Targeting Msx2 as a brake in the fusion fate of osteoclasts and an anabolic therapy in pre-clinical models of osteoporosis

Classified as probably not a substantial claim.

FBXW7 recognizes a conserved S/TxxxS/T motif.

“FBXW7 is known to recognize a conserved S/TxxxS/T motif59,60”

• cites [59] Doble, B. W. & Woodgett, J. R. GSK-3: tricks of the trade for a multi-tasking kinase. J. Cell Sci. 116, 1175–1186 (2003). sourceDisputed

  The Introduction (page not provided) identifies Ser/Thr–X–X–X–Ser/Thr-P as the consensus sequence for GSK-3 substrates, not as a motif recognized by FBXW7. Searches for FBXW7, Fbw7, and Cdc4 found no passage assigning the motif to that protein. (LLM, conf=0.85)

  Checked by a reasoning model — may be wrong.

  Experimental — accuracy not yet measured
• cites [60] Davis, R. J., Welcker, M. & Clurman, B. E. Tumor suppression by the Fbw7Ubiquitin ligase: mechanisms and opportunities. Cancer Cell 26, 455–464 (2014). sourceSupported

Probably not a substantial claim: may not pass S3 (load-bearing).

Multiple homeobox genes have been implicated in bone mass and osteogenesis regulation.

“Multiple homeobox genes have been implicated in the regulation of bone mass and osteogenesis16–19.”

• cites [16] Yu, X. et al. TGFβ-induced factor homeobox 2 blocks osteoblastic differentiation through targeting pSmad3/HDAC4/H4ac/Runx2 axis. J. Cell Physiol. 234, 21284–21293 (2019). sourceNot checked — no full text of the source
• cites [17] Yang, H., Liang, Y., Cao, Y., Cao, Y. & Fan, Z. Homeobox C8 inhibited the osteo-/dentinogenic differentiation and migration ability of stem cells of the apical papilla via activating KDM1A. J. Cell Physiol. 235, 8432–8445 (2020). sourceNot checked — no full text of the source
• cites [18] Heo, J. S., Lee, S. G. & Kim, H. O. Distal-less homeobox 5 is a master regulator of the osteogenesis of human mesenchymal stem cells. Int. J. Mol. Med. 40, 1486–1494 (2017). sourcePartly supported

  The Introduction (page unavailable) says homeobox-containing genes regulate skeletal development and that DLX genes function in osteoblast development; it also links DLX5 to osteogenesis. This supports the broad point about homeobox genes and osteogenesis, but the paper does not specifically address regulation of bone mass. (LLM, conf=0.82)

  Experimental — accuracy not yet measured
• cites [19] Xu, J. et al. Shox2 regulates osteogenic differentiation and pattern formation during hard palate development in mice. J. Biol. Chem. 294, 18294–18305 (2019). sourceNot checked — no full text of the source

Probably not a substantial claim: may not pass S3 (load-bearing).

M-CSF and RANKL activate PU.1 and NFATc1 via MAPKs and NF-κB, initiating osteoclastogenesis.

“Mechanistically, M-CSF and RANKL bind to their receptors CSF1R and RANK, respectively, and stimulate downstream signaling pathways such as MAPKs and NF-κB, leading to the activation of master transcription factors including PU.1 and NFATc1, initiating osteoclastogenesis9,10.”

• cites [9] Jiang, T., et al. Role and regulation of transcription factors in osteoclastogenesis. Int. J. Mol. Sci. 24, 16175 (2023). sourcePartly supported

  Sections 4.1 and 5.8 describe M-CSF binding CSF-1R and RANKL binding RANK, with downstream MAPK and NF-κB signaling. Section 5.1 links NF-κB and p38 signaling to NFATc1 induction. But sections 3 and 5.4 do not establish that those pathways activate PU.1; they describe early PU.1 expression and its role in promoting NFATc1 expression. Page numbers were not provided in our copy. (LLM, conf=0.87)

  Experimental — accuracy not yet measured
• cites [10] Takayanagi, H. RANKL as the master regulator of osteoclast differentiation. J. Bone Min. Metab. 39, 13–18 (2021). sourceNot checked — no full text of the source

May combine two claims.

Global Msx2 knockout impairs osteoclast formation, whereas loss of Irx3 has no effect on osteoclasts.

“According to previous studies, global Msx2 knockout impairs osteoclast formation, whereas loss of Irx3 has no effect on osteoclasts26,27.”

• cites [26] Aïoub, M. et al. Msx2 -/- transgenic mice develop compound amelogenesis imperfecta, dentinogenesis imperfecta and periodental osteopetrosis. Bone 41, 851–859 (2007). sourceNot checked — no full text of the source
• cites [27] Tan, Z. et al. IRX3 and IRX5 inhibit adipogenic differentiation of hypertrophic chondrocytes and promote osteogenesis. J. Bone Miner. Res. 35, 2444–2457 (2020). sourceSupported

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

MSX2 belongs to the Msx family and is involved in bone development.

“MSX2 belongs to the Msx family, Antennapedia (ANTP) class of human homeobox genes, and has been reported to be involved in bone development, including skull and tooth formation20,21.”

• cites [20] Babajko, S., de La Dure-Molla, M., Jedeon, K. & Berdal, A. MSX2 in ameloblast cell fate and activity. Front. Physiol. 6, 1–10 (2015). sourceSupported
• cites [21] Alappat, S., Zhang, Z. Y. & Chen, Y. P. Msx homeobox gene family and craniofacial development. Cell Res. 13, 429–442 (2003). sourceNot checked — no full text of the source

May combine two claims.

Msx2 null mutant mice show osteopetrosis in a RANK-dependent manner, suggesting Msx2 might participate in osteoclastogenesis.

“Although Msx2 is generally considered an important regulator of osteogenesis, Msx2 null mutant mice show an osteopetrosis phenotype in a RANK-dependent manner in skeleton24, suggesting that Msx2 might also participate in osteoclastogenesis, and its regulatory effect in the bone environment could be complex.”

