Disparate Bone Anabolic Cues Activate Bone Formation by Regulating the Rapid Lysosomal Degradation of Sclerostin Protein

  1. Nicole R. Gould
  2. Katrina M. Williams
  3. Humberto C. Joca
  4. Olivia M. Torre
  5. James S. Lyons
  6. Jenna M. Leser
  7. Manasa P. Srikanth
  8. Marcus Hughes
  9. Ramzi J. Khairallah
  10. Ricardo A. Feldman
  11. Christopher W. Ward
  12. Joseph P. Stains

  • Curated by eLife

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    Summary: The article by Gould et al breaks new ground by demonstrating a role for lysosomal-mediated degradation in the mechanosensitive repression of Sclerostin levels in bone. Though the post-translational repression of Sclerostin has long been apparent, no one has yet unraveled the mechanisms. Therefore, this discovery is important to the skeletal biology community - both because of the findings themselves, and because the conditions/models used by this team to make these discoveries will be useful for other investigators, including their ability to manipulate and observe the rapid lysosome-dependent control of Sclerostin levels in vitro and in vivo in response to PTH or mechanical stimulation. In addition to the importance within this field, the work has broad impact on multiple levels including a) the clinical relevance for understanding and potentially treating osteoporosis and the skeletal phenotypes in individuals with lysosomal disease, and b) the mechanoregulation of lysosomal function and its relationships to crinophagy, which has implications not only for the regulation of Sclerostin, but also for other factors in and beyond the skeleton (RANKL, insulin).

    Essential revisions:

    The study is elegantly designed, clearly communicated, and rigorously conducted. However, the reviewers require additional data to support the overall conclusion on the significance of lysosome-mediated degradation of sclerostin in skeletal biology. First, it is important to elaborate on what gives the authors confidence that the inhibitors were effective and act as expected throughout the study - but especially Bafilomycin A1 and Apocynin in vivo. If BafA1 and Apocynin treatment in vivo work as expected, they should prevent the rapid load-dependent repression of Sclerostin levels (shown in Figure 1D). Second, the author's demonstration of mechanical load-dependent changes in sclerostin localization in osteocytes lysosomes in vivo by immunohistochemistry would be important to support the in vivo relevance of this pathway in the acute regulation of sclerostin levels. While the western blotting of mechanically loaded mouse ulnas showing previously-undocumented acute reductions in lysate sclerostin levels is interesting, it is unclear if these changes are caused by mechanical loading-induced lysosomal function.

    Reviewer #1 and Reviewer #2 opted to reveal their name to the authors in the decision letter after review.

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Abstract

The down regulation of sclerostin mediates bone formation in response to mechanical cues and parathyroid hormone (PTH). To date, the regulation of sclerostin has been attributed exclusively to the transcriptional downregulation that occurs hours after stimulation. Here, we describe, for the first time, the rapid post-translational degradation of sclerostin protein by the lysosome following mechanical load or PTH. We present a unifying model, integrating both new and established mechanically- and hormonally-activated effectors into the regulated degradation of sclerostin by lysosomes. Using an in vivo mechanical loading model, we find transient inhibition of lysosomal degradation or the upstream mechano-signaling pathway controlling sclerostin abundance impairs subsequent load-induced bone formation. We also link dysfunctional lysosomes to aberrant sclerostin regulation using Gaucher disease iPSCs. These results inform a paradigm shift in how bone anabolic cues post-translationally regulate sclerostin and expands our understanding of how osteocytes regulate this fundamentally important protein to regulate bone formation.

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  1. eLife

    Reviewer #3:

    This is an outstanding work from the lab of Dr. Stains establishing rapid post-translational regulation of sclerostin, a robust inhibitor of bone formation. They carefully and clearly establish that sclerostin is rapidly degraded by lysosomes in response to mechanical loading, and further link lysosomal abnormalities, using Gaucher iPSCs, to sclerostin levels.

