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. 2023 Jun 28;24(13):10797.
doi: 10.3390/ijms241310797.

Accelerated Bone Loss in Transgenic Mice Expressing Constitutively Active TGF-β Receptor Type I

Parichart Toejing  1 , Nithidol Sakunrangsit  1 , Pinyada Pho-On  1 , Chinnatam Phetkong  1 , Asada Leelahavanichkul  2 , Somyoth Sridurongrit  3 , Matthew B Greenblatt  4 , Sutada Lotinun  1
Affiliations

Accelerated Bone Loss in Transgenic Mice Expressing Constitutively Active TGF-β Receptor Type I

Parichart Toejing et al. Int J Mol Sci. .

Abstract

Transforming growth factor beta (TGF-β) is a key factor mediating the intercellular crosstalk between the hematopoietic stem cells and their microenvironment. Here, we investigated the skeletal phenotype of transgenic mice expressing constitutively active TGF-β receptor type I under the control of Mx1-Cre (Mx1;TβRICA mice). μCT analysis showed decreased cortical thickness, and cancellous bone volume in both femurs and mandibles. Histomorphometric analysis confirmed a decrease in cancellous bone volume due to increased osteoclast number and decreased osteoblast number. Primary osteoblasts showed decreased ALP and mineralization. Constitutive TβRI activation increased osteoclast differentiation. qPCR analysis showed that Tnfsf11/Tnfrsf11b ratio, Ctsk, Sufu, and Csf1 were increased whereas Runx2, Ptch1, and Ptch2 were decreased in Mx1;TβRICA femurs. Interestingly, Gli1, Wnt3a, Sp7, Alpl, Ptch1, Ptch2, and Shh mRNA expression were reduced whereas Tnfsf11/Tnfrsf11b ratio was increased in Mx1;TβRICA mandibles. Similarly, osteoclast-related genes were increased in Mx1;TβRICA osteoclasts whereas osteoblast-related genes were reduced in Mx1;TβRICA osteoblasts. Western blot analysis indicated that SMAD2 and SMAD3 phosphorylation was increased in Mx1;TβRICA osteoblasts, and SMAD3 phosphorylation was increased in Mx1;TβRICA osteoclasts. CTSK was increased while RUNX2 and PTCH1 was decreased in Mx1;TβRICA mice. Microindentation analysis indicated decreased hardness in Mx1;TβRICA mice. Our study indicated that Mx1;TβRICA mice were osteopenic by increasing osteoclast number and decreasing osteoblast number, possibly by suppressing Hedgehog signaling pathways.