• cites [24] Castaneda, B., et al. Role of RANKL (TNFSF11)-dependent osteopetrosis in the dental phenotype of Msx2 null mutant mice. PLoS ONE 8, e80054 (2013). sourceSupported

Classified as probably not a substantial claim. May combine two claims.

FBXW7 participates in osteoclastogenesis, and mice with osteoclast-specific Fbxw7 ablation (Fbxw7f/f Ctsk-Cre) exhibit an osteoporotic phenotype.

“FBXW7 has been reported to participate in osteoclastogenesis and mice with osteoclast-specific Fbxw7 ablation (Fbxw7f/f Ctsk-Cre) exhibit osteoporotic phenotype30.”

• cites [30] Fukushima, H. et al. NOTCH2 Hajdu-Cheney mutations escape SCFFBW7-dependent proteolysis to promote osteoporosis. Mol. Cell 68, 645–658.e5 (2017). sourceSupported

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Msx2 deficient adult mice show a slight osteopetrosis phenotype in the skull, tibia, and mandible, which could be reversed by RANK overexpression.

“It has also been reported that Msx2 deficient adult mice show a slight osteopetrosis phenotype in the skull, tibia, and mandible, which could be reversed by RANK overexpression24.”

• cites [24] Castaneda, B., et al. Role of RANKL (TNFSF11)-dependent osteopetrosis in the dental phenotype of Msx2 null mutant mice. PLoS ONE 8, e80054 (2013). sourcePartly supported

  The Results and Discussion report soft osteopetrosis in the tibia that shifted toward osteopenia with RANK overexpression; they also report partial closure of a skull defect. But the Figure 5 caption says the Msx2-deficient mandible had no major alterations compared with wild type, while RANK overexpression caused marked mandibular bone disruption. The paper therefore does not support reversal of a slight osteopetrosis phenotype across all three sites. Page numbers were not available in our copy. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Msx2 promotes primary murine osteoblast differentiation and calcification in concert with typical osteoblastic transcription factors, and ectopic Msx2 rescues impaired osteoblastogenesis induced by Runx2 deficiency.

“It has been reported that Msx2 promotes primary murine osteoblast differentiation and calcification in concert with typical osteoblastic transcription factors, and ectopic Msx2 rescues the impaired osteoblastogenesis induced by Runx2 deficiency47,48.”

• cites [47] Mirzayans, F., Lavy, R., Penner-Chea, J. & Berry, F. B. Initiation of early osteoblast differentiation events through the direct transcriptional regulation of Msx2 by FOXC1. PLoS ONE 7, 1–8 (2012). sourcePartly supported

  The Results and Discussion (page unavailable) report that ectopic Msx2 initiates osteogenic differentiation in C2C12 mouse myoblasts. They do not establish differentiation or calcification in primary murine osteoblasts, or show that Msx2 rescues osteoblastogenesis impaired by Runx2 deficiency. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites [48] Ichida, F. et al. Reciprocal roles of Msx2 in regulation of osteoblast and adipocyte differentiation. J. Biol. Chem. 279, 34015–34022 (2004). sourceNot checked — no full text of the source

Probably not a substantial claim: may not pass S3 (load-bearing).

Antibodies targeting these molecules have also been developed.

“Antibodies targeting these molecules have also been developed52.”

• cites [52] Zhen, G. et al. An antibody against Siglec-15 promotes bone formation and fracture healing by increasing TRAP+ mononuclear cells and PDGF-BB secretion. Bone Res. 9, 47 (2021). sourceSupported

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Msx2 expression responds to BMP2/4, and further activates canonical Wnt signaling in osteoblast progenitors.

“Mechanistically, Msx2 expression responds to BMP2/4, and further activates canonical Wnt signaling in osteoblast progenitors49,50.”

• cites [49] Cheng, S. L., Shao, J. S., Cai, J., Sierra, O. L. & Towler, D. A. Msx2 exerts bone anabolism via canonical Wnt signaling. J. Biol. Chem. 283, 20505–20522 (2008). sourcePartly supported

  The paper reports that Msx2 increases Wnt7 expression, reduces Dkk1, and enhances canonical Wnt signaling in osteoprogenitor cells (opening summary; page unavailable). It supports the downstream Wnt claim, but the passages found do not establish that Msx2 expression responds to BMP2/4. (LLM, conf=0.87)

  Experimental — accuracy not yet measured
• cites [50] Sun, J., Ishii, M., Ting, M. C. & Maxson, R. Foxc1 controls the growth of the murine frontal bone rudiment by direct regulation of a Bmp response threshold of Msx2. Development 140, 1034–1044 (2013). sourcePartly supported

  The Results (page unavailable) show that BMP2-soaked beads increased Msx2 expression, and expanded Bmp2/Bmp4 expression coincided with an expanded Msx2 domain. The paper does not report that Msx2 activates canonical Wnt signaling in osteoblast progenitors; its Results mention Wnt1-Cre only as a lineage-tracing tool. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

Probably not a substantial claim: may not pass S3 (load-bearing).

Molecules affecting osteoclast fusion and angiogenesis, such as SIGLEC15, L-plastin, and TLR2, have been successively discovered.

“Since then, molecules affecting osteoclast fusion and angiogenesis, such as SIGLEC15, L-plastin, and TLR2, have been successively discovered52–54.”

• cites [52] Zhen, G. et al. An antibody against Siglec-15 promotes bone formation and fracture healing by increasing TRAP+ mononuclear cells and PDGF-BB secretion. Bone Res. 9, 47 (2021). sourceSupported
• cites [53] Li, X. et al. Targeting actin-bundling protein L-plastin as an anabolic therapy for bone loss. Sci. Adv. 6, 1–13 (2020). sourceSupported
• cites [54] Dou, C. et al. Sialylation of TLR2 initiates osteoclast fusion. Bone Res. 10, 1–11 (2022). sourceSupported

Probably not a substantial claim: may not pass S3 (load-bearing).

Homeodomain proteins play an active role in adult bone mass regulation.

“it has been reported that homeodomain proteins play an active role in adult bone mass regulation42,43.”