  2. eLife

    Reviewer #2:

    The article by Gould et al breaks new ground by demonstrating a role for lysosomal-mediated degradation in the mechanosensitive repression of Sclerostin levels in bone. Though the post-translational repression of Sclerostin has long been apparent, no one has yet unraveled the mechanisms. Therefore, this discovery is important to the skeletal biology community - both because of the findings themselves, and because the conditions/models used by this team to make these discoveries will be useful for other investigators, including their ability to manipulate and observe the rapid lysosome-dependent control of Sclerostin levels in vitro and in vivo in response to PTH or mechanical stimulation. In addition to the importance within this field, the work has broad impact on multiple levels including a) the clinical relevance for understanding and potentially treating osteoporosis and the skeletal phenotypes in individuals with lysosomal disease, and b) the mechanoregulation of lysosomal function and its relationships to crinophagy, which has implications not only for the regulation of Sclerostin, but also for other factors in and beyond the skeleton (RANKL, insulin).

    The study is elegantly designed, clearly communicated, and rigorously conducted. The conclusions drawn in the manuscript are mostly supported by the data provided. In general, it is important to elaborate on what gives the authors confidence that the inhibitors were effective and act as expected throughout the study - but especially Bafilomycin A1 and Apocynin in vivo. If BafA1 and Apocynin treatment in vivo work as expected, they should prevent the rapid load-dependent repression of Sclerostin levels (shown in Figure 1D).

    Other revisions or additions, described below, would improve the quality of the study:

    1. Are Sclerostin levels insensitive to FSS or PTH in Gaucher cells (though it understandably may not be feasible to differentiate these cells in microfluidic devices)?

    2. Since a sex-specific effect of exercise on bone anabolism has previously been described, and TRPV4 also has a sexually dimorphic effect on bone, were any differences observed between male and female animals here?

    3. Can the authors discuss where the pathway used by PTH diverges from that activated by FSS/load?

    4. Is it possible to detect load dependent changes in sclerostin localization in lysosomes in vivo?

    5. Given the non-specific effects of hydrogen peroxide, Figure 6D may not add a great deal in light of the other data that was gathered with more rigorous approaches. Additional controls would give more confidence in the efficacy/specificity of this approach.

    6. Please include how long the OCY454 cells were differentiated prior to the treatments applied.

    7. Please identify the route by which inhibitory agents were administered to the mice (i.e. subcutaneous, intraperitoneal).

    8. Please increase the N for experiments in Figure 4A and 5D, or remove these data and the corresponding conclusions.

  3. eLife

    Reviewer #1:

    This manuscript by Gould et al presents highly novel data which is logically presented and is likely to have both clinical and fundamental implications. Of relevance to the bone field, it defines a new mechanism by which one of the most important clinical targets for the treatment of osteoporosis is endogenously regulated. Beyond bone, I am not aware of any other examples of stimulus-directed acute lysosomal degradation of a secreted canonical Wnt antagonist as a mechanism to provide rapid de-repression. What seems lacking is a careful analysis of the physiological consequences of the acute degradation of sclerostin.

    1. A landmark paper which convinced many in the field that sclerostin down-regulation is necessary for osteoanabolic responses to loading was based on a transgenic model from the Bellido lab (Tu et al, Bone, 2012). In that study, expression of Sost from the DMP-1 promoter precluded its transcriptional and protein-level down-regulation at late time points. That was sufficient to largely prevent bone gain following loading. Several other groups interpreted this as indicating Sost transcript regulation is required for bone's adaptation to loading, calling into question the physiological relevance of transient post-translational degradation described here. Can the authors reconcile that study with their own?

    2. One way the authors attempt to demonstrate in vivo relevance is through western blotting of mechanically loaded mouse ulnas, showing previously-undocumented acute reductions in lysate sclerostin levels. It is standard practice in the field to quantify sclerostin positive osteocytes histologically, rather than by western blotting. This is because mechanical loading can rapidly increase blood flow to the limb (even in this study, the authors implicate the vasodilator NO) as well as having inflammatory effects, diluting the proportion of osteocyte-specific proteins in the lysate. Demonstrating protein-level sclerostin down-regulation specifically in osteocytes rapidly following loading would be a major addition to this study.

