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  1. 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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  2. 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.

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  3. 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?

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