• cites [42] Oono, F., Sakamoto, Y., Tachi, Y., Mabashi-Asazuma, H. & Iida, K. Effect of Cdx2 polymorphism on the relationship between dietary calcium intake and peak bone mass in young Japanese women. Nutrients 12, 191 (2020). sourcePartly supported

  The Introduction identifies Cdx2 as a homeobox protein, and the Discussion reports that calcium intake was associated with peak bone mass differently across Cdx2 genotypes in young women (pages unavailable in our copy). But the paper does not establish that homeodomain proteins generally play an active role in regulating adult bone mass; the Discussion says the Cdx2 polymorphism did not affect bone mass on its own. (LLM, conf=0.84)

  Experimental — accuracy not yet measured
• cites [43] Isaac, J. et al. DLX3 regulates bone mass by targeting genes supporting osteoblast differentiation and mineral homeostasis in vivo. Cell Death Differ. 21, 1365–1376 (2014). sourceSupported

Probably not a substantial claim: may not pass S3 (load-bearing).

A few studies have revealed that homeobox genes, such as Lhx2, Dlx5, and Pax6, affect osteoclast formation.

“A few studies have revealed that homeobox genes, such as Lhx2, Dlx5, and Pax6, affect osteoclast formation44–46”

• cites [44] Kim, J. H. et al. Lhx2 regulates bone remodeling in mice by modulating RANKL signaling in osteoclasts. Cell Death Differ. 21, 1613–1621 (2014). sourceSupported
• cites [45] Lézot, F. et al. Dlx homeobox gene family expression in osteoclasts. J. Cell Physiol. 223, 779–787 (2010). sourceNot checked — no full text of the source
• cites [46] Jie, Z. et al. Activating; -catenin/Pax6 axis negatively regulates osteoclastogenesis by selectively inhibiting phosphorylation of p38/MAPK. FASEB J. 33, 4236–4247 (2019). sourceNot checked — no full text of the source

Probably not a substantial claim: may not pass S3 (load-bearing).

Morusinol reportedly regulates beta3-integrin, tumor necrosis factor, and interleukin 1 beta involved in osteoclastogenesis.

“morusinol reportedly regulates beta3-integrin, tumor necrosis factor, and interleukin 1 beta involved in osteoclastogenesis”

• cites [34] Lee, J. J. et al. Morusinol extracted from Morus alba inhibits arterial thrombosis and modulates platelet activation for the treatment of cardiovascular disease. J. Atheroscler. Thromb. 19, 516–522 (2012). sourceCould not verify

  The platelet-aggregation results and Conclusion report effects of morusinol on platelet aggregation and thromboxane formation, not osteoclastogenesis. Searches for β3-integrin, tumor necrosis factor, interleukin-1β, and osteoclastogenesis found no passage saying morusinol regulates those factors in osteoclastogenesis; the paper mentions β3-integrin only as a platelet receptor. (LLM, conf=0.75)

  Experimental — accuracy not yet measured
• cites [35] Kwon, H. W. Pharmacological actions of morusinol on modulation of platelet functions via integrin αIIb/β3 signaling. J. Appl. Biol. Chem. 66, 171–178 (2023). sourcePartly supported

  The Discussion (page unavailable) reports that morusinol inhibits integrin αIIb/β3 activation in human platelets. The paper does not report regulation of tumor necrosis factor or interleukin 1 beta, or examine osteoclastogenesis; searches for those terms returned no relevant passage. (LLM, conf=0.86)

  Experimental — accuracy not yet measured
• cites [36] Zelová, H. et al. Evaluation of anti-inflammatory activity of prenylated substances isolated from Morus alba and Morus nigra. J. Nat. Prod. 77, 1297–1303 (2014). sourceNot checked — no full text of the source

Probably not a substantial claim: may not pass S3 (load-bearing).

Pre-fusion osteoclasts release PDGF-BB, which promotes H-type vessel formation and subsequently stimulates osteogenesis.

“In 2014, Xie et al. first reported that pre-fusion osteoclasts release PDGF-BB, which promotes H-type vessel formation and subsequently stimulates osteogenesis11.”

• cites [11] Xie, H., et al. PDGF-BB secreted by preosteoclasts induces angiogenesis during coupling with osteogenesis. Nat. Med. 20, 1270–1278 (2014). sourceSupported

May combine two claims.

Anti-resorptive drugs such as bisphosphonates and denosumab cause severe adverse events, poor compliance, and easy recurrence.

“However, these drugs cause severe adverse events, poor compliance, and easy recurrence61”

• cites [61] Ramchand, S. K. & Leder, B. Z. Sequential therapy for the long-term treatment of postmenopausal osteoporosis. J. Clin. Endocrinol. Metab. 1–9, https://doi.org/10.1210/clinem/dgad496 (2023). sourcePartly supported

  The Abstract (page not provided) identifies rare but serious side effects as a concern with long-term bisphosphonate and denosumab use. The Denosumab Discontinuation section describes rebound bone loss and increased fracture risk after denosumab is stopped, not easy recurrence with both drugs. The passages do not support the claim of poor compliance. (LLM, conf=0.87)

  Experimental — accuracy not yet measured

May combine two claims.

PU.1 participates in the entire process of osteoclast differentiation and promotes osteoclast commitment and maturity.

“Considering PU.1 participates in the entire process of osteoclast differentiation and promotes osteoclast commitment and maturity of osteoclast33,57,58”

• cites [33] Carey, H. A. et al. Enhancer variants reveal a conserved transcription factor network governed by PU.1 during osteoclast differentiation. Bone Res. 6, 1–12 (2018). sourceSupported
• cites [57] Izawa, N. et al. Cooperation of PU.1 with IRF8 and NFATc1 defines chromatin landscapes during RANKL-induced osteoclastogenesis. J. Bone Miner. Res. 34, 1143–1154 (2019). sourcePartly supported

  The Discussion reports PU.1 motifs in precursor macrophages and osteoclasts, and notes that mice lacking PU.1 lack mature osteoclasts. The paper supports a role in osteoclast differentiation, but its findings do not establish that PU.1 promotes commitment or participates in every stage of the process. (LLM, conf=0.82)

  Experimental — accuracy not yet measured
• cites [58] de la Rica, L. et al. PU.1 target genes undergo Tet2-coupled demethylation and DNMT3b-mediated methylation in monocyte-to-osteoclast differentiation. Genome Biol. 14, 1–21 (2013). sourcePartly supported

  The Discussion identifies PU.1 as an early influence on osteoclast-lineage commitment, and the Background and Results link it to activation of osteoclast-specific genes during differentiation. These passages support a role in commitment and differentiation, but do not establish that PU.1 participates throughout the entire process or promotes osteoclast maturity specifically. Page numbers are unavailable in our copy. (LLM, conf=0.82)

  Experimental — accuracy not yet measured

May combine two claims.

