Bone corticalization requires local SOCS3 activity and is promoted by androgen action via interleukin-6

Long bone strength is determined by its outer shell (cortical bone), which forms by coalescence of thin trabeculae at the metaphysis (corticalization), but the factors that control this process are unknown. Here we show that SOCS3-dependent cytokine expression regulates bone corticalization. Young male and female Dmp1Cre.Socs3 f/f mice, in which SOCS3 has been ablated in osteocytes, have high trabecular bone volume and poorly defined metaphyseal cortices. After puberty, male mice recover, but female corticalization is still impaired, leading to a lasting defect in bone strength. The phenotype depends on sex-steroid hormones: dihydrotestosterone treatment of gonadectomized female Dmp1Cre.Socs3 f/f mice restores normal cortical morphology, whereas in males, estradiol treatment, or IL-6 deletion, recapitulates the female phenotype. This suggests that androgen action promotes metaphyseal corticalization, at least in part, via IL-6 signaling.

Introduction "sex-differences in corticalization may be explained, at least in part, by inhibition of local IL-6 signaling by androgens" Title "Androgen action promotes bone corticalization by inhibiting SOC S 3-dependent interleukin-6 signalling" Those above sentences appear to contrast the following sentences: Figure 7 legend: "In Dmp1C re.Socs3f/f mice, this inhibition is lost, leading to delayed corticalization, which is inhibited by androgens through interleukin 6 (IL-6)" Start of discussion "pure androgen action (mainly mediated by testosterone in vivo) promotes corticalization through an IL-6 dependent pathway" End of discussion "pure androgen action promotes corticalization by a mechanism that is p artially dependent on IL-6 signaling" My confusion as outlined above may very well be a lack of understanding on my part and I would welcome clarification by the authors Minor comments: Is testing the efficacy of SOC S3 deletion by RT-PC R as shown in Figure 1A really the best method? As this shows only a slight reduction in SOC S3 mRNA levels. Page 10: The sentence "Thickened" cortical bone was measured throughout the metaphyseal region and partitioned into the spongy, incompletely coalesced cortical component versus normal compact cortical bone, we observed that the incompletely corticalized (spongy) bone" does not appear to make sense Tb.Sp is not defined Abstract, line 48 "mic" should read "mice" It would be interesting to see if loss of gp130 reverses the phenotype of the SOC S3 KO osteocytes

Reviewer #2 (Remarks to the Author):
The authors of the manuscript study the effect of knockout of SOSC 3 specifically in osteocytes using the DMP1cre. The authors find a very interesting phenotype where there is initially an increase in the trabecular bone phenotype with a delay in corticalization in females that persists. Treating females with androgen rescues the cortical phenotype a nd treating males with estradiol recapitulates the corticalization delay in males. Suggesting that testosterone through IL6 mediates corticalization. The bone phenotype is thoroughly and extensively characterized. The authors use OVX and ORX to thoroughly explore the sex dependent phenotypes. The signaling mechanism is partially shown to be through IL6 by using null mice. Though most of the data is descriptive it is very well written with the study being very novel and general interest to the bone field. I would support publication with the below mentioned revisions. The major drawback is the lack of molecular data showing the mechanism. Having some in vitro data to support the mechanism would be very beneficial and would significantly add to this solid data set. Is there any molecular data showing the involvement of STAT signaling? Some additional information about the DMP1cre would be helpful with the new data showing that the 14kb DMP1cre has a promiscuous expression pattern it would be useful if the au thors could comment on the specificity of the cre line they utilized.