Keywords: TGF-β; bone; hardness; osteoblast; osteoclast.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
PCR of genomic DNA from mouse tails showing bands in Mx1;TβRI mice using pRCA/pPA, pHPRT, and Mx1-Cre primers.
Figure 2
Figure 2
Serum chemistries of Mx1;TβRICA mice. Serum levels of (A) phosphorus (n = 6), (B) calcium (n = 6), and (C) PTH (n = 6) from Mx1;TβRICA mice compared to WT mice. Data are mean ± SEM. * p < 0.05 compared to WT.
Figure 3
Figure 3
Mx1;TβRICA mice are osteopenic. (A) Body weight (n = 6). (B) Femur and mandible length (n = 6). (C) Representative µCT images of femurs and mandibles. (D) µCT analysis of cancellous bone in femurs and mandibles from Mx1;TβRICA mice compared to WT controls (n = 5–6). (E) µCT analysis of cortical bone in femurs and mandibles from Mx1;TβRICA mice compared to WT controls (n= 5–6). Data are mean ± SEM. * p < 0.05 compared to WT. BV/TV; bone volume per tissue volume, Tb.Th; trabecular thickness, Tb.N; trabecular number, Tb.Sp; trabecular separation, Conn.D; connectivity density, SMI; structural model index, and BMD; bone mineral density.
Figure 3
Figure 3
Mx1;TβRICA mice are osteopenic. (A) Body weight (n = 6). (B) Femur and mandible length (n = 6). (C) Representative µCT images of femurs and mandibles. (D) µCT analysis of cancellous bone in femurs and mandibles from Mx1;TβRICA mice compared to WT controls (n = 5–6). (E) µCT analysis of cortical bone in femurs and mandibles from Mx1;TβRICA mice compared to WT controls (n= 5–6). Data are mean ± SEM. * p < 0.05 compared to WT. BV/TV; bone volume per tissue volume, Tb.Th; trabecular thickness, Tb.N; trabecular number, Tb.Sp; trabecular separation, Conn.D; connectivity density, SMI; structural model index, and BMD; bone mineral density.
Figure 4
Figure 4
Constitutive activation of TβRI decreases osteoblast differentiation. (A) ALP staining in osteoblasts derived from the long bones and mandibles of Mx1;TβRICA mice compared to WT controls. 4× Magnification, Scale bar = 200 μm. (B) ALP activity in osteoblasts (n = 4). (C) Alizarin red staining in osteoblasts derived from long bones and mandibles of Mx1;TβRICA mice compared to WT controls. 4× Magnification, Scale bar = 200 μm. (D) Mineralization in osteoblasts (n = 5). Results are mean ± SEM. * p < 0.05 compared to WT.
Figure 5
Figure 5
Constitutive activation of TβRI increases osteoclast differentiation. (A) TRAP staining in osteoclasts. 10x Magnification, Scale bar = 100 μm. (B) Osteoclast number per area (N.Oc/Ar) from long bones and mandibles in Mx1;TβRICA mice compared to WT control (n = 4). Results are mean ± SEM. * p < 0.05 compared to WT controls.
Figure 6
Figure 6
Constitutive TβRI activation increases osteoclast- and decreases osteoblast-related gene expression in femurs and mandibles. (A) qPCR analysis of osteoblast and osteoclast related genes expression in femurs (n = 5–6) and (B) mandibles from Mx1;TβRICA mice compared to WT controls (n = 5–6). Results are mean ± SEM. * p < 0.05 compared to WT.
Figure 7
Figure 7
Constitutive TβRI activation increases osteoclast- and decreases osteoblast-related gene expression in vitro. (A) qPCR analysis of TGF-β and their receptors from osteoblasts and osteoclasts (n = 3). (B) osteoblast and osteoclast related genes expression in vitro derived from long bone of Mx1;TβRICA mice compared to WT controls (n = 3–4). Results are mean ± SEM. * p < 0.05 compared to WT.
Figure 8
Figure 8
Mx1;TβRICA mice induced TGF-β signaling, decreased RUNX2, and PTCH1 expression and increased CTSK expression. (A) SMAD2 and 3 phosphorylation, RUNX2 and PTCH1 protein levels in osteoblasts cells derived from long bones of Mx1;TβRICA mice compared to WT controls (n = 4). (B) SMAD2 and 3 phosphorylation, CTSK protein levels in osteoclast cells derived from long bones of Mx1;TβRICA mice compared to WT controls (n = 4). Results are mean ± SEM. * p < 0.05 compared to WT.
Figure 8
Figure 8
Mx1;TβRICA mice induced TGF-β signaling, decreased RUNX2, and PTCH1 expression and increased CTSK expression. (A) SMAD2 and 3 phosphorylation, RUNX2 and PTCH1 protein levels in osteoblasts cells derived from long bones of Mx1;TβRICA mice compared to WT controls (n = 4). (B) SMAD2 and 3 phosphorylation, CTSK protein levels in osteoclast cells derived from long bones of Mx1;TβRICA mice compared to WT controls (n = 4). Results are mean ± SEM. * p < 0.05 compared to WT.
Figure 9
Figure 9
Mx1;TβRICA mice have decreased bone hardness. (A) Five indents of tibial cortical mid-shaft. (B) Hardness of tibial cortical bone in Mx1;TβRICA mice compared to WT controls (n = 8). Results are mean ± SEM. * p < 0.05 compared to WT. HV; Vickers hardness.
Figure 10
Figure 10
Constitutive activation of TβRI engages TGF-β signaling and affects osteoblast and osteoclast transcriptional profiles, leading to bone loss.
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References

    1. Janssens K., ten Dijke P., Janssens S., Van Hul W. Transforming Growth Factor-β1 to the Bone. Endocr. Rev. 2005;26:743–774. doi: 10.1210/er.2004-0001. - DOI - PubMed
    1. Vaidya A., Kale V.P. TGF-β signaling and its role in the regulation of hematopoietic stem cells. Syst. Synth. Biol. 2015;9:1–10. doi: 10.1007/s11693-015-9161-2. - DOI - PMC - PubMed
    1. Bonewald L.F., Dallas S.L. Role of active and latent transforming growth factor β in bone formation. J. Cell. Biochem. 1994;55:350–357. doi: 10.1002/jcb.240550312. - DOI - PubMed
    1. Edwards J.R., Nyman J.S., Lwin S.T., Moore M.M., Esparza J., O’Quinn E.C., Hart A.J., Biswas S., Patil C.A., Lonning S., et al. Inhibition of TGF-β signaling by 1D11 antibody treatment increases bone mass and quality in vivo. J. Bone Min. Res. 2010;25:2419–2426. doi: 10.1002/jbmr.139. - DOI - PubMed
    1. Karst M., Gorny G., Galvin R.J., Oursler M.J. Roles of stromal cell RANKL, OPG, and M-CSF expression in biphasic TGF-beta regulation of osteoclast differentiation. J. Cell. Physiol. 2004;200:99–106. doi: 10.1002/jcp.20036. - DOI - PMC - PubMed

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