Loss of Msx2 acts as a brake in the fusion fate of osteoclasts, resulting in a larger population of pre-osteoclasts.

“Loss of Msx2 acts as a “brake” in the fusion fate of osteoclasts, resulting in a larger population of pre-osteoclasts.”

May combine two claims.

Msx2 knockout altered PU.1 protein level but not mRNA level.

“After treating Msx2f/f BMMs with Cre or Msx2 adenoviruses, the protein level of PU.1 was altered, whereas the mRNA level of PU.1 remained unchanged (Fig. 3d–f).”

May combine two claims.

Msx2 deficient cells generated reduced resorption pit areas and produced fewer CTSK.

“As shown in Fig. 1h–j, Msx2 deficient cells generated reduced resorption pit areas on bone slices and produced fewer resorptive enzymes, such as CTSK.”

May combine two claims.

Analysis revealed 18 TFs, and MSX2 is not among them.

“This analysis revealed 18 TFs (Supplementary Fig. 4a).”

• Also stated in: § MSX2 promotes pre-osteoclast fusion through stabilizing PU.1 · plain

  “Notably, MSX2 is not among these 18, although it is a well-known TF.”

May combine two claims.

Morusinol can bind to MSX2 and release PU.1, promoting PU.1 degradation.

“Finally, we screened a natural compound library and found that morusinol, a flavonoid isolated from Morus alba root bark, can bind to MSX2 and release PU.1, promoting PU.1 degradation.”

May combine two claims.

Overexpressing PU.1 rescued formation of multinucleated osteoclasts and expression of fusion-related genes.

“Overexpressing of PU.1 indeed rescued the formation of multinucleated osteoclasts and the expression of fusion-related genes (Fig. 3a–c).”

May combine two claims.

Msx2 protein levels peaked on day 3 and remained constant on day 6.

“The protein levels of Msx2 also peaked on day 3 and remained constant on day 6 (Supplementary Fig. 1f, g).”

May combine two claims.

Msx2 deficiency in osteoclast progenitors resulted in higher OCN+ cells, enhanced MAR, and enhanced BFR/BS.

“Here, Msx2 deficiency in osteoclast progenitors also resulted in changes on the bone formation side, with a higher osteocalcin (OCN) -positive cell number, enhanced mineral apposition rate (MAR), and bone formation rate per bone surface (BFR/BS) in Msx2 cKO mice (Fig. 2a–d).”

May combine two claims.

Msx2 deficiency resulted in elevated BV/TV, Tb.N, Ct.Th, and reduced Tb.Sp in males.

“Similar to that in females, Msx2 deficiency resulted in elevated BV/TV, Tb.N, Ct.Th, and reduced Tb.Sp in males (Supplementary Fig. 2).”

May combine two claims.

Msx2 deficiency impairs osteoclast fusion and maintains osteoclasts at pre-fusion stage.

“Msx2 deficiency impairs osteoclast fusion and exerts the role of “brake” in the progression of osteoclast cell fate, which subsequently maintains osteoclasts at the pre-fusion stage.”

May combine two claims.

HUVECs cultured in Msx2 cKO medium exhibited elevated migration and tube formation.

“The wound-healing assay showed that HUVECs cultured in Msx2 cKO medium exhibited elevated migration activity (Supplementary Fig. 3e, f).”

• Also stated in: § Msx2 deficiency promotes vessel formation of endothelial cells in vitro · hedged

  “The Matrigel tube formation assay also indicated that Msx2 cKO conditioned medium enhanced HUVECs tube formation (Supplementary Fig. 3g, h).”

May combine two claims.

Msx2 cKO mice exhibited elevated vessel volume and vessel surface.

“Angiography showed that Msx2 cKO mice exhibited elevated vessel volume and vessel surface in the bones (Fig. 2e, f).”

May combine two claims.

Msx2 cKO cells exhibited higher Pdgfb expression and produced more PDGF-BB.

“Msx2 cKO cells exhibited higher Pdgfb expression levels and produced more PDGF-BB in the medium (Supplementary Fig. 3a–c).”

May combine two claims.

Msx2 mRNA levels were upregulated on day 3 and decreased on day 6 of osteoclastogenesis.

“The mRNA levels were upregulated on day 3 of osteoclastogenesis and decreased on day 6 (Supplementary Fig. 1e).”

May combine two claims.

PU.1 showed higher frequency and binding score in all five fusion-related genes, meaning PU.1 is the most probable TF regulating fusion-related genes.

“Therefore, we analyzed the CHIP-Seq data of fusion-related genes from Genecards, and PU.1 (Spi1) showed a relatively higher frequency and binding score in all five fusion-related genes, which means PU.1 is the most probable TF regulating fusion-related genes (Supplementary Fig. 4b–f).”

May combine two claims.

Msx2 cKO mice exhibited increased BV/TV, Tb.N, Ct.Th, decreased Tb.Sp, and unchanged Tb.Th.

“Micro-computed tomography (Micro-CT) analysis showed that bone volume per tissue volume (BV/TV), trabecular bone number (Tb.N), and cortical bone thickness (Ct.Th) increased, while trabecular bone separation (Tb.Sp) decreased in Msx2 cKO mice; trabecular bone thickness (Tb.Th) remained unchanged (Fig. 1b).”