Reviewer #3 (Remarks to the Author):
This is an interesting and carefully performed study defining the development of metaphyseal cortical bone and the possible role of sex steroids. It shows an important role for the family of cytokines regulated by SOC S3 in this process, and it is possible that targeting this pathway could enhance metaphyseal cortical bone in adults and therefore be of therapeutic importance.
The paper can be difficult to follow in places, as it uses complex histology and imaging. Nonetheless, the data are presented well overall. My main concern pertains to the IL6 story. As I understand this, deleting SOC S3 leads to increased activity of a family of cytokines, including IL6. In females, deleting IL6 does not rescue the phenotype, so IL6 is not the cause. In males, deleting IL6 moves them towards a female phenotype ( Fig 7B) -and yet the authors conclude that DHT suppression of IL6 is part of the reason for the sexual divergence. The main part that does not make sense is how this explanation fits with the data that deleting IL6 in males makes cortical porosity look more like females? If DHT is suppressing IL6, and IL6 is part of the reason for the delayed corticalization, then deleting IL6 in males should have either no effect (IL6 already suppressed) or actually make the males look even more different from the females (IL6 even lower)? It seems I am missing something here… A second point related to cortical bone in the metaphysis vs diaphysis. As shown by C adet et al. (JBJS 85A: 1739(JBJS 85A: , 2003, cortical bone at the metaphysis does develop by trabecular condensation. However, diaphyseal cortical bone develops through sub -periosteal apposition. And yet in Fig 6, the authors present very similar findings at both the metaphysis and diaphysis. Do they have an explanation for this?
Other, minor points: -Were all mice in the C 57BL/6 background? -Given that E2 and perhaps DHT often lead to metaphyseal sclerosis when gi ven by pellet or pump, was this a confounder in the study? How "physiological" were these doses? What about uterine or seminal vesicle weights as biomarkers for replacement? - Fig 1A -the extent of deletion of SOC S3 is surprisingly small. Generally the D MPC re gives better results in terms of reduction of the specific mRNA. Any reasons for what appears to be relatively poor deletion? - Fig 7B -to argue that the IL6 KO males were no longer significantly different from the females may be true in terms of the p value, but they do still look quite different, and may well be with a somewhat larger sample size. So the authors do need to be more cautious regarding this contention.
Introduction "sex-differences in corticalization may be explained, at least in part, by inhibition of local IL-6 signaling by androgens" Title "Androgen action promotes bone corticalization by inhibiting SOCS3-dependent interleukin-6 signalling" Those above sentences appear to contrast the following sentences: Figure 7 legend: "In Dmp1Cre.Socs3f/f mice, this inhibition is lost, leading to delayed corticalization, which is inhibited by androgens through interleukin 6 (IL-6)" Start of discussion "pure androgen action (mainly mediated by testosterone in vivo) promotes corticalization through an IL-6 dependent pathway" End of discussion "pure androgen action promotes corticalization by a mechanism that is partially dependent on IL-6 signaling" My confusion as outlined above may very well be a lack of understanding on my part and I would welcome clarification by the authors.

RESPONSE:
The reviewer has helped us to think about our data in a new way. As the reviewer points out, since deletion of IL-6 in females had no effect on the phenotype, it is more likely that the effect of androgens on corticalisation is due to a stimulation of IL-6 rather than an inhibition. In the original manuscript we showed that androgens block (inhibit) the delay in corticalisation in Dmp1Cre.Socs3 f/f increase in the trabecular bone phenotype with a delay in corticalization in females that persists. Treating females with androgen rescues the cortical phenotype and treating males with estradiol recapitulates the corticalization delay in males. Suggesting that testosterone through IL6 mediates corticalization. The bone phenotype is thoroughly and extensively characterized. The authors use OVX and ORX to thoroughly explore the sex dependent phenotypes. The signaling mechanism is partially shown to be through IL6 by using null mice. Though most of the data is descriptive it is very well written with the study being very novel and general interest to the bone field. I would support publication with the below mentioned revisions.
The major drawback is the lack of molecular data showing the mechanism. Having some in vitro data to support the mechanism would be very beneficial and would significantly add to this solid data set. Is there any molecular data showing the involvement of STAT signaling? RESPONSE: We agree with the reviewer that molecular data would be helpful, but the effect of SOCS3 cytokines on corticalisation are (1) site-specific (only in metaphyseal bone), (2) age-specific (varies with maturity and stages of bone development), (3) modified by systemic sex-steroids, and (4) likely to be modified by mechanical loading. For these reasons, we do not think it is possible to use cultured cells to identify the mechanism for this phenotype; our continuing work is focussing on using in vivo approaches.

