT, R76W and 130-136 mice with or without loading) be compared to each other to determine differences in their ability to mount a response of bone to a mechanical load. The correct statistical test is a two way ANOVA when there are multiple variables (genotype and load). However, multiple t-tests are used to support major conclusions. Since primary data was supplied by the authors in the supplement, we checked this using statistical software. Many of the statistical analyses do not hold up when run through the appropriate statistical test. Thus, the primary findings reported are not supported.

    Two additional significant weaknesses affect the potential quality and impact of this study.

    1. No convincing evidence is presented that the phenotype was rescued by PGE2. In Figure 8 and the corresponding supplement, vehicle treated and PGE2 treated unloaded controls are not shown and are critical to the appropriate interpretation of the experiment. Meaningful bone parameters including bone area and cortical thickness are not affected by the PGE2. Trabecular bone was completely unaffected by PGE2 or even the M1 antibody. Also, a one-way ANOVA is the incorrect measure with which to assess these changes. There are many variables in these mice: treatment with or without M1 antibody, loading or unloading (although not included) and treatment with or without PGE2. These are not accounted for with the statistical models used to assess the data.

    2. No convincing evidence that PGE2 secretion through connexin 43 hemichannels is shown. Instead, Figure 4C shows that a protein (COX2) responsible for producing PGE2 is reduced in the cells that produce PGE2 in the D130-136 mice. Several papers have shown that connexin 43 regulates ptgs2 and could affect PGE2 abundance independent of the ability to pass through connexin 43 hemichannels and others show that PGE2 also regulates connexin 43 abundance and gap junctional communication.

  4. eLife

    Reviewer #1 (Public Review):

    I'm not sure why the authors are not seeing Evans Blue dye entry into the osteocytes of loaded bone from D130-136 transgenic mice. The Augusta, GA very nicely (and it has since been repeated) that osteocyte membrane disruptions occur with much milder loading (e.g., treadmill running) and allow in EB dye. These membrane tears have nothing to do with channel or hemichannel activity. So it is very hard to understand why the D130-136 mice would be spared from membrane tears that should allow copious amounts of EB into the cells. Do certain mutations in connexin prevent membrane tears?

    If the R76W mutation enhances hemichannel function, and the conclusions of the paper are correct that the hemichannels are controlling the response to loading, then why were the R76W mutants not more responsive than WT to mechanical loading?
    Fig 3: How is it justified to say that the D130-D136 mice had increased bone formation response to loading on the periosteum when the relative change between loaded and nonloaded look to be about the same in all three genotypes? Are the authors not adjusting for the higher or lower control leg bone formation measurements?

  5. Version published to 10.7554/elife.74365 on eLife
  6. Version published to 10.1101/2021.10.15.464551v1 on bioRxiv