May combine two claims.

MSX2 and PU.1 are both nuclear.

“Immunofluorescence labeling revealed that MSX2 and PU.1 are both nuclear (Fig. 4a).”

May combine two claims.

Knocking out Msx2 enhanced PU.1 degradation; overexpression of MSX2 suppressed PU.1 degradation.

“Moreover, knocking out Msx2 enhanced PU.1 degradation when treated with cycloheximide (CHX) (Fig. 3g, h), whereas overexpression of MSX2 suppressed PU.1 degradation (Supplementary Fig. 5a, b).”

May combine two claims.

Msx2 cKO mice showed more OCN-positive cells, higher MAR and BFR/BS after OVX.

“On the bone formation side, Msx2 cKO mice showed more OCN-positive cells, higher MAR and BFR/BS (Supplementary Fig. 8f–i).”

May combine two claims.

Protein levels of CTSK and PU.1 were reduced by morusinol.

“The protein level of CTSK and PU.1 was also reduced by morusinol (Fig. 6h, i).”

May combine two claims.

Msx2 cKO mice exhibited increased Tb.N, Ct.Th, and decreased Tb.Sp after OVX.

“Moreover, Msx2 cKO mice exhibited increased Tb.N, Ct.Th, and decreased Tb.Sp after OVX (Supplementary Fig. 8b).”

May combine two claims.

Msx2 cKO pre-osteoclasts exhibited increased PU.1 ubiquitination; overexpression of MSX2 attenuated PU.1 ubiquitination.

“Subsequently, we performed ubiquitination assays and found Msx2 cKO pre-osteoclasts exhibited increased PU.1 ubiquitination (Fig. 3k).”

• Also stated in: § MSX2 promotes pre-osteoclast fusion through stabilizing PU.1 · plain

  “In contrast, overexpression of MSX2 attenuated PU.1 ubiquitination, similar results were obtained in the 293T cell line (Supplementary Fig. 5c, d).”

May combine two claims. The quote only partly states this claim.

Angiogenesis was more active in Msx2 cKO mice, with more EMCNhi CD31hi vessels after OVX.

“Consistently, angiogenesis was more active in Msx2 cKO mice, with more EMCNhi CD31hi vessels (Supplementary Fig. 8j, k).”

May combine two claims.

Myeloid Fbxw7 deficiency (Fbxw7f/f LysM-Cre, Fbxw7 cKO) leads to low bone mass, with reduced BV/TV, Tb.Th, Tb.N, and elevated Tb.Sp.

“Here, we found that myeloid Fbxw7 deficiency (Fbxw7f/f LysM-Cre, Fbxw7 cKO) also led to low bone mass, with reduced BV/TV, Tb.Th, Tb.N, and elevated Tb.Sp (Supplementary Fig. 6a, b).”

May combine two claims.

Exogenous MSX2 reduced PU.1 ubiquitination, rescued by FBXW7 overexpression.

“Expectedly, exogenous MSX2 significantly reduced the ubiquitination level of PU.1, which was rescued by FBXW7 overexpression in HEK-293T cells (Fig. 4e).”

May combine two claims.

FBXW7 promoted PU.1 ubiquitination, canceled by PU.1 mutation.

“We further mutated PU.1-Ser41 and PU.1-Ser142 (PU.1-MUT: PU.1-S41A, S142A) and found that FBXW7 could promote PU.1 ubiquitination, however, this effect was canceled by PU.1 mutation (Fig. 4h).”

May combine two claims.

Msx2 expression is upregulated in PMOP bones and during osteoclastogenesis.

“Msx2 expression is upregulated in PMOP bones and during osteoclastogenesis.”

May combine two claims.

Morusinol targets the MSX2-PU.1 complex and inhibits osteoclast fusion.

“Fig. 6 Morusinol targets MSX2-PU.1 complex and inhibits osteoclast fusion.”

May combine two claims.

Morusinol treatment increased OCN-positive cells and enhanced MAR and BFS/BS.

“In terms of bone formation, morusinol treatment increased OCN-positive cells and enhanced MAR and BFS/BS (Fig. 7i–l).”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Morusinol subtly influenced expression of representative pro-inflammatory cytokines, including Il-1b, Tnf-a, and Il-6, only slightly enhancing Il1-b.

“morusinol subtly influenced the expression of representative pro-inflammatory cytokines, including Il-1b, Tnf-a, and Il-6, only slightly enhancing Il1-b.”

May combine two claims.

OVX mice exhibited increased multi-nuclear osteoclasts and reduced mono-nuclear osteoclasts.

“TRAP staining showed that OVX mice exhibited increased multi-nuclear osteoclasts and reduced mono-nuclear osteoclasts”

May combine two claims.

Molecular docking showed that morusinol forms hydrophobic interactions with Val32 and Leu179, a hydrogen bond interaction with Ser178, and π-cation with Lys33.

“morusinol forms hydrophobic interactions with Val32 and Leu179, a hydrogen bond interaction with Ser178, and π-cation with Lys33 (Fig. 6n).”

May combine two claims.

Morusinol can target MSX2 and disrupt the interaction between MSX2 and PU.1.

“In this study, we found that morusinol can target MSX2 and disrupt the interaction between MSX2 and PU.1.”

May combine two claims.

Morusinol treatment attenuates multi-nuclear osteoclast formation both in vitro and in vivo.

“Morusinol treatment attenuates multi-nuclear osteoclast formation both in vitro and in vivo.”

May combine two claims.

Knocking out Msx2 in osteoclast progenitors inhibited bone resorption, enhanced angiogenesis-mediated bone formation, and rescued bone mass in an OVX-induced osteoporosis mouse model.

“Knocking out Msx2 in osteoclast progenitors inhibited bone resorption, enhanced angiogenesis-mediated bone formation, and rescued bone mass in an OVX-induced osteoporosis mouse model.”

May combine two claims.

Msx2 deficiency in myeloid cells reduces bone resorption and increases bone mass.

“Here, we demonstrate that Msx2 deficiency in myeloid cells reduces bone resorption and increases bone mass.”