COMMENT:
Some additional information about the DMP1cre would be helpful with the new data showing that the 14kb DMP1cre has a promiscuous expression pattern it would be useful if the authors could comment on the specificity of the cre line they utilized. RESPONSE: We have added a short paragraph on the potential limits of this study due to non-specific Dmp1Cre expression (Lines 418-427) Reviewer #3 COMMENT: The paper can be difficult to follow in places, as it uses complex histology and imaging. Nonetheless, the data are presented well overall. My main concern pertains to the IL6 story. As I understand this, deleting SOCS3 leads to increased activity of a family of cytokines, including IL6. In females, deleting IL6 does not rescue the phenotype, so IL6 is not the cause. In males, deleting IL6 moves them towards a female phenotype (Fig 7B) -and yet the authors conclude that DHT suppression of IL6 is part of the reason for the sexual divergence. The main part that does not make sense is how this explanation fits with the data that deleting IL6 in males makes cortical porosity look more like females? If DHT is suppressing IL6, and IL6 is part of the reason for the delayed corticalization, then deleting IL6 in males should have either no effect (IL6 already suppressed) or actually make the males look even more different from the females (IL6 even lower)? It seems I am missing something here… RESPONSE: Please see response to Reviewer 1's major comment.
COMMENT: A second point related to cortical bone in the metaphysis vs diaphysis. As shown by Cadet et al. (JBJS 85A: 1739, 2003, cortical bone at the metaphysis does develop by trabecular condensation. However, diaphyseal cortical bone develops through sub-periosteal apposition. And yet in Fig 6, the authors present very similar findings at both the metaphysis and diaphysis. Do they have an explanation for this? RESPONSE: We observed no difference in periosteal width at the diaphysis, suggesting that subperiosteal apposition is normal in our mouse model. We have added this data to Supplementary Figure  1, and describe it in lines 109-110. Even though periosteal growth was unaffected, femoral strength at the diaphysis was modified in older female mice. This suggests that although sub-periosteal apposition plays a role in diaphyseal cortical development, as suggested by Cadet, it may not be the exclusive mediator of diaphyseal development, and trabecular coalescence may also be involved. We have added a note about this in the discussion (lines 350-357). In Figure 6, the region that we termed "diaphysis" is not the central diaphysis, but a region that is closer to the diaphysis than the metaphyseal region. To avoid confusion, we have changed the terminology used in Figure 6 to "upper metaphysis" and "lower metaphysis" to avoid confusion (line 230, and Figure 6).
Other, minor points: COMMENT: Were all mice in the C57BL/6 background? RESPONSE: Yes, this detail has been added to the Methods section (lines 439 and 444).
COMMENT: Given that E2 and perhaps DHT often lead to metaphyseal sclerosis when given by pellet or pump, was this a confounder in the study? How "physiological" were these doses? What about uterine or seminal vesicle weights as biomarkers for replacement? RESPONSE: There was no metaphyseal sclerosis associated with the doses of E 2 and DHT used in this study. We used doses previously shown to retain uterine and seminal vesicle weights after gonadectomy. We did not measure uterine or seminal vesicle weights in this experiment. We quantified the effects of E 2 and DHT on trabecular bone at the vertebrae because an effect on corticalisation would not impact on the phenotype at this site ( Figure 5) and did not observe sclerosis at that site. We also did not observe metaphyseal sclerosis due to E 2 or DHT in femoral samples, as indicated by the metaphyseal images shown in Figure 6A, C. We have added a note to clarify doses used in the methods, including specific references to those papers that measured the effects of these implants on uterine and seminal vesicle weights (lines 478-480) and have added a note that there was no bone sclerosis in the Results section (lines 191-193).
COMMENT: Fig 1A -the extent of deletion of SOCS3 is surprisingly small. Generally the DMPCre gives better results in terms of reduction of the specific mRNA. Any reasons for what appears to be relatively poor deletion? RESPONSE: Please see our response to Reviewer 1's first minor comment. With ubiquitously expressed genes, the DMP1Cre is generally only about 50% effective at reducing target gene levels in whole mRNA samples of marrow-flushed bones, likely due to contaminating cells. Fig 7B -to argue that the IL6 KO males were no longer significantly different from the females may be true in terms of the p value, but they do still look quite different, and may well be with a somewhat larger sample size. So the authors do need to be more cautious regarding this contention. RESPONSE: This was already noted at the end of the Introduction and Results sections, and in both the first paragraph and latter part of the Discussion, where we said that this was partially rescued, indicating "at least partial" dependency on IL-6. We have now modified the last line of the discussion to further soften this assertion (lines 433-435).