    3. A long-stranding, reproducible finding which has always been very perplexing is that the largest transcriptomic responses to osteogenic mechanical loading occur very quickly, within an hour of loading, before Sost is down-regulated. Even in UMR106 cells in vitro, B-catenin is stabilised before Sost is down-regulated following exposure to substrate strain. The current findings may explain this temporal discrepancy. The authors should responses to sclerostin degradation such as quantifying Wnt target genes to provide physiologically-relevant readouts of their findings.

    4. Figure 3 shows co-localisation of endogenous or ever-expressed sclerostin with lysosomal markers. Does this co-localisation change following FSS or PTH?

    5. It is not clear whether early lysosomal degradation which transiently decreases sclerostin is triggered by the same mechanoresponsive pathways which subsequently down-regulate its RNA levels, or whether the two responses are distinct. Can the authors clarify this? For example, does Sost decrease in the BafA1-treated cells 8 hours after FSS or PTH treatment?

    6. Discussion "that the rapid and transient nature of sclerostin degradation may be critical to the precise anatomical positioning of new bone formation following an anabolic stimulus" is very unclear. How do the authors propose that lysosomal sclerostin degradation produces regionalised responses to a greater degree than the previously-reported transcriptional mechanisms?

    7. The evidence of lysosomal involvement in sclerostin down-regulation is largely based on pharmacological compounds of limited selectivity. A degree of genetic evidence is indirectly provided by the Gaucher cell line, but this is based on a single patient line. Can the authors provide direct genetic evidence that lysosomal function is necessary for sclerostin down-regulation, and ideally for bone formation?

    8. References to previous studies which described mechanisms and relevance of Sost down-regulation are sparse. For example, see previous implications of NO signalling from the Vanderschueren lab (Callewaert et al, JBMR, 2010), protein-level down-regulation of sclerostin in the context of ageing from the Price lab (Meakin et al, JBMR, 2014) relevant to the discussion in the current manuscript, as well as work from the Ferrari lab on sclerostin regulation following both PTH and mechanical loading (e.g. Bonnet et al, JBC 2009; Bonnet et al, PNAS 2012).

  4. eLife

    Summary: The article by Gould et al breaks new ground by demonstrating a role for lysosomal-mediated degradation in the mechanosensitive repression of Sclerostin levels in bone. Though the post-translational repression of Sclerostin has long been apparent, no one has yet unraveled the mechanisms. Therefore, this discovery is important to the skeletal biology community - both because of the findings themselves, and because the conditions/models used by this team to make these discoveries will be useful for other investigators, including their ability to manipulate and observe the rapid lysosome-dependent control of Sclerostin levels in vitro and in vivo in response to PTH or mechanical stimulation. In addition to the importance within this field, the work has broad impact on multiple levels including a) the clinical relevance for understanding and potentially treating osteoporosis and the skeletal phenotypes in individuals with lysosomal disease, and b) the mechanoregulation of lysosomal function and its relationships to crinophagy, which has implications not only for the regulation of Sclerostin, but also for other factors in and beyond the skeleton (RANKL, insulin).

    Essential revisions:

    The study is elegantly designed, clearly communicated, and rigorously conducted. However, the reviewers require additional data to support the overall conclusion on the significance of lysosome-mediated degradation of sclerostin in skeletal biology. First, it is important to elaborate on what gives the authors confidence that the inhibitors were effective and act as expected throughout the study - but especially Bafilomycin A1 and Apocynin in vivo. If BafA1 and Apocynin treatment in vivo work as expected, they should prevent the rapid load-dependent repression of Sclerostin levels (shown in Figure 1D). Second, the author's demonstration of mechanical load-dependent changes in sclerostin localization in osteocytes lysosomes in vivo by immunohistochemistry would be important to support the in vivo relevance of this pathway in the acute regulation of sclerostin levels. While the western blotting of mechanically loaded mouse ulnas showing previously-undocumented acute reductions in lysate sclerostin levels is interesting, it is unclear if these changes are caused by mechanical loading-induced lysosomal function.

    Reviewer #1 and Reviewer #2 opted to reveal their name to the authors in the decision letter after review.

  5. Version published to 10.1101/2020.10.26.355800 on bioRxiv