May combine two claims.

Msx2 and Fbxw7 double knockout rescued PU.1, and subsequently osteoclast fusion, which was also confirmed in vivo.

“Msx2 and Fbxw7 double knockout rescued PU.1, and subsequently osteoclast fusion, which was also confirmed in vivo.”

May combine two claims.

Morusinol promoted vessel formation and osteogenesis.

“Meanwhile, morusinol promoted vessel formation and osteogenesis.”

May combine two claims.

In PMOP, osteoclasts are over-activated and mediate enhanced bone resorption, leading to severe bone loss.

“In the bone environment of PMOP, osteoclasts are over-activated and mediate significantly enhanced bone resorption, leading to severe bone loss7.”

• cites [7] Song, S., Guo, Y., Yang, Y. & Fu, D. Advances in pathogenesis and therapeutic strategies for osteoporosis. Pharm. Ther. 237, 108168 (2022). sourceNot checked — no full text of the source

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Functional haploinsufficiency of MSX2 causes PFM, and whole-body Msx2 knockout mice exhibit pleiotropic defects.

“Functional haploinsufficiency of MSX2 causes parietal foramina (PFM), and whole-body Msx2 knockout mice exhibit pleiotropic defects in bone mass and ectodermal organ formation22,23.”

• cites [22] Satokata, I. et al. Msx2 deficiency in mice causes pleiotropic defects in bone growth and ectodermal organ formation. Nat. Genet. 24, 391–395 (2000). sourceNot checked — no full text of the source
• cites [23] Wilkie, A. O. M. et al. Functional haploinsufficiency of the human homeobox gene MSX2 causes defects in skull ossification. Nat. Genet. 24, 387–390 (2000). sourceNot checked — no full text of the source

May combine two claims.

Loss of Msx2 in myeloid cells increases bone mass by impairing osteoclastogenesis and enhancing angiogenesis and osteogenesis in vivo.

“Collectively, the loss of Msx2 in myeloid cells increases bone mass by impairing osteoclastogenesis and enhancing angiogenesis and osteogenesis in vivo.”

May combine two claims.

Msx2 is upregulated in PMOP and osteoclastogenesis, and may be a regulator of osteoclast formation.

“Together, these findings suggest that Msx2 is upregulated in PMOP and osteoclastogenesis, and may be a potential regulator of osteoclast formation.”

• AssumptionRests on an assumption: The direct role of Msx2 in osteoclasts/osteoclastogenesis lacks solid evidence and its underlying mechanism remains unclear.

May combine two claims.

Restraining osteoclast fusion and maintaining preosteoclast stage could suppress bone resorption while coupling angiogenesis and osteogenesis, with potential for osteoporosis treatment.

“Therefore, restraining osteoclast fusion and maintaining them in the preosteoclast stage could suppress bone resorption while coupling angiogenesis and osteogenesis, exhibiting great potential for osteoporosis treatment.”

May combine two claims.

Myeloid Msx2 deficiency enhances vessel formation of endothelial cells and the observed promoting effect on osteogenesis in vivo is indirect.

“These results suggest that myeloid Msx2 deficiency enhances vessel formation of endothelial cells and that the observed promoting effect on osteogenesis in vivo is indirect.”

May combine two claims.

MSX2 plays a critical role in PMOP pathogenesis and bone metabolism, coupling osteoclastogenesis with angiogenesis and osteogenesis, and may be a target for PMOP treatment.

“Our results suggest that MSX2 plays a critical role in PMOP pathogenesis and bone metabolism, coupling osteoclastogenesis with angiogenesis and osteogenesis, and may be a potential target for the treatment of PMOP.”

• AssumptionRests on an assumption: The current understanding of the role of homeobox genes in PMOP and osteoclastogenesis is superficial and scattered, and the hub homeobox genes that regulate PMOP remain unclear.
• AssumptionRests on an assumption: The direct role of Msx2 in osteoclasts/osteoclastogenesis lacks solid evidence and its underlying mechanism remains unclear.

Probably not a substantial claim: may not pass S3 (load-bearing).

Natural compounds have shown advantages in terms of safety, effectiveness, and convenience in disease treatment.

“Natural compounds have shown advantages in terms of safety, effectiveness, and convenience in disease treatment31”

• cites [31] Rodrigues, T., Reker, D., Schneider, P. & Schneider, G. Counting on natural products for drug design. Nat. Chem. 8, 531–541 (2016). sourceNot checked — no full text of the source

Probably not a substantial claim: may not pass S3 (load-bearing).

All experiments were repeated 3 times independently.

“All experiments were repeated 3 times independently, and the representative images were shown (a–c, e, and h).”

May combine two claims.

F4/80 expression was comparable in Msx2f/f and Msx2 cKO mice, indicating no influence on myeloid differentiation and osteoclast precursor formation.

“The expression level of F4/80 was comparable in Msx2f/f and Msx2 cKO mice, indicating that myeloid Msx2 deficiency does not no influence on myeloid differentiation and osteoclast precursor formation (Supplementary Fig. 5e, f).”

• AssumptionRests on an assumption: PU.1 is crucial for myeloid differentiation.

May combine two claims.

MSX2 promotes expression of osteoclast fusion-related genes by preventing proteasome-mediated degradation of PU.1 without affecting macrophage and osteoclast precursor development.

“In summary, MSX2 promotes the expression of osteoclast fusion-related genes by preventing the proteasome-mediated degradation of PU.1 without affecting macrophage and osteoclast precursor development.”

• AssumptionRests on an assumption: MSX2 serves as a co-transcriptional regulator and affects mRNA expression through other transcription factors.
• AssumptionRests on an assumption: PU.1 is crucial for myeloid differentiation.

Probably not a substantial claim: may not pass S3 (load-bearing).

Osteoblast lineage cells could secrete RANKL and induce osteoclastogenesis.

“The most classic and well-known interaction between bone cells is that osteoblast lineage cells could secrete RANKL and induce osteoclastogenesis51.”

• cites [51] Martin, T. J. & Sims, N. A. RANKL/OPG; Critical role in bone physiology. Rev. Endocr. Metab. Disord. 16, 131–139 (2015). sourceNot checked — no full text of the source

May combine two claims.

Morusinol may inhibit osteoclastogenesis by interrupting the combination of MSX2 and PU.1 and promoting PU.1 degradation.

“thus, morusinol may inhibit osteoclastogenesis by interrupting the combination of MSX2 and PU.1 and promoting PU.1 degradation.”

May combine two claims.

Pre-osteoclasts secrete PDGF-BB and enhance angiogenesis-mediated bone formation.

“Pre-osteoclasts secrete PDGF-BB and enhance angiogenesis-mediated bone formation (upper part of Fig. 8).”

Probably not a substantial claim: may not pass S2 (checkable). May combine two claims.

The role of Msx2 in different types of bone cells is complex, and conditional Msx2 deficient models are warranted.

“Therefore, the role of Msx2 in different types of bone cells is complex, and conditional Msx2 deficient models are warranted.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

MSX2 mutations cause Boston-type craniosynostosis, and haploinsufficiency of human MSX2 causes deficient ossification around the parietal notch.

“MSX2 mutations cause Boston-type craniosynostosis, and haploinsufficiency of human MSX2 causes deficient ossification around the parietal notch23.”

• cites [23] Wilkie, A. O. M. et al. Functional haploinsufficiency of the human homeobox gene MSX2 causes defects in skull ossification. Nat. Genet. 24, 387–390 (2000). sourceNot checked — no full text of the source

Probably not a substantial claim: may not pass S3 (load-bearing).

Angiogenesis plays an important role in mediating bone cell crosstalk and regulating bone mass.

“These studies emphasize the important role of angiogenesis in mediating bone cell crosstalk and regulating bone mass.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Msx2 deficient P30 mice exhibit an osteopenia phenotype with predominant osteoblast defects.

“Msx2 deficient P30 mice exhibit an osteopenia phenotype with predominant osteoblast defects.”

• cites [22] Satokata, I. et al. Msx2 deficiency in mice causes pleiotropic defects in bone growth and ectodermal organ formation. Nat. Genet. 24, 391–395 (2000). sourceNot checked — no full text of the source

May combine two claims.

Knocking out Msx2 increases bone mass by attenuating osteoclastogenesis and enhancing angiogenesis-mediated bone formation.

“As shown in Fig. 8, knocking out Msx2 increases bone mass by attenuating osteoclastogenesis and enhancing angiogenesis-mediated bone formation.”

Probably not a substantial claim: may not pass S4 (based).

MSX2 is important in regulating Wnt signaling and bone formation in concert with osteogenic transcription factors.

“voluminous prior studies have reported that MSX2 is important in regulating Wnt signaling and bone formation in concert with osteogenic transcription factors”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Whole-body Msx2 deficient mice are viable but with a variety of malformations, including PFM, defective cerebellum, tooth, hair follicle, and mammary gland development.

“In mouse models, whole-body Msx2 deficient mice are viable but with a variety of malformations, including PFM, defective cerebellum, tooth, hair follicle, and mammary gland development22.”

• cites [22] Satokata, I. et al. Msx2 deficiency in mice causes pleiotropic defects in bone growth and ectodermal organ formation. Nat. Genet. 24, 391–395 (2000). sourceNot checked — no full text of the source

May combine two claims.

Msx2 null mutation leads to periodontal osteopetrosis, with impaired osteoclast generation in alveolar bones.

“In a Msx2−/− transgenic mouse model, Msx2 null mutation leads to periodontal osteopetrosis, with impaired osteoclast generation in alveolar bones26.”

• cites [26] Aïoub, M. et al. Msx2 -/- transgenic mice develop compound amelogenesis imperfecta, dentinogenesis imperfecta and periodental osteopetrosis. Bone 41, 851–859 (2007). sourceNot checked — no full text of the source

May combine two claims.

In wild-type myeloid cells, MSX2 binds PU.1 and prevents PU.1 from FBXW7 mediated ubiquitination, which safeguards fusion cell fate of osteoclast and results in bone resorption.

“In wild-type myeloid cells, MSX2 binds PU.1 and prevents PU.1 from FBXW7 mediated ubiquitination, which safeguards fusion cell fate of osteoclast and results in bone resorption.”

May combine two claims.

Targeting Msx2 promotes PU.1 degradation and acts as a "brake" in the progression of the fusion fate of osteoclast, resulting in a larger population of pre-osteoclasts.

“Targeting Msx2 promotes PU.1 degradation and acts as a “brake” in the progression of the fusion fate of osteoclast, resulting in a larger population of pre-osteoclasts.”

May combine two claims.

High levels of PDGF-BB promote H-type vessel formation and subsequently stimulate osteogenesis.

“High levels of PDGF-BB promote H-type vessel formation and subsequently stimulate osteogenesis.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Adipocytes in bone marrow could couple angiogenesis through estrogen-related receptor α.

“Moreover, a recent study also demonstrated that adipocytes in bone marrow could couple angiogenesis through estrogen-related receptor α55.”

• cites [55] Huang, T. et al. Targeting adipocyte ESRRA promotes osteogenesis and vascular formation in adipocyte-rich bone marrow. Nat. Commun. 15, 1–20 (2024).Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

May combine two claims.

Targeting MSX2 with morusinol inhibits bone resorption, enhances bone and vessel formation, and protects bone mass.

“Overall, targeting MSX2 with morusinol inhibits bone resorption, enhances bone and vessel formation, and protects bone mass.”

Probably not a substantial claim: may not pass S3 (load-bearing).

The reduction of beta3-integrin by morusinol may be a downstream effect of PU.1.

“it may also be a downstream effect of PU.1, which has been reported to transactivate beta3-integrin in concert with NFATc137.”

• cites [37] Crotti, T. N. et al. PU.1 and NFATc1 mediate osteoclastic induction of the mouse β3 integrin promoter. J. Cell Physiol. 215, 636–644 (2008). sourceNot checked — no full text of the source

May combine two claims.

Morusinol directly disrupts the MSX2-PU.1 complex and serves as an anabolic therapy for osteoporosis.

“identify morusinol, which directly disrupts the MSX2-PU.1 complex and serves as an anabolic therapy for osteoporosis.”

• AssumptionRests on an assumption: Compared to synthetic compounds and biological agents, natural products have greater molecular rigidity, which makes them more valuable in tackling protein-protein interactions.

May combine two claims.

Knocking out Msx2 in osteoclast progenitors improves bone mass, with impaired osteoclastogenesis mainly manifesting as impaired osteoclast fusion rather than reduced osteoclast numbers.

“We generated myeloid-specific Msx2 deficiency mice and demonstrated that knocking out Msx2 in osteoclast progenitors improves bone mass, with impaired osteoclastogenesis mainly manifesting as impaired osteoclast fusion rather than reduced osteoclast numbers.”

May combine two claims.

Targeting MSX2 primarily affects the osteoclast fusion stage, which evades the potential risk of PU.1 deficiency in myeloid development.

“the expression pattern of Msx2 indicates that targeting MSX2 primarily affects the osteoclast fusion stage, which evades the potential risk of PU.1 deficiency in myeloid development.”

Probably not a substantial claim: may not pass S3 (load-bearing).

All animal procedures were conducted in compliance with the approved ethical guidelines.

“All animal procedures were ethically approved by Sir Run Run Shaw Hospital Committee for Animal Resources, and were conducted in compliance with the approved ethical guidelines.”

Probably not a substantial claim: may not pass S4 (based).

Ni-1 precisely reflects the number of fusion events to form a syncytium with Ni nuclei, regardless of whether the syncytium was generated by fusion between two mono-nuclear cells, one mono-nuclear cell and one multi-nuclear cell, or two multi-nuclear cells.

“Ni-1 could precisely reflect the number of fusion events to form a syncytium with Ni nuclei irrespective of whether the syncytium was generated by fusion between two mono-nuclear cells, one mono-nuclear cell and one multi-nuclear cell or two multi-nuclear cells”

Classified as probably not a substantial claim.

Positively stained cells were quantified in five randomly selected fields of the distal metaphysis of femurs using Halo software.

“we quantified the positively stained cells in five randomly selected fields of the distal metaphysis of femurs using Halo software.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Teriparatide and romosozumab have been developed and applied clinically, drawing more attention to anabolic therapeutic strategies for osteoporosis treatment.

“Recently, drugs such as teriparatide and romosozumab have been developed and applied clinically, drawing more attention to anabolic therapeutic strategies for osteoporosis treatment62.”

• cites [62] Langdahl, B. L. et al. Romosozumab (sclerostin monoclonal antibody) versus teriparatide in postmenopausal women with osteoporosis transitioning from oral bisphosphonate therapy: a randomised, open-label, phase 3 trial. Lancet. 390, 1585–1594 (2017). sourceNot checked — no full text of the source

Probably not a substantial claim: may not pass S3 (load-bearing).

All animal procedures were ethically approved by the Sir Run Run Shaw Hospital Committee for Animal Resources.

“All animal procedures were ethically approved by Sir Run Run Shaw Hospital Committee for Animal Resources, and were conducted in compliance with the approved ethical guidelines.”

Probably not a substantial claim: may not pass S3 (load-bearing).

All animal procedures were ethically approved by Sir Run Run Shaw Hospital Committee for Animal Resources.

“All animal procedures were ethically approved by Sir Run Run Shaw Hospital Committee for Animal Resources”

Probably not a substantial claim: may not pass S3 (load-bearing).

It is difficult to rule out the direct transcriptional effect of MSX2 on fusion-related genes.

“it is still difficult to rule out the direct transcriptional effect of MSX2 on these genes.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Primary antibodies from different sources and a specific secondary antibody were used in the Co-IP assay to avoid heavy/light chain interference.

“Primary antibodies from different sources and specific secondary antibody (AS062, Abclonal and abs20213, Absin) were used to avoid heavy/light chain interference.”

May combine two claims.

Msx2 deficiency impairs osteoclast fusion and exerts the role of "brake" in the progression of osteoclast cell fate, leading to increased pre-osteoclast numbers and PDGF-BB secretion.

“Specifically, Msx2 deficiency impairs osteoclast fusion and exerts the role of “brake” in the progression of osteoclast cell fate, leading to increased pre-osteoclast numbers and PDGF-BB secretion.”

May combine two claims.

Targeting MSX2 acts as a "brake" in the progression of osteoclast cell fate by promoting PU.1 degradation, which subsequently enhances angiogenesis-mediated bone formation.

“Targeting MSX2 acts as a “brake” in the progression of osteoclast cell fate by promoting PU.1 degradation, which subsequently enhances angiogenesis-mediated bone formation.”

Classified as probably not a substantial claim.

Thirty mice were randomly categorized into three groups (Sham, OVX, and OVX + morusinol), with 10 mice per group.

“For the morusinol treatment, 30 mice were randomly categorized into three groups: Sham, OVX, and OVX + murosinol, with 10 mice per group.”

Classified as probably not a substantial claim.

The normalized number of fusion events, calculated as Σ(Ni-1)/Ntotal, precisely evaluates the fusion efficiency of osteoclasts.

“To precisely evaluate the fusion efficiency of osteoclasts, we calculated the normalized number of fusion events according to a previous report.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Ntotal normalizes the subtle discrepancy of cell density among samples.

“Ntotal was used to normalize the subtle discrepancy of cell density among samples.”

May combine two claims.

Morusinol targets MSX2 and serves as an anabolic therapy for osteoporosis.

“Morusinol targets MSX2 and serves as an anabolic therapy for osteoporosis.”

• AssumptionRests on an assumption: Compared to synthetic compounds and biological agents, natural products have greater molecular rigidity, which makes them more valuable in tackling protein-protein interactions.

Probably not a substantial claim: may not pass S3 (load-bearing).

β-actin was used as the internal control for relative gene expression calculations.

“The relative gene expressions were calculated with the −ΔCT method, and β-actin was used as the internal control.”